M. THEVIS H. GEYER U. MARECK (EDITORS) RECENT ADVANCES IN DOPING ANALYSIS (28) Proceedings of the Manfred Donike Workshop 38th Cologne Workshop on Dope Analysis 9th to 14th February 2020 SPORTVERLAG Strauß - Hellenthal 2020 1 Bibliografische Information Der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über abrufbar. Thevis, Mario; Geyer, Hans; Mareck, Ute (Eds.) Recent Advances in Doping Analysis (28). Proceedings of the Manfred Donike Workshop, 38th Cologne Workshop on Dope Analysis, 9th to 14nd February 2020 / [MDI e.V.] – 2020 Sportverlag Strauß. ISBN 978-3-86884-046-9 ©SPORTVERLAG Strauß Neuhaus 12 – 53940 Hellenthal Tel. +49 (0)2448/2470040 – Fax +49 (0)2448/9195610 E-mail: info@sportverlag-strauss.de www.sportverlag-strauss.de Satz: Autorensatz 2 MANFRED DONIKE WORKSHOP 2020 TABLE OF CONTENTS PAGE LECTURES Marchand A, Ericsson M, Audran M, Fenaille F, Capdeville P, Martin L: EPO Biosimilars with unusual profiles: characterization and detection .................................... 13-22 Heiland CE, Masquelier M: Sepharose gel beads: an effective technique for recombinant erythropoietin immuno- purification from blood ................................................................................................................ 23-27 POSTER PRESENTATIONS Sigmund G, Geyer H, Goergens C, Thevis M: Prevalence of the imidazole derivatives naphazoline, oxymetazoline and xylometa- zoline in doping control samples ................................................................................................. 28-31 Wicka M, Kaliszewski P, Grucza K, Stanczyk D, Drapala A, Konarski P, Zalewska Z, Siek P, Michalak D, Kwiatkowska D: Elimination profile of triamcinolone acetonide and its metabolite in human urine after multiple transdermal administrationed stimulants .................................................................... 32-36 Barbán Duarte L, Martínez Brito D, Correa Vidal T, Montes de Oca Porto R: Trend analysis and decision making in the Havana Anti-Doping Laboratory ............................. 37-40 Rodriguez Fernandez A, Laffita D, López Castillo D, Terrero O, Montes de Oca R: Increase in the incidence of THC in urine samples of athletes in 2019 ....................................... 41-45 Biasini GM, Donati F, Rutigliano L, de La Torre X, Botrè F: Proteomic analysis of biomarkers of red blood cells storage: towards the development of direct detection of autologous blood transfusion ................................................................... 46-51 Donati F, Botrè F, de La Torre X, Sian V, Biasini GM, Concetti L, Rutigliano L: Evaluation of the impact of genetic polymorphisms on basal values of human serum and urinary biomarkers: implications for anti-doping analyses ................................................. 52-58 3 PAGE Momobayashi A, Kageyama S, Okano M: Detection of homologous blood transfused erythrocytes by means of a two color fluorescence flow cytometric analysis ......................................................................................... 59-62 Zhou X, He S, Zhang L, Shen L, He C: Administration Study of rEPOs circulated in China ................................................................................... 63-67 He S, Zhou X, Zhang L, Shen L, He C: Troubleshooting for doping analysis of EPO ................................................................................ 68-70 Somay Selbes Y, Onol S, Kacar T, Demirel AH, Aytekin S: Effects of protease inhibitors on stability of urine hCG and LH in doping analyses ................... 71-73 Iannone M, Botrè F, de La Torre X: Direct and indirect detection of pseudo-endogenous steroids in plasma .................................. 74-79 Son J, Park JW, Lee SH: Multiple Biomarkers of Anabolic Agent Administration in Urine as Candidates for Doping Diagnosis ........................................................................................................................... 80-84 Danila G, Pop A, Toboc A, Stan C: 7-Keto-DHEA – a case study .......................................................................................................... 85-89 Geyer H, Fusshöller G, Haenelt N, Mareck U, Blatt C, Gougoulidis V, Hülsemann F, Piper T, Thevis M: The ratio 5aAdiol/E as indicator for the detection of testosterone doping in athletes with naturally low T/E – a case study ........................................................................................... 90-93 Wen C, Liu X, Tianshuo Z, Jingzhu W: Isolation of boldenone and its metabolite in urine sample and further determination of the C/ C ratios by gas chromatographycombustion-isotope ratio mass spectrometry ............. 94-97 Mabusa I, Du Preez H, van der Merwe T: Proposed suppression of phentermine derivatization by concomitant medication: A GC-MS case study and suggested solution ............................................................................. 98-102 Kaewklum M, Kaewklum S, Dithayam S, Wilairat P, Kongpatanakul S: Separation and identification of regioisomers of fluoroamphetamine and fluorometh- amphetamine in doping control by gas chromatography-mass spectrometry ...................... 103-107 Stacchini C, Dima AP, de La Torre X, Mazzarino M, Botrè F: In vitro studies of the metabolic profile of different classes of SARMs using liquid chromatographic-mass spectrometric techniques .................................................................. 108-112 Songsaeng R, Saardpun N, Kusamran T, Kongpatanakul S: Method Development for Identification of Growth Hormone Releasing Peptides (GHRPs) in Doping Control by UFLC/MS-IT-TOF ....................................................................... 113-117 4 PAGE Ahi S, Dubey S, Bhardwaj A, Sahu PL: Fast and sensitive detection of small peptides in human urine using liquid chroma- tography tandem mass spectrometry .............................................................................................. 118-123 Berghes B, Radu M, Cristea CD, Toboc A, Stan C: Validation of HIF activating agents confirmation methods ......................................................... 124-128 Grucza K, Wicka M, Drapala A, Konarski P, Stanczyk D, Michalak D, Kaliszewski P, Kwiatkowska D: Impact of various purity grades of acetonitrile and different mass spectrometry ionization methods on limits of detection of dopingsubstances by means of the “Dilute- and-Shoot” approach .......................................................................................................................... 129-133 Alechaga E, Terrero A, Monfort N, Ventura R: UHPLC-HRMS multi-screening method for doping control analyses in urine samples ........... 134-138 de la Cal A, Espinosa P, Blázquez I, Fernández B, García L, de Pedraza L, Carreras D, Muñoz G: Solving problems with HILIC columns: Compounds with difficult chromatographic behavior................................................................................................................................................. 139-143 Fernández-Álvarez M, Cuervo D, Baltazar-Martins JG, Plata MDM, Muñoz-Guerra JA, Lara B, Del Coso J, Muñoz G, Carreras D: Determination of tramadol and its metabolites in plasma by using liquid chroma- tography quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) ................................. 144-148 Cuervo D, Fernández-Álvarez M, Baltazar-Martins JG, Plata MDM, Muñoz-Guerra JA, Lara B, Del Coso J, Muñoz G, Carreras D: Determination of tramadol and its metabolites in urine via accuratemass quadrupole time-of-flight (Q-TOF) LC/MS ............................................................................................................ 149-152 PRESENTATIONS ABSTRACTS - LECTURES Gotzmann A: "Operation Aderlass”: Legislation as a cornerstone of anti-doping .............................................. 153 Rossi F, Saugy M: Dried Blood Spots: Alternative matrix to deter and detect Tramadol abuse in road cycling competitions ........................................................................................................................ 154 Knoop A, Planitz P, Wuest B, Thevis M: Evaluation of cobalt in doping control samples: Discrimination of different cobalt sources .............................................................................................................................................. 155 Paßreiter A, Thomas A, Walpurgis K, Thevis M: First steps towards uncovering gene doping with CRISPR/Cas9 .................................................... 156 5 PAGE Coll S, Monfort N, Alechaga &, Matabosch X, Pérez-Mañà C, Ventura R: Evaluation of the reporting level to detect betamethasone misuse in sports: additional studies after intranasal and intramuscular administrations .......................................................... 157 Walpurgis K, Rubio A, Wagener F, Krug O, Knoop A, Görgens C, Guddat S, Thevis M: Elimination profiles of microdosed ostarine mimicking contaminated products ingestion .................................................................................................................................................... 158-159 Garzinsky A, Thomas A, Krug O, Thevis M: Exhaled breath in doping controls: characterization of SensAbues® sampling devices using an aerosol test generator ....................................................................................................... 160 Mongongu C, Ericsson M, Madi Moussa E, Audran M, Coudoré F, Marchand A, Buisson C: Quantitative determination of IGF-I in dried blood using Volumetric Absorptive Micro- sampling and Top-down analysis with LC-HRMS ..................................................................... 161-162 Miller G, Madrigal K, Lamm J, Husk J, Eichner D: Detecting growth hormone abuse in dried capillary blood spots: results from a large- scale field trial and a controlled administration study ................................................................... 163 Dib J, Thevis M, Thomas A: Fully-automated "offline" dried blood spot (DBS) extraction and its implementation into doping control analysis ............................................................................................................. 164 Lange T, Thomas A, Walpurgis K, Thevis M: Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide and non-peptide doping agents by means of LC-HRMS ......................... 165 Solheim SA, Stillesby Levernæs MC, Mørkeberg J, Juul A, Upners EN, Nordsborg NB, Dehnes Y: Dried Blood Spot analysis of testosterone esters is applicable in doping analysis ....................... 166 Padilha M, Cavalcanti G, Gomes M, Carneiro G, Oliveira F, Pereira H: Can Graviola juice result an AAF for higenamine? .......................................................................... 167 de Wilde L, Roels K, van Eenoo P, Deventer K: Update on the use of 2D-LC-MS/MS in doping analysis ................................................................. 168 de La Torre X, Colamonici C, Molaioni F, Jardines D, Iannone M, Botrè F: Intentional or accidental clostebol detection. An Italian Story ..................................................... 169 Görgens C, Walker K, Boeser C, Wijeratne N, Martins C, Guddat S, Thevis M Paperspray mass spectrometry – A potential novel technique for the detection of polar compounds in sports drug testing? A pilot study ........................................................................... 170 6 PAGE Kraiem S, Al-Mahamed H, Al-Menhali A, Al-Thani N, Helaleh M, Samsam W, Al-Jaber MY, Bouabdallah S, Touil S, Georgakopoulos C, Beotra A, Mohamed Ali V, Al Maadheed M: Analytical strategy for the detection of ecdysterone and its metabolites in vivo in uPA(+/+)-SCID mice with humanised liver, human urine samples and estimation of the prevalence of its use in anti-doping samples ........................................................................... 171-172 Salamin O, Nicoli R, Schweizer C, Xu C, Pitteloud N, Saugy M, Kuuranne T: Longitudinal steroid profiling in healthy women: impact of transdermal testosterone administration .................................................................................................................................. 173 Zanitzer K, Erceg D, Forsdahl G, Gmeiner G: Comparison of the ratios of sulpho-conjugated and glucuroconjugated phase II meta- bolites of endogenous steroids in urine samples after administration of testosterone preparations via UHPLCHRMS .................................................................................................. 174-175 de Wilde L, Roels K, van Renterghem P, van Eenoo P, Deventer K: Steroid profiling in urine of intact glucuronidated and sulfated steroids using liquid chromatography-mass spectrometry .............................................................................................. 176 Albertsdóttir AD, van Gansbeke W, Coppieters G, Balgimbekova K, Polet M, van Eenoo P: Searching for new long term metabolites of metenolone anddrostanolone by gas chromatography-mass spectrometry with a focus on non-hydrolysed sulphates ........................ 177 Putz M, Piper T, Thevis M: Metabolite identification studies of trenbolone ............................................................................ 178 Piper T, Heimbach S, Adamczewski M, Thevis M: Investigations on the potential impact of different doping agents on the steroid profile by means of a steroidogenesis assay .............................................................................................. 179 Polet M, de Wilde L, van Renterghem P, van Eenoo P: A fast GC-C-IRMS method for the detection of epiandrosterone sulfate in urine ........................ 180 Hülsemann F, Fußhöller G, Lehn C, Thevis M: Excretion of 19-norandrosterone after consumption of boar meat .............................................. 181 Piper T, Rubio A, Thevis M: Investigations on carbon isotope ratios of potential novel long term metabolites of testo- sterone and testosterone prohormone misuse - 5α- and 5β-androstane-3α,17α-diol ......... 182-183 Iannella L, Botrè F, Colamonici C, Curcio D, Ciccarelli C, de La Torre X: Potential masking effect of different prednisolone based pharmaceutical formulations on IRMS confirmation analysis ........................................................................................................ 184 Piper T, Fußhöller G, Geyer H, Danila M, Toboc A, Thevis M: Detecting the misuse of 7-oxo-DHEA by means of carbon isotope ratio mass spectro- metry in doping control analysis ..................................................................................................... 185 7 PAGE Coppieters G, Judák P, van Haecke N, van Renterghem P, van Eenoo P, Deventer K: A high-throughput assay for the quantification of intact Insulin-like Growth Factor I in human serum using online SPE-LC-HRMS ....................................................................................... 186 Renovato-Martins M, Amorim C, Magdalena I, Dudenhoeffer-Carneiro AC, López S, Nunes I, Pizzatti L, Aquino Neto F, Mirotti L, Pereira HM: Monocytes as sensors of autologous blood transfusion detection ............................................... 187 Judák P, Coppieters G, Deventer K, Van Eenoo P: Urinary detection of insulin analogues: Improvements in the Ghent laboratory ......................... 188 Dehnes Y, Bogetveit S, Solheim SA, Sletten C, Mørkeberg J, Baastrup Nordsborg N: Effect of testosterone administration on GH-biomarkers IGF-1 and PIIINP .................................. 189 Renovato-Martins M, Dudenhoeffer-Carneiro AC, Du Rocher-Silva B, Soares F, Aquino Neto FR, Hasan M, Rahaman KA, Muresan A, Oh-Seung K, Mirotti L, Pereira HM: Erythrocyte phenotyping of international athletes by flow cytometry ......................................... 190 Narduzzi L, Buisson C, Morvan M, Marchand A, Audran M, Le Bouc Y, Ericsson M, Le Bizec B, Dervilly G: ABP-omics: a combined use of the haematological module and the “steroidomics passport” to track GH users ...................................................................................................... 191-192 Voss SC, Elsaftawy W, Saghbazarian S, Al-Kaabi A, Mohamed Ali V, Georgakopoulos C, Al Maadheed M, Orie N: Testing the applicability of HRP-conjugated Anti-EPO antibodies for direct rhEPO detection – proof of concept ........................................................................................................... 193 Dehnes Y, Miller G, Naud J, Martin L, Reihlen P, Reichel C: Inter-laboratory validation of biotinylated clone AE7A5EPO-antibody for EPO detection by single blotting of urine and blood samples ................................................................................ 194 Reichel C, Erceg D, Lorenc B, Scheiblhofer V, Farmer L, Zanitzer K, Geisendorfer T, Gmeiner G: Data from a low-dosed rEPO administration study ................................................................. 195-196 Leuenberger N, Salamin O, Gottardo E, Schobinger C, Reverter-Branchat G, Segura J, Saugy M, Tissot J, Kuuranne T: Detection of stimulated erythropoiesis using the transcriptomic ALAS2 biomarker in dried blood spot samples ................................................................................................................. 197 Walpurgis K, Weigand T, Knoop A, Thomas A, Reichel C, Dellanna F, Thevis M: Detection of follistatin-based inhibitors of the TGF-β signaling pathways in serum/ plasma by means of LC-HRMS/MS & Western blotting ................................................................. 198 8 PAGE Miller G, Cox H, Manandhar A, Husk J, Eichner D: Evaluation of CD71, Band3, and serum markers as evidence of recombinant EPO use: results from a non-drug longitudinal study and a blinded, placebo-controlled admini- stration study ................................................................................................................................... 199 PRESENTATIONS ABSTRACTS - POSTERS Nazir J, Cowan D, Caldwell R, Walker C, Bartlett C, Brailsford A, Wolff K: A Menu Driven Laboratory Information Management System (DCLIMS) for Anti-Doping Laboratories ...................................................................................................................................... 200 Son J, Cho Y: Model System for Brain Doping: Identification of the Correlation between Performance Enhancing and Alterations of Neurochemical Profiling .................................................................. 201 Araujo A, da Costa Nunes IK, Sardela V, Pereira H, Cabral L, Anselmo C: Application of the Zebrafish Water Tank Model to investigate the budesonide metabolic profile .............................................................................................................................. 202 Prado E, Gomes G, Lima de Sá C, Matos R, Sardela V, Anselmo C, Nunes I, Ramalho G, Vanini G, Padilha M, Oliveira MA, Oliveira A, Canuto C, Amaral L, Siqueira D, Aquino Neto FR, Pereira HM: Study of the metabolism of the cathinones α-PVP, methylone and 4-chloro- dimethylcathinone through the Zebrafish Water Tank Model ............................................... 203-204 Pettersson-Bohlin K, Sjöö M, Sjöö J: The sniffing dog/anti-doping dog Molly - A new dimension of doping controls ........................... 205 Mazzarino M, Ambrosio G, Grieco A, Parr MK, de La Torre X, Botrè F: Ecdysterone: A snapshot of use in the athlete’s population .......................................................... 206 Ahi S, Nimker V, Sharma R, Narula B, Sahu PL: Continuous Quality Improvement in doping control laboratories: Implementation of ISO/IEC 17025:2017 and WADA ISL 2019, Challenges and Future Perspective ............................. 207 Vargas García-Tenorio S, Serrano Garde E, Soldevilla Navarro AB, Cortes Carrillo N, Martín Escudero P, Muñoz-Guerra JA, Galindo Canales M, del Prado N, Fuentes Ferrer M, Fernández Pérez C, Brouwer B, Benish P: Indirect detection of steroids abuse in sports based on androgenic activity measure- ment by AR Calux bioassay ....................................................................................................... 208-209 Voss SC, Elsaftawy W, Saghbazarian S, Al-Kaabi A, Al-Hamad K, Al Maadheed M, Nofal A, Georgakopoulos C: IAAF Doha 2019 World Championships – Sysmex XN-1000 Hematology Results ......................... 210 9 PAGE Mazzarino M, di Costanzo L, Comunità F, de La Torre X, Botrè F: Dried blood spot in doping control test: evaluation of different blood collection device ............ 211 Reihlen P, Blobel M, Völker-Schänzer E, Schänzer W, Geyer H, Thevis M: Case reports of re-analyzed rEPO suspicious doping control urine samples from 2011-2014...... 212 Ericsson M, Bhuiyan H, Ekström L: GHRH daily administration can be determined with longitudinally monitoring of IGF-I .............. 213 Alechaga E, Pont L, Terrero A, Monfort N, Ventura R: Optimization of an immunopurification method for the screening of growth hormone- releasing hormones in urine doping control ............................................................................ 214-215 Martin L, Maurice T, Audran M, Ericsson M, Marchand A: Detection of a non-erythropoietic EPO, Neuro-EPO, in blood after intra-nasal administration in rat ................................................................................................................. 216-217 Kaliszewski P, Siek P, Zalewska Z, Michalak D, Grucza K, Wicka M, Kwiatkowska D: Method development for the detection of carbamylated erythropoietin (CEPO) for doping control purposes .................................................................................................................. 218 Zhou X, He S, Zhang L, Shen L, He C: Assessment on rat EPO as Internal Standard in Doping ControlSamples for EPO Detection with SAR-PAGE using Biotinylated Primary Antibody ................................................... 219 Reihlen P, Weiß P, Blobel M, Walpurgis K, Thevis M: Evaluation of PEGylated EPO-conjugates as internal standards for EPO analysis in doping controls ................................................................................................................................. 220 Kiousi P, Fragkaki AG, Lyris E, Petrou M, Georgakopoulos C, Angelis YS: Novel Long-Term Markers for Methylnortestosterone Misuse in Human Urine .......................... 221 Martinez Brito D, Botrè F, de La Torre X: Mass spectrometric analysis of 7-oxygenated androst-5-ene steroids - an in vitro study ........... 222 Martinez Brito D, Iannone M, Tatangelo M, Molaioni F, Botrè F, de La Torre X: Old compounds, new alertness: In vitro and in vivo experiments using methyl- testosterone .............................................................................................................................. 223-224 Deventer K, van Gansbeke W, van Eenoo P: Alternative approach for the detection of aminoglutethimide ..................................................... 225 Andersson A, Elings Knutsson J, Ekström L, Lindén Hirschberg A: ABP profile in relation to transdermal testosterone administration in women ........................... 226 Suominen T, Ericsson M, Ekström L, Schulze J: ABP-profile in relation to the menstrual cycle ................................................................................ 227 10 PAGE Andersson A, Eklund E, Hirschberg AL, Ekström L: ABP urinary metabolites and how they correlate to serum hormone levels in female athletes ............................................................................................................................................. 228 Brailsford A, Wojek N, Chih-Wei C, Walker C, Cowan D, Wolff K: Determination of anabolic steroids in dried blood using microsampling and gas chromatography-mass spectrometry: application to a testosterone gel administration study ................................................................................................................................................. 229 Cavalcanti G, Zhu Y, Wijeratne N, Martins C, Padilha M, Pereira H: Application of VeriSpray ion source Triple Quadrupole Mass Spectrometry for stimulants and narcotics analysis in oral fluid ................................................................................ 230 Kowalczyk K, Torres Elguera JC, Konopka A, Jarek A, Stanczyk D, Wicka M, Kwiatkowska D, Bulska E: In vitro metabolic studies of emerging selective androgen receptor modulators (PF-0620414, TFM-4-AS-1, BMS 564929 and GSK 2881078) and implementation of the results into LC-MS/MS-based dopingcontrol analysis ............................................................. 231-232 Sato M, Kageyama S, Okano M: Detection of the selective estrogen receptor modulator Bazedoxifene in human urine by LC-MS/MS .................................................................................................................................... 233 Dubey S, Sah S, Sahu PL: Comprehensive screening of doping agents by ‘dilute and shoot’ urine analysis using high resolution/high accuracy mass spectrometry ......................................................................... 234 Mongongu C, Marchand A, Buisson C, Coudoré F, Audran M, Ericsson M, Madi Moussa E: A sensitive detection of synthetic IGF-I analogs using a mass spectrometric immuno- assay and nano-ultra-high-performance liquid chromatography coupled to high resolution mass spectrometry ......................................................................................................... 235 Rahaman KA, Muresan AR, Rafique FB, Kim KH, Lee KM, Min H, Kim HJ, Sung C, Son J, Lee J, Kwon OS: Metabolites of TB-500 peptide in various in-vitro enzyme systems by UHPLC-MS/MS ............... 236 Rafique FB, Muresan AR, Rahaman KA, Kim KH, Lee KM, Min H, Kim HJ, Sung C, Son J, Lee J, Kwon OS: In vitro phase I metabolism study of the diuretic clopamide by LCMS/MS .................................. 237 Pettersson-Bohlin K, Oesterlind L, Börjesson A, Ekström L: Bis-(4-fluouphenyl)-methylpiperazine detection in urine from female and male Caucasians after oral lomerizine intake .......................................................................................... 238 Silveira C, Sardela V, Anselmo C, Pereira HM: Chiral analysis of amphetamine and methamphetamine with Mosher derivatization by LC-MS2 ........................................................................................................................................ 239-240 11 PAGE He C, Shen L, Zhou X, He S: Quantification of intact human IGF-I in serum by LC-MS/MS ........................................................ 241 Fragkaki A, Kiousi P, Paloumpis N, Kioukia-Fougia N, Panderi I, Angelis I: MS Behavior of Intact Phase-II Metabolites of Anabolic Androgenic Steroids after Derivatization for their Detection by LC/(MS)MS .................................................................... 242-243 Görgens C, Guddat S, Bosse C, Knoop A, Geyer H, Thevis M: Implementation of the HIF activator IOX-2 in routine doping controls – pilot study data ........... 244 SCIENTIFIC BOARD B. Ahrens, Los Angeles (USA) S. Chundela, Kreischa (Germany) H. Cox, Los Angeles (USA) X. de la Torre, Rome (Italy) H. Geyer, Cologne (Germany) G. Gmeiner, Seibersdorf (Austria) R. Kazlauskas, Sydney (Australia) T. Kuuranne, Lausanne (Switzerland) U. Mareck, Cologne (Germany) J-F. Naud, Montreal (Canada) T. Piper, Cologne (Germany) C. Reichel, Seibersdorf (Austria) W. Schänzer, Cologne (Germany) J. Schulze, Stockholm (Sweden) M. Thevis, Cologne (Germany) A. Thomas, Cologne (Germany) P. van Eenoo, Ghent (Belgium) R. Ventura, Barcelona (Spain) 12 Marchand A , Ericsson M , Audran M , Fenaille F , Capdeville P , Martin L EPO Biosimilars with unusual profiles: characterization and detection Département des Analyses, AFLD, Châtenay-Malabry, France ; Laboratoire d'Etude du Métabolisme des Médicaments, CEA, Saclay, France Abstract Biosimilars of recombinant erythropoietin (rEPO) can present some small structural differences compared to the reference product. Three biosimilars, with apparent lighter molecular weight than the genuine rEPO (Eprex ), were selected for further investigation: Jimaixin™ authorized in China, and Hemax and Epotin™ authorized in Algeria. The aims of this research were: i) to study the electrophoretic EPO profiles obtained after a spike of the three biosimilars in urine and plasma by using improved IEF-PAGE and SDS- PAGE methods and to evaluate their identification using reevaluated criteria ii) to test complementary strategies to improve their detection: a two-dimensional electrophoresis approach (SDS separation following IEF) or a neuraminidase treatment of the sample (proposed to increase the separation between recombinant and endogenous EPO by SDS-PAGE), iii) to compare using MALDI-TOF the specific N- glycosylation pattern of each biosimilar with the original rEPO Eprex . Experiments with spiked samples indicated that the biosimilars were more difficult to detect at low doses than Eprex , in particular, Epotin™ and Jimaixin™ by IEF-PAGE in urine and Hemax by SDS-PAGE in plasma. IEF-PAGE for plasma samples and SDS-PAGE for urine samples had the highest identification rates considering the three biosimilars. Two-dimensional electrophoresis experiments did not improve the detection. Samples pre-treatment with neuraminidase gave more promising results for low doses as it slightly increased the detection. Compared to Eprex N-glycosylations, all three biosimilars presented a decrease in the most complex forms (loss of sugar antennae and sialic acids) while bi and tri antennae forms were enriched. Introduction Since expiration of the patents for the first generation of recombinant erythropoietin (rEPO) drugs, pharmaceutical companies world-wide have begun to produce rEPOs copies. All licensed EPO biosimilars shall demonstrate the same safety and efficacy for therapeutic use than the original drug but they can present some small structural differences compared to the reference product due to some variations in the production process [1]. Consequently the electrophoretic profile of a biosimilar can be slightly different from a conventional rEPO one and its detection in a doping control sample might be challenging. After analyzing various EPO biosimilars, three drugs were selected for further characterization: Jimaixin™ (Jintan Ltd, China), Hemax (Biosidus, Argentina) and Epotin™ (Gulf Pharmaceutical Industries, United Arab Emirates). They presented slight differences compared to the original epoetin alpha Eprex using SDS-PAGE (lower molecular weight) and IEF-PAGE (more isoforms in the basic area). 1 1 1 2 1 1 1 2 ® ® ® ® ® ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-913 The aims of this research were: i) to study EPO electrophoretic profiles obtained by IEF-PAGE and SDS-PAGE for the 3 biosimilars after a spike in urine and plasma and to evaluate their detection following WADA’s criteria [2], ii) to test potential complementary strategies to improve detection: a two-dimensional electrophoresis approach or a neuraminidase treatment of the sample (cleavage of sialic acids) before electrophoretic separation, iii) to evaluate the specific N-glycosylation pattern of each biosimilar (Jimaixin™, Hemax and Epotin™) and compare it with Eprex to identify differences with the original epoetin alpha drug. Experimental Detection of rEPOs by IEF and SDS-PAGE analysis Spikes of Jimaixin™, Hemax , Epotin™ and Eprex were performed in 6 different urine samples (starting volume 9 mL) and 3 different plasma samples (starting volume 500 µL). After an estimation of endogenous EPO content in each sample (using human EPO Quantikine ELISA kit from R&D Systems), addition of rEPO was performed to obtain mixes of rEPO/endogenous EPO with approximate abundances of 35/65, 50/50 and 65/35 respectively. EPO analyses were performed as in Martin et al. [4] using optimized protocols to improve sensitivity of detection and, for IEF-PAGE method, criteria slightly modified compared to WADA’s TDEPO to recruit more suspicious samples. All urine and plasma samples were immuno-purified using an anti-EPO coated well from a human EPO ELISA detection kit (STEMCell Technologies) for urine and magnetic beads coated with anti-human EPO antibody 9C21D11 (R&D systems) for plasma. Eluates were analyzed. A single-blot was performed after IEF-PAGE and SDS-PAGE, and EPO immuno-detection was conducted using a biotinylated anti-EPO antibody (clone AE7A5, R&D Systems). Complementary tests Neuraminidase treatment: Biosimilars and Dynepo standards as well as retentates resulting from the ultrafiltration of urine samples (spiked with the biosimilars (50/50 mix) or not) were incubated in wells of a Stemcell EPO ELISA plate for 2 hours at 37°C. After binding of EPO, desialylation was performed by addition of a 5 mU neuraminidase solution and incubation 1 h at room temperature as previously described [3]. After elution, SDS-PAGE analysis was performed as usual and IEF-PAGE using a 2-10 pH gradient instead of 2-6. 2D-gel: Standards and eluates from urine samples with or without spike of biosimilars (50/50 mix) and with or without neuraminidase treatment were analyzed as in Desharnais et al. [3]. For the IEF part, samples without neuraminidase were separated on a 2-6 pH gradient gel and samples treated with neuraminidase on a 3-11 pH gradient gel. Biosimilar glycan characterization by MALDI-TOF/TOF rEPO standards (Eprex and the three biosimilars) N-glycosylations were characterized by mass spectrometry using a previously published protocol [5,6]. Briefly, after protein denaturation, 4 µg of each rEPO were treated with 2 units of N-Glycosidase F (Roche) overnight at 37°C to release N-glycans. After acidification, proteins were precipitated using ice-cold ethanol and resulting N-linked glycans were then ® ® ® ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-914 purified on a porous graphitic carbon SPE cartridge using a mixture of 40% acetonitrile, 0.1% trifluoro‐ acetic acid for elution. After a permethylation step, N-glycans were finally purified on C-18 spin columns (Pierce) and eluted in 80% acetonitrile. After evaporation, final reconstitution was made in 50% methanol. 0.5 µL were mixed with 0.5 µL DHB matrix (10 mg/mL 2,5 dihydroxybenzoic acid (DHB) in methanol/H2O (50/50, v/v) containing 1 mM sodium acetate). Analysis were performed using a MALDI- TOF/TOF instrument UltrafleXtreme instrument (Bruker Daltonics). Resulting spectra were then analyzed using flexAnalysis software for identification of N-glycan specific rEPO structures. Three separated experiments with 2 or 3 technical replicates each time were performed and the mean of these replicates was considered representative of the glycosylations of each rEPO. The relative abundance of each glycan structure was estimated. Results and Discussion Standards characterization The formulation composition indicated the presence of human albumin in all three biosimilars contrary to Eprex . This was corroborated by Coomassie blue staining after SDS-PAGE analysis. Strong signals around 60 kDa attributable to albumin and other bands probably due to other serum proteins were detected (Figure 1A). After immuno-extraction (data not shown), almost all additional protein signals were eliminated. After immuno-detection of EPO, only a clear rEPO signal was found. Compared to Eprex , Jimaixin™ and Epotin™ had broader bands by SDS-PAGE with half of the signal below the Dynepo reference corresponding to the endogenous EPO zone. Hemax signal showed a more compact band with an apex close to the Dynepo reference. By IEF-PAGE (Figure 1B) all three biosimilars had a basic profile clearly indicative of a recombinant EPO. Epotin™ and Jimaixin™ presented more isoforms than Eprex in the basic area, in particular an intense fifth band, and Jimaixin™ and Hemax had respectively two and one isoforms more acidic than Eprex and located in the endogenous area of the gel. Two batches of each biosimilar were tested and showed similar profiles but batch-to-batch variability might exist. ® ® ® ® ® ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-915 Figure 1. Analysis of the three biosimilars by SDS-PAGE and IEF-PAGE. A. SDS-PAGE Left part: Coomassie staining- Right part: EPO immuno-detection after Western-Blot. Bovine serum albumin (BSA), Eprex and urinary EPO (uEPO) were added for comparison. B. IEF-PAGE. The most intense bands in each profile are indicated by black arrows. EPO references (REF): BRP and NESP as well as Eprex and urine (endogenous EPO) were added for comparison. Detection in urine and plasma after spiking rEPOs were identified according to the following criteria. For SDS-PAGE, the presence of a smear above endogenous EPO and Dynepo apex position was indicative of the presence of rEPO. For IEF-PAGE, a ratio between the second most intense band in the basic area of the gel and the most intense band in the endogenous area (2B/N) was calculated. The criteria for a suspicious sample was 2B/N > 0.85 for urine, and the criteria used for plasma samples was 2B/N ≥ 1.30. These criteria were slightly modified compared to WADA’s TDEPO criteria after a reevaluation study in order to recruit more suspicious samples without increasing the number of false suspicions [4]. For urines using this new criteria 2B/N > 0.85 instead of TDEPO's criteria (summarized as 2B/N > 1) led to 11 more samples classified as suspicious on 72 spiked samples analysed (+15%). Similarly for plasma samples using 2B/N ≥ 1.30 instead of TDEPO's criteria (summarized as 2B/N ≥ 2) led to 10 more samples classified as suspicious on 36 spiked samples analysed (+28%). All non spiked samples were negative with the new criterias applied. Increasing concentrations of biosimilar were reflected in the increasing 2B/N ratios by IEF-PAGE and increasing smear intensity above endogenous EPO signal by SDS-PAGE (Figure 2 and 3). ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-916 Figure 2. rEPO analysis of one urine spiked with increasing concentrations of each Biosimilar and Eprex . The endogenous EPO in the urine without spike (uEPO) and a standard of each biosimilar were also analyzed. A. SDS-PAGE method: REF corresponded to Dynepo (lower band) and NESP (upper band). The results of identification are indicated (– = negative results, +/– = suspicious, + = positively identified). B. IEF- PAGE method: REF corresponded to BRP (upper bands) and NESP (lower bands). The results of the 2B/N ratio ( > 0.85 = suspicious) and results of identification are indicated. ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-917 Figure 3. rEPO analysis of one plasma spiked with increasing concentrations of each biosimilar and Eprex . The endogenous EPO in the plasma without spike (plasma EPO) and a standard of each biosimilar were also analyzed. A. SDS-PAGE method: REF corresponded to Dynepo (lower band) and NESP (upper band). The results of identification are indicated ( – = negative results, +/– = suspicious, + = positively identified). B. IEF- PAGE method: REF corresponded to BRP (upper bands) and NESP (lower bands). The results of the 2B/N ratio ( ≥ 1.30 = suspicious) and results of identification are indicated. The results (summarized in Table 1) demonstrated that while Eprex was efficiently identified by both IEF-PAGE and SDS-PAGE methods, the identification of the biosimilars was more challenging in particular for the lowest dose (35:65 mixes of rEPO:endogenous EPO). The two different methods of separation were complementary because, depending on the biosimilar, one or the other was more efficient. Jimaixin™ and Epotin™ were more easily identified by SDS-PAGE while Hemax was better identified by IEF-PAGE. SDS-PAGE method applied on urine samples or IEF-PAGE on plasma samples appeared the best options to obtain the greatest number of suspicions. ® ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-918 Table 1. Summary of the results obtained for the REPO detection in spiked urine and plasma using either IEF-PAGE or SDS-Page methods. Six urines and 3 plasma samples were spiked at three different ratios of recombinant EPO to endogenous EPO (35/65, 50/50 and 65/35). Percentage of suspicious samples identified by each method are indicated (upper table). For IEF-PAGE the percentages obtained when using the new criteria for suspicions and those obtained when using TDEPO's criteria are indicated. In addition percentage of samples with clear positive identification in case of confirmation by SDS-PAGE are also indicated (lower table). The best conditions are shown in bold. Complementary tests to increase identification Neuraminidase treatment: Neuraminidase cleaves the sialic acids at the end of the N-glycan chains and this was suggested as a way to increase the distance of migration between recombinant EPO and endogenous EPO (which contains more sialic acids)[3]. - SDS-PAGE results (Figure 4A and 4B): All EPO profiles after treatment by neuraminidase had lower molecular weight and wider signal. Dynepo treated by neuraminidase was evaluated: the Dynepo +neuraminidase band was just above the endogenous EPO+neuraminidase signal and presented a sharp band: it could be used as a marker to separate the endogenous zone from the exogenous zone. With this marker, urines spiked with biosimilars were slightly more suspicious than before neuraminidase treatment in particular for Hemax . However a loss of intensity could sometimes be observed after neuraminidase. - IEF-PAGE results (Figure 4C): Due to the cleavage of sialic acids, neuraminidase treatment induced a shift of the EPO profiles towards the basic part of the gel and a 2-10 pH gel gradient was necessary. After spike, for all three biosimilars, three small bands could be observed interspaced between the three most basic isoforms of EPO even at low amounts. This specific pattern was a distinctive sign and increased the sensitivity of detection compared to the conventional IEF-PAGE method. Samples analyzed by both IEF-PAGE and SDS-PAGE before and after treatment by neuraminidase indicated that the detection of biosimilars by IEF-PAGE method (intact EPO) was less performant than the IEF-PAGE protocol with neuraminidase-treated samples. IEF-PAGE on neuraminidase treated samples had similar sensitivity than the SDS-PAGE method on untreated samples. Finally SDS-PAGE method on neuraminidase treated samples was a bit more performant to identify rEPO biosimilars at low doses and could be used in case of a suspicious sample. ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-919 Figure 4. Effect of neuraminidase on EPO profiles of endogenous EPO (urine), Eprex and biosimilars. A. SDS-PAGE. Urine EPO without spike (uEPO) or spiked with each rEPO at 50:50 with or without neuraminidase treatment were analyzed. B. SDS-PAGE: uEPO or urine spiked with increasing amount of Hemax as well as Dynepo without and and with neuraminidase treatment were analyzed. Dynepo was used to delimitate endogenous and exogenous zone for EPO and results of identification are shown. C. IEF-PAGE. uEPO standard or urine spiked with increasing amount of each biosimilar treated by neuraminidase were analyzed on a 2-10 pH gradient gel. Black arrows indicate newly identified bands specific of rEPOs and well expressed in biosimilars. Two dimension gel electrophoresis : The first attempts confirmed the previously published observations with Eprex [3], but the 2D-separation did not improve biosimilars identification compared to IEF-PAGE or SDS-PAGE (data not shown). This method is also technically complex and no clear criteria were identified for biosimilar detection. Analysis of N-Glycosylations As shown in Figure 5, all profiles were qualitatively similar between Eprex and the 3 biosimilars: they all exhibited the same N-glycan forms. This indicates that EPO was correctly produced. However, there were significant differences in relative abundance for some specific hyposialylated N-glycan forms. In particular we identified a significant enrichment in bi-antennary and tri-antennary forms compared to Eprex and a decrease of the tetra-antennae tetrasialylated form (the main N-glycan form of Eprex ) towards an increase of the tetra-antennae N-acetyl-lactosamine (LacNAc) bisialylated form for Epotin™ and Jimaixin™. This might happen due to the conditions of culture or during the purification process [7- 9]. Epotin™ and Jimaixin™ presented the most pronounced differences while Hemax had a ® ® ® ® ® ® ® ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-920 glycosylation profile closer to Eprex . This was in good agreement with the IEF-PAGE profiles of all 3 biosimilars. Figure 5. Graphical representation of the relative abundance of each N-glycan EPO structures for Eprex and the three biosimilars. Mean+/-SD of all replicates obtained for each immuno-purified biosimilar (n=5 for Epotin™, n=6 for Hemax and Jimaixin™) and for Eprex (n=6) are presented. Major differences with Eprex® are shown in red squares. Statistical comparison with Eprex for each N-glycan structure were performed: *: p < 0.05, **: p < 0.01. Green circles, mannoses; yellow circles, galactoses; blue squares, N-acetyl- glucosamines; red triangles, fucoses; purple diamonds, sialic acids. Conclusions In this study, 3 biosimilars of rEPO (Epotin™, Hemax and Jimaixin™) were characterized and presented unusual profiles by IEF-PAGE and/or SDS-PAGE. After a spike in urine and plasma, the detection was slightly more difficult than conventional rEPO Eprex in particular with low doses. SDS-PAGE analysis and urine matrix were the best option for the detection of all biosimilars. In addition, this method avoid the risk of false suspicions due to active/effort urines that might happen with IEF-PAGE. Using a 2D-gel analysis approach was not useful but a treatment with neuraminidase slightly improved the detection. Comparison of N-glycosylations between the biosimilars and Eprex indicated an increase in less acidic and less complex glycan forms the three biosimilars in line with their different electrophoretic profiles. These findings needs to be confirmed by an appropriate administration study in healthy men. References 1. Thomson RI, Gardner RA, Strohfeldt K, et al. Analysis of Three Epoetin Alpha Products by LC and LC-MS Indicates Differences in Glycosylation Critical Quality Attributes, Including Sialic Acid Content. Anal Chem. 2017;89(12):6455-6462. ® ® ® ® ® ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-921 2. TD2014EPO. Harmonization of analysis and reporting of erythropoiesis stimulating agents (ESAs) by electrophoretic techniques. www.wada-amaorg/sites/default/files/resources/files/WADA-TD2014EPO-v1- Harmonization-of-Analysis-and-Reporting-of-ESAs-by-Electrophoretic-Techniques-ENpdf. 2014 3. Desharnais P, Naud JF, Ayotte C. Desialylation improves the detection of recombinant erythropoietins in urine samples analyzed by SDS-PAGE. Drug Test Anal. 2013;5(11-12):870-876. 4. Martin L, Martin J, Collot D, et al. Improved detection methods significantly increase the detection window for EPO microdoses. Drug Test Anal. 2020;in press. 5. Bruneel A, Cholet S, Drouin-Garraud V, et al. Complementarity of electrophoretic, mass spectrometric, and gene sequencing techniques for the diagnosis and characterization of congenital disorders of glycosylation. Electrophoresis. 2018;39(24):3123-3132. 6. Goyallon A, Cholet S, Chapelle M, Junot C, Fenaille F. Evaluation of a combined glycomics and glycoproteomics approach for studying the major glycoproteins present in biofluids: Application to cerebrospinal fluid. Rapid Commun Mass Spectrom. 2015;29(6):461-473. 7. Nam JH, Zhang F, Ermonval M, Linhardt RJ, Sharfstein ST. The effects of culture conditions on the glycosylation of secreted human placental alkaline phosphatase produced in Chinese hamster ovary cells. Biotechnol Bioeng. 2008;100(6):1178-1192. 8. Gupta SK, Shukla P. Glycosylation control technologies for recombinant therapeutic proteins. Appl Microbiol Biotechnol. 2018;102(24):10457-10468. 9. Zhang P, Woen S, Wang T, et al. Challenges of glycosylation analysis and control: an integrated approach to producing optimal and consistent therapeutic drugs. Drug Discov Today. 2016;21(5):740-765. Acknowledgements This project has been carried out with the support of the World Anti-Doping Agency (grant 18B04AM) and AFLD’s own resources. MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-922 Heiland CE, Masquelier M Sepharose gel beads: an effective technique for recombinant erythropoietin immunopurification from blood Doping Control Laboratory Karolinska University Hospital, Stockholm, Sweden Abstract We have previously shown that the performance of the immunopurification step used to concentrate erythropoietin (EPO) and its recombinants before electrophoretic analysis has an impact on the detection of EPO. Although urine is the most common doping control matrix, blood seems to be a better alternative for abuse detection as highlighted by WADA’s published statistics for EPO between 2015 and 2018. In this study, an EPO Purification Gel Kit for Blood (EPGK for Blood; MAIIA Diagnostics) containing anti-EPO antibody-linked sepharose gel beads was used to immunopurify blood samples fortified with four erythropoiesis stimulating agents (BRP, NESP, EPO-Fc and CERA) and the results were validated, with special attention to detection limit and eluate stability determination. Lately, dried blood spots (DBS), an alternative collection method and matrix, has been attractive for the anti-doping scene. Therefore, the same immunopurification technique was also applied to extract ESA from DBS to evaluate the impact of low blood volume of the spots on the detection limit and if multiple spots can compensate and increase the method sensitivity. Introduction Between 2015 and 2018, erythropoiesis-stimulating agent (ESA) adverse analytical findings (AAFs) in blood represented over 20% of the total number of ESA AAFs, although blood tests made up only about 8% of the total ESA tests taken [1], showing the importance of blood as a matrix for recombinant erythropoietin (rEPO) detection. This could be attributed to blood stabilizing effects from high protein concentration and to the cold transport requirement reducing erythropoietin (EPO)-degrading bacteria in blood. Consequently, more blood analysis should result in better detection of rEPO. Choosing an effective immunopurification method is also crucial, as shown previously [2]. The EPO Purification Kit (EPK; MAIIA Diagnostics), commonly used in anti-doping laboratories, has caused filtration issues in some urine, therefore MAIIA developed a gel kit (EPGK) using sepharose gel beads coupled to anti-EPO antibodies, aiming to simplify purification, increase sample throughput, and reduce sample handling, while keeping a good EPO detection both in urine [3] and in blood. The possibility of using DBS for EPO detection after ELISA immunopurification has been described by Reverter-Branchat et al. [4], thus initiating interest in the EPGK’s capabilities for this matrix. Therefore, in this study, serum and DBS were spiked and immunopurified with the EPGK to evaluate and validate the kit for EPO blood analysis. The validation evaluated the detection limit and immunoextract stability, the application of this technique to DBS, and the impact of DBS sample volume on EPO detection. MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-923 Experimental Materials Reference standards used for spiking were: Biological Reference Protein (BRP) (European Directorate for the Quality of Medicines and Healthcare; Strasbourg, France); NESP (Aranesp; Amgen, Thousand Oaks, CA, USA); CERA (Mircera; Roche, Mannheim, Germany); and EPO-Fc (Prospec, Ness-Ziona, Israel). EPO Purification Gel Kit (EPGK) for Blood was provided by MAIIA Diagnostics (Uppsala, Sweden). Whatman 903 protein saver cards were from GE Healthcare (Uppsala, Sweden). Trayster Digital (IKA; Staufen, Germany) was used for EPO extraction. Biotinylated AE7A5 monoclonal antibody from R&D Systems (MN, USA) was used as the detection antibody. Streptavidin-HRP was from Calbiochem (CA, USA). Method Sample collection: Serum samples were collected from volunteers, anonymized, and pooled. Samples were either kept blank or fortified with four ESAs. Whole blood was collected from volunteers, anonymized, spiked and pipetted onto Whatman 903 protein saver cards (50 µL/spot). Immuno-extraction and -purification: The method was performed according to the manufacturer’s instructions [5]. Briefly, 0.5 mL of serum or 1 whole blood spot (50 µL) and Sample Buffer were placed in a column attached to a funnel, which was then inserted into a Falcon tube, and incubated end-over-end. The sample was filtered with vacuum pressure, the gel beads were washed, and the EPO was eluted with Elution Buffer (35 µL for serum and 50 µL for DBS). SAR-PAGE and Western blot: A single blot analysis was done as previously described [3], with a few changes to the sample volume used for analysis and the detection antibody. 8 µL (serum) or 16 µL (DBS) of sample were loaded onto the electrophoretic gel. Immunodetection was performed as follows: biotiny‐ lated antibody (0.5 µg/mL) overnight, wash, streptavidin-HRP (0.5 µg/mL) for one hour, and a wash. Serum stability: The stability of spiked serum eluates was investigated at -20°C, 4°C, and room temperature for 0, 1, 7, and 30 days after immunopurification. Serum samples were spiked with BRP (150 pg/mL serum), NESP (30 pg/mL serum), CERA (1000 pg/mL serum), and EPO-Fc (500 pg/mL serum). Limit of detection for serum: Five decreasing concentrations of the four ESAs were analyzed in 1 mL of serum by spiking the samples as follows: 2000 – 18.8 pg CERA; 1000 – 1.8 pg EPO-Fc; 60 – 3.8 pg NESP; 300 – 18.8 pg BRP. Limit of detection for DBS: Six decreasing concentrations of the 4 ESAs were analyzed in 50-µL DBS by spiking whole venous blood as follows: 200 – 6.3 pg CERA; 50 – 1.6 pg EPO-Fc; 5 – 0.2 pg NESP; 25 – 0.8 pg BRP. DBS sample volume: Venous blood was spiked with 4 ESAs (126 pg BRP, 50 pg NESP, 1000 pg CERA, and 250 pg EPO-Fc per mL whole blood) and pipetted onto a filter paper support and four 50-µL spots were immunopurified together and compared with immunopurification of one spot and 100 µL of serum spiked with the same ESA concentrations. Purification of four 50-µL DBS discs is equivalent to approximately 100 µL of serum. The volumes of immunopurified sample were adjusted for loading on the electrophoretic gel. MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-924 Results and Discussion The stability of spiked serum eluates is presented in Figure 1. All ESAs spiked in serum samples were quite stable at all temperatures for at least 30 days, with a decrease mainly for CERA and EPO-Fc at room temperature. Therefore, the immunopurified samples should preferably be kept at 4°C or -20°C. Figure 1. Stability experiment of eluates from serum samples spiked with 4 ESAs and immunopurified with EPGK. Samples were stored at -20 °C, 4 °C, and room temperature for 1, 7, and 30 days and compared to freshly purified sample (Day 0). (A) Gel image of the eluate stability experiment. (B) Graphs of the variation in percentage of band intensity of stored samples compared to freshly purified sample. For serum spiked and immunopurified by EPGK, LOD was determined to be approximately 31.2 pg for CERA, 15.6 pg for EPO-Fc, 3.8 pg for NESP, and 18.8 pg for BRP per mL serum. However, this is calculated assuming a recovery range of 100% and for application of only 8 µL of immunopurified eluate on the gel. The LOD obtained for DBS was determined to be approximately 125 pg for CERA, 62.5 pg for EPO-Fc, 12.5 pg for NESP, and 62.5 pg for BRP per mL whole blood. The detection limits determined by Reverter-Branchat et al. (1200 pg CERA, 60 pg for NESP, and 60 pg for BRP per mL whole blood) and obtained using 25-µL spots of modeled-blood [4] were similar for BRP but higher for NESP and CERA MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-925 compared to our results. Indeed, for DBS our LOD was comparable or better, but it was still 3 to 4 times higher than the LOD we obtained for serum immunopurification, as could be expected, which was confirmed in Figure 2. When comparing band intensities of these two matrices, spiked alike, it is apparent that the ESA detection is comparable to each other (Figure 2A and B). Therefore, it seems that multiple DBS are an effective approach in order to reach the same sensitivity as in serum. The importance of sample volume for EPO analysis was further demonstrated when 4 DBS were compared to only 1 DBS (Figure 2C). There is a noticeable improvement in EPO detection as the number of discs increases. Figure 2. Sample volume comparison for serum and DBS. (A) Serum samples (two replicates of 100 µL each) spiked and immunopurified with the EPGK. (B) DBS samples (two replicates of four 50-µL spots each) spiked and immunopurified with the EPGK. (C) DBS samples (two replicates of one 50-µL spot each) spiked and immunopurified with the EPGK. Concentrations (pg/mL whole blood): BRP: 125 pg, NESP: 50 pg, CERA: 1000 pg, EPO-Fc: 250 pg. Volumes loaded onto electrophoretic gel: 11.2 µL for serum and 16 µL for DBS. Since sample volume is a limitation for optimal EPO detection in DBS, there is a need for sufficient sample volumes and sensitive methods when DBS are added to the anti-doping sample matrix repertoire. Therefore, future experiments will include efforts to improve sensitivity by concentrating the DBS eluate during immunopurification and loading the entire volume onto the electrophoretic gel in order to compensate for the small initial volume of whole blood. This will hopefully optimize ESA detection in DBS. Other possible solutions might be to optimize transfer during the blotting step by performing a discon‐ tinuous semi-dry transfer as described [6], instead of using the typical continuous transfer system. While the possibility of detecting EPO in patient samples has been demonstrated by Reverter-Branchat et al. [4], it would be interesting to investigate lower doses of rEPO that an athlete might potentially use, and this investigation is currently underway. Conclusions The EPO Purification Gel Kit for Blood can easily be used to immunopurify serum spiked with ESAs, giving MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-926 stable eluates and detection comparable to the previous method. Moreover, it could be successfully applied to extract EPO from dried blood spots. More investigations are needed to further evaluate the detection limits, such as a large-scale study where DBS samples would be collected from participants administered with rEPO. References 1. World Anti-Doping Agency. 2018 Anti-Doping Testing Figures. (2018) https://www.wada-ama.org/ en/resources/laboratories/anti-doping-testing-figures-report (access date 01.2020). 2. Masquelier M, Heiland C, Bhuiyan H, Erlandsson G, Molin A, Ericsson M. (2018) Optimization of ESAs detection in urine and blood: comparison of different immunopurification techniques. In: Thevis M, Geyer H, Mareck U (eds.) Recent advances in doping analysis (26). Sportverlag Strauß, Köln 58-65. 3. Heiland CE, Masquelier M, Bhuiyan H, Ericsson M. (2019) A simple method to immunopurify erythropoiesis stimulating agents from urine, aiming to optimize erythropoietin screening by SAR-PAGE. Drug Test Anal. 11 (11-12), 1666-1674. 4. Reverter-Branchat G, Ventura R, Ezzel Din M, Mateus J, Pedro C, Segura J. (2018) Detection of erythropoiesis-stimulating agents in a single dried blood spot. Drug Test Anal. 10 (10), 1496-1507. 5. MAIIA Diagnostics. EPO Purification Gel Kit – For Blood. (2019) http://media.maiiadiagnostics.com/2020/ 01/DfU-EPGK-for-Blood.pdf (access date: 30.07.2020) 6. Martin L, Martin J-A, Collot D, Hoang O, Audran M, Ericsson M, Marchand A. (2020) Improved detection methods significantly increase the detection window for EPO microdoses. Drug Test Anal. 2020;1-12. doi.org/10.1002/dta.2904 Acknowledgements We would like to thank MAIIA for providing the immunopurification kits for this project and for being readily available for questions and advice concerning the purification procedure. MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-927 Sigmund G, Geyer H, Goergens C, Thevis M Prevalence of the imidazole derivatives naphazoline, oxymetazoline and xylometazoline in doping control samples Institute of Biochemistry, German Sport University Cologne, Cologne, Germany Abstract In 2017 an AAF for the imidazole derivative naphazoline was reported the first time in the WADA testing figures since their implementation in 2003. This reporting of an AAF was particularly noteworthy as the Cologne laboratory frequently detects imidazole derivatives in in-competition samples, but these findings are not reported as AAFs due to the remark in the prohibited list that imidazole derivatives are permitted for topical/ophthalmic use. To assess the prevalence of imidazole derivatives among German and international elite athletes, the Digital Matrix (full scan data of GC/MS analyses) of 5500 in-competition (IC) doping control samples from different sports, collected from January till June 2017, were evaluated for the imidazole derivatives naphazoline, oxymetazoline and xylometazoline. Out of the 5500 evaluated samples, 199 samples contained imidazole derivatives (3.6 %). Out of the 199 samples, xylometazoline was detected in 178 samples (89.4%), oxymetazoline in 11 samples (5.5 %) and naphazoline in 10 samples (5.0 %). The majority of the target analytes (45.2 %) was detected at concentrations between 101-500 ng/mL. Twelve samples (6.0 %) showed concentrations above 1000 ng/mL. Introduction According to the WADA Prohibited List, S6 stimulants, imidazole derivatives are prohibited except for dermatological, nasal or ophthalmic use [1]. In 2017 an AAF for the imidazole derivative naphazoline was reported the first time in the WADA testing figures since their implementation in 2003 [2]. Details about the reporting of the naphazoline (e.g. extraordinary high concentrations, missing declaration in DCF, missing TUE etc.) were not available. This reporting of an AAF was particularly noteworthy as the Cologne laboratory frequently detects imidazole derivatives in in-competition samples, but these findings are not reported as AAFs due to the remark in the prohibited list that imidazole derivatives are permitted for topical/ophthalmic use. The time interval from January till June was chosen to cover different seasons of the year. The DCFs were not evaluated for the declaration of imidazol derivatives. Experimental The Digital Matrix, i.e. full scan data of GC/MS analyses, of 5500 in-competition (IC) doping control samples from different sports were evaluated for the presence of the imidazole derivatives naphazoline, oxymetazoline and xylometazoline. Additionally, the concentrations of these derivatives were estimated. MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-928 In order to cover different seasons, the samples analyzed came from the period from January to June 2017. The DCFs were not evaluated for the declaration of imidazol derivatives. Samples were analyzed according to the method described by Thevis et al. in 2007 [3]. The used analytical method in brief: LLE of 5 mL urine at pH 14 with TBME, injection of 5 µL of organic layer into a GC/MS/NPD system. The mass spectrometer was operated with EI and full scan analysis m/z 40-400, 4 scans/s. Results and Discussion Out of the 5500 evaluated samples, 199 samples contained imidazole derivatives (3.6 %). 10 samples contained 2 different imidazole derivatives. Out of the 199 samples, xylometazoline was detected in 178 samples (89.4%), oxymetazoline in 11 samples (5.5 %) and naphazoline in 10 samples (5.0 %). The majority of the target analytes (45.2 %) was detected at concentrations between 101-500 ng/mL (see Figure 1). Twelve samples (6.0 %) showed concentrations above 1000 ng/mL. Corresponding sample details are presented in Table 1. The imidazole derivatives were detected in 46 different sports. Most findings were detected in football (Table 2). Figure 1. Estimated concentrations of the imidazole derivatives in the 199 findings MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-929 Table 1: Samples with concentrations of imidazole derivatives > 1000 ng/mL Table 2: Sports with most frequent findings of imidazole derivatives Conclusions This is the first time that the frequency of the use of imidazole derivatives in elite athletes has been evaluated. More than 3% of all tested IC samples showed the presence of imidazole derivatives. Xylometazoline was the most frequently detected imidazole derivative. These data should be presented to the WADA Prohibited List expert group to facilitate the decision on whether or not a reporting limit should be implemented for these substances (e.g. > 1000 ng/mL). If it is agreed to implement such a reporting level, as next step excretion studies with imidazole derivatives MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-930 have to be conducted, to evaluate whether the detected concentrations are consistent with a permitted topical/ophthalmic use or prohibited routes of administration. References 1. WADA prohibited List 2017, accessdate : 05.10.2018 2. WADA 2107 Anti-Doping Testing Figures. Laboratory Report, page 23 of 26., access date: 05.10.2018 3. Thevis M, Sigmund G, Koch A, Schänzer W. Determination of tuaminoheptane in doping control urine samples. Eur. J. Mass Spectrom 13, 213 (2007) 2013-221 Acknowledgements The presented work was conducted with financial support of NADA Germany (Bonn, Germany), the Federal Ministry of the Interior, Building and Community of the Federal Republic of Germany and the Manfred-Donike Institute for Doping Analysis (Cologne, Germany). MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-931 Wicka M, Kaliszewski P, Grucza K, Stanczyk D, Drapala A, Konarski P, Zalewska Z, Siek P, Michalak D, Kwiatkowska D Elimination profile of triamcinolone acetonide and its metabolite in human urine after multiple transdermal administration Polish Anti-Doping Laboratory, Warsaw, Poland Abstract Triamcinolone acetonide (TA) is prohibited in competition and only for oral, intravenous, intramuscular or rectal administration. In order to distinguish whether TA was administered by one of the prohibited routes or not, a specific reporting limit for urinary concentrations of the parent compound and its metabolite was established at 30 ng/mL. Triamcinolone acetonide is a synthetic corticosteroid applied topically to treat various skin conditions, intra-articularly to treat joint injuries, intramuscularly for the control of severe allergic states. In nasal spray form, it is used to treat allergic rhinitis. The aim of the present work was to study the metabolic profile of Triamcinolone acetonide in urine following transdermal administration. Four healthy male volunteers applied 4 g of Pevisone (econazole nitrate 10 mg/g + Triamcinolone acetonide 1.1 mg/g) once a day and twice a day throughout three consecutive days. Four spontaneous urine samples were collected per day i.e. in the morning, midday, afternoon and evening. Urine samples were analyzed by liquid chromatography combined with mass spectrometry to measure TA and 6β-OH triamcinolone acetonide (6β-OH TA), the main TA metabolite. After administration, the concentration of TA was significantly lower than of 6β-OH TA. For 6β-OH TA, a greater concentration was obtained: 0.10 - 4.36 ng/mL. However, concentrations of TA and 6β-OH TA were lower than 30 ng/mL for all volunteers. Introduction Triamcinolone acetonide is asynthetic corticosteroid used as anti-inflammatory drug to treat various diseases. It is applied topically to treat various skin conditions (e.g., severe erythema multiforme and pemphigus), intra-articularly to treat joint injuries (e.g., gout, psoriatic arthritis and systemic lupus erythematosus), intramuscularly for the control of severe allergic states. In nasal spray form, it is used to treat allergic rhinitis. It is used to treat many different conditions such as gastrointestinal diseases (e.g., ulcerative colitis)[1]. Triamcinolone acetonide is widely used by athletes to treat articular pain, injures and sprains [2]. According to the WADA Prohibited List [3], glucocorticosteroids are prohibited in competition and only when administered by oral, intravenous, intramuscular or rectal routes. In order to distinguish whether glucocorticosteroids were administered by one of the prohibited routes or not, a specific reporting limit for urinary concentrations of parent compounds and their metabolites was established at 30 ng/mL [4]. MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-932 Experimental Four male healthy subjects, aged 28, 29, 41 and 60 years, height and weight respectively 179 cm/84 kg, 175 cm/67 kg, 178 cm/83 kg, 178 cm/90 kg (subject 1M, 2M, 3M and 4M), applied 4 g of Pevisone (econazole nitrate 10 mg/g + Triamcinolone acetonide 1.1 mg/g) once a day for three days and then twice a day for three consecutive days – transdermal administration. One dose of the preparation that was applied to the skin weighed 4 grams and provided a one-time dose of 4.4 mg of the active substance acetonide triamcinolone. All volunteers applied the preparation to the sacrolumbar part of the spine. Four spontaneous urine samples were collected per day, i.e. in the morning, midday, afternoon and evening. Urine samples were collected for 5 days after the final administration (Figure 1). Figure 1. Scheme of dosage of Pevisone and administration The sample preparation is a two-step procedure involving enzymatic deconjugation of glucuronides and then liquid-liquid extraction with methyl tert-butyl ether. Chromatographic separation was conducted using a Waters Acquity UPLC System liquid chromatograph with BEH C18 (1.7 µm, 100 mm x 2.1 mm) from Waters. The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B), and LC gradient was employed at the constant flow rate of 300 µL/min at 45°C. Triamcinolone acetonide (TA) retention time was 4.43 min, while that of 6β-OH triamcinolone acetonide (6β-OH TA) was 3.10 min. MRMs of the studied substances were traced with a Micromass Quattro Premier XE mass spectrometer equipped with an ESI source. All analytes were investigated in the ESI mode. Desolvation gas flow was set at 643 L/h at 450°C with ion source temperature at 120°C. Capillary voltage was 3.0 kV. + MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-933 Results and Discussion Characteristics of the calibration curves are shown in Table 1. Table 1. TA and 6β-OHa curve parameters The LOD for TA and 6β-OH TA were estimated at 0.05 ng/mL. The linear regression curve ranged from 0.5 to 30 ng/mL and had a satisfactory coefficient of correlation. Typical MRM chromatograms generated from a urine sample spiked with 30 g/mL of Triamcinolone acetonide and the metabolite 6β-OH Triamcinolone acetonide are shown in Figure 2. Blank urine control samples which were analyzed in each series of the validtion procedure (n=6) were free of compounds wich might be eluted in the retention times of TA and 6β-OH TA. As shown in Figure 2, TA and 6β-OH TA could be identified in human urine after transdermal administration at low concentrations. MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-934 Figure 2. Typical MRM chromatograms: (A) spiked urine 30 ng/mL; (B) urine sample pre and 3 day after transdermal administration In our study, 4 g per day for three days, and 8 g per day of TA cream for three consecutive days (total 36 mg of cream, equivalent 39.6 mg of the active substance acetonide triamcinolone) were administered to the volunteers. The topical dose was administered for six consecutive days, to determine if repeated doses lead to accumulation of TA in urine. Figure 3 shows the changes in the concentrations of TA and 6β-OH TA after administration. The maximum concentration of TA were in the range of 0.7 – 0.8 ng/mL. However, the maximum concentation of 6β-OH TA was in the range of 1.93 – 4.36 ng/mL.The low concentration values obtained for TA, as in case [1], were resulted from a low absorption of TA by the skin. There was also a visible lack of TA accumulation after multiple administrations. The high inter- individual variability in concentrations between volunteers was probably depending on the age and the metabolism associated with it. rd MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-935 Figure 3. Changes in the concentrations of TA and 6β-OH TA in urine after administration Conclusions 1. During monitoring of the profile excretion of Triamcinolone acetonide, the concentration values ranged from 0.10 ng/mL to 0.80 ng/ml. While for the metabolite of Triamcinolone acetonide (6β-OH Triamcinolone acetonide), the concentration values ranged from 0.10 ng/mL to 4.36 ng/mL. 2. After the end of the last dose of Pevisone, the presence of both substances was observed for an average of 2 days. 3. Based on the data collected, the Triamcinolone acetonide and 6β-OH-Triamcinolone acetonide were not detected above the reporting limit. References 1. Xavier Matabosh, Oscar J. Pozo, Clara Pérez-Mañá, Esther Papaseit, Josep Marcos, Jordi Segura, Rosa Ventura. Evaluation of the reporting level to detect triamcinolone acetonide misuse in sports. J. Steroid Biochem. Mol. Biol. 145 (2015) 94-102 2. Chih-Wei Chang, Tai-Yuan Huang, Yi-Chun Tseng, Guo-Ping Chang-Chien, Su-Fan Lin, Mei-Chich Hsu. Positive doping results caused by the single-dose local injection of triamcinolone acetonide. Forensic Sci. Int. 244 (2014) 1-6 3. World Anti-Doping Agency. The 2020 Prohibited List, https://www.wada-ama.org/sites/default/ files/wada_2020_english_prohibited_list_0.pdf.Accessed January 2020 4. World Anti-Doping Agency TD2019MRPL, MINIMUM REQUIRED PERFORMANCE LEVELS FOR DETECTION AND IDENTIFICATION OF NONTHRESHOLD SUBSTANCES. https://www.wada-ama.org/sites/default/files/ resources/files/td2019mrpl_eng.pdf. accessed January 2020 Acknowledgements Financial support from the Ministry of Sport and Tourism of the Republic of Poland under the project numer 2019.0432/1575/UDot/BM is gratefully acknowledged. MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-936 Barbán Duarte L, Martínez Brito D, Correa Vidal T, Montes de Oca Porto R Trend analysis and decision making in the Havana Anti-Doping Laboratory Havana Antidoping Laboratory, La Habana, Cuba Abstract Trend analysis is a technique widely used in the fields of finance, marketing, and decision making in general. The Havana Anti-Doping Laboratory, accredited uninterruptedly since 2003, possesses and applies internal procedures to internally calibrate the measurement instruments. The objective of the present study was to evaluate the trends of nine calibration parameters of three Agilent Technologies 5973N Simple Quadrupole, in order to provide data for a feasible decision making. Three tests were applied to the data (nine parameters evaluated annually from 2003 to 2019) in order to check the presence of trends: Mann-Kendall trend test, Spearman test and Kendall tau test. Then, a prediction was estimated by the slope, intercept and, the residues values. The study was carried out for gas chromatograph instruments and for the calibration parameters as injector and detector linearity, flow and injector accuracy and uncertainty. The results allowed to obtain the approximate date that the instruments could reach a maximum value of the calibration acceptance criteria. This criterion could (should) be used by the Laboratory Manager in order to plan their replacement and to plan the use of the instrument. One of the results was that quantification assays should be suspended for the instrument coded as MSD4 with the aim of maintaining the reliability of the results reported by the Laboratory. Introduction The decision process has characteristics that make it continuous, interrelated and the result of a statistical process. According to Fundamental and General Metrology Terms, “calibration” is the set of operations with which, under specified conditions, the correspondence between the values indicated in the instrument, equipment or measurement system and the values are established represented by a reference material or by known values corresponding to a measurement or standard. The equipment and patterns responses, vary over time due to different causes, i.e. aging, deterioration, improper cleaning and chemical reactions [1,2]. The main objective was to evaluate trends based on the data obtained in the calibration processes of three GC/MS in the Havana Antidoping Laboratory during sixteen years. Experimental Data and methods applied Three Agilent Technologies 5973N Simple Quadrupole coded as MSD2, MSD3 and MSD4 were studied through nine parameters that define the instrument performance. The verification of these parameters is MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-937 carried out every year and the present study included the results obtained from 2003 to 2019. Table 1 describes the parameters and the acceptance criteria applied in the evaluation of the instruments. Statistics. Data obtained in the lapse from 2003 and 2019 was statistically evaluated through three tests in order to check the presence of trends: Mann-Kendall trend test; Spearman test and Kendall tau test with 95% confidence interval [3,4]. After to applied the tests to the data, it was selected the parameters that show trends in their behaves between 2003 and 2019 and finally; a long term prediction by the application of the “automatic forecasting” (by the slope, intercept and, the residues values) was made and, it was estimated the year in which the maximum value of the Calibration Acceptance Criteria should be achieve. Software used were STATGRAPHICS centurion XV, version 15.1.02, StatPoint Inc, 2008 and; PAST 3.26 (Hammer, Ø, Harper, DAT Ryan PD 2001. PAST: paleontological statistics software package for education and data analysis Paleontological Electronica 4 (1): 9pp. Table 1 . Parameters evaluated during the calibration process of three single quadrupole gas chromatograph- mass spectrometer (GC/MS) and the acceptance criteria in the Havana Anti-Doping Laboratory Results and Discussion Annually, the verification of the calibration status for each instrument is perform using the “accepted” or “no accepted” criteria. This does not allow to evaluate trends in the data. After 17 working years, out of the nine parameters verified, the flow (in)accuracy and detector carryover showed values permanently close to zero and for that, they remained out of the analysis. The Injector Precision (PI) evaluates the precision of the injector by calculating the coefficient of variation of the chromatographic peaks areas of the reference material obtained after to inject consecutively 5 replicates of the 1 µL injection volume. This parameter showed a marked trend for all three instruments (Spearman rho, Kendall tau and Mann-Kendall trend, α = 0.05, Table 2). Because it is a significatively contributor to the uncertainty value, also this parameter showed a significant trend for all three instruments [5]. Additionally to injector precision (PI) and uncertainty (uc), the instrument MSD4 showed a significant trend for the parameter Injector Linearity (LI). MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-938 Table 2 . Statistical evaluation of the GC/MS calibration data during 17 years. The bold and underlined values represent a statistically significant trend (α = 0.05). Table 3 shows the results obtained for the prediction analysis. As expected, the uncertainty was the “warning parameter” because its maximum accepted value should be reached firstly that others. Being the Injector the main instrument part linked to the obtained results, the Manager may consider the possibility to replace only this part and no the all instrument. The instruments included in this trend analysis are GC/MS single quadrupole that are used today to quantified substances with a threshold at high concentrations values (per example Salbutamol or Tetrahydrocannabinol carboxylic acid) and also, they are used for research tasks. After these results, the laboratory routine should be re-organized in order to avoid the use of the instruments at least for quantitation purposes. Table 3 . Description of the prediction line obtained for each instrument and the year when the instrument should reach the maximum acceptance criteria in the calibration process MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-939 Figure 1. Prediction line obtained for the parameters that showed a statistical trend from 2003 to 2019 Conclusions The analysis of trends using the data obtained from the calibration process showed results that could (should) be used by the Laboratory Management in order to plan and execute the budget of the laboratory. It is important to take into account that the prediction proposed here are valid as long as the same level of labor and frequency of maintenance operations are maintained. These predictions allow an adequate and prioritized budget enforcement as well as the re-organization of the tasks performed in each instrument. References 1. Bureau International des Poids et Mesures (BIPM), JCGM 200: 2012, International Vocabulary of Metrology – Basic and General Concepts and Associated Terms. Third edition, 2008 version with minor corrections, 2012. 2. Guerra Bretaña, Rosa Mayelin, Meizoso Valdés, María del Carmen. Gestión de Calidad: Conceptos, Talleres y Herramientas. Editorial Universidad de la Habana, p. 219, 2012. 3. Øyvind Hammer. Natural History Museum, University of Oslo PAST PAleontological Statistics Version 3.25. Reference manual. 1999-2019. 4. Richard O. Gilbert. Statistical Methods for Environmental Pollution Monitoring. February 1987. ISBN: 978-0- 471-28878-7. http://www.osti.gov/scitech/biblio/7037501/. 5. Vicki J. Barwick. Review Sources of uncertainty in gas chromatography and high-performance liquid chromatography. Journal of Chromatography A, 849 (1999) 13–33. MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-940 Rodriguez Fernandez A, Laffita D, López Castillo D, Terrero O, Montes de Oca R Increase in the incidence of THC in urine samples of athletes in 2019 Havana Antidoping Laboratory, Sports Medicine Institute, La Habana, Cuba Abstract According to regulations of the World Anti-Doping Agency (WADA), the use of cannabinoids is forbidden in competition [1]. In doping controls, the detection of cannabinoid misuse is based on the analysis of the pharmacologically inactive metabolite 11-nor-Δ -carboxy-tetrahydrocannabinol-9-carboxylic acid (THC- COOH), and urinary concentrations greater 150 ng/mL constitute as an adverse analytical finding (AAF) [2,3]. This threshold level was only 15 ng/mL until 2013. The objective of this study is to describe the presence of THC-COOH in samples of athletes received in the Havana Anti-doping Laboratory between 2017 and 2019. Samples were classified by sports, gender, and country, as well as by means of the collection mode In Competition (IC) and Out of Competition (OOC). In 2019, 40.0% of the collected IC samples showed concentrations between 15 and 150 ng/mL. The sports Judo, Football, Sambo and Para Table Tennis were showing concentrations higher than 150 ng/mL. Three of these samples were reported as Adverse Analytical Finding after adjusting the decision limits by specific gravity. In the same year (2019), OOC samples collected from eight different sports showed THC-COOH levels above 150 ng/mL. 18.9% of these samples came from Chile. This data show the increase of THC-COOH consumption during 2019 compared to 2017 and 2018. Introduction Tetrahydrocannabinol (THC), also known as Δ -tetrahydrocannabinol, is the main psychoactive constituent of cannabis [3,4]. THC is one of the substances of the S8 cannabinoid group that is prohibited in competition; the threshold level of the relvant analyte THC-COOH is 150 ng/mL [1,2]. Years ago this level was only 15 ng/mL. However, in May 2013, the WADA Executive Committee decided to increase the threshold level after considering the numerous presentations received from interested parties during the Code review process. Cannabis data from all sources has been analyzed for the potential benefits and hazards for athletes related to the abuse of cannabinoids for the purpose of doping in sports [3]. The objective of this study was to describe the presence of THC-COOH in samples of athletes between 2017 and 2019 and the increase in consumption in the last year. Experimental Data: A total of 13328 urine samples were processed between 2017 and 2019. Samples were collected according to the Initial Testing Procedure which is applied to all doping control samples in our laboratory in Havana. Sample preparation was done as described by Martínez-Brito [5]. Briefly, 2.5 mL of urine were hydrolyzed with β-glucuronidase, after previous addition of ISTD and pH correction to 7. Afterwards, 9 9 MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-941 liquid-liquid extraction using tert-butylmethyl-ether was done. Then, TMS derivatives were analyzed using an Agilent 7890B gas chromatograph coupled to a mass spectrometer triple quadrupole Agilent 7000. One quality control was added in each analysis batch. Reagents: THC-COOH was supplied by Cerilliant. All reagents and solvents (sodium bicarbonate, potassium carbonate, sodium phosphate, sodium hydrogen phosphate) were of analytical grade. Tert- butylmethyl-ether was HPLC grade. β-glucuronidase (Escherichia coli K12) from Roche Diagnostic (Mannheim, Germany). Mercaptoethanol, Ammonium Iodide and MSTFA were purchase by Sigma-Aldrich. Results and Discussion From all processed samples between 2017 and 2019, 1.9% showed the presence of THC-COOH in 2017, 1.8% in 2018 and 2.8% in 2019, showing an increase during the last year. The higher incidence of this substance was observed in males with 81.2%. These findings results from the screening and identification was done using a positive THC-COOH control. If the THC-COOH concentration, estimated with the positive control, is greater than 75 ng/mL (50% of the threshold), THC-COOH was quantified with three new aliquots of the sample. If we compare the presence of THC-COOH among sports in 2019, an increase was seen in some of them (Figure 1). Weight lifting, Judo, Athletics, and Tae-Kwon-Do were those with the highest levels. Figure 1. Sports with a greater number of samples where the presence of THC-COOH was detected (2017-2019) MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-942 In 2019, there was an increase of IC and OOC samples with THC-COOH concentration levels above 15 ng/mL (Figure 2). From the IC samples showing THC-COOH, 55.7% had a concentration higher than 15 ng/mL. This value was only 39.1% in 2018 and 26.8% in 2017. Out of 61 IC samples with presence of THC-COOH in 2019, 29 showed concentration levels between 15 and 150 ng/mL, representing 47.5%. In this study, concentration ranges of 15 ng/mL, 15-150 ng/mL and more than 150 ng/mL were determined, since the quantification thresholds set by WADA before and after 2013 were 15 ng/mL and 150 ng/mL. Figure 2. Representation for years of the concentration of THC-COOH detected by number of samples MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-943 In 2019, Athletics, Basketball, Fencing, Weight lifting and Volleyball had more OOC samples with concentration levels of THC-COOH above 15 ng/mL. On the other hand, Athletics, Football, Judo and Weight lifting were the sports with the highest incidence of this indicator for IC testing controls (Figure 3). After the adjustment of the decision limits for the specific gravity, the Adverse Analytical Findings reported in 2019 correspond to Sambo (219.8 ng/mL), Football (790.0 ng/mL) and Para Table tennis (1456.0 ng/mL). Figure 3. Sports with the highest incidence of THC-COOH in samples collected IC (a) and OOC (b) in 2019 MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-944 In 2019, the countries with the highest presence of THC-COOH in the OOC controls were Chile (26 out of 137 analyzed), Mexico (23 out of 1019 analyzed) and Cuba (8 out of 593 analyzed) (Figure 4). Figure 4. Countries with the highest presence of THC-COOH in the controls OOC in 2019 Conclusions In 2019, the presence of THC-COOH increased in the total samples received. Compared to previous years, 70% of sports showed an increase in THC-COOH detection. In 2019, IC samples from Judo, Football, Sambo and Para Table Tennis athletes were analyzed with THC-COOH levels higher than 150 ng/mL. Three of them were reported as Adverse Analytical Finding. During the same year, OOC samples were positive for THC-COOH levels higher than 150 ng/mL in 8 sports. Chile shows the highest presence of THC-COOH in OOC samples collected in 2019, representing 18.9 % of the samples received. References 1. World Anti-Doping Agency, The 2019 Prohibited List. International Standard., 2019, https://www.wada- ama.org/sites/default/files/wada_2019_english_prohibited_list.pdf 2. World Anti-Doping Agency, WADA Technical Document - TD2019DL v. 2.0: Decision Limits for the Confirmatory Quantification of Threshold Substances. 2019, (04.05.2020), https://www.wada- ama.org/sites/default/files/resources/files/td2019dl_v2_finalb.pdf 3. M. A. Huestis, I. Mazzoni, O. Rabin. Cannabis in Sport: Anti-Doping Perspective. Sports Med. 2011, 41, 949. 4. U. Mareck, et al. Temporal Indication of Marijuana Use. Recent advances in doping analysis, 2016, 14, Köln, 101-105 5. A. Rodríguez Fernández, D. Martínez Brito, T. Correa Vidal, D. López Castillo, R. Montes de Oca Porto. Evaluation of the steroid profile in Latin-American population of male football players. Recent advances in doping analysis, 2019, 27, Köln, 39-44 MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-945 Biasini GM, Donati F, Rutigliano L, de La Torre X, Botrè F Proteomic analysis of biomarkers of red blood cells storage: towards the development of direct detection of autologous blood transfusion Antidoping Laboratory of Rome, FMSI, Rome, Italy Abstract In recent years one of the greatest challenges for antidoping in sports has been to develop a direct method to detect the misuse of autologous blood transfusions (ABT) that can be tempted by cheating athletes to increase oxygen delivery to muscles so enhancing sport performance. Despite being prohibited by the World Anti-Doping Agency (WADA), there is currently no direct method to tackle ABT doping strategies. Storage of erythrocytes, under standard blood banking conditions, for transfusion purpose, induces biochemical and physiological changes that affect red blood cells (RBCs) shape, proteome, metabolism as well as affecting oxidative stress. At the protein level, alterations of the morphological structure of erythrocytes involve Band 3, the most abundant RBC transmembrane protein that plays a key role in membrane integrity and cytoskeletal organization. Moreover, loss of the oxidant- antioxidant balance mostly depends on Peroxiredoxin-2 (PRDX2), one of the primary RBC antioxidant enzymes whose effect is to limit hydrogen peroxide (H O )-mediated toxicity. The aim of the present project is to evaluate alterations occurring in RBC proteome during storage, comparing their proteome before and after different periods of time, under standard blood banking conditions. In this study, it has been demonstrated that levels of Band 3 decrease over storage time, highlighting its role in the structural and morphological organization of RBCs; moreover, levels of PRDX2 are shown to considerably increase over storage time with the consequence of the increased oxidative stress in erythrocytes. Introduction ABT is a banned method by the WADA [1] and consists of the withdrawal of a sufficient volume of blood from an individual, its storage, and its reinfusion. During storage, under standard blood banking conditions, erythrocytes undergo several modifications: loss of shape and membrane integrity [2,3], increased oxidative stress [4] and changes in metabolome and proteome [5]. Phenomena of aggregation and degradation occur to proteins over time, representing a "signature" of RBCs storage. We selected two new biomarkers: Band 3, a transmembrane protein of RBCs involved in cytoskeleton organization [6,7], and PRDX2, the main erythrocytes peroxidase [8,9], to evaluate their levels before and after different periods of storage, to assess the suitability of these biomarkers for the development of a direct method for ABT detection. 2 2 MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-946 Experimental Preparation of samples Whole blood samples were analyzed fresh at T0 and divided into two aliquots. Aliquoted samples were stored at 4°C in K EDTA tubes and stained for flow cytometry (FC) and western blotting (WB) applications after 20 (T1) and 40 days (T2). For the WB assay, blood samples (n=2) were washed twice with phosphate buffer (PB). Pellet was resuspended in N-ethylmaleimide (NEM) 0.1 M in PB solution and incubated for 15 minutes at RT. After incubation, 0.1 M of NEM in PB/2% cOmplete Protease Inhibitor Cocktail (Roche, Switzerland) solution was added to samples, and cells were lysed mechanically through a glass homogenizer. Samples were washed twice, and the pellet was resuspended in phosphate- buffered saline (PBS). Protein concentration was estimated through Bradford assay. Samples were denaturated at 70°C for 10 minutes. SDS-PAGE and Western Blotting Denatured protein samples (n=2) were analyzed through SDS-PAGE. 5 µg or 300 ng of each sample, respectively for PRDX2 and Band 3 analysis, were loaded in the gel (NuPAGE 4-12% Bis-Tris Gel, Novex- life technologies). Band 3 and PRDX2 standards were loaded as controls. The WB analyses were performed on PVDF membranes against PRDX2 (Anti-h/m/r Peroxiredoxin2 at 1/1600), Band 3 (SLC4A1 Monoclonal Antibody-BIII136 at 1/100000). The membrane was incubated with the secondary antibody (Gt anti-MS IgG (H+L) cross-adsorbed secondary antibody, HRP-conjugate) for 90 minutes. Images were acquired through the ImageQuant LAS4000. Bands of interest were quantified by densitometry by means of the GASepo software. Flow Cytofluorimeter assay Whole blood samples (n=5) were washed twice with saline. Band 3 antibody (CD233, Band 3 extra‐ cellular domain) was added to samples and incubated for 45 minutes. Samples were washed twice to remove the excess of antibody. Glycophorin A (GYPA) antibody as control was added and incubated for 30 minutes. Samples were rewashed to allow FC acquisition (FC500 Beckman Coulter). Results and Discussion Our results demonstrated a trend towards the increase of PRDX2 protein amount during storage, while β- actin, the normalizing control, remains stable over storage conditions. Levels of PRDX2 increased progressively after 20 days (p=0.09) and after 40 days (p=0.07) of storage compared to freshly collected blood samples. 2 MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-947 Figure 1a and b. The amount of PRDX2, present in erythrocytes, changes during storage. (a) Representative western immunoblots analysis of blood samples against PRDX2 and β-actin (b) Different levels of PRDX2 after different periods of storage (T1=20 days, T2=40 days) normalized with levels of β -actin, the loading control. PRDX2 levels progressively increase after 20 days (p=0.09) and after 40 days (p=0.07). The storage causes damages to the structure of PRDX2 that aggregates in high molecular weight complexes migrating from the cytosol to the membrane of the erythrocytes and, consequently, it becomes more detectable in RBCs membranes [9]. In parallel, there is a trend towards the decrease of Band 3 levels over storage time. The total amount of Band 3 decreases progressively after 20 days (p=0.99) and after 40 days (p=0.62) of storage compared to freshly collected blood samples. MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-948 Figure 2a and b. The amount of Band 3, the major integral RBCs protein, changes during storage. (a) Representative western immunoblots analysis of blood samples against Band 3 and β-actin. (b) Different levels of Band 3 after different periods of storage (T1=20 days, T2=40 days) normalized with levels of β -actin, the loading control. Band 3 levels progressively decrease after 20 days (p=0.99) and after 40 days (p=0.62). To better assess the alterations that occur to Band 3 due to the storage of RBCs, we also conducted a dual-color staining FC assay. Figure 3a, b, and c shows the progressive decrease in both Band 3, GYPA levels, and their ratio during storage. As we can see, the levels of Band 3 decrease over storage time with a percentage of 19% after 20 days (p=0.06) and 39% after 40 days (p

M. THEVIS H. GEYER U. MARECK (EDITORS) RECENT ADVANCES IN DOPING ANALYSIS (28) Proceedings of the Manfred Donike Workshop 38th Cologne Workshop on Dope Analysis 9th to 14th February 2020 SPORTVERLAG Strauß - Hellenthal 2020 1 Bibliografische Information Der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über abrufbar. Thevis, Mario; Geyer, Hans; Mareck, Ute (Eds.) Recent Advances in Doping Analysis (28). Proceedings of the Manfred Donike Workshop, 38th Cologne Workshop on Dope Analysis, 9th to 14nd February 2020 / [MDI e.V.] – 2020 Sportverlag Strauß. ISBN 978-3-86884-046-9 ©SPORTVERLAG Strauß Neuhaus 12 – 53940 Hellenthal Tel. +49 (0)2448/2470040 – Fax +49 (0)2448/9195610 E-mail: info@sportverlag-strauss.de www.sportverlag-strauss.de Satz: Autorensatz 2 MANFRED DONIKE WORKSHOP 2020 TABLE OF CONTENTS PAGE LECTURES Marchand A, Ericsson M, Audran M, Fenaille F, Capdeville P, Martin L: EPO Biosimilars with unusual profiles: characterization and detection .................................... 13-22 Heiland CE, Masquelier M: Sepharose gel beads: an effective technique for recombinant erythropoietin immuno- purification from blood ................................................................................................................ 23-27 POSTER PRESENTATIONS Sigmund G, Geyer H, Goergens C, Thevis M: Prevalence of the imidazole derivatives naphazoline, oxymetazoline and xylometa- zoline in doping control samples ................................................................................................. 28-31 Wicka M, Kaliszewski P, Grucza K, Stanczyk D, Drapala A, Konarski P, Zalewska Z, Siek P, Michalak D, Kwiatkowska D: Elimination profile of triamcinolone acetonide and its metabolite in human urine after multiple transdermal administrationed stimulants .................................................................... 32-36 Barbán Duarte L, Martínez Brito D, Correa Vidal T, Montes de Oca Porto R: Trend analysis and decision making in the Havana Anti-Doping Laboratory ............................. 37-40 Rodriguez Fernandez A, Laffita D, López Castillo D, Terrero O, Montes de Oca R: Increase in the incidence of THC in urine samples of athletes in 2019 ....................................... 41-45 Biasini GM, Donati F, Rutigliano L, de La Torre X, Botrè F: Proteomic analysis of biomarkers of red blood cells storage: towards the development of direct detection of autologous blood transfusion ................................................................... 46-51 Donati F, Botrè F, de La Torre X, Sian V, Biasini GM, Concetti L, Rutigliano L: Evaluation of the impact of genetic polymorphisms on basal values of human serum and urinary biomarkers: implications for anti-doping analyses ................................................. 52-58 3 PAGE Momobayashi A, Kageyama S, Okano M: Detection of homologous blood transfused erythrocytes by means of a two color fluorescence flow cytometric analysis ......................................................................................... 59-62 Zhou X, He S, Zhang L, Shen L, He C: Administration Study of rEPOs circulated in China ................................................................................... 63-67 He S, Zhou X, Zhang L, Shen L, He C: Troubleshooting for doping analysis of EPO ................................................................................ 68-70 Somay Selbes Y, Onol S, Kacar T, Demirel AH, Aytekin S: Effects of protease inhibitors on stability of urine hCG and LH in doping analyses ................... 71-73 Iannone M, Botrè F, de La Torre X: Direct and indirect detection of pseudo-endogenous steroids in plasma .................................. 74-79 Son J, Park JW, Lee SH: Multiple Biomarkers of Anabolic Agent Administration in Urine as Candidates for Doping Diagnosis ........................................................................................................................... 80-84 Danila G, Pop A, Toboc A, Stan C: 7-Keto-DHEA – a case study .......................................................................................................... 85-89 Geyer H, Fusshöller G, Haenelt N, Mareck U, Blatt C, Gougoulidis V, Hülsemann F, Piper T, Thevis M: The ratio 5aAdiol/E as indicator for the detection of testosterone doping in athletes with naturally low T/E – a case study ........................................................................................... 90-93 Wen C, Liu X, Tianshuo Z, Jingzhu W: Isolation of boldenone and its metabolite in urine sample and further determination of the C/ C ratios by gas chromatographycombustion-isotope ratio mass spectrometry ............. 94-97 Mabusa I, Du Preez H, van der Merwe T: Proposed suppression of phentermine derivatization by concomitant medication: A GC-MS case study and suggested solution ............................................................................. 98-102 Kaewklum M, Kaewklum S, Dithayam S, Wilairat P, Kongpatanakul S: Separation and identification of regioisomers of fluoroamphetamine and fluorometh- amphetamine in doping control by gas chromatography-mass spectrometry ...................... 103-107 Stacchini C, Dima AP, de La Torre X, Mazzarino M, Botrè F: In vitro studies of the metabolic profile of different classes of SARMs using liquid chromatographic-mass spectrometric techniques .................................................................. 108-112 Songsaeng R, Saardpun N, Kusamran T, Kongpatanakul S: Method Development for Identification of Growth Hormone Releasing Peptides (GHRPs) in Doping Control by UFLC/MS-IT-TOF ....................................................................... 113-117 4 PAGE Ahi S, Dubey S, Bhardwaj A, Sahu PL: Fast and sensitive detection of small peptides in human urine using liquid chroma- tography tandem mass spectrometry .............................................................................................. 118-123 Berghes B, Radu M, Cristea CD, Toboc A, Stan C: Validation of HIF activating agents confirmation methods ......................................................... 124-128 Grucza K, Wicka M, Drapala A, Konarski P, Stanczyk D, Michalak D, Kaliszewski P, Kwiatkowska D: Impact of various purity grades of acetonitrile and different mass spectrometry ionization methods on limits of detection of dopingsubstances by means of the “Dilute- and-Shoot” approach .......................................................................................................................... 129-133 Alechaga E, Terrero A, Monfort N, Ventura R: UHPLC-HRMS multi-screening method for doping control analyses in urine samples ........... 134-138 de la Cal A, Espinosa P, Blázquez I, Fernández B, García L, de Pedraza L, Carreras D, Muñoz G: Solving problems with HILIC columns: Compounds with difficult chromatographic behavior................................................................................................................................................. 139-143 Fernández-Álvarez M, Cuervo D, Baltazar-Martins JG, Plata MDM, Muñoz-Guerra JA, Lara B, Del Coso J, Muñoz G, Carreras D: Determination of tramadol and its metabolites in plasma by using liquid chroma- tography quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) ................................. 144-148 Cuervo D, Fernández-Álvarez M, Baltazar-Martins JG, Plata MDM, Muñoz-Guerra JA, Lara B, Del Coso J, Muñoz G, Carreras D: Determination of tramadol and its metabolites in urine via accuratemass quadrupole time-of-flight (Q-TOF) LC/MS ............................................................................................................ 149-152 PRESENTATIONS ABSTRACTS - LECTURES Gotzmann A: "Operation Aderlass”: Legislation as a cornerstone of anti-doping .............................................. 153 Rossi F, Saugy M: Dried Blood Spots: Alternative matrix to deter and detect Tramadol abuse in road cycling competitions ........................................................................................................................ 154 Knoop A, Planitz P, Wuest B, Thevis M: Evaluation of cobalt in doping control samples: Discrimination of different cobalt sources .............................................................................................................................................. 155 Paßreiter A, Thomas A, Walpurgis K, Thevis M: First steps towards uncovering gene doping with CRISPR/Cas9 .................................................... 156 5 PAGE Coll S, Monfort N, Alechaga &, Matabosch X, Pérez-Mañà C, Ventura R: Evaluation of the reporting level to detect betamethasone misuse in sports: additional studies after intranasal and intramuscular administrations .......................................................... 157 Walpurgis K, Rubio A, Wagener F, Krug O, Knoop A, Görgens C, Guddat S, Thevis M: Elimination profiles of microdosed ostarine mimicking contaminated products ingestion .................................................................................................................................................... 158-159 Garzinsky A, Thomas A, Krug O, Thevis M: Exhaled breath in doping controls: characterization of SensAbues® sampling devices using an aerosol test generator ....................................................................................................... 160 Mongongu C, Ericsson M, Madi Moussa E, Audran M, Coudoré F, Marchand A, Buisson C: Quantitative determination of IGF-I in dried blood using Volumetric Absorptive Micro- sampling and Top-down analysis with LC-HRMS ..................................................................... 161-162 Miller G, Madrigal K, Lamm J, Husk J, Eichner D: Detecting growth hormone abuse in dried capillary blood spots: results from a large- scale field trial and a controlled administration study ................................................................... 163 Dib J, Thevis M, Thomas A: Fully-automated "offline" dried blood spot (DBS) extraction and its implementation into doping control analysis ............................................................................................................. 164 Lange T, Thomas A, Walpurgis K, Thevis M: Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide and non-peptide doping agents by means of LC-HRMS ......................... 165 Solheim SA, Stillesby Levernæs MC, Mørkeberg J, Juul A, Upners EN, Nordsborg NB, Dehnes Y: Dried Blood Spot analysis of testosterone esters is applicable in doping analysis ....................... 166 Padilha M, Cavalcanti G, Gomes M, Carneiro G, Oliveira F, Pereira H: Can Graviola juice result an AAF for higenamine? .......................................................................... 167 de Wilde L, Roels K, van Eenoo P, Deventer K: Update on the use of 2D-LC-MS/MS in doping analysis ................................................................. 168 de La Torre X, Colamonici C, Molaioni F, Jardines D, Iannone M, Botrè F: Intentional or accidental clostebol detection. An Italian Story ..................................................... 169 Görgens C, Walker K, Boeser C, Wijeratne N, Martins C, Guddat S, Thevis M Paperspray mass spectrometry – A potential novel technique for the detection of polar compounds in sports drug testing? A pilot study ........................................................................... 170 6 PAGE Kraiem S, Al-Mahamed H, Al-Menhali A, Al-Thani N, Helaleh M, Samsam W, Al-Jaber MY, Bouabdallah S, Touil S, Georgakopoulos C, Beotra A, Mohamed Ali V, Al Maadheed M: Analytical strategy for the detection of ecdysterone and its metabolites in vivo in uPA(+/+)-SCID mice with humanised liver, human urine samples and estimation of the prevalence of its use in anti-doping samples ........................................................................... 171-172 Salamin O, Nicoli R, Schweizer C, Xu C, Pitteloud N, Saugy M, Kuuranne T: Longitudinal steroid profiling in healthy women: impact of transdermal testosterone administration .................................................................................................................................. 173 Zanitzer K, Erceg D, Forsdahl G, Gmeiner G: Comparison of the ratios of sulpho-conjugated and glucuroconjugated phase II meta- bolites of endogenous steroids in urine samples after administration of testosterone preparations via UHPLCHRMS .................................................................................................. 174-175 de Wilde L, Roels K, van Renterghem P, van Eenoo P, Deventer K: Steroid profiling in urine of intact glucuronidated and sulfated steroids using liquid chromatography-mass spectrometry .............................................................................................. 176 Albertsdóttir AD, van Gansbeke W, Coppieters G, Balgimbekova K, Polet M, van Eenoo P: Searching for new long term metabolites of metenolone anddrostanolone by gas chromatography-mass spectrometry with a focus on non-hydrolysed sulphates ........................ 177 Putz M, Piper T, Thevis M: Metabolite identification studies of trenbolone ............................................................................ 178 Piper T, Heimbach S, Adamczewski M, Thevis M: Investigations on the potential impact of different doping agents on the steroid profile by means of a steroidogenesis assay .............................................................................................. 179 Polet M, de Wilde L, van Renterghem P, van Eenoo P: A fast GC-C-IRMS method for the detection of epiandrosterone sulfate in urine ........................ 180 Hülsemann F, Fußhöller G, Lehn C, Thevis M: Excretion of 19-norandrosterone after consumption of boar meat .............................................. 181 Piper T, Rubio A, Thevis M: Investigations on carbon isotope ratios of potential novel long term metabolites of testo- sterone and testosterone prohormone misuse - 5α- and 5β-androstane-3α,17α-diol ......... 182-183 Iannella L, Botrè F, Colamonici C, Curcio D, Ciccarelli C, de La Torre X: Potential masking effect of different prednisolone based pharmaceutical formulations on IRMS confirmation analysis ........................................................................................................ 184 Piper T, Fußhöller G, Geyer H, Danila M, Toboc A, Thevis M: Detecting the misuse of 7-oxo-DHEA by means of carbon isotope ratio mass spectro- metry in doping control analysis ..................................................................................................... 185 7 PAGE Coppieters G, Judák P, van Haecke N, van Renterghem P, van Eenoo P, Deventer K: A high-throughput assay for the quantification of intact Insulin-like Growth Factor I in human serum using online SPE-LC-HRMS ....................................................................................... 186 Renovato-Martins M, Amorim C, Magdalena I, Dudenhoeffer-Carneiro AC, López S, Nunes I, Pizzatti L, Aquino Neto F, Mirotti L, Pereira HM: Monocytes as sensors of autologous blood transfusion detection ............................................... 187 Judák P, Coppieters G, Deventer K, Van Eenoo P: Urinary detection of insulin analogues: Improvements in the Ghent laboratory ......................... 188 Dehnes Y, Bogetveit S, Solheim SA, Sletten C, Mørkeberg J, Baastrup Nordsborg N: Effect of testosterone administration on GH-biomarkers IGF-1 and PIIINP .................................. 189 Renovato-Martins M, Dudenhoeffer-Carneiro AC, Du Rocher-Silva B, Soares F, Aquino Neto FR, Hasan M, Rahaman KA, Muresan A, Oh-Seung K, Mirotti L, Pereira HM: Erythrocyte phenotyping of international athletes by flow cytometry ......................................... 190 Narduzzi L, Buisson C, Morvan M, Marchand A, Audran M, Le Bouc Y, Ericsson M, Le Bizec B, Dervilly G: ABP-omics: a combined use of the haematological module and the “steroidomics passport” to track GH users ...................................................................................................... 191-192 Voss SC, Elsaftawy W, Saghbazarian S, Al-Kaabi A, Mohamed Ali V, Georgakopoulos C, Al Maadheed M, Orie N: Testing the applicability of HRP-conjugated Anti-EPO antibodies for direct rhEPO detection – proof of concept ........................................................................................................... 193 Dehnes Y, Miller G, Naud J, Martin L, Reihlen P, Reichel C: Inter-laboratory validation of biotinylated clone AE7A5EPO-antibody for EPO detection by single blotting of urine and blood samples ................................................................................ 194 Reichel C, Erceg D, Lorenc B, Scheiblhofer V, Farmer L, Zanitzer K, Geisendorfer T, Gmeiner G: Data from a low-dosed rEPO administration study ................................................................. 195-196 Leuenberger N, Salamin O, Gottardo E, Schobinger C, Reverter-Branchat G, Segura J, Saugy M, Tissot J, Kuuranne T: Detection of stimulated erythropoiesis using the transcriptomic ALAS2 biomarker in dried blood spot samples ................................................................................................................. 197 Walpurgis K, Weigand T, Knoop A, Thomas A, Reichel C, Dellanna F, Thevis M: Detection of follistatin-based inhibitors of the TGF-β signaling pathways in serum/ plasma by means of LC-HRMS/MS & Western blotting ................................................................. 198 8 PAGE Miller G, Cox H, Manandhar A, Husk J, Eichner D: Evaluation of CD71, Band3, and serum markers as evidence of recombinant EPO use: results from a non-drug longitudinal study and a blinded, placebo-controlled admini- stration study ................................................................................................................................... 199 PRESENTATIONS ABSTRACTS - POSTERS Nazir J, Cowan D, Caldwell R, Walker C, Bartlett C, Brailsford A, Wolff K: A Menu Driven Laboratory Information Management System (DCLIMS) for Anti-Doping Laboratories ...................................................................................................................................... 200 Son J, Cho Y: Model System for Brain Doping: Identification of the Correlation between Performance Enhancing and Alterations of Neurochemical Profiling .................................................................. 201 Araujo A, da Costa Nunes IK, Sardela V, Pereira H, Cabral L, Anselmo C: Application of the Zebrafish Water Tank Model to investigate the budesonide metabolic profile .............................................................................................................................. 202 Prado E, Gomes G, Lima de Sá C, Matos R, Sardela V, Anselmo C, Nunes I, Ramalho G, Vanini G, Padilha M, Oliveira MA, Oliveira A, Canuto C, Amaral L, Siqueira D, Aquino Neto FR, Pereira HM: Study of the metabolism of the cathinones α-PVP, methylone and 4-chloro- dimethylcathinone through the Zebrafish Water Tank Model ............................................... 203-204 Pettersson-Bohlin K, Sjöö M, Sjöö J: The sniffing dog/anti-doping dog Molly - A new dimension of doping controls ........................... 205 Mazzarino M, Ambrosio G, Grieco A, Parr MK, de La Torre X, Botrè F: Ecdysterone: A snapshot of use in the athlete’s population .......................................................... 206 Ahi S, Nimker V, Sharma R, Narula B, Sahu PL: Continuous Quality Improvement in doping control laboratories: Implementation of ISO/IEC 17025:2017 and WADA ISL 2019, Challenges and Future Perspective ............................. 207 Vargas García-Tenorio S, Serrano Garde E, Soldevilla Navarro AB, Cortes Carrillo N, Martín Escudero P, Muñoz-Guerra JA, Galindo Canales M, del Prado N, Fuentes Ferrer M, Fernández Pérez C, Brouwer B, Benish P: Indirect detection of steroids abuse in sports based on androgenic activity measure- ment by AR Calux bioassay ....................................................................................................... 208-209 Voss SC, Elsaftawy W, Saghbazarian S, Al-Kaabi A, Al-Hamad K, Al Maadheed M, Nofal A, Georgakopoulos C: IAAF Doha 2019 World Championships – Sysmex XN-1000 Hematology Results ......................... 210 9 PAGE Mazzarino M, di Costanzo L, Comunità F, de La Torre X, Botrè F: Dried blood spot in doping control test: evaluation of different blood collection device ............ 211 Reihlen P, Blobel M, Völker-Schänzer E, Schänzer W, Geyer H, Thevis M: Case reports of re-analyzed rEPO suspicious doping control urine samples from 2011-2014...... 212 Ericsson M, Bhuiyan H, Ekström L: GHRH daily administration can be determined with longitudinally monitoring of IGF-I .............. 213 Alechaga E, Pont L, Terrero A, Monfort N, Ventura R: Optimization of an immunopurification method for the screening of growth hormone- releasing hormones in urine doping control ............................................................................ 214-215 Martin L, Maurice T, Audran M, Ericsson M, Marchand A: Detection of a non-erythropoietic EPO, Neuro-EPO, in blood after intra-nasal administration in rat ................................................................................................................. 216-217 Kaliszewski P, Siek P, Zalewska Z, Michalak D, Grucza K, Wicka M, Kwiatkowska D: Method development for the detection of carbamylated erythropoietin (CEPO) for doping control purposes .................................................................................................................. 218 Zhou X, He S, Zhang L, Shen L, He C: Assessment on rat EPO as Internal Standard in Doping ControlSamples for EPO Detection with SAR-PAGE using Biotinylated Primary Antibody ................................................... 219 Reihlen P, Weiß P, Blobel M, Walpurgis K, Thevis M: Evaluation of PEGylated EPO-conjugates as internal standards for EPO analysis in doping controls ................................................................................................................................. 220 Kiousi P, Fragkaki AG, Lyris E, Petrou M, Georgakopoulos C, Angelis YS: Novel Long-Term Markers for Methylnortestosterone Misuse in Human Urine .......................... 221 Martinez Brito D, Botrè F, de La Torre X: Mass spectrometric analysis of 7-oxygenated androst-5-ene steroids - an in vitro study ........... 222 Martinez Brito D, Iannone M, Tatangelo M, Molaioni F, Botrè F, de La Torre X: Old compounds, new alertness: In vitro and in vivo experiments using methyl- testosterone .............................................................................................................................. 223-224 Deventer K, van Gansbeke W, van Eenoo P: Alternative approach for the detection of aminoglutethimide ..................................................... 225 Andersson A, Elings Knutsson J, Ekström L, Lindén Hirschberg A: ABP profile in relation to transdermal testosterone administration in women ........................... 226 Suominen T, Ericsson M, Ekström L, Schulze J: ABP-profile in relation to the menstrual cycle ................................................................................ 227 10 PAGE Andersson A, Eklund E, Hirschberg AL, Ekström L: ABP urinary metabolites and how they correlate to serum hormone levels in female athletes ............................................................................................................................................. 228 Brailsford A, Wojek N, Chih-Wei C, Walker C, Cowan D, Wolff K: Determination of anabolic steroids in dried blood using microsampling and gas chromatography-mass spectrometry: application to a testosterone gel administration study ................................................................................................................................................. 229 Cavalcanti G, Zhu Y, Wijeratne N, Martins C, Padilha M, Pereira H: Application of VeriSpray ion source Triple Quadrupole Mass Spectrometry for stimulants and narcotics analysis in oral fluid ................................................................................ 230 Kowalczyk K, Torres Elguera JC, Konopka A, Jarek A, Stanczyk D, Wicka M, Kwiatkowska D, Bulska E: In vitro metabolic studies of emerging selective androgen receptor modulators (PF-0620414, TFM-4-AS-1, BMS 564929 and GSK 2881078) and implementation of the results into LC-MS/MS-based dopingcontrol analysis ............................................................. 231-232 Sato M, Kageyama S, Okano M: Detection of the selective estrogen receptor modulator Bazedoxifene in human urine by LC-MS/MS .................................................................................................................................... 233 Dubey S, Sah S, Sahu PL: Comprehensive screening of doping agents by ‘dilute and shoot’ urine analysis using high resolution/high accuracy mass spectrometry ......................................................................... 234 Mongongu C, Marchand A, Buisson C, Coudoré F, Audran M, Ericsson M, Madi Moussa E: A sensitive detection of synthetic IGF-I analogs using a mass spectrometric immuno- assay and nano-ultra-high-performance liquid chromatography coupled to high resolution mass spectrometry ......................................................................................................... 235 Rahaman KA, Muresan AR, Rafique FB, Kim KH, Lee KM, Min H, Kim HJ, Sung C, Son J, Lee J, Kwon OS: Metabolites of TB-500 peptide in various in-vitro enzyme systems by UHPLC-MS/MS ............... 236 Rafique FB, Muresan AR, Rahaman KA, Kim KH, Lee KM, Min H, Kim HJ, Sung C, Son J, Lee J, Kwon OS: In vitro phase I metabolism study of the diuretic clopamide by LCMS/MS .................................. 237 Pettersson-Bohlin K, Oesterlind L, Börjesson A, Ekström L: Bis-(4-fluouphenyl)-methylpiperazine detection in urine from female and male Caucasians after oral lomerizine intake .......................................................................................... 238 Silveira C, Sardela V, Anselmo C, Pereira HM: Chiral analysis of amphetamine and methamphetamine with Mosher derivatization by LC-MS2 ........................................................................................................................................ 239-240 11 PAGE He C, Shen L, Zhou X, He S: Quantification of intact human IGF-I in serum by LC-MS/MS ........................................................ 241 Fragkaki A, Kiousi P, Paloumpis N, Kioukia-Fougia N, Panderi I, Angelis I: MS Behavior of Intact Phase-II Metabolites of Anabolic Androgenic Steroids after Derivatization for their Detection by LC/(MS)MS .................................................................... 242-243 Görgens C, Guddat S, Bosse C, Knoop A, Geyer H, Thevis M: Implementation of the HIF activator IOX-2 in routine doping controls – pilot study data ........... 244 SCIENTIFIC BOARD B. Ahrens, Los Angeles (USA) S. Chundela, Kreischa (Germany) H. Cox, Los Angeles (USA) X. de la Torre, Rome (Italy) H. Geyer, Cologne (Germany) G. Gmeiner, Seibersdorf (Austria) R. Kazlauskas, Sydney (Australia) T. Kuuranne, Lausanne (Switzerland) U. Mareck, Cologne (Germany) J-F. Naud, Montreal (Canada) T. Piper, Cologne (Germany) C. Reichel, Seibersdorf (Austria) W. Schänzer, Cologne (Germany) J. Schulze, Stockholm (Sweden) M. Thevis, Cologne (Germany) A. Thomas, Cologne (Germany) P. van Eenoo, Ghent (Belgium) R. Ventura, Barcelona (Spain) 12 Marchand A , Ericsson M , Audran M , Fenaille F , Capdeville P , Martin L EPO Biosimilars with unusual profiles: characterization and detection Département des Analyses, AFLD, Châtenay-Malabry, France ; Laboratoire d'Etude du Métabolisme des Médicaments, CEA, Saclay, France Abstract Biosimilars of recombinant erythropoietin (rEPO) can present some small structural differences compared to the reference product. Three biosimilars, with apparent lighter molecular weight than the genuine rEPO (Eprex ), were selected for further investigation: Jimaixin™ authorized in China, and Hemax and Epotin™ authorized in Algeria. The aims of this research were: i) to study the electrophoretic EPO profiles obtained after a spike of the three biosimilars in urine and plasma by using improved IEF-PAGE and SDS- PAGE methods and to evaluate their identification using reevaluated criteria ii) to test complementary strategies to improve their detection: a two-dimensional electrophoresis approach (SDS separation following IEF) or a neuraminidase treatment of the sample (proposed to increase the separation between recombinant and endogenous EPO by SDS-PAGE), iii) to compare using MALDI-TOF the specific N- glycosylation pattern of each biosimilar with the original rEPO Eprex . Experiments with spiked samples indicated that the biosimilars were more difficult to detect at low doses than Eprex , in particular, Epotin™ and Jimaixin™ by IEF-PAGE in urine and Hemax by SDS-PAGE in plasma. IEF-PAGE for plasma samples and SDS-PAGE for urine samples had the highest identification rates considering the three biosimilars. Two-dimensional electrophoresis experiments did not improve the detection. Samples pre-treatment with neuraminidase gave more promising results for low doses as it slightly increased the detection. Compared to Eprex N-glycosylations, all three biosimilars presented a decrease in the most complex forms (loss of sugar antennae and sialic acids) while bi and tri antennae forms were enriched. Introduction Since expiration of the patents for the first generation of recombinant erythropoietin (rEPO) drugs, pharmaceutical companies world-wide have begun to produce rEPOs copies. All licensed EPO biosimilars shall demonstrate the same safety and efficacy for therapeutic use than the original drug but they can present some small structural differences compared to the reference product due to some variations in the production process [1]. Consequently the electrophoretic profile of a biosimilar can be slightly different from a conventional rEPO one and its detection in a doping control sample might be challenging. After analyzing various EPO biosimilars, three drugs were selected for further characterization: Jimaixin™ (Jintan Ltd, China), Hemax (Biosidus, Argentina) and Epotin™ (Gulf Pharmaceutical Industries, United Arab Emirates). They presented slight differences compared to the original epoetin alpha Eprex using SDS-PAGE (lower molecular weight) and IEF-PAGE (more isoforms in the basic area). 1 1 1 2 1 1 1 2 ® ® ® ® ® ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-913 The aims of this research were: i) to study EPO electrophoretic profiles obtained by IEF-PAGE and SDS-PAGE for the 3 biosimilars after a spike in urine and plasma and to evaluate their detection following WADA’s criteria [2], ii) to test potential complementary strategies to improve detection: a two-dimensional electrophoresis approach or a neuraminidase treatment of the sample (cleavage of sialic acids) before electrophoretic separation, iii) to evaluate the specific N-glycosylation pattern of each biosimilar (Jimaixin™, Hemax and Epotin™) and compare it with Eprex to identify differences with the original epoetin alpha drug. Experimental Detection of rEPOs by IEF and SDS-PAGE analysis Spikes of Jimaixin™, Hemax , Epotin™ and Eprex were performed in 6 different urine samples (starting volume 9 mL) and 3 different plasma samples (starting volume 500 µL). After an estimation of endogenous EPO content in each sample (using human EPO Quantikine ELISA kit from R&D Systems), addition of rEPO was performed to obtain mixes of rEPO/endogenous EPO with approximate abundances of 35/65, 50/50 and 65/35 respectively. EPO analyses were performed as in Martin et al. [4] using optimized protocols to improve sensitivity of detection and, for IEF-PAGE method, criteria slightly modified compared to WADA’s TDEPO to recruit more suspicious samples. All urine and plasma samples were immuno-purified using an anti-EPO coated well from a human EPO ELISA detection kit (STEMCell Technologies) for urine and magnetic beads coated with anti-human EPO antibody 9C21D11 (R&D systems) for plasma. Eluates were analyzed. A single-blot was performed after IEF-PAGE and SDS-PAGE, and EPO immuno-detection was conducted using a biotinylated anti-EPO antibody (clone AE7A5, R&D Systems). Complementary tests Neuraminidase treatment: Biosimilars and Dynepo standards as well as retentates resulting from the ultrafiltration of urine samples (spiked with the biosimilars (50/50 mix) or not) were incubated in wells of a Stemcell EPO ELISA plate for 2 hours at 37°C. After binding of EPO, desialylation was performed by addition of a 5 mU neuraminidase solution and incubation 1 h at room temperature as previously described [3]. After elution, SDS-PAGE analysis was performed as usual and IEF-PAGE using a 2-10 pH gradient instead of 2-6. 2D-gel: Standards and eluates from urine samples with or without spike of biosimilars (50/50 mix) and with or without neuraminidase treatment were analyzed as in Desharnais et al. [3]. For the IEF part, samples without neuraminidase were separated on a 2-6 pH gradient gel and samples treated with neuraminidase on a 3-11 pH gradient gel. Biosimilar glycan characterization by MALDI-TOF/TOF rEPO standards (Eprex and the three biosimilars) N-glycosylations were characterized by mass spectrometry using a previously published protocol [5,6]. Briefly, after protein denaturation, 4 µg of each rEPO were treated with 2 units of N-Glycosidase F (Roche) overnight at 37°C to release N-glycans. After acidification, proteins were precipitated using ice-cold ethanol and resulting N-linked glycans were then ® ® ® ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-914 purified on a porous graphitic carbon SPE cartridge using a mixture of 40% acetonitrile, 0.1% trifluoro‐ acetic acid for elution. After a permethylation step, N-glycans were finally purified on C-18 spin columns (Pierce) and eluted in 80% acetonitrile. After evaporation, final reconstitution was made in 50% methanol. 0.5 µL were mixed with 0.5 µL DHB matrix (10 mg/mL 2,5 dihydroxybenzoic acid (DHB) in methanol/H2O (50/50, v/v) containing 1 mM sodium acetate). Analysis were performed using a MALDI- TOF/TOF instrument UltrafleXtreme instrument (Bruker Daltonics). Resulting spectra were then analyzed using flexAnalysis software for identification of N-glycan specific rEPO structures. Three separated experiments with 2 or 3 technical replicates each time were performed and the mean of these replicates was considered representative of the glycosylations of each rEPO. The relative abundance of each glycan structure was estimated. Results and Discussion Standards characterization The formulation composition indicated the presence of human albumin in all three biosimilars contrary to Eprex . This was corroborated by Coomassie blue staining after SDS-PAGE analysis. Strong signals around 60 kDa attributable to albumin and other bands probably due to other serum proteins were detected (Figure 1A). After immuno-extraction (data not shown), almost all additional protein signals were eliminated. After immuno-detection of EPO, only a clear rEPO signal was found. Compared to Eprex , Jimaixin™ and Epotin™ had broader bands by SDS-PAGE with half of the signal below the Dynepo reference corresponding to the endogenous EPO zone. Hemax signal showed a more compact band with an apex close to the Dynepo reference. By IEF-PAGE (Figure 1B) all three biosimilars had a basic profile clearly indicative of a recombinant EPO. Epotin™ and Jimaixin™ presented more isoforms than Eprex in the basic area, in particular an intense fifth band, and Jimaixin™ and Hemax had respectively two and one isoforms more acidic than Eprex and located in the endogenous area of the gel. Two batches of each biosimilar were tested and showed similar profiles but batch-to-batch variability might exist. ® ® ® ® ® ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-915 Figure 1. Analysis of the three biosimilars by SDS-PAGE and IEF-PAGE. A. SDS-PAGE Left part: Coomassie staining- Right part: EPO immuno-detection after Western-Blot. Bovine serum albumin (BSA), Eprex and urinary EPO (uEPO) were added for comparison. B. IEF-PAGE. The most intense bands in each profile are indicated by black arrows. EPO references (REF): BRP and NESP as well as Eprex and urine (endogenous EPO) were added for comparison. Detection in urine and plasma after spiking rEPOs were identified according to the following criteria. For SDS-PAGE, the presence of a smear above endogenous EPO and Dynepo apex position was indicative of the presence of rEPO. For IEF-PAGE, a ratio between the second most intense band in the basic area of the gel and the most intense band in the endogenous area (2B/N) was calculated. The criteria for a suspicious sample was 2B/N > 0.85 for urine, and the criteria used for plasma samples was 2B/N ≥ 1.30. These criteria were slightly modified compared to WADA’s TDEPO criteria after a reevaluation study in order to recruit more suspicious samples without increasing the number of false suspicions [4]. For urines using this new criteria 2B/N > 0.85 instead of TDEPO's criteria (summarized as 2B/N > 1) led to 11 more samples classified as suspicious on 72 spiked samples analysed (+15%). Similarly for plasma samples using 2B/N ≥ 1.30 instead of TDEPO's criteria (summarized as 2B/N ≥ 2) led to 10 more samples classified as suspicious on 36 spiked samples analysed (+28%). All non spiked samples were negative with the new criterias applied. Increasing concentrations of biosimilar were reflected in the increasing 2B/N ratios by IEF-PAGE and increasing smear intensity above endogenous EPO signal by SDS-PAGE (Figure 2 and 3). ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-916 Figure 2. rEPO analysis of one urine spiked with increasing concentrations of each Biosimilar and Eprex . The endogenous EPO in the urine without spike (uEPO) and a standard of each biosimilar were also analyzed. A. SDS-PAGE method: REF corresponded to Dynepo (lower band) and NESP (upper band). The results of identification are indicated (– = negative results, +/– = suspicious, + = positively identified). B. IEF- PAGE method: REF corresponded to BRP (upper bands) and NESP (lower bands). The results of the 2B/N ratio ( > 0.85 = suspicious) and results of identification are indicated. ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-917 Figure 3. rEPO analysis of one plasma spiked with increasing concentrations of each biosimilar and Eprex . The endogenous EPO in the plasma without spike (plasma EPO) and a standard of each biosimilar were also analyzed. A. SDS-PAGE method: REF corresponded to Dynepo (lower band) and NESP (upper band). The results of identification are indicated ( – = negative results, +/– = suspicious, + = positively identified). B. IEF- PAGE method: REF corresponded to BRP (upper bands) and NESP (lower bands). The results of the 2B/N ratio ( ≥ 1.30 = suspicious) and results of identification are indicated. The results (summarized in Table 1) demonstrated that while Eprex was efficiently identified by both IEF-PAGE and SDS-PAGE methods, the identification of the biosimilars was more challenging in particular for the lowest dose (35:65 mixes of rEPO:endogenous EPO). The two different methods of separation were complementary because, depending on the biosimilar, one or the other was more efficient. Jimaixin™ and Epotin™ were more easily identified by SDS-PAGE while Hemax was better identified by IEF-PAGE. SDS-PAGE method applied on urine samples or IEF-PAGE on plasma samples appeared the best options to obtain the greatest number of suspicions. ® ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-918 Table 1. Summary of the results obtained for the REPO detection in spiked urine and plasma using either IEF-PAGE or SDS-Page methods. Six urines and 3 plasma samples were spiked at three different ratios of recombinant EPO to endogenous EPO (35/65, 50/50 and 65/35). Percentage of suspicious samples identified by each method are indicated (upper table). For IEF-PAGE the percentages obtained when using the new criteria for suspicions and those obtained when using TDEPO's criteria are indicated. In addition percentage of samples with clear positive identification in case of confirmation by SDS-PAGE are also indicated (lower table). The best conditions are shown in bold. Complementary tests to increase identification Neuraminidase treatment: Neuraminidase cleaves the sialic acids at the end of the N-glycan chains and this was suggested as a way to increase the distance of migration between recombinant EPO and endogenous EPO (which contains more sialic acids)[3]. - SDS-PAGE results (Figure 4A and 4B): All EPO profiles after treatment by neuraminidase had lower molecular weight and wider signal. Dynepo treated by neuraminidase was evaluated: the Dynepo +neuraminidase band was just above the endogenous EPO+neuraminidase signal and presented a sharp band: it could be used as a marker to separate the endogenous zone from the exogenous zone. With this marker, urines spiked with biosimilars were slightly more suspicious than before neuraminidase treatment in particular for Hemax . However a loss of intensity could sometimes be observed after neuraminidase. - IEF-PAGE results (Figure 4C): Due to the cleavage of sialic acids, neuraminidase treatment induced a shift of the EPO profiles towards the basic part of the gel and a 2-10 pH gel gradient was necessary. After spike, for all three biosimilars, three small bands could be observed interspaced between the three most basic isoforms of EPO even at low amounts. This specific pattern was a distinctive sign and increased the sensitivity of detection compared to the conventional IEF-PAGE method. Samples analyzed by both IEF-PAGE and SDS-PAGE before and after treatment by neuraminidase indicated that the detection of biosimilars by IEF-PAGE method (intact EPO) was less performant than the IEF-PAGE protocol with neuraminidase-treated samples. IEF-PAGE on neuraminidase treated samples had similar sensitivity than the SDS-PAGE method on untreated samples. Finally SDS-PAGE method on neuraminidase treated samples was a bit more performant to identify rEPO biosimilars at low doses and could be used in case of a suspicious sample. ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-919 Figure 4. Effect of neuraminidase on EPO profiles of endogenous EPO (urine), Eprex and biosimilars. A. SDS-PAGE. Urine EPO without spike (uEPO) or spiked with each rEPO at 50:50 with or without neuraminidase treatment were analyzed. B. SDS-PAGE: uEPO or urine spiked with increasing amount of Hemax as well as Dynepo without and and with neuraminidase treatment were analyzed. Dynepo was used to delimitate endogenous and exogenous zone for EPO and results of identification are shown. C. IEF-PAGE. uEPO standard or urine spiked with increasing amount of each biosimilar treated by neuraminidase were analyzed on a 2-10 pH gradient gel. Black arrows indicate newly identified bands specific of rEPOs and well expressed in biosimilars. Two dimension gel electrophoresis : The first attempts confirmed the previously published observations with Eprex [3], but the 2D-separation did not improve biosimilars identification compared to IEF-PAGE or SDS-PAGE (data not shown). This method is also technically complex and no clear criteria were identified for biosimilar detection. Analysis of N-Glycosylations As shown in Figure 5, all profiles were qualitatively similar between Eprex and the 3 biosimilars: they all exhibited the same N-glycan forms. This indicates that EPO was correctly produced. However, there were significant differences in relative abundance for some specific hyposialylated N-glycan forms. In particular we identified a significant enrichment in bi-antennary and tri-antennary forms compared to Eprex and a decrease of the tetra-antennae tetrasialylated form (the main N-glycan form of Eprex ) towards an increase of the tetra-antennae N-acetyl-lactosamine (LacNAc) bisialylated form for Epotin™ and Jimaixin™. This might happen due to the conditions of culture or during the purification process [7- 9]. Epotin™ and Jimaixin™ presented the most pronounced differences while Hemax had a ® ® ® ® ® ® ® ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-920 glycosylation profile closer to Eprex . This was in good agreement with the IEF-PAGE profiles of all 3 biosimilars. Figure 5. Graphical representation of the relative abundance of each N-glycan EPO structures for Eprex and the three biosimilars. Mean+/-SD of all replicates obtained for each immuno-purified biosimilar (n=5 for Epotin™, n=6 for Hemax and Jimaixin™) and for Eprex (n=6) are presented. Major differences with Eprex® are shown in red squares. Statistical comparison with Eprex for each N-glycan structure were performed: *: p < 0.05, **: p < 0.01. Green circles, mannoses; yellow circles, galactoses; blue squares, N-acetyl- glucosamines; red triangles, fucoses; purple diamonds, sialic acids. Conclusions In this study, 3 biosimilars of rEPO (Epotin™, Hemax and Jimaixin™) were characterized and presented unusual profiles by IEF-PAGE and/or SDS-PAGE. After a spike in urine and plasma, the detection was slightly more difficult than conventional rEPO Eprex in particular with low doses. SDS-PAGE analysis and urine matrix were the best option for the detection of all biosimilars. In addition, this method avoid the risk of false suspicions due to active/effort urines that might happen with IEF-PAGE. Using a 2D-gel analysis approach was not useful but a treatment with neuraminidase slightly improved the detection. Comparison of N-glycosylations between the biosimilars and Eprex indicated an increase in less acidic and less complex glycan forms the three biosimilars in line with their different electrophoretic profiles. These findings needs to be confirmed by an appropriate administration study in healthy men. References 1. Thomson RI, Gardner RA, Strohfeldt K, et al. Analysis of Three Epoetin Alpha Products by LC and LC-MS Indicates Differences in Glycosylation Critical Quality Attributes, Including Sialic Acid Content. Anal Chem. 2017;89(12):6455-6462. ® ® ® ® ® ® ® ® MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-921 2. TD2014EPO. Harmonization of analysis and reporting of erythropoiesis stimulating agents (ESAs) by electrophoretic techniques. www.wada-amaorg/sites/default/files/resources/files/WADA-TD2014EPO-v1- Harmonization-of-Analysis-and-Reporting-of-ESAs-by-Electrophoretic-Techniques-ENpdf. 2014 3. Desharnais P, Naud JF, Ayotte C. Desialylation improves the detection of recombinant erythropoietins in urine samples analyzed by SDS-PAGE. Drug Test Anal. 2013;5(11-12):870-876. 4. Martin L, Martin J, Collot D, et al. Improved detection methods significantly increase the detection window for EPO microdoses. Drug Test Anal. 2020;in press. 5. Bruneel A, Cholet S, Drouin-Garraud V, et al. Complementarity of electrophoretic, mass spectrometric, and gene sequencing techniques for the diagnosis and characterization of congenital disorders of glycosylation. Electrophoresis. 2018;39(24):3123-3132. 6. Goyallon A, Cholet S, Chapelle M, Junot C, Fenaille F. Evaluation of a combined glycomics and glycoproteomics approach for studying the major glycoproteins present in biofluids: Application to cerebrospinal fluid. Rapid Commun Mass Spectrom. 2015;29(6):461-473. 7. Nam JH, Zhang F, Ermonval M, Linhardt RJ, Sharfstein ST. The effects of culture conditions on the glycosylation of secreted human placental alkaline phosphatase produced in Chinese hamster ovary cells. Biotechnol Bioeng. 2008;100(6):1178-1192. 8. Gupta SK, Shukla P. Glycosylation control technologies for recombinant therapeutic proteins. Appl Microbiol Biotechnol. 2018;102(24):10457-10468. 9. Zhang P, Woen S, Wang T, et al. Challenges of glycosylation analysis and control: an integrated approach to producing optimal and consistent therapeutic drugs. Drug Discov Today. 2016;21(5):740-765. Acknowledgements This project has been carried out with the support of the World Anti-Doping Agency (grant 18B04AM) and AFLD’s own resources. MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-922 Heiland CE, Masquelier M Sepharose gel beads: an effective technique for recombinant erythropoietin immunopurification from blood Doping Control Laboratory Karolinska University Hospital, Stockholm, Sweden Abstract We have previously shown that the performance of the immunopurification step used to concentrate erythropoietin (EPO) and its recombinants before electrophoretic analysis has an impact on the detection of EPO. Although urine is the most common doping control matrix, blood seems to be a better alternative for abuse detection as highlighted by WADA’s published statistics for EPO between 2015 and 2018. In this study, an EPO Purification Gel Kit for Blood (EPGK for Blood; MAIIA Diagnostics) containing anti-EPO antibody-linked sepharose gel beads was used to immunopurify blood samples fortified with four erythropoiesis stimulating agents (BRP, NESP, EPO-Fc and CERA) and the results were validated, with special attention to detection limit and eluate stability determination. Lately, dried blood spots (DBS), an alternative collection method and matrix, has been attractive for the anti-doping scene. Therefore, the same immunopurification technique was also applied to extract ESA from DBS to evaluate the impact of low blood volume of the spots on the detection limit and if multiple spots can compensate and increase the method sensitivity. Introduction Between 2015 and 2018, erythropoiesis-stimulating agent (ESA) adverse analytical findings (AAFs) in blood represented over 20% of the total number of ESA AAFs, although blood tests made up only about 8% of the total ESA tests taken [1], showing the importance of blood as a matrix for recombinant erythropoietin (rEPO) detection. This could be attributed to blood stabilizing effects from high protein concentration and to the cold transport requirement reducing erythropoietin (EPO)-degrading bacteria in blood. Consequently, more blood analysis should result in better detection of rEPO. Choosing an effective immunopurification method is also crucial, as shown previously [2]. The EPO Purification Kit (EPK; MAIIA Diagnostics), commonly used in anti-doping laboratories, has caused filtration issues in some urine, therefore MAIIA developed a gel kit (EPGK) using sepharose gel beads coupled to anti-EPO antibodies, aiming to simplify purification, increase sample throughput, and reduce sample handling, while keeping a good EPO detection both in urine [3] and in blood. The possibility of using DBS for EPO detection after ELISA immunopurification has been described by Reverter-Branchat et al. [4], thus initiating interest in the EPGK’s capabilities for this matrix. Therefore, in this study, serum and DBS were spiked and immunopurified with the EPGK to evaluate and validate the kit for EPO blood analysis. The validation evaluated the detection limit and immunoextract stability, the application of this technique to DBS, and the impact of DBS sample volume on EPO detection. MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-923 Experimental Materials Reference standards used for spiking were: Biological Reference Protein (BRP) (European Directorate for the Quality of Medicines and Healthcare; Strasbourg, France); NESP (Aranesp; Amgen, Thousand Oaks, CA, USA); CERA (Mircera; Roche, Mannheim, Germany); and EPO-Fc (Prospec, Ness-Ziona, Israel). EPO Purification Gel Kit (EPGK) for Blood was provided by MAIIA Diagnostics (Uppsala, Sweden). Whatman 903 protein saver cards were from GE Healthcare (Uppsala, Sweden). Trayster Digital (IKA; Staufen, Germany) was used for EPO extraction. Biotinylated AE7A5 monoclonal antibody from R&D Systems (MN, USA) was used as the detection antibody. Streptavidin-HRP was from Calbiochem (CA, USA). Method Sample collection: Serum samples were collected from volunteers, anonymized, and pooled. Samples were either kept blank or fortified with four ESAs. Whole blood was collected from volunteers, anonymized, spiked and pipetted onto Whatman 903 protein saver cards (50 µL/spot). Immuno-extraction and -purification: The method was performed according to the manufacturer’s instructions [5]. Briefly, 0.5 mL of serum or 1 whole blood spot (50 µL) and Sample Buffer were placed in a column attached to a funnel, which was then inserted into a Falcon tube, and incubated end-over-end. The sample was filtered with vacuum pressure, the gel beads were washed, and the EPO was eluted with Elution Buffer (35 µL for serum and 50 µL for DBS). SAR-PAGE and Western blot: A single blot analysis was done as previously described [3], with a few changes to the sample volume used for analysis and the detection antibody. 8 µL (serum) or 16 µL (DBS) of sample were loaded onto the electrophoretic gel. Immunodetection was performed as follows: biotiny‐ lated antibody (0.5 µg/mL) overnight, wash, streptavidin-HRP (0.5 µg/mL) for one hour, and a wash. Serum stability: The stability of spiked serum eluates was investigated at -20°C, 4°C, and room temperature for 0, 1, 7, and 30 days after immunopurification. Serum samples were spiked with BRP (150 pg/mL serum), NESP (30 pg/mL serum), CERA (1000 pg/mL serum), and EPO-Fc (500 pg/mL serum). Limit of detection for serum: Five decreasing concentrations of the four ESAs were analyzed in 1 mL of serum by spiking the samples as follows: 2000 – 18.8 pg CERA; 1000 – 1.8 pg EPO-Fc; 60 – 3.8 pg NESP; 300 – 18.8 pg BRP. Limit of detection for DBS: Six decreasing concentrations of the 4 ESAs were analyzed in 50-µL DBS by spiking whole venous blood as follows: 200 – 6.3 pg CERA; 50 – 1.6 pg EPO-Fc; 5 – 0.2 pg NESP; 25 – 0.8 pg BRP. DBS sample volume: Venous blood was spiked with 4 ESAs (126 pg BRP, 50 pg NESP, 1000 pg CERA, and 250 pg EPO-Fc per mL whole blood) and pipetted onto a filter paper support and four 50-µL spots were immunopurified together and compared with immunopurification of one spot and 100 µL of serum spiked with the same ESA concentrations. Purification of four 50-µL DBS discs is equivalent to approximately 100 µL of serum. The volumes of immunopurified sample were adjusted for loading on the electrophoretic gel. MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-924 Results and Discussion The stability of spiked serum eluates is presented in Figure 1. All ESAs spiked in serum samples were quite stable at all temperatures for at least 30 days, with a decrease mainly for CERA and EPO-Fc at room temperature. Therefore, the immunopurified samples should preferably be kept at 4°C or -20°C. Figure 1. Stability experiment of eluates from serum samples spiked with 4 ESAs and immunopurified with EPGK. Samples were stored at -20 °C, 4 °C, and room temperature for 1, 7, and 30 days and compared to freshly purified sample (Day 0). (A) Gel image of the eluate stability experiment. (B) Graphs of the variation in percentage of band intensity of stored samples compared to freshly purified sample. For serum spiked and immunopurified by EPGK, LOD was determined to be approximately 31.2 pg for CERA, 15.6 pg for EPO-Fc, 3.8 pg for NESP, and 18.8 pg for BRP per mL serum. However, this is calculated assuming a recovery range of 100% and for application of only 8 µL of immunopurified eluate on the gel. The LOD obtained for DBS was determined to be approximately 125 pg for CERA, 62.5 pg for EPO-Fc, 12.5 pg for NESP, and 62.5 pg for BRP per mL whole blood. The detection limits determined by Reverter-Branchat et al. (1200 pg CERA, 60 pg for NESP, and 60 pg for BRP per mL whole blood) and obtained using 25-µL spots of modeled-blood [4] were similar for BRP but higher for NESP and CERA MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-925 compared to our results. Indeed, for DBS our LOD was comparable or better, but it was still 3 to 4 times higher than the LOD we obtained for serum immunopurification, as could be expected, which was confirmed in Figure 2. When comparing band intensities of these two matrices, spiked alike, it is apparent that the ESA detection is comparable to each other (Figure 2A and B). Therefore, it seems that multiple DBS are an effective approach in order to reach the same sensitivity as in serum. The importance of sample volume for EPO analysis was further demonstrated when 4 DBS were compared to only 1 DBS (Figure 2C). There is a noticeable improvement in EPO detection as the number of discs increases. Figure 2. Sample volume comparison for serum and DBS. (A) Serum samples (two replicates of 100 µL each) spiked and immunopurified with the EPGK. (B) DBS samples (two replicates of four 50-µL spots each) spiked and immunopurified with the EPGK. (C) DBS samples (two replicates of one 50-µL spot each) spiked and immunopurified with the EPGK. Concentrations (pg/mL whole blood): BRP: 125 pg, NESP: 50 pg, CERA: 1000 pg, EPO-Fc: 250 pg. Volumes loaded onto electrophoretic gel: 11.2 µL for serum and 16 µL for DBS. Since sample volume is a limitation for optimal EPO detection in DBS, there is a need for sufficient sample volumes and sensitive methods when DBS are added to the anti-doping sample matrix repertoire. Therefore, future experiments will include efforts to improve sensitivity by concentrating the DBS eluate during immunopurification and loading the entire volume onto the electrophoretic gel in order to compensate for the small initial volume of whole blood. This will hopefully optimize ESA detection in DBS. Other possible solutions might be to optimize transfer during the blotting step by performing a discon‐ tinuous semi-dry transfer as described [6], instead of using the typical continuous transfer system. While the possibility of detecting EPO in patient samples has been demonstrated by Reverter-Branchat et al. [4], it would be interesting to investigate lower doses of rEPO that an athlete might potentially use, and this investigation is currently underway. Conclusions The EPO Purification Gel Kit for Blood can easily be used to immunopurify serum spiked with ESAs, giving MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-926 stable eluates and detection comparable to the previous method. Moreover, it could be successfully applied to extract EPO from dried blood spots. More investigations are needed to further evaluate the detection limits, such as a large-scale study where DBS samples would be collected from participants administered with rEPO. References 1. World Anti-Doping Agency. 2018 Anti-Doping Testing Figures. (2018) https://www.wada-ama.org/ en/resources/laboratories/anti-doping-testing-figures-report (access date 01.2020). 2. Masquelier M, Heiland C, Bhuiyan H, Erlandsson G, Molin A, Ericsson M. (2018) Optimization of ESAs detection in urine and blood: comparison of different immunopurification techniques. In: Thevis M, Geyer H, Mareck U (eds.) Recent advances in doping analysis (26). Sportverlag Strauß, Köln 58-65. 3. Heiland CE, Masquelier M, Bhuiyan H, Ericsson M. (2019) A simple method to immunopurify erythropoiesis stimulating agents from urine, aiming to optimize erythropoietin screening by SAR-PAGE. Drug Test Anal. 11 (11-12), 1666-1674. 4. Reverter-Branchat G, Ventura R, Ezzel Din M, Mateus J, Pedro C, Segura J. (2018) Detection of erythropoiesis-stimulating agents in a single dried blood spot. Drug Test Anal. 10 (10), 1496-1507. 5. MAIIA Diagnostics. EPO Purification Gel Kit – For Blood. (2019) http://media.maiiadiagnostics.com/2020/ 01/DfU-EPGK-for-Blood.pdf (access date: 30.07.2020) 6. Martin L, Martin J-A, Collot D, Hoang O, Audran M, Ericsson M, Marchand A. (2020) Improved detection methods significantly increase the detection window for EPO microdoses. Drug Test Anal. 2020;1-12. doi.org/10.1002/dta.2904 Acknowledgements We would like to thank MAIIA for providing the immunopurification kits for this project and for being readily available for questions and advice concerning the purification procedure. MANFRED DONIKE WORKSHOP 2020 Lecture RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-927 Sigmund G, Geyer H, Goergens C, Thevis M Prevalence of the imidazole derivatives naphazoline, oxymetazoline and xylometazoline in doping control samples Institute of Biochemistry, German Sport University Cologne, Cologne, Germany Abstract In 2017 an AAF for the imidazole derivative naphazoline was reported the first time in the WADA testing figures since their implementation in 2003. This reporting of an AAF was particularly noteworthy as the Cologne laboratory frequently detects imidazole derivatives in in-competition samples, but these findings are not reported as AAFs due to the remark in the prohibited list that imidazole derivatives are permitted for topical/ophthalmic use. To assess the prevalence of imidazole derivatives among German and international elite athletes, the Digital Matrix (full scan data of GC/MS analyses) of 5500 in-competition (IC) doping control samples from different sports, collected from January till June 2017, were evaluated for the imidazole derivatives naphazoline, oxymetazoline and xylometazoline. Out of the 5500 evaluated samples, 199 samples contained imidazole derivatives (3.6 %). Out of the 199 samples, xylometazoline was detected in 178 samples (89.4%), oxymetazoline in 11 samples (5.5 %) and naphazoline in 10 samples (5.0 %). The majority of the target analytes (45.2 %) was detected at concentrations between 101-500 ng/mL. Twelve samples (6.0 %) showed concentrations above 1000 ng/mL. Introduction According to the WADA Prohibited List, S6 stimulants, imidazole derivatives are prohibited except for dermatological, nasal or ophthalmic use [1]. In 2017 an AAF for the imidazole derivative naphazoline was reported the first time in the WADA testing figures since their implementation in 2003 [2]. Details about the reporting of the naphazoline (e.g. extraordinary high concentrations, missing declaration in DCF, missing TUE etc.) were not available. This reporting of an AAF was particularly noteworthy as the Cologne laboratory frequently detects imidazole derivatives in in-competition samples, but these findings are not reported as AAFs due to the remark in the prohibited list that imidazole derivatives are permitted for topical/ophthalmic use. The time interval from January till June was chosen to cover different seasons of the year. The DCFs were not evaluated for the declaration of imidazol derivatives. Experimental The Digital Matrix, i.e. full scan data of GC/MS analyses, of 5500 in-competition (IC) doping control samples from different sports were evaluated for the presence of the imidazole derivatives naphazoline, oxymetazoline and xylometazoline. Additionally, the concentrations of these derivatives were estimated. MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-928 In order to cover different seasons, the samples analyzed came from the period from January to June 2017. The DCFs were not evaluated for the declaration of imidazol derivatives. Samples were analyzed according to the method described by Thevis et al. in 2007 [3]. The used analytical method in brief: LLE of 5 mL urine at pH 14 with TBME, injection of 5 µL of organic layer into a GC/MS/NPD system. The mass spectrometer was operated with EI and full scan analysis m/z 40-400, 4 scans/s. Results and Discussion Out of the 5500 evaluated samples, 199 samples contained imidazole derivatives (3.6 %). 10 samples contained 2 different imidazole derivatives. Out of the 199 samples, xylometazoline was detected in 178 samples (89.4%), oxymetazoline in 11 samples (5.5 %) and naphazoline in 10 samples (5.0 %). The majority of the target analytes (45.2 %) was detected at concentrations between 101-500 ng/mL (see Figure 1). Twelve samples (6.0 %) showed concentrations above 1000 ng/mL. Corresponding sample details are presented in Table 1. The imidazole derivatives were detected in 46 different sports. Most findings were detected in football (Table 2). Figure 1. Estimated concentrations of the imidazole derivatives in the 199 findings MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-929 Table 1: Samples with concentrations of imidazole derivatives > 1000 ng/mL Table 2: Sports with most frequent findings of imidazole derivatives Conclusions This is the first time that the frequency of the use of imidazole derivatives in elite athletes has been evaluated. More than 3% of all tested IC samples showed the presence of imidazole derivatives. Xylometazoline was the most frequently detected imidazole derivative. These data should be presented to the WADA Prohibited List expert group to facilitate the decision on whether or not a reporting limit should be implemented for these substances (e.g. > 1000 ng/mL). If it is agreed to implement such a reporting level, as next step excretion studies with imidazole derivatives MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-930 have to be conducted, to evaluate whether the detected concentrations are consistent with a permitted topical/ophthalmic use or prohibited routes of administration. References 1. WADA prohibited List 2017, accessdate : 05.10.2018 2. WADA 2107 Anti-Doping Testing Figures. Laboratory Report, page 23 of 26., access date: 05.10.2018 3. Thevis M, Sigmund G, Koch A, Schänzer W. Determination of tuaminoheptane in doping control urine samples. Eur. J. Mass Spectrom 13, 213 (2007) 2013-221 Acknowledgements The presented work was conducted with financial support of NADA Germany (Bonn, Germany), the Federal Ministry of the Interior, Building and Community of the Federal Republic of Germany and the Manfred-Donike Institute for Doping Analysis (Cologne, Germany). MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-931 Wicka M, Kaliszewski P, Grucza K, Stanczyk D, Drapala A, Konarski P, Zalewska Z, Siek P, Michalak D, Kwiatkowska D Elimination profile of triamcinolone acetonide and its metabolite in human urine after multiple transdermal administration Polish Anti-Doping Laboratory, Warsaw, Poland Abstract Triamcinolone acetonide (TA) is prohibited in competition and only for oral, intravenous, intramuscular or rectal administration. In order to distinguish whether TA was administered by one of the prohibited routes or not, a specific reporting limit for urinary concentrations of the parent compound and its metabolite was established at 30 ng/mL. Triamcinolone acetonide is a synthetic corticosteroid applied topically to treat various skin conditions, intra-articularly to treat joint injuries, intramuscularly for the control of severe allergic states. In nasal spray form, it is used to treat allergic rhinitis. The aim of the present work was to study the metabolic profile of Triamcinolone acetonide in urine following transdermal administration. Four healthy male volunteers applied 4 g of Pevisone (econazole nitrate 10 mg/g + Triamcinolone acetonide 1.1 mg/g) once a day and twice a day throughout three consecutive days. Four spontaneous urine samples were collected per day i.e. in the morning, midday, afternoon and evening. Urine samples were analyzed by liquid chromatography combined with mass spectrometry to measure TA and 6β-OH triamcinolone acetonide (6β-OH TA), the main TA metabolite. After administration, the concentration of TA was significantly lower than of 6β-OH TA. For 6β-OH TA, a greater concentration was obtained: 0.10 - 4.36 ng/mL. However, concentrations of TA and 6β-OH TA were lower than 30 ng/mL for all volunteers. Introduction Triamcinolone acetonide is asynthetic corticosteroid used as anti-inflammatory drug to treat various diseases. It is applied topically to treat various skin conditions (e.g., severe erythema multiforme and pemphigus), intra-articularly to treat joint injuries (e.g., gout, psoriatic arthritis and systemic lupus erythematosus), intramuscularly for the control of severe allergic states. In nasal spray form, it is used to treat allergic rhinitis. It is used to treat many different conditions such as gastrointestinal diseases (e.g., ulcerative colitis)[1]. Triamcinolone acetonide is widely used by athletes to treat articular pain, injures and sprains [2]. According to the WADA Prohibited List [3], glucocorticosteroids are prohibited in competition and only when administered by oral, intravenous, intramuscular or rectal routes. In order to distinguish whether glucocorticosteroids were administered by one of the prohibited routes or not, a specific reporting limit for urinary concentrations of parent compounds and their metabolites was established at 30 ng/mL [4]. MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-932 Experimental Four male healthy subjects, aged 28, 29, 41 and 60 years, height and weight respectively 179 cm/84 kg, 175 cm/67 kg, 178 cm/83 kg, 178 cm/90 kg (subject 1M, 2M, 3M and 4M), applied 4 g of Pevisone (econazole nitrate 10 mg/g + Triamcinolone acetonide 1.1 mg/g) once a day for three days and then twice a day for three consecutive days – transdermal administration. One dose of the preparation that was applied to the skin weighed 4 grams and provided a one-time dose of 4.4 mg of the active substance acetonide triamcinolone. All volunteers applied the preparation to the sacrolumbar part of the spine. Four spontaneous urine samples were collected per day, i.e. in the morning, midday, afternoon and evening. Urine samples were collected for 5 days after the final administration (Figure 1). Figure 1. Scheme of dosage of Pevisone and administration The sample preparation is a two-step procedure involving enzymatic deconjugation of glucuronides and then liquid-liquid extraction with methyl tert-butyl ether. Chromatographic separation was conducted using a Waters Acquity UPLC System liquid chromatograph with BEH C18 (1.7 µm, 100 mm x 2.1 mm) from Waters. The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B), and LC gradient was employed at the constant flow rate of 300 µL/min at 45°C. Triamcinolone acetonide (TA) retention time was 4.43 min, while that of 6β-OH triamcinolone acetonide (6β-OH TA) was 3.10 min. MRMs of the studied substances were traced with a Micromass Quattro Premier XE mass spectrometer equipped with an ESI source. All analytes were investigated in the ESI mode. Desolvation gas flow was set at 643 L/h at 450°C with ion source temperature at 120°C. Capillary voltage was 3.0 kV. + MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-933 Results and Discussion Characteristics of the calibration curves are shown in Table 1. Table 1. TA and 6β-OHa curve parameters The LOD for TA and 6β-OH TA were estimated at 0.05 ng/mL. The linear regression curve ranged from 0.5 to 30 ng/mL and had a satisfactory coefficient of correlation. Typical MRM chromatograms generated from a urine sample spiked with 30 g/mL of Triamcinolone acetonide and the metabolite 6β-OH Triamcinolone acetonide are shown in Figure 2. Blank urine control samples which were analyzed in each series of the validtion procedure (n=6) were free of compounds wich might be eluted in the retention times of TA and 6β-OH TA. As shown in Figure 2, TA and 6β-OH TA could be identified in human urine after transdermal administration at low concentrations. MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-934 Figure 2. Typical MRM chromatograms: (A) spiked urine 30 ng/mL; (B) urine sample pre and 3 day after transdermal administration In our study, 4 g per day for three days, and 8 g per day of TA cream for three consecutive days (total 36 mg of cream, equivalent 39.6 mg of the active substance acetonide triamcinolone) were administered to the volunteers. The topical dose was administered for six consecutive days, to determine if repeated doses lead to accumulation of TA in urine. Figure 3 shows the changes in the concentrations of TA and 6β-OH TA after administration. The maximum concentration of TA were in the range of 0.7 – 0.8 ng/mL. However, the maximum concentation of 6β-OH TA was in the range of 1.93 – 4.36 ng/mL.The low concentration values obtained for TA, as in case [1], were resulted from a low absorption of TA by the skin. There was also a visible lack of TA accumulation after multiple administrations. The high inter- individual variability in concentrations between volunteers was probably depending on the age and the metabolism associated with it. rd MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-935 Figure 3. Changes in the concentrations of TA and 6β-OH TA in urine after administration Conclusions 1. During monitoring of the profile excretion of Triamcinolone acetonide, the concentration values ranged from 0.10 ng/mL to 0.80 ng/ml. While for the metabolite of Triamcinolone acetonide (6β-OH Triamcinolone acetonide), the concentration values ranged from 0.10 ng/mL to 4.36 ng/mL. 2. After the end of the last dose of Pevisone, the presence of both substances was observed for an average of 2 days. 3. Based on the data collected, the Triamcinolone acetonide and 6β-OH-Triamcinolone acetonide were not detected above the reporting limit. References 1. Xavier Matabosh, Oscar J. Pozo, Clara Pérez-Mañá, Esther Papaseit, Josep Marcos, Jordi Segura, Rosa Ventura. Evaluation of the reporting level to detect triamcinolone acetonide misuse in sports. J. Steroid Biochem. Mol. Biol. 145 (2015) 94-102 2. Chih-Wei Chang, Tai-Yuan Huang, Yi-Chun Tseng, Guo-Ping Chang-Chien, Su-Fan Lin, Mei-Chich Hsu. Positive doping results caused by the single-dose local injection of triamcinolone acetonide. Forensic Sci. Int. 244 (2014) 1-6 3. World Anti-Doping Agency. The 2020 Prohibited List, https://www.wada-ama.org/sites/default/ files/wada_2020_english_prohibited_list_0.pdf.Accessed January 2020 4. World Anti-Doping Agency TD2019MRPL, MINIMUM REQUIRED PERFORMANCE LEVELS FOR DETECTION AND IDENTIFICATION OF NONTHRESHOLD SUBSTANCES. https://www.wada-ama.org/sites/default/files/ resources/files/td2019mrpl_eng.pdf. accessed January 2020 Acknowledgements Financial support from the Ministry of Sport and Tourism of the Republic of Poland under the project numer 2019.0432/1575/UDot/BM is gratefully acknowledged. MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-936 Barbán Duarte L, Martínez Brito D, Correa Vidal T, Montes de Oca Porto R Trend analysis and decision making in the Havana Anti-Doping Laboratory Havana Antidoping Laboratory, La Habana, Cuba Abstract Trend analysis is a technique widely used in the fields of finance, marketing, and decision making in general. The Havana Anti-Doping Laboratory, accredited uninterruptedly since 2003, possesses and applies internal procedures to internally calibrate the measurement instruments. The objective of the present study was to evaluate the trends of nine calibration parameters of three Agilent Technologies 5973N Simple Quadrupole, in order to provide data for a feasible decision making. Three tests were applied to the data (nine parameters evaluated annually from 2003 to 2019) in order to check the presence of trends: Mann-Kendall trend test, Spearman test and Kendall tau test. Then, a prediction was estimated by the slope, intercept and, the residues values. The study was carried out for gas chromatograph instruments and for the calibration parameters as injector and detector linearity, flow and injector accuracy and uncertainty. The results allowed to obtain the approximate date that the instruments could reach a maximum value of the calibration acceptance criteria. This criterion could (should) be used by the Laboratory Manager in order to plan their replacement and to plan the use of the instrument. One of the results was that quantification assays should be suspended for the instrument coded as MSD4 with the aim of maintaining the reliability of the results reported by the Laboratory. Introduction The decision process has characteristics that make it continuous, interrelated and the result of a statistical process. According to Fundamental and General Metrology Terms, “calibration” is the set of operations with which, under specified conditions, the correspondence between the values indicated in the instrument, equipment or measurement system and the values are established represented by a reference material or by known values corresponding to a measurement or standard. The equipment and patterns responses, vary over time due to different causes, i.e. aging, deterioration, improper cleaning and chemical reactions [1,2]. The main objective was to evaluate trends based on the data obtained in the calibration processes of three GC/MS in the Havana Antidoping Laboratory during sixteen years. Experimental Data and methods applied Three Agilent Technologies 5973N Simple Quadrupole coded as MSD2, MSD3 and MSD4 were studied through nine parameters that define the instrument performance. The verification of these parameters is MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-937 carried out every year and the present study included the results obtained from 2003 to 2019. Table 1 describes the parameters and the acceptance criteria applied in the evaluation of the instruments. Statistics. Data obtained in the lapse from 2003 and 2019 was statistically evaluated through three tests in order to check the presence of trends: Mann-Kendall trend test; Spearman test and Kendall tau test with 95% confidence interval [3,4]. After to applied the tests to the data, it was selected the parameters that show trends in their behaves between 2003 and 2019 and finally; a long term prediction by the application of the “automatic forecasting” (by the slope, intercept and, the residues values) was made and, it was estimated the year in which the maximum value of the Calibration Acceptance Criteria should be achieve. Software used were STATGRAPHICS centurion XV, version 15.1.02, StatPoint Inc, 2008 and; PAST 3.26 (Hammer, Ø, Harper, DAT Ryan PD 2001. PAST: paleontological statistics software package for education and data analysis Paleontological Electronica 4 (1): 9pp. Table 1 . Parameters evaluated during the calibration process of three single quadrupole gas chromatograph- mass spectrometer (GC/MS) and the acceptance criteria in the Havana Anti-Doping Laboratory Results and Discussion Annually, the verification of the calibration status for each instrument is perform using the “accepted” or “no accepted” criteria. This does not allow to evaluate trends in the data. After 17 working years, out of the nine parameters verified, the flow (in)accuracy and detector carryover showed values permanently close to zero and for that, they remained out of the analysis. The Injector Precision (PI) evaluates the precision of the injector by calculating the coefficient of variation of the chromatographic peaks areas of the reference material obtained after to inject consecutively 5 replicates of the 1 µL injection volume. This parameter showed a marked trend for all three instruments (Spearman rho, Kendall tau and Mann-Kendall trend, α = 0.05, Table 2). Because it is a significatively contributor to the uncertainty value, also this parameter showed a significant trend for all three instruments [5]. Additionally to injector precision (PI) and uncertainty (uc), the instrument MSD4 showed a significant trend for the parameter Injector Linearity (LI). MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-938 Table 2 . Statistical evaluation of the GC/MS calibration data during 17 years. The bold and underlined values represent a statistically significant trend (α = 0.05). Table 3 shows the results obtained for the prediction analysis. As expected, the uncertainty was the “warning parameter” because its maximum accepted value should be reached firstly that others. Being the Injector the main instrument part linked to the obtained results, the Manager may consider the possibility to replace only this part and no the all instrument. The instruments included in this trend analysis are GC/MS single quadrupole that are used today to quantified substances with a threshold at high concentrations values (per example Salbutamol or Tetrahydrocannabinol carboxylic acid) and also, they are used for research tasks. After these results, the laboratory routine should be re-organized in order to avoid the use of the instruments at least for quantitation purposes. Table 3 . Description of the prediction line obtained for each instrument and the year when the instrument should reach the maximum acceptance criteria in the calibration process MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-939 Figure 1. Prediction line obtained for the parameters that showed a statistical trend from 2003 to 2019 Conclusions The analysis of trends using the data obtained from the calibration process showed results that could (should) be used by the Laboratory Management in order to plan and execute the budget of the laboratory. It is important to take into account that the prediction proposed here are valid as long as the same level of labor and frequency of maintenance operations are maintained. These predictions allow an adequate and prioritized budget enforcement as well as the re-organization of the tasks performed in each instrument. References 1. Bureau International des Poids et Mesures (BIPM), JCGM 200: 2012, International Vocabulary of Metrology – Basic and General Concepts and Associated Terms. Third edition, 2008 version with minor corrections, 2012. 2. Guerra Bretaña, Rosa Mayelin, Meizoso Valdés, María del Carmen. Gestión de Calidad: Conceptos, Talleres y Herramientas. Editorial Universidad de la Habana, p. 219, 2012. 3. Øyvind Hammer. Natural History Museum, University of Oslo PAST PAleontological Statistics Version 3.25. Reference manual. 1999-2019. 4. Richard O. Gilbert. Statistical Methods for Environmental Pollution Monitoring. February 1987. ISBN: 978-0- 471-28878-7. http://www.osti.gov/scitech/biblio/7037501/. 5. Vicki J. Barwick. Review Sources of uncertainty in gas chromatography and high-performance liquid chromatography. Journal of Chromatography A, 849 (1999) 13–33. MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-940 Rodriguez Fernandez A, Laffita D, López Castillo D, Terrero O, Montes de Oca R Increase in the incidence of THC in urine samples of athletes in 2019 Havana Antidoping Laboratory, Sports Medicine Institute, La Habana, Cuba Abstract According to regulations of the World Anti-Doping Agency (WADA), the use of cannabinoids is forbidden in competition [1]. In doping controls, the detection of cannabinoid misuse is based on the analysis of the pharmacologically inactive metabolite 11-nor-Δ -carboxy-tetrahydrocannabinol-9-carboxylic acid (THC- COOH), and urinary concentrations greater 150 ng/mL constitute as an adverse analytical finding (AAF) [2,3]. This threshold level was only 15 ng/mL until 2013. The objective of this study is to describe the presence of THC-COOH in samples of athletes received in the Havana Anti-doping Laboratory between 2017 and 2019. Samples were classified by sports, gender, and country, as well as by means of the collection mode In Competition (IC) and Out of Competition (OOC). In 2019, 40.0% of the collected IC samples showed concentrations between 15 and 150 ng/mL. The sports Judo, Football, Sambo and Para Table Tennis were showing concentrations higher than 150 ng/mL. Three of these samples were reported as Adverse Analytical Finding after adjusting the decision limits by specific gravity. In the same year (2019), OOC samples collected from eight different sports showed THC-COOH levels above 150 ng/mL. 18.9% of these samples came from Chile. This data show the increase of THC-COOH consumption during 2019 compared to 2017 and 2018. Introduction Tetrahydrocannabinol (THC), also known as Δ -tetrahydrocannabinol, is the main psychoactive constituent of cannabis [3,4]. THC is one of the substances of the S8 cannabinoid group that is prohibited in competition; the threshold level of the relvant analyte THC-COOH is 150 ng/mL [1,2]. Years ago this level was only 15 ng/mL. However, in May 2013, the WADA Executive Committee decided to increase the threshold level after considering the numerous presentations received from interested parties during the Code review process. Cannabis data from all sources has been analyzed for the potential benefits and hazards for athletes related to the abuse of cannabinoids for the purpose of doping in sports [3]. The objective of this study was to describe the presence of THC-COOH in samples of athletes between 2017 and 2019 and the increase in consumption in the last year. Experimental Data: A total of 13328 urine samples were processed between 2017 and 2019. Samples were collected according to the Initial Testing Procedure which is applied to all doping control samples in our laboratory in Havana. Sample preparation was done as described by Martínez-Brito [5]. Briefly, 2.5 mL of urine were hydrolyzed with β-glucuronidase, after previous addition of ISTD and pH correction to 7. Afterwards, 9 9 MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-941 liquid-liquid extraction using tert-butylmethyl-ether was done. Then, TMS derivatives were analyzed using an Agilent 7890B gas chromatograph coupled to a mass spectrometer triple quadrupole Agilent 7000. One quality control was added in each analysis batch. Reagents: THC-COOH was supplied by Cerilliant. All reagents and solvents (sodium bicarbonate, potassium carbonate, sodium phosphate, sodium hydrogen phosphate) were of analytical grade. Tert- butylmethyl-ether was HPLC grade. β-glucuronidase (Escherichia coli K12) from Roche Diagnostic (Mannheim, Germany). Mercaptoethanol, Ammonium Iodide and MSTFA were purchase by Sigma-Aldrich. Results and Discussion From all processed samples between 2017 and 2019, 1.9% showed the presence of THC-COOH in 2017, 1.8% in 2018 and 2.8% in 2019, showing an increase during the last year. The higher incidence of this substance was observed in males with 81.2%. These findings results from the screening and identification was done using a positive THC-COOH control. If the THC-COOH concentration, estimated with the positive control, is greater than 75 ng/mL (50% of the threshold), THC-COOH was quantified with three new aliquots of the sample. If we compare the presence of THC-COOH among sports in 2019, an increase was seen in some of them (Figure 1). Weight lifting, Judo, Athletics, and Tae-Kwon-Do were those with the highest levels. Figure 1. Sports with a greater number of samples where the presence of THC-COOH was detected (2017-2019) MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-942 In 2019, there was an increase of IC and OOC samples with THC-COOH concentration levels above 15 ng/mL (Figure 2). From the IC samples showing THC-COOH, 55.7% had a concentration higher than 15 ng/mL. This value was only 39.1% in 2018 and 26.8% in 2017. Out of 61 IC samples with presence of THC-COOH in 2019, 29 showed concentration levels between 15 and 150 ng/mL, representing 47.5%. In this study, concentration ranges of 15 ng/mL, 15-150 ng/mL and more than 150 ng/mL were determined, since the quantification thresholds set by WADA before and after 2013 were 15 ng/mL and 150 ng/mL. Figure 2. Representation for years of the concentration of THC-COOH detected by number of samples MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-943 In 2019, Athletics, Basketball, Fencing, Weight lifting and Volleyball had more OOC samples with concentration levels of THC-COOH above 15 ng/mL. On the other hand, Athletics, Football, Judo and Weight lifting were the sports with the highest incidence of this indicator for IC testing controls (Figure 3). After the adjustment of the decision limits for the specific gravity, the Adverse Analytical Findings reported in 2019 correspond to Sambo (219.8 ng/mL), Football (790.0 ng/mL) and Para Table tennis (1456.0 ng/mL). Figure 3. Sports with the highest incidence of THC-COOH in samples collected IC (a) and OOC (b) in 2019 MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-944 In 2019, the countries with the highest presence of THC-COOH in the OOC controls were Chile (26 out of 137 analyzed), Mexico (23 out of 1019 analyzed) and Cuba (8 out of 593 analyzed) (Figure 4). Figure 4. Countries with the highest presence of THC-COOH in the controls OOC in 2019 Conclusions In 2019, the presence of THC-COOH increased in the total samples received. Compared to previous years, 70% of sports showed an increase in THC-COOH detection. In 2019, IC samples from Judo, Football, Sambo and Para Table Tennis athletes were analyzed with THC-COOH levels higher than 150 ng/mL. Three of them were reported as Adverse Analytical Finding. During the same year, OOC samples were positive for THC-COOH levels higher than 150 ng/mL in 8 sports. Chile shows the highest presence of THC-COOH in OOC samples collected in 2019, representing 18.9 % of the samples received. References 1. World Anti-Doping Agency, The 2019 Prohibited List. International Standard., 2019, https://www.wada- ama.org/sites/default/files/wada_2019_english_prohibited_list.pdf 2. World Anti-Doping Agency, WADA Technical Document - TD2019DL v. 2.0: Decision Limits for the Confirmatory Quantification of Threshold Substances. 2019, (04.05.2020), https://www.wada- ama.org/sites/default/files/resources/files/td2019dl_v2_finalb.pdf 3. M. A. Huestis, I. Mazzoni, O. Rabin. Cannabis in Sport: Anti-Doping Perspective. Sports Med. 2011, 41, 949. 4. U. Mareck, et al. Temporal Indication of Marijuana Use. Recent advances in doping analysis, 2016, 14, Köln, 101-105 5. A. Rodríguez Fernández, D. Martínez Brito, T. Correa Vidal, D. López Castillo, R. Montes de Oca Porto. Evaluation of the steroid profile in Latin-American population of male football players. Recent advances in doping analysis, 2019, 27, Köln, 39-44 MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-945 Biasini GM, Donati F, Rutigliano L, de La Torre X, Botrè F Proteomic analysis of biomarkers of red blood cells storage: towards the development of direct detection of autologous blood transfusion Antidoping Laboratory of Rome, FMSI, Rome, Italy Abstract In recent years one of the greatest challenges for antidoping in sports has been to develop a direct method to detect the misuse of autologous blood transfusions (ABT) that can be tempted by cheating athletes to increase oxygen delivery to muscles so enhancing sport performance. Despite being prohibited by the World Anti-Doping Agency (WADA), there is currently no direct method to tackle ABT doping strategies. Storage of erythrocytes, under standard blood banking conditions, for transfusion purpose, induces biochemical and physiological changes that affect red blood cells (RBCs) shape, proteome, metabolism as well as affecting oxidative stress. At the protein level, alterations of the morphological structure of erythrocytes involve Band 3, the most abundant RBC transmembrane protein that plays a key role in membrane integrity and cytoskeletal organization. Moreover, loss of the oxidant- antioxidant balance mostly depends on Peroxiredoxin-2 (PRDX2), one of the primary RBC antioxidant enzymes whose effect is to limit hydrogen peroxide (H O )-mediated toxicity. The aim of the present project is to evaluate alterations occurring in RBC proteome during storage, comparing their proteome before and after different periods of time, under standard blood banking conditions. In this study, it has been demonstrated that levels of Band 3 decrease over storage time, highlighting its role in the structural and morphological organization of RBCs; moreover, levels of PRDX2 are shown to considerably increase over storage time with the consequence of the increased oxidative stress in erythrocytes. Introduction ABT is a banned method by the WADA [1] and consists of the withdrawal of a sufficient volume of blood from an individual, its storage, and its reinfusion. During storage, under standard blood banking conditions, erythrocytes undergo several modifications: loss of shape and membrane integrity [2,3], increased oxidative stress [4] and changes in metabolome and proteome [5]. Phenomena of aggregation and degradation occur to proteins over time, representing a "signature" of RBCs storage. We selected two new biomarkers: Band 3, a transmembrane protein of RBCs involved in cytoskeleton organization [6,7], and PRDX2, the main erythrocytes peroxidase [8,9], to evaluate their levels before and after different periods of storage, to assess the suitability of these biomarkers for the development of a direct method for ABT detection. 2 2 MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-946 Experimental Preparation of samples Whole blood samples were analyzed fresh at T0 and divided into two aliquots. Aliquoted samples were stored at 4°C in K EDTA tubes and stained for flow cytometry (FC) and western blotting (WB) applications after 20 (T1) and 40 days (T2). For the WB assay, blood samples (n=2) were washed twice with phosphate buffer (PB). Pellet was resuspended in N-ethylmaleimide (NEM) 0.1 M in PB solution and incubated for 15 minutes at RT. After incubation, 0.1 M of NEM in PB/2% cOmplete Protease Inhibitor Cocktail (Roche, Switzerland) solution was added to samples, and cells were lysed mechanically through a glass homogenizer. Samples were washed twice, and the pellet was resuspended in phosphate- buffered saline (PBS). Protein concentration was estimated through Bradford assay. Samples were denaturated at 70°C for 10 minutes. SDS-PAGE and Western Blotting Denatured protein samples (n=2) were analyzed through SDS-PAGE. 5 µg or 300 ng of each sample, respectively for PRDX2 and Band 3 analysis, were loaded in the gel (NuPAGE 4-12% Bis-Tris Gel, Novex- life technologies). Band 3 and PRDX2 standards were loaded as controls. The WB analyses were performed on PVDF membranes against PRDX2 (Anti-h/m/r Peroxiredoxin2 at 1/1600), Band 3 (SLC4A1 Monoclonal Antibody-BIII136 at 1/100000). The membrane was incubated with the secondary antibody (Gt anti-MS IgG (H+L) cross-adsorbed secondary antibody, HRP-conjugate) for 90 minutes. Images were acquired through the ImageQuant LAS4000. Bands of interest were quantified by densitometry by means of the GASepo software. Flow Cytofluorimeter assay Whole blood samples (n=5) were washed twice with saline. Band 3 antibody (CD233, Band 3 extra‐ cellular domain) was added to samples and incubated for 45 minutes. Samples were washed twice to remove the excess of antibody. Glycophorin A (GYPA) antibody as control was added and incubated for 30 minutes. Samples were rewashed to allow FC acquisition (FC500 Beckman Coulter). Results and Discussion Our results demonstrated a trend towards the increase of PRDX2 protein amount during storage, while β- actin, the normalizing control, remains stable over storage conditions. Levels of PRDX2 increased progressively after 20 days (p=0.09) and after 40 days (p=0.07) of storage compared to freshly collected blood samples. 2 MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-947 Figure 1a and b. The amount of PRDX2, present in erythrocytes, changes during storage. (a) Representative western immunoblots analysis of blood samples against PRDX2 and β-actin (b) Different levels of PRDX2 after different periods of storage (T1=20 days, T2=40 days) normalized with levels of β -actin, the loading control. PRDX2 levels progressively increase after 20 days (p=0.09) and after 40 days (p=0.07). The storage causes damages to the structure of PRDX2 that aggregates in high molecular weight complexes migrating from the cytosol to the membrane of the erythrocytes and, consequently, it becomes more detectable in RBCs membranes [9]. In parallel, there is a trend towards the decrease of Band 3 levels over storage time. The total amount of Band 3 decreases progressively after 20 days (p=0.99) and after 40 days (p=0.62) of storage compared to freshly collected blood samples. MANFRED DONIKE WORKSHOP 2020 Poster RECENT ADVANCES IN DOPING ANALYSIS (28) ISBN 978-3-86884-046-948 Figure 2a and b. The amount of Band 3, the major integral RBCs protein, changes during storage. (a) Representative western immunoblots analysis of blood samples against Band 3 and β-actin. (b) Different levels of Band 3 after different periods of storage (T1=20 days, T2=40 days) normalized with levels of β -actin, the loading control. Band 3 levels progressively decrease after 20 days (p=0.99) and after 40 days (p=0.62). To better assess the alterations that occur to Band 3 due to the storage of RBCs, we also conducted a dual-color staining FC assay. Figure 3a, b, and c shows the progressive decrease in both Band 3, GYPA levels, and their ratio during storage. As we can see, the levels of Band 3 decrease over storage time with a percentage of 19% after 20 days (p=0.06) and 39% after 40 days (p

M. THEVIS H. GEYER U. MARECK (EDITORS) RECENT ADVANCES IN DOPING ANALYSIS (31) Proceedings of the Manfred Donike Workshop 41st Cologne Workshop on Dope Analysis 27th February to 3rd March 2023 SPORTVERLAG Strauß - Hellenthal 2023 RECENT ADVANCES IN DOPING ANALYSIS (31) 2 ISBN 978-3-86884-049-0 Bibliografische Information Der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über abrufbar. Thevis, Mario; Geyer, Hans; Mareck, Ute (Eds.) Recent Advances in Doping Analysis (31). Proceedings of the Manfred Donike Workshop, 41st Cologne Workshop on Dope Analysis, 27th February to 3rd March 2023/ [MDI e.V.] – 2023 Sportverlag Strauß. ISBN 978-3-86884-049-0 ©SPORTVERLAG Strauß Neuhaus 12 – 53940 Hellenthal Tel. +49 (0)2448/2470040 – Fax +49 (0)2448/9195610 E-mail: info@sportverlag-strauss.de www.sportverlag-strauss.de Satz: Autorensatz RECENT ADVANCES IN DOPING ANALYSIS (31) 3 ISBN 978-3-86884-049-0 MANFRED DONIKE WORKSHOP 2023 TABLE OF CONTENTS PAGE LECTURES Fedoruk M, Breuer J, Thevis M, Silk N: Investigating claims of sexual contamination by analysis for the presence of semeno- gelin in athlete urine samples ...................................................................................................... 14-15 POSTER PRESENTATIONS Athanasiadou I, Tsivou M, Geisendorfer T, Gmeiner G: Occurrence of ecdysteroids in doping control urine samples analysed by the Seibersdorf Laboratory ................................................................................................................ 16-21 Soni A, Rani S, Sahu PL: Effect of mefenamic acid on the urinary endogenous anabolic androgenic steroid profile ............................................................................................................................................ 22-26 Suominen T, Piirainen M, Vanne H: Analysis of steroid esters and quantitation of endogenous steroids in blood as Girard P derivatives .................................................................................................................................... 27-31 Wu Q, Yan K, Dong T, Liu L, Wang Y, Wang Z: Quantification of testosterone and androstenedione in human serum by HRMS ..................... 32-36 Fußhöller G, Geyer H, Görgens C, Guddat S, Jübner M, Lucuta L, Thevis M: Excretion pattern of urinary metabolites of dehydrochloromethyltestosterone after transdermal application ................................................................................................................ 37-40 Guddat S, Fußhöller G, Geyer H, Görgens C, Jübner M, Lucuta L, Thevis M: Excretion pattern of urinary metabolites of metandienone after transdermal application ..... 41-44 Montes de Oca Porto R, Martinez Brito D, Lopez Castillo D, Rodriguez Fernandez A, Laffita Jorrin D: Behaviour of 19-norandrosterone levels in negative samples ........................................................ 45-49 RECENT ADVANCES IN DOPING ANALYSIS (31) 4 ISBN 978-3-86884-049-0 PAGE Kwon OS, Muresan AR, Rahaman KA, Xu Y, Jeon MJ, Kim MY, Jung S, Chin AL, Min H, Kim HJ, Sung C, Kim KH, Lee KM, Kang MJ, Lee J, Son J: The proposed mass fragmentation pathways for bolasterone metabolite identification in rats by high resolution LC-HRMS and GC-MS/MS .................................................................... 50-56 Cutler C, Viljanto M, Hincks P, Habershon-Butcher J, Scarth J, van Eenoo P: Comparison of the human and equine metabolism of the SARM S-23 in vitro and in urine .............................................................................................................................................. 57-60 Loungyot T, Thangboonjit W, Prompittayayut P, Yaikwawong M, Songsaeng R, Panan S, Thitamadee S, Kongpatanakul S: Alterations of muscular atrophy gene expressions in clenbuterol induced human myotubes ....................................................................................................................................... 61-65 Grucza K, Wicka M, Drapala A, Konarski P, Stanczyk D, Kaliszewski P, Kwiatkowska D: Furosemide glucuronide as a valuable biomarker for the interpretation of results for furosemide in doping control analysis s ....................................................................................... 66-70 Grucza K, Wicka M, Drapala A, Konarski P, Stanczyk D, Kaliszewski P, Kwiatkowska D: Occurrence of higenamine and its sulfate conjugate in routine doping control analysis - interpretation of results ............................................................................................................. 71-75 Görgens C, Guddat S, Bosse C, Thevis M: Enantiomeric separation of various stimulants in urine after chiral derivatization using Marfey’s reagent for doping control purposes ............................................................................ 76-80 Sahu PL, Handa T, Sahu K, Soni A, Kori RK, Mahajan S, Padder AH, Nandi U, Ahmed QN, Reddy DS: Synthesis and characterization of 17α-hydroxymethyl-17β-methyl-18-nor-2-oxa-5α- androst-13-en-3-one (Oxandrolone M2), a long-term metabolite via a Kratena’s reaction approach ........................................................................................................................................ 81-85 Okano M, Akiyama K, Sato M, Mombayashi A: Can current WADA-approved direct detection methods identify the use of GH analogue Somatrogon in doping control? .................................................................................................... 86-90 Saavedra MJ, Sánchez-Wandelmer J, Polo ML, Rodríguez-de Francisco G, Muñoz G: Bandmate™ as a good alternative to BlotCycler™ for erythropoietin detection by Western blot .................................................................................................................................. 91-96 Mc Pherson Medina A, Fiallo Fernandez T, Martinez Brito D, Montes de Oca Porto R: Stability of the biotinylated monoclonal antibody BAM2871 in the 1% non-fat milk solution ........................................................................................................................................ 97-101 da Costa S, Ruivo J, Casimiro T, Pereira L, Salema B: Long-term stability study of erythropoietin and erythropoietin receptor agonists detection in dried blood spots and comparison of different absorbent sample supports .... 102-106 RECENT ADVANCES IN DOPING ANALYSIS (31) 5 ISBN 978-3-86884-049-0 PAGE Stojanovic B, Pecher D, Geisendorfer T, Tsivou M, Gmeiner G: Dried blood spot microsampling as a suitable tool to monitor diuretics misuse in sport ...... 107-111 Yan X, Yan S, Chang W, Zhang L, Wang Z, Yang S: A comparative study of DFT calculated and experimental UV/Visible spectra for the E/Z testosterone ester oximes at trace levels ....................................................................................... 112-116 Chang W, Yan X, Yan S, Liu Y, Yang S: Pilot study of small peptide analysis in dried blood spots using automated SPE and UPLC-HRMS without DMSO ............................................................................................................... 117-121 Thomas A, Garzinsky A, Görgens C, Guddat S, Geyer H, Thevis M: Carbonic anhydrase inhibitors brinzolamide and dorzolamide in dried blood spots .............. 122-125 Görgens C, Geyer H, Guddat S, Machnik M, Schenk I, van de Bovenkamp A, Wagener F, Thevis M: Trimetazidine: pilot study data illustrating approaches to evaluate a potential contamination scenario ...................................................................................................................... 126-128 Radu M, Catana D, Berghes B, Cristea C, Toboc A, Stan C: Development and validation of a chromatographic separation method for betametha- sone and dexamethasone .................................................................................................................. 129-133 Wicka M, Konarski P, Drapala A, Kaliszewski P, Grucza K, Stanczyk D, Kwiatkowska D: LC-MS/MS method for the detection of synthetic cathinones in urine ..................................... 134-138 Sobolevsky T, Ahrens B: Simplified method for detection of urinary cobalt by LCMS ....................................................... 139-142 Liu L, Wang Z, Yan K, He G, Xing Y, Wu Q: Doping control analysis of morphine in human urine by ultraperformance liquid chromatographic-electrospray ionization tandem mass spectrometry? .................................. 143-148 Grainger J, Agon V, Croft M, Goebel C: Stability assessment of two small peptide ( < 2kDa) mixed standard ........................................ 149-153 Yan X, Yan S, Chang W, Yang S: A warning list to athletes of musk as ingredients from Traditional Chinese Medicine .......... 154-157 Wicka M, Drapala A, Konarski P, Zyfka-Zagrodzinska E, Golaszewska A, Marusza M, Gorczyca D, Kwiatkowska D: Hemp oils and potential for unintentional anti-doping rule violations ..................................... 158-162 Wicka M, Drapala A, Konarski P, Grucza K, Siek P, Kopczynska M, Kwiatkowska D: BPC-157 – case study ........................................................................................................................... 163-168 RECENT ADVANCES IN DOPING ANALYSIS (31) 6 ISBN 978-3-86884-049-0 PRESENTATIONS ABSTRACTS - LECTURES Ahi S, Wolff K: An overview of doping control analysis at the Birmingham 2022 Commonwealth Games ............................................................................................................................................... 169 Al-Qahtani A, Abushareeda W, Vonaparti A, Kraiem S, Elsaftawy W, Saad K, Al-Wahaibi A, Hilal N, Dbes N, Nandlal V, Beotra A, Al Maadheed M: Dope Testing: Overview of FIFA World Cup 2022 .................................................................... 170-171 Al-Mohannadi I, Samsam W, Al-Darwish S, Cherif A, Al-Jaber M, Abushreeda W, Al-Qahtani A, Kraiem S, Al-Mohammed H, Farooq A, Nimker V, Stuart M, Robinson N, Zangenfeind G, Rovira D, Beotra A, Al-Maadheed M, Mohamed-Ali V: First drone delivery of anti-doping samples: safe, secure, rapid, reliable and robust ........... 172-173 Brooker L, Shimko K, Speers N, Goebel C, Thomas K: Summary and evaluation of the Australian PIED wastewater-based epidemiology collaboration ............................................................................................................................. 174-175 Torres C, Oliveira F, Jooris LF, Padilha M, Pereira H: The presence of doping agents in dietary supplements: A glimpse into the Brazilian situation ..................................................................................................................................... 176-177 Rubio A, Thomas A, Euler L, Geyer H, Krug O, Reis G, Costa Padilha M, Pereira HMG, MunizSantos R, Cameron LC, Stojanovic B, Kuehne D, Lagojda A, Mc Leod MD, Thevis M: Investigations into Annona fruit consumption as a potential source of dietary higenamine intake in the context of sports drug testing ........................................................ 178-179 Kwon OS, Rahaman KA, Muresan AR, Xu Y, Jeon MJ, Kim MY, Jung S, Chin AL, Min H, Kim HJ, Sung C, Kim KH, Lee KM, Kang MJ, Lee J, Son J: The urinary metabolite Ac-Tβ1-14 in huvec cells and rats treated with thymosin β4: A potential biomarker for doping tests ........................................................................................... 180 Colpaert T, de Hon O, Duiven E, Olijhoek M, T'sjoen G, van Eenoo P, Deventer K, Coppieters G, van Uytfanghe K: Detection of thyroid hormones T3 and T4 in serum samples after T4 administration in healthy volunteers .................................................................................................................... 181-182 Krug O, Guddat S, Möller T, Piper T, Görgens C, Thevis M: Determination of urinary deanol-N-oxide excretion profiles after ingestion of nutritional supplements containing deanol ............................................................................. 183-184 Santos V, Carneiro G, Braz B, Santelli R, Machado S, Pereira H Coordination chemistry applied to antidoping science: cobalt quantification as diethyldithiocarbamate complex by LC-HRMS ............................................................................... 185-186 RECENT ADVANCES IN DOPING ANALYSIS (31) 7 ISBN 978-3-86884-049-0 PAGE Paßreiter A, Naumann N, Thomas A, Grogna N, Delahaut P, Thevis M: How to detect CRISPR with CRISPR - employing SHERLOCK for doping control purposes ........... 187 Wagener F, Naumann N, Leciejewska N, Görgens C, Guddat S, Thevis M: Using organ-on-a-chip for predicting the metabolism of the selective androgen receptor modulator RAD140 in comparison to established in vitro approaches .......................... 188 Naumann N, Wapurgis K, Rubio A, Thomas A, Paßreiter A, Thevis M: Detection of doping control sample substitutions via SNP-based ID typing ................................. 189 Euler L, Deinert K, Thomas A, Thevis M: Mass spectrometric characterization and development of detection methods for in vitro synthesized metabolites of the troponin activators reldesemtiv and tirasemtiv for doping control purposes ................................................................................................................. 190 Krombholz S, Thomas A, Delahaut P, Miller GD, Bidlingmaier M, Thevis M: A combined top-down and bottom-up LC-HRMS/MS method for the quantification of human growth hormone in plasma ................................................................................................. 191 Möller T, Naumann N, Krug O, Thevis M: Identification and synthesis of selected in vitro generated metabolites of the novel selective androgen receptor modulator (SARM) 2f ........................................................................ 192 Coll S, Bressan C, Alechaga E, Monfort N, Carbó M, Ventura R: Elimination profile of methylprednisolone in urine and plasma after oral administration: Evaluation of the reporting level and washout period ........................................ 193 Alechaga E, Bressan C, Coll S, Carbó M, Monfort N, Ventura R: Glucocorticoids and DBS I. Concentrations of glucocorticoids in DBS after oral administrations ................................................................................................................................ 194 Ventura R, Bressan C, Coll S, Alechaga E, Carbó M, Monfort N: Glucocorticoids and DBS II. Correlation of glucocorticoid concentrations in DBS and plasma samples ................................................................................................................................ 195 Langer T, Nicoli R, Kuuranne T, Musenga A: Comparison of serum and urine analysis for the confirmation of oral testosterone undecanoate administration .................................................................................................... 196-198 Thilmany S, Hardy T, Thomas A, Vogel M, Scholl C, Thevis M: Transcriptomic analysis of the effects of steroid hormones on neuronal cells ...................... 199-200 Breuer J, Thomas A, Delahaut P, Schänzer W, Geyer H, Thevis M: Investigations into the concentration and metabolite profiles of stanozolol and LGD- 4033 in blood plasma and seminal fluid using liquid chromatography high-resolution mass spectrometry ........................................................................................................................... 201 RECENT ADVANCES IN DOPING ANALYSIS (31) 8 ISBN 978-3-86884-049-0 PAGE Aguilera R: Athlete isotopic signature of δ C values. Complementary data from testosterone gel application (males and females) ..................................................................................................... 202 Piper T, Krombholz S, Thevis M: Carbon isotope ratios of phenethylamine and phenylacetylglutamine and their applicability to doping control ........................................................................................................ 203 Nair M, Howa J, Kucherova Y, Ahrens B, Pereira HMG, Nicoli R, Kuuranne T, Fedoruk M: Applicability of longitudinal monitoring of IRMS values - A case study with unusual values ................................................................................................................................................ 204 Piper T, Thevis M: Investigations into the human metabolism of ecdysterone ................................................... 205-206 de La Torre X, Colamonici C, Curcio D, Botrè F: 19-Norandrosterone confirmation by GC-C-IRMS: 10 years of experience ................................... 207 Piper T, Thevis M: Employing 11-ketotestosterone as target analyte for adrenosterone (11OXO) administrations in doping control ................................................................................................... 208 Goodrum J, Nair V, Moore C, Eichner D, Miller G: Effects of heavy biotin supplementation on detection of hCG after Ovidrel® administration to males ................................................................................................................... 209 Marchand A, Roulland I, Semence F, Jaffredo F, Dehainault C, Mekacher LR, Ericsson M: Detection of the homologous transfusion of a red blood cell concentrate in vivo: evaluation of flow cytometry and DNA analysis methods ...................................................... 210-211 Loria F, Breenfeldt Andersen A, Bejder J, Bonne T, Grabherr S, Kuuranne T, Leuenberger N, Baastrup Nordsborg N: Erythropoiesis-related RNAs biomarkers are sensitive and specific to low-dose erythropoietin treatment at sea level and altitude ........................................................................ 212 Dørum S, Sletten C, Oll Ø, Dehnes Y: EPO screening with K9 ..................................................................................................................... 213 Leuenberger N, Kuuranne T, Castella V: Sequencing c.577del in EPO gene in urine and dried blood samples...................................... 214-215 Heiland CE, Leuenberger N, Ericsson M, Marchand A, Martin L: DBS in practice for EPO doping control analysis: in vivo evaluation ...................................... 216-218 RECENT ADVANCES IN DOPING ANALYSIS (31) 9 ISBN 978-3-86884-049-0 PAGE Requena-Tutusaus L, Anselmo I, Alechaga E, Bergés R, Ventura R: Detection of ERAs in a single-spot DBS sample at MRPL level: improving applicability of DBS in doping control .................................................................................................................. 219 Reichel C, Gmeiner G, Thevis M: Cost minimized immunoaffinity purification of EPO and its analogs in doping control – A step-by-step protocol for human blood and urine ............................................................... 220-221 Miller G, Goodrum J, Campbell T, Eichner D: Assessing the stability of recombinant EPO in urine ...................................................................... 222 Garzinsky A, Harth J, Reihlen P, Leipp F, Thomas A, Thevis M: Can the detection of EPO and its derivatives be manipulated by the addition of oral fluid to doping control urine samples? ........................................................................................... 223 PRESENTATIONS ABSTRACTS - POSTERS Martinez Brito D, de La Torre X, Botrè F: Analysis of testosterone esters by gas chromatography-highresolution mass spectrometry with low-energy electron ionization ................................................................. 224-225 Martinez Brito D, de La Torre X, Botrè F: Correlation of androstenedione and testosterone measurements by LC-MS/MS and GC-LE-TOF .................................................................................................................................. 226-227 Martinez Brito D, de La Torre X, Botrè F: Effect of thyroid hormones administration on the urinary endogenous steroid profile of the athlete biological passport ............................................................................................. 228-229 Sobolevsky T, Walpurgis K, Görgens C, Fedoruk M, Lewis L, Ahrens B, Thevis M: Detection of capromorelin in urine following different routes of administration ........................ 230 Andersson A, Pohanka A, Ekström L: 18-Methyl-19-noretiocholanolone detected after use of emergency contraceptive ................... 231 Euler L, Mürdter T, Heinkele G, Schwab M, Miller GD, Eichner D, Thomas A, Thevis M: Identification of (Z )-3‘-hydroxy clomiphene as a new potential dopingrelevant metabolite of clomiphene ............................................................................................................... 232 Wen H, Wagener F, Neudörfl J, Thevis M, Schäfer M: Investigation of the phase I metabolism of a new 20-keto-steroid S42 by C-6 oxidation and GC-MS analysis ................................................................................................................... 233-234 RECENT ADVANCES IN DOPING ANALYSIS (31) 10 ISBN 978-3-86884-049-0 PAGE Peters I, Naumann N, Görgens C, Guddat S, Thevis M: 3D cultivation of HepaRG cells for metabolite prediction of the calstabin-ryanodine receptor complex stabilizer S107 in preventive doping research .................................................. 235 Keiler AM, Rzeppa S, Voss SC, Thieme D: In vitro biotransformation of voxelotor .......................................................................................... 236 Sahu PL, Matsa R, Pawar S, Seth K, Radhakrishnanand P, Murty U, Handa T, Sahu K, Kori RK, Kumar P: Synthesis and characterization of ostarine metabolite: applicable for TL 12 ............................... 237 Walpurgis K, Agricola J, Thomas A, Thevis M: Myostatin inhibitory peptides in sports drug testing ..................................................................... 238 Walpurgis K, Gäde A, Thomas A, Gochard S, Delahaut P, Thevis M: Detection of extracellular hemoglobin from Arenicola marina in doping control serum and plasma samples by means of LC-HRMS/MS ............................................................................. 239 Fiallo Fernandez T, Mc Pherson Medina A, Martinez Brito D, Montes de Oca Porto R: Evaluation of the Cuban CBSSEPO monoclonal antibody for the detection of erythropoiesis-stimulating agents in doping control ..................................................................... 240 He S, Liu X, Zhou X: Administration study of rEPO on c.577del carriers ......................................................................... 241 Joonyeop Y, Minyoung K, Mijin J, Hophil M, Byung-Gee K, Junghyun S, Changmin S: Direct multiplex-PCR based gene doping detection for human growth hormone and its isoform using CRISPR/Cas12a-assisted HOLMES ............................................................................ 242 Mongongu C, Madi Moussa E, Marchand A, Buisson C, Ericsson M: Cubosomic supramolecular solvents: synthesis, characterization and potential for high throughput multiclass testing of banned substances in urine ....................................................... 243 Somay Selbes Y, Demirel AH, Elaldi K: Comparison of Insulin-like growth factor 1 measured between Siemens Immulite and IDS-iSYS ............................................................................................................................................. 244 Donati F, de La Torre X, Botrè F: Screening of exogenous hemoglobin with Sysmex XN analyzer .................................................... 245 Donati F, de La Torre X, Botrè F: Real-Time PCR SNP genotyping for the detection of homologous blood transfusion in dried blood spots .............................................................................................................................. 246 RECENT ADVANCES IN DOPING ANALYSIS (31) 11 ISBN 978-3-86884-049-0 PAGE Ponzetto F, Settanni F, Nonnato A, Nicoli R, Ghigo E, Kuuranne T, Mengozzi G: Volumetric absorptive microsampling (VAMS) for targeted LC-MS/MS measurement of novel blood markers of EAAS doping ....................................................................................... 247-248 Guo J, Goucher E, Patterson C, Gibson R, van Natta K, Samra S: Direct quantitation of 77 drugs of abuse in dried blood spots using the fully automated transcend DSX-1 system ................................................................................................................... 249 Buisson C, Smires S, Ericsson M: Dried blood spots for doping controls – Validation of a quantification procedure for corticosteroids ........................................................................................................................... 250-251 Alechaga E, Bressan C, Coll S, Monfort N, Ventura R: Multiclass method for the screening of doping agents in dried blood spot .................................. 252 Musenga A, Langer T, Salamin O, Nicoli R, Kuuranne T: A fast screening of doping agents in DBS by LC-MS/MS ................................................................. 253 Miyamoto A, Ota M, Sato M, Okano M: Simultaneous detection method of 26 testosterone, nandrolone and boldenone esters in dried blood spots by LC-MS/MS .................................................................................................. 254 Sletten C, Solheim SA, Bækken LV, Dehnes Y: Effect of Sustanon® administration on ALAS2 expression ............................................................. 255 Heiland CE, Mongongu C, Semence F, Pohanka A, Ericsson M, Marchand A, Ekström L: IGF-I variation in DBS and serum .............................................................................................. 256-257 Brockbals L, Thomas A, Schneider TD, Kraemer T, Steuer AE, Thevis M: LC-MS/MS-based peptide analysis for dried blood spot sampling time point estimation ........... 258 Kim KH, Jeong W, Eun Byul C, Min H, Son J: Development of an online immunopurification assay for urinary insulin and insulin mimetics using AF4-ESI-MS/MS ....................................................................................................... 259 González-Rubio S, Caballero-Casero N, Ballesteros-Gómez A, Cuervo D, Muñoz G, Rubio S: A general screening method for testing of banned substances in urine based on cubosomic supramolecular solvents and liquid chromatography/time-of-flight mass spectrometry .................................................................................................................................... 260 Keiler AM, Derwand D, Rzeppa S, Voss SC, Thieme D: QuEChERS as alternative extraction procedure in doping analyses .............................................. 261 RECENT ADVANCES IN DOPING ANALYSIS (31) 12 ISBN 978-3-86884-049-0 PAGE Cristea C, Radu M, Catana D, Berghes B, Toboc A, Stan C: Triple quadrupole UHPLC-MS/MS method as an alternative of orbitrap UHPLC-HRMS method used for detection of growth hormone-releasing hormones ........................................... 262 Upadhyay AK, Bhardwaj A, Sahu P: Comprehensive identification method for hypoxia-inducible factor (HIF) activating agents................................................................................................................................................ 263 Naumann N, Vollmert C, Krajina M, Thevis M: Evaluation of a novel gene doping detection approach via high multiplex MALDI-TOF MassARRAY analysis ........................................................................................................................ 264 Akiyama K, Momobayashi A, Okano M: Individual identification method using samples related to doping tests: a comparison of mitochondrial and nuclear genetic data ..................................................................................... 265 Son J, Park H, Cho Y, Sung C, Min H, Lee KM, Bahn Y: Enhancement of anti-doping strategy using artificial intelligence (AI): A study on improving the accuracy and efficiency of detecting performance-enhancing drugs in athletics as a proposed nextgeneration doping diagnosis strategy ............................................... 266 Son J, Cho Y, Park H, Jeon S, Sung DJ, Jeong JH, Sung C, Min H, Lee KM, Kim H, Yoon SS: Clinical study of brain doping diagnosis (bD ): Confirmation of sports performance enhancement and neurochemical alterations via brain stimulation ............................................. 267 Aldubayyan A, Castrignano' E, Abbate V: Short- and long-term stability of synthetic cathinones and dihydrometabolites in human urine samples: considerations for antidoping and forensic toxicology applications ...................................................................................................................................... 268 Al-Naimi N, Kraiem S, Samsam W, Beotra A, Orie N, Mohamed-Ali V, Almaadheed M: Turkesterone a noval ecdysteroid: High resolution orbitrap mass spectrometry identification of turkesterone in supplements and urine samples ......................................... 269-270 Pettersson-Bohlin K, Pohanka A, Ekström L: Detection of SARMs in the Swedish society ............................................................................. 271-272 Leogrande P, Jardines D, Martinez Brito D, de La Torre X, Botrè F, Luch A, Diel P, Parr MK: Physiological and post-training effects of ecdysteroid supplements. A multivariate study of the human serum metabolome ........................................................................................ 273 Saad K, Vonaparti A, Al-Mohannadi I, Khelifi S, Salma S, Mahieddine S, Saleh A, Lommen A, Horvatovich P, Beotra A, Abushareeda W, Al Maadheed M, Georgakopoulos C: Preliminary study of MetAlign software automatic LC/MS screening data evaluation ......... 274-275 RECENT ADVANCES IN DOPING ANALYSIS (31) 13 ISBN 978-3-86884-049-0 SCIENTIFIC BOARD F. Botrè, Rome (Italy) D. Cowan, London (UK) X. de la Torre, Rome (Italy) H. Geyer, Cologne (Germany) G. Gmeiner, Seibersdorf (Austria) A. Keiler, Dresden (Germany) U. Mareck, Cologne (Germany) M. Okano, Tokyo (Japan) H. Pereira, Rio de Janeiro (Brazil) T. Piper, Cologne (Germany) C. Reichel, Seibersdorf (Austria) W. Schänzer, Cologne (Germany) M. Thevis, Cologne (Germany) A. Thomas, Cologne (Germany) P. van Eenoo, Ghent (Belgium) R. Ventura, Barcelona (Spain) S. Rzeppa, Dresden (Germany) Fedoruk M , Breuer J , Thevis M , Silk N Investigating claims of sexual contamination by analysis for the presence of semenogelin in athlete urine samples Science & Research, U.S. Anti-Doping Agency (USADA), Colorado Springs, United States of America ; Centre for Preventive Doping Research, Institute of Biochemistry, German Sport University Cologne, Cologne, Germany Abstract In cases where athletes allege sexual contamination has resulted in a positive test, semenogelin has emerged as an important potential marker of semen exposure from sexual contact that could help to corroborate an athlete's claim. Methods to identify semenogelin I, a major and specific protein constiuent of semen, in urine samples collected during doping control have been published using two analytical approaches, a laminar flow immunological test strip (initial testing procedure) and a bottom-up LC- HRMS/MS-based test (confirmation) method, specific for semenogelin I. Four previous cases were examined where positive samples were kept in long-term storage to determine whether the presence or absence of semenogelin I could corroborate the athlete's version of events. Based on these further analyses, it was determined that the presence or absence of semenogelin in the urine does not provide unequivocal evidence as to how a drug or its metabolites are introduced into an athlete's urine sample, but could provide helpful supporting evidence. Specific factors surrounding seminal exposure and intimate contact may influence the ability to detect semenogelin in an athlete's urine sample. Introduction The athlete exposome has recently become an important focus of anti-doping research due to the likelihood that athletes are exposed to prohibited substances through circumstances that may not be related to doping. Contamination scenarios based on the ingestion of food, cosmetics, dietary supplements, medications, uncommon metabolic drug interactions, and physical contact are all proven avenues for adverse analytical findings (AAFs)[1]. The differentiation between intentional drug use and all the various contamination scenarios is critical in order to ensure that athletes are able to effectively defend themselves, as the burden remains on the athlete to demonstrate the source of a positive doping test. Using urine in long-term storage, urine samples were analyzed for the presence of sperm residue marker, semenogelin, to examine the possibility of a prohibited substance being present in a female athlete’s urine sample due to residual ejaculate being introduced by recent sexual intercourse into the vaginal area of an athlete and contaminating the urine sample collected during doping control. Trace amounts of semenogelin could be used to support an athlete’s claim of contamination due to their doping male partner transferring a prohibited substance to their body. Experimental Athlete samples were tested using a protocol of a commercially available lateral flow immuno‐ 1 2 2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-014 chromatographic test directed against semenogelin. Secondly, a liquid chromatography/tandem mass spectrometry (LC-MS/MS)-based method was used, employing solid-phase extraction of urine, followed by trypsinization of the retained protein content and subsequent detection of semenogelin I-specific peptides [2]. Results and Discussion Signals were not observed for semenogelin in an athlete’s urine sample that reported sexual intercourse with her male partner shortly before sample collection. Prior further investigation of this case demonstrated that the male partner was doping with letrozole and GW-1516 at the time of multiple unprotected sexual encounters with the athlete. Similiarly, no semenogelin was detected in an athlete’s sample that reported unprotected sexual intercourse two days prior to sample collection; however, the athlete tested positive for LGD-4033 metabolite dihydroxy-LGD-4033. An investigation in this case demonstrated her male partner was using therapeutic doses of LGD-4033 at the time of intimate contact. Two further samples contained semenogelin, however the athletes in these cases admitted to using oral dehydroepiandrosterone (DHEA), or sexual contamination was not communicated as an explanation for the positive test. Two other samples tested negative for the presence of semenogelin where athletes claimed sexual contamination as a possible source of both stanozolol and ostarine positive tests. During the results management of these doping cases, sexual contamination ruled out as a plausible route of exposure based on a comprehensive investigation of the facts. Importantly, it appears additional consideration must be given to the specific circumstances and timing of sexual contact relative to sample collection, the viracity of alleged use by the partner of prohibited substances and the estimated concentrations of prohibited substances and/or their metabolites observed in the athlete's urine sample. Conclusions Semenogelin presence can be used as strong supporting evidence for prohibited substance exposure via the introduction of male ejaculate ‘dripping’ into a female athlete’s doping control urine sample, however there are some important limitations, and the presence (or absence) cannot be used as unequivocal evidence of prohibited substance exposure via sexual transmission. References 1. Thevis M, Kuuranne T, Fedoruk M, Geyer H. Sports drug testing and the athletes' exposome. Drug Test Anal. 2021 Nov;13(11-12):1814-1821 2. Breuer, J, Thomas, A, Geyer, H, Thevis, M. Probing for the presence of semenogelin in human urine by immunological and chromatographic-mass spectrometric methods in the context of sports drug testing. Anal Sci Adv. 2022; 3: 21– 28 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-015 Athanasiadou I, Tsivou M, Geisendorfer T, Gmeiner G Occurrence of ecdysteroids in doping control urine samples analysed by the Seibersdorf Laboratory Doping Control Laboratory, Seibersdorf Labor GmbH, Seibersdorf, Austria Abstract In 2020, ecdysterone was included in the monitoring program of the World Anti-Doping Agency (WADA) to monitor its prevalence of misuse in professional sports, in and out of competition. Ecdysterone has been suggested to be included in WADA’s Prohibited List in class S.1.2 Other Anabolic Agents with a reporting cut-off limit [1].The screening of 20525 doping control samples' “digital matrix” data was used to extract information regarding ecdysterone and turkesterone use in relation to the athlete's gender, type of competition and sport. The occurrence of ecdysteroids in the athlete population was estimated to be approx. 1% with a higher prevalence in OOC samples from strength and endurance sports originating from Balkan and Eastern European countries. Furthermore, a controlled administration study with a single oral dose of approx. 50 mg of a beta-ecdysone supplement was conducted to monitor the excretion profiles of ecdysterone and its main metabolite 14-deoxy-ecdysterone and to investigate any effects on steroid profile parameters (testosterone, epitestosterone, androsterone, etiocholanolone, 5α- androstane-3α,17β-diol (5αAdiol), 5β-androstane-3α,17β-diol (5βAdiol) and ratios. All the steroid profile markers followed the normal urinary circadian rhythm. No effect was observed on steroid profile markers or the relevant respective ratios in the presence of ecdysterone. Introduction In sports, ecdysteroids are widely marketed to athletes as dietary supplements advertised to increase strength and muscle mass during resistance training, to reduce fatigue and to ease recovery. Several studies have reported a wide range of pharmacological effects of ecdysteroids in mammals, most of them beneficial to the organism [2]. The most active phytoecdysteroid, ecdysone (also known as “Russian secret”), was suspected to be used by Olympic athletes back in the 1980s. Recently, ecdysterone was reported to enhance performance in sports [3] and to promote an anabolic effect even stronger than that of the anabolic steroid metandienone [4-9]. In addition, a global interest in turkesterone supplements distributed online was observed in the last couple of years, especially in countries such as Brazil, Australia, Norway, the United States, and Canada. “Digital matrix” of the analysed doping control samples containing a significant amount of information (e.g. gender, type of competition, sport discipline, etc) adds a tremendous value for retrospective data mining purposes [10,11]. The screening of the “digital matrix” of doping control data could lead to useful conclusion regarding the prevalence and patterns of the use of substances based on different aspects of the athlete population. In this context, the current project exploited the “digital matrix” of 20525 doping control urine samples for the retrospective data mining of ecdysterone and turkesterone presence to obtain a snapshot of the athlete population for the period between June 2020 and September 2021. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-016 Experimental Oral administration of ecdysterone and urine collection A controlled excretion study was performed after the administration of a single oral dose of approx. 50 mg (20-beta-hydroxy-ecdysone, Westpharmaceuticals LLC, Indiana, USA) to one male volunteer (55 yrs, weight of 95 kg, and height of 193 cm). Supplement analysis The content of one capsule (100 mg as referred in the product label) was diluted with 10 mL MeOH and then incubated in the ultrasound bath for 10 min. 10 µL of the diluted capsule were transferred in a conical tube, spiked with 50 µL internal standard and diluted up to 1 mL with MeOH/Water (10/90). 100 µL of the prepared mixture were transferred into an autosampler vial and 10 μL was injected to the LC-MS system for analysis. Table 1. Mass spectrometric information for the analytes under investigation Evaluation of excretion profile in urine In this study, the excretion profiles of ecdysterone and its main metabolite after a single-dose administration of 50 mg of pure ecdysterone were evaluated. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-017 Evaluation of the steroid profile markers Steroid profile markers including testosterone, epitestosterone, androsterone, etiocholanolone, 5α- androstane-3α,17β-diol (5αAdiol), 5β-androstane-3α,17β-diol (5βAdiol) and the respective ratios were evaluated in the excreted urine samples as mentioned in the WADA technical document [12]. Urine concentrations of the steroid profile markers were adjusted based on the specific gravity (SG) measurements of the excretion samples. Retrospective analysis using TraceFinder software Retrospective analysis was performed on all urine samples analysed by the Seibersdorf Doping Control Laboratory between June 2020 and September 2021 (in total 20525 data files evaluated). Using the Tracefinder software (TraceFinderTM 5.1), a Compound Database and a Master Method created based on the mass spectrometric info presented for the target analytes. A statistical evaluation based on the athlete's gender, the type of competition and the sport was conducted to obtain a snapshot of ecdysterone and turkesterone use in the athlete population. Results and Discussion Supplement analysis Supplement analysis showed ~ 50 mg ecdysterone per capsule (exact amount 48 mg/capsule). However, the amount of ecdysterone on the product label was equal to 100 mg/capsule. Evaluation of excretion profile of ecdysterone and its main metabolite Following a single-dose administration of 50 mg ecdysterone, the parent compound resulted to be the most abundant analyte in all post-administration urine samples. C (2221 ng/mL) was detected 3.67 h after administration (TAD), whereas the main metabolite (14-deoxy-ecdysterone) was detectable from 9.83 h up to 3 days with C (743 ng/mL) observed at 43 h TAD. Excretion profiles of ecdysterone and its metabolite are presented in Figure 1. Figure 1. Urinary excretion profile (concentration-time curve) of ecdysterone (left) and 14-deoxy-ecdysterone (right), following a single-dose administration of 50 mg of pure ecdysterone in a male volunteer max max MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-018 Evaluation of the steroid profile markers No effect was observed on steroid profile markers or the relevant respective ratios. The testosterone excretion profile and the T/E ratio are depicted as examples in Fiure 2. All the steroid profile markers followed the normal urinary circadian rhythm. Figure 2. Urinary excretion profile (concentration-time curve) of endogenous testosterone (left) and testosterone/ epitestosterone (T/E) (right) Retrospective and statistical analysis The occurrence of ecdysteroids in the athlete population tested was equal to be approx. 1%. Declared nutritional supplements containing ecdysterone and/or turkesterone include but are not limited to: Retibol Eiselt Research, Mega Ecdysterone, Ecdysterone-S, Beta Ecdyx Pure, Eksumid (Exumid), EcDyBol, etc. Russia, Estonia, Ukraine, Greece, Hungary, Lithuania, Moldova and Serbia were among the countries with the highest number of declared nutritional supplements containing ecdysteroids. As shown in Figure 3a, findings were distributed as 61% out of competition (OC) and 39% in-competition (IC) samples. Ecdysterone findings were distributed as 62% in males and 38% in females. Weightlifting, canoe/kayak, athletics and aquatics were the sports with the highest prevalence of ecdysterone and turkesterone. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-019 Figure 3. Graphic distribution of ecdysterone and turkesterone findings based on the type of distribution (a), the gender (b) and sport (c) Proof of concept The 2020 WAADS Educational Samples (pooled urine from an excretion study after ingestion of a supplement containing ecdysterone and separately turkesterone – according to the provider), were used to prove the fit for purpose of the developed retrospective method. Samples were processed with the retrospective method and all analytes were successfully detected. Conclusions The current study underlines the high occurrence of ecdysteroids in the athlete population (approx. 1%). In addition, the hereby presented retrospective approach can be a useful tool [11] - despite the limitation of the raw data files (approx. 1 Terabyte), when investigating the abuse of substances, that are not yet included in the prohibited menu of accredited laboratories but are available on the black market. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-020 References 1. WADA The 2021 Monitoring Program https://www.wada-ama.org/sites/default/files/resources/files/2021list_monitoring_program_en_0.pdf 2. M. Mazzarino, G. Ambrosio, A. Grieco, et al. Ecdysterone: a snapshot of use in the athlete’s population. Poster presented at the 38 Manfred Donike Workshop on Doping Analysis, Cologne, Germany, 2020. 3. E. Isenmann, G. Ambrosio, JF. Joseph, et al. Ecdysteroids as non-conventional anabolic agent: performance enhancement by ecdysterone supplementation in humans. Archives of Toxicology (2019), 93:7, 1807- 1816. 4. M. Parr, G. Ambrosio, B. Wuest, et al. Targeting the administration of ecdysterone in doping control samples. Forensic Toxicology (2020), 38:1, 172-184. 5. MK. Parr, F. Botre, A. Nass, et al. (2015) Ecdysteroids: a novel class of anabolic agents? Biol Sport 32(2):169–173. 6. NN. Orie, A. Raees, MY. Aljaber, et al. 20-Hydoxyecdysone dilates muscle arterioles in a nitrc oxide- dependent, estrogen ER-and beta receptor-independent manner. Poster presented at the 40 Manfred Donike Workshop on Doping Analysis, Cologne, Germany, 2022. 7. D. Kwiatkowska, K. Grucza, K. Chajewska, et al. Ecdysterone - possible sources of origin in urine. Poster presented at the 40 Manfred Donike Workshop on Doping Analysis, Cologne, Germany, 2022. 8. S. Kraiem, S. Bouabdallah, S. Touil, et al. Elimination profile of 20-hydroxyecdysone (20-OHE) in urine - Liquid-Liquid extraction and dilute and inject methodology using UHPLC/HRMS. A comparative study. Poster presented at the 40 Manfred Donike Workshop on Doping Analysis, Cologne, Germany, 2022. 9. D. Kwiatkowska, K. Grucza, K. Kowalczyk, et al. Ecdysterone - excretion study after ingestion of spinach. Poster presented at the 39 Manfred Donike Workshop on Doping Analysis, Cologne, Germany, 2021. 10. S. Khelifi, K. Saad, A. Vonaparti, et al. Ultra-Fast retroactive processing by MetAlign of LC-Chromatography High-resolution Mass Spectrometry Data in WADA Human Urine Sample Monitoring Program. Poster presented at the 39 Manfred Donike Workshop on Doping Analysis, Cologne, Germany, 2021. 11. M. Thevis, K. Walpurgis, A. Thomas. Analytical Approaches in Human Sports Drug Testing: RecentAdvances, Challenges, and Solutions. Anal.Chem. (2020), 92, 506−523. 12. WADA Measurement and Reporting of Endogenous Anabolic Androgenic Steroid (EAAS) Markers of the Urinary Steroid Profile; www.wada-ama.org/sites/default/files/2022-01/td2021eaas_final_eng_v_2.0.pdf Acknowledgements The current work was supported by the Austrian Ministry of Sports. The authors would like to acknowledge the Cologne Laboratory and Dr. Geyer for the ecdysterone supplement. th th th th th th MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-021 Soni A, Rani S, Sahu PL Effect of mefenamic acid on the urinary endogenous anabolic androgenic steroid profile National Dope Testing Laboratory, New Delhi, India Abstract Endogenous anabolic androgenic steroids (EAAS) are prohibited in sports when administered exogenously. The World Anti-Doping Agency (WADA) accredited laboratories monitor the steroid profile markers (concentrations and concentration ratios of “pseudo-endogenous” steroid, especially testosterone, its precursors and metabolites) in urine samples collected from athletes in and out of competition. The Athlete Biological Passport (ABP) has been used for long-term monitoring of alterations of the intra-individual steroid concentrations and ratios in anti-doping analysis. As steroid metabolism is very complex and certain non-prohibited drugs may influence the concentrations of the urinary steroid profile or its interpretation. Aldo-keto-reductases (AKRs), particularly AKR1C3, are a significant group of enzymes that are crucial for steroidogenesis. It was discovered that NSAIDs are powerful inhibitors of AKR1C3 enzymes. Thus, the aim of this work was to investigate the effect of mefenamic acid on the urinary steroid profile. A urinary excretion study after drug administration for two consecutive days (twice a day) was carried out using a validated GC-MS/MS method of the laboratory. Data analysis was performed by using Agilent Mass-Hunter Quantitative software. As expectetd, significant changes in a few steroid concentrations and their ratios were observed in some volunteers after the intake of mefenamic acid. The significant changes in the steroid profile were detected especially the concentration of androsterone and the Andro/T ratio. Introduction Endogenous anabolic androgenic steroids (EAAS) abuse in sports is indicated by changes in steroid profile markers [1]. The steroidal module of the Athlete Biological Passport has been used to monitor steroid profiles and it compares individual reference ranges established from several test results of the same athlete [2]. But recent studies have shown, that some other (neither prohibited nor monitored) drugs, so called confounding factors, can alter the individual's steroid profile. Mefenamic acid (trade name in India, e.g., Meftal) is a nonsteroidal analgesic (NSAID) approved by the FDA for the treatment of mild to moderate acute pain. A previous study reports that mefenamic acid inhibits the aldo-keto reductase 1C2 (AKR1C2) enzyme in vitro [4]. This enzyme plays a key role in endogenous steroid metabolism (e.g., conversion of potent 5a-dihydrotestosterone (5aDHT) to inactive 5a-androstane- 3a,17b-diol (ADIOL)) [3]. This inhibition may cause changes in the steroid profile and a misleading interpretation by the ABP module could lead to unnecessary but very costly confirmative analyses by GC/C/IRMS. This work shows an in vivo excretion study on two volunteers followed by analysis of urinary steroids included in the steroid profile using GC-MS/MS. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-022 Experimental Materials Reference standards of endogenous steroids and deuterated internal standards were procured from Sigma-Aldrich and National Measurement Institute, Australia. Inert-sep HLB sample preparation cartridges were procured from GLSciences, the enzyme β-glucuronidase from E. coli was purchased from Roche, USA. MSTFA and all other solvents and chemicals were of high-performance liquid chromatography (HPLC) grade and analytical grade, respectively, from Merck and Sigma-Aldrich mainly. Selection of volunteers and in vivo administration trial Two volunteers (1 female and 1 male) were selected by screening of their urinary steroid profile. The age range of selected volunteers was 18-30 years. A 500 mg mefenamic acid tablet (manufactured by Blue Cross Laboratories Pvt. Limited) was provided twice a day for two consecutive days to each volunteer. Urine samples were collected in the following manner: - 4 urine samples before drug administration (sampling points: 07:00 h, 12:00 h, 17:00 h and 21:00 h) - 4 urine samples for 2 days during the administration (same time-points as before) - 2 urine samples for next 4 days - 1 sample for further 4 days after drug administration. All samples were anonymized, collected in sterile plastic tubes and stored at -20 °C until analysis. The study was approved by the ethics committee of the National Dope Testing Laboratory (NDTL), India and all volunteers signed the informed consent. Urine sample preparation Extraction of endogenous steroids (as free and glucuronide fraction) was performed using an already established protocol for steroid profiling of endogenous steroids as per TD2021EAAS which is routinely used in the dope testing laboratory at New Delhi, India. In brief, 2 mL of urine, 50 µL of internal standard mixture 1 mL of 0.2 M phosphate buffer (pH 7.0) and 50 µL of β-glucuronidase enzyme (E. coli) were added, followed by incubation of the sample at 60 °C for 60 minutes. These pre-treated samples were placed on Gilson ASPEC, eluted with 2 mL of 5% methanol in ethyl acetate into the labelled tubes. We dried the elute(s) using a nitrogen evaporator at 60 °C and dissolved the dried elute with 50 µL of MSTFA/ IODO –TMS/DTE mixture (1000/2/2: v/v/w).The tubes were incubated at 60 °C for 30 minutes. The derivatized extract was injected directly into the GC-MS/MS. Steroid profile measurements on GC-MS/MS A previously described method as per TD2021EAAS, which is validated, accredited under ISO17025, and currently used in routine samples analysis for the detection and quantification of endogenous steroids was used. In brief, analysis was performed on an Agilent GC–MS/MS 7890 A/7000C with an Agilent HP Ultra-1 column (length: 17 m; diameter: 0.2 mm; film-thickness: 0.11 µm) and 7683B Automatic Liquid Sampler. Carrier gas was helium, flow rate 2.25 mL/min, injection and transfer line temperature were set to 280 °C and injection was performed in split mode with a ratio of 11:1. The oven program was as follows: initial temparature of 180 °C hold for 1 min, ramp 1: 3 °C/min to 229 °C, ramp 2: 40 °C/min to 300 °C; final temparature was hold for 4 min. The ionization energy was set to 70 eV. The acquisition was carried out in multiple-reaction monitoring (MRM) mode. Quantitation of the urinary steroids was MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-023 performed based on the peak area ratio of the analyte to the corresponding internal standard. Calibration and quality control samples were prepared in steroid free urine. Results and Discussion Steroid profile parameters including markers and their ratios were calculated as per method characterization given in TDEAAS and validated excel sheets being used for routine sample reporting [1]. Mefenamic acid metabolites and endogenous steroids under current study are depicted in Table 1. The measured concentrations of the markers of the steroid profile were reported versus the collection time to evaluate their physiological fluctuation. All data were normalized for the specific gravity applying the following formula: (concentration ng/mL) x (1:020-1)/ (specific gravity sample-1). Table 1. List of Endogenous Steroids under investigation As mefenamic acid is an inhibitor of AKR1C3, the most logical assumption was that the concentrations and ratios of the steroid profile that contains 3-hydroxy-steroids should increase. For both volunteers, changes in the steroid profile were observed and these changes were evaluated statistically using one- way analysis of variance (ANOVA) using metaboanalyst software. In the male volunteer significant changes in the steroid profile were detected for three steroid-profile markers: the concentration of androsterone and the ratios Andro/T and Andro/Etio (boxplots with significance levels represented in Figure 1, A-C). For Andro, a significant decrease from difference in mean values; P = 3×10 was observed, similarly, the ratio Andro/T decreased from difference in mean values; P = 0.014 in the concentration was detected during the administration of mefenamic acid compared to pre- administration, whereas no significant difference was detectable between the samples collected during and after the application of mefenamic acid. For the female volunteer significant changes were detected for Andro/T (P = 6×10 ), 5A-diol/5B-diol (P = 1.6×10 ), 5A-diol/E (P = 9.7×10 ) and E (P = 0.01). Boxplots with the changes in steroid concentrations are displayed in Figure 1, D-H. Since the steroid concentrations investigated in the steroid profile are much lower in females compared to males, interpretation is more challenging and a lot of factors should be taken in to account when interpreting the results [5]. As assumed, concentrations and concentration ratios of the steroid profile involving one - 3 -7 -4 -4 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-024 3-hydroxy-steroid (Andro, 5A-diol, Etio, 5B-diol, Andro/T and Andro/E) are reduced. Unfortunately, the picture is not so clear from the two volunteers in this study. Figure 1. Boxplots of urinary steroid concentrations and ratios included in the steroid profile from the male and female volunteer with significant changes MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-025 Furthermore, the metabolite of mefenamic acid (3-OH methylmefenamic acid) was reported significantly till 10 days after stopping drug intake in both of the volunteers (Figure 2. A-B). Figure 2. Excretion profile of 3-hydroxy methyl mefenamic acid in male (A) and Female (B) volunteers Conclusions An inhibition of aldo-keto-reductases (AKRs), especifically AKR1C3, by non-steroidal anti-inflammatory drugs (NSAIDs), which are neither prohibited nor monitored, but frequently used drugs in sports, should be demonstrated. Mefenamic acid (NSAID) is an easy-over the counter accessible pain killer. This study shows the relevance of AKR1C3 inhibition by mefenamic acid (NSAIDs). Changes in the steroidal profile caused by the intake of therapeutic doses of mefenamic acid over two consecutive days (twice a day) were shown by anaylzing the urine samples using a validated method of the laboratory. In the male volunteer significant changes in the steroid profile were detected for the concentration of androsterone and the ratios Andro/T and Andro/Etio and in the female volunteer significant changes were detected for Andro/T, 5A-diol/5B-diol, 5A-diol/E and E. Since the inhibition by mefenamic acid is significanntly causing changes in some steroid profile markers, unnecessary GC/C/IRMS may be avoided during interpreting atypical steroid profiles in the ABP’s steroidal module. Furthermore, this study proposes an administration trail with a larger number of volunteers to elucidate the role of mefenamic acid and NSAIDs as confounding substances for steroid profiling. References 1. 2021 WADA Technical Document – TD2021EAAS. 2. 2021 WADA Technical Document – TD2021APMU. 3. Gobec S, Brozic P, Rizner TL. Nonsteroidal anti-inflammatory drugs and their analogues as inhibitors of aldo-keto reductase AKR1C3: new lead compounds for the development of anticancer agents. Bioorg Med Chem Lett. 2005 Dec 1;15(23):5170-5. doi: 10.1016/j.bmcl.2005.08.063.. 4. Shiiba M, Yamagami H, Yamamoto A, Minakawa Y, Okamoto A, Kasamatsu A, Sakamoto Y, Uzawa K, Takiguchi Y, Tanzawa H. Mefenamic acid enhances anticancer drug sensitivity via inhibition of aldo-keto reductase 1C enzyme activity. Oncol Rep. 2017 Apr;37(4):2025-2032. doi: 10.3892/or.2017.5480 5. Mareck U, Geyer H, Opfermann G, Thevis M, Schänzer W. Factors influencing the steroid profile in doping control analysis. J Mass Spectrom. 2008 Jul;43(7):877-91. doi: 10.1002/jms.1457 Acknowledgements The authors acknowledge financial support from Government of India. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-026 Suominen T, Piirainen M, Vanne H Analysis of steroid esters and quantitation of endogenous steroids in blood as Girard P derivatives Doping Control Laboratory Helsinki, Finnish Institute for Health and Welfare (THL), Helsinki, Finland Abstract A wide range of testosterone as well as other steroid formulations (e.g. nandrolone and trenbolone) with different structures and thus differing pharmacokinetic parameters are available. As most steroids are not well absorbed as oral formulations, the most widely used forms of administration are intramuscular injections of steroid ester preparations. Detection of intact steroid esters in blood provides indisputable proof of steroid misuse. Steroid esters are present at low concentrations in blood, and therefore the method used for their analysis shall be able to detect the compounds at low pg/ml concentrations. In this work, an LC-MS/MS method suitable for the quantitative analysis of testosterone and androstenedione and for the quantitative analysis of steroid esters was developed and validated. Introduction Detection of steroid misuse in sport is mainly based on the urinary steroid profile, followed by possible GC-C-IRMS confirmation of samples with atypical profiles. An alternative approach is to detect steroid esters in blood, requiring less time-consuming sample preparation compared to IRMS analysis. Steroid esters have been analyzed with different methods, such as GC-MS and more commonly by LC-MS, usually as oxime or Girard derivatives. For extraction of the steroid esters from the sample matrix, solvents such as MTBE, cyclohexane, toluene, mixtures of hexane and ethyl acetate have been successfully used. The aim of this work was to develop a LC-MS/MS method suitable for the quantitative analysis of testosterone and androstenedione, and for the quantitative analysis of steroid esters. Experimental Sample preparation ISTDs d -T (Cerilliant), d -A4 (TRC) and d -testosterone undecanoate (Alsachim), were added to 200 µL of serum or plasma, followed by extraction with 3 mL of TBME. After evaporation to dryness, 50 µL of 10 % acetic acid in methanol and 20 µL of Girard P reagent (4 mg/mL in H O) were added, followed by incubation at 60 °C for 10 min and transfer to autosampler vials. For oxime derivatization, 75 µL of hydroxylamine (100 mM in meOH/H O) was added, followed by incubation at 60 °C for 30 min. LC-MS/MS Analysis was performed using Agilent 1290 Infinity autosampler and 6460 Triple Quadrupole MS with Agilent Zorbax Eclipse Plus C18 (2.1 x 50 mm, 1.8 µm) column operated at 55 °C. 2.5 mM ammonium formate in 0.1 % formic acid (A) and 2.5 mM ammonium formate in 0.1 % formic acid in 95 % methanol 3 3 4 2 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-027 (B) were used as mobile phases. The flow rate was 0.3 mL/min and injection volume 5 µL. All compounds were monitored in positive ionization (SRM mode). The capillary voltage, drying gas flow, drying gas temperature, nebulizer, nozzle voltage, sheath gas flow and sheath gas temperature were set at 3500 V, 8 L/min, 200 °C, 35 psi, 500 V, 12 L/min, 350 °C. To wash out remaining Girard P reagent, the eluent was directed to waste for the first 3 minutes. Validation The aim of the method was to a) quantitate T and A4 in serum samples and b) detect exogenous steroid esters in serum and plasma samples according to the WADA Guideline Quantification of Endogenous Steroids in Blood for the Athlete Biological Passport and the WADA TN on Analytical Method validation. The validation included establishing repeatability, selectivity, LOD, carryover and extraction recovery for all analytes, and additionally LOQ, MU and linear range for the quantitation of T and A4. For the quantitation of T and A4, commercial calibrators (Chromsystems Multilevel Serum Calibration Set Steroid Panel 2), as well as in-house calibrators, were used. The in house-calibrators were prepared by spiking T and A4 into a buffer (0.1 BSA/PBS). Results and Discussion Method development: The intensities of the protonated molecules were compared for non-derivatized, oxime derivatization and Girard P derivatization. For most analytes, the Girard P derivatization provided the best intensity of the protonated ion. For some steroid esters the oxime derivative was more intensive, but the S/N were similar, and the Girard P derivatization was selected for use in the validation. The two most prominent SRM transitions for the compounds were chosen for the ITP method. The protonated compounds have a target ion consisting of M+N C H . Figure 1. Comparison of the ionization of selected steroid esters with no derivatisation, oxime derivatisation and Girard P derivatisation 3 7 8 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-028 Table 1. Compounds and SRM transitions used for ITP Different HPLC columns were tried during method development, but the best separation of the analytes was with a Agilent Zorbax Eclipse Plus C18 column. Linearity was evaluated using two independent sets of calibrators; the Chromsystems Multilevel Serum Calibrator Set, and in-house calibrators spiked into PBS-buffer. The linearity was evaluated according to the WADA Guideline “Quantification of Endogenous Steroids in Blood for the Athlete Biological Passport” from 0.05-15 ng/mL for both T and A4. With both calibrators, the linearity was excellent for both T and A4 (r > 0.999). The extraction recoveries were between 70-100%. 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-029 Figure 2. Linearity of T and A4 using in-house calibrators For T and A4, the LOQ was defined as the lowest concentration with a S/N of 6 and acceptable bias ( < 30%). For both T and A4, the LOQ was at 0.05 ng/mL. For the steroid esters, the LODs were established by spiking the compounds into six different serum and plasma samples, serially diluted with the same plasma/serum, and set at the concentration with a s/n of ≥ 3. Based on the analysis of parallel serum and plasma samples, either blank samples or samples to which the exogenous steroid esters had been spiked, the method was proven to be repeatable and selective. The MU was evaluated based on the analysis of EQAS samples; for T uc = 7.2% and for A4 uc = 7.7 %. Table 2. LODs, LOQs and extraction recoveries Conclusions With the developed and validated method, the quantitation of testosterone and androstenedione as well as the qualitative analysis of exogenous steroid esters can be done. The method was developed and validated according to the Guideline (WADA) criteria. The method consists of a fairly simple extraction protocol, followed by derivatization, and the chromatographic conditions have been optimized to wash out the derivatization reagent in the beginning of the chromatographic run, thus making any extra clean- up steps unnecessary. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-030 References 1. Forsdahl, G et al (2015). Detection of testosterone esters in blood. Drug Test Anal, 7, 983-989 2. Van Renterghem, P et al (2020). Validation of an ultra-sensitive detection method for steroid esters in plasma for doping analysis using positive chemical ionization GC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci 15(1141:122026) 3. Keski-Rahkonen, P, Huhtinen, K, Poutanen, M and Auriola, S (2011). Fast and sensitive liquid chromatography–mass spectrometry assay for seven androgenic and progestagenic steroids in human serum. Journal of Steroid Biochemistry & Molecular Biology 127, 396–404 4. Häkkinen, M R et al (2019). Simultaneous analysis by LC–MS/MS of 22 ketosteroids with hydroxylamine derivatization and underivatized estradiol from human plasma, serum and prostate tissue. Journal of Pharmaceutical and Biomedical Analysis 164, 642–652 5. de la Torre, X, Iannone, M, Botré, F (2021). Improving the detection of anabolic steroid esters in human serum by LC–MS. Journal of Pharmaceutical and Biomedical Analysis 194 (2021) 113807. 6. Frey, A J et al (2016). Validation of highly sensitive simultaneous targeted and untargeted analysis of keto- steroids by Girard P derivatization and stable isotope dilution-liquid chromatography-high resolution mass spectrometry. Steroids 116, 60–66 7. Langer, T et al (2022). A comprehensive UHPLC–MS/MS method for the analysis of endogenous and exogenous steroids in serum for anti-doping purposes. Drug Test Anal. 14, 1904–1919 8. World Anti-Doping Agency, Laboratory Guidelines (2023). Quantification of Endogenous Steroids in Blood for the Athlete Biological Passport Acknowledgements The authors would like to acknowledge Dr. Günther Gmeiner for valuable advice with the method development. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-031 Wu Q , Yan K , Dong T , Liu L , Wang Y , Wang Z Quantification of testosterone and androstenedione in human serum by HRMS Beijing Sport University, Beijing, People's Republic of China ; Beijing Anti-Doping Laboratory, Beijing Sport University, Beijing, People's Republic of China Abstract The identification and judgement of whether the atypical change of the concentrations of endogenous steroids originates from exogenous steroid drug abuse by athletes is a complicated and difficult topic in doping-control. Through the ‘steroidal module’ of the Athlete Biological Passport by GC-MS (/MS), the quantification of selected endogenous anabolic androgenic steroids (EAAS) from athletes’ urine is the major monitoring method. However, many confounding factors such as ethnicity, individual difference, micro-biological degradation, gene polymorphism, intaking of non-prohibit substances or alcohol cause some limitations of the urinary steroid profile. Quantification of endogenous steroids, especially testosterone and androstenedione from human serum was considered as a possible and effective complementory program. In this study we developed an ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) method for quantifying testosterone and androstenedione in human serum. The method involves precipitating proteins (PPT) from a 50 µL serum sample and optimizing the concentration steps for micro aliquots to meet the LOQ requirement of 0.1 ng/mL. All measurements were conducted under UPLC-HRMS routine conditions. Method validation included recovery at various concentrations, specificity, intra-day and inter-day precision, accuracy, and uncertainty, with a linear range of 0.1 to 10 ng/mL. This method allows the quantification of testosterone and androstenedione in human serum at low cost and microsample volume, and results also shown high accuracy and stability. Introduction Testosterone and androstenedione are endogenous steroids. Especially testosterone plays a key role in regulating effects for human body muscle content, aggression, physical strength and resilience. Due to the identification and judgement of whether the atypical change of endogenous steroid originates from exogenous steroid drug abuse by athlete is a complicated and difficult topic in doping-control. An UPLC- HRMS method for quantifying the concentration of testosterone and androstenedione in human serum was developed and validated in this study. The method demonstrats to quantify testosterone and androstenedione levels in human serum accurately and it may become the indirect proof to monitor athlete’s behaviour in the future 1 2 2 2 2 2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-032 Experimental Charcoal stripped human serum In this work, charcoal stripped human serum (CSHS) was used to evaluate linearity, specificity, recovery and precision. It was made by dextran coated charcoal (Merck, Sigma-Aldrich, C6241), followed by at least two rounds of centrifugation to collect the supernatant. Each batch of CSHS was evaluated regarding its purity before it was employed in the experiment process. Calibrators 1 mL of Cal 1 to Cal 5 and controls were prepared by CSHS which was finished evaluation of purification and reference materials in each batch. Then 50 µL of single serum was separated into 1.5 mL centrifuge tubes and stored at -20 °C before use. The concentrations of the calibrators were 0.1, 0.5, 1.0, 5.0 and 10.0 ng/mL of testosterone (T) and androstenedione (AD), respectively. Sample preparation All serum samples were prepared as follows: 50 µL of serum was transferred into 1.5 mL centrifuge tube, 5 µL of d -T (0.01 ng/µL in methanol) and 5 µL of d -AD (0.01 ng/µL in methanol), which were internal standard, were added into the tube. 140 µL of acetonitrile, used for protein precipitation, was added to the tube and the solution was thoroughly mixed by vortex mixing. All samples were centrifuged for 10 minutes at 12000 rpm, 170 µL of supernatant was transferred and evaporated to dryness by vacuum concentrator (Eppendorf concentrator plus, Germany) at 45 °C. The samples were reconstituted with 100 µL of initial mobile phase and centrifuged again for 10 minutes. Finally, 90 µL of supernatant was transferred to a vial prior to analysis. UPLC-HRMS Thermo Q-Exactive Plus with Dionex UltiMate 3000 was used for sample analysis.The LC conditions did not require switching between this method and the routine urine analysis method. The instrument conditions are shown in Table 1. Table 1. UPLC-HRMS instrument conditions for the quantification method 3 7 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-033 Results and Discussion In this work, solid-phase extraction (SPE), solid-liquid extraction (SLE) and protein precipitation (PPT) were initially compared. The responses show that SPE and SLE are much higher than PPT, but the response of PPT prepared at LOQ of 0.1 ng/mL also met the quantitative requirements. Compared to other options, PPT is the least expensive and the method is simple and fast to operate. Therefore, the PPT pre-processing method was selected for development and optimization. Selectivity, LOQ, recovery, precision, accuracy, and uncertainty were evaluated. The results were favourable and are shown in Figure 1 and Table 2. Figure 1. Selectivity and specificity of testosterone and androstenedione CSHS+LOQ: Spike 0.1 ng/mL to charcoal stripped human serum; HS: Human serum; CSHS: Charcoal stripped human serum Table 2. Validation of the quantification method 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-034 As shown in Figure 2, the R of T and AD linearity ranges was over 0.995. The response of the LOQ at 0.1 ng/mL was consistent with the quantification requirements. The recovery at different concentrations was exceeding 60%. The coefficient of variation was less than 15% at each concentration from inter- and intra-precision. The bias of accuracy was less than 12%. The uncertainty was less than 30% the LOQ level, and less than 20% for the medium and high concentrations. This method was capable of quantifying testosterone and androstenedione in human serum under the condition of low cost and low sample volume, and the result showed high accuracy and good stability. Figure 2. Calibration curves of testosterone and androstenedione. A: Testosterone; B: Androstenedione Testosterone: Y = 0.9932 X + 0.01924; R : 0.9989; Origin: Ignore; W: 1/X; Area Androstenedione: Y = 1.24 X – 0.008512; R : 0.9988; Origin: Ignore; W: 1/X; Area In this method validation experiment, recovery, linearity, and precision density focus on the stability of the quantitative method, so CSHS was selected for evaluation with RM (Figure 1 and Table 2). Accuracy and uncertainty are closely related to the matrix, CSHS no longer has the ability to access the deviation caused by different matrices due to its purification process, therefore, the different normal human serum (HS) was selected (Table 2). The synthetic uncertainty was calculated by Sw from HS and Bias (Ext) from “Multilevel Serum Calibrator Set” (Chromsystem, Munich, Germany). 2 2 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-035 Conclusions In this study, we developed and validated a UPLC-HRMS method for the quantification of testosterone and androstenedione from human serum in accordance with the World Anti-Doping Agency (WADA) guidelines. Based on the optimized protein precipitation technology, the quantitative results show high accuracy and good stability at low cost and microsample volume. By providing additional proof beyond the urine steroid profile, this method has the potential to improve the monitoring of exogenous steroid drug abuse in athletes and contribute to the fairness and integrity of sports competitions. References 1. Salamin O, Ponzetto F, Cauderay M, Boccard J, Rudaz S, Saugy M, Kuuranne T, Nicoli R. Development and validation of an UHPLC-MS/MS method for extended serum steroid profiling in female populations. Bioanalysis. 2020 Jun;12(11):753-768. doi: 10.4155/bio-2020-0046. 2. Sobolevsky T, Samara V, Ahrens B. Detection of testosterone and androstenedione in serum by LC-HRMS. In: Thevis M, Geyer H, Mareck U (eds.) Recent advances in doping analysis (30). Sportverlag Strauß, Hellenthal (2022).63-66 3. Ponzetto F, Boccard J, Baume N, Kuuranne T, Rudaz S, Saugy M, Nicoli R. High-resolution mass spectrometry as an alternative detection method to tandem mass spectrometry for the analysis of endogenous steroids in serum. J Chromatogr B Analyt Technol Biomed Life Sci. 2017 May 1;1052:34-42. doi: 10.1016/j.jchromb.2017.03.016 4. Ponzetto F, Parasiliti-Caprino M, Gesmundo I, Marinelli L, Nonnato A, Nicoli R, Kuuranne T, Mengozzi G, Ghigo E, Settanni F. Single-run UHPLC-MS/MS method for simultaneous quantification of endogenous steroids and their phase II metabolites in serum for anti-doping purposes. Talanta. 2023 Apr 1;255:124218. doi: 10.1016/j.talanta.2022.124218 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-036 Fußhöller G , Geyer H , Görgens C , Guddat S , Jübner M , Lucuta L , Thevis M Excretion pattern of urinary metabolites of dehydrochloromethyl‐ testosterone after transdermal application Institute of Biochemistry, German Sport University, Cologne, Germany ; Center of Preventive Doping Research, German Sport University, Cologne, Germany ; Department of Toxicology, Institute of Legal Medicine, Faculty of Medicine and University Hospital, University of Cologne, Cologne, Germany Abstract The aim of the present study was to investigate whether the transdermal application of dehydrochloromethyltestosterone (DHCMT) leads to a characteristic excretion pattern of urinary metabolites, differing from those known to exist after oral administration of the drug. Six volunteers received transdermal administrations of 10 mg of DHCMT dissolved in 100 μL DMSO on different skin regions. Urine samples were collected before and till 330 hours after the application and were analysed with GC-MS/MS and LC-HRMS for 5 different DHCMT metabolites. Compared with literature data obtained after oral administration of DHCMT, it could be shown that, in case of transdermal application, DHCMT glucuronide constitutes a long-term metabolite, whereas after oral application it is commonly categorized under the group of short-term metabolites. Introduction Several studies have shown that the transdermal administration of testosterone leads to excretion patterns of urinary testosterone metabolites that differ from those observed after oral administration or injection of testosterone due to an increased 5a-reductase activity of the skin [1,2]. In a recent study with clostebol, such distinct differences between the transdermal and oral administration of clostebol were not corroborated [3]. Based on data obtained from investigations of Gessner et al. [4], the present study aimed at assessing whether the transdermal application of dehydrochloromethyltestoterone (DHCMT) leads to a characteristic excretion pattern of urinary metabolites differing from those known to exist after oral administration of the drug. Experimental The experimental details are described in the publication of Gessner et al. [4]. In short, 6 volunteers received transdermal administrations of 10 mg DHCMT dissolved in 100 µL DMSO. Two volunteers each received the substance/DMSO mixture on the back of the hand, on the top of the lower arm and on the neck. Urine samples were collected before and till 330 hours after the application. The urine samples were analysed with GC-MS/MS and LC-HRMS for the DHCMT metabolites DHCMT glucuronide, 6-OH- DHCMT and M1, M2 and M3 (nomenclature of M1-M3 according to Sobolevsky and Rodchenkov [5]). 1,2 1,2 1,2 1,2 3 3 1,2 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-037 Results and Discussion The detection windows for the different DHCMT metabolites obtained with LC-MS/MS and GC-MS/MS after transdermal administration of DHCMT are presented in Figure 1a-c (two volunteers for each skin region). The data originate from initial testing procedures, i.e. for some presumptive findings it is not guaranteed that a confirmation of the target analyte in further procedures would be successful. For comparison, data of one volunteer (volunteer 5) after an oral administration of 5 mg DHCMT from an investigation of Loke et al. [6] are presented in Figure 2. After transdermal application, the longest detection window for all volunteers and all skin regions could be observed for the DHCMT glucuronide (Figure 1a-c). This is different to the oral application, where one of the metabolites M1-M3 has the longest detection window and DHCMT glucuronide is only a short term metabolite (Figure 2 [6]). In none of the samples after transdermal administration, the metabolites M1 and M2 were detected. Figure 1a-b. Detection windows for the different DHCMT metabolites after transdermal administration of DHCMT on the hand (a) and lower arm (b) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-038 Figure 1c. Detection windows for the different DHCMT metabolites after transdermal administration of DHCMT on the neck Figure 2. Detection windows for the different DHCMT metabolites after oral administration of DHCMT [6] Conclusions The metabolic pattern after the transdermal application of DHCMT is different from the pattern after oral application. In case of transdermal application the DHCMT glucuronide is a long-term metabolite. Therefore the sole presence of DHCMT glucuronide in a doping control sample may be possible and may be an indication of a transdermal application of DHCMT. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-039 References 1. Geyer H, Flenker U, Haenelt N, Mareck U, Piper T, Schmechel A, Schrader Y, Platen P, Schänzer W. Steroid profiling and GC/C/IRMS for the detection of the misuse of testosterone gel. Schänzer W, Geyer H, Gotzmann A, Mareck U (eds.) Recent advances in doping analysis (15). Sportverlag Strauß, Köln (2007) 133-142 2. Mullen J, Börjesson A, Hopcraft O, Schulze JJ, Ericsson M, Rane A, Lehtihet M, Ekström L. Sensitivity of doping biomarkers after administration of a single dose testosterone gel. Drug Test Anal. 2018 May;10(5):839-848.Piper T, Emery C, Saugy M. Norandrosterone analysis by GC/C/IRMS. In: Schänzer W, Thevis M, Geyer H, Mareck U (eds.) Recent advances in doping analysis (20). Sportverlag Strauß, Köln (2012) 201-204 3. de la Torre X, Colamonici C, Iannone M, Jardines D, Molaioni F, Botrè F. Detection of clostebol in sports: Accidental doping? Drug Test Anal. 2020 Nov;12(11-12):1561-1569. 4. Gessner L, Thevis M, Rothschild MA, Jübner M. Detectability of oxandrolone, metandienone, clostebol and dehydrochloromethyltestosterone in urine after transdermal application. Drug Test Anal. 2022 Oct;14(10):1744-1761 5. Sobolevsky T, Rodchenkov G. Detection and mass spectrometric characterization of novel long-term dehydrochloromethyltestosterone metabolites in human urine. J Steroid Biochem Mol Biol. 2012 Feb;128(3- 5):121-7. 6. Loke S, de la Torre X, Iannone M, La Piana G, Schlörer N, Botrè F, Bureik M, Parr MK. Controlled administration of dehydrochloromethyltestosterone in humans: Urinary excretion and long-term detection of metabolites for anti-doping purpose. J Steroid Biochem Mol Biol. 2021 Nov;214:105978. Acknowledgements The authors acknowledge support from Sport Ireland (Dublin, Ireland), the Federal Ministry of the Interior, Community and Building of the Federal Republic of Germany (Berlin, Germany) and the Manfred Donike Institute for Doping Analysis (Cologne, Germany). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-040 Guddat S , Fußhöller G , Geyer H , Görgens C , Jübner M , Lucuta L , Thevis M Excretion pattern of urinary metabolites of metandienone after transdermal application Institute of Biochemistry, German Sport University, Cologne, Germany ; Department of Toxicology, Institute of Legal Medicine, Faculty of Medicine and University Hospital, University of Cologne, Cologne, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract In the present study, the urinary excretion pattern of metandienone metabolites after transdermal application of the drug was investigated and the presence of potentially characteristic excretion profiles differing from those known to exist after oral administration of the drug was assessed. Six volunteers received transdermal administrations of 10 mg of metandienone dissolved in 100 µL DMSO on different skin regions. Urine samples were collected before and till 330 hours after the application and were analysed with GC-MS/MS and LC-HRMS for 8 different metandienone metabolites. Compared with literature data after oral administration it could be shown that, in case of transdermal application, 6-OH- metandienone constitutes a long-term metabolite, whereas after oral application it is commonly categorized under the group of short-term metabolites. Introduction Several studies have shown that the transdermal administration of testosterone leads to excretion patterns of urinary testosterone metabolites that differ from those observed after oral administration or injection of testosterone due to an increased 5a-reductase activity of the skin [1,2]. In a recent study with clostebol, such distinct differences between the transdermal and oral administration of clostebol were not corroborated [3]. Based on data obtained from investigations of Gessner et al. [4], the present study aimed at assessing whether the transdermal application of metandienone leads to a characteristic excretion pattern of urinary metabolites differing from those known to exist after oral administration of the drug. Experimental The experimental details are described in the publication of Gessner et al. [4]. In short, 6 volunteers received transdermal administrations of 10 mg of metandienone dissolved in 100 µL DMSO. Two volunteers each received the substance/DMSO mixture on the back of the hand, on the top of the lower arm and on the neck. Urine samples were collected before and till 330 hours after the application. The urine samples were analysed with GC-MS/MS and LC-HRMS for the metandienone metabolites 6-OH- metandienone (6OH-Metand), 17α-methyl-5α-androstane-3α,17β-diol (tetrahydromethyltestosterone, THMT), 18-norepimetendiol (NorEMD), epimetendiol (EMD), epi-LTM (epi-long-term metabolite [5]), LTM (long-term metabolite [5]), epimetandienone (epiMD) and metandienone (MD). 1 1 1 1 2 2 1,3 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-041 Results and Discussion The detection windows for the different metandienone metabolites obtained with LC-MS/MS and GC- MS/MS after transdermal administration of metandienone are presented in Figures 1a-c (two volunteers for each skin region). The data originate from initial testing procedures, i.e. for some presumptive findings it is not guaranteed that a confirmation of the target analyte in further procedures would be successful. For comparison, data gathered after oral administration of 5-10 mg metandienone from Polet et al. [6] are presented in Figure 2. After transdermal application, the longest detection windows for all volunteers and all skin regions could be observed for the metandienone LTM and 6OH-metandienone (Figure1a-c). This is different to the oral application, where 6OH-metandienone is considered as a short-term metabolite (Figure 2 [6]). Figure 1a-b. Detection windows for the different DHCMT metabolites after transdermal administration of metandienone on the hand (a) and lower arm (b) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-042 Figure 1c. Detection windows for the different Metandienone metabolites after transdermal administration of metandienone on the neck Figure 2. Detection windows for the different metandienone metabolites after oral administration of metandienone [6] Conclusions The metabolic pattern after the transdermal application of metandienone is different from the pattern after oral application. In case of transdermal application, 6OH-metandienone constitutes a long-term metabolite, whereas after oral application it is commonly categorized under the group of short-term metabolites. The presence of 6OH-metandienone together with the long-term metabolite of metandienone (LTM) may be an indication of a transdermal application of metandienone. Similar to MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-043 scenarios where the LTM of metandienone is exclusively detected after oral administration, the sole presence of 6OH-metandienone after transdermal application of metandienone cannot be excluded. References 1. Geyer H, Flenker U, Haenelt N, Mareck U, Piper T, Schmechel A, Schrader Y, Platen P, Schänzer W. Steroid profiling and GC/C/IRMS for the detection of the misuse of testosterone gel. Schänzer W, Geyer H, Gotzmann A, Mareck U (eds.) Recent advances in doping analysis (15). Sportverlag Strauß, Köln (2007) 133-142 2. Mullen J, Börjesson A, Hopcraft O, Schulze JJ, Ericsson M, Rane A, Lehtihet M, Ekström L. Sensitivity of doping biomarkers after administration of a single dose testosterone gel. Drug Test Anal. 2018 May;10(5):839-848.Piper T, Emery C, Saugy M. Norandrosterone analysis by GC/C/IRMS. In: Schänzer W, Thevis M, Geyer H, Mareck U (eds.) Recent advances in doping analysis (20). Sportverlag Strauß, Köln (2012) 201-204 3. de la Torre X, Colamonici C, Iannone M, Jardines D, Molaioni F, Botrè F. Detection of clostebol in sports: Accidental doping? Drug Test Anal. 2020 Nov;12(11-12):1561-1569. 4. Gessner L, Thevis M, Rothschild MA, Jübner M. Detectability of oxandrolone, metandienone, clostebol and dehydrochloromethyltestosterone in urine after transdermal application. Drug Test Anal. 2022 Oct;14(10):1744-1761 5. Schänzer W, Geyer H, Fußhöller G. Mass spectrometric identification and characterization of a new long- term metabolite of metandienone in human urine. Rapid Commun Mass Spectrom. 2006;20(15):2252- 2258.. 6. Polet M, Van Gansbeke W, Van Eenoo P, Deventer K. Efficient approach for the detection and identification of new androgenic metabolites by applying SRM GC-CI-MS/MS: a methandienone case study. J Mass Spectrom. 2016 Jul;51(7):524-34. Acknowledgements The authors acknowledge support from Sport Ireland (Dublin, Ireland), the Federal Ministry of the Interior, Community and Building of the Federal Republic of Germany (Berlin, Germany) and the Manfred Donike Institute for Doping Analysis (Cologne, Germany). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-044 Montes de Oca Porto R, Martinez Brito D, Lopez Castillo D, Rodriguez Fernandez A, Laffita Jorrin D Behaviour of 19-norandrosterone levels in negative samples Antidoping Laboratory, Havana, Cuba Abstract Background: One of the pathways for the formation of 19-norandrosterone (19-NA) in the human organism is through the transformation of testosterone to estradiol with the participation of the aromatase enzyme. Several authors have described the inhibition or increase of the activity of this enzyme due to physiological processes or interactions with other drugs, even in different metabolic pathways. This study describes the behavior of 19-norandrosterone levels in samples analyzed from 2017 to 2022. Experimental: The study included samples with the presence of 19-NA in female (n = 217) and male (n = 239) samples below 2.5 ng/mL (adjusted for specific gravity). The samples were classified by sports groups (n = 4) according to control of sports training literature. Statistical analysis of the data distribution was performed (Shapiro-Wilk and Anderson-Darling tests, α = 0.05) and statistical differences between groups were evaluated (Kruskal-Wallis test, α = 0.05). Results: Non-parametric distributions were observed for all the groups evaluated except for the male group in competitive arts sports, probably influenced by the number of samples. Significant differences were observed between the female and male groups for the concentration of 19-NA (p = 5.3 x 10 ) with higher values for the male group. Likewise, significant differences were found between the sexes for the group of sports with balls (p = 0.00037). Specifically, within the male group, significant differences were observed in the ball sports group compared to the rest. While in the female group there are no differences between the sport groups studied. Introduction One of the pathways for the formation of 19-norandrosterone (19-NA) in the human organism is through the transformation of testosterone to estradiol with the participation of the aromatase enzyme. Several authors have described the inhibition or increase of the activity of this enzyme due to physiological processes or interactions with other drugs, even in different metabolic pathways [1-3]. To maintain an observation on this affirmation, the goal of this work was to describe the behavior of 19-norandrosterone levels in female and male samples analyzed from 2017 to 2022. Experimental Urine samples of athletes received in the Havana Antidoping Laboratory from 2017 to 2022 that presented 19-norandrosterone below 2.5 ng/mL (after the concentration was adjusted by SG) were classified, and statistically compared. All samples included in the study met the NA identification criteria according to the TD-IDCR technical document. The group was divided into females (F, n = 217) and -6 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-045 males (M, n = 239). Then, they were classified by sports groups (n = 4) according to control of sports training literature into ball sports (F: n = 50, M: n = 154), endurance sports (F: n = 68, M: n = 42), combat sports (F: n = 56, M: n = 32) and competitive art sports (F: n = 33, M: n = 7). Sample preparation under routine conditions were done following a solid phase extraction on C18 cartridges, enzymatic hydrolysis (50 µL β-glucuronidase Roche, pH 6.8) and then liquid-liquid extraction (TBME, 5 mL, pH 10). Trimethylsylil derivatives (MSTFA: NH I: 2-ME, 100:2:6, m:V:m) were analysed on an Agilent 7890A gas chromatograph coupled to an Agilent 7000B triple quadrupole. The column used was a HP1MS methyl fused-silica capillary column (17 m x 0.2 mm, 0.11 μm film thickness). The data acquisition was performed in multiple reaction monitoring (MRM). Statistical analyses were done by using MedCalc Statistical Software version 20.2 (MedCalc Software Ltd, Ostend, Belgium; https://www.medcalc.org; 2022). Data distribution was checked by Shapiro-Wilk and Anderson-Darling tests (p < 0.05). Statistical differences between groups were checked by applying Kruskal-Wallis test, p < 0.05. Results and Discussion Except males in competitive art sports, no data showed normal distribution (Shapiro-Wilk and Anderson- Darling tests, p 20 ng/mL) and furosemide glucuronide (est. conc. > 20 ng/mL) This is the most frequent situation in routine doping control analysis so far. The exemplary chromatograms from a CP analysis are shown in Figure 1. Figure 1. Chromatograms of athlete's sample from confirmatory analysis performed after sample preparation involving double LLE SCENARIO 2: Furosemide (est. conc. > 20 ng/mL) and furosemide glucuronide (est. conc. < 20 ng/mL) According to the TL-24, this situation constitutes an AAF, as well. This situation was observed e.g. in December 2022. Two samples with Specific Gravities of 1.003 and 1.004 were collected from one Athlete competing in Powerlifting. The confirmation procedure of the first sample showed furosemide and its metabolite at concentrations of 24 ng/mL and 6 ng/mL, respectively. In turn, in the second sample, the estimated concentrations of furosemide and its metabolite were 25 ng/mL and 6 ng/mL, respectively. SCENARIO 3: Furosemide (est. conc. < 20 ng/mL) and furosemide glucuronide (est. conc. < 20 ng/mL) To date, for the correct interpretation and reporting of results from the furosemide analysis, the WADA TL-24 shall be applied. Should a modification of this TL consider the inclusion of phase-II metabolites such as the furosemide glucuronide in the future, the second-phase metabolite of furosemide could be MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-068 used as a target compound for the Initial Testing Procedure (ITP) and Confirmation Procedures (CP) for furosemide analysis, as well. This would then also be compatible with WADA TD 2022 MRPL (point "c" in the MRL table) [5] stating "when the Analytical Method used includes also the determination of phase-II Metabolites (e.g., glucuronides, sulfates) of the specific target substance, the MRL is applied to the total concentration (i.e., free and conjugated fractions) of the substance. This estimation is obtained either by separate determination of the molecular species (e.g., by LC-MS analysis) or following the de-conjugation of the phase-II Metabolite(s) […]” SCENARIO 4: Only Furosemide presence in a sample (est. conc. > 20 ng/mL or < 20 ng/mL) According to TL-24, the presence of furosemide in each case at an estimated concentration greater than (> ) 20 ng/mL, shall be reported as an AAF, in turn, ≤ 20 ng/mL, shall be reported as a Negative. As the sole exception to this new MRL, the presence of this compound in a sport or discipline which are listed in Annex 1. SCENARIO 5: Only metabolite presence in a sample (est. conc. > 20 ng/mL or < 20 ng/mL) The situation is similar to Scenario 4. Due to the limited number of papers concerned with the elimination process of furosemide, the exclusive presence of the furosemide metabolite in a sample might necessitate consideration for doping control samples collected from defined sport disciplines. Conclusions The WADA TL-24 harmonized the scheme for the reporting of furosemide as AAF, ATF, or Negative finding. According to this document, the presence of furosemide (parent compound only), in each case at an estimated concentration greater than (> ) 20 ng/mL, shall be reported as an AAF. The presented data show that the second-phase metabolite of furosemide is a valuable biomarker in furosemide detection (especially as it confirms the administration, biotransformation, and elimination of the drug). Including this metabolite in routine operations could be valuable for the interpretation of results for furosemide analysis purposes also, e.g. with furosemide glucuronide potentially being an indicator of the time point of application of furosemide. In addition, targeting furosemide glucuronide could be of added value when screening doping control samples of sport disciplines listed in Annex 1 WADA TL-24 (sport/disciplines with relevant weight classes). In order to assess the extent of this added value, the monitoring of furosemide glucuronide could be initialized by the volunteer laboratories for some time to evaluate a scale of the presence of this metabolite in athlete samples. Undeniably, the identification of new metabolites of prohibited substances is a crucial aspect of developing the anti-doping policy, as well as research concerning the elimination process of furosemide seems to be necessary. References 1. M. Espinosa Bosch, A.J. Ruiz Sanchez, F. Sanchez Rojas, C. Bosch Ojeda. Recent developments in analytical determination of furosemide. J.Pharm. Biomed. Anal. (2008) 48, 519–532. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-069 2. World Anti-Doping Agency. Prohibited List 2023. Available at https://www.wada- ama.org/sites/default/files/2022-09/2023list_en_final_9_september_2022.pdf [26 January 2023]. 3. World Anti-Doping Agency. WADA Technical Letter – TL24. Available at https://www.wada- ama.org/sites/default/files/2022-01/tl24_diuretics_v2.0.pdf [26 January 2023]. 4. Grucza K., Wicka M., Kowalczyk K., StaÅ„czyk D., Kaliszewski P., Michalak D., Szutowski M., Kwiatkowska D.: Identification of glucuronide conjugate of furosemide in routine doping control analysis. Poster presented at the 37 Manfred Donike Workshop on Doping Analysis, Cologne, Germany, 17.02. - 22.02. 2019 5. World Anti-Doping Agency. TD2022MRPL. Available at https://www.wada-ama.org/sites/default/files/2022- 01/td2022mrpl_v1.1_eng_0.pdf [31 January 2022]. Acknowledgements Financial support from the Ministry of Health of the Republic of Poland under the project number 1/85195/PLAD/2022/4186/406 is gratefully acknowledged. th MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-070 Grucza K , Wicka M , Drapala A , Konarski P , Stanczyk D , Kaliszewski P , Kwiatkowska D Occurrence of higenamine and its sulfate conjugate in routine doping control analysis - interpretation of results Polish Anti-Doping Laboratory, Warsaw, Poland ; Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland Abstract Higenamine (Norcoclaurine) is a very popular substance in Chinese medicine present in many plants and is one of the substances occurring in dietary supplements. Moreover, it is included in Class S3 (β-2- agonists) of the WADA 2023 Prohibited List. This poster describes instances where performing hydrolysis would cause an AAF status and indicates that change in the current principles of interpretations of results should be considered. Introduction Higenamine (Norcoclaurine) is a very popular substance in Chinese medicine present in many plants and is one of the substances commonly occurring in dietary supplements [1]. Although extracts of these plants are listed as ingredients in many pre-workout and fat-burner products, several products were contaminated, without notice on the label [2]. Higenamine is prohibited in sport at all times and included in Class S3 (β-2-agonists) of the WADA 2023 Prohibited List [3]. The presence of higenamine in urine samples at concentrations greater than 10 ng/mL constitutes an Adverse Analytical Finding (AAF) [4]. This poster shows instances where performing hydrolysis would cause an AAF status and indicates that change of the current principles of interpretations of results should be considered. Experimental Urine samples were prepared by 2 protocols: Dilute-and-Shoot (DaS) approach (200 µL of urine and 800 µL of water) and a method involving acid hydrolysis and double extraction as described previously [5]. Analysis was performed on a UPLC™ Acquity chromatograph (Waters, Milford Massachusetts, USA) equipped with an HSST3 column (1.8 μm, 2.1 × 100 mm). The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B) and the LC gradient was employed at a constant flow rate of 300 μL/min at 45 °C. The concentration of acetonitrile was gradually increased in a linear manner: from 0% to 15% within the first 6 minutes, and from 15% to 100% in 1 minute. Then it was kept constant for additional 2 minutes. Finally, the column was re-equilibrated for 2 minutes with the mobile phase of the initial composition. Samples were stored at 5 °C in the autosampler prior to analysis and the injection volume was fixed at 5 μL. Multiple reaction monitoring (MRMs) of higenamine and metabolite were traced with Xevo TQ-S (Waters, USA) mass spectrometer equipped with a new atmospheric pressure ionization source commercialized as UniSpray™. Analytes were investigated in the positive (+) mode. The 1 1 1 1 1 1 1,2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-071 desolvation gas flow was set at 800 L/h at 500 °C and the source temperature was 150 °C. The impactor voltage applied was 3.0 kV. The cone and collision gas flows were set at 150 L/h and 0.20 mL/min, respectively. Traced MRMs and their corresponding MS settings are listed in Table 1. Table 1. MS settings (MRMs traced, Cone [v], Collision [eV]) of the substances tested Results and Discussion At the WADA-accredited laboratory in Warsaw, higenamine as well as higenamine sulfate are detected in routine anti-doping analysis by the DaS approach. In August 2016, WADA obliged all accredited laboratories to detect higenamine as soon as possible. In turn, afterward, the metabolite of higenamine was included in the ITP in 2017 [5]. Since then, cases where only the metabolite was present in a sample were observed 18 times: 4 times in 2017, 4 times in 2018, 4 times in 2019, 1 time in 2020, 1 time in 2021, 3 times in 2022, and 1 time in 2023 (so far) (Figure 1). For these cases, the ratios for higenamine metabolite to meldonium-D3 were calculated as well. As a result, samples prepared according to the second protocol showed the presence of the higenamine at estimated concentrations ranging between 1 ng/mL and 50 ng/mL as the total concentration (sum of free form, glucuronide conjugation, and sulfate conjugation)(Figure 2). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-072 Figure 1. Chromatograms of the Initial Testing Procedure (DaS approach) for higenamine sulfate (only detection window): (a) blank urine, (b) athlete`s sample. The presence of higenamine (parent compound) was not observed in the athlete`s sample Moreover, higenamine was identified at the estimated concentration above MRL in seven cases. Undoubtedly, the above concentration range does not allow us to indicate whether an athlete consumed higenamine for doping purposes or whether it was an unintentional doping case by an athlete, as well. The first information about reporting only the free form of Higenamine was included in WADA TD2018MRPL as: „The reporting limits specified for salmeterol and higenamine apply to the determination of the free parent compound only” and is present in WADA TD2022MRPL as well [4]. This clearly indicates that hydrolysis shall not be performed. As a consequence of this principle of interpretation of results, such an approach may cause an underestimation of the concentration of higenamine in urine samples. In turn, the WADA threshold concentration is based on the sum of free form and the glucuronide conjugate for salbutamol and formoterol. As it is well known, higenamine is excreted in unchanged form, and as second-phase metabolites(both glucuronide and sulfate conjugates), and consequently hydrolysis of a sample should be considered for the proper estimation of the higenamine. Looking more closely, the principle of higenamine detection as „the determination of the free (non- conjugated) parent compound" [4] does not eliminate the risk of reporting unintentional doping by athletes. Conversely, not carrying out sample hydrolysis may affect that laboratories may not report „intentional doping” (an AAF for Higenamine), especially, in cases where the estimated concentration of higenamine after performed sample hydrolysis will exceed MRL. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-073 Figure 2. Chromatograms of analysis performed after sample preparation involving acid hydrolysis and double LLE: (a) blank urine, (b) athlete's sample, and (c) quality control sample (10 ng/mL) Conclusions Undeniably, monitoring of second-phase metabolites of prohibited substances is a crucial activity for doping control purposes. It seems to be undoubted that point "c" in the MRL table of the WADA TD2022MRPL should be applied for all β-2-agonist without sole exceptions to avoid reporting false negative findings for higenamine. Therefore, a change of the current principles of interpretation of the results for higenamine should be considered as soon as possible. Moreover, research concerning the presence of higenamine in nutritional supplements should be conducted to set a new MRL for higenamine metabolites or verify general MRL for higenamine. References 1. Cohen PA, Travis JC, Keizers PHJ, Boyer FE, Venhuis BJ. The stimulant higenamine in weight loss and sports supplements. Clin Toxicol. 2019;57(2):125-130. 2. Comunità F, de la Torre X, Mazzarino M, Botrè F. Higenamine and coclaurine positive doping case associated massage cream use. In: Schänzer W, Thevis M, Geyer H, Mareck U, eds. Recent Advances in Doping Analysis. Köln: Sportverlag Strauss; 2017:47-50. 3. World Anti-Doping Agency. Prohibited List 2023. Available at https://www.wada- ama.org/sites/default/files/2022-09/2023list_en_final_9_september_2022.pdf [03 February 2023] 4. World Anti-Doping Agency. TD2022MRPL. Available at https://www.wada-ama.org/sites/default/files/2022- 01/td2022mrpl_v1.1_eng_0.pdf [03 February 2023] MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-074 5. Grucza K., Kwiatkowska D., Kowalczyk K., Wicka M., Szutowski M., Cholbinski P.: Analysis for higenamine in urine by means of UHPLC/MS/MS: Interpretation of results. Drug Test Anal. 2017;10(6):1017-1024. Acknowledgements Financial support from the Ministry of Health of the Republic of Poland under the project number 1/85195/PLAD/2022/4186/406 is gratefully acknowledged. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-075 Görgens C , Guddat S , Bosse C , Thevis M Enantiomeric separation of various stimulants in urine after chiral derivatization using Marfey’s reagent for doping control purposes Institute of Biochemistry, German Sport University, Cologne, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract Enantiomers of chiral drugs often provide different pharmacological and pharmacodynamic properties due to their stereoselectivity. In many instances, the desired pharmacological activity is primarily attributed to one specific enantiomer, whereas the other enantiomer may exhibit reduced activity or even possesses toxic properties. For metamfetamine, the detected enantiomer determines the sub- classification by the World Anti-Doping Agency (WADA) as a “specified” or a “non-specified” stimulant, which is essential for the sanctioning process of the athlete. Therefore, the use of confirmation assays, which are able to distinguish between different enantiomers are essential for the compound’s unequivocal identification. Besides different LC-MS/MS-based approaches using chiral chromatographic analysis, derivatization strategies with chiral derivatizing agents followed by GC-MS/MS or LC-MS/MS detection also proved capable. The aim of the study was to develop a simple and fast LC-MS/MS-based assay for the enantioselective identification of some of the most frequently detected stimulants in sports drug testing after derivatization using Marfey’s reagent. Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide) is used to separate and to determine enantiomeric, amino-group containing compounds. The reagent reacts stoichiometrically with primary or secondary amino groups to produce stable diastereomeric derivatives, which can readily be separated by conventional reversed-phase HPLC without the need of a special and cost-intensive chiral column. The developed enantioselective assay is highly specific and reproducible, providing limits of identification (LOI) fulfilling WADA requirements for stimulants assigned with a minimum reporting level (MRL) of 50 ng/mL. As proof-of-concept, authentic urine samples containing the target compounds were analyzed and their enantiomeric composition was assessed. The approach proved suitable for the chiral separation of a total of six selected enantiomeric stimulants prohibited in sports. Introduction In recent years, a trend towards more efficient initial testing procedures (ITP), using LC-MS/MS methods covering hundreds of prohibited compounds as well as their metabolites in one single chromatographic run has been observed in sports drug testing. Using these high-throughput analytical assays, some crucial characteristics of specific analytes, e.g. chirality or isomerism of small polar compounds (m/z 100 – m/z 200) often remain undetected in the ITP, according to a non-enantioselective separation on common reversed-phase analytical columns and/or a limited number of product ions using electrospray ionization mass spectrometry. However, for some stimulants, e.g. metamfetamine, the detected enantiomer determines the sub-classification by the World Anti-Doping Agency (WADA) as “specified” or 1 1 1 1,2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-076 “non-specified”, which is essential for the sanctioning process of the athlete [1]. Moreover, the enantiomers of chiral drugs often provide different pharmacological and pharmacodynamic properties due to their stereoselectivity. In many instances, the desired pharmacological activity is primarily attributed to one specific enantiomer, whereas the other enantiomer may exhibit reduced activity or even possesses toxic properties. Therefore, the use of confirmation assays, which are able to distinguish between different enantiomers are essential for the compound’s unequivocal identification. Besides different LC-MS/MS-based approaches using chiral chromatographic analysis, derivatization strategies with chiral derivatizing agents followed by GC-MS/MS or LC-MS/MS detection proved valuable [2-4]. The aim of the study was to develop a simple and fast LC-MS/MS-based assay for the enantioselective identification of some of the most frequently detected stimulants in sports drug testing after derivatization using Marfey’s reagent. Figure 1. Nucleophilic substitution reaction of l-amfetamine (I) and Marfey’s reagent (II) to yield the corresponding l,l-diastereomer derivative (III) with elimination of hydrogen fluoride Experimental Materials and methods The reference materials of d-amfetamine, l-amfetamine, d-metamfetamine, l-metamfetamine and 4- methylhexan-2-amine were obtained from from Cerilliant (Round Rock, TX, USA). 5-methylhexan-2-amine and 1,3-dimethylbutylamine were purchased from Sigma-Aldrich (Deisendorf, Germany), while 3-methyl- hexan-2-amine was bought from Curpys Chemicals (Kyiv, Ukraine). Experiments were carried out using an Exploris 480 orbitrap mass spectrometer operating in negative-ionization full scan and parallel reaction monitoring (PRM) acquisition mode coupled to a Vanquish UHPLC (Thermo Scientific, Bremen, Germany), equipped with a Nucleoshell RP 18 (100 x 2 mm, 2.7 µm) analytical column (Macherey Nagel, Düren, Germany). The mobile phase composed of 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B). Initial conditions were 60% of solvent B at a flow rate of 250 µL/min. After 5 min of isocratic elution, the content of solvent B was increased linear from 60% to 80% within 10 min. After another one-minute of isocratic elution at 98% B, re-equilibration at 60% B started for 4 min, resulting in an overall runtime of 20 min. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-077 Samlpe preparation To 50 µL of urine sample, 10 µL of the racemic internal standard amfetamine-D11 and 20 µL of 1 M NaHCO was added. After mixing, 100 µL of a 0.1% ( w/v) solution of Marfey's reagent (1-fluoro-2-4- dinitrophenyl-5-L-alanine amide) dissolved in acetone, was added to the mixture, vortexed and heated at 45 °C for 1 hour. The reaction was stopped by adding 40 µL of 1 M HCl in water and evaporation to dryness under nitrogen at 60°C. The samples were reconstituted in 100 µL of 40:60 water:methanol (v/v) before injection into the instrument. Results and Discussion As demonstrated in Figure 2, analysis of the aforementioned stimulants after derivatization using Marfey’s reagent and LC-HRMS/MS detection provides excellent enantioselective separation with almost zero biological noise. As demonstrated in Table 1, the assay was found the be highly specific and reproducible, providing limits of identification (LOI) fulfilling WADA requirements for stimulants assigned with a minimum reporting level (MRL) of 50 ng/mL. As a proof-of-concept, authentic urine samples containing the target compounds were analyzed and their enantiomeric composition was assessed. The approach proved suitable for the chiral separation of a total of six selected enantiomeric stimulants prohibited in sports. Table 1. Method validation results 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-078 Figure 2. Extracted ion chromatogram of spiked urine samples with (A) 3-methylhexan-2-amine, (B) 4- methylhexan-2-amine, (C) 5-methylhexan-2-amine, (D) metamfetamine and amfetamine, and (E) 1,3- dimethylbutylamine (in each case 50 ng/mL) after derivatization using Marfey‘s reagent MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-079 Conclusions Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide) is used to separate and to determine enantiomeric, amino-group containing compounds. The reagent reacts stoichiometrically with primary or secondary amino groups to produce stable diastereomeric derivatives, which can readily be separated by conventional reversed-phase HPLC without the need of a special and cost-intensive chiral column (Figure 1). The developed sample preparation is simple and fast and fulfills WADA requirements for MRL compounds. References 1. World Anti-Doping Agency. The 2023 Prohibited List. 2023, https://www.wada- ama.org/sites/default/files/2022-09/2023list_en_final_9_september_2022.pdf (24-01-2023) 2. Thomas A, Thevis M. Stereoisomers in sports drug testing: Analytical strategies and applications. J Chromatogr A. 2022; 1674:463154. doi: 10.1016/j.chroma.2022.463154. Epub 2022 May 17. 3. Newmeyer MN, Concheiro M, Huestis MA, Rapid quantitative chiral amphetamines liquid chromatography- tandem mass spectrometry method in plasma and oral fluid with a cost-effective chiral derivatizing reagent, J Chromatogr A. 2014; 1358 (5): 68–74. https://doi.org/10.1016/j.chroma.2014.06.096 4. Foster BS, Gilbert DD, Hutchaleelaha A, Mayersohn M, Enantiomeric determination of amphetamine and methamphetamine in urine by precolumn derivatization with Marfey's reagent and HPLC, J Analytical Toxicology. 1998; 22 (4): 265-269. https://doi.org/10.1093/jat/22.4.265 Acknowledgements The presented work was conducted with support of the Federal Ministry of the Interior, Building and Community, of the Federal Republic of Germany, and the Manfred-Donike Institute for Doping Analysis (Cologne, Germany). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-080 Sahu PL , Handa T , Sahu K , Soni A , Kori RK , Mahajan S , Padder AH , Nandi U , Ahmed QN , Reddy DS Synthesis and characterization of 17α-hydroxymethyl-17β-methyl-18- nor-2-oxa-5α-androst-13-en-3-one (Oxandrolone M2), a long-term meta​bolite via a Kratena’s reaction approach National Dope Testing Laboratory, New Delhi, India ; CSIR-Indian Institute of Integrative Medicine, Jammu, India, Jammu, India Abstract The oxandrolone long-term urinary metabolite (OxM2) belongs to S1.1 Anabolic Androgenic Steroids under S1 Anabolic Agents class listed in the WADA Prohibited List. Despite its potent anabolic activity and less side effects, anabolic steroids like oxandrolone have limited positive findings, making it a popular choice for athletes who cheat. Oxandrolone marketed under the brand name Oxandrin and Anavar is an Androgenic Anabolic Steroid (AAS). Despite of its therapeutic usage, it is associated with one or more side effects such as liver cell tumors, peliosishepatis, intra-abdominal hemorrhage, liver failure, fatal and blood lipid changes associated with increased risk of atherosclerosis, cholestasis hepatitis, increased risk for the development of prostatic hypertrophy and prostatic carcinoma in geriatric patients and hypercalcemia in patients with breast cancer. So, FDA announced the final decision on June 28, 2023 to withdraw approval of brand and generic Oxandrin and decided that the drug products should be removed from the market [1]. For the synthesis of OxM2, literature report by Kratena et al. was followed and due to various bottle neck challenges in the reported method, series of optimizations were performed to successfully achieve the formation of OxM2. So, in the present report, an optimized procedure for the synthesis of OxM2 along with its spectral analysis is reported. Keywords : oxandrolone, long-term metabolite, OxM2 Introduction Anabolic Androgenic Steroids are still popular prohibited substances among athletes and are the most frequently found drug in tests, as stated by the statistics provided by WADA [2]. Oxandrolone has two long-term urinary metabolites which are C-17-epimeric substances, i.e 17α-hydroxymethyl-17β-methyl- 18-nor-2-oxa-5α-androst-13-en-3-one (Ox M2) and its epimer 17β-hydroxymethyl-17α-methyl-18-nor-2- oxa-5α-androst-13-en-3-one (Ox M1). The merit of these metabolites for the anti-doping community is the prolonged detection period, which significantly increases the effectiveness of sports drug testing. In the present work, some optimizations of the approach of Kratena et al. are carried out to access the OxM2 metabolite [3], which is required as reference material in anti-doping laboratories for the detection of Oxandrolone misuse by athletes. 1 1 1 1 1 2 2 2 2 2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-081 Experimental Procedure for the synthesis of OxM2 Oxandrolone 1 (0.900 g) was dissolved in dry pyridine (20 mL) and POCl (2 mL) was added at -78 °C and stirred overnight for 48 h at room temperature. After cooling the mixture to room temperature, it was carefully poured into ice cold water. The suspension thus obtained was extracted with dichloromethane (DCM), the organic phases were dried over Na SO and evaporated in vacuo to obtain the crude product which was recrystallized in DCM and hexane. Solid thus obtained was filtered and characterized as mixture of isomers 2. Further 2 (0.889 g) was dissolved in 10 mL dry DCM, 1.2 equiv. m-CPBA (0.639 g) was added portion wise at 0 °C and then stirred at room temperature for 3 h. The solution was then quenched with 40 mL saturated sodium thiosulfate and 40 mL NaHCO solutions and extracted with DCM. The crude product thus obtained was purified by column chromatography (230-400 mesh silica gel, neutralized with triethylamine) using 15% ethyl acetate:hexane as eluent to give 0.443 g mixture of epoxides 3 and 4. The mixture of 3 and 4 (0.436 g) was further dissolved in glacial acetic acid (9.6 mL) and stirred overnight at room temperature for 24 hours. The reaction mixture was then treated with 20 mL water, neutralized with saturated solution of NaHCO and extracted with DCM. The combined organic extracts were washed with brine and dried over Na SO . The crude product obtained by evaporation of the combined organic extracts was purified using column chromatography (230-400 mesh silica gel, neutralized with triethylamine) with 17% EtOAc:hexane as eluent. For achieving 95% pure desired OxM2 metabolite, it was further purified using column chromatography (100-200 mesh silica gel, neutralized with triethylamine) with 24% EtOAc:hexane as eluent to obtain the desired OxM2 metabolite (30 mg) as a white waxy solid. Results and Discussion Initially, Oxandrolone 1 was dehydrated as reported by Kratena to obtain a crude mixture of regioisomers 2. As reported, it was used without purification for epoxidation but some undesired products were obtained, when the rearrangement of isolated mixture of epoxides was performed using Kratena’s approach. So we modulated the reaction conditions and performed the dehydration by adding POCl at - 78 °C followed by overnight stirring at room temperature for 48 h to obtain 2 after recrystallization in DCM with hexane (Scheme 1, 50% yield). 2 was further subjected to epoxidation with portion wise addition of 1.2 equiv. of m-CPBA at 0 °C and then at room temperature stirring for 3 h in the absence of Na HPO /KH PO buffer. After 3 h, the formation of the desired mixture of epoxides 3 and 4 was observed along with unreacted reactant (Scheme 2) which was isolated using column chromatography. 3 2 4 3 3 2 4 3 2 4 2 4 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-082 Figure 1. Scheme 1 and Scheme 2 Mixture of 3 and 4 was then stirred for 24 h in glacial acetic acid ( Scheme 3) which resulted in the rearrangement of 3 to yield OxM2 which was purified using column chromatography. Figure 2. Scheme 3 1 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-083 H NMR (400 MHz, CDCl ): δ 4.24 (d, J = 10.7 Hz, 1H), 3.90 (d, J =10.7 Hz, 1H), 3.29 (dd, J =35.1, 10.5 Hz, 2H), 2.47 (dd, J =18.7, 5.8 Hz, 1H), 2.21-2.13 (m, 2H), 2.09-1.96 (m, 3H), 1.95-1.84 (m, 3H), 1.82-1.73 (m, 1H), 1.68-1.62 (m, 1H), 1.56-1.46 (m, 3H), 1.30-1.23 (m, 1H), 1.18-1.14 (m, 1H), 1.05 -0.96 (m, 2H), 0.91 (s, 3H), 0.89 (s, 3H). C NMR (101 MHz, CDCl ): δ 170.57, 139.98, 136.51, 81.00, 68.96, 51.60, 47.28, 40.21, 36.07, 34.68, 33.92, 33.72, 30.56, 30.41, 27.51, 22.85, 22.17, 21.68, 9.84. IR (KBr) ν cm : 3441.91, 2926.41, 2858.69, 1734.03, 1447.24, 1409.10, 1384.19, 1265.3, 1208.65, 1037.66, 917.9, 800.57, 665.84. Figure 3. H (CDCl , 400 MHz) and C NMR (CDCl , 101 MHz) spectrum of OxM2 Figure 4. HRMS analysis of OxM2 1 3 13 3 -1 1 3 13 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-084 Conclusions The isomeric metabolite of oxandrolone i.e. OxM2 has been successfully synthesized via revisiting Kratena's approach and fully characterized using H, C NMR and mass spectrometry to improve sports drug testing in respect to develop comprehensive analytical strategies for the effective detection of oxandrolone abuse. References 1. Federal Register / Vol. 88, No. 123 / Wednesday, June 28, 2023 / Notices https://www.regulations.gov/document/FDA-2023-N-2226-0001. 2. List_en_final_9_september_2022.pdf; https://www.wada-ama.org/sites/default/files/2022-09/2023 3. Kratena N, Stöger B, Weil M, Enev VS, Gärtner P. Synthesis of two epimeric long-term metabolites of oxandrolone. Tetrahedron Letters, 2017, 58;13:1316-1318. doi.org/10.1016/j.tetlet.2017.02.048. Acknowledgements Authors are thankful to CSIR-Indian Institute of Integrative Medicine, Canal Road Jammu for providing all the necessary research facilities and National Dope Testing Laboratory (NDTL), New Delhi for providing the funding. 1 13 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-085 Okano M, Akiyama K, Sato M, Mombayashi A Can current WADA-approved direct detection methods identify the use of GH analogue Somatrogon in doping control? Anti-Doping Laboratory LSI Medience Corporation, Tokyo, Japan Abstract Somatrogon is a long-acting human growth hormone (GH) with a C-terminal peptide domain derived from human chorionic gonadotropin (hCG). In 2022, somatrogon was added as an example of GH analogues on the S2.2.3 of the World Anti-Doping Agency (WADA) Prohibited List. The study purpose is to verify whether the detection of somatrogon use is possible using the current WADA–approved direct methods (i.e. GH isoform test and urinary hCG tests). An injectable solution of somatrogon (0.66 mg/kg) was subcutaneously injected to six healthy subjects (three males and three females). The detectability of somatrogon use was dependent on each GH isoform kit (Kit1 and Kit2). The Pit_kit1 showed a dose response, but the Rec_kit1 did not show that, resulting in a profile in which the Rec/Pit1 ratio decreased from the baseline. In contrast to Kit1, the Rec_kit2 showed post-dose response. The Rec/Pit2 ratio exceeded the WADA decision limit in all subjects. It was suggested that antigen-antibody interaction at the epitope of Rec_kit1 was disrupted by hCG β-subunit. Since Kit2 can effectively detect the use of somatrogon, it is recommended to perform the further GH biomarkers test in cases where Kit1 shows an abnormally low Rec/Pit1 ratio. Total hCG above 5 IU/L was detected in two of three male subjects after administration. On the other hand, the result of the intact hCG analysis was negative in all subjects as expected. This suggests that hCG β-subunit is excreted into urine as a metabolite/degradation product of somatrogon. Introduction The illicit use of human growth hormone (hGH) has been prohibited by the World Anti-Doping Agency (WADA)[1]. Somatrogon is a recombinant fusion glycoprotein composed of C-terminal peptides of the β- subunit of human chorionic gonadotropin (hCG) at positions 1-28, 220-247 and 248-275 and hGH at positions 29-219, which was designed/developed to have a long action compared with the conventional recombinant hGH [2]. In 2022, somatrogon was added as an example of GH analogues on the S2.2.3 of the WADA Prohibited List [1]. The study purpose is to verify whether the detection of somatrogon use is possible using the current WADA–approved direct methods (i.e. GH isoform test and urinary hCG tests). Experimental Materials Somatrogon (Ngenla ) was purchased from Pfizer Inc. (Tokyo, Japan). ® MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-086 GH isoform differential immunoassay The detection method was a quantitative analysis of recombinant GH (Rec) and pituitary GH (Pit) using immunoassay kits provided by CMZ-Assays GmbH (Berlin, Germany). The kits consist of two parts: Kit1 and Kit2. The assay was fully validated and performed according to the WADA technical document [3]. Determination of urinary hCG The initial testing procedure (ITP) was performed using Immulite 2000 (Siemens, IL, USA ), which is specific for total hCG. The limit-of-quantification (LOQ) was set at 1.00 IU/L. The confirmation analysis (CP) using an assay-specific α /β heterodimeric hCG was performed. A Roche Cobas e411 (Roche Diagnostics, In, USA) using the Elecsys hCG STAT assay. The LOQ for each test was 1.00 IU/L. Both assays were fully validated in accordance with WADA technical document – TD2021CGLH [4]. Cross-reactivity test The somatrogon formulation was prepared at 3 ng/mL and 30 ng/mL in sheep serum and analyzed to confirm cross-reactivity with each assay system. Human subjects An injectable solution of somatrogon (0.66 mg/kg) was subcutaneously injected to six healthy volunteers (three males: BB01-03, three females: BB04-06) at 9:00 am. The administration study was reviewed and approved by the Ethical Review Board of LSI Medience Corporation. The time courses of sample collection were -24, -20, -16, -12, 0 (pre-administration), 2, 4, 6, 8, 10, 12, 24, 28, 32, 36, 48, 52, 56, 60 and 96 h. Serum and urine samples collected were stored at -20°C until analysis. Results and Discussion Cross-reactivity As shown in Table 1, the Rec_kit2 showed a strong reactivity, but the Rec_kit1 did not. It was suggested that steric hindrance disrupts antigen-antibody interaction at the epitope of Rec_kit1. Neither the intact hCG kit nor the total hCG kit showed cross-reactivity with intact somatotrogon. GH isoform test (Kit1) As shown in Figure 1, the Pit_kit1 showed a dose response, but the Rec_kit1 did not show that, resulting in a profile in which the Rec/Pit1 ratio decreased from the baseline. It was consistent with the results of cross-reactivity test (Table 1). GH isoform test (Kit2) In contrast to Kit1, the Rec_kit2 showed post-dose response (Figure 2). The cross-reactivity test shown in Table 1 also indicated the full reactivity with the Rec_kit2. The Rec/Pit2 ratio exceeded the WADA decision limit (DL) in all 6 subjects (Table 2). In all female cases, it did not return to baseline even 96 h after administration, indicating a long-acting profile. In the future, it may be possible to detect even male by setting individual reference values based on athlete biological passport instead of DL judgment. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-087 Table 1. Cross-reactivity test; Rate (%) = (Measured value / Target conc.) x 100 Figure 1. GH isoform test result after administration of somatrogon (Kit1) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-088 hCG tests As shown in Table 2, total hCG above 5 IU/L was detected in 2 of 3 male subjects after administration (Table 2). On the other hand, the result of intact hCG measurement was negative in all subjects as expected. Cross-reactivity testing of the formulation showed no reactivity with either the Immulite2000 kit or the intact hCG kit (Table 1). This suggests that hCG β-subunit is excreted into urine as a metabolite/degradation product of somatrogon. Figure 2. GH isoform test result after administration of somatrogon (Kit2) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-089 Table 2. Detectability of GH isoform test (Kit2) and total hCG (urine) Conclusions The detectability of somatrogon use was dependent on each GH isoform kit. Since Kit2 can effectively detect the use of somatrogon, it is recommended to perform the further GH biomarkers test in cases where kit1 shows an abnormally low Rec/Pit ratio. As expected, an administration of somatrogon did not affect the CP for urinary intact hCG (no false positive of hCG), whereas the ITP for total hCG might detect some somatrogon fragments/metabolites having β-subunit of hCG. If ITP with Immulite2000 results in suspected total hCG in urine, it may be beneficial to recommend additional GH testing, although limited to male athletes, due to the possibility of using somatrogon. References 1. World Anti-Doping Agency. The 2023 Prohibited list. International Standard, Montreal (2023) https://www.wada-ama.org/sites/default/files/2023-05/2023list_en_final_9_september_2022.pdf (access date 21.08.2023) 2. Hershkovitz O, Bar-Ilan A, Guy R, Felikman Y, Moschcovich L, Hwa V, Rosenfeld RG, Fima E, Hart G. (2016) In vitro and in vivo characterization of MOD-4023, a long-acting carboxy-terminal peptide (CTP)-Modified human growth hormone. Mol Pharm. 13(2), 631-9 3. World Anti-Doping Agency. WADA Technical Document – TD2021GH, Montreal (2020) https://www.wada- ama.org/sites/default/files/2022-01/td2021gh_final_eng_0.pdf (access date 21.08.2023) 4. World Anti-Doping Agency. WADA Technical Document – TD2021CG/LH, Montreal (2020) https://www.wada- ama.org/sites/default/files/2022-01/td2021cg-lh_final_eng_0.pdf (access date 21.08.2023) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-090 Saavedra MJ , Sánchez-Wandelmer J , Polo ML , Rodríguez-de Francisco G , Muñoz G Bandmate™ as a good alternative to BlotCycler™ for erythropoietin detection by Western blot Madrid Anti Doping Laboratory, Health Institute Carlos III, Madrid, Spain ; Madrid Antidoping Laboratory, Tragsatec, Madrid, Spain Abstract Analysis of erythropoietin receptor agonists (ERAs) is crucial in antidoping control, being the Western blot analysis the most extended method for their detection [1,2]. The robustness in the performance of this kind of analytical technique is key to warrant fiability of results. Manual methods are often inconsistent and show high variability. Due to that, and also with the aim to increase productivity, most of the antidoping laboratories decided to use an automatized system for the analysis of erythropoietins by Western blot with BlotCycler as the system of choice [3,4]. This system allows the simultaneous incubation (first with the primary antibody and then with the secondary antibody or streptavidin-POD) of up to 4-midi gels, making possible to process up to 56 samples. However, and probably due to the need of working under cold conditions, this instrument does not seem to be robust enough for the requirements and use in antidoping laboratories, suffering an important deteriorating in very short time. In this work, the alternative system called Bandmate Western blot Processor (from Invitrogen) has been tested for automatization of ERAs analysis. The system has been tested in Madrid Doping Control Laboratory in the analysis of several urine samples spiked at 50% of MRPL of the more relevant erythropoietins (recombinant EPO, NESP, CERA and EPO-Fc). All the species were properly detected according TD2022EPO requirements [5]. Introduction Erythropoietin (EPO) is a glycoprotein hormone that promotes the production of red blood cells in response to cellular hypoxia. This biological function has made erythropoietin a good candidate for the development of therapeutic analogues. Both recombinant erythropoietins (EPOs) and erythropoietin receptor agonists (ERAs) have been extensively used for the treatment of several clinical conditions such as anemia or renal insufficiency. The stimulation of erythropoiesis and hence, of oxygen transport to tissues by these drugs has also made them attractive for athletes participating in endurance sports. EPOs and agents affecting erythropoiesis are therefore listed in section S2 of the World Anti-Doping Agency (WADA) Prohibited List and the number of adverse analytical findings (AFF) for these substances is always the highest in S2 Prohibited List. Exogenous EPO and ERAs differ from endogenous erythropoietin in molecular weight. Therefore, current methods to detect them are based on these variations, using polyacrylamide gel electrophoresis (SAR- PAGE) followed by immunoblotting, which is the only method accepted in the current WADA Technical Document for the analysis and reporting of EPOs and ERAs (TD2022EPO). Although both SDS and SAR- PAGE can be used for EPO detection, SAR-PAGE showed better sensitivity for PEGylated EPOs than SDS- 1 2 1 1 1 1 2 TM TM MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-091 PAGE [6] and it is currently the method of choice for analysis of EPOs in urine samples. Selectivity and sensitivity of this analysis are not only affected by the electrophoresis conditions, but also immunodetection is an important factor to be considered as well. Automatization of this process would certainly contribute to decrease variability due to manual intervention and contributes to intra and interlaboratory standardization of methodology to ensure reliable results. In this study, the Bandamate Western blot processor (Invitrogen) has been tested to further automate of the analysis of ERAs in urine samples as an alternative to using the BlotCycler , which was already reported to decrease membrane background and increase sensitivity [3] compared to manual processing. Here, we confirmed that by performing Western blot on the Bandmate . We were able to detect recombinant EPO, NESP, CERA and EPO-Fc in positive urine QCs, and in urine contaminated at both MRPL and 50% MRPL. In addition, the use of Bandmate offered several advantages such as a significant reduction in antibody volume without affecting detection sensitivity and low maintenance demands. Experimental Materials EPO-BRP (European Pharmacopoeia), NESP (Darbepoein alfa, Amgen), Mircera (CERA, Roche) Dynepo (DYN, Shire Pharmaceutical Ibérica S.L), and EPO-Fc (Prospec), ELISA Kit (Stemcell Technologies), EPO NuPAGE 10% Bis-Tris Midi gels (ThermoFisher), EPO clone AE7A5 (R&D Systems BAM2871), Streptavidin- POD Conjugate (Roche), SuperSignal West Femto (ThermoFisher). Samples In all cases two sets of three different urine samples from healthy volunteers were spiked with different types of ERAs at MRPL or below: BRP-EPO, Aranesp (NESP), Mircera (CERA) or EPO-Fc (ProSpec). The ERA- Mix standards are DYN-NESP-CERA in 0.1% casein in PBS. Urine ultrafiltration The samples were concentrated by ultrafiltration [2]. 15 mL of urine, 1.5 mL Tris- HCl (3.75 mM), and 200 µL complete solution (1 tablet / 2 mL water) were mixed and centrifuged (3900 rpm, 20 min). The supernatant was transferred to an Amicon Ultra 15 filter (30 kDa MWCO) and centrifuged (3900 rpm, 20 min). The retentate was washed (15 mL aprox. 50 mM Tris-HCl buffer and 200 µL complete and centrifuged (3900 rpm, 20 min). Finally, the retentate was transferred to an Amicon Ultra 0.5 filter (14000 rpm, 15 min) and recovered by reserved spinning at 1000 RCF for 2 min. Immunopurification All urine retentates were incubated on the ELISA plate overnight at 4 °C, next washed five times with 300 µL PBS, eluted with 15 µL of sample elution buffer and heated for 5 min at 95 ºC under shaking (650 rpm; ThermoMixer). Cool down, and centrifuge (3900 rpm, 1 min). SAR-PAGE, single-blotting, and detection Samples were loaded onto the electrophoretic gel, followed by SAR-PAGE electrophoresis at 125V / 25W / constant voltage for 3 h in a cold bath. Transfer was performed at 200 mA / 25 V for 45 min, the PVDF TM TM TM TM ® ® TM MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-092 membrane was blocked with 5% milk and incubations were performed overnight in a BlotCycler and Bandmate . Chemiluminescent signals were detected using a camera (Amersham Imager 680) and analyzed with GASepo software [7,8]. Results and Discussion Figure 1 and Figure 2 show the immunoblot of the two sets of samples performed in BlotCycler or Bandmate . In both cases, membranes were blocked offline and subsequently incubated with 20 mL of primary antibody (1:1000 dilution in 1% milk in PBS) for 12 hours and with 20 mL of Streptavidin (1:2000 dilution in 1% milk in PBS) for 5 hours in the automated Western blot systems evaluated. Every set of samples was composed of a set of positive quality controls (negative urine samples spiked with every type of the ERAs at MRPL). Two different stock solutions of EPO-Fc were used (1 and 2) in the positive controls and two different urine samples spiked with a mix of ERAs at MRPL and 50% MRPL. The comparison of the images processed using GASepo software showed that the results obtained in both systems were comparable in terms of sensitivity and background. Figure 1. Immunoblot of the indicated samples performed in BlotCycler TM TM TM TM TM MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-093 Figure 2. Immunoblot of the indicated samples performed in Bandmate In addition, in the case of Bandmate , it was also tested the online blocking of the membrane and the possibility of incubation using half the volume of the primary antibody (10 mL instead of 20 mL of 1:1000 in dilution in 1% milk in PBS). Figure 3 shows that the results for the positive quality control samples were similar to the ones obtained in the previous case. TM TM MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-094 Figure 3. Immunoblot of the indicated samples performed in Bandmate . The membrane was blocked in the instrument and subsequently incubated with 10 mL of primary antibody (1:1000 dilution in 1% milk in PBS) and with 10 mL of streptavidin (1:2000 dilution in 1% milk in PBS) Conclusions Bandmate™ acts as the BlotCycler™ for the detection of ERAs by immnunodetection. Its productivity is reduced compared to BlotCyclerrTM, as only 2 midi gels can be processed in the same run. However, it increases cost-effectiveness, as the volume of antibody solutions can be reduced, leading to significant savings in both primary and secondary antibodies or streptavidin. In addition, Bandmate™ easily allows the recovery of antibodies for reuse and, in contrast to BlotCycler™, Bandmate™ only requires a quick flush with water after use and very low maintenance operations. In summary, we conclude that Bandmate™ is a good alternative to BlotCycler™ for immunodetection of ERAs. References 1. Reichel C Detection of peptidic erythropoiesis-stimulating agents in sport. Br J Sports Med. 2014 May;48(10):842-7. doi: 10.1136/bjsports-2014-093555. Epub 2014 Mar 27. 2. Masquelier M, Heiland C, Bhuiyan H, Erlandsson G, Molin A, Magnus E. Optimization of ESAs detection in urine and blood: comparison of different immunopurification techniques. In: Thevis M, Geyer H, Mareck U (eds.) Recent Advances In Doping Analysis (26). Sport und Buch Strauß - Köln 2018, pp 58-65 3. Reichel C, Gmeiner G, Reihlen P, Thevis M, Schänzer W. SARCOSYL-PAGE: Optimized Protocols for the Separation and Immunological Detection of PEGylated Proteins. Methods Mol Biol. 2019;1855:131-149. doi: 10.1007/978-1-4939-8793-1_14 TM MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-095 4. Reichel C, Erceg D, Lorenc B, Scheiblhofer V, Farmer L, Zanitzer K, Geisendorfer T, Gmeiner G, Thevis M. Data from a microdosed recombinant human erythropoietin administration study applying the new biotinylated clone AE7A5 antibody and a further optimized sarcosyl polyacrylamide gel electrophoresis protocol. Drug Test Anal. 2023 Feb;15(2):163-172. doi: 10.1002/dta.2989. Epub 2021 Jan 21. PMID: 33450134. 5. WADA Technical Document TD EPO: Harmonization of Analysis and Reporting of EPO and other Erythropoietin Receptor Agonists (ERAs) by Polyacrylamide Gel Electrophoretic (PAGE) Analytical Methods. 6. Reihlen P, Blobel M, Kempkes R, Reichel C, Völker Schänzer E, Majer B, Schänzer W. Optimizing SAR-PAGE. Drug Test Anal. 2015 Nov-Dec;7(11-12):1014-6. doi: 10.1002/dta.1915. 7. Bajla I, Holländer I, Minichmayr M, Gmeiner G, Reichel Ch. GASepo-a software solution for quantitative analysis of digital images in Epo doping control. Comput Methods Programs Biomed. 2005 Dec;80(3):246- 70. doi: 10.1016/j.cmpb.2005.09.005 8. Bajla I, Holländer I, Gmeiner G, Reichel Ch. Quantitative analysis of images in erythropoietin doping control. Med Biol Eng Comput. 2005 May;43(3):403-9. doi: 10.1007/BF02345819. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-096 Mc Pherson Medina A, Fiallo Fernandez T, Martinez Brito D, Montes de Oca Porto R Stability of the biotinylated monoclonal antibody BAM2871 in the 1% non-fat milk solution Antidoping Laboratory, Havana, Cuba Abstract Clone AE7A5 anti-EPO antibody recognizes an epitope within the first 26 amino acids of the amino terminal chain of human and recombinant erythropoietin. Its biotinylated version (BAM2871) improves the detection with respect to its non-biotinylated version (MAB2871). This work evaluates the stability of BAM2871 incubation solution considering the conductivity of water and the origin of the non-fat milk. Three BAM2871 solutions were prepared with Millipore non-fat milk for microbiological use prepared with water MQ (conductivity values of 0.1 [S1] and 0.5 μS/cm [S2]) and Nestlé commercial non-fat milk prepared with water MQ (conductivity of 0.1 μS/cm [S3]). Gels with quality negative and positive controls in urine, at 50% MRPL, were analyzed simultaneously. Analyses were carried out from 0 to 35 days and the 1% non-fat milk solutions were conserved at 4 °C during this time. Values of pH were measured, and EPO-Fc, rEPO, CERA, NESP and Dynepo were tested. From day 15, turbidity and sediment were observed i n S3. Also, the pH started to increase from day 5 and, an excessive background on the membrane difficulted the interpretation of the bands. From day 20, a decrease in the intensity of the bands was observed when S2 was used and a stench was detected at day 25 after the preparation and the pH value started to increase before day 15. On the contrary, results obtained with S1 were repeatable up to day 30. Its pH value was stable until day 30. Introduction Clone AE7A5 anti-EPO antibody recognizes an epitope within the first 26 amino acids at the N-terminus of human urinary and human recombinant EPO [1]. Its biotinylated version (BAM2871, R&D Systems) has allowed compliance with the MRPL described in the TD2022EPO [2]. Usually, the antibody is added in amounts that exceed those necessary to guarantee a quantitative reaction [3]. Furthermore, it binds only to regions of the membrane where a related protein is found, therefore, after incubation, the remanent of the solution containing the antibody should be usable again. This work aimed to evaluate the stability of the BAM2871 monoclonal antibody incubation solution taking into consideration the conductivity of MQ water and the origin of the non-fat dry milk. Experimental BAM2871 was purchased from R&D System; Milk powder fat-free suitable for microbiology (1.15363, Millipore) was from SIGMA and Nestlé Carnation was from Nestle. The quality of all reagents and materials meets the criteria for immunoassays and are described in the SOP to detect and to confirm ESAs accredited by the requirements of ISO/IEC 17025 [4]. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-097 BAM2871 was prepared in three different non-fat milk solutions at a concentration of 1%. Non-fat milk were dissolved in PBS solution and the water used was microfiltrated to exclude the presence of bacteria. The solutions were identified as: Millipore 1.15363, water conductivity: 0.1 μS/cm Millipore 1.15363, water conductivity: 0.5 μS/cm Nestlé Carnation, water conductivity: 0.1 μS/cm Three gels were analyzed simultaneously every 5 days (from to 0 to 35 days) using the solutions S1, S2 and S3 with the monoclonal antibody BAM2871 to detect EPO-Fc, rEPO, CERA, NESP and Dynepo at 50% of the MRPL reference material and urine. The volume absolute of the bands obtained by the GASepo software [5] was plotted for each ESA and for each solution S1, S2 and S3. After the incubation was finished, solutions S1, S2 and S3 were conserved at 4 °C until the next analysis. Results and Discussion The results for each solution S1, S2 and S3 are represented in Figure 1. Figure1. Results of the band densities for EPO-Fc, rEPO, CERA, NESP and Dynepo during the experiment from day 0 – 35 for Millipore and water conductivity 0.1 μS/cm (S1); Millipore and water conductivity 0.5 μS/cm (S2); Nestlé Carnation and water conductivity 0.1 μS/cm (S3) and behavior of the pH values for S1, S2 and S3 (pH) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-098 The solution S3, prepared in the non-fat milk from Nestlé, started to increase values of pH from day fifth after the preparation. The maximum pH value of 8.5 was reached on day 20 and it was stable until the end of the experiment. After fifteen days, the solution showed turbidity, stench, and sediments, which provoked the presence of spots and unspecific bands that increased the background, making interpretation difficult and decreased the quality of the image. The most affected ESAs were NESP and EPO-Fc. Figure 2 shows an example of gels on days 5 and 25. Figure 2. GASepo image for the gels after 5 and 25 days incubating BAM2871 in a solution of non-fat milk Nestlé Carnation prepared with a water conductivity of 0.1 μS/cm (S3) The solution S2, containing BAM2871 dissolved in the non-fat milk from Millipore and water conductivity of 0.5 μS/cm, showed a decrease in the intensity of the bands from day 20 after preparation. A stench was detected at day 25 after the preparation and the pH value started to increase before day 15, showing a maximum value of pH of 8.3 after day 25. Although to a lesser extent than for S3, the presence of spots and unspecific bands that increased the background was also observed (Figure 3). The solution S1, containing BAM2871 solved in the non-fat milk from Millipore and water conductivity of 0.1 μS/cm, showed repeatable results from day 0 to the end of the experiments on day 35. Only the last measurement showed a decrease on the band intensities except for EPO-Fc, that showed a decreased intensity after day 25. The pH of the solution was also stable and until day 35 when the value increased up to 8, and no sediments were observed. As Figure 4 shows, also the quality of the image was stable during all the experiment. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-099 Figure 3. GASepo image for the gels after 5 and 30 days incubating BAM2871 in a solution of non-fat milk Millipore prepared with a water conductivity of 0.5 μS/cm (S2) Figure 4. GASepo image for the gels after 5 and 35 days incubating BAM2871 in a solution of non-fat milk Millipore prepared with a water conductivity of 0.1 μS/cm (S1) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0100 Conclusions The stability of the solutions of non-fat milk used in the incubations with BAM2871 to detect ESAs is not the same depending on the water quality. The preparations of solutions of Millipore milk in water with a conductivity of 0.1 μS/cm showed no alterations of the image background. It seemed to be stable at least for 30 days conserved at 4 °C. On the contrary, preparations of non-fat milk with water showing higher levels of conductivity favoured the presence of sediments, stench, elevation of pH value, and presence of spots and/or unspecific, that can make image interpretation difficult. References 1. W. Jelkmann. Erythropoiesis Stimulating Agents and Techniques: A Challenge for Doping Analysts. Curr. Med. Chem., 2009, 16, 1236–1247. 2. World Anti-Doping Agency. WADA Technical Document – TD2022EPO. Harmonization of analysis and reporting of erythropoietin (EPO) and other EPO-receptor agonists (ERAs) by Polyacrylamide gel electrophoretic (PAGE) Analytical Methods. Version 1.0, January 2022. 3. R. F. Ochoa-Azze. Bases Metodológicas para la evaluación de Anticuerpos en Ensayos Clínicos de Vacunas Mediante Técnicas Inmunoenzimáticas, Finlay Ediciones, Ciudad de La Habana, 2008. 4. ISO/IEC 17025:2017 General requirements for the competence of testing and calibration laboratories. 5. Bajlaa, I., Holländer, I., Minichmayr, M., Gmeiner, G., Reichel, Ch. GASepo - a software solution for quantitative analysis of digital images in Epo doping control. Computer Methods and Programs in Biomedicine 80, 246-270, 2005. doi:10.1016/j.cmpb.2005.09. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0101 da Costa S, Ruivo J, Casimiro T, Pereira L, Salema B Long-term stability study of erythropoietin and erythropoietin receptor agonists detection in dried blood spots and comparison of different absorbent sample supports Laboratório de Análises de Dopagem, INSA, Lisbon, Portugal Abstract The use of DBS for the analysis of doping substances has been increasing in anti-doping field. For that reason, the present study aims to understand whether capillary blood in DBS is a viable complement [1] for the detection of EPO and ERAs. This study involved 30 volunteers, from whom capillary blood and venous blood samples were taken. Urine and blood samples were collected from 4 volunteers. DBS samples were immunopurified (Gel Purification Kit – MAIIA nº 1430) prior to sodium N-lauroysarcosinate polyacrylamide gel electrophoresis (SAR-PAGE) and Western Blot using the biotinylated monoclonal mouse anti-human EPO clone AE7A5 (R&D Systems BAM2871). Limits of detection (LODs) were determined for the main ERAs: BRP (3.15 mUI/mL), NESP (3.15 pg/mL), EPO-Fc (31 pg/mL) and CERA (31.3 pg/mL). The comparison study of 6 different absorbent sample supports was also carried out to assess EPO and ERAs recovery. DBS Collection Card for erythropoietin analysis from MAIIA proved to be the support that shows best results for both endogenous EPO and spiked ERAs. Stability tests performed on DBS, serum and urine samples, stored at different temperatures (room temperature (RT), 4 °C and ‑20 °C showed promising results for DBS. DBS samples were re-analyzed 1 year after collection and all ERAs were present. This study reinforces the utility of DBS as a possible complementary biological matrix for the detection of EPO and ERAs especially when long transport times and limited storage capacities are expected. Introduction Requirements for the analysis of EPO and ERAs are described, in TD2022EPO, for urine and serum/plasma [2]. WADA published, in 2021, a specific document for the analysis of DBS, as a result of its increasing applicability, outlined in the most recent Testing Figures Report [3]. Since it is currently possible to perform capillary blood analysis by DBS for ERAs detection, it has become essential to carry out more studies to understand if DBS is a viable complement matrix [1] for the detection of EPO and ERAs. Several parameters were tested, being the stability study and comparison of different absorbent sample supports the most prominent. Useful information was gathered for collection, storage and stability time and temperatures. Experimental Subjects Capillary finger blood and venous blood samples (EDTA tube) were collected from 30 healthy subjects MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0102 and applied on the 903 Protein Saver card (air dried for at least 1h). From 4 of the 30 volunteers, urine and serum were also collected (samples used on the stability test). Questionnaire 3 questions related to pain level and collection preference among the 3 matrices: urine, venous or capillary blood were asked after the collections. Procedure 60 µL of capillary blood on DBS were immunopurified using Gel Purification Kit – MAIIA nº1430. Retentate was concentrated in Ultracell YM-30 tubes prior to sodium N-lauroysarcosinate polyacrylamide gel electrophoresis (SAR-PAGE) and Western Blot using the biotinylated monoclonal mouse anti-human EPO clone AE7A5 (R&D Systems BAM2871) and Streptavidin-HRP (Biospa) using Blotcycler during about 19 hours. Detection was carried out using the LAS-4000 camera (Fujifilm) and the images were processed using the Gasepo software. For urine, 10 mL was immunopurified using StemCell ELISA and the retentate was concentrated in Amicon Ultra-0.5 (30 K). For serum, 500 µL were immunopurified using EPO Purification Kit - MAIIA nº1390 and for both matrices the procedure was the same as described above for DBS. Selectivity and Limits of Detection (LODs) 60 µL of capillary blood from 30 volunteers were analyzed for selectivity and seven different concentrations of the 4 main ERAs (BRP, NESP, EPO-Fc and CERA) were tested to establish the LOD. Comparison of different absorbent sample supports Comparison of 6 brands of filter paper/DBS/Volumetric Absorptive Microsampling (VAMS) to understand the impact on recovery of bEPO and main ERAs. Filter paper: Whatman #1 and Macherey-Nagel DBS: 903 Protein Saver Card (Whatman), DBS Collection Card for EPO analysis (MAIIA) and Microsampling HemaXis DB10 Kits (DBS Systems SA) VAMS: Neoteryx Long-term stability study Carried out with capillary blood in DBS, serum and urine samples, stored at different temperatures: room temperature (RT), 4 °C and -20 °C and analyzed after 1, 2 and 3 months. DBS were also re-analyzed 1 year after collection. Results and Discussion Questionnaire Urine is the 1 option by 73.3% volunteers and capillary blood is the 2 most chosen option by 63.3%. On a pain scale of 1 to 10, the average pain rating for venous blood collection was 3.8 (mild-to-moderate pain), while for capillary blood was 2.3 (no pain-mild). Selectivity and limits of detection (LODs) Currently, the requirements for the analysis of EPO and ERAs in anti-doping laboratories are only st nd MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0103 described for urine and serum/plasma. It was possible to observe all bEPO bands with adequate intensities in the 30 samples analyzed with 60 µL. Compared to the MRPL for serum/plasma defined in TD2022EPO, values below 50% MRPL were achieved for rEPO (BRP) and NESP, and values around 125% MRPL were detected for CERA and EPO-Fc, resulting in limits higher than those defined for serum/plasma (Table 1). Table 1. Estimated limits of detection (LODs) for main ERAs in DBS Comparison of different absorbent sample supports Figure 1 shows visible bEPO on all supports with minimal differences in relative band volumes. The results showed that for filter paper #1 (Whatman) and DBS Collection Card for EPO (MAIIA) more intense bands were obtained when compared to other supports (Figure 1). Figure 1. Comparison of 6 different absorbent sample supports Long-term stability study After 1 month, degradation can be observed in serum and urine samples at all temperatures, although in the DBS (Figure 2) promising results were obtained as it is the only one that allows the preservation of all ERAs after 2 months at all storage temperatures analyzed. Similar results can be observed after 3 months of storage. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0104 Figure 3 shows DBS samples analyzed after 12 months demonstrating that some steps allowed to optimize the extraction of ERAs. After this period, for the 3 temperatures studied, samples showed greater intensity when stored at -20 °C. Figure 2. Stability study after 2 months storage at room temperature, 4 °C and ?20 °C of capillary blood in DBS, serum and urine spiked with the main ERAs at the same concentration MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0105 Figure 3. Stability study after 12 months storage at room temperature, 4 °C and -20 °C of capillary blood in DBS spiked with the main ERAs Conclusions According to the LODs obtained, an optimization of the method should be made to decrease the values for CERA and EPO-Fc, upon a possible scenario of the establishment os specific MRPL for capillary blood on DBS in TDEPO. The analysis of the three matrices stored at different temperatures (RT, 4 °C, ‑20 °C) after 3 months, allowed to demonstrate the ability of preservation of all ERAs in DBS, even after 12 months, when compared to serum and urine. The DBS Collection Card for EPO analysis (MAIIA) proved to be the one with the best results. This study confirms the usefulness of DBS as a possible complementary biological matrix for the detection of EPO and ERAs in doping control analysis. References 1. Heiland CE, Ericsson M, Pohanka A, Ekström L, Marchand A. (2022) Optimizing detection of erythropoietin receptor agonists from dried blood spots for anti-doping application. Drug Test Anal. Aug;14(8):1377-1386. 2. World Anti-Doping Agency. Technical Document. TD2022EPO - Harmonization of analysis and reporting of Erythropoietin (EPO) and other EPO-Receptor Agonists (ERAs) by polyacrylamide gel electrophoretic (PAGE) analytical methods (2022); https://www.wada-ama.org/sites/default/files/2023- 02/td2022epo_v1.0_final_eng.pdf (acess date 31.08.2023) 3. World Anti-Doping Agency. Testing Figures Report (2021); https://www.wada- ama.org/sites/default/files/2023-01/2021_anti-doping_testing_figures_en.pdf (acess date 31.08.2023) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0106 Stojanovic B, Pecher D, Geisendorfer T, Tsivou M, Gmeiner G Dried blood spot microsampling as a suitable tool to monitor diuretics misuse in sport Doping Control Laboratory, Seibersdorf Labor GmbH, Seibersdorf, Austria Abstract In recent years, the implementation of dried blood spot (DBS) microsampling increased significantly in the field of antidoping. This is supported by the World Antidoping Agency (WADA) TD2023DBS [1] as well as numerous publications [2,3]. However, the high number of substances in the WADA Prohibited List [4], their different physico-chemical properties, the small sampling volume of available devices as well as the complex matrix represent a challenge when developing comprehensive analytical methods for DBS samples. In the present study, the suitability of non-volumetric (DBS cards) and volumetric dried blood spotting devices (TASSO and volumetric absorptive microsampling (VAMS)) for selected substances from S5. class of the WADA Prohibited List was investigated. For this purpose, a sensitive liquid chromatography high resolution mass spectrometry (LC-HRMS) method was developed, validated in accordance with the current International Standard for Laboratories (ISL) [5] and applied to real sample analysis. As proof-of- concept, samples obtained from 2 healthy volunteers after administration of a single dose of a two- component medication (amiloride 2.5 mg/chlortalidone 25 mg) were used. Amiloride was identified in samples collected up to 24 h after administration, while its presence in the 48 h samples was confirmed in TASSO and VAMS devices only. Chlortalidone was identified in all collected samples. Consequently, dried blood microsampling is proved to be a suitable tool for the monitoring of diuretic misuse in sport. Introduction DBS microsampling is a complementary technique to traditional sample collection methods, which is recently gaining popularity and significance in the doping community [2,3,6]. The well-known benefits of a small amount of blood dried on a collection device forces the development of new methods. However, to perform the two-step protocol in doping analysis – initial testing procedure (ITP) and confirmation procedure (CP) – using one device can be challenging. Therefore, multi-component CP for substance groups such as diuretics may be a suitable strategy to cope with this challenge. In the current study, a protocol for multi-component LC-HRMS analysis for selected diuretics in 3 types of DBS collection devices was developed and applied to samples collected after administration of selected diuretics (amiloride and chlortalidone). Experimental Sample preparation for ITP and CP Fortified blood (20 µL) was spotted onto the DBS cards (FTA DMPK cards, Merck, Germany), VAMS® MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0107 (20 mL Mitra Clamshell, Neoteryx, The Netherlands) and TASSO-M20 (Tasso, Inc. USA) and allowed to dry for minimum 2 h. A 6 mm punch from DBS cards was taken from the center of the spot. For ITP: Substances were extracted from the spot using the extraction solvent - mixture of methanol- acetonitrile (4:1 v/v; 2 mL) containing ISTD. The samples were shaken in a water bath (50 °C) for 30 minutes and centrifuged at 2100 rpm for 5 minutes. Afterwards, 5 µL of 3 M HCl was added. The solvent was transferred into a glass test tube and evaporated. Samples were reconstituted in 100 µL of a mixture of methanol-water (3:7 v/v) and injected into the LC/MS/MS system. For CP: Methanol-acetonitrile (1:1 v/v;1 mL) containing ISTD was used as extraction solvent. The extraction protocol was the same as for ITP but only without adding 3 M HCl. Finally, after reconstitution in a mixture of methanol-water (3:7 v/v), samples were injected into the LC/HRMS system. Chromatographic conditions are presented in Table 1. Table 1. Chromatographic conditions Administration study As proof-of-concept, samples obtained from 2 healthy volunteers (1 male, 1 female) after oral administration of a single dose of a two-component medication (amiloride 2.5 mg/chlortalidone 25 mg) were used. Samples were collected at 0, 2, 4, 6, 8, 10, 12, 24, and 48 h after administration, dried for a minimum of 2 h at room temperature, and stored at 4 °C until analysis. Samples were initially analyzed using the ITP for DBS analysis. Subsequently, only the samples in which the administered diuretics were detected, were subjected to the CP analysis. Results and Discussion In the current study, an LC-HRMS method suitable for the confirmation of 20 substances from substance group S5 of the WADA Prohibited List was developed. The method was successfully validated for all 3 tested microsampling devices (DBS, VAMS, and TASSO). The method proved to be selective, robust, and no carry over was observed. The obtained limits of identification (LOI) of this method for each device are presented in Table 2. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0108 Table 2. Performance parameters of the developed LC/HRMS confirmation method In this administration study, amiloride was detected in samples collected up to 24 h post administration, while its presence in the 48 h sample was confirmed in the TASSO and VAMS devices only. Chlortalidone was identified in all collected samples. The estimated concentrations of amiloride and chlortalidone obtained when using the TASSO device are depicted in Table 3. The c was obtained 4 h post administration for amiloride and 4-10 h post administration for chlortalidone. m a x MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0109 Table 3. Estimated concentrations 0-48 h after administration for amiloride (top) and chlortalidone (bottom) The results obtained for amiloride are comparable to the already published data [7]. Chlorthalidone is concentrated in erythrocytes which resulted in a concentration in blood 7-10 times higher compared to plasma as reported in a previous study [8]. Data gathered in the present study could assist in definition of the minimal required performance level (MRPL) for diuretics in DBS, but an administration study with more volunteers should also be considered. Conclusions Dried blood microsampling proved to be a suitable tool for the monitoring of diuretics misuse in sport. The capability of non-volumetric and volumetric devices for selected diuretic analysis was comparable and the obtained results have shown good compliance. Results obtained for samples collected in an administration study confirmed the applicability of the tested devices for the analysis of diuretics, but also confirmed that the developed and validated multi-component LC-HRMS confirmation method is fit for purpose. References 1. World Antidoping Agency. DRIED BLOOD SPOTS (DBS) FOR DOPING CONTROL TD2023DBS | World Anti- Doping Agency (wada-ama.org) (access date 27.01.2023) 2. Thevis M, Walpurgis K, Thomas A. (2023) DropWise: current role and future perspectives of dried blood spots (DBS), blood microsampling, and their analysis in sports drug testing. Crit Rev Clin Lab Sci. 60 (1) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0110 https://doi.org/10.1080/10408363.2022.2103085 3. Mazzarino M, Di Costanyo L, Comunita F, Stacchini C, Torre X, Botre F. (2022) UHPLC−HRMS Method for the Simultaneous Screening of 235 Drugs in Capillary Blood for Doping Control Purpose: Comparative Evaluation of Volumetric and Non-volumetric Dried Blood Spotting Devices. ACS Omega 7, 31845-31868. 4. World Antidoping Agency. The 2023 Prohibited list. International Standard, 2023 Prohibited List | World Anti-Doping Agency (wada-ama.org) (access date 27.01.2023) 5. World Antidoping Agency. International Standard for Laboratories 2021, https://www.wada- ama.org/sites/default/files/resurces/files/isl_2021.pdf (access date 14.07.2022) 6. Garzinsky A-M, Thomas A, Guddat S, Görgens C, Dib J, Thevis M. (2023) Dried blood spots for doping controls – Development of a comprehensive initial testing procedure with fully automated sample preparation. Biomed Chrom. DOI: 10.1002/bmc.5633 7. Sun Q, Sever P. (2020) Amiloride: a review Journal of the Renin-Angiotensin-Aldosterone System (1-9) doi.org/10.1177/1470320320975893 8. Carter BL, Ernst ME, Cohen JD (2004) Hydrochlorothiazide Versus Chlorthalidone Hypertension. 43:4–9 DOI: 10.1161/01.HYP.0000103632.19915.0E MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0111 Yan X, Yan S, Chang W, Zhang L, Wang Z, Yang S A comparative study of DFT calculated and experimental UV/Visible spectra for the E/Z testosterone ester oximes at trace levels Beijing Anti-Doping Laboratory, Beijing Sport University, Beijing, People's Republic of China Abstract Dried blood spot (DBS) is a new alternative matrix used in 2022 Beijing Winter Olympics and Paralympics. It can realize the unequivocally distinguish the exogenous testosterone preparations without the aid of GC-C-IRMS confirmation. The detective method used methoxylamine reacting with the testosterone undecanoate, which formed the E and Z isomers. However, the absolute configurations of the oxime products have not been elucidated. This paper used a density function theory (DFT) calculation method to get the theoretical UV spectra data. The basis set was B3LYP, 6-31G (d,p) for the geometry optimizations. The Polarizable Continuum Model (PCM) was used. The actual UV spectra were determined by HPLC with DAD detector. The wavelength was set from 190 nm to 400 nm. By comparison the results, the E or Z configuration of testosterone undecanoate oxime can be deduced at nanogram levels (ng/mL). Introduction The prohibition history of testosterone is dated back to 1976 [1]. Dried blood spot (DBS) is recently a new alternative matrix used in anti-doping analysis [2-4]. It can distinguish exogenous testosterone preparations without the aid of GC-C-IRMS. Thus, it is recognized as a powerful technique introduced by WADA and played an important role in the 2020 Tokyo Olympics [5], 2022 Beijing Winter Olympics and Paralympics [6]. The method used methoxylamine reagent reacting with the eight testosterone esters, which all formed the E and Z isomers. However, the absolute configurations of the oxime products have not been elucidated. This paper focuses on using a density function theory (DFT) approach combined with experimental UV visible spectra to tentatively solve this problem at nanogram levels (ng/mL). Experimental Reagents and Materials Testosterone acetate (R1), testosterone propionate (R2), testosterone benzoate (R3), testosterone phenylpropionate (R4), testosterone cypionate (R5), testosterone enanthate (R6), testosterone decanoate (R7) and testosterone undecanoate (R8) are purchased from Sigma-Aldrich. The chemical structures are shown in Figure 1. Methanol, tert-butyl methyl ether and formic acid are HPLC grade, and the D3-testosterone is purchased from Sigma-Aldrich. Ammonium formate is purchased from Fluca Inc. Sodium bicarbonate, sodium carbonate and sodium hydroxide were purchased from Sinopharm Reagent Co., Ltd. Methoxylamine is purchased from Sigma-Aldrich. DMPK-C filter card was purchased from GE Healthcare company (USA). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0112 Figure 1. The chemical structures of the testosterone esters: testosterone acetate (R1), testosterone propionate (R2), testosterone benzoate (R3), testosterone phenylpropionate (R4), testosterone cypionate (R5), testosterone enanthate (R6), testosterone decanoate (R7) and testosterone undecanoate (R8) Sample preparation A mixture of the eight testosterone esters are diluted with methanol at a concentrations of 2.5 ng/μL. 50 μL of the standard solution was aliquoted, and 100 μL 10 mM methoxylamine methanol solution was added. The derivatization was conducted in capped glass tubes heated for 30 mins at 80 °C. After centrifugation at 4000 rpm for 3 mins, the residues were transferred to vials for injection. A volume of 10 μL was injected. Mass spectrometry and HPLC-UV spectrum For DBS samples, an ESI-LC-HRMS analysis was performed on a Vanquish Q Exactive HF orbitrap mass spectrometer (Thermofisher, Bremen, Germany). The UV-visible spectrum was determined on an Agilent 1200 HPLC (Agilent, USA). The conditions and parameters are listed in Table 1. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0113 Table 1. Instruments parameters used in Q Exactive HRMS and Agilent 1200 HPLC DFT theoretical calculation The theoretical calculation was performed using Gaussian 16 software (Gaussian Inc, Wallingford, CT, USA). The geometries of E/Z testosterone oxime isomers’ ground and excited states, excitation energy and UV absorption spectra were calculated by using the DFT functional, B3LYP, 6-31G (d,p) basis set [7- 8]. For the atoms in geometry optimizations, the Polarizable Continuum Model (PCM) was used [9]. Vertical excitation energy calculation was carried out by using time-dependent density functional theory (TD-DFT). Results and Discussion The testosterone oxime isomers have characterizations of double peaks in the extraction chromatograph by mass spectrometry. After the process of heating at 80 °C, one low abundance and one high abundance peak with 1:3 (peak area ratio) were observed. The E/Z isomers have the same mass spectra and ion fragments for each testosterone ester oxime. There is no difference observed from the mass spectra. Main fragment ions are m/z of 126.0913 and 138.0913, with mass accuracy error less than 3 ppm. The mass spectrum of testosterone undecanoate is shown in Figure 2. The UV-HPLC is improved by using acetonitrile as the B mobile phase, and a baseline separation is achieved, which is better than methanol as B phase. For testosterone undecanoate oxime isomers, the MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0114 retention times are 19.339 and 19.866 min, respectively. The UV spectrum of the peaks showed the absorption wavelength at 258 nm for the peak RT = 19.339, and 248 nm for the RT = 19.866, respectively (Figure 3). The theoretical calculation of testosterone undecanoate E isomer shows that the first excitation wavelength is 252.2 nm, while for the Z isomer it is 258 nm. The differences or errors are 4.2 nm and 0.4 nm. Therefore, the E and Z isomers are tentatively identified by comparing the theoretical calculations with the actual UV spectrum determinations. Figure 2. Mass spectra of testosterone undecanoate oxime: mass spectrum of the peak at RT = 12.66 min (top); mass spectrum of the peak at RT = 12.79 min (bottom) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0115 Figure 3. UV-HPLC analysis of the testosterone undecanoate oxime E/Z isomers. The upper chromatograph shows the UV spectra of the low abundance peak at RT = 19.33 min Conclusions The E or Z isomer of testosterone undecanoate oxime can be separated by HPLC and the UV spectrum determined. A DFT calculation using each previously determined isomer can give the theoretical results of the UV-Vis spectrum. And the E or Z isomer can be assigned based on these spectrometric data. Therefore, this method can tentatively distinguish betrween the E or Z isomers of the oxime products. References 1. World Anti-Doping Agency. The 2023 Prohibited List. International Standard, Montreal (2023) https://www.wada-ama.org/sites/default/files/2023-05/2023list_en_final_9_september_2022.pdf 2. Thomas A, Geyer H, Guddat S, et al. (2011) Dried blood spots (DBS) for doping control analysis. Drug Test Anal. 3(11-12), 806-813 3. Tretzel L, Thomas A, Geyer H, et al. (2014) Use of dried blood spots in doping control analysis of anabolic steroid esters. J Pharm Biomed Anal. 96(5), 21-30 4. Forsdahl G, Erceg D, Geisendorfer T, et al. (2015) Detection of testosterone esters in blood. Drug Test Anal. 7(11-12), 983-989 5. Okano M, Ikekita A, Sato M, et al.(2022) Doping control analyses during the Tokyo 2020 Olympic and Paralympic Games. Drug Test Anal. 14(11-12), 1836-1852 6. Wang Y, Zhang L, Xing Y. (2022) Operation of the anti-doping laboratory for the Beijing 2022 Olympic and Paralympic Winter Games. Drug Test Anal. 14(11-12), 1853-1863 7. Tirado-Rives J, Jorgensen W. (2008) Performance of B3LYP density functional methods for a large set of organic molecules. J Chem Theory Comput. 4(2), 297-306 8. Frisch M, Trucks G, Schlegel H, et al. Gaussian 16, Gaussian, Inc., Wallingford CT, 2016. 9. Cossi M, Barone V, Cammi R, Tomasi J. (1996) Ab initio study of solvated molecules: a new implementation of the polarizable continuum model. Chemical Physics Letters. 255(4-6), 327-335 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0116 Chang W, Yan X, Yan S, Liu Y, Yang S Pilot study of small peptide analysis in dried blood spots using automated SPE and UPLC-HRMS without DMSO Beijing Anti-Doping Laboratory, Beijing Sport University, Beijing, People's Republic of China Abstract Small peptides are banned by WADA in sports because they can either increase the performance or be used as masking agents. Compared to the mature methods used for small peptide analysis in human urine, the detection protocols for small peptides in dried blood spots (DBS) have not yet been carried out in many anti-doping laboratories. In this pilot study, an optimized automated SPE pretreatment and LC- HRMS method is presented to detect 48 types of small peptides in DBS without the assistance of DMSO. Validation was performed including selectivity, LOD, extraction stability and carryover. The data showed that the method herein is selective for small peptides without obvious interference at the retention times of target analytes. All peptide analytes could be detected at a concentration of 20 ng/mL. In addition, carryover was not observed at a concentration of 80 ng/mL, and all small peptide analytes were detectable at least for up to 72 h in the autosampler. It is believed that the method has the potential to reach LODs below 20 ng/mL after future formal evaluation. It could be deduced that, even without DMSO enhancing the ionization efficiency, this method can still be used to detect small peptides in DBS samples, which is helpful to further realize the simultaneous detection of small peptides and small molecular compounds in a single injection. Introduction While the detection methods for small peptides in human urine are well performed in routine analysis [1- 4], the mature protocols for the dried blood spots (DBS) detection have not carried out in many anti- doping laboratories. It is noted that several clinical trials have investigated the administration of small peptides, and these studies have reported peak concentrations of certain peptide analytes in blood samples exceeding 20 ng/mL [5-6]. Thus, Lange et. al reported a promising method to screen 46 small peptide analytes with the assistance of DMSO, and the LODs range between 0.5 and 20 ng/mL [7]. However, DMSO will cause adverse influence when small molecular compounds are detected together with peptide analytes. This pilot work thus presents an optimized automated SPE pretreatment and LC- HRMS method to detect 48 types of small peptides in DBS without the aid of DMSO, which is likely to provide prospective LOD levels below 20 ng/mL. Experimental Sample preparation Stock solutions of small peptide analytes and their mixed solution of 1 mg/mL were prepared in 1% formic acid in acetonitrile/water (v/v= 1/1) mixture in low-binding microtubes and stored in the freezer. A MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0117 bovine insulin solution of 50 μg/mL was prepared with the above-mentioned solvent mixture used to prepare the stock solutions, and a working solution of the peptide analytes at 0.1 ng/μL was prepared in the bovine insulin solution. A shell device developed by CHINADA was used to obtain triplicate 20 μL spots on filter paper once (Figure 1)[8]. To check LOD levels and extraction stability for the peptide analytes, the pre-extraction DBS samples were prepared by adding 4 μL of the working solution onto the center of a DBS. In addition, 16 μL of the working solution was added onto the center of the DBS to check carryover. Figure 1. DBS device developed by CHINADA (left); triplicate 20 μL spots are collected from a drop of fingertip blood (right) Sample extraction Samples were extracted by adding 0.4 mL of bovine insulin solution, then 0.8 mL water and 20 μL IS solution. After vortexing, 1.2 mL of phosphate buffer (0.2 M, pH 6) was added, followed by centrifuging at 10,000 rpm for 10 mins. Afterwards, 2 mL of the supernatant was loaded onto an Oasis WCX cartridge (30 mg, 1 cc) previously conditioned with 1 mL of methanol and 1 mL of water by Biotage Automated Sample Preparation System. The cartridge was washed with 1 mL water and 0.5 mL methanol and then analytes were eluted with 1 mL methanol containing 5% formic acid. After evaporation, the dry residues were dissolved with 100 μL of 2% acetic acid and injected into the UPLC-HRMS. UPLC-HRMS parameters are shown in Table 1, the ion transitions and the corresponding normalized collision energy (NCE) have been listed in Table 2. The injection volume was 10 μL. ® MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0118 Table 1. Dionex Ultimate 3000 LC/Q Exactive Plus mass spectrometer parameters for the detection of small peptide analytes Table 2. Retention times, monitored ion transitions and normalized collision energies for the detection of small peptide analytes Results and Discussion The analytical method of small peptides in DBS samples needs to eliminate most of the interferences in the matrix and get LODs at an appropriate level. As these are non-threshold substances, the developed method for small peptides in DBS was validated with regard to the following parameters: selectivity, limit MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0119 of detection (LOD), carryover and sample extract stability in the autosampler. For selectivity, ten blank DBS samples were analyzed to distinguish analytes from matrix compounds present in the DBS samples. There was no obvious interference at the retention times for each analyte, indicating that the presented method herein is selective for the small peptides. For the LODs, ten above-mentioned blank DBS samples, fortified with 4 μL of working solution of the small peptides at a level of 20 ng/mL, were analyzed. As shown in Figure 2, all of the peptide peaks at this level could exhibit a signal to noise ratio significantly higher than 3. The ion transitions and the corresponding normalized collision energies (NCE) for all analytes are listed in Table 2. Furthermore, carryover was evaluated with the consecutive analysis of a DBS sample fortified with peptide analytes at 80 ng/mL and two following blank DBS samples. No carryover was observed. In addition, ten above-mentioned urine samples for checking LODs were re-analyzed on the instrument autosampler after 72 h, and all small peptide analytes were still detectable. Conclusions This study presents a detection method for small peptides in DBS including sample extraction and UPLC- HRMS detection. All peptide analytes could be detected at concentrations of 20 ng/mL, and the method has the potential to reach LODs below 20 ng/mL after future formal evaluation. The performance of the presented method is comparable with that reported by Lange et.al [7], which is possibly caused by the optimized sample extraction to increase recoveries of the target analytes. The validation data show that our method can be used for the detection of small peptides in DBS samples without DMSO in the UPLC mobile phase, which is helpful to further realize the simultaneous detection of small peptides and small molecular compounds in one injection. Considering that DBS analysis is already a specific test, the com‐ bined analysis of small peptides and other low molecular substances is meaningful, as analyzing DBS for multiple prohibited substance categories would significantly increase the cost of testing. References 1. Görgens C, Guddat S, Thomas A, Thevis M. (2018) Recent improvements in sports drug testing concerning the initial testing for peptidic drugs ( < 2 kDa) - sample preparation, mass spectrometric detection, and data review. Drug Test Anal. 10(11-12), 1755-1760 2. Coppieters G, Deventer K, Van Eenoo P and Judák P. (2021) Combining direct urinary injection with automated filtration and nanoflow LC-MS for the confirmatory analysis of doping-relevant small peptide hormones. J Chromatogr B: Anal Technol Biomed Life Sci. 1179, 122842 3. Jing J, Tian T, Wang Y, Xu X, Shan Y. (2022) Multi-analyte screening of small peptides by alkaline pre- activated solid phase extraction coupled with liquid chromatography-high resolution mass spectrometry in doping controls. J Chromatogr A. 1676, 463272 4. Chang W, He G, Yan K, Wang Z, Zhang Y, Dong T, Liu Y, Zhang L, Hong L. (2021) Doping control analysis of small peptides in human urine using LC-HRMS with parallel reaction monitoring mode: screening and confirmation. Anal Methods.13(48), 5838-5850 5. Roumi M, Lenaerts V, Boutignon F, Wuthrich P, Deghenghi R, Bellemare M, Adam A, Ong H. (1995) Radioimmunoassay for hexarelin, a peptidic growth hormone secretagogue, and its pharmacokinetic studies. Peptides. 16(7), 1301-1306 6. Zdravkovic M, Olsen A, Christiansen T, Schulz R, Taub M, Thomsen M, Rasmussen M, Ilondo M. (2003) A clinical study investigating the pharmacokinetic interaction between NN703 (tabimorelin), a potential inhibitor of CYP3A4 activity, and midazolam, a CYP3A4 substrate. Eur J Clin Pharmacol. 58(10), 683-688. 7. Lange T, Thomas A, Walpurgis K, Thevis M. (2020) Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide and non-peptide doping agents by means of LC- HRMS. Anal Bioanal Chem. 412(15), 3765-3777 8. Wen H, Gao J, Wang X, Yu L. (2021) Application of dried blood spot sampling equipment and method in doping control. Chin. Sport Sci. 41(11), 30-37. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0120 Figure 2. Extracted ion chromatogram (EIC) of small peptide analytes in the DBS sample at a concentration of 20 ng/mL MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0121 Thomas A, Garzinsky A, Görgens C, Guddat S, Geyer H, Thevis M Carbonic anhydrase inhibitors brinzolamide and dorzolamide in dried blood spots Institute of Biochemistry, German Sport University, Cologne, Germany Abstract Dried blood spots as a possible sample matrix in doping control has been approved since 2022 and is becoming increasingly popular. DBS are currently applicable to the analysis of prohibited substances that are banned at all times and are not subject to any threshold or MRL. This is the case, for example, for diuretics and masking agents (S5 of the WADA Prohibited List), where the two carbonic anhydrase inhibitors brinzolamide (BA) and dorzolamide (DA) are also listed. With the exception of a local ophthalmic application, BA and DA are prohibited in sports. The retrospectivity achievable with DBS is usually lower compared to urine samples, because urine represents a quasi-integral of plasma concentrations. Thus, the detection window for drugs of low molecular mass is usually higher than in the respective blood samples. However, in the case of the two carbonic anhydrase inhibitors brinzolamide (BA) and dorzolamide (DA), this is exactly the opposite. Due to the rapid incorporation of these substances into erythrocytes (with an average lifetime of 120 days), BA and DA are detectable for much longer in whole blood samples (such as DBS) than in urine. A half-life of 24 weeks in whole blood has been described, so that a detectability of months, if not years, can be assumed. DA and BA are carbonic anhydrase inhibitors and often used by topical administration to treat glaucoma. Although locally applied, BA and DA will also enter the circulation via the blood-retinal-barrier and are enriched in the erythrocytes. Two post administration samples (DBS and urine, 2 and 8 hours after one drop of BA 10 mg/mL) yielded BA concentrations of approx. 10 resp. 100 ng/mL in urine and 19 resp. 17 µg/mL in the corresponding DBS samples. This phenomenon might explain a comparably high percentage of BA and DA observations in DBS. After one year of DBS doping control sampling BA and DA showed a significant prevalence most likely due to the prevalence of glaucoma in athletes. Introduction Dried blood spots as a possible sample matrix in doping control has been approved since 2022 and is becoming increasingly popular. DBS are currently applicable to the analysis of prohibited substances that are banned at all times and are not subject to any threshold or MRL [1]. This is the case, for example, for diuretics and masking agents (S5 of the WADA Prohibited List), where the two carbonic anhydrase inhibitors brinzolamide (BA) and dorzolamide (DA) are also listed (see Figure 1). With the exception of a local ophthalmic application, BA and DA are prohibited in sports. The retrospectivity achievable with DBS is usually lower compared to urine samples, because urine represents a quasi-integral of plasma concentrations. Thus, urine concentrations (and detcetion windows) for drugs of low molecular mass are usually higher than in the respective blood samples. However, in the case of the two carbonic anhydrase inhibitors brinzolamide (BA) and dorzolamide (DA), this is exactly the opposite. Due to the rapid MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0122 incorporation of these substances into erythrocytes (with an average lifetime of 120 days), BA and DA are detectable for much longer in whole blood samples (such as DBS) than in urine. A half-life of 24 weeks in whole blood has been described, so that a detectability of months, if not years, can be assumed [2]. DA and BA are carbonic anhydrase inhibitors and often used by topical administration to treat glaucoma (see Figure 2). Although locally applied, BA and DA will also enter the circulation via the blood-retinal-barrier and are enriched in the erythrocytes [3]. Figure 1. Structure of a) brinzolamide (BA) and b) dorzolamide (DA) Figure 2. Ocular drug administration following a) topical and b) intracameral administration Experimental Sample preparation For DBS sampling DMPK cellulose cards as well as TASSO devices were used and found to be appropriate. Both were loaded with 20 µL of capillary whole blood (venous EDTA blood for validation). The cards were extracted fully automated by means of the Gerstel DBS autosampler (Mülheim, Germany) following the protocol described elsewhere [4]. The TASSO samples were extracted following an adapted manual protocol with the nearly identical conditions. In brief, samples were fortified with ISTD mixture (stable isotope labelled clenbuterol, dexamethasone, hydrochlorothiazide, amphetamine) solution and extracted with a mixture of methanol/water (80/20). The extract is acidified with 5 µL of hydrochloric acid (0.3 N) prior to evaporation and reconstituted in acetonitrile/water (80/20) before injection into the LC-MS system. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0123 Post administration samples Two paired post administation samples (one male, one female) were collected after topical ophthalmic administration of one drop of BA (10 mg/mL, twice a day, as permanent glaucom treatment > 2 weeks). DBS and urine samples were collected 2 resp. 8 hours after the last administration. The urine samples were analysed according to the established direct urine injection with the validated LC-HRMS initial testing procedure. Here the urinary concentrations were estimated at approx. 13 ng/mL (after 2 h) resp. 107 ng/mL (after 8h). The corresponding DBS samples showed concentrations of 19 µg/mL (after 8 h) resp. 17 µg/mL (after 2 h). These findings strongly confirm the very long detection window of BA in DBS samples. Especially, considering the described half-life of approx. 24 weeks [2]. Results and Discussion The prevalence of glaucoma increases with age from 0% for babies to approximately 2% for patients with 45 years of age or older. Because DA and BA are common therapeutic drugs used to treat glaucoma, a comparatively high prevalence is also expected in samples from athletes accordingly. This is especially given in consideration of the expected long detectability in DBS samples. Direct differentiation between permitted topical- and prohibited systemic administration, is not enabled with the present method, because both will yield high whole blood levels with low (undetectable) urine concentrations. For BA it is described, that topic administration will produce lower levels of the known metabolite N-Desethyl-BA in the erythrocytes, but this phenomenon has to be confirmed with controlled administration samples yet [2]. Table 1 shows the validation results for the confirmation of BA and DA in dried blood spots according to the current requirements of the WADA. Table 1. Validation results (ITP/CP non-threshold) Conclusions In 2022, the Cologne laboratory had two cases of confirmed BA samples with DBS. The estimated concentrations of BA were 5 resp. 9 ng/mL and the corresponding urine samples were negativ (no BA detectable). In addition, several other samples showed traces of BA and DA, which were not reported due to the low concentration of BA and DA (below the LOI of 5 ng/mL). Figure 3 shows the extracted ion chromatograms of the product ion experiments used for confirmatory analysis. Due to the prevalence of glaucoma in the population as well as in competing athletes and the long detection window, the analysis of DBS for BA and DA will yield a considerable high number of analytical findings. In comparison, the 2021 anti-doping testing figure report of the WADA shows 28 DA and 23 BA cases considering > 200,000 urine samples [5]. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0124 Figure 3. Extracted ion chromatograms from a) a blank DBS sample, b) a DBS from an athlete (AAF) and c) a DBS fortified at 5 ng/mL with BA References 1. WADA, TD2023 DBS, https://www.wada-ama.org/sites/default/files/2022-11/td2023dbs_cleanversion.pdf, 2023 2. Fachinformation Brinzolamide, https://www.ema.europa.eu/en/documents/product-information/azopt-epar- product-information_de.pdf, 2023 3. Suzuki G, Kunikane E, Shigemi W, Shinno K, Kozai S, Kurata M, Kawamura A. Ocular and systemic pharmacokinetics of brimonidine and brinzolamide after topical administration in rabbits: comparison between fixed-combination and single-drug formulations. Curr Eye Res. 2021 Mar;46(3):380-386. doi: 10.1080/02713683.2020.1800046. 4. Garzinsky AM, Thomas A, Guddat S, Görgens C, Dib J, Thevis M. Dried blood spots for doping controls- Development of a comprehensive initial testing procedure with fully automated sample preparation. Biomed Chromatogr. 2023 Aug;37(8):e5633. doi: 10.1002/bmc.5633. 5. WADA, Anti-Doping Testing Figure Report 2021, https://www.wada-ama.org/sites/default/files/2023- 01/2021_anti-doping_testing_figures_en.pdf Acknowledgements The study was carried out with support of the Manfred Donike Institute for Doping Analysis (Cologne, Germany) and the Federal Ministry of the Interior, Community and Building of the Federal Republic of Germany (Bonn, Germany). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0125 Görgens C , Geyer H , Guddat S , Machnik M , Schenk I , van de Bovenkamp A , Wagener F , Thevis M Trimetazidine: pilot study data illustrating approaches to evaluate a potential contamination scenario Institute of Biochemistry, Center of Preventive Doping Research, German Sport University, Cologne, Germany ; Amsterdam Universitair Medische Centra, Amsterdam, Netherlands ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract Triggered by an AAF for trimetazidine, allegedly caused by a contamination scenario, several pilot studies were conducted to provide data for RMAs to evaluate such scenarios in the future. The pilot studies consisted of the LC/HRMS analyses of hair and oral fluid samples collected from patients on a therapeutic trimetazidine regimen as well as urine samples of a volunteer, who orally administered a microdose (5 µg) of trimetazidine. The obtained data of the pilot studies provide first insights regarding concentrations of trimetazidine in oral fluid and hair samples of patients as well as urinary concentrations and detection windows after microdosing of trimetazidine. Introduction In connection with an AAF for trimetazidine in 2022, it was published that the AAF was presumably attributable to a contamination scenario. Trace amounts of the substance may have been unknowingly ingested by the athlete, sharing a glass of water with a person who used trimetazidine therapeutically. The approximate concentration of trimetazidine in the doping control urine sample, resulting in the AAF, was reported to be 2.1 ng/mL [1]. Analytical data supporting the evaluation of such scenarios are scarce, and pilot studies were conducted to answer the following questions: 1. At which magnitude do concentrations of trimetazidine exist in the oral fluid (OF) of patients receiving (repeated) therapeutic doses of trimetazidine? 2. Which urinary concentrations of trimetazidine are obtained after the administration of a microdose (simulating a contamination) of trimetazidine (5 µg)? 3. Is it possible to detect trimetazidine in hair of patients receiving therapeutic doses of trimetazidine? Experimental Hair samples (strand of hair, with a diameter of ca. 0.5 cm from the back of head) and OF samples (2 mL) were collected from two patients (54 and 52 years old) who had been taking trimetazidine at a daily dose of 2 x 35 mg/day of slow-release trimetazidine dihydrochloride for 11 months. The samples were collected 2 hours after the last trimetazidine application. Urine samples were obtained from a male volunteer (49 years) who orally administered 5 µg of (free) trimetazidine. Therefore, trimetazidine dihydrochloride was dissolved in methanol and an aliquot corresponding to 5 µg of dissociated 1 1 1 1 1 2 1 1,3 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0126 trimetazidine was evaporated and consumed after solution in vodka. Urine samples were collected before and till 24 hours after the drug administration. The urine and OF samples were analyzed with LC/HRMS for trimetazidine using an established routine doping control analytical method reported elsewhere [2]. The validated LOD was 2.1 ng/mL. The trimetazidine concentration in the urine samples were not corrected for specific gravity. The hair samples were pulverized, 50 mg of each sample were extracted according to a method of Nguyen [3], and the extracts were analyzed by LC/HRMS with the same method as the urine and OF samples. Results and Discussion Oral fluid samples: in the OF samples of the two patients, trimetazidine was detected at roughly estimated levels of 540 ng/mL and 1130 ng/mL, respectively. Hair samples: in the hair samples of both patients trimetazidine was identified. The roughly estimated concentrations of trimetazidine were 880 pg/mg and 350 pg/mg, respectively. It is not known, if a single application of trimetazidine in doses in the low microgram range can be detected in hair. Urine samples: after the oral application of 5 µg of trimetazidine, a maximum value of trimetazidine of ca. 4.7 ng/mL was obtained after 13.5 h (concentrations not corrected for specific gravity (SG)). After 19.5 h trimetazidine could no longer be detected (Figure 1). Figure 1. Urinary concentrations after oral administration of 5 µg trimetazidine (conc. not corrected for SG) Conclusions These pilot study data provide first insights into drug deposition and elimination profiles, which might assist RMAs in future case evaluations concerning trimetazidine findings (if hair samples are available and collected in a timely manner). Follow-up investigations considering e.g. hair segment analyses and/or additional microdosings simulating exposure appear warranted. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0127 References 1. CAS award OG 22/08 -CAS OG 22/09 -CAS OG 22/10. https://www.tas- cas.org/fileadmin/user_upload/OG_22_08-09-10_Arbitral_Award__publication_.pdf; access: 02.02.2023 2. Görgens, C, Guddat, S, Bosse, C, Knoop, A, Geyer, H, Thevis, M. Implementation of the HIF activator IOX-2 in routine doping controls – Pilot study data. Drug Test Anal. 2020; 12: 1614– 1619. 3. Nguyen V.M.C. (2020): Entwicklung und Validierung eines Nachweisverfahrens für ausgewählte Dopingsubstanzen im Pferdehaar unter Berücksichtigung verschiedener Einflussfaktoren wie Farbe, Morphologie und Vorbehandlung der Haare. Köln, Deutsche Sporthochschule Köln, Institut für Biochemie, 1. Staatsexamen Lebensmittelchemie Acknowledgements The authors acknowledge support from the Manfred Donike Institute for Doping Analysis (Cologne, Germany) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0128 Radu M , Catana D , Berghes B , Cristea C , Toboc A , Stan C Development and validation of a chromatographic separation method for betamethasone and dexamethasone LC-MS, Romanian Doping Control Laboratory, Bucharest, Romania ; Romanian Doping Control Laboratory, Bucharest, Romania Abstract Betamethasone and dexamethasone are prohibited in sports, in competition only, and are included in the World Anti-Doping Agency (WADA) Prohibited List, class S.9 Glucocorticoids. The two compounds are epimers, the only difference between them being the orientation of the methyl group at carbon 16. This results in similar spectra and a very difficult chromatographic separation. Our study proposes a method for the identification of betamethasone and dexamethasone by liquid chromatography in tandem with mass spectrometry (LC-MS/MS) technique after investigating several chromatographic conditions and using a C8 reversed phase chromatographic column for separation. The method was validated for specificity, limit of identification, carry-over and robustness. The results indicat, that this method is specific and ensures reliable analytical results without interferences. Therefore, it is suitable for the identification of betamethasone and dexamethasone in accordance with the current WADA Technical Documents TD2023IDCR and TD2022MRPL. Introduction Betamethasone and dexamethasone are included in the World Anti-Doping Agency (WADA)’s Prohibited List in class S9. Glucocorticoids and are prohibited in competition only [1]. Due to their structural similarity, their MS-MS spectra have the same fragments that differ only by abundance [2], and are difficult to separate by chromatography. Various studies regarding analysis of betamethasone and dexamethasone in pharmaceutical products [3,4] and biological samples [5,6] were performed, the best results being obtained using chiral columns [7]. This study proposes a method for the separation and identification of betamethasone and dexamethasone in urine samples using a C8 reversed phase chromatographic column and detection by tandem mass spectrometry. Experimental Betamethasone and dexamethasone were purchased from LGC Standards (Wesel, Germany). Tert-butyl methyl ether, acetonitrile potassium bicarbonate and potassium carbonate were purchased from Merck (Redox Romania). Ultrapure water was obtain using a Milli-Q Q-POD equipment from Merck. Sample preparation method is presented in Figure 1. 1 2 2 2 2 2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0129 Figure 1. Sample preparation scheme Equipment used was AB SCIEX 6500 / EXION LC AD. The chromatographic column was Zorbax Eclipse XDB-C8 (4.6 x 150 mm, 5 µm). The A solvent was 5 mM amonium formate, 1‰ formic acid in water, B solvent 5 mM amonium formate, 1‰ formic acid in 90% acetonitrile and 10% water. LC flow was 450 µL/min, injection volume 2 µL and the column was thermostated at 30 °C. The MS parameters were the following: acquisition mode MRM, polarity positive, curtain gas 35 psi, ionization voltage 5500 V, source temperature 550 °C, nebulizer gas 60 psi, auxiliary gas 60 psi, collision cell gas (CAD) 12. B gradient and MRM transitions are presented in Table 1. Table 1. LC program and MRM transitions The methods were investigated for matrix effects, limit of identification, carry-over, and robustness: Matrix effects: 10 negative control samples were injected and then the presence of interferences at the retention times of the analyte of interest was checked. + MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0130 Limit of identification (LOI): scalar dilutions in urine to obtain the lowest concentration for substances identification (S/N> 3). 10 negative urine samples from different sources were fortified with the standard betamethasone and dexamethasone, establishing that the identification limit has the value of 15 ng/mL and 30 ng/mL. Carry-over was evaluated with the consecutive injection of a sample fortified at 300 ng/mL and blank samples to investigate if there are any contamination. Robustness was evaluated by modifying column temperature, source temperature, flow, injection volume, TBME volume, reconstitution volume, hydrolysis time. Results and Discussion The following columns and gradients were tested (data not shown): Zorbax SB-C18 (2.1 x 50 mm, 5 µm) with the following gradient: flow 0.3 mL/min, 0 - 20 min 22% B, flow 0.50 mL: 21 - 26 min 70% B, and four minutes reequilibration at 22% B Poroshell 120 EC-C18 (3 x 50 mm, 2.7 µm) with the following gradient flow 0.3 mL/min, 0 - 2 min 0% B, 2 - 22 min 45% B, 22 - 25 min 45% - 100% B, 25 - 28 min 100% B, 28.1 - 31 min 0% B Zorbax Eclipse XDB-C18 (4.6 x 150 mm, 5 µm) and Zorbax Eclipse XDB-C8 (4.6 x 150 mm, 5 µm) with the following gradients: flow 0.5 mL/min gradient 1: 0 - 5 min 25% B, 5.5 - 12 min 32% B, 12.5 - 17 min 48% B, 17.5 - 25 min 70% B then reequilibration for 5 min at 25% B flow 0.45 mL/min gradient 2: 0 - 5 min 25% B, 5.5 - 12 min 32% B, 12.5 - 17 min 48% B, 17.5 - 25 min 70% B then reequilibration for 5 min at 25% B flow 0.45 mL/min gradient 3: 0 - 5 min 20% B, 5.5 - 12 min 25% B, 12.5 - 17 min 40% B, 17.5 - 25 min 65% B then reequilibration for 5 min at 20% B flow 0.40 mL/min gradient 4: 0 - 23 min 0% B, 23.1 - 27 min 90% B, 27.1 - 32 min 0% B flow 0.35 mL/min gradient 5: 0 - 1 min 0% B, 1 - 23 min 0-70% B, 23 - 27 min 70-100% B, 27 - 29 min 100% B, then reequilibration for 5 min at 0% B (0.5 mL/min on reequilibration step) flow 0.40 mL/min gradient 6: 0 - 5 min 20% B, 5.5 - 12 min 30% B, 12.5 - 17 min 45% B, 17.5 - 25 65% B, then reequilibration for 5 min at 20% B (0.5 mL/min on reequilibration step) The best separation was achieved under the conditions presented in Table 1. Although baseline separation was not achieved, the method is capable to differentiate between beta‐ methasone and dexamethasone when are present in the urine sample simultaneously. The separation between betamethasone and dexamethasone proved to be sufficient to meet the WADA chromatographic identification criteria only for betamethasone when it is tested against a dexamethasone individual standard and vice versa. Chromatograms of a urine fortified with betamethasone and dexa​methasone and individual standards of betamethasone and dexa​methasone are presented in Figure 2. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0131 Figure 2. Chromatograms for A: negative urine sample; B: urine sample spiked with betamethasone and dexamethasone at 15 ng/mL; C: individual standards of betamethasone and dexamethasone in H O spiked at 30 ng/mL The results obtained during validation showed that the method is specific for identification of betamethasone and dexamethasone and no interference signals were present in blank samples. The limit of identification was established at 15 ng/mL and it is compliant with the WADA TD2022MRPL document. The method presents no significant carry-over and is robust for investigated parameters for both compounds. 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0132 Conclusions This study presents a method for the separation and identification of betamethasone and dexamethasone in urine samples using a reversed phase C8 chromatographic column and detection by tandem mass spectrometry. Even though baseline separation was not achieved, the retention time difference between the two compounds is enough to meet the WADA identification criteria, so that betamethasone can’t be misidentified as dexamethasone or vice versa. The method has a limit of identification of 15 ng/mL and is suitable for the identification of betamethasone and dexamethasone in doping control urine samples in compliance with the WADA technical documents TD2023IDCR and TD2022MRPL. References 1. World Anti-Doping Agency. The 2023 Prohibited List. https://www.wada-ama.org/en/resources/world-anti- doping-program/2023-prohibited-list#resource-download (access date 23.01.2023). 2. De Wasch K, De Brabander HF, Van de Wiele M, Vercammen J, Courtheyn D, (2001) Differentiation between dexamethasone and betamethasone in a mixture using multiple mass spectrometry. J. Chromatogr. A. 926, 79–86. 3. Xiao, KP, Xiong, Y, & Rustum, AM (2008) Quantitation of trace betamethasone or dexamethasone in dexamethasone or betamethasone active pharmaceutical ingredients by reversed-phase high-performance liquid chromatography. J. Chromatogr. Sci. 46(1), 15-22. 4. Arthur, KE, Wolff, JC, Carrier, DJ (2004) Analysis of betamethasone, dexamethasone and related compounds by liquid chromatography/electrospray mass spectrometry. RCM 18(6), 678-684. 5. Cirimele, V, Kintz, P, Dumestre, V, Goulle, JP, Ludes, B (2000) Identification of ten corticosteroids in human hair by liquid chromatography–ionspray mass spectrometry. Forensic Sci. Int. 107(1-3), 381-388. 6. Deventer, K, Delbeke, FT (2003) Validation of a screening method for corticosteroids in doping analysis by liquid chromatography/tandem mass spectrometry. RCM. 17(18), 2107-2114. 7. Karatt, TK., Sayed, R, Nalakath, J, Perwad, Z, Albert, PH, & Khader, KA (2018) Separation and identification of the epimeric doping agents–Dexamethasone and betamethasone in equine urine and plasma: A reversed phase chiral chromatographic approach. Steroids 140, 77-82. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0133 Wicka M , Konarski P , Drapala A , Kaliszewski P , Grucza K , Stanczyk D , Kwiatkowska D LC-MS/MS method for the detection of synthetic cathinones in urine Polish Anti-Doping Laboratory, Warsaw, Poland ; Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland Abstract Synthetic cathinones (SCs) are a broad group of chemicals, which have a very similar structure. Cathinones are derivatives of the parent compound cathinone, which is one of the active compounds found in the khat plant (Catha edulis). The cathinone structure is strictly related to amphetamine. However, synthetic cathinones can be much more potent than the drugs that they are intended to mimic, which increases the risk of overdose and death. In the WADA Prohibited List, SCs are classified in section S6 (stimulants) and are prohibited in competition only. The aim of the study was to develop an initial testing analytical method that would allow the detection of 25 cathinones in urine to be used for routine testing of athletes by means of ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The method was verified for selectivity, the limit of detection (LOD), recovery (RE), matrix effects (ME), and process efficiency (PE). The presented method allows the simultaneous detection and identification of 25 cathinones in urine by means of the LC-MS/MS approach and is fit-for-purpose for routine analysis of doping samples. Introduction Synthetic cathinones (SCs) are β-keto phenethylamine derivatives, which have the same core structure [1,2]. The cathinone structure is strictly related to amphetamine, with the difference being the presence of a keto group [2-5]. Cathinone derivatives can be synthesized by the addition of several substituents at different sites of the cathinone scaffold as represented in Figure 1. Synthetic cathinones are widely abused due to their hallucinogenic and stimulant effects. SCs are replacing cocaine and MDMA (3,4- methylenedioxymethamphetamine), which are much more expensive. However, synthetic cathinones can be much more potent than the drugs that they are intended to mimic, which increases the risk of overdose and death [2]. In the WADA Prohibited List, SCs are classified in section S6 (stimulants) and are prohibited in competition only [6]. The aim of the presented work was to develop and validate an LC- MS/MS method for the detection of 25 cathinones in urine to be used for routine testing of athletes. The described method is suitable for use on complex biological matrices. 1 1 1 1 1 1 1,2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0134 Figure 1. The general structure of cathinone derivatives Experimental Sample pre-treatment The sample preparation is a two-step procedure involving enzymatic deconjugation of glucuronides and then liquid-liquid extraction with 6 mL of methyl tert-butyl ether. The residue was reconstituted in 100 µL of mobile phase (acetonitrile/water, 1/1, v/v), transferred in a vial and 2 µL was injected into the LC- MS/MS system. Instrumental analysis Chromatographic separation was conducted by means of a Waters Acquity I-Class UPLC System liquid chromatography with BEH C18 column (1.7 µm, 100 mm x 2.1 mm, Waters). The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B), and the LC gradient was employed at a constant flow rate of 300 µL/min at 45 °C. MRMs of the studied substances were traced with a Xevo TQ-XS mass spectrometer equipped with an electrospray source. All analytes were investigated in the ESI mode. Desolvation gas flow was set at 800 L/h at 600 °C with ion source temperature at 150 °C. The capillary voltage was 1.0 kV (Table 1). Method validation The validation process was performed in accordance with the WADA technical document TD2023IDCR [7]. The method was verified for selectivity, limit of detection (LOD), retention time (RT), extraction recovery (RE), process efficiency (PE), and matrix effects (ME). + MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0135 Table 1. Selected tandem mass spectrometry transitions for analysis Results and Discussion The extraction recovery of the method showed that, with two exceptions (15.3% for 4-CEC and 28.3% for cathinone), the percentages of recovery ranged from 31.6% to 95.3%. The process efficiency was 5.3 % to 82.9%. The matrix effect for the analyzed compounds ranged from 18.2% to 96.5% (Table 2). Selectivity was assessed by analyzing 6 blank samples from different individuals (3 males and 3 females). Figure 2 shows an example chromatogram for the strongest MRM for each cathinone (for the final concentration of 10 ng/mL). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0136 Table 2. The value obtained from LOD, RT, RE, ME, and PE of cathinones in urine Figure 2. Typical MRM chromatograms of urine samples – blank urine and analyzed cathinone at a concentration of 10 ng/mL MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0137 Conclusions All compounds were successfully fragmented into ions . All the selected ionic transitions have proved to be specific. No interfering signals from the matrix were observed for any of the MRMs - high selectivity of the method. The method is suitable for use with complex biological matrices (% RSD 408.0 (CE 75 V) for Co-5-Br-PADAP (target, blue) and 665.1 > 362.1 (CE 65 V) for Co-5-Cl-PADAP (ISTD, yellow) Lastly, as sediment is often present in urine samples, it is important to know whether analyte losses can occur due to coprecipitation. This may be critical in cases where secondary aliquot is taken after urine had sufficient time to settle, or when sediment is intentionally removed prior to SPE. Here, 22 randomized urine samples containing visually different amounts of sediment were spiked with cobalt ions, mixed thoroughly, and refrigerated. After 72 h the samples (both spiked and non-spiked) were carefully resuspended and secondary aliquots were immediately taken for analysis. The remaining MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0141 volume was centrifuged and an aliquot of supernatant was processed as well. Using a single-point calibration, concentration of cobalt was found to be not significantly different between aliquots taken with and without sediment (Table 2). Table 2. Negative and spiked urine analyzed before and after centrifugation (n = 22) Conclusions The method presented herein can be suggested for use as an ITP in those laboratories which do not have the ICP-MS capability. Given the short LC run time ( < 3 min per sample), this method can easily be adapted to a 96-well plate format, if high productivity is desired. Although a reporting threshold has yet to be established by WADA, broader testing for cobalt is vital to the effectiveness of the global anti- doping program. References 1. Sobolevsky T, Ahrens B. (2021) Measurement of urinary cobalt as its complex with 2-(5-chloro-2- pyridylazo)-5-diethylaminophenol by liquid chromatography-tandem mass spectrometry for the purpose of anti-doping control. Drug Test Anal. 13(6), 1145-1157 2. Dos Santos VF, Carneiro GRA, Braz BF, Santelli RE, de Paula Machado S, Gualberto Pereira HM. (2023) Coordination chemistry applied to anti-doping analysis: Cobalt quantification as its diethyldithiocarbamate complex by liquid chromatography coupled to high resolution tandem mass spectrometry. Drug Test Anal. doi: 10.1002/dta.3571. Epub ahead of print MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0142 Liu L, Wang Z, Yan K, He G, Xing Y, Wu Q Doping control analysis of morphine in human urine by ultra- performance liquid chromatographic-electrospray ionization tandem mass spectrometry Beijing Sport University, Beijing Anti-Doping Laboratory, Beijing, People´s Republic of China Abstract Morphine is a narcotic analgesic commonly used in clinics, while prohibited in the time of sports competition. Morphine could be metabolized in the urine from codeine and ethylmorphine. Nowadays, the use of codeine and ethylmorphine is allowed, while the threshold for total morphine is 1 μg/mL, which is regulated by the latest valid WADA technical document (TD) on decision limits (DL). Based upon these conditions, an ultra-performance liquid chromatographic-electrospray ionization tandem mass spectrometry (UPLC–ESI-MS/MS) method for three prohibited morphine substances was developed and validated, including morphine, morphine-3β-D-glucuronide and morphine-6β-D-glucuronide in human urine in the concentration ranges between 100 and 1000 ng/mL. The quantitative bias caused by the pretreatment conditions and the different matrix was reduced by a straightforward sample preparation of the dilute and shoot method. The compounds were analyzed directly without the need for hydrolysis, solvent transfer, evaporation or reconstitution. Since codeine is not included in the prohibited list by WADA, it is important to determine whether a positive morphine finding is a result of codeine metabolism. Hence, the codeine and codeine-6β-D-glucuronide were monitored simultaneously in the single injection. The method proved to be specific, accurate and precise for linearity; the limit of quantification, intermediate precision, repeatability, matrix effects and uncertainty have been evaluated. The Beijing anti-doping laboratory has applied this method in routine tests and reported adverse analytical finding for many years. The relative comparison experiment results from WADA-external quality assessment scheme have been further discussed. Introduction Morphine is a narcotic analgesic commonly used in clinics, while prohibited in the time of sports competition. Morphine could be metabolize from codeine and ethylmorphine in urine [1]. Codeine is widely used as a milder analgesic and antitussive, whereas ethylmorphine is used to treat scleritis [2]. Nowadays, the use of codeine and ethylmorphine is permitted, and a concentration of morphine only above the relevant threshold (or decision limit) results in an AAF [3], if the criteria involving the consideration of codeine or ethylmorphine and corresponding metabolites are met [4]. Based upon these conditions, it is necessary to establish a simple and accurate quantitative method fot the simultaneous determination of the concentration of total morphine (M) including the parent and glucuronide metabolites (M3G & M6G) in human urine, which has been applied in routine testing and reported adverse analytical findings. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0143 Experimental 2.1 Instrumental analysis The UPLC-MS/MS system consisted of an Agilent 1295 LC system, and a 6470 triple-quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source. The MS/MS parameters including the MRM transitions, collision energy (CE) and retention times are presented in Table 1. Other mass spectrometry parameters were as follows: capillary voltage, 3.5 kV; gas temperature, 300 °C; sheath gas temperature, 350 °C and nebulizer pressure, 45 psi. High purity nitrogen was employed as drying and heating gas with a flow rate of 11 L/min. High purity argon was employed as nebulizing gas with a flow rate of 6.0 L/min. The chromatographic separation was achieved on an ACE Excel 3 Super C column (150 x 4.6 mm) with the temperature maintained at 30 °C. The mobile phase using a flow rate maintained 0.8 mL/mL, was a mix of 0.1% ammonia and 10 mmol/L ammonium formate solution (A) and MeOH (B). The eluting conditions were optimized as follows: isocratic at 20% B (0–3 min), 20% to 40% B (3–4 min), isocratic at 40% B (4–15 min), 40-100% B (15–20 min), 100-20% B (20–20.01 min), isocratic at 20% B (20–25 min). Table 1. method validation parameters 2.2 Sample pretreatment procedure The LC/MS method has the ability to determine the parent and glucuronide metabolites simultaneously. However, it has been reported that the optimization of hydrolysis conditions is difficult and the hydrolysis rates vary according to the type of enzyme, temperature, incubation time, etc. [5-6]. The dilute and shoot method was applied in this quantitative method, while hydrolysis and extraction steps of the normal screening procedure have been replaced by a single centrifugation and filtering. More specifically, for the quality control samples including the calibrations, standard solutions are spiked separately and dried under nitrogen; then 1 mL of blank urine is spiked with 50 μL of D -mophine at 10 μg/mL. After fully mixing and centrifugation, the supernatant is injected or diluted with water if needed. For the blank, negative control and actual urine samples, the pretreatment procedure is similar without the dried step. 2.3 Method validation The limit of quantification (LOQ) for 100 ng/mL, linearity ranging from 100 ng/mL to 1000 ng/mL, the intermediate precision and repeatability preformed at 500 ng/mL of M, M3G and M6G were fully validated and evaluated. The uncertainty (Uc) evaluation was performed using the methods B and D 18 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0144 recommended in WADA-TD-DL [3]. The intermediate precision and Uc data could validate the method stability comprehensively. Results and Discussion 3.1 Method validation The established method demonstrated satisfactory linearity, intermediate precision, LOQ, and repeatability within the measurement range of 100 to 1000 ng/mL. The relevant validation parameters are presented in Table 2. Table 2. MRM transitions and ESI-MS/MS parameters 3.2 Experimental results The structures of morphine substances are similar, so to obtain the suitable product ions, the parameters for the fragmentor and the collision energies were optimized. The representative mass spectra and fracture manners of these components are listed in Figure 1a. Typical chromatograms of QCP added with the 3 analytes and IS and the actual urine sample are illustrated in Figure 1b. No coeluting peaks or interfering signals were detected at the retention times of the targets operated at their specific mass transition. Therefore, it can be concluded that the present method is selective for the determination of morphine substances. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0145 Figure 1. (a) MS/MS spectra; (b) chromatograms of QCP and actual urine sample 3.3 Comparison results The measurement of uncertainty is evaluated according to the results from relevant rounds of the EQAS, and partial results are listed in Figure 2a and 2b. In the practical application process, except for the quantitative process of morphine, the accurate assessment of codeine concentration is equally necessary. Codeine could undergo metabolism to morphine in the liver via CYP2D6, and affect the metabolite profiling in a certain extent. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0146 Figure 2. (a) Application results of morphine; (b) comparison results; (c) QC chart of morphine 3.4. Discussion It is worth noting, that during the method validation process the peak shape and ionization efficiency of M3G and M6G could be easily influenced by the concentration. To avoid this, the linearity was designed to range from 100 ng/mL to 1000 ng/mL when the concentration of morphine substances larger than 1000 ng/mL; the dilution operation is necessary to ensure the accuracy. Beyond that, 0.1% ammonia and 10 mmol/L ammonium formate solution was used to improve peak shape and enhance ESI. The results showed that the uncertainties of the morphine substances were all below the maximum uncertainties specified by WADA, as shown in Figure 2c. Due to the Olympic requirement of fast turn-around analysis time, a convenient and reliable quantitative method is essential. And there was one positive case for morphine reported during the XIII Paralympic Winter Games. Conclusions The measurement of morphine in human urine is influenced by many factors, such as individuals with ultra-rapid CYP2D6 metabolism [7], the parallel and efficiency of pretreatment procedures, or taking several non-prohibited drugs simultaneously. Determining the origin of morphine found in a doping control urine sample of athletes is the basic requirement for anti-doping laboratories. In this report, urine samples were diluted and directly injected into the UHPLC-MS/MS system without any sample pretreatment, also the use of deuterated internal standard was necessary to reduce the matrix effects. The QC chart indicates that the method allows retrospective data analysis, which is strong in operability and convenient for the application in routine testing and reporting of adverse analytical findings. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0147 References 1. Kolmonen M, Leinonen A, Kuuranne T, Pelander A, Ojanper I. (2010). Hydrophilic interaction liquid chromatography and accurate mass measurement for quantification and confirmation of morphine, codeine and their glucuronide conjugates in human urine. J Chromatogr B Analyt Technol Biomed Life Sci, 878(29), 2959-2966. 2. Vree T B, Wissen V V.(2010). Pharmacokinetics and metabolism of codeine in humans. Biopharm. Drug Dispos., 13(6), 445-460. 3. World Anti-Doping Agency. Decision Limits for the Confirmatory Quantification of Threshold Substances. (2022). https://www.wada-ama.org/en/resources/ lab-documents /td2022dl#resourcedownload 4. World Anti-Doping Agency. WADA Technical Letter - TL22 Ethylmorphine. (2020). https://www.wadaama.org/en/resources/tl22#resource-download 5. Hackett L P, Dusci L J, Ilett K F, Chiswell G M. (2002). Optimizing the hydrolysis of codeine and morphine glucuronides in urine. Ther Drug Monit. 24(5), 652. 6. Wang P, Stone J A, Chen K H, Gross S F, Haller C A, Wu A H. (2006). Incomplete recovery of prescription opioids in urine using enzymatic hydrolysis of glucuronide metabolites. J Anal Toxicol, 30(8):570-5. 7. He YJ, Brockmöller J, Schmidt H, Roots I, Kirchheiner J. (2008). CYP2D6 ultrarapid metabolism and morphine/codeine ratios in blood: was it codeine or heroin? J Anal Toxicol. Mar;32(2):178-82. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0148 Grainger J, Agon V, Croft M, Goebel C Stability assessment of two small peptide ( < 2kDa) mixed standard Australian Sports Drug Testing Laboratory (ASDTL), Sydney, Australia Abstract ASDTL commissioned Auspep Pty Ltd to synthesise and prepare two small peptide mixed standards. SPMix-1 and SPMix-2 contained the same 41 small peptides and metabolites with the addition of bovine insulin to SPMix-2 to act as a carrier peptide. The homogeneity and stability of the two mixes were evaluated. Three peptides for which isotopically labelled internal standards were available – Leuprolide, GHRP-2 and GHRP-2(1-3)-OH were used to evaluate homogeneity as they provided the most precise data sets. Analysis of Variance (ANOVA) of the three data sets obtained for the duplicate analysis of 14 vials determined the between vial variability to be ≤ 2.50% for SPMIX-1 and ≤ 2.16% for SPMix-2. Peptide stability was assessed over a 12-month period under four storage conditions: in solution or dried at either -20 °C or -80 °C. Most peptides in both SPMixes were found to be best stored at -80 °C whether dried or in solution. Drying caused losses of hexarelin and its metabolites in both SPMixes, with decreases of 10-30% observed when stored at -20 °C. Losses of around 15% were observed for AOD-9604(7-16) when stored in solution at -20 °C. Both SPMixes were distributed to all WADA laboratories for assessment. Of the laboratories that responded, all found the quality of the SPMixes to be comparable to in-house standard mixes but would prefer additional peptides to be included. Introduction The peptides and metabolites in WADA’s 2020 Prohibited List [1] cover a range of sub-classes including LH releasing hormones, GH secretagogues, GH releasing peptides, GH fragments, masking peptides and Tβ4 derivatives. Reliable reference standards are an essential component of multi-residue anti-doping testing procedures. Over 50 peptides/metabolites custom synthesised for ASDTL are available to WADA laboratories. This study aimed to produce a multi-peptide mixed standard to aid the harmonisation of peptide testing across WADA laboratories. Including all peptides/metabolites in the mix would have been cost prohibitive, so peptide metabolism [2-4] was considered. GHRP-1 and alexamorelin, for example, were excluded because they are completely metabolised and not found in urine [3,4], while metabolites detected in urine for longer periods, were included. Experimental Auspep Pty Ltd (Melbourne, Australia) was commissioned to synthesise and prepare two SPMixes, each containing 41 peptides (Table1) with SPMix-2 also containing bovine insulin to act as carrier. Two hundred vials of each mix were produced. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0149 Table 1. List of small peptides included in the SPMixes prepared by Auspep Pty Ltd (25 µg net of each peptide) Homogeneity and stability assessment was performed using 14 randomly selected vials of each SPMix. After reconstitution, 200 μL aliquots from each vial were transferred into five tubes. One tube was used for homogeneity assessment. The remaining tubes were placed into four different long-term storage (LTS) conditions – in solution at -20 °C; in solution at -80 °C; dried at -20 °C; and dried at -80 °C. After 1, 2, 3, 4, 5, 6 and 12 months tubes were removed and stored at -80 °C until tested. Sample analysis was performed by LC-HRMS. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0150 The long-term stability of the peptides after reconstitution was assessed using the isochronous approach described in ISO Guide 35 [5]. Aliquots stored under the same storage conditions were analysed by LC- HRMS in a single batch under repeatability conditions at the completion of the stability period to minimise analytical variance. Assessment of the homogeneity of the mass of peptides across individual vials was based on the recommendations of ISO Guide 35 [5]. Isotopically labelled analogues of GHRP-2, GHRP-2(1-3)-OH and leuprolide were used in the measurement procedure. This resulted in more precise data sets for these analytes, which were then used to estimate between vial homogeneity by ANOVA. Results and Discussion Homogeneity ANOVA of the concentrations of the three representative peptides measured from 14 vials was used to indicate if, at a level of confidence of 95%, the variance observed between vials was significantly different to the variance observed within vials (duplicate analysis). The between vial variability for leuprolide, GHRP-2(1-3)-OH and GHRP-2 was 1.80%, 1.67% and 2.5% respectively in SPMix-1 and 2.05%, 2.16% and 2.14% respectively in SPMix-2. Long Term Stability In-Solution (1 mL 0.2% FA) Assessment of both SPMixes stored in solution at -20 °C showed an overall decrease in concentration of all peptides over 12 months. AOD-9604(7-16) and LHRH(2-10) decreased by 10-15% in both SPMixes (Figure 1), while hexarelin and hexarelin(1-3)-OH had losses of approximately 8% (Figure 2). When stored in solution at -80 °C the decrease over the same period was less. AOD-9604(7-16), hexarelin and hexarelin(1-3)-OH decreased by approximately 5%, while LHRH(2-10) had negligible losses. Figure 1. Stability of LHRH(2-10) and AOD-9604(7-16) in SPMIX-1 and SPMIX-2 when stored in solution at -20 °C MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0151 Dried (vacuum concentrator, 55 °C) With the exception of hexarelin and hexarelin(1-3)-OH, the majority of peptides in both SPMixes were slightly more stable dried and stored at -20 °C or -80°C, than stored in solution at -20 °C or -80 °C. Hexarelin and hexarelin(1-3)-OH were severely affected when dried with losses of 10-30% in both SPMixes stored at -20 °C (Figure 2) and losses of approximately 6% at -80 °C, over the study period. Figure 2. Comparison of stability of hexarelin and metabolite in SPMIX-1 over 12-month period when stored in solution or dried at -20 °C Assessment by WADA Laboratories All WADA laboratories (~30) were given vials of both SPMixes for assessment. The 11 labs that responded found the quality of the SPMixes compared well with in-house mixes but most would prefer the mixes include additional peptides. Only two laboratories preferred the addition of bovine insulin. The majority of laboratories were prepared to purchase the SPMixes noting the advantages of time-saving and harmonisation across laboratories for peptide testing. Conclusions The production and supply of suitable mixed peptide standards offers an efficient and cost-effective means by which laboratories can satisfy WADA requirements for peptide reference materials. This study demonstrated the viability of producing a suitable material. Two mixed peptide standards were prepared by Auspep Pty Ltd, each containing 41 peptides. Homogeneity testing determined between bottle variability was ≤ 2.5% for SPMix-1 and ≤ 2.2% for SPMix-2 over the 200 vial production batch. Stability testing over 12 months determined the best storage conditions for the SPMixes after reconstitution to be in solution at -80 °C. The majority of WADA laboratories responding to the assessment survey of these materials noted the time-saving and harmonisation advantages provided and were interested in using the SPMixes. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0152 References 1. World Anti-Doping Agency. The 2020 Prohibited List. International Standard, Montreal (2020). https://www.wada-ama.org/sites/default/files/wada_2020_english_prohibited_list_0.pdf (access date 1/07/2020) 2. Esposito S, Deventer K, Geldof L, Van Eenoo P. In vitro models for metabolic studies of small peptide hormones in sport drug testing. J Pept Sci. 2015;21(1):1-9. 3. Semenistaya E, Zvereva I, Thomas A, Thevis M, Krotov G, Rodchenkov G. Determination of growth hormone releasing peptides metabolites in human urine after nasal administration of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin. Drug Test Anal. 2015;7(10):919-25. 4. Thomas A, Delahaut P, Krug O, Schänzer W, Thevis M. Metabolism of Growth Hormone Releasing Peptides. Anal Chem. 2012;84(23):10252-9. 5. International Organisation of Standardisation (2017). Reference materials - Guidance for characterisation and assessment of homogeneity and stability (ISO Guide 35:2017). Acknowledgements This study was carried out with the support of the National Measurement Institute (Sydney, Australia) and the World Anti-Doping Agency (WADA, 17A11CG). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0153 Yan X, Yan S, Chang W, Yang S A warning list to athletes of musk as ingredients from Traditional Chinese Medicine Beijing Anti-Doping Laboratory, Beijing Sport University, Beijing, People's Republic of China Abstract Musk, as a traditional Chinese medicine, can be used to treat traumatic injuries and cardiovascular disease. Numerous steroid compounds have been found in musk. Anabolic androgenic steroids feature prominently in the doping analysis field. Here we report an adverse analytical finding resulting from a previous administration of musk-containing drugs. This article sorts a list of medicines containing musk as an ingredient by the Pharmacopoeia of the People’s Republic of China (Chinese Pharmacopoeia). In summary, there are 74 traditional Chinese medicines containing musk extracts in the Chinese Pharmacopoeia. We suggest that athletes do not administrate those drugs to avoid triggering Adverse Analytical Findings (AAFs). Introduction Steroids, due to their anabolic effects, have been banned by WADA and IOC for many years. In recent years, various steroidal components including endogenous steroids were detected in musk, one of traditional Chinese medicines (TCMs), indicating a potential risk of triggering AAFs in doping tests, if athletes use a drug containing musk unintentionally [1]. Therefore, this article summarizes 74 traditional Chinese medicines containing musk from the Chinese Pharmacopoeia as a checklist for athletes and their team staff. Occurence of musk deer and composition of musk Musk deer are widespread in China, which is one of the original distribution areas around the world (Figure 1). However, the distribution area in China has been greatly reduced compared with ancient times, mainly distributed in Sichuan, Tibet, Qinghai, and Gansu provinces, and other places like Heilongjiang and Jilin also have small amounts. In addition to the wild, musk deer are widely domestically raised in recent years. As in 2020, there are about 30,000 domestic musk deer, mainly Moschus berezovskii Flerov, distributed in Shaanxi, Sichuan, Gansu, and Hubei provinces. Reports were also made in Shanxi, Fujian, and Shanghai. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0154 Figure 1. The distribution area of musk deer in China The chemical composition of musk is very complex. At present, the quality of traditional Chinese medicines containing musk is determined by the concentration of muscone. Amino acids, peptides, proteins, and steroidal compounds are also considered as main components of musk. Anabolic steroids found in musk ingredients were testosterone (T), epitestosterone (ET), dehydroepiandrosterone (DHEA), androsterone (An), etiocholanolone (Etio), and epiandrosterone (Epi-A). In addition, musk grains also contain small amounts of cholesterol, fatty acids, and esters (Table 1)[1]. Table 1. Steroids have been identified in Musk MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0155 Musk containing medicaments Musk extract has been used as a TCM for more than 2,000 years. Its medical application was first published in the “Shennong Ben Cao Jing”. This ancient Pharmacopoeia was created in the Qin-Han dynasty (about 200 years B.C.) in China [2]. Musk grains could be obtained from the pod of mature male musk deer (Moschus berezovskii Flerov, Moschus sifanicus Przewalski, Moschus Moschiferus Linnaeus ). In terms of the Chinese Pharmacopoeia, musk can be used for the treatment of cardiovascular diseases and traumatic injuries [3]. A list of traditional Chinese medicines containing musk is shown in Table 2. In the Chinese Pharmacopoeia 2020 edition, the number of traditional Chinese medicines containing musk has increased to 74, with up to 12 preparations, including 13 varieties of natural musk. Table 2. List of Traditional Chinese Medicines containing musk (from Chinese Pharmacopoeia, 2020 edition) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0156 Results and Discussion There have been approximately 20 steroidal compounds identified in musk extract so far, and nine steroids among them are listed in the WADA Prohibited List [1,4,5]. The use of musk extract has contributed to the triggering of AAFs at the 2011 FIFA Women World Cup [1]. It was reported that musk- containing drugs can alter steroid profile in human urine samples which could make athletes be tested positive in anti-doping analysis [6,7]. According to Gong Xiaoli et al., there are 469 kinds of traditional Chinese medicines containing musk in China [8]. The majority are used to affect the nervous system, and there are 101 drugs among them that are applied to the musculoskeletal system [9]. There are crucial differences between natural musk and artificial musk, especially the variety and content of macrocyclic ketones and androgens [10]. The relative content of natural musk components in different varieties and habitats also variy greatly. All kinds of TCM containing musk are complicated, and quality control is an essential task in the pharmaceutical factories in China nowadays. Conclusions A portion of anabolic androgenic steroids found in musk deer extracts are of the same kind as those in the human body, the main differences are that the concentrations may vary. Many more anabolic androgenic steroids have been identified in musk (Table 1)[1]. The prohibited endogenous steroids in musk can be abused as doping. This poster provides a list of traditional Chinese medicines containing musk. It is suggested that athletes should check the ingredients and keep alert to avoid Adverse Analytical Findings. References 1. Thevis M, Schänzer W, Geyer H, Thieme D, Grosse J, Rautenberg C, Flenker U, Beuck S, Thomas A, Holland R, Dvorak J. (2012) Traditional Chinese medicine and sports drug testing: identification of natural steroid administration in doping control urine samples resulting from musk (pod) extracts. Br J Sports Med. 47, 109–114 2. Ren L, Gao H, Zheng L, Peng C, Hou X. (2022) History and literature review of Moschus. Mod Chin Med. 25(3), 608-615 3. Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China. The medicine science and technology press of China, Beijing (2020), 402-403 4. World Anti-Doping Agency. The 2023 Prohibited List. International Standard, Montreal 5. (2023). https://www.wada-ama.org/sites/default/files/2023-05/2023list_en_final_9_september_2022.pdf (access date 05.08.2023) 6. Zhou J, Li N, Xie W, Song M, Han F, Li F, Li G. (2022) Advance in study on chemical constituents of natural musk and their pharmacological effects. Lishizhen Med Mater Med Res. 33(1), 185-188 7. He Y, Wang J, Liu X, Xu Y, He Z. (2013) Influences of musk administration on the doping test. Steroids. 78, 1047–1052 8. Wang J, He Y, Liu X, Yang Z, Yang W. (2017) Steroid profile and IRMS analysis of musk administration for doping control. Drug Test Anal. 9, 1779–1787 9. Gong L, Wang B, Luo S, You Z, Wang X, Li L, Zhao N. (2022) Research Status of Quality of Chinese Patent Medicines Containing Moschus. Mod Chin Med. 25(5), 1155-1162 10. Zhang Z, Huang H, Luo Y, Tan T, Yang M. (2019) Chemical Constituents from Musk of Moschus berezovskii. J Chin Med Mater. 42(2), 331-332 11. Jiang Y, Luo Y, Tan T, Yang M, Liao G. (2018) Comparison of Volatile Chemical Compositions of Moschus from Different Species and Producing Areas by GC-MS and Chemometric Analysis. Chin J Exp Tradit Med Formulae. 24(3), 49-55 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0157 Wicka M , Drapala A , Konarski P , Zyfka-Zagrodzinska E , Golaszewska A , Marusza M , Gorczyca D , Kwiatkowska D Hemp oils and potential for unintentional anti-doping rule violations Polish Anti-Doping Laboratory, Warsaw, Poland ; Lazarski University, Warsaw, Poland ; LymeLab Pharma, Warsaw, Poland ; Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland Abstract All natural and synthetic cannabinoids with one exception – cannabidiol (CBD) - are prohibited in competition and are included in section S8 in the WADA Prohibited List. CBD products are widely available on the commercial market. It is highly probable that the use of this group of preparations could cause unintentional violations of anti-doping regulations. Moreover, CBD preparations from cannabis plants may contain additional minor cannabinoids. The aim of the study was to analyze products containing CBD (hemp teas and oil) and urine samples collected after taking these preparations. Afterwards, the laboratory tested 15 oils from dried hemp available on the commercial market. Furthermore, urine samples from routine doping control have been analyzed for the presence of CBD. The presented study was conducted by means of two different LC- MS/MS systems. Introduction Cannabidiol is popularly known as CBD, a substance that is part of the cannabinoids, chemical components extracted from the cannabis or hemp plant [1]. All natural and synthetic cannabinoids are prohibited in sport in-competition with one exception - cannabidiol - and are classified under section S8 of the WADA Prohibited List [2]. CBD can be consumed in multiple products, in drops of oil, processed foods, drinks, and other products that athletes can find in a supermarket or specialized sports store, and that is why the consumption of CBD has increased significantly among athletes. It is highly probable that the use of this group of preparations could cause unintentional violations of anti-doping regulations [3]. Moreover, CBD preparations from cannabis plants may contain additional minor cannabinoids [1,4,5]. CBD could become an alternative to non-steroidal anti-inflammatory drugs, opioids or corticosteroids [6]. The aim of the study was to analyze products containing CBD (hemp teas and oil) and urine samples after taking these preparations. Moreover, the laboratory tested 15 oils from dried hemp available on the commercial market. In addition, urine samples from routine doping control have been analyzed for the presence of CBD. The presented study was conducted by means of two different LC-MS/MS systems. 1 1 1 2,3 3 2 2,3 1,4 1 2 3 4 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0158 Experimental The samples were prepared using two different methods described below. Method 1 (for urine) The sample preparation is a two-step procedure involving enzymatic deconjugation of glucuronides and then liquid-liquid extraction with 6 mL of methyl tert-butyl ether. The residue was reconstituted in 100 µL of mobile phase (acetonitrile/water, 1/1, v/v), transferred in a vial, and then 2 µL was injected into the LC-MS/MS system. Method 2 (for tested oils) 10 µL of the test sample was weighed into a centrifuge tube. Next, 400 µL of isopropanol was added and the sample was vortexed for 30 seconds. After the dissolution of the sample, 400 µL methanol was added and the sample was vortexed again for 30 seconds. Finally, the sample was filtered through a 0.2 µm PTFE syringe filter into a brown glass HPLC vial. Instrumental analysis Figure 1. Chromatographic separation and MS settings MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0159 Results and Discussion Fifteen oils from dried hemp, available on the commercial market were tested. For the analysis of the cannabinoids, aliquots of the stock solutions were diluted with ethanol 10 and 100 times and injected in duplicates in randomized orders onto the LC-MS for analysis. Some of the 15 oils had a full spectrum of cannabinoids, as the producer declared, and some of them contained only isolates of selected cannabinoids (mainly CBD, and CBG). Olive oil was used as a blank sample. The results of the analysis are shown in Table 1. Table 1. Results of analysis of 15 oils – Method 2 and Instrumental Analysis II Three different products - two teas and one oil - were purchased (Figure 2). The studies were performed on three healthy volunteers (1 female and 2 males) who consumed the hemp products as recommended by the manufacturer for 3 consecutive days (Table 2). Two spontaneous urine samples were collected per day i.e. in the morning and evening (the first one 8 hours after the first administration). Urine samples were collected for 3 consecutive days after the first administration. What’s more, urine samples from routine doping control (male and female, in- and out- of- competition) were analyzed for the presence of CBD. Concentrations of cannabinoids in urine samples were not adjusted for specific gravity. Results of the analysis are shown in Table 3A and Table 3B. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0160 Table 3 . A – Results of analysis of urine samples from routine doping control – Method 1 and Instrumental Analysis 1, B – Results of analysis of urine samples after administration of hemp products (excretion study) and analysis of taken hemp products for cannabinoids – Method 1 and Instrumental Analysis 2 Conclusions The consumption of hemp products could lead to the detection of prohibited cannabinoids in urine. According to WADA, CBD is not banned, but commercial products often contain banned substances such as THC and other cannabinoids. All CBD and hemp products should be avoided to prevent accidental doping from these often contaminated products. References 1. Rojas-Valverde D, Fallas-Campos A. Cannabidiol in sports: insights on how CBD could improve performance and recovery. Front Pharmacol. 2023; 14: 1210202, doi: 10.3389/fphar.2023.1210202 2. World Anti-Doping Agency. The 2023 Prohibited List, https://www.wada-ama.org/en/resources/world-anti- doping-program/2023-prohibited-list 3. Mareck U, Fusshöller G, Schertel T, Petring S, Huestis MA, Thevis M. Risk of unintentional antidoping rule violations by consumption of hemp products. Drug Test Anal. 2023; 15:27-41. DOI: 10.1002/dta.3327 4. Mareck U., Fussholler G., Geyer H., Huestis M., Scheiff A. B., Thevis M. Preliminary data on the potential for unintentional antidoping rule violations by permitted cannabidiol (CBD) use. Drug Test Anal. 2021; 13:539- 549. DOI: 10.1002/dta.2959 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0161 5. Ramos-Guerrero L., Montalvo G., Cosmi M., Garcia-Ruiz C., Ortega-Ojeda F.E. Classification of Various Marijuana Varieties by Raman Microscopy and Chemometrics. Toxics 2022, 10, 115, https://doi.org/10.3390/toxics10030115 6. Gamelin F-X et al. Cannabidiol in sport: Ergogenic or else? Pharmacol Res. 2020 Jun:156:104764. doi: 10.1016/j.phrs.2020.104764. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0162 Wicka M , Drapala A , Konarski P , Grucza K , Siek P , Kopczynska M , Kwiatkowska D BPC-157 – case study Polish Anti-Doping Laboratory, Warsaw, Poland ; Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland Abstract BPC-157 is a stable gastric pentadecapeptide composed of 15 amino acids – Gly-Glu-Pro-Pro-Pro-Gly-Lys- Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val. It is an experimental compound that has been tested for the treatment of ulcerative colitis, multiple sclerosis, and acute liver lesions. According to the World Anti-Doping Agency (WADA) Prohibited List from 2022, BPC-157 is included in section S0 (non-approved substances). Nowadays, BPC-157 has become an easily available supplement that brings beneficial effects in sports, for example accelerated healing of muscle tissue and joints (e.g. after injuries), and improved functioning of the brain. The presented case study resulted from the investigation following the detection of GHRP-2 and its metabolite in a urine sample undergoing routine doping control analysis. The athlete declared the administration of only one supplement labeled BPC-157, which he delivered for further analysis. Analysis was conducted by means of ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Introduction BPC-157, or body-protective compound 157, is a peptide isolated from human gastric juice, composed of 15 amino acids – Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val [1,2]. It is an experimental compound, that has been tested for the treatment of ulcerative colitis, multiple sclerosis, and acute liver lesions [1,3]. BPC-157 is stable in human gastric juice, and thus it has good oral bioavailability (always given alone) [3]. It can also be administered intramuscularly and subcutaneously. Moreover, BPC157 has demonstrated remarkable potential as a therapeutic agent for severe trauma and stress damage and can promote the healing of wounds, ligament injuries, tendon injuries, and fractures [4]. The beneficial effects of its use may cause athletes more often to decide to use BPC-157. Hitherto, there is not enough information from clinical trials to assess the possible harmful effects of the use of BPC-157. Therefore, BPC-157 is included in section S0 (non-approved substances) of the WADA Prohibited List from 2022 [5]. The presented case study indicates possible consequences after the administration of the supplement containing BPC-157 (as of 2021, BPC-157 was not namely including on the list). In this case, it was the detection of substances from the WADA prohibited list - GHRP-2 and its metabolite. This paper describes also the validation of an LC-MS/MS method for the detection of BPC-157 in human urine. 1 1 1 1 1 1 1,2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0163 Experimental The samples were prepared using three different methods described below. Method A 200 µL of urine sample was transferred to 1.5 mL tube (Eppendorf), spiked with mildronate-d (internal standard, ISTD), and diluted with 800 µL of water. Samples were strongly centrifuged and 200 µL of supernatant was transferred to a vial. 10 µL was injected into the LC-MS/MS system. Method B 10 µL of ISTD ([Lys-8]-vasopressin, d -GRP 2 (1-3), FA) and 200 µL of phosphate buffer (1 M, pH 6.5) were added to 2 mL urine transferred to Protein LoBindTubes (Eppendorf), and centrifuged. Samples were loaded onto an Oasis WCX – microelution plate (2 mg, Waters), previously conditioned with 1 mL of methanol and 1 mL of water, and washed with 1 mL of water. Afterwards, the target base fraction analytes were eluted with 1 mL of 25% ammonium hydroxide in water/methanol (1:9, v/v), and the target acid fraction analytes were eluted with 1 mL of 10% formic acid in water/methanol (1:9, v/v) into a round 96-well collection plate (Waters). Both fractions have been evaporated (SpeedVac) and reconstituted in 200 µL of mobile phase (acetonitrile/water, 1/1, v/v). 10 µL was injected into the LC- MS/MS system. Method C 10 µL of ISTD ([Lys-8]-vasopressin, d -GRP 2 (1-3), FA) and 1 mL 2% of acetic acid in water were added to 1 mL urine transferred to Protein LoBindTubes (Eppendorf), and centrifuged. Samples were loaded onto an Oasis WCX – microelution plate (2 mg, Waters), previously conditioned with 1 mL of methanol and 1 mL of water, and washed with 2% carbonate buffer (0.5 mL, pH ca.11), 1 mL of water, and 1 mL of methanol. Afterwards, the target base fraction analytes were eluted with 1 mL of 10% formic acid in water/methanol (1:9, v/v) into a round 96-well collection plate (Waters). The fraction has been evaporated (SpeedVac) and reconstituted in 150 µL of mobile phase (acetonitrile/water, 1/1, v/v). 10 µL was injected into the LC-MS/MS system. Instrumental analysis Chromatographic separation was conducted by means of a Waters Acquity I-Class UPLC System liquid chromatograph with HSS T3 column (1.8 µm, 100 mm x 2.1 mm, Waters). The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B), and the LC gradient was employed at the constant flow rate of 300 µL/min at 45 °C. MRMs of the studied substances were traced with a Xevo TQ-S mass spectrometer equipped with a UniSpray source. All analytes were investigated in the US mode. Desolvation gas flow was set at 300 L/h at 500 °C with ion source temperature at 150 °C. The capillary voltage was 3.0 kV. Method validation The validation process was performed in accordance with the WADA technical document TD2023IDCR [6]. The method was verified for selectivity, linearity, limit of detection (LOD), limit of identification (LOI), intra-day precision, and carryover (Figure 1). 3 4 4 + MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0164 Figure 1. Characteristics of the method validation performed and mass spectrometry parameters for BPC-157 Results and Discussion The validation was conducted for three different methods and for two of them (A and C) correlation coefficients were > 0.997 (covering a concentration range from 0.1 ng/mL to 100 ng/mL), with LOD at 0.5 ng/mL (Figure 1). The results and parameters of the initial test procedure for the athlete's urine sample in which GHRP-2 and its metabolite were detected and the results and parameters for the confirmation procedure are shown in Figure 2. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0165 Figure 2. Mass spectrometry parameters for ITP and CP detection of GHRP-2 and its metabolite The laboratory received a vial of white lyophilized powder called BPC-157 for analysis. The concentration declared by the producer was 10 mg per vial and was intended for intramuscular injections. The actual concentration of BPC 157 in the received vial was not estimated in. The analysis of the supplement labeled BPC-157, which indicated the presence of not declared peptide GHRP-2, is shown in Figure 3. Figure 3. Supplement analysis for BPC-157 and GHRP-2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0166 A reanalysis of the athlete’s urine sample has not confirmed the presence of BPC-157 (Figure 4). Figure 4. Reanalysis of the urine sample after investigation of administration of the contaminated supplement named BPC-157 Conclusions According to the published data [7,8], it is unlikely to detect BPC-157 (parent compound) in urine. Therefore, for detecting the presence of BPC 157 or its metabolites, it is important to specify a period of time from peptide administration to sample collection. Based on pharmacokinetics, it would be worth to consider monitoring BPC-157 metabolites. The above case is another example that indicates the problem of contamination of permitted supplements with prohibited compounds. References 1. Sikiric P., Seiwerth S., Grabarevic Z., Rucman R., Petek P., Rotkvic I. et.al. Hepatoprotective effect of BPC 157, A 15-aminoacid peptide, on liver lesions induced by either restraint stress or bile duct and hepatic artery ligation or CCl4 administration. A comparative study with dopamine agonists and somatostatin. Life Sciences, v.53 (18), 1993, pages: PL291-PL296. 2. Jelovac N., Sikiric P., Rucman R., Petek M., Marovic A., Perovic D., Prkacin, I. Pentadecapeptide BPC 157 attenuates disturbances induced by neuroleptics: the effect on catalepsy and gastric ulcers in mice and rats. Eur. J. Pharmacol. 1999; 379(1), 19-31. 3. Vukojevic J., Milavic M., Perovic D., IIic S., Cillic A. Z., Duran N., Strbe S., Zoricic Z., Filipcic I., Brecic P., Siverth S., Sikiric P. Pentadecapeptide BPC 157 and the central nervous system. Neural Regen Res. 2022 Mar; 17(3): 482-487. 4. Lei He et. al. Pharmacokinetics, distribution, metabolism, and excretion of body-protective compound 157, a potential drug for treating various wounds, in rats and dogs. Front Phrmacol 2022; 13: 1026182. doi: 10.3389/fphar.2022.1026182 5. World Anti-Doping Agency. The 2023 Prohibited List, https://www.wada-ama.org/en/resources/world-anti- MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0167 doping-program/2023-prohibited-list 6. The World Anti-Doping Agency (WADA). Minimum Criteria for Chromatographic-Mass Spectrometric Confirmation of The Identity of Analytes for Doping Control Purposes. Available online: https://www.wada- ama.org/en/resources/lab-documents/td2023idcr 7. Cox HD, Miller GD, Eichner D. Detection and in vitro metabolism of the confiscated peptides BPC 157 and MGF R23H. Drug Test Anal. 2017 Oct;9(10):1490-1498. 8. Lei He, et. al. Pharmacokinetics, distribution, metabolism, and excretion of body-protective compound 157, a potential drug for treating various wounds, in rats and dogs. Front. Pharmacol., 2022; doi: https://doi.org/10.3389/fphar.2022.1026182 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0168 Ahi S, Wolff K An overview of doping control analysis at the Birmingham 2022 Commonwealth Games King's College London, Drug Control Centre, London, United Kingdom Abstract Doping control analysis is an integral part of any sporting event. The XXII Commonwealth Games took place in Birmingham, United Kingdom between 28 July and 8 August 2022. The collaborative work and effective communication between Commonwealth Games Federation, UK Anti Doping (UKAD) and the Drug Control Center (DCC) made the event a success. This presentation would summarize the doping control analysis performed by the DCC, King’s College London as per the guidelines stipulated in the World Anti Doping Agency (WADA) International Standard for Laboratories (ISL, 2021) incuding details of laboratory infrastructure, staffing, training, methods and result management. The DCC operated continuously during the games period to provide services as per an agreed turn-around-time (TAT). Of all the samples tested during the event the presence of some prohibited substances was confirmed, resulting in 1 atypical finding (ATF) and 4 adverse analytical findings (AAFs) which would also be discussed. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0169 Al-Qahtani A , Abushareeda W , Vonaparti A , Kraiem S , Elsaftawy W , Saad K , Al-Wahaibi A , Hilal N , Dbes N , Nandlal V , Beotra A , Al Maadheed M Dope Testing: Overview of FIFA World Cup 2022 Doping Analysis Lab, Antidoping Lab Qatar, Doha, Qatar ; Institute of Doping Analysis and Sports Biochemistry (IDAS) - Dresden, Kreischa, Germany Abstract The FIFA World Cup, one of the major football events in the world was played in the State of Qatar in 2022 (November 18 to December 19 ). For the first time in history, the games were hosted in one of the GCC countries, the State of Qatar. A stringent Test Distribution Plan (TDP) was implemented by the FIFA Medical Committee in agreement with the requirements of the World Anti-Doping Agency (WADA). A total of 1612 samples (623 urine and 622 blood) were collected during in and out of competition. In addition, a total of 367 blood samples were collected for the hematological passport (ABP) during out of competition only and were analyzed within 24 hrs. Additional analysis for erythropoiesis stimulating agents (ESAs), small peptides and IRMS were carried out as per the requests received. The Test Distribution Plan (TDP) for the FIFA World Cup 2022 was challenging as there was an increased number of tests for human growth hormone (hGH) and the Athlete Biological Passport (ABP). The reporting of results was done within 24 to 72 hours for all the samples including ESAs analysis. A strict chain of custody was followed by the FIFA medical doctors/DCOs to hand over the samples to ADLQ to analyze athlete samples and safeguard the integrity of the samples. The FIFA Medical Committee was prompt in responding to any irregularity or clarification requested by the laboratory related to the samples. Figure 1 shows the distribution of samples received each day (1a) and the comparison of hGH isoform tests performed in various Olympic games with FIFA 2022 (1b). A thorough risk assessment and its mitigation was done prior to the event to ensure effective controls for a high number of hGH isoform test (622) and the overall conduct of the testing for the event. However, proper planning and collaboration together with the FIFA Medical Committee led to successful completion of testing. In this paper, an overview of the logistics, challenges, and statistics of FIFA World Cup 2022 testing was presented. The laboratory handled the event as per the scope of accredited methods by ISO/IEC 17025:2017 [1] and in accordance with the WADA International Standard for Laboratories (ISL [2]). The testing of the event was supported by 27 permanent scientists, 9 temporary scientific staff, 2 secretaries and 2 laboratory assistants. The Laboratory was supported by 6 international scientific experts from other WADA-accredited laboratories. The international experts were chosen to complement the existing staff and allow high-level scientific expertise during the event. 1 1 2 1 1 1 1 1 1 1 1 1 1 2 th th MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0170 Figure 1. a) Distribution of samples and analysis performed during in an out of competition, b) comparison of hGH isoform tests performed in various Olympic games with FIFA 2022 References 1. SO/IEC 17025:2017. General requirements for the competence of testing and calibration laboratories 2. WADA International Standard for Laboratories (ISL) https://www.wada-ama.org/en/resources/laboratories/international-standard-for-laboratories-isl) MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0171 Al-Mohannadi I , Samsam W , Al-Darwish S , Cherif A , Al-Jaber M , Abushreeda W , Al-Qahtani A , Kraiem S , Al-Mohammed H , Farooq A , Nimker V , Stuart M , Robinson N , Zangenfeind G , Rovira D , Beotra A , Al-Maadheed M , Mohamed-Ali V First drone delivery of anti-doping samples: safe, secure, rapid, reliable and robust Doping Analysis Lab, Antidoping Lab Qatar, Doha, Qatar ; Aspetar - Qatar Orthopaedic and Sports Medicine Hospital, Doha, Qatar ; International Testing Authority, Lausanne, Switzerland ; Rigi Technologies, Prilly, Switzerland ; Centre for Metabolism and Inflammation, Division of Medicine, University College London, London, UK Abstract As drone technology advances, its diverse applications, from medical logistics to parcel delivery, have rapidly expanded. However, transporting anti-doping samples during major sports events still needs to be explored. Although drones promise speed and efficiency, concerns regarding the stability of biochemical and haematological parameters post-transport, especially under adverse conditions, remain. The potential and challenges of drone applications in medical logistics are evident from contemporary research, including studies by Zailani et al. (2021) and Johannessen (2022). In this collaborative project between the International Testing Authority (ITA), Anti-Doping Lab Qatar (ADLQ), and Rigitech, a Swiss start-up, the study investigated the efficacy and compliance of drone delivery with the World Anti-Doping Agency (WADA) Code & WADA International Standard for Testing and Investigations (ISTI) requirements. The central objective of this research was to validate the hypothesis that drone transport does not compromise the integrity of blood, urine, and DBS samples, in alignment with Article 9.3 of the WADA ISTI. The study lasted four days, during which Eiger VTOL drones from Rigitech were used to transport samples in temperature-regulated compartments over distances ranging from 5 km to 20 km. Samples obtained from 47 healthy volunteers (including three females) before and after ingesting caffeine or placebo tablets were transported by either car or drone. The study was authorized by a nationally registered ethical committee in Doha, Qatar (IRB-AOSM-2020-007). In this research, the evaluation of blood parameters was conducted using Sysmex. Gas and liquid chromatography, along with mass spectrometry, were employed to detect steroid profiles and caffeine levels. Additionally, Metabolon's hardware and software aided in the analysis of metabolites through Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). Key results indicated that the drone was twice as fast as the car for short distances, and three times faster for a longer distance. Most haematological and clinical chemistry profiles retained consistency post-transport, irrespective of mode. The haematological factors and urinary steroid profile were stable, independent of mode of transport, over both distances (5 and 20 km). For example, when investigating differences between car and drone in testosterone, the p-values for the 5 km and 20 km groups were 0.11 and 0.13, respectively. For epitestosterone, the values were 0.48 and 0.65; for HGB, the values were 0.78 and 0.52. Following metabolomics analysis, concentrations of most metabolites (drone: 92%, 1 1 1 1 1 1 1 1 1 2 1 3 3 4 4 1 1,5 1,5 1 2 3 4 5 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0172 car: 83%) were stable regardless of the means of transport. Thus, for the current testing compounds, drone delivery seems a secure, faster and environmentally safer mode of transport. Multi-omics data, metabolites in this study, also seem largely stable but needs further validation. In conclusion, integrating drone transport with both the current and advanced analytical techniques offers promising avenues for future anti-doping measures. However, comprehensive research, accounting for diverse conditions and sample types, remains crucial. References 1. Zailani, M.A., Azma, R.Z., Aniza, I. et al. Drone versus ambulance for blood products transportation: an economic evaluation study. BMC Health Serv Res 21, 1308 (2021). 2. Johannessen KA. A Conceptual Approach to Time Savings and Cost Competitiveness Assessments for Drone Transport of Biologic Samples with Unmanned Aerial Systems (Drones). Drones. 2022; 6(3):62. 3. Perlee D, van der Steege KH, den Besten G. The effect of drone transport on the stability of biochemical, coagulation and hematological parameters in healthy individuals. Clin Chem Lab Med. 2021;59(11):1772- 1776. 4. Amukele TK, Hernandez J, Snozek CLH, et al. Drone Transport of Chemistry and Hematology Samples Over Long Distances. Am J Clin Pathol. 2017;148(5):427-435. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0173 Brooker L , Shimko K , Speers N , Goebel C , Thomas K Summary and evaluation of the Australian PIED wastewater-based epidemiology collaboration Australian Sports Drug Testing Laboratory (ASDTL), National Measurement Institute, North Ryde, Australia ; Queensland Alliance for Environmental Health Sciences, University of Queensland, Brisbane, Australia ; Science and Medicine, Sport Integrity Australia, Canberra, Australia Abstract Wastewater-based epidemiology (WBE) is a proven methodology that can provide spatial and temporal information on the use of and exposure to chemicals within the general population. It has been suggested that the illegal use of performance and image enhancing drugs (PIEDs) such as anabolic steroids and SARMs is increasing in general society. In this collaboration between the Queensland Alliance for Environmental Health Sciences (QAEHS, University of Queensland), Sport Integrity Australia (SIA) and the Australian Sports Drug Testing Laboratory (ASDTL), WBE methodology was developed and validated for AAS and SARMs, and applied to authentic archived wastewater samples to provide an insight into the emergence and occurrence of PIED use in Australia. To evaluate the results from this comprehensive project and to reflect on the effectiveness of modifications to initial testing procedures (ITP) over time at the ASDTL, reported findings from non- athletes (so-called workplace samples) over the period 2011-2022 were collated and tabulated graphically. For traditional S1.1 class substances such as testosterone, nandrolone, metandienone, trenbolone, stanozolol and clenbuterol, overall trends to fewer reported AAFs in recent years could be seen. This was potentially due to reduced sample numbers submitted for analysis through the COVID pandemic period or possibly users were moving to other forms of PIED use. When substances such as SARMs and also related PIEDs like ibutamoren (MK677) and cardarine (GW1516) were inspected, distinct trends could be observed. The first detections occurred from 2015 onwards, peaking through 2017 and 2019, with a decline through the years 2022-2022 (Figure 1). These trends from the findings of non-athletes urine samples analysed at ASDTL correlate remarkably well with the obtained WBE results. It can be noted that ASDTL did not report any enobosarm (ostarine) through the years 2011-2015 though WBE has shown that this substance was in use in the Australian community (Figure 1, yellow box). However, for more recent substances such ligandrol (LGD4033) it seems as if the laboratory was able to implement the appropriate target compound into the ITP prior to widespread use in the community starting. Lastly, it can be remarked how trends in analytical findings at anti-doping laboratories, especially for non-athlete samples, is useful information for others in the academic field of the study of the illegal use of performance and image enhancing drugs. 1 2 3 1 2 1 2 3 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0174 Figure 1. A side-by-side comparison of ASDTL non-athlete findings (2011-2022) and wastewater-based epidemiology results (2009-2021) for SARMs and related PIEDs This work has been published in the following articles: K. Shimko, T. Piatkowski, K. Thomas, N. Speers, L. Brooker, B. Tscharke, J. O’Brien. (2021) Performance and image enhancing drug use in the community: use prevalence, user demographics and the potential role of wastewater-based epidemiology. Journal of Hazardous Materials, 419, 126340 (1-12). K. Shimko, J. O’Brien, J. Li, B. Tscharke, L. Brooker, P.K. Thai, P.M. Choi, S. Samanipour and K. Thomas. (2022) In-sewer stability assessment of anabolic steroids and selective androgen receptor modulators. Environmental Science and Technology, 56, 3, 1627-1638. K. Shimko, J. O’Brien, X. Lin, B. Tscharke and K. Thomas. (2023) In-Sample Stability of 52 Performance- and Image-Enhancing Drug Biomarkers in Wastewater, ACS EST Water, 3, 3, 669–678. K. Shimko, J. O’Brien, B. Tscharke, L. Brooker, C. Goebel, R. Shiels, N. Speers, J, Mueller, K. Thomas. (2023) Emergence and Occurrence of Performance-Enhancing Substance Use in Australia Determined by Wastewater Analysis. Nature Water, in press. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0175 Torres C , Oliveira F , Jooris LF , Padilha M , Pereira H The presence of doping agents in dietary supplements: A glimpse into the Brazilian situation Chemistry Institute - UFRJ, Rio de Janeiro, Brazil ; Bazilian Doping Control Laboratory - LBCD, Rio de Janeiro, Brazil Abstract Dietary supplements (DS) are intended for healthy people to maintain or improve their overall health. According to the Brazilian Health Regulatory Agency (ANVISA), DS are products that are orally taken to provide nutrients, substances, enzymes, or probiotics missing in diets. Its consumption is widespread in a large part of the general population and at all levels of athletes aiming to optimize physical performance. Nevertheless, the DS intake can also pose health risks and may lead to Adverse Analytical Findings (AAF) due to the possibility of its contamination or adulteration with doping agents. Recent studies have shown an increasing number of doping cases associated with, a priori, the ingestion of contaminated DS with prohibited substances [1–3]. The detection of such substances in athletes' samples is considered a violation of the World Anti-Doping Code [4]. In this context, to raise awareness regarding the risks associated with DS consumption by athletes, this work aims to present the descriptive data concerning the presence of prohibited substances [5] in DS products reported by the Brazilian Doping Control Laboratory (LBCD) after Testing Authorities (TAs) analysis requests. All analyzed DS samples were related to AAF cases, and product contaminations were claimed during the results management or in the sports tribunal to justify the adverse findings. All DS samples (n=140) received by the LBCD between 2017 and 2022 (up to May) were included in the study. Most of the samples were presented as solid contents (capsules, tablets, and powder). For liquid samples, 1 mL was transferred and diluted in 9 mL of the same solvent. The materials were extracted with 10 mL of methanol. An aliquot of 1 ml of the methanolic solution was used for the analyses. The sample extracts were screened by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high resolution mass spectrometry (LC-HRMS), using similar method conditions applied to the anti-doping samples routine analysis. The findings of DS analyzed by the LBCD showed an alarming number of tainted samples. Diuretics were the most common adulterants found in all supplement types. However, the profile of prohibited substances in manufactured and compounded dietary supplements (MDS and CDS, respectively) were distinct, with stimulants being most prevalent in MDS, mainly due to the slimming product profiles and anabolic agents in CDS products. Additionally, MDS samples generally presented higher estimated concentrations of banned substances (mg/g), while CDS samples revealed lower estimated concentrations of prohibited substances (µg/g). The common practice of DS intake by athletes continues to be of great concern for a doping-free sport, given the high prevalence of prohibited substances detected in the analyzed samples by the LBCD. The current outlook regarding detecting doping agents in supplement samples reinforces the importance of raising awareness for the sports community of the possible consequences of an inadvertent doping case linked to DS use. 1 2 2 2 2 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0176 The topic of this workshop presentation was published as: Torres CL, de Oliveira FAG, Jooris LF, Padilha MC, Pereira HMG. The presence of doping agents in dietary supplements: A glimpse into the Brazilian situation. Drug Test Anal. 2024 Jan;16(1):38-48. doi: 10.1002/dta.3517. References 1. V. R. Kozhuharov, K. Ivanov, S. Ivanova. Dietary Supplements as Source of Unintentional Doping. Biomed Res. Int., 2022, 2022, 8387271. 2. U. Mareck, H. Geyer, T. Schertel, S. Petring, O. Krug, M. Thevis. Detection of undeclared doping substances in nutritional supplements in the context of follow-up investigations concerning adverse analytical findings. Drug Test. Anal., 2021, 13, 1911–1914. 3. N. M. Mathews. Prohibited Contaminants in Dietary Supplements. Sports Health, 2018, 10, 19–30. 4. World Anti-Doping Agency - WADA. “World Anti-Doping Code,” available at: https://www.wada-ama.org/sites/default/files/resources/files/2021_wada_code.pdf, 2021. 5. World Anti-Doping Agency. “The 2022 prohibited list,” available at: https://www.wada-ama.org/en/news/wada-publishes-2022-prohibited-list, 2022. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0177 Rubio A , Thomas A , Euler L , Geyer H , Krug O , Reis G , Costa Padilha M , Pereira HMG , Muniz- Santos R , Cameron LC , Stojanovic B , Kuehne D , Lagojda A , Mc Leod MD , Thevis M Investigations into Annona fruit consumption as a potential source of dietary higenamine intake in the context of sports drug testing Laboratory Medicine, Hospital Universitario Son Espases, Palma, Spain ; Center for Preventive Doping Research - Institute of Biochemistry, German Sport University, Cologne, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne / Bonn, Germany ; Brazilian Doping Control Laboratory (LBCD - LADETEC / IQ - UFRJ) - Chemistry Institute, Rio de Janeiro, Brazil ; Laboratory of Protein Biochemistry, The Federal University of State of Rio de Janeiro, Rio de Janeiro, Brazil; Seibersdorf Labor GmbH, Seibersdorf, Austria ; Bayer AG, Crop Science Division, Bayer AG, Monheim, Germany ; Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory, Australia Abstract Higenamine is a non-selective β -agonist prohibited in sports at all times by the World Anti-Doping Agency (WADA). Since it has been described as a key component of a great variety of natural plants, including the Annonaceae family, one aim of this research project was to evaluate whether the ingestion o f Annona fruit could lead to higenamine adverse analytical findings (AAF) in sports. Single-dose administration studies including three of the most widely consumed Annona species (i.e. A. muricata, A. cherimola, and A. squamosa) were conducted, leading to higenamine findings below the established minimum reporting level (MRL) of 10 ng/mL in urine, and supporting the position that single-dose administrations, considering the ingested amount, are rather unlikely to lead to an AAF in sports. In consideration of C values (7.8 ng/mL) and t values (up to 24 h) observed for higenamine after single-dose administration of Annona fruit, a multidose administration study was also conducted, indicating cumulative effects under the chosen conditions, and thus increasing the possibility of an AAF for higenamine by Annona fruit ingestion. However, the MRL was also not exceeded at any time point. Further, as both administration studies showed a major urinary excretion of higenamine in its conjugated forms, especially at the expense of the sulfo-conjugates, and the MRL refers to the free compound only, the estimation of contributions of higenamine phase-II metabolite to the substance´s elimination was assessed by enzymatic hydrolysis. The contribution of the glucuronide conjugates represented only 1.7– 2.5% of the sulfo-conjugates, and the unconjugated form represented 1.1–1.9% of the sulfo-conjugates, corroborating the major urinary excretion of higenamine in its sulfo-conjugated form. In the light of the herein presented results, a stability study concerning higenamine and its intact major phase-II metabolite (higenamine sulfate) in urine was performed. Higenamine was found to be stable under all storage conditions for up to 90 days, except for one volunteer at room temperature, and +4 °C. Higenamine sulfo-conjugate was found to be stable under all storage conditions at all different studied times, indicating that it does not liberate higenamine over time. Therefore, higenamine sulfo-conjugates were also considered optimal biomarkers for indicating higenamine intake. In the absence of reference material, higenamine sulfo-conjugates were synthesized and comprehensively characterized by means of LC-HRMS(/MS) and H-NMR, suggesting the predominant presence of higenamine 7-sulfate. 1 2 2 2,3 2,3 4 4 4 5 5 6 7 7 8 2,3 1 2 3 4 5 6 7 8 2 max max 1 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0178 In addition, due to the reported potential of higenamine-containing sports supplements to result in anti- doping rule violations, the option to include complementary biomarkers of diet-related higenamine intake into routine doping controls was also investigated. This research was based on the monitoring of intermediate compounds of the benzylisoquinoline alkaloids (BIAs) biosynthetic pathway. For that purpose, urine samples collected after single- and multi-dose administration of three different higenamine-containing supplements were also evaluated. A characteristic urinary pattern attributed to isococlaurine, reticuline, and yet not fully characterized bismethylated higenamine glucuronide was observed after Annona fruit ingestion but not after supplement use (Figure 1), providing a promising dataset of a total of five urinary biomarkers, which supports the discrimination between different sources of urinary higenamine detected in sports drug testing programs. Published as: Rubio A, Thomas A, Euler L, Geyer H, Krug O, Reis G, Padilha MC, Pereira HMG, Muniz-Santos R, Cameron LC, Stojanovic B, Kuehne D, Lagojda A, McLeod MD, Thevis M. Investigations into Annona fruit consumption as a potential source of dietary higenamine intake in the context of sports drug testing. Drug Test Anal. 2023 Nov-Dec;15(11-12):1488-1502. doi: 10.1002/dta.3558. Figure 1. Extracted ion chromatograms with diagnostic precursor-product ion pairs for the target analytes (MS/MS experiment) are shown as an example for one volunteer. The data originated from the analyses of urine samples collected before application (A) and 8 h following single-administration of supplement 1 (B), supplement 2 (C), A. muricata (D), or A. cherimola (E). Noteworthy differences in the urinary metabolite pattern were observed after Annona fruit ingestion when monitoring specific glucuronide conjugates such as the tentatively identified isococlaurine-, reticuline-, and N,X-bismethylhigenamine glucuronides MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0179 Kwon OS , Rahaman KA , Muresan AR , Xu Y , Jeon MJ , Kim MY , Jung S , Chin AL , Min H , Kim HJ , Sung C , Kim KH , Lee KM , Kang MJ , Lee J , Son J The urinary metabolite Ac-Tβ in huvec cells and rats treated with thymosin β4: A potential biomarker for doping tests Doping Control Center, Korea Institute of Science and Technology (KIST), Seoul, South Korea ; Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul, South Korea ; Center for Advanced Molecular Recognition, KIST, Seoul, South Korea Abstract Thymosin β4 (Tβ4) was reported to exert various beneficial bioactivites such as tissue repair and regeneration, anti-inflmmation, and reduced scar formation, and it is listed on the prohibited substnaces by the World Anti-Doping Agency. No metabolism studies of Tβ4 were not reported yet. Previously, our lab reported that total 13 metabolites were found by using multiple enzymes, and 6 metabolites (Ac-Tβ31-43, Ac-Tβ 1-11, Ac-Tβ 17-43, Ac-Tβ 1-14, Ac-Tβ 1-15, and β1-17) were confirmed by comparing with the synthetic standards. The study was aimed at identifying new metabolites of Tβ4 in biological systems such as leucine aminopeptidase, human kidney microsomes, huvec cells and rats to develope biomarkers for detecting doping in sports. A method for detecting and quantifying Ac-Tβ1-14 was developed and validated using Q-Exactive orbitrap mass spectrometry. LOD and LOQ of the Ac-Tβ 1-14 were 0.19 and 0.58 ng/mL and showed a good linearity (r =0.9998). This work was conducted in in vitro pure enzyme and microsome systems, cultured huvec cells, and rats after administration of parent protein. Among 6 metabolites above, Ac-Tβ was found in huvec cells exposed to 10 μg/mL and in urine of rats intraperitoneally treated with 20 mg/kg Tβ4. As a result, Ac-Tβ , the metabolite of Tβ4, was detected in all in vitro biological systems including pure enzyme and microsome systems, huvec cells, and rat urine samples. In addition, the metabolite Ac-Tβ was quantitatively determined to 48 hr in rats, with the highest concentration occurred between 0- 6 hr. Ac-Tβ was not detected in non-treated control groups including human blank urine. This results suggest that Ac-Tβ in urine is a potential biomarker for screening the parent Tβ4 in doping tests. Published as: Rahaman KA, Muresan AR, Hasan ML, Joung YK, Min H, Son J, Kang MJ, Kwon OS. Detection and quantification of the metabolite Ac-Tβ in in vitro experiments and urine of rats treated with Ac-Tβ4: A potential biomarker of Ac-Tβ4 for doping tests. Drug Test Anal. 2023 Nov-Dec;15(11-12):1454-1467. doi: 10.1002/dta.3552. 1,2 1,2 1,2 1 1 1 1 1 1 1 1 1 1 2,3 1 1 1-14 1 2 3 2 1-14 1-14 1- 14 1-14 1-14 1-14 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0180 Colpaert T , de Hon O , Duiven E , Olijhoek M , T'sjoen G , van Eenoo P , Deventer K , Coppieters G , van Uytfanghe K Detection of thyroid hormones T3 and T4 in serum samples after T4 administration in healthy volunteers DoCoLab Universiteit Gent-UGent, Ghent, Belgium ; Doping Authority Netherlands, Capelle aan den IJssel, Netherlands ; Ghent University Hospital, Ghent, Belgium ; Laboratory of Toxicology - UGent, Ghent, Belgium Abstract Thyroid hormones triiodothyronine (T3) and thyroxine (T4) are iodine-containing hormones involved in a wide range of metabolic processes. Protein synthesis/catabolism, lipid catabolism, and cardiac output are all influenced by T4 and the more active, yet less abundant, T3. There are rumours that some athletes started supplementing these hormones to lose weight, due to their catabolic effect on fat mass. Currently, thyroid hormones are not mentioned on the World Anti-Doping Agency (WADA) Prohibited List, even though multiple anti-doping organizations have lobbied for their inclusion. To further broaden the knowledge on thyroid hormone supplementation in a doping-related context, an administration study was performed where 1 mg Levothyroxine was administered to six healthy volunteers. Urine, serum, and dried blood spot (DBS) excretion samples were collected up to seven days prior to T4 administration to evaluate baseline T3 and T4 concentration levels. Post-administration samples were collected to investigate the intensity and duration of potential changes in these levels. To analyse these samples, a quantitative LC-HRMS/MS-based method was developed and validated for serum. The same method, with slight changes to the extraction protocol, was validated for urine. Figure 1 provides an overview of all serum post-administration samples. Samples are marked in yellow if their value exceeded the 95% confidence interval (CI) upper limit (average + 2 * standard deviation of pre-administration samples) or in green if their value exceeded the 99.7% CI upper limit (average + 3 * standard deviation of pre-administration samples). On the left side of the table, CI values were calculated for each volunteer individually (like a biological passport), whereas on the right side, CI values were calculated for all six volunteers together (like a population). Not shown in Table 1 are the baseline (pre- administration) samples that were marked in yellow. Using an individual 95% CI, 1 serum T4 and 2 serum T3 values would be incorrectly marked as significantly elevated. Using a group-based 95% CI, 2 serum T3, and 2 serum T4/T3 ratio values would be incorrectly marked as significantly elevated. It is clear that T4 supplementation only affects serum T4 levels, with serum T3 levels remaining constant throughout the duration of the study. Since T4 is converted to T3 by enzymes (deiodinases) within target cells, T4 supplementation would only raise intracellular T3 concentrations, not seen in the bloodstream. Our findings support this claim. Both T4 and T4/T3 ratio appear to be acceptable metrics for detecting the administration of T4, showing significant elevations up to one day post-administration. Unfortunately, no later samples were collected, making it unknown how long the elevations last. Since a 95% CI threshold results in some false positives, a more strict 99.7% CI threshold must be used. Comparing the 1 2 2 2 3 1 1 1 4 1 2 3 4 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0181 findings from individually calculated thresholds (biological passport) and one single group-based threshold, the former results in a more sensitive method. Nonetheless, all volunteers had multiple true positives using the single population threshold, something which is more easily implemented in routine doping control. Figure 1. Results for post-T4 administration serum samples from six volunteers. Yellow and green bars respectively indicate samples exceeding the 95% and 99.7% confidence interval upper limit, calculated using individual (left) or group (right) baseline values Full paper citation: TBA MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0182 Krug O , Guddat S , Möller T , Piper T , Görgens C , Thevis M Determination of urinary deanol-N-oxide excretion profiles after ingestion of nutritional supplements containing deanol Center for Preventive Doping Research - Institute of Biochemistry, German Sport University, Cologne, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne / Bonn, Germany Abstract 2-(Dimethylamino)ethan-1-ol (Deanol) is a widely produced chemical used by both industry and consumers in a variety of applications. Meclofenoxate, a stimulant classified on the World Anti-Doping Agency Prohibited List, metabolizes into deanol and, presumably, its main metabolite deanol-N-oxide. Hence, using liquid chromatography-tandem mass spectrometry, a quantitative detection method for deanol-N-oxide in urine was developed. Subsequently, the urinary excretion of deanol-N-oxide after oral application of 130 mg of deanol was determined in 6 volunteers, and urine samples of a cohort of 180 male and female athletes from different sports were analyzed. In addition, urinary deanol-N-oxide was determined in an exploratory study with one volunteer ingesting 250 mg of meclofenoxate. The developed test method allowed for limits of detection and quantification for deanol-N-oxide at 0.05 and 0.15 µg/mL, respectively. Urinary deanol-N-oxide c levels were found between 100 and 250 µg/mL 2-5 h post-administration of 130 mg of deanol. Similarly, urine samples collected after the administration of 250 mg of meclofenoxate exhibited c levels of 115 µg/mL (Figure 1). In contrast, deanol-N-oxide urine concentrations of pre-administration specimens and 180 routine doping control urine sample were found between 0.3 and 1.3 µg/mL, and below LOQ and 1.8 µg/mL, respectively. The study suggests that the use of deanol and meclofenoxate result in significantly elevated urinary deanol-N-oxide levels. Whether or not monitoring deanol-N-oxide in doping controls can support decision- making processes concerning the detection of meclofenoxate use necessitates further investigations taking into consideration the elimination kinetics of 4-chlorophenoxyacetic acid, the main metabolite of meclofenoxate, and deanol-N-oxide. Figure 1. Urinary elimination profiles of deanol-N-oxide in 6 volunteers after ingestion of 130 mg deanol and one volunteer after ingestion of 250 mg meclofenoxate (red line) 1,2 1 1 1 1 1,2 1 2 max max MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0183 Published as: Krug O, Guddat S, Görgens C, Piper T, Möller T, Thevis M. Determination of urinary deanol-N-oxide excretion profiles after ingestion of nutritional supplements containing deanol. Drug Test Anal. 2023 Nov- Dec;15(11-12):1312-1318. doi: 10.1002/dta.3580. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0184 Santos V , Carneiro G , Braz B , Santelli R , Machado S , Pereira H Coordination chemistry applied to antidoping science: cobalt quantification as diethyldithiocarbamate complex by LC-HRMS Brazilian Doping Control Laboratory - LBCD, Rio de Janeiro, Brazil ; Chemistry Institute - UFRJ, Rio de Janeiro, Brazil Abstract Cobalt, known as a producer of a chemical or metal-induced hypoxia, was included in the WADA’s Prohibited List in 2015 due to its effect on stimulus of erythropoiesis via stabilization of hypoxia-inducible factor. Although its proven benefits as a performance enhancement agent [1,2], especially in endurance sports, relatively low attention has been provided to the cobalt issue on last years. One factor that discourage research and improvements on this topic is the unavailability of inductively coupled plasma– mass spectrometry (ICP-MS) in most of the WADA-accredited laboratories. Therefore, the main objective of the study was to develop and validate, based on the WADA guidelines, an analytical method for cobalt quantification as its diethyldithiocarbamate (C H NCS , DDC) complex by liquid-chromatography coupled to high resolution tandem mass spectrometry (LC-HRMS/MS). As cobalt is a naturally occurring trace element in the human body and its typical urinary concentrations are reported in ranges between 0.1 and 2 ng/mL [3], a study with ICP-MS was conducted to access possible matrix influences. Aliquots of homogenized urine samples from fifteen volunteers returned a cobalt concentration in the range of (0.23 – 2.72) ng/mL in the total fraction (i.e., including both sediments and its supernatant) and a range of (0.23 – 2.88) ng/mL only in the supernatant fraction. However, for one volunteer, approximately 52.5% (i.e., 1.58 ng/mL) of the measured cobalt were aggregated to sediments. Therefore, a sample digestion is highly recommended for cobalt quantification by LC-HRMS/MS to avoid possible matrix effects. In the LC-HRMS/MS analysis, palladium was proposed as internal standard and was included in the first step of sample preparation. A microwave-assisted digestion with purified concentrated HNO was essential to liberate cobalt from possible matrix components. After the samples’ mineralization, a neutralization and pH adjustment to pH 6, a complexation with DDC and an oxidation with citric acid were conducted. Then, the samples were subjected to the liquid-liquid extraction (LLE) with tert-butyl methyl ether, and, after centrifugation and separation of the organic phases, rhodium was included as a complexation control to guarantee that there was excess of ligand even after the complexation reaction. The reconstituted samples were injected in a Dionex Ultimate 3000 ultra-high performance liquid chromatography system coupled to a QExactive Plus hybrid quadrupole-Orbitrap mass spectrometer. As validation, the method showed adequate selectivity in differentiating typical urinary cobalt concentrations found in population in relation to supraphysiological levels. The limit of identification (LOI) was estimated in 0.21 ng/mL, the samples were stable in autosampler at 20 °C for at least 48 hours and the method was not prone to carryover with spiked cobalt concentration at 100 ng/mL. Also, the method presented equivalent performance for small changes in complexation, time for LLE and time for evaporation to dryness. For the quantitative confirmation, the limit of quantification (LOQ) was estimated 1 1 2 2 2 1 1 2 4 10 2 3 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0185 at 2.5 ng/mL, a coefficient of variation (CV%) of 7.76% was obtained on day 1 and 12.8% on day 2, resulting in an intermediate precision of 10.2%. Finally, a combined standard uncertainty (u ) of 11.4% was obtained for the developed quantification method. Therefore, the method for cobalt quantification in urine showed an adequate precision and accuracy and represents a feasible way to encourage more anti-doping laboratories to start monitoring this analyte in routine analysis. Also, a risk assessment from the international anti-doping system regarding the use of cobalt as doping agent becomes necessary, as well as the evaluation of a threshold value. For more information, please refer to: dos Santo, VF, Carneiro GRA, Braz BF, Santelli RE, de Paula Machado S, Gualberto Pereira HM. Coordination chemistry applied to anti-doping analysis: Cobalt quantification as its diethyldithio‐ carbamate complex by liquid chromatography coupled to high resolution tandem mass spectrometry. Drug Test Anal. 2023; 1-11. doi:10.1002/dta.3571 References 1. Hoffmeister T, Schwenke D, Wachsmuth N, Krug O, Thevis M, Byrnes WC, Schmidt WFJ. Erythropoietic effects of low-dose cobalt application. Drug Test Anal. 2019 Feb;11(2):200-207. doi: 10.1002/dta.2478 2. Hoffmeister T, Schwenke D, Krug O, Wachsmuth N, Geyer H, Thevis, M, Byrnes WC, Schmidt WFJ. Effects of 3 Weeks of Oral Low-Dose Cobalt on Hemoglobin Mass and Aerobic Performance. Front Physiol. 2018 Sep 19;9:1289. doi: 10.3389/fphys.2018.01289. 3. Unice KM, Monnot AD, Gaffney SH, Tvermoes BE, Thuett KA, Paustenbach DJ, Finley BL. Inorganic cobalt supplementation: prediction of cobalt levels in whole blood and urine using a biokinetic model. Food Chem Toxicol. 2012 Jul;50(7):2456-61. doi: 10.1016/j.fct.2012.04.009 Acknowledgements The authors thank CNPq (PhD scholarship: 162996/2018-7) and Autoridade Brasileira de Controle de Dopagem for financial support, and Juliana Scofano Barrabin for the statistical support. c MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0186 Paßreiter A , Naumann N , Thomas A , Grogna N , Delahaut P , Thevis M How to detect CRISPR with CRISPR - employing SHERLOCK for doping control purposes Institute of Biochemistry, German Sport University, Cologne, Germany ; Département Santé, CER Groupe, Marloie, Belgium Abstract The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) tool kit constitutes one of today’s most frequently used gene editing techniques. Editing of virtually any DNA sequence can be realised, due to the quickly progressing research into different Cas effectors and their ever-expanding range of targets. Moreover, the simplicity and cost-effectiveness of those CRISPR tools can, unfortunately, also facilitate the illicit utilisation of CRISPR/Cas in order to achieve performance enhancements amongst athletes. Consequently, there is an urgent need for the direct detection of illegally applied CRISPR/Cas methods in doping control samples, for which a promising strategy is presented herein employing Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) for targeted nucleic acid detection. An analytical method was developed that enables the detection of sgRNA associated with Cas9 from Streptococcus pyogenes (SpCas9) in serum samples by means of reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection via SHERLOCK in combination with a complementary gel-based screening procedure in order to uncover doping attempts with lipid mediated CRISPR ribonucleoprotein (RNP) complexes. Initial qualitative method characterisation confirmed the specificity of both procedures and established a detection sensitivity of 10 nM uncomplexed target sequence and 100 pM sgRNA in the form of RNP complexes. Furthermore, a proof-of-concept in vivo administration study simulating a hypothetical gene doping scenario employing a mouse model revealed a detection window of 8 h after intravenous injection, supporting the principal applicability of the test strategy to authentic doping control samples in the future and encouraging progressing method optimisation. Published as: Paßreiter A, Naumann N, Thomas A, Grogna N, Delahaut P, Thevis M. How to detect CRISPR with CRISPR - employing SHERLOCK for doping control purposes. Analyst. 2022 Nov 21;147(23):5528-5536. doi: 10.1039/d2an01318e 1 1 1 2 2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0187 Wagener F , Naumann N , Leciejewska N , Görgens C , Guddat S , Thevis M Using organ-on-a-chip for predicting the metabolism of the selective androgen receptor modulator RAD140 in comparison to established in vitro approaches Institute of Biochemistry, German Sport University, Cologne, Germany ; University of Life Sciences, Poznan, Poland ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne, Germany Abstract The identification of metabolites for the expansion of possible targets for anti-doping analysis is an important field of research to ensure the fairness of sporting competitions. Especially for novel substances such as selective androgen receptor modulators (SARMs), information on metabolic fate is scarce. Human in vivo experiments yield samples with the highest applicability for anti-doping analysis but require administration studies, which can be difficult to conduct with pharmacologically relevant doses. Animal models provide information about the metabolism of substances but can be of limited use due to inter-species variability in metabolism and should be reduced due to animal-welfare concerns. In vitro models using liver enzymes are accessible and fast ways to produce metabolites, yet lack the full complexity of human biology. Novel approaches such as the organ-on-a-chip technology may provide a metabolic profile that resembles human in vivo samples more closely than approaches that rely on human liver enzymes only. Liver spheroids formed using HepaRG cells and human stellate cells were cultivated in an organ-on-a-chip platform and subjected to the SARM RAD140. Samples were collected over a three-week period and analyzed using HPLC-HRMS/MS. The detected metabolites resulting from the organ-on-a-chip experiment were compared to a human urine sample containing RAD140 as well as in vitro samples after incubation with human liver enzymes. A total of 15 metabolites was detected during this study, including five formerly unknown biotransformation products. Both organ-on-a-chip and established in vitro approaches yielded metabolites also found in human urine. Because some metabolites were exclusively detected in samples of one of the employed techniques, the approaches can be viewed as complementary to one another. Published as: Wagener F, Naumann N, Göldner V, Görgens C, Guddat S, Karst U, Thevis M. Comparison of in vitro approaches for predicting the metabolism of the selective androgen receptor modulator RAD140. Anal Bioanal Chem. 2023 Sep;415(23):5657-5669. doi: 10.1007/s00216-023-04835-z. 1 1 2 1 1 1,3 1 2 3 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0188 Naumann N , Wapurgis K , Rubio A , Thomas A , Paßreiter A , Thevis M Detection of doping control sample substitutions via SNP-based ID typing Center for Preventive Doping Research / Institute of Biochemistry, German Sport University Cologne, Cologne, Germany ; Laboratory Medicine, Hospital Universitario Son Espases, Palma de Mallorca, Spain Abstract The authenticity of a doping control sample is a key element of sports drug testing programs. Doping control sample manipulation by providing another individual’s urine or blood (instead of the tested athlete’s sample) has been observed in the past and is an unequivocal violation of the World Anti-Doping Agency (WADA) anti-doping rules. To determine attempts of manipulations by sample swapping, the utility of a single nucleotide polymorphism (SNP)-based sample authentication with a multi-target SNP panel was assessed. The panel comprises detection assays for 44 different SNPs, three gender markers, and five quality control markers for DNA-profile determination. Sample analysis is based on a multiplex polymerase chain reaction (PCR)-step followed by a multiplex single base extension (SBE)-reaction and subsequent SBE-product detection by MALDI-TOF MS. Panel performance was evaluated for urine and dried blood spot (DBS) samples. Urine (8 mL) and DBS (20 µL) test samples were reliably typed and matched to whole blood reference samples, while efficient typing of urine samples correlated with sample quality and input amounts. Robust profiling of urine doping control specimens was confirmed with an assay input of 12 mL. Samples can be processed in a high-throughput format with an overall assay turnaround time of approximately 11 hours. SNP-based DNA typing via MALDI-TOF MS thus represents a high throughput-capable possibility for doping control sample authentication. SNP-profiling of samples could offer the opportunity to complement existing steroid profile analytics to substantiate sample manipulations and to support quality control processes in high throughput routine settings. Published as: Naumann N, Walpurgis K, Rubio A, Thomas A, Paßreiter A, Thevis M. Detection of doping control sample substitutions via single nucleotide polymorphism-based ID typing. Drug Test Ana l. 2023 Nov-Dec;15(11- 12):1521-1533. doi: 10.1002/dta.3597. 1 1 2 1 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0189 Euler L, Deinert K, Thomas A, Thevis M Mass spectrometric characterization and development of detection methods for in vitro synthesized metabolites of the troponin activators reldesemtiv and tirasemtiv for doping control purposes Institute of Biochemistry, German Sport University, Cologne, Germany Abstract The fast skeletal muscle troponin activators (FSTAs) reldesemtiv and tirasemtiv were developed for patients suffering from a non-curable degenerative disease of the motor nervous system (e.g. ALS). The drug candidates are able to increase the sensitivity of troponin C to calcium by selectively activating the troponin complex. This slows down the rate of dissociation of calcium from the troponin complex and sensitizes the sarcomere to calcium, resulting in increased skeletal muscle contraction. This effect of FSTAs on the human body could be abused by athletes to enhance performance. The aim of the present study was to identify and characterize metabolites of reldesemtiv and tirasemtiv in order to ensure their reliable identification in doping control analysis, if the class of substances is considered as relevant by the World Anti-Doping Agency (WADA). The biotransformation was studied in vitro using pooled human liver microsomes. The metabolites were analyzed by means of HPLC-HRMS. In total, six phase I and three phase II for reldesemtiv and three phase I and two phase II metabolites for tirasemtiv were formed by the liver microsomes. To obtain further information on the metabolic fate of reldesemtiv and tirasemtiv, in vitro experiments were carried out using the human hepatic cell line HepaRG. For a potential implementation in doping control analysis, a HPLC-MS/MS method was developed to detect reldesemtiv and tirasemtiv in urine. As a proof of concept, urine samples were spiked using in vitro-generated phase II metabolites and subsequently processed and analyzed according to the developed method. The details of this study will be published elsewhere. * The 2023 Manfred Donike Award for the best oral presentation went to Luisa Euler, graduate student at the Institute of Biochemistry of the German Sport University, Cologne. Mrs. Euler provided first information on the human metabolism of the troponin activators reldesemtiv and tirasemtiv, which belong to a new class of substances featured on the 2024 WADA Prohibited List. Knowledge about the metabolism and, hence, suitable target analytes for routine doping controls is vital for effective anti- doping work concerning new drugs and drug candidates. This information was produced thoroughly and presented comprehensively regarding phase-I and phase-II metabolism, allowing the implementation of these compounds into initial testing procedures in sports drug testing laboratories. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0190 Krombholz S , Thomas A , Delahaut P , Miller GD , Bidlingmaier M , Thevis M A combined top-down and bottom-up LC-HRMS/MS method for the quantification of human growth hormone in plasma Institute of Biochemistry, German Sport University, Cologne, Germany ; Departement Sante, CER Groupe, Marloie, Belgium ; Sports Medicine Research and Testing Laboratory, Salt Lake City, Utah, USA ; Ludwig-Maximilians-University, Medizinische Klinik?Innenstadt, Munich, Germany Abstract The precise determination of human growth hormone (GH) levels in serum/ plasma is crucial for the diagnosis and control of diseases like GH-deficiency or acromegaly, as well as in the field of sports drug testing. This can, however, be challenging due to the pulsatile character and the heterogeneous isoform composition of the protein, in addition to inter-assay variations in the ligand-binding assays (LBA) currently used for routine tests. Quantitative determination of proteins by top-down liquid chromatography-mass spectrometry (LC-MS) can provide a valuable alternative to LBA, offering improved specificity, selectivity and precision, as well as the opportunity to determine several analytes simultaneously. Here, we present a combined top-down and bottom-up LC-HRMS/MS approach for the quantification of human growth hormone in plasma. Extraction from 200 µL of plasma was achieved by protein precipitation, followed by an immunocapture step using Protein-A-coupled magnetic beads and a polyclonal anti-GH antibody. Analysis by 2D-LC-HRMS/MS on an orbitrap system enabled the selective quantification of the intact monomeric 22 kDa-Isoform of GH in a representative working range of 0.5 - 10 ng/mL. Subsequent bottom-up analysis of the eluate involving trypsin digestion provided additional data on total plasma GH and the second most abundant isoform 20 kDa GH. Growth hormone concentrations in plasma samples of healthy adults, as well as acromegaly patients and in samples obtained after administration of recombinant human growth hormone were determined using the presented approach, demonstrating the proof-of-principle and how the selectivity of this method can offer further information in case results obtained from routine immunoassays are contradictory or ambiguous. Published as: Krombholz S, Thomas A, Delahaut P, Bidlingmaier M, Schilbach K, Miller G, Thevis M. A combined top- down and bottom-up LC-HRMS/MS method for the quantification of human growth hormone in plasma and serum. Growth Horm IGF Res. 2023 Oct-Dec;72-73:101560. doi: 10.1016/j.ghir.2023.101560. 1 1 2 3 4 1 1 2 3 4 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0191 Möller T , Naumann N , Krug O , Thevis M Identification and synthesis of selected in vitro generated metabolites of the novel selective androgen receptor modulator (SARM) 2f Institute of Biochemistry, German Sport University, Cologne, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract Among anabolic agents, selective androgen receptor modulators (SARMs) represent a new class of potential drugs that can exhibit anabolic effects on muscle and bone with reduced side effects due to a tissue-selective mode of action. Besides possible medical applications, SARMs are used as performance- enhancing agents in sports. Therefore, they are prohibited by the World Anti-Doping Agency (WADA) in and out of competition. Since their inclusion into the WADA Prohibited List in 2008, there has been an increase in not only the number of adverse analytical findings, but also the total number of SARMs, making continuous research into SARMs an ongoing topic in the field of doping controls. 4-((2R,3R)-2- Ethyl-3-hydroxy-5-oxopyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile (SARM 2f) is a novel SARM candidate and is therefore of particular interest for sports drug testing. This study describes the synthesis of SARM 2f using a multi-step approach, followed by full characterization using liquid chromatography–high- resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR). To provide the first insights into its biotransformation in humans, SARM 2f was metabolized using human liver microsomes and the microsomal S9 fraction. A total of seven metabolites, including phase I and phase II metabolites, were found, of which three metabolites were chemically synthesized in order to confirm their structure. Those can be employed in testing procedures for routine doping controls, further improving anti-doping efforts. Published as: Möller T, Wen H.-C, Naumann N, Krug O, Thevis M. Identification and Synthesis of Selected In Vitro Generated Metabolites of the Novel Selective Androgen Receptor Modulator (SARM) 2f. Molecules. 2023 Jul; 28(14):5541. https://doi.org/10.3390/molecules28145541 1 1 1,2 1,2 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0192 Coll S , Bressan C , Alechaga E , Monfort N , Carbó M , Ventura R Elimination profile of methylprednisolone in urine and plasma after oral administration: Evaluation of the reporting level and washout period Catalonian Antidoping Laboratory Fundació Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain ; Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain Abstract Methylprednisolone (MP) is prohibited in sports competitions when administered by all injectable, oral or rectal routes, and is permitted at all times when dermatologically administered. A general minimum reporting level (MRL) of 30 ng/mL was initially established to discriminate between allowed and prohibited administrations of all glucocorticoids. Since 2022, new compound-specific MRLs have been established for some GC. For MP, although low concentrations were reported after dermatological applications, the MRL was maintained at 30 ng/mL because the sensitivities after oral and injectable administrations were considered adequate. The aim of the present work is to study the urinary and plasma profiles of MP after single and multiple oral administrations in order to provide additional data to evaluate the current regulations to detect MP misuse in sports. MP was administered to healthy volunteers using a single-dose oral treatment (12 mg, n=8 males) and multiple-dose oral treatment (12 mg/day for 3 days, n=8 males). Urine and plasma samples collected before and after administration were analysed using a liquid chromatography-tandem mass spectrometry method. The current reporting level and wash-out period used to detect MP misuse in sports will be evaluated by assessing the urinary profile of MP obtained after different oral administrations. Furthermore, MP and cortisol concentrations obtained in plasma will be presented and discussed. 1 1 1,2 1 2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0193 Alechaga E , Bressan C , Coll S , Carbó M , Monfort N , Ventura R Glucocorticoids and DBS I. Concentrations of glucocorticoids in DBS after oral administrations Catalonian Antidoping Laboratory Fundació Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain ; Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain Abstract There is an interest in the evaluation of alternative sampling techniques for anti-doping purposes. Dried Blood Spots (DBS) present several advantages compared to conventional doping control matrices such as urine and blood or serum, as they facilitate sample collection, storage and transport. Conversely, the reduced sample volume available represents a limitation to reach the necessary detection limits and requires the use of highly sensitive instrumentation. Another limitation is the difficulty for quantitation of drugs due various factors such as the heterogeneous spreading of blood on the support or the necessity of controlling spot volume. In this work, a quantitative method for the determination of dexamethasone (DEX) and methyl- prednisolone (MP) in DBS samples has been developed and validated. Sample preparation consisted of a two-step extraction procedure with methanol:methyl tert-butyl ether 1:4 and acetone. The developed method was applied to samples collected in clinical studies after oral administrations of these glucocorticoids to healthy volunteers. For DEX, single dose (4 mg, n = 8 males) or multiple dose (2x 2 mg/day, 5 days, n = 8 males) treatments were applied, while for MP, a single dose administration of 12 mg (n=8 males) was studied. In both cases, DBS samples from capillary blood were collected at different time points covering up to 3 days after administration and submitted to analysis. Results from quantitative validation were satisfactory in terms of linearity, accuracy and precision. The relevance of the different factors that might affect quantitation will be assessed. Elimination profiles of DEX and MP in DBS samples will be presented. In a following presentation, correlations of plasma and DBS concentrations will be discussed. 1,2 1 1 2 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0194 Ventura R , Bressan C , Coll S , Alechaga E , Carbó M , Monfort N Glucocorticoids and DBS II. Correlation of glucocorticoid concentrations in DBS and plasma samples Catalonian Antidoping Laboratory Fundació Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain ; Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain Abstract Among the different advantages that the use of dried blood sports (DBS) has for anti-doping analyses, the possibility to correlate DBS concentrations with plasma concentrations is one of the most relevant, specially when minimum reporting levels (MRL) have to be defined for specific groups for forbidden substances prohibited only in competition. There are several known factors that might explain differences between plasma and DBS concentrations for a particular compound, e.g., blood-to-plasma ratios of the analytes, hematocrit level, differences in composition of capillary and venous blood, etc. This work is a follow-up of previous studies presenting concentrations of dexamethasone (DEX) and methylprednisolone (MP) in plasma, DBS and urine samples obtained after oral administrations. Here, the correlation of the concentrations of the aforementioned glucocorticoids in plasma and DBS has been evaluated in samples collected in two clinical studies. DEX and MP were administered orally to healthy volunteers in single-dose (4 mg, n = 8 males, and 12 mg, n=8 males, respectively) and multiple-dose (2x2 mg/day, 5 days, n = 8 males, only for DEX) treatments. Plasma and DBS samples were collected at the same time points and analyzed by liquid chromatography coupled to tandem mass spectrometry. Correlation between the concentrations in both matrices will be evaluated. Statistical comparison of the results will be performed by paired t-test, Passing-Bablok regression and Bland-Altman plots. Finally, the comparability of plasma and DBS results according to different factors will be discussed. Based on the results obtained, MRLs for DEX and MP in DBS will be proposed. Results from the analysis of urine samples also collected during the same studies will be also evaluated to assess the correspondence of all matrices and the agreement with the defined MRLs. 1 1 1 1,2 2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0195 Langer T, Nicoli R, Kuuranne T, Musenga A Comparison of serum and urine analysis for the confirmation of oral testosterone undecanoate administration Laboratoire Suisse d'Analyse du Dopage Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Epalinges, Switzerland Abstract Uncovering doping practices with pseudo-endogenous substances is one of the most important challenges in the fight for clean competitions. The Athlete Biological Passport (ABP) has proven to be an invaluable tool for the indirect detection of this kind of doping practices. However, for the detection of testosterone doping, the longitudinal monitoring of the urinary steroid profile is not always specific enough to report adverse analytical findings (AAF). To increase the likelihood of a doping scenario, urine samples that are considered suspicious are most often confirmed with a method that is capable of directly and unequivocally identify exogenous origin of testosterone and its metabolites. For urine samples, this method is isotope ratio mass spectrometry (IRMS). However, despite its specificity, this process is tedious, resource- and time-consuming. Similar to the urinary steroid profile, endogenous steroids can be measured in serum samples, allowing longitudinal monitoring [1]. While it has been shown that the determination of the origin of endogenous steroids in serum is possible [2], this approach is very recent and not yet available for all laboratories. However, when testosterone is administered in the form of esters, the direct detection of the intact esters in serum samples by LC-MS/MS is currently a much simpler approach compared to IRMS analysis. We developed a method that is capable of analysing a simplified steroid profile containing androstenedione (A4), testosterone (T), and dihydrotestosterone (DHT) in serum as well as detecting steroid esters in the same sample aliquot [3]. Serum samples from a testosterone undecanoate (TU) administration study including 19 male volunteers [4] were analysed, highlighting the influence on the steroid profile and demonstrating the detectability of intact TU after the administration. During this administration study, urine samples were collected as well, so urinary steroid profiles could be established, and the urine samples of nine volunteers were analysed by IRMS according to the routine laboratory procedure [5]. All results were compared to propose an efficient process for the detection of oral TU administration. Steroid profiles in urine and serum were influenced depending on the amount of detected TU. While the urinary ratio of testosterone to epitestosterone (T/E) was generally a good indicator of the administration, the ratio of 5α-androstane-3α,17β-diol to epitestosterone (5αAdiol/E) was more indicative for the volunteers with the UGT2B17 del/del polymorphism. Serum steroid profiles were less affected than urinary steroid profiles with DHT being a better marker than T or T/A4. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0196 Figure 1. Comparison of average urinary IRMS results with the average TU concentration in serum after the administration. Serum TU is shown as black line, the Δ C-values of T, androsterone (A), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5αAdiol) and 5β-androstane-3α,17β-diol (5βAdiol) are indicated as bar-graphs and were calculated using pregnanediol (PD) as endogenous reference. The standard deviations are indicated in the table below the average values. The colour and pattern of the bars correspond to the background colour of the steroids in the table. Full-grey cells indicate that the average value was above the threshold, hatched grey cells indicate, that the average is below the threshold, but individual values were above. Results showed that, by analysing serum samples, we were able to detect intact TU up to 24 h post- administration, although with high inter-individual variability. For half of the volunteers, TU was detectable 12 h after the administration or even longer. In comparison, using IRMS on the corresponding urine samples, it was also possible to determine the exogenous origin of testosterone and its metabolites, leading to AAF up to 24 h after the administration and to atypical findings (ATF) up to 48 h after the administration. In conclusion, the detection window in IRMS is slightly longer than the detection 13 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0197 window of TU in serum, but directly proportional to the detected TU concentration. However, the workload needed for the two methods is very different. Steroid esters in up to 80 serum samples can be analysed within 1-2 days, while the IRMS analysis of just 8 samples takes a full week, giving a slightly extended detection window. In the case that the urinary steroid profile is flagged as atypical and both urine and serum samples are available, it could be beneficial to perform the fast and simple steroid esters analysis in serum by LC-MS/MS before considering IRMS analysis of steroids in urine. Published as: Langer T, Nicoli R, Schweizer-Grundisch C, Grabherr S, Kuuranne T, Musenga A. Comparison of analytical approaches for the detection of oral testosterone undecanoate administration in men. Drug Test Anal. 2024 Jan 11. doi: 10.1002/dta.3638. References 1. Ponzetto F, Mehl F, Boccard J, et al. Longitudinal monitoring of endogenous steroids in human serum by UHPLC-MS/MS as a tool to detect testosterone abuse in sports. Anal Bioanal Chem. 2016;408(3):705-719. 2. Piper T, Geyer H, Nieschlag E, Bally L, Thevis M. Carbon isotope ratios of endogenous steroids found in human serum-method development, validation, and reference population-derived thresholds. Anal Bioanal Chem. 2021;413(22):5655-5667. 3. Langer T, Salamin O, Nicoli R, Grabherr S, Kuuranne T, Musenga A. A comprehensive UHPLC-MS/MS method for the analysis of endogenous and exogenous steroids in serum for anti-doping purposes. Drug Test Anal. 2022;14(11-12):1904-1919. 4. Badoud F, Boccard J, Schweizer C, Pralong F, Saugy M, Baume N. Profiling of steroid metabolites after transdermal and oral administration of testosterone by ultra-high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. J Steroid Biochem Mol Biol. 2013;138:222-235. 5. Piper T, Emery C, Thomas A, Saugy M, Thevis M. Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sports drug testing. Anal Bioanal Chem. 2013;405(16):5455-5466. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0198 Thilmany S , Hardy T , Thomas A , Vogel M , Scholl C , Thevis M Transcriptomic analysis of the effects of steroid hormones on neuronal cells Institute of Biochemistry, German Sport University Cologne, Cologne, Germany ; Research Department, Federal Institute for Drugs and Medical Devices, Bonn, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract Introduction Steroid hormone use is still popular among athletes; according to the 2021 testing figures of the World Anti-Doping Agency (WADA), almost 900 samples tested positive for steroids [1]. Steroids are also widely used for medical reasons, e.g., hormonal contraception [2], hormone replacement therapy [3], or cancer treatment [4,5]. In addition to the desired therapeutic effect, steroids can cause numerous undesirable side effects. For instance, an influence of hormonal contraceptives on the development of mental illnesses has been controversially discussed for years [6–12], and several reports are suggesting a similar effect with corticoids [13,14]. Consequently, the entire class of steroids and steroid receptor ligands is suspected of negatively influencing users’ mental health. So far, the findings are based on clinical and epidemiological studies; to the best of our knowledge, no evidence has been obtained under controlled laboratory conditions. This project aims to investigate the influence of steroids on the transcriptome of neuronal cells. Methods Human neuroblastoma cells were treated with different steroid hormone receptor ligands in cell culture. Samples were collected over the duration of the treatment, and transcriptome analysis was performed using DNA microarrays. Statistical analysis of gene expression changes over time identified genes affected by the treatment. Results Significant changes in gene expression were detected for all steroid hormone receptor ligands tested. These differentially expressed genes support the theory that steroid receptor ligands may have a negative impact on mental health as comparisons with published data from depression research show that there are numerous similarities to already known potential depression markers. The most significant changes involved pathways of proliferation, the neuronal system, neuronal development, neuronal degeneration, neurotransmission, neuroinflammation, and the immune system. Outlook Since the proteome is closer to biological function and thus allows more precise conclusions to be drawn about changes in the organism and the mechanism of action of steroids, the transcriptome analysis results are to be expanded in the further course of the project by analyses at the proteome level. For this purpose, a bottom-up proteomics platform is currently being established, with the help of which the neuronal proteome can be relatively quantified by means of isobaric labeling. 1,2 2 1 2 2 1,3 1 2 3 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0199 References 1. World Anti-Doping Agency. 2021 Anti-Doping Testing Figures. Report. 2023. 2. United Nations and Department of Economic and Social Affairs and Population Division. World Contraceptive Use 2022. Dataset. 2022. 3. Deutsche Gesellschaft für Gynäkologie und Geburtshilfe (DGGG), Östereichische Gesellschaft für Gynäkologie und Geburtshilfe (OEGGG) und Schweizerische Gesellschaft für Gynäkologie und Geburtshilfe (SGGG). S3-Leitlinie Peri- und Postmenopause - Diagnostik und Interventionen. Version 2.3. Guideline 015- 062. 2020. 4. Leitlinienprogramm Onkologie der Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften e.V. (AWMF), Deutschen Krebsgesellschaft e.V. (DKG) und Deutschen Krebshilfe (DKH). S3-Leitlinie Früherkennung, Diagnostik, Therapie und Nachsorge des Mammakarzinoms. Version 4.4. Guideline 032-045OL. 2021. 5. Leitlinienprogramm Onkologie der Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften e.V. (AWMF), Deutschen Krebsgesellschaft e.V. (DKG) und Deutschen Krebshilfe (DKH). S3-Leitlinie Prostatakarzinom. Version 6.2. Guideline 043-022OL. 2021. 6. Gingnell, M. et al. „Oral contraceptive use changes brain activity and mood in women with previous negative affect on the pill–a double-blinded, placebo-controlled randomized trial of a levonorgestrel- containing combined oral contraceptive“. In: Psychoneuroendocrinology 38 (7). 2013. DOI: 10.1016/j.psyneuen.2012.11.006. 7. Skovlund, C. W. et al. „Association of Hormonal Contraception With Suicide Attempts and Suicides“. In: Am J Psychiatry 175 (4). 2018. DOI: 10.1176/appi.ajp.2017.17060616. 8. Skovlund, C. W. et al. „Association of Hormonal Contraception With Depression“. In: JAMA Psychiatry 73 (11). 2016. DOI: 10.1001/jamapsychiatry.2016.2387. 9. Toffol, E. et al. „Hormonal contraception and mental health: results of a population-based study“. In: Hum Reprod 26 (11). 2011. DOI: 10.1093/humrep/der269. 10. Hamstra, D. A. et al. „Oral contraceptives positively affect mood in healthy PMS-free women: A longitudinal study“. In: J Psychosom Res 103. 2017. DOI: 10.1016/j.jpsychores.2017.10.011. 11. Scheuringer, A. et al. „Use of an estradiol-based combined oral contraceptives has no influence on attentional bias or depressive symptoms in healthy women“. In: Psychoneuroendocrinology 113. 2020. DOI: 10.1016/j.psyneuen.2019.104544. 12. Duke, J. M. et al. „Is there an association between the use of oral contraception and depressive symptoms in young Australian women?“ In: Contraception 75 (1). 2007. DOI: 10.1016/j.contraception.2006.08.002. 13. Brown, E. S. et al. „Mood and Cognitive Changes During Systemic Corticosteroid Therapy“. In: Prim Care Companion J Clin Psychiatry 3 (1). 2001. DOI: 10.4088/pcc.v03n0104. 14. Brown, E. S. „Effects of glucocorticoids on mood, memory, and the hippocampus. Treatment and preventive therapy“. In: Ann N Y Acad Sci 1179. 2009. DOI: 10.1111/j.1749-6632.2009.04981.x. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0200 Breuer J , Thomas A , Delahaut P , Schänzer W , Geyer H , Thevis M Investigations into the concentration and metabolite profiles of stanozolol and LGD-4033 in blood plasma and seminal fluid using liquid chromatography high-resolution mass spectrometry Institute of Biochemistry, German Sport University, Cologne, Germany ; CER Groupe, Marloie, Belgium ; European Monitoring Center for Emerging Doping Agents, Cologne/Bonn, Germany Abstract Potential scenarios as to the origin of minute amounts of banned substances detected in doping control samples have been a much-discussed problem in anti-doping analysis in recent years. One such debated scenario has been the contamination of female athletes’ urine with ejaculate containing doping agents and/or their metabolites. The aim of this work was to obtain complementary information on whether relevant concentration ranges of doping substances are excreted into the ejaculate and which metabolites can be detected in the seminal fluid (sf) and corresponding blood plasma (bp) samples. A method was established to study the concentration and metabolite profiles of stanozolol and LGD-4033 – substances listed under anabolic substances (S1) on the World Anti-Doping Agency’s Prohibited List – in bp and sf using liquid chromatography high-resolution mass spectrometry (LC-HRMS). For sf and bp, methods for detecting minute amounts of these substances were developed and tested for specificity, recovery, linearity, precision, and reliability. Subsequently, sf and bp samples from an animal administration study, where a boar orally received stanozolol at 0.33 mg/kg and LGD-4033 at 0.11 mg/kg, were measured. The developed assays proved appropriate for the detection of the target substances in both matrices with detection limits between 10 and 40 pg/mL for the unmetabolized drugs in sf and bp, allowing to estimate the concentration of stanozolol in bp (0.02–0.40 ng/mL) and in sf (0.01– 0.25 ng/mL) as well as of LGD-4033 in bp (0.21–2.00 ng/mL) and in sf (0.03–0.68 ng/mL) post- administration. In addition, metabolites resulting from different metabolic pathways were identified in sf and bp, with sf resembling a composite of the metabolic profile of bp and urine. Published as: Breuer, J., Thomas, A., Delahaut, P. Schänzer, W., Geyer, H., Thevis, M. Investigations into the concentration and metabolite profiles of stanozolol and LGD-4033 in blood plasma and seminal fluid using liquid chromatography high-resolution mass spectrometry. Anal Bioanal Chem 415, 669–681 (2023). https://doi.org/10.1007/s00216-022-04456-y 1 1 2 1 1,3 1,3 1 2 3 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0201 Aguilera R Athlete isotopic signature of δ C values. Complementary data from testosterone gel application (males and females) Drug Control Centre King's College, London, United Kingdom Abstract Isotope Ratio Mass Spectrometry continues to be the most powerful technique to confirm the use of synthetic endogenous steroid in the doping sport arena. The technique is based on the fact that pharmaceutical steroids have a lower C ratio than endogenous naturally produced steroids. Today these isotope ratios of C/ C can be measured with high accuracy and precision. Nonetheless, there is a significant natural variation of d C values within different populations and analytical issues related to calibration and sample preparation within laboratories. This final research project was focused on the variable d C values reported from different analytical laboratories and the micro doses used during recovery time by athletes. Some of these differences can be due to differences in an individual’s steroid metabolism variation. In order to avoid these inconsistencies in d C values between analytical groups and individual populations we have established the Athlete Internal Metabolic Reference by use of an isotopic signature or isotopic fingerprint including the detection of testosterone gel. 13 13 13 12 13 13 13 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0202 Piper T, Krombholz S, Thevis M Carbon isotope ratios of phenethylamine and phenylacetylglutamine and their applicability to doping control Institute of Biochemistry, German Sport University, Cologne, Germany Abstract Phenethylamine (PEA) is a naturally occurring trace amine that acts as a modulator in the central nervous system. It is widely sold as a dietary supplement and advertised for its mood enhancing effects and should support weight loss. It is prohibited in sports and itemized as a stimulant on the Prohibited List issued by the World Anti-Doping Agency (WADA). After oral administration of PEA, its urinary concentration is found only slightly elevated while metabolites of PEA show a significant increase [1,2]. Besides 2-(2-hydroxyphenyl)acetamide sulfate, especially phenylacetylglutamine (PAG) was found at significantly elevated urinary concentrations after the administration [2]. Due to large inter- and intra- individual variations in urinary concentrations of all metabolites, establishing a concentration or concentration ratio-based threshold remained complicated to unambiguously identify post-administration samples [2]. In accordance with the approach employed in detecting testosterone misuse, the applicability of isotope ratio mass spectrometry to differentiate between endogenously elevated concentrations and PEA administrations was investigated. A method encompassing solid-phase extraction combined with acetylation and high-performance liquid chromatography (HPLC)-based clean- up was developed and validated for PEA. The more abundant metabolite PAG was purified by a direct injection approach on the HPLC and could be analysed without the need for derivatization. Both methods were validated considering applicable WADA regulations. A reference population was investigated to establish population-based thresholds considering the carbon isotope ratios (CIR) found at natural abundance for PAG. The derived threshold was tested for its applicability by re-analysis of numerous post-administration samples encompassing single- and multi-dose trials [2]. Published as: Piper T, Krombholz S, Thevis M. Carbon isotope ratios of phenethylamine and its urinary metabolite phenylacetylglutamine. Drug Test Anal. 2023 Dec 4. doi: 10.1002/dta.3616 References 1. G. Sigmund, J. Dib, L. Tretzel, T. Piper, C. Bosse, W. Schänzer, M. Thevis. Monitoring 2-phenylethanamine and 2-(3-hydroxyphenyl)acetamide sulfate in doping controls. Drug Test Anal 2015, 7, 1057-1062. 2. S. Krombholz, A. Thomas, T. Piper, A. Lagojda, D. Kühne, M. Thevis. Urinary phenylethylamine metabolites as potential markers for sports drug testing purposes. Biomedical Chromatography 2021; e5274. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0203 Nair V , Howa J , Kucherova Y , Ahrens B , Pereira HMG , Nicoli R , Kuuranne T , Fedoruk M Applicability of longitudinal monitoring of IRMS values - A case study with unusual values The Sports Medicine Research and Testing Laboratory (SMRTL), South Jordan, United States ; UCLA Olympic Analytical Laboratory, Los Angeles, USA ; Brazilian Doping Control Laboratory, Rio de Janeiro, Brazil ; Laboratoire Suisse d?Analyse du Dopage Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland ; United States Anti-Doping Agency, Colorado Springs, United States Abstract Longitudinal monitoring of IRMS values has been previously suggested as a way to increase the sensitivity of the IRMS assay. Accordingly, data from athletes who received multiple IRMS tests over a period of three years were evaluated longitudinally. The overall variability of the Δδ C values was low. However, possible dietary changes caused noticeable fluctuation in the absolute δ C values for some athletes. While these changes occur across all assay analytes, it is possible that there could be transient perturbation in the subject's Δδ C profile. It is extremely unlikely that these changes will exceed the threshold set in the IRMS technical document but may flag a sample in a longitudinal model. A possible application of longitudinal monitoring may be for samples where measured isotope values do not meet criteria for positivity according to the IRMS technical document but are inconsistent with physiological variability. A case study with multiple unusual isotope measurements will be presented. No exogenous substances were detected in urine but analysis of serum and DBS samples from the same athlete for the presence of exogenous steroids and esters produced suspicious but as yet inconclusive results. A satisfactory explanation for these results has not yet been elucidated. 1 1 2 2 3 4 4 5 1 2 3 4 5 13 13 13 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0204 Piper T, Thevis M Investigations into the human metabolism of ecdysterone Institute of Biochemistry, German Sport University, Cologne, Germany Abstract The possible performance-enhancing effects and medical benefits of ecdysterone (ECDY) have been discussed several times throughout the last decades [1-4]. In 2020, WADA decided to include ECDY in their monitoring program and continued this prevalence study until now. Only little is known about the metabolism of ECDY. The first study performed on human subjects in the field of sports drug testing was already conducted in 2001 [5]. Besides the parent compound, 2-deoxyecdysterone and deoxyecdysone were identified in post-administration urine samples. These results were recently corroborated by two different studies, adding 14-deoxy-poststerone (14DP) as a potential urinary ECDY metabolite to the scope of investigations [6,7]. Aim of this study was the in-depth investigation on human ECDY metabolism to improve its detectability and to support the decision-making processes as to how ECDY can be implemented most effectively into sports drug testing regulations. In a first trial, one male volunteer was administered with deuterated ECDY to enable the detection and potential identification of all urinary metabolites still comprising the deuterium label by employing hydrogen isotope ratio mass spectrometry and high resolution/high accuracy mass spectrometry [8]. Samples were collected for up to 14 days and metabolites excreted unconjugated, glucuronidated and sulphated were investigated separately. The detected deuterated metabolites were confirmed in a second administration trial encompassing 3 male and female volunteers. After the administration of 50 mg ECDY, urine samples were collected for up to 7 days. Besides the already described urinary metabolites of ECDY, more than 20 new metabolites were detected encompassing all expected metabolic conversions including the side chain cleavage described for 14DP. A significant inter-individual variation in the amounts of excreted metabolites was noted. Published as: Piper T, Thevis M. Investigations into the human metabolism of ecdysterone. Drug Test Anal. 2023 Nov- Dec;15(11-12):1503-1520. doi: 10.1002/dta.3582. References 1. Bucci LR. Selected herbals and human exercise performance. Am. J. Clin. Nutr. 2000, 72(suppl), 624S– 636S. 2. Le Bizec B, et al. Ecdysteroids: one potential new anabolic family in breeding animals. Anal. Chim. Acta 2002, 473, 89-97. 3. Simnadis TG, et al. Physiological Effects Associated with Quinoa Consumption and Implications for Research Involving Humans: a Review. Plant Foods Hum. Nutr. 2015, 70, 238–249. 4. Isenmann E, et al. Ecdysteroids as non-conventional anabolic agent: performance enhancement by ecdysterone supplementation in humans. Arch. Toxicol. 2019, 93(7), 1807-1816. 5. Tsitsimpikou C, et al. Study of excretion of ecdysterone in human urine. Rapid Commun. Mass Spectrom. 2001, 15, 1796-1801. 6. Kraiem S, et al. Analytical strategy for the detection of ecdysterone and ist metabolites in vivo in uPA(+/+)- SCID mice with humanized liver, human urine samples, and estimation of prevalence of ist use in anti- doping samples. Drug Test Anal. 2021, 13, 1341–1353. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0205 7. Ambrosio G, et al. Urinary Elimination of Ecdysterone and Its Metabolites Following a Single-Dose Administrations in Human. Metabolites, 2021, 11, 366. 8. Thevis M, et al. Hydrogen isotope ratio mass spectrometry and high-resolution/ high-accuracy mass spectrometry in metabolite identification studies: Detecting target compounds for sports drug testing. Rapid Commun. Mass Spectrom. 2013, 27, 1904–1912. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0206 de La Torre X, Colamonici C, Curcio D, Botrè F 19-Norandrosterone confirmation by GC-C-IRMS: 10 years of experience Laboratorio Antidoping FMSI, Federazione Medico sportiva Italiana, Rome, Italy Abstract The use of GC-C-IRMS in antidoping analysis to determine the origin of urinary 19-norandrosterone was introduced at the beginning of 2000s and implemented in WADA Technical documents in 2010. The method should be able to distinguish between 19-norandrosterone produced in vivo in specific physiological conditions or ex-vivo in unstable urine samples (the so-called active urines) where some microbial activity is suspected. As with many other IRMS methods, this is based on the principle that the in vivo or ex vivo “endogenous” delta values can be distinguished from the values obtained from the pharmaceutical preparations or dietary supplements containing nandrolone precursors potentially used by athletes. The method is time- consuming and requires a large volume of urine (usually > 15-20 mL) since the amounts to be detected are low ( < 2-5 mL). This is not a mandatory procedure to be available in all WADA accredited laboratories and can be subcontracted to another WADA laboratory. The work dealt with the evolution of the confirmation procedure for 19-NA by IRMS, described the experience of more than 150 analyses in our laboratory and raised some difficulties that the antidoping community may encounter. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0207 Piper T, Thevis M Employing 11-ketotestosterone as target analyte for adrenosterone (11OXO) administrations in doping control Institute of Biochemistry, German Sport University, Cologne, Germany Abstract The human metabolism of 11-ketotestosterone (11KT) was recently investigated to enable the detection of 11KT administrations in doping control samples [1]. For both, the initial testing procedure (ITP) based on urinary concentrations and the confirmation procedure (CP) based on carbon isotope ratios (CIR), 11KT itself was the most promising candidate to detect 11KT administrations. Due to the structural correspondence between 11KT and 11OXO (adrenosterone, androst-4-ene-3,11,17-trione) and the described metabolism of 11OXO towards 11KT, the investigation on 11KT as potential target for 11OXO administrations was warranted [2]. In the context of sports drug testing, 11OXO was already investigated more than 10 years ago [3,4]. Urinary concentrations of 11-hydroxy-androsterone (11OHA) greater 10,000 ng/mL or a ratio of 11OHA divided by 11-hydroxy- etiocholanolone were suggested as potential markers during ITP. Samples found above these thresholds should be forwarded to isotope ratio mass spectrometry-based determinations of 11OHA. In the Cologne laboratory, the suggested ITP-thresholds were found to be suboptimal as samples with elevated 11OHA concentrations were extremely uncommon, while samples with elevated ratios are observed comparably often (approx. 10 % of all samples). Therefore, one male volunteer administered 100 mg 11OXO (2 capsules of a nutritional supplement) and collected urine sample before and up to 45 h after administration. All samples were subjected to the developed sample preparation, and both urinary concentrations and CIR were determined for relevant metabolites [1]. For both the ITP and the CP 11KT showed superior capabilities in detection of 11OXO administration compared to the already described target analytes. Employing 11KT as target analyte at a urinary concentration of 100 ng/mL may simplify and improve the detection for both, 11OXO and 11KT. Further investigations in a larger reference population encompassing n = 2000 routine doping control samples are ongoing to improve the statistical power of the threshold suggested for the urinary concentration of 11KT. Additionally, the CIR of urine samples found with elevated concentrations of 11KT will be investigated to establish a reliable Δ-value for this target analyte. As soon as these investigations are finished, the results will be published in detail elsewhere. References 1. Piper T, Thevis M. Development of mass spectrometry-based methods for the detection of 11- ketotestosterone and 11-keto​dihydro​testosterone. Drug Test. Analysis 2023, accepted 2. Pretorius E, Arlt W, Storbeck KH. A new dawn for androgens: Novel lessons from 11-oxygenated C19 steroids. Molecular and Cellular Endocrinology 2017, 441, 76-85. 3. Brooker L, Parr MK, Cawley A, Flenker U, Howe C, Kazlauskas R, Schänzer W, George A. Developement of criteria for the detection of adrenosterone administration by gas chromatography-mass spectrometry and GC/C/IRMS for doping control. Drug Test. Analysis 2009, 1, 587-595. 4. Brooker L, Cawley A, Kazlauskas R, Goebel C, George A. Carbon isotope ratio analysis of endogenous glucocorticoid urinary metabolites after cortisone acetate and adrenosterone administration for doping control. Drug Test. Analysis 2012, 4, 951–961. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0208 Goodrum J, Nair V, Moore C, Eichner D, Miller G Effects of heavy biotin supplementation on detection of hCG after Ovidrel administration to males The Sports Medicine Research and Testing Laboratory (SMRTL), Salt Lake City, United States Abstract Human chorionic gonadotropin (hCG) is a protein hormone that can be used as a performance enhancing substance by male athletes to re-establish or increase endogenous testosterone production. Doping control testing for hCG is conducted in urine, often on a variety of immunoanalyzer platforms, some of which utilize biotin-streptavidin dependent sandwich immunoassays where biotin is a well-known confounding factor. The extent of biotin interference in serum samples has been well studied, however biotin interference in urine samples has yet to be characterized. The goal of this study was to understand the extent of biotin interference in urine samples on biotin-dependent anti-doping hCG testing platforms to determine if biotin could act as a masking agent for hCG abuse. Ten recreationally active, male individuals self-administered hCG for 3 weeks concurrent with either heavy biotin or placebo supplementation. Urinary hCG detectability was evaluated using a biotin-dependent assay on the Roche Cobas e411 immunoanalyzer and a biotin-independent assay on the Siemens Advia Centaur XPT immunoanalyzer. Additionally, biotin concentration in urine samples from both groups was quantified. Finally, urinary steroid profiles were generated for each individual throughout the administration period. Data from these analyses will be presented. ® MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0209 Marchand A , Roulland I , Semence F , Jaffredo F , Dehainault C , Mekacher LR , Ericsson M Detection of the homologous transfusion of a red blood cell concentrate in vivo: evaluation of flow cytometry and DNA analysis methods Laboratoire AntiDopage Francais (LADF), Paris, France ; IGNA, Nantes, France ; CHU Tizi-Ouzou, Tizi-Ouzou, Algeria Abstract Two doping cases of homologous blood transfusion (HBT) during Tokyo 2020 Summer Olympics have shown that more controls are needed. The method of detection using flow cytometry to evaluate the expression of minor blood group antigens from red blood cells (RBC) and identify different RBC populations is efficient but still complex to perform with multiple antigens detection. Recently the interest of using forensic DNA analysis was also highlighted as a potential new method to detect HBT, with possibility to start from dried blood spots (DBS) instead of fresh blood. After a first phase of development a protocol was validated for HBT detection using DNA profiling analysis after extraction from DBS. Presence of a second DNA was clear down to 2% of donor blood in vitro. A flow cytometry protocol was also developed with preparation and analysis in 96-well plates and detection of 9 different antigens in total (C ,c, E, Jka, Jkb, Fya, Fyb, S,s) with two different primary antibodies per well whenever possible recognized by two different secondary antibodies (IgM coupled to a fluorescent 488 tag for detection of anti-C, anti-c, anti-E, anti -Jka and anti Jkb ; IgG coupled to a fluorescent RPE tag for detection of anti-S, anti-s, anti-Fya and anti-Fyb). The objective of the project was to evaluate the window of detection of an HBT performed in vivo with 150 mL of RBC concentrate prepared from whole blood without a deleukocytation step on two healthy volunteers. Blood samples obtained over 7 weeks post-transfusion were analyzed. While a positive control (mix of blood with 2% minor blood) was correctly identified with a DNA mix, DNA profiling from DBS was not sensitive enough to detect the presence of a second DNA in the sample obtained from the transfusion study even one day after transfusion, possibly due to the fast elimination of the donor white blood cells (which are the only blood cells containing DNA). On the contrary the flow cytometry protocol was very efficient and allowed identification of double populations (DP) of RBC (mix of expressing/ non expressing RBC). This was detected by the presence of a minor peak beside the major peak in one histogram of fluorescence (PE or AF-488 depending on the secondary antibody used for each antigen detection) and by the presence of a second cloud of points in the associated dot-plots PE versus AF-488 fluorescence.). Three to four different antigens with DP were detected in each sample post-transfusion until day 50 post-transfusion. The confirmation procedure (CP) was performed by using a new aliquot of the blood conserved at 4 °C: for each antigen with a suspected double population, three different dilutions of the primary antibody were tested (the same used for the screening analysis and 2-fold more and 2-fold less). Presence of a clear DP in at least 2 of the 3 antibody dilutions confirmed the DP for one antigen and if two antigens presented a DP in the same sample, HBT was confirmed. Reanalysis of the 1 1 1 2 2 3 1 1 2 3 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0210 blood samples stored at 4 °C for more than 30 days, despite hemolysis and a lower sensitivity for some antigens still allowed identification of double populations of RBC and identification of HBT. This protocol can be fully validated for a future application to doping control samples. For more details see publication: Marchand A, Roulland I, Semence F, et al. (2023) Evaluation of the detection of the homologous transfusion of a red blood cell concentrate in vivo for antidoping. Drug Test Anal . 2023; 1-13. doi:10.1002/dta.3448. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0211 Loria F , Breenfeldt Andersen A , Bejder J , Bonne T , Grabherr S , Kuuranne T , Leuenberger N , Baastrup Nordsborg N Erythropoiesis-related RNAs biomarkers are sensitive and specific to low-dose erythropoietin treatment at sea level and altitude Swiss Laboratory for Doping Analyses, Lausanne University Hospital, Lausanne, Switzerland ; Department of Nutrition, Exercise and Sports, University of Copenhagen, Copenhagen, Denmark ; University Center of Legal Medicine, Lausanne University Hospital, Lausanne, Suisse Abstract Background Indirect detection of recombinant human erythropoietin (rhEPO) abuse in Anti-Doping world is done by hematological module of athlete biological passport (ABP). However, actual markers (reticulocytes (%RET) and hemoglobin (HGB)) of this module are not enough specific and have difficulties to distinguish rhEPO administration from altitude sojourn especially if both condition are merged. Previous study have demonstrated that erythropoiesis-related RNAs, genes 5’-aminolevulinate synthase 2 (ALAS2) and carbonic anhydrase 1 (CA1) in capillary dried blood spots (DBS) could be potential biomarkers to increased specificity and sensitivity of the ABP in case of hypoxic conditions. Study design and method In the present study, we monitored ALAS2 and CA1 expression in DBS after injection of 20 IU kg bw epoetin alpha at sea level and the effect merged with altitude exposure. For this purpose, DBSs samples collected from 39 volunteers’ males and females were analyzed. Participants were divided in two groups (EPO and placebo) and subjects from both groups sojourned at sea level and at altitude. The purpose was to evaluate the performance of RNAs biomarkers for the detection of rHEPO injection in a longitudinal blinded setting. Results Our results illustrated a clear significant increase of ALAS2 until to 300% when treated at sea level and at altitude. Regarding CA1 the increase is up to 200% in both conditions. Interestingly, our results suggested no combined effect of altitude and rhEPO injection on ALAS2 and CA1 biomarkers as previously demonstrated with %RET values. In placebo group. Moreover, no effect of altitude on ALAS2 and CA1 was observed in contrast to %RET parameter. Results from blind setting test demonstrated a performance of interpretation of 92% regarding ALAS2 and CA1 monitoring. Conclusion Erythropoiesis-related RNAs in capillary DBS such as ALAS2 and CA1 were sensitive and specific biomarkers for low-dose rhEPO misuse at sea level and altitude. Interestingly, altitude seemed not to be a confounding factor for ALAS2 and CA1 biomarkers in contrast to %RET. Furthermore, this investigation provide evidence that low-dose of rhEPO injections can be detected in a longitudinal blinded setting with RNA biomarkers in DBS. 1 2 2 2 3 1 1 2 1 2 3 -1 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0212 Dørum S , Sletten C , Oll Ø , Dehnes Y EPO screening with K9 Norwegian Doping Control Laboratory, Oslo University Hospital, Oslo, Norway ; Asker Dog Centre, Oslo, Norway Abstract Doping with recombinant EPO (rEPO) has a long history of abuse in endurance sports. rEPO is considered to be a doping agent with a potentially high performance enhancing effect, making it an attractive doping agent for cheating athletes. To date, there is no fast and sensitive screening method for detection of rEPO. The method implemented in the WADA laboratories to detect misuse of rEPO is both sensitive and specific, but it is time-consuming and expensive, and only a small percentage of all samples are selected for EPO analysis. The aim of this project was to develop a rapid pre-screening method for detection of rEPO in urine from doped athletes, by taking advantage of the exceptional nose of the dogs. With such a method, a far higher amount of urine samples could be pre-screened for rEPO to select suspicious samples for further analysis in the laboratory. This would save both time and money, and it could potentially increase the number of positive rEPO samples identified. Together with Asker Dog Centre, we trained dogs to recognize low amounts of rEPO in urine. Here we will show the training strategy and present results from this highly challenging project. 1 1 2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0213 Leuenberger N , Kuuranne T , Castella V Sequencing c.577del in EPO gene in urine and dried blood samples Laboratoire Suisse d'Analyse du Dopage Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Epalinges, Suisse ; Forenic genetic unit, Epalinges, Suisse Abstract Human erythropoietin (hEPO), which increases the human red blood cell count, has been banned in sports since the 1990s. To detect recombinant EPO (rEPO), an analytical method has been set up using an electrophoretic approach, such as sarcosyl-polyacrylamide gel electrophoresis (SAR-PAGE) or sodium dodecyl sulfate-PAGE (SDS-PAGE), which separates the proteins by size in consideration of the molecular weight difference between the rEPO and the endogenous hEPO protein. Recently, a single nucleotide polymorphism (SNP) for EPO was detected in the East Asian population c.577del in the EPO gene, which can produce EPO with different molecular weights. This single mutation results in size increase of approximately 2.9 kDa. Therefore, the current confirmation method for rEPO with electrophoretic separation can lead to misinterpretation of the protein pattern of the EPO variant (VAR-EPO) for rEPO. The prevalence of the mutation is low (an allele frequency of approximately 0.5 – 1 %) and limited to East Asian populations. Nevertheless, to strengthen the analytical method, in October 2021, the WADA published a new version of the technical document for EPO analysis, including gene sequencing, in Annex B of TD2022EPO. According to this document, EPO sequencing shall be performed as a further investigation if double-band (heterozygous of EPO c.577del variant) or single band (homozygous of EPO c.577del variant) at a position higher than wild-type EPO is observed in blood samples with SDS-/SAR- PAGE analytical results. However, the detection of rEPO abuse for an athlete carrying the VAR-EPO is currently not possible with this approach. Collecting and transporting blood samples may be logistically difficult from certain areas, slowing down the overall process and delaying the reporting of the results. Different approaches, still based on the collection of blood samples, have already been proposed for the analyses, using either SDS-PAGE or CRISPR/dCas9 analyses. Urines are currently used as a source of DNA for genetic analyses. Urine contains about 1-20 ng/mL DNA from epithelial cells (shed from the urinary tract for both sexes plus the vagina for the females), leukocytes, and cell-free DNA. Blood contains about 20,000-40,000 ng/mL DNA from white blood cells and cell-free DNA. Considering equal volumes, urine contains about 1,000 to 40,000 times less DNA than blood. However, contrary to blood, urine can be collected using non-invasive methods. Therefore, urine can be used as an alternative source of DNA for the detection of the EPO c.577 del variant, provided the success rate is high. In recent years, considerable anti-doping research and development have focused on the dried blood samples (DBS). Moreover, DBS has been used for years in forensic genetics. DBS testing exhibits advantages in collection, transportation, and storage compared to traditional anti-doping testing matrices such as venous blood. For the practicalities of sample collection, an important benefit with DBS is the minimally invasive collection of capillary blood by puncture from a finger-prick or upper arms. In this study, we first analyzed 20 fresh urine samples from volunteers (10 females and 10 males) to define the amount of DNA needed to obtain a conclusive sequence of the EPO exon 5. Then, using this threshold, we 1 1 2 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0214 determined the proportions of conclusive EPO sequences that could be expected from urine and DBS samples by considering 191 urine samples with varying storage duration that were analyzed for anti- doping cases and 20 cellulose paper and polymer resin DBS (10 Whatman 903 cards and 10 Tasso-M20, respectively) from female and male volunteers. The amount of DNA recovered and the analytical success rate of urine samples were compared with those from published studies. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0215 Heiland CE , Leuenberger N , Ericsson M , Marchand A , Martin L DBS in practice for EPO doping control analysis: in vivo evaluation Stockholm Doping Control Laboratory, Karolinska Univeristy Hospital, Stockholm, Sweden ; Swiss Laboratory for Doping Analyses, Lausanne University Hospital, Lausanne, Switzerland ; Laboratoire AntiDopage Français, Université Paris-Saclay, Paris, France Abstract Background The applicability of dried blood spots (DBS) to erythropoiesis stimulating agent (ESA) detection has been evaluated previously using 20 to 60 µL sample volumes and various immunopurification methods [1,2], but it is still debated, particularly due to the risk of reduced sensitivity. Here, we demonstrate the suitability of DBS for ESA analysis via athlete samples and administration studies using Tasso-M20, Whatman 903 filter paper, and Mitra VAMS. Samples Detection limit samples: Venous blood (K EDTA, BD Vacutainer, NJ, USA) was collected from healthy volunteers with approval from the Bioethics Department of the French Ministère de l’Education de l’Enseignement Supérieur et de la Recherche (DC-2019-3644). Samples were stored at 4 °C. In-competition samples: DBS samples were collected from 111 athletes (with written consent) during two competitions organized by the Professional Triathletes Organization (PTO) in 2022. One Tasso-M20 (Tasso Inc., WA, USA) was used for blood collection from the upper arm skin and two of the four blood spots were used for analysis (about 35 µL capillary blood in total). Also, 34 urine samples were collected from some of these athletes simultaneously with the DBS samples. DBS samples were stored at room temperature and urine was stored at -20 °C until analysis. Aranesp administration study: Venous blood was collected from a patient (with verbal consent) in Switzerland undergoing Aranesp (NESP) treatment (30 µg/kg) every two weeks. The blood was pipetted onto Whatman 903 (Cytiva, MA, USA) filter paper (20 µL/spot), dried, sent to LADF, and stored at room temperature until analysis. Samples from pre-injection, days 4, 7, 14, and 28 were analysed (two spots/analysis). Eprex administration study: At University Hospital Tizi-Ouzou, Algeria, two healthy volunteers (one male, one female) received a micro-dose of Eprex (10 IU/kg) and venous blood was collected pre- injection, and 24-, 48-, and 72-hours post-injection (Ethical permit 006/CAN/Int/TECH/2021). Samples were sent to LADF, where the blood was applied to Mitra VAMS (Neoteryx, CA, USA), dried, and stored at room temperature until analysis. Methodology Spiking: To determine the detection limits of recombinant EPO (rEPO; NeoRecormon ), NESP, CERA, and EPO-Fc from analysis of two Tasso spots, venous blood was spiked at various concentrations (rEPO: 5-40 IU/mL; NESP: 5-80 pg/mL; CERA: 50-400 pg/mL; EPO-Fc: 25-400 pg/mL) and pipetted into the Tasso device. 1 2 3 3 3 1 2 3 2 ® ® ® ® ® MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0216 Immunopurification: Starting from two spots of 17.5-20 µL each collected on Tasso devices, Whatman filter paper, or Mitra VAMS, EPO was extracted using the EPO Purification Gel Kit (EPGK; MAIIA Diagnostics, Uppsala, Sweden). Eluates of 200 µL were concentrated down to ~15 µL using Amicon with 30kDa cut-off, and then analysed with polyacrylamide gel electrophoresis (PAGE). The other two immunopurification methods tested were extractions with buffer from EPGK combined with anti-EPO antibody coupled magnetic beads, and 1% tween solution combined with antibody coupled magnetic beads, based on a previously published method [3] For urine samples, EPO was quantified, and samples were immunopurified by previously published methods [3]. PAGE and Western blot: DBS samples were analysed with SAR-PAGE (and SDS-PAGE CAPS discontinuous transfer for detection limit tests) with Western blot, based on previously described methods [1,3]. Urine samples were analysed with SDS-PAGE according to a previously published method [3]. Results and conclusions The detection limits determined for the ESAs were: 10 IU/L rEPO, 10 pg/mL NESP, 250 pg/mL CERA, and 100 pg/mL EPO-Fc. High sensitivity and selectivity were achieved with samples immunopurified with EPGK and analysed with SAR-PAGE. Therefore, this method was applied to the in-competition and administration study samples. For the in-competition samples, no urine or DBS samples showed the presence of any prohibited ESAs. Endogenous EPO signals were identified in all DBS samples, whereas in 38% of the corresponding urine samples, EPO was undetectable. DBS samples on Whatman filter paper collected from a patient treated with Aranesp demonstrated that a detection window of at least two weeks can be obtained after use of therapeutic doses. A longer detection window is likely at such a dose since NESP signals were still strong two weeks after Aranesp® administration. Study subjects who received one Eprex micro-dose showed that this rEPO was detectable in Mitra VAMS for at least three days (Figure 1). However, differences between the two individuals were seen in signal strength, where the female subject (Subject 2) had generally fainter rEPO signals than the male subject (Subject 1). All these data are in favour of implementing DBS (polymer and filter paper supports) for EPO analysis from 35-40 µL dried blood as a supplemental routine analysis. Figure 1. Detection of Eprex micro-dose from DBS by SAR-PAGE: A single administration of 10 IU/kg Eprex was performed in 2 volunteers. One baseline (D0) and three post-administration (24, 48, and 72 h later, D1-3) samples were analysed for each subject: a male (Subject 1) and female (Subject 2). DBS samples were spotted from venous blood applied to Mitra VAMS. Two spots of 20 µL were used for analysis. Reference standard mixes (Ref), a negative control (Neg), and Eprex reference standard (Eprex ref) were also loaded. ® ® ® ® ® ® MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0217 Details published in: Heiland CE, Martin L, Zhou X, Zhang L, Ericsson M, Marchand A. Dried blood spots for erythropoietin analysis: Detection of micro-doses, EPO c.577del variant and comparison with in-competition matching urine samples. Drug Test Anal. 2023 Nov 9. doi: 10.1002/dta.3596. References 1. Heiland CE, Ericsson M, Pohanka A, Ekstrom L, Marchand A. (2022) Optimizing detection of erythropoietin receptor agonists from dried blood spots for anti-doping application. Drug Test Anal. 14(8):1377-86 2. Reverter-Branchat G, Ventura R, Din ME, Mateus J, Pedro C, Segura J. (2018) Detection of erythropoiesis- stimulating agents in a single dried blood spot. Drug Test Anal. 10(10):1496-507 3. Martin L, Martin JA, Audran M, Marchand A. (2022) An optimized SDS-PAGE protocol with a new blotting system for the initial testing procedure of ESAs in doping control. Drug Test Anal. 14(1):181-7 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0218 Requena-Tutusaus L , Anselmo I , Alechaga E , Bergés R , Ventura R Detection of ERAs in a single-spot DBS sample at MRPL level: improving applicability of DBS in doping control Catalonian Antidoping Laboratory Fundació Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain ; Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain ; Catalonian Antidoping Laboratory Fundació Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain Abstract Erythropoietin (EPO) receptor agonists (ERAs) are a group of substances prohibited by the World Antidoping Agency (WADA), that includes rEPO, NESP, EPO-Fc and CERA. ERAs are generally monitored in urine and blood samples (either serum or plasma), for which sampling, transportation and storage may be costly. For this reason, there is a great interest in the development of analytical procedures for doping control in new matrices such as dried blood spots (DBS). In this study a direct method for the detection of ERAs in DBS samples using one single spot of 25 µL has been evaluated. Two different immunopurification methods, MAIIA EPGK and StemCell EPO ELISA kit, were adapted to achieve maximum sensitivity and minimize background. Several steps of the immunopurification process were optimized, and the MAIIA kit was selected as it presented superior performance overall in terms of sensitivity and recovery. After immunopurification, automated SAR-PAGE western blot using primary biotinylated antibody anti-EPO (BAM2871) was applied to achieve excellent sensitivity and lower background. A complete validation of selectivity, limit of detection, recovery, stability, robustness and carryover was conducted for the optimized procedure. Results of the validation protocol will be presented. Stability of the four ERAs in this matrix was evaluated at different storage temperatures for up to 5 months, showing promising results regarding long-term storage of DBS samples. Performance of the developed method will be compared with the requirements established for conventional liquid matrices such as urine, serum or plasma. Particularly, the capability of detecting ERAs in a single low volume spot at the Minimum Required Performance Level (MRPL) established in blood will be assessed. 1,2 3 2,3 3 3 1 2 3 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0219 Reichel C , Gmeiner G , Thevis M Cost minimized immunoaffinity purification of EPO and its analogs in doping control – A step-by-step protocol for human blood and urine Seibersdorf Labor GmbH Doping Control Laboratory, Seibersdorf, Austria ; Institute of Biochemistry/Center for Preventive Doping Research, German Sport University Cologne, Cologne, Germany Abstract During the COVID-19 pandemic, many doping control laboratories were facing difficulties in obtaining consumables required for EPO analysis (e.g. ultrafilters, materials for immunoaffinity purification, sample tubes). While these shortages have been largely overcome, costs for materials and reagents drastically increased between 2020 and 2023 (e.g. +42% for the biotinylated clone AE7A5 EPO-antibody, +27% for clone 9C21D11 EPO capture antibody, + 21% for the EPO immunoaffinity isolation plate). Sample preparation of urine and blood specimens for the detection of EPO and its analogs is quite costly due to the requirement of immunoaffinity purification (IP) before electrophoretic separation (TD2022EPO, chapter 2.2).[1] Additionally, the “CP should differ, where necessary, from the ITP” (chapter 2.2.1.2 TD2022EPO), e.g. by application of an IP method using a different capture antibody. While for urine samples sufficient commercial kits fulfilling this requirement are available (i.e. EPO-ELISA plate from Stemcell Technologies [2], EPO purification products from MAIIA Diagnostics [3-5]), for blood only the kits from MAIIA Diagnostics exist.[3, 5] However, they use the same capture antibody (clone 3F6). We focused on a protocol requiring no covalent immobilization procedure and significantly modified it in order to increase its sensitivity and specificity. Furthermore, it is now universally applicable for blood and urine samples, performs with the same sensitivity as the commercial products but at a fraction of their costs. Figure 1 shows the protocol for urine. For more details, please refer to the full article published in Drug Testing and Analysis [6]. References 1. World Anti-Doping Agency (WADA). Technical Document TD2022EPO. Harmonization of analysis and reporting of erythropoietin (EPO) and other EPO-receptor agonists (ERAs) by polyacrylamide gel electrophoretic (PAGE) analytical methods. https://www.wada- ama.org/sites/default/files/resources/files/td2022epo_v1.0_final_eng.pdf. Accessed 1 September 2023. 2. Stemcell Technologies. EPO Immunoaffinity Isolation Plate. https://www.stemcell.com/products/epo- immunoaffinity-isolation-plate.html. Accessed 17 March 2023. 3. MAIIA Diagnostics. EPO Purification Kit (Art. 1390). https://maiiadiagnostics.com/wp- content/uploads/2022/06/DfU-EPK.pdf. Accessed 17 March 2023. 4. MAIIA Diagnostics. EPO Purification Gel Kit for Urine (Art. 1410). https://maiiadiagnostics.com/wp- content/uploads/2022/06/DfU-EPGKU-2022.pdf. Accessed 17 March 2023. 5. MAIIA Diagnostics. EPO Purification Gel Kit for Blood (Art. 1430). https://maiiadiagnostics.com/wp- content/uploads/2022/06/DfU-EPGKB-2022.pdf. Accessed 17 March 2023. 6. Reichel C, Gmeiner G, Thevis M. Cost minimized immunoaffinity purification of EPO and its analogs in doping control – A step-by-step protocol for human urine and blood. Drug Test Anal. (in press) 1 1 2 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0220 Figure 1. Protocol for immunopurification of EPO and its analogs from urine Acknowledgements The project was carried out with support of the Austrian Ministry of Sports and the Federal Ministry of the Interior of the Federal Republic of Germany. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0221 Miller G, Goodrum J, Campbell T, Eichner D Assessing the stability of recombinant EPO in urine The Sports Medicine Research and Testing Laboratory (SMRTL), Salt Lake City, United States Abstract The WADA Technical Document for EPO analysis includes a section on the reporting of samples that show no electrophoretic band for either endogenous or recombinant EPO. Although these samples are reported negative, we cannot be certain that the urine sample was provided by an athlete clean from EPO. And while the TD lists specifically samples with no endogenous or exogenous EPO signal, Western blot interpretation also becomes difficult with samples showing signs of degradation and in samples with very low total EPO content. It is unclear exactly what causes this EPO instability, so we designed a three-part study to further understand the stability of endogenous, and, more importantly, recombinant EPO in human urine. First, samples showing no EPO bands or degraded EPO bands analyzed at SMRTL in 2022 were examined to understand trends or similarities amongst the samples. Analysis variables included sport/discipline, sample pH, sample SG, presence of microbial activity according to the steroid profile, shipping time/distance, time of year, and geographic location of sample collections. Second, anti-doping urine samples showing either no endogenous EPO or degraded endogenous EPO after screening were spiked with a combination of recombinant EPO, darbepoetin, and Mircera. Western blotting results from the spiking of 100 samples will be presented. The third portion of the study included a single-dose administration of EPO to twelve male and female volunteers. Each volunteer was administered 40 IU/kg of EPOGEN (epoetin alfa) by subcutaneous injection. A bulk urine collection was performed at an assigned, specific time point following the injection (up to 48 hours). Aliquots from the bulk collection were stored under various time and temperature conditions, designed to mimic shipping conditions at various times in the year. Samples were then analyzed for the presence of endogenous and recombinant EPO, and any potential degradation products. Data from the administration study will be presented, in addition to suggested guidance on the collection, handling and EPO analysis of urine samples. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0222 Garzinsky A , Harth J , Reihlen P , Leipp F , Thomas A , Thevis M Can the detection of EPO and its derivatives be manipulated by the addition of oral fluid to doping control urine samples? Institute of Biochemistry, German Sport University, Cologne, Germany ; Ecole Nationale Superieure de Chimie de Paris, Universite Paris Sciences et Lettres, Paris, France Abstract In the field of sports drug testing, so called “no-shows” represent a well-known problem when it comes to the analysis of erythropoiesis-stimulating agents (ESAs) by means of SAR-PAGE and Western blotting. In these specimens, endogenous EPO is not identifiable. Past studies already demonstrated, that proteases were found to contribute to this observation by leading to the degradation of ESAs. Since oral fluid (OF) comprises proteases, among others, it was assumed that OF could also lead to the degradation of ESAs and thus to impaired EPO analyses, resulting in a readily accessible method for urine sample tampering. The aim of this study was to investigate this assumption and to develop a detection assay in order to identify present OF in urine doping control samples. For this purpose, a total of 1080 urine samples were subjected to EPO analysis and evaluated with regard to variations due to the subject, sex, volume of OF, time point of OF-sampling, and storage conditions. The results showed, that OF can indeed lead to masking of ESA abuse. In particular, interindividual differences as well as the sex and the timing of OF-sampling (pre- and post-prandial) were observed to have an impact on the analysis. In addition, the volume of OF in urine is of major relevance, but realistic amounts, which can be achieved e.g. by spitting once or twice, were found to impair the EPO analysis in a significant number of cases. In order to identify contaminations or urine samples with OF, detection methods targeting human salivary α-amylase (sAA) were developed, as it was found to be a specific and most abundant protein in OF. For this purpose, both a lateral flow strip test (rapid test) and a bottom-up proteomic assay involving tryptic digestion followed by LC-HRMS/MS analysis were evaluated in terms of selectivity, sensitivity, and stability. Carry-over effect as well as linearity were additionally assessed for the bottom-up proteomic approach. Both approaches successfully identified sAA in urine, and the negative controls and OF- enriched samples could be clearly distinguished from each other. However, the naturally excreted level of sAA in urine presented a major challenge. A proof-of-concept study revealed an intersection between individuals with naturally occurring high levels of sAA in urine and those with low levels despite contaminations with OF, e.g. due to degradation processes caused by high concentrations of proteases in OF. First follow-up experiments demonstrated that peptides of the protein “salivary acidic proline-rich phosphoprotein ½” could be used as complementary biomarkers, but further research is required to confirm and subsequently optimize this approach in terms of its applicability. The details of this study will be published elsewhere. 1 1 1 2 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0223 Martinez Brito D, de La Torre X, Botrè F Analysis of testosterone esters by gas chromatography-high- resolution mass spectrometry with low-energy electron ionization Laboratorio Antidoping FMSI, Federazione Medico sportiva Italiana, Rome, Italy Abstract Rationale High-resolution mass spectrometry (HRMS) has demonstrated to be an alternative platform for quantitative analyses, identifying unknown compounds and gathering information for elucidation of chemical structures. This work describes a method to detect thirteen esters of testosterone (T) in 0.1 mL of human serum by gas chromatography coupled to HRMS. Method Analytes were extracted from serum after deproteinization and liquid-liquid extraction. The trimethylsilyl derivatives were analyzed using a gas chromatograph coupled to HRMS at low electron energy to minimize molecule fragmentation. The acquisition in profiling full scan mode was applied with a resolving power of 30,000 at m/z 400. The method was evaluated for linearity, lower limit of quantitation (LOQ) and measurement uncertainty. Mass accuracy (MA) and mass extraction window (MEW) were also assessed. Results The T esters showed a linear response between 0.25-10 ng/mL (except for undecanoate, enanthate and propionate that showed linear responses between 0.5-10 ng/mL and isocaproate between 2-10 ng/mL). Detection limits remained between 0.1-0.5 ng/mL and accuracy between 81-119%. The MA (MEW = 10 ppm) was maintained between -2.4 and 4.8 ppm. Conclusions The method to detect T esters in serum using gas chromatography coupled to HRMS showed linear responses up to 10 ng/mL with adequate linearity, precision, and accuracy. It was possible to distinguish cholesterol from T-isocaproate based on the MEW of 10 ppm preventing false positives. The difference between the mass 458.32176 of T-isocaproate and the mass 458.39415 of cholesterol is higher than 100 ppm (Figure 1). Besides, this method in full scan acquisition allows searching for other biomarkers and/or unknown metabolites (and other ester forms not included here) but at a later stage if necessary. Since the results were encouraging, the need to look for criteria to identify the analytes when HRMS is used, is imposed. Published as: Martínez Brito D, Leogrande P, Jardines D, de la Torre X, Botrè F. Simultaneous quantification of endogenous biomarkers and detection of testosterone esters in human serum using gas chromatography-high-resolution mass spectrometry. Rapid Commun Mass Spectrom. 2024 Mar 30;38(6): e9685. doi: 10.1002/rcm.9685. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0224 Figure 1. Extracted ion chromatogram m/z 458.32 ± 0.5 Da in a positive control (A); extracted ion chromatogram m/z 458.3944 ± 10 ppm for cholesterol (B); reduced mass spectra for cholesterol trimethylsilyl derivative (B1); extracted ion chromatogram m/z 458.3216 ± 10 ppm for T-isocaproate and -caproate (C); reduced mass spectra for T-isocaproate trimethylsilyl derivative (C1); overlapping chromatograms B and C and mass spectra at the RT at which the two compounds coelute (A1). Also, chemical structures, formula and accurate mass are presented MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0225 Martinez Brito D, de La Torre X, Botrè F Correlation of androstenedione and testosterone measurements by LC-MS/MS and GC-LE-TOF Laboratorio Antidoping FMSI, Federazione Medico sportiva Italiana, Rome, Italy Abstract In the frame of the implementation of the steroidal module of the athlete biological passport (ABP) in serum, the World Anti-Doping Agency (WADA) has established the minimum requirements to quantify testosterone (T) and androstenedione (A4) in serum. The method should be based on liquid chromatography combined with tandem Mass Spectrometry (LC-MS ). This work evaluated an alternative method to quantify T and A4 in 0.1 mL of human serum using gas chromatography coupled to high- resolution mass spectrometry (HRMS) taking advantage of an assay designed to detect T-esters. Both procedures using LC-MS/MS and GC-HRMS instruments were validated using the 6PLUS1 Multilevel Serum Calibrator Set MassChrom Steroid Panel 2. Positive and negative quality controls and human serum samples were used to check the correlation between these two approaches. Correlation, regression analysis, and Bland-Altman tests showed a good correlation between these two procedures. Only the T limit of quantification did not meet the requirement established for this parameter. LOD of T was 0.2 ng/mL instead of 0.1 ng/mL as required. Figure 1. Scatter diagram of the regression analysis for A4 and T (upper panel) and Bland-Altman plots for A4 and T (lower panel) n ® ® MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0226 As required by WADA technical document TD-IDCR, the identification criteria are based on the fact that each measured mass used for identification shall be within ± 0.5 Da of the corresponding mass of the same diagnostic ion acquired from a reference analyzed in the same analytical batch. Also, when using single-stage MS, at least three diagnostic ions shall be acquired. The identification of analytes using HRMS instrumentation is not yet regulated in the technical document but given the increase in the use of this type of platforms in recent years in anti-doping laboratories, it is a situation that should be addressed. The data evaluation of T and A4 by GC-TOF was done with a MEW of ± 3 ppm from the accurate mass of A4 (m/z 430.2723) and T (m/z 432.2879), which represents a range of ± 0.000697 Da from the accurate mass of the substance. The second evaluated fragment was the [M -CH ] characteristic of the trimethylsilyl derivatives. In this case, given the characteristics of the mass spectra of both compounds (intense molecular ion and low fragmentation), the error of the second fragment was ± 8 ppm (± 0.00186 Da), which is more than 250 times lower than the value of 0.5 Da established in the technical document TD-IDCR. The TD-IDCR does not include the acceptance criteria when HRMS platforms are used, but it should be considered because of the expansion of its use in antidoping laboratories. + 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0227 Martinez Brito D, de La Torre X, Botrè F Effect of thyroid hormones administration on the urinary endogenous steroid profile of the athlete biological passport Laboratorio Antidoping FMSI, Federazione Medico sportiva Italiana, Rome, Italy Abstract This work focused on the possible alterations of the markers of the steroidal module of the Athlete Biological Passport, considering samples of athletes declaring and not-declaring the supplementation with thyroid hormones (TH) in the Doping Control Form (DCF). Concentrations of 5α-androstane-3α,17β-diol (5α-Adiol), 5β-androstane-3α,17β-diol (5β-Adiol), testosterone (T), androsterone (A), etiocholanolone (Etio), epitestosterone (E), pregnanediol (PD), dehydroepiandrosterone (DHEA) and 11β-hydroxy-androsterone (OHA) were established by gas chromatography-tandem mass spectrometry. Metabolic ratios between the above biomarkers were also estimated. The data set was composed of samples from females and males declaring (FD and MD) and not-declaring (FND and MND) TH supplementation in the DCF. To corroborate these observations, a controlled urinary excretion study was carried out with multiple doses of sodium liothyronine (T3) in a healthy female volunteer. Data from females showed significant differences for the concentrations of 5α-Adiol, A, DHEA, E, OHA, and T and the ratio A/Etio between FD and FND groups (Figure 1), while the male groups only showed significant differences in OHA concentration. In both cases, males and females declaring the consumption of levothyroxine showed a narrower data distribution and diminished percentiles from 17 to 67% with respect to the non-declaring corresponding groups. Concentrations of 5α-metabolites showed a higher depression in the FND, and both FD and MD groups showed an odd behavior for the PD concentrations. The controlled urinary excretion study agreed with the observations, mainly for the female group. Significant differences were observed for concentrations of E, Etio, 5α-Adiol, 5β-Adiol. The interpretation of the steroid markers of the ABP steroidal module should consider TH administrations, due to the actions and metabolism of TH that involve androgen and estrogen metabolism. Published as: Martínez Brito D, de la Torre X, Botrè F. Effect of thyroid hormones administration on urinary endogenous steroid profile of the athlete biological passport. Drug Test Anal. 2023 Nov-Dec;15 (11-12):1361-1370. doi: 10.1002/dta.3534. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0228 Figure 1. Box-and-whisker plot showing the minimum, maximum and median value, and the first and third quartiles of the data for the female groups. Data identified as white boxes correspond to the group not declaring consumption of TH and orange boxes correspond to the group declaring the consumption of TH for testosterone, epitestosterone, androsterone and etiocholanolone concentrations, and T/E and And/Etio ratios. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0229 Sobolevsky T , Walpurgis K , Görgens C , Fedoruk M , Lewis L , Ahrens B , Thevis M Detection of capromorelin in urine following different routes of administration UCLA Olympic Analytical Laboratory, Los Angeles, United States ; Institute of Biochemistry, German Sport University Cologne, Cologne, Germany ; U.S. Anti-Doping Agency, Colorado Springs, United States Abstract Capromorelin (CP-424,391) is a non-peptidic ghrelin receptor agonist that has been shown to stimulate production of growth hormone (GH) and insulin growth factor-1 in humans and other animal species. It was originally developed by Pfizer as a potential treatment for cachexia and frailty in the elderly, but its development was eventually discontinued. Subsequent studies demonstrated capromorelin’s ability to increase food intake in animals, leading to approval in the U.S. and Europe as an appetite stimulant for cats (Elura) and dogs (Entyce). Capromorelin is prohibited in sports due to its ability to stimulate GH production and enhance performance, with any amount detected in an athlete’s sample to be reported as an adverse analytical findings (AAF).Given that its veterinary preparation is formulated as a highly concentrated solution (20 or 30 mg/mL) delivered orally, incidental ingestion or dermal absorption may result in an AAF by way of direct exposure during oral administration to a pet. An administration study was conducted by either oral or transdermal application of capromorelin solution to mimic the scenario of inadvertent exposure to the drug. Ingestion of 30 µg of capromorelin orally (equivalent to 1 µL of Entyce) resulted in detectable amounts of capromorelin in urine for up to 48 hours after administration with a maximum urinary concentration of 7 ng/mL. Importantly, when applied directly to the skin on the hands in larger quantities mimicking a pet administration exposure scenario (30 mg, or 1 mL of Entyce), capromorelin was also detected reaching a maximum urinary concentration of 0.7 ng/mL. Athletes and testing authorities should be aware of the risk of an AAF arising due to incidental exposure to veterinary preparations of capromorelin. To our knowledge, before 2022, no positive test for capromorelin had ever been reported. Published as: Sobolevsky T, Walpurgis K, Goergens C, Fedoruk M, Lewis L, Ahrens B, Thevis M. Detection of capromorelin in urine following oral and dermal routes of administration. Drug Test Anal. 2023 Nov-Dec; 15(11-12):1449-1453. doi: 10.1002/dta.3573. 1 2 2 3 3 1 2 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0230 Andersson A , Pohanka A , Ekström L 18-Methyl-19-noretiocholanolone detected after use of emergency contraceptive Department of Laboratory Medicine, Karolinska Institut, Stockholm, Sweden ; Doping Control Laboratory, Karolinska University Hospital, Stockholm, Sweden Abstract The world anti-doping agency puts forth a list each year which prohibits substances to keep sports clean. Athletes are inherently responsible for everything they consume, which becomes an issue when the metabolic pathways of prohibited and non-prohibited compound intersect. This was the case when an 18- methylnandrolone metabolite was detected in a routine sample of an athlete that had recently ingested an emergency contraceptive pill. Our aim was to study the metabolism and excretion of levonorgestrel in urine. Five women were recruited into a study to elucidate the link between 18-methyl-19- noretiocholanolone and levonorgestrel. After providing a pre-treatment sample, NorLevo was ingested and six additional samples were collected. The sample were analysed with GC-MS/MS after extraction and derivatisation. This study demonstrates a metabolic link between 18-methyl-19-noretiocholanolone and levonorgestrel. Confirming the need to exclude a levonorgestrel intake before reporting an adverse analytical finding. If not, women might be penalised for the use of hormonal contraceptives. 1,2 1,2 1,2 1 2 ® MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0231 Euler L , Mürdter T , Heinkele G , Schwab M , Miller GD , Eichner D , Thomas A , Thevis M Identification of (Z)-3‘-hydroxy clomiphene as a new potential doping- relevant metabolite of clomiphene Institute of Biochemistry, German Sport University, Cologne, Germany ; Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany ; Sport Medicine Research & Testing Laboratory, Salt Lake City, USA Abstract The anti-estrogen clomiphene is used therapeutically to induce ovulation in women. This effect has also been observed in animals. For instance, studies with laying hens that received orally administered clomiphene have shown a significantly increased egg production rate but, as a consequence, eggs were found to incorporate residues of clomiphene. A recent study has shown that consumption of just two eggs containing clomiphene residues could result in an AAF for clomiphene in a doping control. To protect athletes from sanctions due to anti-doping rule violations arising from an unintentional ingestion of the doping agent, a method was developed to distinguish whether clomiphene was ingested in its pharmaceutical form or as residues of the substance in contaminated eggs. After consumption of clomiphene-containing eggs, (Z)-4-hydroxy clomiphene accounted for over 90% of the hydroxy metabolites. In contrast, in urine from 8 out of 9 participants who had received a microdose of (E/Z)-clomiphene citrate, (Z)-3-hydroxy clomiphene, a metabolite of (E)- clomiphene, accounted for > 80% of the mono-hydroxylated metabolites. Re-analyses of doping control samples with clomiphene AAFs demonstrated that the major hydroxy clomiphene isomer could not be assigned to any of the reference compounds available in this study. Comparison with the microdose excretion study samples suggested that this hydroxy metabolite may be a long-term metabolite. Ion mobility experiments were performed to obtain preliminary information on the structure of this metabolite. Based on this data, new reference compounds were synthesized to be included in the differentiation method. Consequently, (Z)-3'-hydroxy clomiphene was identified as the potential long- term metabolite. Using this updated method, samples from a previous study were re-analyzed in which male study participants had received therapeutic doses for 30 days. The excretion profiles of two volunteers were measured. In both cases, (Z)-3'-hydroxy clomiphene was excreted and the relative concentration increased only after peak concentrations. Published as: Euler L, Mürdter T, Heinkele G, Schwab M, Miller GD, Eichner D, Thomas A, Thevis M. Identification and synthesis of (Z)-3'-hydroxy clomiphene as a new potential doping-relevant metabolite of clomiphene. Rapid Commun Mass Spectrom. 2023 Sep 15;37(17):e9599. doi: 10.1002/rcm.9599. 1 2 2 2 3 3 1 1 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0232 Wen H , Wagener F , Neudörfl J , Thevis M , Schäfer M Investigation of the phase I metabolism of a new 20-keto-steroid S42 by C-6 oxidation and GC-MS analysis Institute of Organic Chemistry, University of Cologne, Cologne, Germany ; Institute of Biochemistry, German Sport University Cologne, Cologne, Germany Abstract Introduction Selective androgen receptor modulators (SARMs) are relevant as illicit drugs for performance enhancement in the sports doping context due to their anabolic properties combined with less side effects based on their tissue selectivity [1]. In 2009, a new synthetic 20-keto-steroid S42 was introduced as a new SARM candidate [2]. Since S42 showed anabolic and anti-catabolic effects on cultured myotubes, it was proposed to be applied as a cure for muscle-related diseases [3], triggering also use in sports doping [4]. However, fundamental understanding of the GC-MS behavior of S42 and its metabolism is indispensable for reliable qualitative and quantitative analysis of this pharmaceutical and of its metabolites in body fluid matrices. Preliminary Data S42-mono-OH, S42-bis-OH and S42-tris-OH were found by GC-MS analysis after S42 phase I metabolism. The structure of S42-mono-OH was closely analyzed, and we assign the hydroxylation position to be in ring A or B based on 3 experimental findings. Firstly, the mass difference of S42-TMS isomers and S42- d -TMS isomers decreased from 6 Da to 5 Da after phase I metabolism. This implies that the possible hydrolysis position is at C-1 to C-3, C-6 or C-7. Secondly, the dominating signals in the GC-Mass spectrum of TMS derivatized S42-mono-OH (1) are found at m/z 219.1200, at m/z 245.1359 and at m/z 297.1671 suggesting that the TMSO functional group is attached to either rings A+B or A+B+C (Figure 1b). This assumption rests on the significantly mass shifted fragment ions of the respective TMS-S42 plus 88 Da ([M-H+TMSO]) molecular ions. Furthermore, [TMSOH] and [TMSOD] peaks were found of the ions noted above, indicating the TMS moiety in tandem MS experiments. However, the TMSO group may not be at the A ring since this elimination reaction is not favorable at an aromatic ring. Thirdly, an indicator ion at m/z 143.0887 which originated from the underivatized D-ring of TMS-S42, was observed. Therefore, a hydroxylation at the D-ring is ruled out. In addition, a S42-C6-ketone was synthesized and the TMS derivatized substance (2) showed similar fragment ion formation with a mass difference of 2 Da to TMS- S42-mono-OH (1) (Figure 1). These observations indicate that the B-ring hydroxylation in the positions C-6 or C-7. 1 2 1 2 1 1 2 6 • + • + MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0233 Figure 1. GC-MS spectra of TMS derivatized S42-C6-ketone (2) and S42-mono-OH (1) References 1. Solomon ZJ, Mirabal JR, Mazur DJ, Kohn TP, Lipshultz LI, Pastuszak AW. Selective Androgen Receptor Modulators: Current Knowledge and Clinical Applications. Sex Med Rev. 2019 Jan;7(1):84-94. doi: 10.1016/j.sxmr.2018.09.006. 2. Min L, Yanase T, Tanaka T, Fan W, Nomura M, Kawate H, Okabe T, Takayanagi R, Nawata H. A novel synthetic androgen receptor ligand, S42, works as a selective androgen receptor modulator and possesses metabolic effects with little impact on the prostate. Endocrinology. 2009 Dec;150(12):5606-16. doi: 10.1210/en.2009-0405. 3. Muta Y, Tanaka T, Hamaguchi Y, Hamanoue N, Motonaga R, Tanabe M, Nomiyama T, Nawata H, Yanase T. Selective androgen receptor modulator, S42 has anabolic and anti-catabolic effects on cultured myotubes. Biochem Biophys Rep. 2019 Jan 15;17:177-181. doi: 10.1016/j.bbrep.2019.01.006. 4. Thevis M, Walpurgis K, Thomas A. Analytical Approaches in Human Sports Drug Testing: Recent Advances, Challenges, and Solutions. Anal Chem. 2020 Jan 7;92(1):506-523. doi: 10.1021/acs.analchem.9b04639. 5. Uyanik C, Malay A, Hanson JR, Hitchcock PB, Tiryakioglu S. Ring a Aromatic Steroids in the Pregnane Series. Journal of Chemical Research. 2006;(7):417-419. doi:10.3184/030823406777980619 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0234 Peters I , Naumann N , Görgens C , Guddat S , Thevis M 3D cultivation of HepaRG cells for metabolite prediction of the calstabin-ryanodine receptor complex stabilizer S107 in preventive doping research Center for Preventive Doping Research - Institute of Biochemistry, German Sport University, Cologne, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne, Germany Abstract In preventive doping research, the prediction of potential metabolites of substances banned in sport is essential. This applies to non-approved substances that are assigned to the category S0 of the World Anti-Doping Agency (WADA) Prohibited List, such as the calstabin-ryanodine receptor complex stabilizer S107. In general, various in vivo and in vitro approaches can be applied to predict metabolite formation and secretion of these substances into urine. Human excretion studies provide the most accurate prediction of metabolites; however, these in vivo studies are often difficult to perform with non-approved substances. Another in vivo option is to perform animal studies, where transferability from animal model systems to humans is usually limited. 2D in vitro cell culture models with human cell lines or experiments with human liver microsomes and S9 liver fractions can provide first indications of metabolites, but are not complex enough to mimic human physiological conditions. Therefore, in the present study, a 3D-liver model was applied for metabolite prediction of the drug candidate S107 by using HepaRG liver spheroids cultured in an ultra-low attachment multi-well plate. The metabolic transformation of S107 was analyzed during a three-week experimental period with repetitive substance application. Supernatants at the experimental time points d3 to d21 were analyzed by HPLC-HRMS/MS to identify the metabolites formed. It can be shown that the metabolic profile found is comparable to that one detected in previous in vitro studies. This leads to the conclusion that HepaRG liver spheroids are an appropriate model system to further investigate the metabolic biotransformation of relevant substances in preventive doping research. In addition, the 3D cultivation of HepaRG cells is planned to be used in the so-called organ-on-a-chip technology to even better mimic physiological conditions in humans. The details of this study will be published elsewhere. 1 1 1 1 1,2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0235 Keiler AM , Rzeppa S , Voss SC , Thieme D In vitro biotransformation of voxelotor Institute of Doping Analysis and Sports Biochemistry (IDAS) - Dresden, Kreischa, Germany ; Environmental Monitoring & Endocrinology, TU Dresden University of Technology, Faculty of Biology, Dresden, Germany Abstract Voxelotor (GBT440) has been integrated in the List of Prohibited Substances and Methods under category M 1 Manipulation of Blood and Blood components only on January 1 2023 [1]. This haemoglobin S polymerisation inhibitor is a therapeutic treatment of patients with sickle cell disease. Voxelotor increases haemoglobin’s oxygen binding affinity and thereby enhances arterial oxygen saturation. In addition, first-in-human studies showed increased serum erythropoietin levels and haemoglobin concentrations in healthy adults as side effects [2]. The present study aimed at the identification and characterization of voxelotor metabolites for doping control purposes. In vitro biotransformation was performed using human HepG2 cells and human pooled liver microsomes. For analysis, we performed high-performance liquid chromatography coupled to tandem mass spectrometry or to high-resolution mass spectrometry. The HepG2 cells generated three phase I metabolites resulting from mono- hydroxylation and reduction, and six phase II metabolites resulting from mono-hydroxylation, reduction and O-methylation or glucuro-conjugation in different combinations. Moreover, the glucuro-conjugate of voxelotor was also formed as a minor metabolite. The pooled human liver microsomal incubation only yielded two phase I metabolites, one also formed by the HepG2 cells. With the spectrometric behaviour of voxelotor and its in vitro metabolites described herein, an implementation in doping control screening and consequently a detection of an abuse in an athlete urine sample might be possible. The presence of the metabolites identified in vitro in human urine has to be proven in future studies. Published as: Rzeppa S, Voss SC, Thieme D, Keiler AM. Identification of human in vitro metabolites of the haemoglobin S polymerization inhibitor voxelotor for doping control purposes. Drug Test Anal. 2023 Nov-Dec;15(11- 12): 1403-1409. doi: 10.1002/dta.3489. References 1. World Anti-Doping Agency. List of Prohibited Substances and Methods. World Anti-Doping Code Int Stand. 2023 2. Hutchaleelaha A, Patel M, Washington C, Siu V, Allen E, Oksenberg D, Gretler DD, Mant T, Lehrer-Graiwer J. Pharmacokinetics and pharmacodynamics of voxelotor (GBT440) in healthy adults and patients with sickle cell disease. Br J Clin Pharmacol. 2019 Jun;85(6):1290-1302. doi: 10.1111/bcp.13896. 1,2 1 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0236 Sahu PL , Matsa R , Pawar S , Seth K , Radhakrishnanand P , Murty U , Handa T , Sahu K , Kori RK , Kumar P Synthesis and characterization of ostarine metabolite: applicable for TL 12 National Dope Testing Laboratory, New Delhi, India ; National Institute of Pharmaceutical Education and Research, Guwahati, India, Guwahati, India Abstract The World Anti-Doping Agency (WADA) publishes and updates a prohibited substance list annually. It has a variety of substances and metabolites that are rarely found. Ostarine, which would further metabolize to O-dephenyl ostarine, is one of the substances in the “Other Anabolic Steroid” category. Furthermore, since O-dephenyl ostarine could also be present in urine samples as a contaminant/impurity and/or minor metabolite of the permitted drug bicalutamide, this metabolite shall not be considered as the sole criterion for the reporting of an Adverse Analytical Finding (AAF) for ostarine (TL 12). Hence, this metabolite plays a significant role for the reporting of AAF. Subsequently, it was planned to synthesize reference material (RM) of the ostarine metabolite. The present poster describes the synthetic route of O-dephenyl ostarine and its characterization studies. Keywords: O-dephenyl Ostarine, Synthesis, Technical Letter (TL 12), The prohibited list. 1 2 2 2 2 2 1 1 1 2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0237 Walpurgis K , Agricola J , Thomas A , Thevis M Myostatin inhibitory peptides in sports drug testing Institute of Biochemistry/Center for Preventive Doping Research, German Sport University Cologne, Cologne, Germany ; Division of BioAnalytical Chemistry, Faculty of Science, Vrije Universiteit Amsterdam, Amsterdam, Netherlands ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract Across species, skeletal muscle mass is negatively regulated by the TGF-β cytokine myostatin (MSTN). Inhibitors of this growth factor and its signaling pathways are therefore not only promising therapeutics for muscular diseases but also potential performance-enhancing agents in sports. Within this study, protein precipitation and liquid chromatography high-resolution tandem mass spectrometry (LC- HRMS/MS) were employed to develop a detection method for six novel MSTN inhibitory peptides derived from the regulatory MSTN propeptide and the natural MSTN inhibitor follistatin (FST) from doping control serum samples. The approach was comprehensively characterized and found to allow for a specific detection down to concentrations of 3–9 ng/mL. Moreover, several potential metabolites of the drug candidates referred to as DF-3, DF-25, and Peptide 7 were identified as valuable complementary analytical targets for doping control analytical assays. Overall, the acquired data pave the way for an implementation of MSTN inhibitory peptides into routine sports drug testing. Even though no drug candidate has obtained clinical approval yet, a proactive development of detection assays is of utmost importance to deter athletes from misusing such compounds, which are readily available for research purposes and on the black market. Published as: Walpurgis K, Agricola J, Thomas A, Thevis M. Myostatin inhibitory peptides in sports drug testing. Drug Test Anal. 2023 Nov-Dec;15(11-12):1477-1487. doi: 10.1002/dta.3473. 1 2 1 1,3 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0238 Walpurgis K , Gäde A , Thomas A , Gochard S , Delahaut P , Thevis M Detection of extracellular hemoglobin from Arenicola marina in doping control serum and plasma samples by means of LC-HRMS/MS Institute of Biochemistry/Center for Preventive Doping Research, German Sport University Cologne, Cologne, Germany ; Pig for Life SA, Marche-en-Famenne, Belgium ; CER Groupe, Marche-en-Famenne, Belgium ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract The manipulation of blood and blood components in sports is prohibited at all times and besides blood transfusions, also hemoglobin-based oxygen carriers (HBOCs) can be employed to artificially improve the oxygen transport capacity of the blood. As cell-free hemoglobin (Hb) is not only very ineffective in reversible oxygen binding and release, but further exhibits toxic effects, different strategies for Hb stabilization were employed during HBOCs development. Unfortunately, the in vivo use of these products was still found to be associated with serious side effects such as vasoconstriction and hypertension. However, the natural giant extracellular Hb from the marine invertebrate Arenicola marina (lugworm) could be a promising alternative for both transfusion medicine and cheating athletes, as it was found to be well tolerated in pre-clinical animal studies. Within this research project, lugworm Hb was implemented into the existing doping control detection method for bovine HBOCs based on ultrafiltration (cut-off: 100 kDa), tryptic digestion, and LC-HRMS/MS. For the mass spectrometric detection of lugworm Hb, two precursor-product ion pairs for a total of four tryptic peptides originating from subunits hbA2 (T ), hbB1 (T & T ) and the linker chain (T ) were employed. The modified approach was comprehensively characterized and found to allow for a both specific and sensitive detection of lugworm Hb down to concentrations of 10 µg/mL from 50 µL of serum or plasma. Therefore, it can serve as confirmation procedure for Lugworm Hb following visual or electrophoretic screening. Moreover, a proof-of-concept rat administration study was conducted, and the observed detection windows of at least 4 (dose: 200 mg/kg) and 8 hours (dose: 600 mg/kg) demonstrated, that the presented approach can be readily employed to efficiently test in-competition doping control samples for the presence of the drug. Published as: Walpurgis K, Gäde A, Thomas A, Gochard S, Delahaut P, Thevis M. Detection of extracellular hemoglobin from Arenicola marina in doping control serum samples by means of liquid chromatography and high- resolution tandem mass spectrometry. Drug Test Anal . 2023 Nov-Dec;15(11-12):1430-1438. doi: 10.1002/dta.3591. 1 1 1 2 3 1,4 1 2 3 4 7 3 6 17 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0239 Fiallo Fernandez T, Mc Pherson Medina A, Martinez Brito D, Montes de Oca Porto R Evaluation of the Cuban CBSSEPO monoclonal antibody for the detection of erythropoiesis-stimulating agents in doping control Antidoping Laboratory, Havana, Cuba Abstract Background Erythropoietin levels are one of the essential factors for the homeostasis of the human organism. Since its cloning and commercialization as recombinant human erythropoietin in its different glycosylation variants (ESAs), its use for doping purposes has been increasing. After their inclusion in the Prohibited List, WADA issues and periodically updates a technical document (TD-EPO) that establishes the analytical conditions for the detection of these compounds in urine and blood. Looking for an alternative to the primary monoclonal antibody established in TD-EPO, the Cuban CBSSEPO monoclonal antibody, initially designed for quality control activities in the industry, was evaluated. Experimental Different concentrations were used for CBSSEPO (200, 500, 1000 ng/mL) and the analytical signals for each of the ESAs were compared taking into consideration those obtained with the monoclonal antibody BAM2871. Time for the lecture on the camera Image Quant LAS 4000 mini and the quality of images were assessed by using the GASepo software quantitative analysis of digital images. EPO-Fc, rEPO, CERA and NESP were tested at 0.25(MRPL), 0.5(MRPL), MRPL, 2(MRPL) and 3(MRPL) levels. Results The optimal working concentration of the Cuban CBSSEPO was 1 µg/mL, five times more than the concentration used for the BAM2871. The exposure time varied from 60 to 300 seconds depending on the epoetin tested. The minimum concentration observed in urine matrix for rEPO and EPO-Fc was the MRPL level, while for CERA was 3(MRPL) level. It was not possible to detect NESP in any of the concentrations studied since an analytical signal that corresponds to the current technical document was not obtained. For that reason, Cuban CBSSEPO monoclonal antibody does not meet the requirements to detect ESAs with doping purposes at least in his current design. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0240 He S, Liu X, Zhou X Administration study of rEPO on c.577del carriers Beijing Anti-Doping Laboratory, Beijing Sport University, Beijing, People's Republic of China Abstract The issue of the variant c.577del in the human EPO gene has been found in recent years. Although WADA has published an annex to the TD2022EPO (Annex B) to direct how to deal with the rEPO positive samples, complicated and time-consuming procedure made further analysis inefficiently, on the other hand, the abuse of rEPO by the variant c.577del carriers is difficult to detect. In order to solve this issue, the method of analyzing de-N-glycosylated EPO in blood samples was developed, both VAR-EPO and rEPO can be determined using this method, however, the method cannot be applied until it is evaluated with rEPO excreted samples from variant carriers. For this purpose, five heterozygous carriers of the variant c.577del were recruited in an administration study of rEPO, urine and blood samples were collected at different time before and after subcutaneous injection with single-dose, and the samples were conserved appropriately until analysis. A series of experiments were designed, the urine samples collected from this administration study, including blank samples and washed-out samples, were analyzed for intact EPO with SAR-PAGE, while the serum samples were analyzed for both intact EPO and de-N-glycosylated EPO with SDS-PAGE. The result of urine samples showed that the typical smear band was detected in both blank samples and washed- out samples, which were difficult to distinguish. The result of serum samples showed that, for the analysis of both intact EPO and de-N-glycosylated EPO, double bands with similar intensity were detected in blank samples. While, for the washed-out serum samples, the intact EPO in samples collected in the first few days after injection showed a typical smear band , then two separated bands were detected vaguely several days after injection. The de-N-glycosylated EPO in all washed-out samples showed two well-separated bands with different intensity. The intensity ratio of lower band to upper band decreased with wash-out periods. Due to this result, we can not only determine whether the blood sample is collected from the variant c.577del carrier, detect rEPO according to the ratio of lower band to upper band, so the de-N-glycosylated EPO could be an effective complementary method for confirmation of rEPO in blood samples. Published as: He S, Liu X, Song H, Zhou X. Administration study of recombinant erythropoietin on the carriers of variant c.577del in EPO gene. Drug Test Anal. 2023 Nov-Dec;15(11-12):1410-1416. doi: 10.1002/dta.3566. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0241 Joonyeop Y , Minyoung K , Mijin J , Hophil M , Byung-Gee K , Junghyun S , Changmin S Direct multiplex-PCR based gene doping detection for human growth hormone and its isoform using CRISPR/Cas12a-assisted HOLMES Korea Institute of Science and Technology, Doping Control Centre, Seoul, Korea ; Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul, Korea ; Bio-MAX/N-Bio Institute, Seoul National University, Seoul, Korea Abstract With the recent development of gene therapy technology, the unethical genetic attempt to improve athletic performance became an immediate threat to the field of anti-doping. This kind of genetic approach is called gene doping and is prohibited by the World Anti-Doping Agency. Various DNA analysis techniques such as quantitative PCR, digital PCR, real-time PCR, loop-mediated isothermal amplification (LAMP), and targeted next-generation sequencing (NGS) have been developed, and research on the development of exogenous gene detection methods using these technologies is also attempted. One of these techniques, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based detection methods such as Specific High Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR), and one-HOur Low-cost Multipurpose highly Efficient System (HOLMES) has demonstrated its high sensitivity in nucleic acid detection. These methods utilize the target-dependent trans-cleavage activity of the CRISPR-associated proteins (e.g., Cas12a, Cas13, etc.) along with fluorescence-labeled single-stranded DNA reporter (ssDNA), allowing for highly sensitive and versatile applications in genetic diagnosis. In this study, we aimed to develop a direct multiplex-PCR based gene doping detection assay using CRISPR/Cas12a-assisted HOLMES for human growth hormone (hGH) and its isoform. The gene of hGH is named 22K-GH and lacks the first 45 nucleotides in exon 3 of 22 kDa- G H is named 20K-GH, were selected as potential targets due to their short half-life in serum and well-known metabolic effects such as muscle mass increasing. During the direct multiplex-PCR, each 22K-GH and 20K-GH could be selectively amplified from 5 μL of whole blood sample using an exon-exon junction spanning primers and the Locus Control Region (LCR) is also amplified as an internal control of PCR. In HOLMES, if amplified exogenous 22K-GH and 20K-GH genes exist in the reaction, the Cas12a/crRNA binary complex forms a ternary complex with PCR products, which in non-target ssDNA reporter (5’FAM-3’BHQ1) illuminates fluorescence by trans-cleavage. To confirm the exogenous GH gene and isoform, all PCR products were separated and visualized on a 2.5% agarose gel. As a result, 2.5~5 copies of the exogenous GH gene could be detected through our assay, and there were no cross-reactions and non-specific amplifications found. Our proposed method is highly convenient as it does not require pretreatment of blood and purification after PCR, making it a feasible gene doping detection assay with high sensitivity and specificity. 1,2 1 1 1 3 1 1 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0242 Mongongu C, Madi Moussa E, Marchand A, Buisson C, Ericsson M Top-down IGF-I quantification in serum using LC-HRMS analysis for longitudinal follow-up: impact of calibration on measurement variability Laboratoire AntiDopage Francais (LADF), Paris, France Abstract The determination of Insulin Growth factor-I (IGF-I) concentration is used for the diagnosis of Growth Hormone-related disorders. This growth factor is synthesized by the liver under the biological effect of GH (growth hormone). IGF-I circulates in the blood as a complex bound to specific high affinity proteins (IGFBPs), regulating its excretion and tissue availability. The quantification of IGF-I is widely performed using immunoassays for clinical purposes but some concerns about the reproducibility between assays have emerged. In recent years, IGF-I analysis using mass spectrometry (MS) has been developed (bottom-up and top-down analysis), to benefit from MS advantages in terms of accuracy, precision and the possibility to differentiate IGF-I from its variants (endogenous and exogenous). In the anti-doping field, IGF-I serum concentration is used as a biomarker of GH doping by athletes. In order to overcome interindividual variability, longitudinal monitoring of GH biomarkers will soon be implemented in the "Athlete's Biological Passport (ABP)" by the World Anti-Doping Agency (WADA). In this work, we evaluated different quantification strategies to determine IGF-I concentration based on a top-down analysis of the intact protein in serum. The sample preparation consists of a microelution solid phase extraction after IGF-I release from its binding proteins (protein precipitation). After dilution, the eluate is then directly analysed by LC-HRMS. Using 20 µL of serum, the developed method was successfully validated according to WADA requirements. The impact of different calibration strategies on the variability of IGF-I concentrations determined in intermediate precision tests for a low and a high quality control (QC) was evaluated. We tested either a calibration curve or a single point calibrator using IGF-I NIST SRM 2926 standard prepared freshly, or alternatively an endogenous human serum used as a single point calibrator. This latest was validated against IGF-I NIST SRM 2926 standard, aliquoted and stored frozen at -80 °C until analysis. Our results supports the use of a single point human serum- based calibrator as it allows the lowest variability in QCs IGF-I concentrations over time, which would be an asset for effective longitudinal monitoring. In addition, a second batch of endogenous human serum calibrator was prepared, calibrated against the first single point human serum-based calibrator and used to quantify IGF-I in the two QCs. The measured IGF-I values were similar to the ones obtained with the first single point human serum-based calibrator showing a very low variability over time. That validated the possibility of easily implementing this approach in anti-doping laboratories. A good practice would be that all laboratories use the same WADA-approved human serum-based calibrator to attribute the value of their own endogenous human serum used thereafter as calibrator. ® ® ® ® MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0243 Somay Selbes Y, Demirel AH, Elaldi K Comparison of Insulin-like growth factor 1 measured between Siemens Immulite and IDS-iSYS Turkish Doping Control Center (TDKM), Ankara, Turkey Abstract Insulin-like growth factor-1 (IGF-1) is a known key hormone that mediates the physiological response to both endogenous and exogenous growth hormone (GH). IGF-1, along with the N-terminal pro-peptide of type III collagen (PIIINP), has been used to detect the abuse of hGH, GH secretagogues, and IGF-I in sports. There are two WADA-defined immunoassays for the detection of IGF-1 levels in anti-doping laboratories. The dependence of these tests on a specific device and occasional problems in kit procurement raises the question of whether other devices and IGF-1 test kits can be considered as acceptable testing techniques in anti-doping laboratories. Therefore, in this study, IGF-1 results obtained with IDS iSYS were compared with Immulite 2000 IGF-I test results. Spearman's rank correlation coefficient showed a moderate correlation between the two measurements (ρ =0,473; p 0.98 are achieved using a weighting factor of 1/x, and the limit of quantification (LOQ) values are established with % RSD and % CV < 15, │% Diff│ < 20, and relative ion ratio < %20. The LOQ values are largely in the low ng/mL levels, which meet the screening and confirmation sensitivity needs of analytical methodologies in labs that routinely monitor sports anti-doping. Conclusion Transcend DSX-1 combines a dried spot autosampler and TurboFlow LC-MS/MS and provides a complete workflow for fast and robust quantification of 77 drug-of-abuse analytes in dried blood spots. 1 2 1 1 1 1 1 2 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0249 Buisson C, Smires S, Ericsson M Dried blood spots for doping controls – Validation of a quantification procedure for corticosteroids Université Paris-Saclay, Laboratoire AntiDopage Français, Chatenay-Malabry, France Abstract Introduction To improve doping control in sport, analytical procedures must constantly evolve to get ahead of new doping techniques and to meet unique challenges of sensitivity. Lately, a particular interest has been shown in dried blood spots (DBS) which simply consists of a drop of blood deposited on an appropriate paper for further analysis. Indeed, compared to conventional samples, like venous blood and urine, a DBS sampling can be less invasive, and reduce storage and shipping costs. Moreover, regarding doping control, DBS could be useful as a complementary matrix to urine for the detection and quantification of doping substances, enabling the correlation of pharmacological effects to urinary concentration. According to the World Anti-Doping Agency (WADA), glucocorticoids, prohibited only in competition by all routes of administration, are one of the most commonly used classes of prohibited substances by athletes. Therefore, the objective of this work is to develop and validate a DBS assay for glucocorticoids such as Triamcinolone acetonide (TA), 6βOH-Triamcinolone acetonide (6βOH-TA), and Betamethasone by LC-MS/MS. Methodology In order to develop a DBS assay, Capitainer® devices were spotted with simulated drops of fortified blood samples and the following parameters were evaluated in accordance with WADA technical documents: recovery, LOQ and dynamic ranges, repeatability and intermediate precision, measurement uncertainty (MU) and selectivity. The extraction method developed is divided into three steps. Firstly, 10 µL of the internal standard are put in a tube with the 10 µL spot, and the spot is diluted twice in a mix of 250 µL H O/CH CN (30:70). Secondly, a liquid-liquid extraction is realized with 400 µL of H O and 800 µL of TBME. Finally, the dry residue is taken up in 50 µL of mobile phase. The doping agents are separated along a gradient within 9.5 min on a Kinetex XB-C18 column. The mobile phase is constituted of H O 10 mM HCOONH /CH CN (initially 70:30), the flow is set up at 0.4 ml/min, and the volume of injection at 10 µL. Results For TA the dynamic range has been validated from 1 ng/mL to 20 ng/mL, for 6bOH-TA from 1.5 ng/mL to 20 ng/mL, and for Betamethasone from 2.5 ng/mL to 25 ng/mL. Performed tests showed a relative standard deviation (RSD) of repeatability lower than 10%, a RSD of intermediate precision lower than 15%, and measurement uncertainty lower than 15% for the three molecules for quality controls (QC) at low, middle and high level of concentration. The extraction yield has been rated greater than 75% for the three molecules. The study of blank samples made it possible to establish the specificity of the method. 2 3 2 2 4 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0250 At least two precursor-product ion transitions (SRM transitions) were validated to guarantee the identification capacity of the method. Conclusion The DBS assay developed is validated for the three glucocorticoids studied with LOQs < 2.5 ng/mL (10 µL of blood). The extraction method had a recovery higher than 85% with CV for repeatability < 10% for the three glucocorticoids, reinforcing the usefulness of volumetric DBS devices for quantification methods. The proposed protocol can thus be used in clinical studies required to establish MRL in blood. However, the need to prepare DBS devices spotted with fortified blood as calibrant may pose challenges for the implementation in routine. Therefore, in parallel of this validation, tests have been done on DBS cards spotted with fortified serum in order to simplify the practical application. However, the need to prepare DBS devices spotted with fortified blood as calibrant may pose challenges for the implementation in routine. Therefore, in parallel of this validation, tests have been done on DBS cards spotted with fortified serum in order to simplify the practical application. The results of these tests show a bias < 20% for the three glucocorticoids with a systematic underestimation for two of them (6βOH-TA and Betamethasone). The use of a correction factor could be considered to enable the use of serum as a matrix for calibration curves. Further tests are thus needed to validate this approach. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0251 Alechaga E , Bressan C , Coll S , Monfort N , Ventura R Multiclass method for the screening of doping agents in dried blood spots Catalonian Antidoping Laboratory Fundació Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain ; Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain Abstract Dried Blood Spots (DBS) are a sampling technique routinely used for newborn screening, and recently evaluated as an alternative to blood matrices such as serum or plasma. DBS possess several advantages with respect to conventional liquid biological samples: they simplify sample collection, storage and transport, and they generally present increased analyte stability. For these reasons, there is an increasing interest in adapting analytical methodologies for doping control to this new matrix. However, they require the use of highly sensitive methodology, due to the very low volume of sample (usually < 30 µL) collected in each spot. This work presents the development of a multi-class screening method for compounds belonging to different groups of the World Anti Doping Agency (WADA) prohibited list, including anabolic agents, growth hormone secretagogues, beta-2 agonists, aromatase inhibitors, anti-estrogenic substances and hypoxia-inducible factor activating agents. DBS samples for method development were obtained depositing 20 µL of venous blood and letting it to dry on pure cellulose cards. The whole DBS spot was then punched out and extracted prior to the analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. The chromatographic separation was carried out with a C18 column and a binary mobile phase of acetonitrile:water, both with 0.01% formic acid. The methodology was validated for qualitative purposes, and different parameters were evaluated, such as limit of detection, selectivity, recovery, matrix effects and intra-assay precision. The vast majority of the compounds could be reliably detected at sub-ng/mL level. Satisfactory results were obtained in terms of recovery and precision, considering only non-threshold compounds without Miminum Reporting Level (MRL). Also, matrix effects were negligible for most compounds, as expected considering the low amount of sample analyzed. Additionally, samples after the administration of boldenone, oxandrolone and tamoxifene to healthy volunteers were analyzed. The method showed good performance and robust results, making it fit-for-purpose for its application to doping control. 1,2 1 1 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0252 Musenga A, Langer T, Salamin O, Nicoli R, Kuuranne T A fast screening of doping agents in DBS by LC-MS/MS Swiss Laboratory for Doping Analyses, University Center of Legal Medicine Lausanne-Geneva, Lausanne University Hospital and University of Lausanne, Switzerland, Epalinges, Switzerland Abstract Doping control is mostly based on the analysis of urine samples, thanks to the relatively low-invasiveness of sample collection. However, this analysis mainly targets the metabolites of the prohibited substances. Blood samples, on the other hand, can provide a more representative information on the presence of substances at the time of collection as well as the possibility to target the parent compound for detection, though sample collection requires trained personnel (e.g. a phlebotomist), is more invasive and the shipment and storage requirements are more demanding (e.g. temperature control). Widely used for the screening of new-borns, the analysis of DBS has recently gained interest in the anti-doping community, since it can overcome some of the difficulties associated with blood samples [1]. This collection method is minimally invasive and the process of drying stops the degradation processes so that samples can be transported at room temperatures as long as the humidity is kept to a minimal level [2]. To regulate the use of this matrix for doping control, the World Anti-Doping Agency (WADA) has issued a technical document [3], which commands that the analysis can only be conducted on substances without threshold or reporting limits. Therefore, DBS qualitative analysis can be applied to the classes of substances that are prohibited in sports at all time, like anabolic agents, b2-agonists, diuretics and hormone modulators. Nevertheless, the DBS analysis of substances belonging to other classes such as stimulants or narcotics is feasible [4], even though the small sample volumes require sensitive techniques. We have developed a fast and comprehensive method for the detection in DBS of over 100 substances across all WADA classes S1-S9. Compounds are quickly extracted from either a cellulose or a polymer based support material using methanol. After the addition of hydrochloric acid to limit the evaporation of basic volatile compounds, the sample extract is dried under a flow of nitrogen and finally reconstituted with mobile phase. The analysis is performed with a 10 min gradient by UHPLC- MS/MS using a mobile phase composed of water, acetonitrile and formic acid. The method has been validated according to ISO 17025 and the International Standard for Laboratories and is currently used in routine for the analysis of non-threshold substances without MRL in agreement with WADA requirements. However, our validation also included substances belonging to the WADA classes S6-S9, demonstrating the capability to expand the analysis to these compounds in case new guidelines are released. References 1. Thevis M, Geyer H, Tretzel L, Schänzer W. Sports drug testing using complementary matrices: Advantages and limitations. J Pharm Biomed Anal. 2016 Oct 25;130:220-230. doi: 10.1016/j.jpba.2016.03.055. 2. World Anti-Doping Agency: International Standard for Testing and Investigations, 2023 3. World Anti-Doping Agency: Technical document TD2023DBS, 2023 4. Salamin O, Garcia A, González-Ruiz V, Rossi F, Bigard X, Déglon J, Daali Y, Faiss R, Saugy M, Rudaz S. Is pain temporary and glory forever? Detection of tramadol using dried blood spot in cycling competitions. Drug Test Anal. 2020 Nov;12(11-12):1649-1657. doi: 10.1002/dta.2923. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0253 Miyamoto A, Ota M, Sato M, Okano M Simultaneous detection method of 26 testosterone, nandrolone and boldenone esters in dried blood spots by LC-MS/MS Anti-Doping Laboratory, LSI Medience Corporation, Tokyo, Japan Abstract WADA approved the use of dried blood spots (DBS) as a sample collection method in doping control in 2021. Since testosterone esters are not directly excreted into urine, the IRMS analysis is essential for detecting the testosterone doping. On the other hand, testosterone esters are stable in DBS, and if they can be detected, IRMS analysis for their metabolites in urine is not required. It has been reported that direct detection of steroid esters in DBS is useful for detecting doping. In this study, a simultaneous detection method was developed for 11 testosterone esters, 9 nandrolone esters and 6 boldenone esters in DBS. Absorption devices for DBS were evaluated with DMPK-C (fingertip sampling) and Tasso M20 (brachial capillary sampling). The method allowed the detection of concentration levels at 0.5 ng/mL for all 26 steroid esters. In addition, the method allowed for confirmation analysis that all evaluated testosterone esters met the TDIDCR at 0.8 ng/mL. For applicability test, the capillary blood samples were collected using both devices after administration of testosterone enanthate (100 mg, i.m.) and testosterone undecanoate (80 mg, p.o.). Testosterone enanthate was detectable up to 96 h and testosterone undecanoate was detectable up to 6 h at least in DBS after each administration. If the MRPL is set at 1 ng/mL for testosterone esters, the method can be applied to doping control sampling using Tasso M20 and DMPK-C card. The full paper will be published elsewhere. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0254 Sletten C , Solheim SA , Bækken LV , Dehnes Y Effect of Sustanon administration on ALAS2 expression Norwegian Doping Control Laboratory, Oslo University Hospital, Oslo, Norway ; Anti Doping Denmark, Brøndby, Denmark Abstract ALAS2 mRNA has previously been proposed as a biomarker for blood doping-stimulated erythropoiesis. In this project we wanted to investigate the intra-individual variation and whether injection of testosterone esters (Sustanon ) affects ALAS2 expression. Capillary blood from fingerprick was collected on Whatman™ DMPK-C cards (GE Healthcare) 14 days before the injection (baseline sample) and 1, 3, 5, 10 and 14 days post injection. DBS cards were stored for 4-5 years at -80 °C before analysis. Quantitative PCR was performed on a an StepOne Plus Real-Time PCR System (Applied Biosystems) using Comparative C analysis with baseline samples as calibrators. Our preliminary results show that RNA can successfully be isolated from DBS after several years in storage. A marked intra-individual variation in the relative expression of ALAS2 was observed in both the treatment group and the placebo group. The CV variance in the relative expression of ALAS2 was higher after use of testosterone ester, although we could not find a significant difference in mean between the two groups, probably due to too few subjects. 1 1,2 1 1 ® 1 2 ® T MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0255 Heiland CE , Mongongu C , Semence F , Pohanka A , Ericsson M , Marchand A , Ekström L IGF-I variation in DBS and serum Doping Control Laboratory Karolinska University Hospital, Stockholm, Sweden ; Laboratoire AntiDopage Français, Université Paris-Saclay, Paris, France ; Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Abstract Background The athlete biological passport (ABP) now includes an endocrine module to longitudinally follow the endogenous growth hormone marker insulin-like growth factor 1 (IGF-I). However, DBS are not established for monitoring this biomarker. Here, we examine the correlation and inter-individual variability of the IGF-I concentration in DBS and serum samples for six weeks. Experimental Samples: DBS Mitra VAMS (Neoteryx, CA, USA) and serum (Becton Dickson, NJ, USA) were collected from 14 subjects (10 males, 4 females; median age 28 years old) over a six-week collection period (Swedish Ethical Review Committee permit Dnr 2020-04258). DBS samples were stored for about six months at room temperature before analysis took place and serum samples at -20 °C. Serum samples (n = 41) were analyzed in duplicate in the LC analysis. DBS samples were analyzed once with LC (n=63) and serum samples were analyzed once with the IDS-iSYS Multi-disciplined Automated System (n = 40). Reference standards, quality controls, calibrators: IGF-I SRM 2926 (NIST, Gaithersburg, MD, USA) stock solution (305 µg/µL IGF-I) was diluted to make an IGF-I reference stand solution (10 ng/µL), also containing 37 ng/µL IGFBP-3 (mole-to-mole; Sigma-Aldrich, Saint-Quentin Fallavier, France). The internal standard N-IGF-I working solution (5 ng/µL) was diluted from SRM 2927 (285 ng/µL N-IGF-I; NIST, Gaithersburg, MD, USA). IGF-II was used at a concentration of 50 ng/µL. Rat serum was used to prepare the working solutions. Methodology: DBS samples were sonicated for 2 hours and both DBS and serum underwent protein precipitation before extraction of IGF-I using Biotage Extrahera LV based on a previously described method (1). A calibration curve was prepared in the same way. LC analysis for DBS used a Zorbax SC18 column, 25-µL injection volume, and a flow rate of 500 µL/min (mobile phase A: 0.1% formic acid/H O, B: 0.1% formic acid/acetonitrile). LC analysis for serum used a Triart C18 column, 5-µL injection volume, and a flow rate of 400 µL/min (same mobile phases). IDS-iSYS was used as previously described (2). Results and discussion There was a strong correlation in IGF-I concentration between serum and DBS (rs = 0.86, p < 0.0001), particularly at lower concentrations in DBS (Figure 1A). The concentration of IGF-I in DBS was about 5x lower than that in serum (Figure 1B). The CV% of IGF-I in DBS was 14-34% (n = 63), while in serum it was 4-12% (n = 24). Serum IGF-I concentrations measured with LC were similar to those measured with IDS- iSYS. After storing the samples for several months at room temperature, it is possible that IGF-I degraded 1 2 2 1 2 2 3 1 2 3 15 15 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0256 slightly and that the desorption was inefficient, thus contributing to lower detection levels in DBS. Overall, DBS may be used for longitudinal IGF-I monitoring when an inter-laboratory harmonization of sample storage, calibrator preparation, and methodology is performed. Figure 1. IGF-I concentrations in blood matrices. (A) Correlation in endogenous IGF-I concentration between DBS and serum from 9 subjects. (B) Intra-individual variation of IGF-I concentration in one participant over a six-week period References 1. Mongongu C, Moussa EM, Semence F, Roulland I, Ericsson M, Coudoré F, et al. (2020) Use of capillary dried blood for quantification of intact IGF-I by LC-HRMS for antidoping analysis. Bioanalysis. 12(11):737- 52. 2. Marchand A, Martin JA, Collot D, Hoang O, Roulland I, Semence F, et al. (2019) Combined administration of microdoses of growth hormone and erythropoietin: Effects on performance and evaluation of GH detection capability using anti-doping methods. Drug Test Anal. 11(11- 12):1698-713. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0257 Brockbals L , Thomas A , Schneider TD , Kraemer T , Steuer AE , Thevis M LC-MS/MS-based peptide analysis for dried blood spot sampling time point estimation Institute of Biochemistry, German Sport University, Cologne, Germany ; Department of Forensic Pharmacology, University of Zurich, Zurich, Switzerland Abstract Along with the recent acknowledgement of the World Anti-Doping Agency to use dried blood spot (DBS) samples for routine doping control purposes, there have been propositions to use DBS as a matrix that allows regular proactive remotely supervised self-sampling, providing potential longitudinal monitoring of an athlete’s exposure to doping agents. However, several organisational aspects have to be considered before implementation, such as the verification of the sample collections time-point. Based on a previous untargeted proteomics workflow utilizing liquid chromatography-high resolution mass spectrometry (LC- HRMS) to identify protein/peptide markers to define the time since deposition of a bloodstain, the aim of the current study was to develop a targeted LC-HRMS/MS analytical method for promising peptidic target analytes. A long-term DBS storage experiment was carried out over a three-month period (sample collection time-points: 0, 2, 4, 7, 14, 21, 28, 42, 56, 70, 84 and 91 days) with DBS samples of 10 volunteers for longitudinal investigation of signal abundance changes of targeted peptide sequences at different storage temperatures (room temperature (RT), 4 °C and -20 °C). Prior to experimental analysis, LC-HRMS/MS method characteristics were successfully assessed, including intra-day precision, carryover and sample extract stability. For estimation of DBS sample collection time points, ratios of two peptides that originate from the same protein prior to tryptic digestion were created. Two targeted peptide area ratios were found to significantly increase after stored at RT for 28 days, representing potential markers for future use in routine doping controls that contribute to advancing complementary avenues in anti- doping. Published as: Brockbals L, Thomas A, Schneider TD, Kraemer T, Steuer AE, Thevis M. Do dried blood spots have the potential to support result management processes in routine sports drug testing? – Part 3: LC-MS/MS- based peptide analysis for dried blood spot sampling time point estimation. Drug Test Anal. 2023 Feb 24. doi: 10.1002/dta.3463. 1 1 2 2 2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0258 Kim KH , Jeong W , Eun Byul C , Min H , Son J Development of an online immunopurification assay for urinary insulin and insulin mimetics using AF4-ESI-MS/MS Korea Institute of Science and Technology, Doping Control Centre, Seoul, Korea ; Chemistry, Sogang University, Seoul, Korea Abstract Insulins and its mimetics could perform a wide range of roles in the body such as a hormone that regulates blood sugar levels. Among these, the anabolic and anticatabolic effects of insulin drugs, related to muscle strength and endurance, may influence improving elite athletes’ performance. Therefore, they were prohibited by WADA as metabolic modulators, and it has been shown to be detected in urine metabolism, so the analysis of insulin in urine is receiving a great deal of attention. In this study, we demonstrated a new online immunopurification analysis method based on asymmetrical flow field-flow fractionation (AF4-ESI-MS/MS) for urinary insulin and six insulin mimetics: Lispro, Glulisine, Aspart, Degludec, Detemir, and Glargine. AF4 is a separation technique based on analytes size by the flow of a carrier solution, and the analytes are focused in a narrow lane on the device then small matrix could be removed through the membrane pores. In order to achieve immunopurification in AF4, the PVDF membrane and RC membrane were used for protein immobilization and filtration, and the pH of the mobile phase was adjusted to control the antigen-antibody reaction. As a result, the mass spectrum of analytes through immunopurification was confirmed. An increase in sensitivity was observed when protein A, which improves antibody orientation, was added to the immunopurification step. Moreover, the optimization was completed within 5 and 10 minutes, which is considerably shorter than the general method. The developed assay was validated for limit of detection (LOD), linearity, matrix effect, intra- day precision, and inter-day precision. Furthermore, studies were conducted on the enrichment performance of AF4 and its potential was confirmed. The AF4-ESI-MS/MS is able to perform the three steps including concentration, immunopurification, and mass spectrometry in a single run, and the immunopurification hyphenated mass spectrometry platform will be useful in the analysis of proteins from a wide variety of biological samples. 1 1,2 1,2 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0259 González-Rubio S , Caballero-Casero N , Ballesteros-Gómez A , Cuervo D , Muñoz G , Rubio S A general screening method for testing of banned substances in urine based on cubosomic supramolecular solvents and liquid chromato- graphy/time-of-flight mass spectrometry Department of Analytical Chemistry, Institute of Chemistry for Energy and the Environment, University of Córdoba, Córdoba, Spain ; Madrid Anti Doping Laboratory, Health Institute Carlos III, Madrid, Spain Abstract Multiclass screening methods in doping control should be comprehensive, allow high throughput sample processing and be cost-effective and green. In this respect, one of the greatest challenges of screening methods is how to extract efficiently the target compounds and reduce matrix effects while preventing the loss of chemicals with very different physicochemical properties during sample extraction and purification. Here, cubosomic supramolecular solvents (SUPRASs), able to extract efficiently multiclass substances while removing matrix interferences were firstly investigated for the multiclass extraction of banned substances in urine prior to their screening analysis by LC-QTOF/MS. For this purpose, eighty prohibited substances and/or their metabolites, selected from the 10 categories (S1-S9, P1) of the WADA list were tested. These substances included a wide range of polarity (log P from -2.4 to 9.2) and chemical structures (e.g. compounds with alcohol, amine, carboxyl, ether, ester, ketone, sulfonyl, etc. functional groups). The SUPRAS selected was synthesized directly in the urine by the spontaneous self-assembly and coacervation of 1,2-hexanediol, induced by sodium sulfate. Method validation was carried out according to WADA guidelines. No interfering peaks were observed for any of the 80 substances investigated. Around 84-93% of drugs were efficiently extracted (recoveries 70-120%) and 83-94% of the analytes did not show matrix effects (±20 %) in the ten urines tested. The only substance with recoveries below 30% for some urine samples was ecgonine methyl ester. It is worth noting that ritalinic acid (log P -2.4) was extracted with recoveries within the range 72.6-93.3%. Method detection and quantification limits for the drugs selected were in the intervals 0.002-12.9 ng mL and 0.007-43.2 ng mL , respectively. The applicability of the method was evaluated by the screening of thirty-six blinded and anonymized urine samples previously analyzed by conventional methods by the Doping Control Laboratory of the Health Institute Carlos III in Madrid. 1 1 1 2 2 1 1 2 -1 -1 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0260 Keiler AM , Derwand D , Rzeppa S , Voss SC , Thieme D QuEChERS as alternative extraction procedure in doping analyses Institute of Doping Analysis and Sports Biochemistry (IDAS) - Dresden, Kreischa, Germany ; Environmental Monitoring & Endocrinology, TU Dresden, Faculty of Biology, Dresden, Germany Abstract QuEChERS is a well-known extraction method in food analysis used for residue analyses, such as pesticides and mycotoxins [1]. The name reflects the advantages of this extraction procedure: Quick, Easy , Cheap, Efficient, Rugged, Safe. The procedure is based on a liquid-liquid extraction with acetonitrile in the presence of a buffer/salt-mixture, which leads to a phase separation. Aim of the present study was to investigate the extractability of the small molecules from groups S1 to S9 of the WADA Prohibited List. As a starting point, the parameters for the extraction were first optimized with regard to pH and buffer/salt ratio. With the optimized parameters, we were able to extract approximately 90 % of the small molecules targeted in our routine GC-MS/MS and LC-MS/MS screening procedure using the QuEChERS method. Furthermore, we observed significant improvements for individual substances and substance groups compared to our established methods. As example, the extractability of stanozolol- N-glucuronides and HIF modulators was significantly improved. Overall, this pilot study demonstrates the potential of QuEChERS as alternative extraction procedure for screening and confirmatory methods in doping analysis. Published as: Derwand D, Rzeppa S, Voss SC, Zschiesche A, Keiler AM. QuEChERS as alternative extraction procedure in doping analyses. Drug Test Anal. 2023 Dec 3. doi: 10.1002/dta.3610. References 1. Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and „dispersive solid-phase extraction“ for the determination of pesticide residues in produce. J AOAC Int. 2003;86(2):412–31. 1,2 1,2 1 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0261 Cristea C , Radu M , Catana D , Berghes B , Toboc A , Stan C Triple quadrupole UHPLC-MS/MS method as an alternative of orbitrap UHPLC-HRMS method used for detection of growth hormone-releasing hormones Romanian Doping Control Laboratory, Bucharest, Romania ; University of Bucharest, Faculty of Chemistry, Bucharest, Romania Abstract The use of growth hormone-releasing hormone (GHRHs) is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). Considering the complexity of urine samples and the low concentrations at which these analytes should be detected, analysis of GHRHs is a challenging task. In most of the studies, GHRHs are analyzed using UHPLC-HRMS with orbitrap. The aim of the current study was to develop and validate a method for the detection of growth hormone-releasing hormones (Tesamorelin, CJC-1295, CJC-1295 DAC, Sermorelin (1-29), Sermorelin (3-29)-NH2, Somatorelin) using triple quadrupole UHPLC/MS-MS method with solid phase extraction (SPE), based on weak cation exchange, which is able to detect concentrations as low as 0.2 ng/mL (Figure 1). This is due to the high extraction selectivity of the sorbent’s surface with carboxylic acid groups, which enables to target the analyte of interest removing interferences that can lead to misidentification of GHRH compounds and further false positive results, as well as due to the specificity of the UHPLC/MS-MS system, which ensure reliable analytical results, without interferences. The method developed by our laboratory was validated according to WADA technical documents for specificity, limit of detection, recovery, carry-over and robustness. The results show that the method has adequate recoveries and sensitivity, hence, it can be employed for routine screening in anti-doping laboratories. Published as: Cristea CD, Radu M, Toboc A, Stan C, David V. Cationic exchange SPE combined with triple quadrupole UHPLC-MS/MS for detection of GHRHs in urine samples. Anal Biochem. 2023 Dec 1;682:115336. Figure 1. UHPLC-MS/MS chromatograms recorded for: A. Urine samples spiked at 0.2 ng/mL with all compounds, B. Negative urine. 1,2 1 1 1 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0262 Upadhyay AK, Bhardwaj A, Sahu P Comprehensive identification method for hypoxia-inducible factor (HIF) activating agents National Dope Testing Laboratory, New Delhi, India Abstract Hypoxia-inducible factors (HIFs) activating agents are included in the Prohibited List of the World Anti- Doping Agency (WADA) because they can induce hypoxia, that results in increased red blood cells and erythropoietin (EPO) production. The detection of HIFs in doping control urines using a single method poses a challenge for anti-doping laboratories due to the required detection limit and structural variability in prohibited HIFs. The aim of this study is to develop a comprehensive identification method for the detection of HIFs in doping control urine samples. The sample preparation includes enzymatic hydrolysis followed by liquid-liquid extraction in alkaline and acidic phase using TBME and ethyl acetate. The method was developed on Liquid chromatography- tandem mass spectrometry (LC-MS/MS, 5500 Qtrap) coupled with Waters UPLC. The results of the method were found fit for the purpose of the identification of all prohibited HIFs in a single method. The limit of identification of the developed method is found from half MRPL to below 10 of MRPL for targeted HIFs as per the prohibited list. The LOI and specificity of the method demonstrate that it can identify targeted HIF-activating agents below half of MRPL in doping control samples. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0263 Naumann N , Vollmert C , Krajina M , Thevis M Evaluation of a novel gene doping detection approach via high multiplex MALDI-TOF MassARRAY analysis Center for Preventive Doping Research / Institute of Biochemistry, German Sport University Cologne, Cologne, Germany ; Agena Bioscience GmbH, Hamburg, Germany Abstract Since 2003, gene doping has been prohibited under the regulations of the World Anti-Doping Agency (WADA). In this project, a new approach for high multiplex detection of seven potential human gene doping targets in one reaction and analysis is presented. The approach is based on a multiplex PCR amplification step and subsequent single-base extension (SBE)-procedure with a final detection step via Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) on a MassARRAY System. In a proof-of-principle study, a gene doping panel prototype has been developed, which demonstrates the capability of simultaneously detecting transgenic DNA encoding for human EPO, FST, GH-1, IGF-1, MSTN (Propeptide), VEGF-A, and VEGF-D via at least two exon-exon junctions per target transcripts. First tests of the panel prototype provided promising results with high specificity and sensitivity comparable to qPCR approaches, underlining the opportunity to establish a novel time- and cost-effective approach for gene doping detection. Next steps and focus of the project is the in depth- validation of the panel prototype and test-runs concerning the method’s applicability in routine screening procedures. In addition, suitable reference material via a SNP-based detection approach will be generated and evaluated in this project. The topic of this poster will be published elsewhere. 1 2 2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0264 Akiyama K, Momobayashi A, Okano M Individual identification method using samples related to doping tests: a comparison of mitochondrial and nuclear genetic data Anti-Doping Laboratory, LSI Medience Corporation, Tokyo, Japan Abstract Doping offences include sample substitution. Therefore, it has been recommended that identification by STR (short tandem repeat) analysis should confirm that the doping control samples collected belong to the same athlete. However, it may be difficult to obtain full STR analysis using degraded or a very small amount of samples. Mitochondrial DNA (mtDNA) is characterized molecular stability and high cellular copy number. Therefore, the use of mtDNA in urine might be suitable for individual identification in doping controls. In this study, we conducted sensitivity, concordance, and case-type studies in order to compare mtDNA testing with STR analysis. In sensitivity studies, mtDNA testing was a highly sensitive analytical method compared to STR analysis. Concordance studies confirmed that all samples were concordant with mtDNA sequences and STR profiles. However, in some urine samples examined by STR analysis, allelic drop-out occurred as expected. In the case-type samples study, assuming actual testing, we found that mtDNA testing could be used to obtain DNA profiles of test samples such as urine, needle and blood stains, similar to STR analysis. We conclude that mtDNA testing would be suitable for analysis of low-concentration and highly degraded DNA samples, such as urine samples, compared to STR analysis. Since mtDNA is inherited maternally, we propose that mtDNA testing using urine samples is conducted for the initial testing procedure. Further STR analysis in blood may be required in cases where the DNA match is detrimental to the athlete. The full paper will be published elsewhere. *This year's Manfred Donike Award for the best poster presentation went to Kentaro Akiyama of the Tokyo Anti-Doping Laboratory. His work on analyzing mitochondrial DNA from urine and blood offered a complementary and robust option to individualize doping control urine samples, especially when low concentration and/or extensive DNA degradation (e.g. in urine samples) is observed. Comparing urinary mtDNA profiles with DBS-derived profiles was shown to be fit for purpose, adding a new tool to uncover sample manipulation attempts. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0265 Son J , Park H , Cho Y , Sung C , Min H , Lee KM , Bahn Y Enhancement of anti-doping strategy using artificial intelligence (AI): A study on improving the accuracy and efficiency of detecting performance-enhancing drugs in athletics as a proposed next- generation doping diagnosis strategy Doping Control Center, Korea Institute of Science and Technology, Seoul, Korea ; Department of Biotechnology, Yonsei University College of Life Science and Biotechnology, Seoul, Korea ; Department of Microbiology and Immunology, Yonsei University College of Medicine, Seoul, Korea Abstract The list of prohibited substances in anti-doping is constantly evolving as new performance-enhancing drugs and methods are developed, making it a challenging task to keep up with the latest developments. Additionally, the manual evaluation of large amounts of biological sample data is time-consuming and prone to human error, making it necessary to explore alternative methods of analysis. We propose that the use of artificial intelligence (AI) technologies in anti-doping efforts can significantly improve the accuracy, efficiency and speed of the testing process. A total of 145,544 data collected over 4 years at the Doping Control Center (South Korea) were used for supervised machine learning, and the integrated dataset consisted of a total of 4 datasets including retention times and MRM pairs for 153 doping agents. The accuracy of doping diagnosis was confirmed with 10 classification algorithm models, and the optimal algorithm model for each dataset was determined by naive Bayes and SMOTE-Tomek for the whole group and beta group, and naive Bayes for alpha and gamma groups. The results of the study, with an accuracy of 93% for the whole group and 99% for the alpha, beta, and gamma groups, demonstrate the potential of AI to accurately diagnose doping in athletes. The high accuracy of the model suggests that this new approach could be an effective next-generation anti-doping strategy. 1 1,2 1,3 1 1 1 2 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0266 Son J , Cho Y , Park H , Jeon S , Sung DJ , Jeong JH , Sung C , Min H , Lee KM , Kim H , Yoon SS Clinical study of brain doping diagnosis (bD ): Confirmation of sports performance enhancement and neurochemical alterations via brain stimulation Doping Control Center, Korea Institute of Science and Technology, Seoul, Korea ; Department of Microbiology and Immunology, Yonsei University College of Medicine, Seoul, Korea ; Department of Biotechnology, Yonsei University College of Life Science and Biotechnology, Seoul, Korea ; Bionics Research Center, Korea Institute of Science and Technology, Seoul, Korea Abstract A novel form of doping, known as “brain doping”, involves temporarily activating certain brain regions to enhance sports performance. However, there is no clear regulation or process for detecting it. We have continued our research to establish a brain doping diagnosis based on the concept that neurochemicals mediate brain stimulation to improve sports performance. In this study, a clinical study was conducted that focused on the effects of transcranial direct current stimulation and tested whether receiving stimulation prior to exercise enhanced sports performance and produced neurochemical alterations. Fifteen volunteers participated in a single-blind crossover test and performed a maximal incremental exercise test until volitional exhaustion on a treadmill after stimulation. In order to identify changes in neurochemicals, urine was collected at a certain time point from participants and analyzed using an ultra-sensitive analysis method developed in a previous study. Compared to sham conditions, brain stimulation led to changes in movement-related cortical potentials and increases in sports performance. As a result of analyzing the urine of the subjects, neurotransmitters having a significant difference were identified according to the sports performance, and six substance ratios were suggested as diagnostic criteria for brain doping. In this study, by detecting a whole shift in patterns for neurochemicals, we carried out research to establish a diagnosis strategy for brain doping for the first time. In addition, the analysis method developed in this research is effective for the simultaneous measurement of neurochemicals in biological samples and is anticipated to be applicable in several sectors, including doping control. 1 1,2 1,3 1 4 4 1 1 1 4 2 2 1 2 3 4 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0267 Aldubayyan A , Castrignano' E , Abbate V Short- and long-term stability of synthetic cathinones and dihydro- metabolites in human urine samples: considerations for antidoping and forensic toxicology applications Department of Analytical, Environmental & Forensic Sciences, School of Cancer and Pharmaceutical Sciences, King's College London, London, United Kingdom ; Drug Control Centre, Department of Analytical, Environmental & Forensic Sciences, School of Cancer and Pharmaceutical Sciences, King's College London, London, United Kingdom Abstract Synthetic cathinones, among the most prevalent classes of new psychoactive substances, pose a large analytical and interpretative challenge for forensic toxicology and antidoping laboratories. Cathinone and its analogues, e.g. mephedrone, methedrone, and α-pyrrolidinovalerophenone, along with other substances having a similar chemical structure or biological effect(s), are prohibited specified stimulants in-competition from class S6 of the World Anti-Doping Code International Standard Prohibited List 2023. Various factors may influence the stability of synthetic cathinones in diverse degrees between sampling and analysis, and therefore stability studies are of great value to bring insight into the best storage conditions and extend period of detection. The study involved 16 synthetic cathinones and 10 dihydro- metabolites spiked in human urine to evaluate the stability under common storage conditions to imitate real forensic toxicology/antidoping samples submitted for synthetic cathinone’s analysis. The samples were stored at either room temperature (22–23 °C) up to 3 days, refrigerated (4 °C) up to 14 days or frozen (–40 °C) up to 12 months and analysed in triplicated using a validated liquid chromatography– tandem mass spectrometry method. Analytes’ concentrations decreased over time, although slower when stored frozen. All analytes remained stable (> 80%) for 1 month when stored frozen before losses in content were more apparent for some analytes, depending on their chemical structure. Under all storage conditions, the highest instability was observed for analytes containing halogens (i.e., chlorine or fluorine). Irrespective of parent analytes, dihydro-metabolites had improved stability at each tested temperature, which highlights their importance as potential biomarkers when retesting is required after a long period of storage. It is therefore suggested that biological evidence containing synthetic cathinones should be analysed immediately, and when storage is required then caution must be taken after extended period of time as it may directly impact the accurate determination of analyte’s concentration. 1 1,2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0268 Al-Naimi N , Kraiem S , Samsam W , Beotra A , Orie N , Mohamed-Ali V , Almaadheed M Turkesterone a noval ecdysteroid: High resolution orbitrap mass spectrometry identification of turkesterone in supplements and urine samples Doping Analysis Lab, Antidoping Lab Qatar, Doha, Qatar ; Centre for Metabolism and Inflammation, Division of Medicine, University College London, London, UK Abstract Ecdysteroids are polyhydroxylated ketosteroids, which were initially isolated from arthropods, and thought to play a role in the molting process. They are now known to be abundant in plants and can be detected in the tissues and body fluids of mammals after dietary consumption [1]. This family of phytosteroids, especially 20-hydroxyecdysone, have gained research interest in recent years because of their anabolic potential in mammals and growing popularity among athletes, with prevalence of use by elite athletes estimated at 0.4–5% [2,3]. Unlike typical anabolic androgenic steroids (AASs), ecdysteroids do not bind to the androgenic receptor (AR), which makes them perfect anabolic agents for use by athletes without androgenic distraction. 20-hydroxyecdysone, the most studied of these compounds is now on the Monitoring List of the World Anti-Doping Agency (WADA). Since the listing, the method of detection and biological effects of 20-hydroxyecdysone have been established and reported [3,4]. However, Turkesterone, an analogue of 20-hydroxyecdysone with an additional hydroxyl group at position C 11 of its structure, is thought to be more potent and is not on the WADA list, therefore more likely to be abused by athletes. There is, as yet no validated method to detect the steroid, and little is known about its pharmacokinetic properties. Therefore, this project aimed to develop a method for the identification and quantification of Turkesterone and its metabolites in different biological matrices, including samples collected with micro-sampling devices. The developed method was applied to detect and quantify the presence of Turkesterone in commercially available dietary supplements, as well as in urine of volunteers after its ingestion. The method used was based on UHPLC high resolution mass spectrometry. A method was developed which was fit for the purpose of detecting Turkesterone. Optimization of various parameters, such as mobile phase gradient, flow rate, run time, injection time, the orbitrap resolution, the automatic gain control (AGC), injection time (ms), and collision energy, resulted in the method development. Mass accuracy less than 2 ppm was obtained with UHPLC/HRMS. LOD of 3 ng/mL was obtained when analyzed on UHPLC/ HRMS, applying the dilute-and-shoot procedure. Out of the four dietary supplements obtained through online suppliers and tested for the presence of Turkesterone, only one showed the presence of the ecdysteroid, but a concentration (33.8 mg/g) far less than claimed on the label (500 mg/g). However, all the four supplements tested showed the presence of 20- hydroxyecdysone (see Figure 1). 1,2 1 1 1 2 1,2 1,2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0269 Figure 1. A) Chromatograms of turkesterone and B) 20HE in each supplement; Suppl. 1: pro force- super-bol, Suppl. 2: pro force l-citrulline turkesterone, Suppl. 3: pro force T40 turkesterone, Suppl. 4: s5 supplement labs turkesterone. Turkesterone was eluted at RT 5.67 and 20HE was eluted at RT 6.05 Urine from 2 volunteers (one male and one female) given Turkesterone (5 mg each), showed peak levels of the compound in the urine within 8 hours of administration in both the volunteers.In conclusion, a method was successfully developed for the detection of Turkesterone. As for other ecdysteroids, while dietary supplements do contain this phytoestrogen its level are significantly lower than the claims made by the manufacturers There may be a gender specific difference in the metabolism of this ecdysteroid, that is currently being investigated. Finally, the prevalence of Turkesterone in stored urine samples, from elite athletes, previously analyzed for 20-hydroxyecdystrone (20HE), will be reanalyzed and compared with the use of 20HE. References 1. Lafont R, Dinan L. Practical uses for ecdysteroids in mammals including humans: an update. J Insect Sci. 2003;3:7. doi: 10.1093/jis/3.1.7. 2. Parr, M.K., Ambrosio, G., Wuest, B. et al. Targeting the administration of ecdysterone in doping control samples. Forensic Toxicol 38, 172–184 (2020). doi.org/10.1007/s11419-019-00504-y 3. Kraiem S, Al-Jaber MY, Al-Mohammed H, Al-Menhali AS, Al-Thani N, Helaleh M, Samsam W, Touil S, Beotra A, Georgakopoulas C, Bouabdallah S, Mohamed-Ali V, Al Maadheed M. Analytical strategy for the detection of ecdysterone and its metabolites in vivo in uPA(+/+)-SCID mice with humanized liver, human urine samples, and estimation of prevalence of its use in anti-doping samples. Drug Test Anal. 2021 Jul;13(7):1341-1353. doi: 10.1002/dta.3032. 4. Nelson N Orie, Asmaa Raees, Maneera Y. Aljaber, Nada Mohamed-Ali, Halima Bensmail, Mostafa M. Hamza, Nasser Al-Ansari, Alka Beotra, Vidya Mohamed-Ali, Mohammed Almaadheed. 20-Hydroxyecdysone dilates muscle arterioles in a nitric oxide-dependent, estrogen ER-β receptor-independent manner. Phytomedicine Plus, 1(3), 100078. doi.org/10.1016/j.phyplu.2021.100078 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0270 Pettersson-Bohlin K , Pohanka A , Ekström L Detection of SARMs in the Swedish society Laboratory Medicine, Karolinska Institute, Doping Control Laboratory Karolinka University Hospital, Stockholm, Sweden ; Doping Control Laboratory Karolinka University Hospital, Stockholm, Sweden Abstract Selective Androgen Receptor Modulators (SARMs) are a heterogenous group of substances that all binds to the androgen receptor. SARMs are marketed as having less androgenic effect and more potent anabolic effect compared to AAS [1]. SARMS are since 2008 forbidden by WADA. According to the Swedish doping law (1991:1969) all anabolic androgenic steroids (AAS) and substances increasing the testosterone levels are juridically forbidden [2]. Subsequently, SARMS are not tested for outside WADA community and the prevalence of SARMs in society is unknown. The aim of this study was to assess the prevalence of SARMs in Swedish health care samples originally subjected to AAS testing. The samples analyzed in this study were urine leftovers from AAS testing done at Drug Abuse Laboratory, Medical Unit of Clinical Pharmacology, Karolinska University Hospital collected between 1 September 2021 and 29 August 2022. In total 651 samples were analyzed, the only information following the samples were gender. The analysis was done using our accredited WADA LC-HRMS methods for detecting the prevalence of SARMs included in the prohibited list [3]. The SARMs found in this study are shown in (Figure 1.), LGD-4033 was in top followed by RAD140 and ostarine. These findings match the findings in sports testing, i.e., according to WADA statistics (2019- 2021) these three SARMs were the most detected. None of the female samples (n=100) contained any SARM even though one might speculate that SARMs may be favorable in females due to minimizing the risk of having irreversible masculinizing effects that are associated with AAS use [4]. In 47% of the samples screened for SARMs, LGD-4033, and/or only the LGD-4033 long lasting metabolite (LGD-4033 LL met) [5,6] were detected, highlighting the importance of including metabolites prolonging the detection window. In the SARM positive samples, two or more SARMs were found in 45% of the cases. There could be several reasons for this, stacking of several SARMs to increase performance outcome can be one reason [7]. Another reason can be contamination problems with the bought products, since studies has shown that labeling and found concentration can vary a lot [8-10]. The variability in concentration of SARM products increases the risk of consuming a toxic dose. SARMs can give acute liver damage when consumed in high doses which has been reported in case reports [11,12]. A limitation herein is that the concentrations of SARMs could not be evaluated, and hence it is not possible to estimate if toxic doses have been used by the study subjects. Another limitation of the study design is that some individuals might have been tested several times during the collection time. The tested individuals are mainly people from abuse treatment programs or having a suspicious AAS use, which makes this group unique. When collecting the next round of samples, the same individual will only be collected once and the concentration of the found SARMs will be evaluated. 1 2 2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0271 To conclude, SARMs were found in samples taken outside WADA community. Therefore, there is a need of educating healthcare and sport organizations not associated to WADA about their potential liver toxicity and to inform that patients using SARMs in Sweden will not be positive if conducted to a AAS test. Figure 1. Top three SARMs found in the healthcare samples, LGD-4033 followed by RAD140 and ostarine. Presented in % with (the number of detects) References 1. Narayanan R, Coss CC, Dalton JT. (2018) Development of selective androgen receptor modulators (SARMs). Mol Cell Endocrinol. 465:134-42. 2. Riksdagen. Lag (1991:1969) om förbud mot vissa dopningsmedel https://www.riksdagen.se/sv/dokument- lagar/dokument/svensk-forfattningssamling/lag-19911969-om-forbud-mot-vissa-dopningsmedel_sfs-1991- 1969. (access date 09.12.2022) 3. Pettersson-Bohlin K, Pohanka A, Andersson A, Ekström L. Detection of Anabolic Agents including Selective Androgen Receptor Modulators in samples outside of sport. DTA-23-0203. 4. Havnes IA, Jorstad ML, Innerdal I, Bjornebekk A. (2021) Anabolic-androgenic steroid use among women - A qualitative study on experiences of masculinizing, gonadal and sexual effects. Int J Drug Policy. 95:102876. 5. Thevis M, Lagojda A, Kuehne D, Thomas A, Dib J, Hansson A, et al. (2015) Characterization of a non- approved selective androgen receptor modulator drug candidate sold via the Internet and identification of in vitro generated phase-I metabolites for human sports drug testing. Rapid Commun Mass Spectrom. 29(11):991-9. 6. Pitsinos EN, Angelis YS, Petrou M. (2022) Structure revision and chemical synthesis of ligandrol's main bishydroxylated long-term metabolic marker. Org Biomol Chem. 20(46):9112-6. 7. Bonnecaze AK, O’Connor T, Burns CA. (2021) Harm Reduction in Male Patients Actively Using Anabolic Androgenic Steroids (AAS) and Performance-Enhancing Drugs (PEDs): a Review. Journal of General Internal Medicine. 36(7):2055-64. 8. Chakrabarty R, Grainger J, Goebel C, Brooker L, George A. (2021) “For research use only”: A comprehensive analysis of SARMs and related IPEDs purchased on local Australian websites between 2017 and 2018. Performance Enhancement & Health. 9(3-4). 9. Van Wagoner RM, Eichner A, Bhasin S, Deuster PA, Eichner D. (2017) Chemical Composition and Labeling of Substances Marketed as Selective Androgen Receptor Modulators and Sold via the Internet. JAMA. 318(20):2004-10. 10. Leaney AE, Beck P, Biddle S, Brown P, Grace PB, Hudson SC, et al. (2021) Analysis of supplements available to UK consumers purporting to contain selective androgen receptor modulators. Drug Test Anal. 13(1):122-7. 11. Weinblatt D, Roy S. (2022) Drug-Induced Liver Injury Secondary to Enobosarm: A Selective Androgen Receptor Modulator. J Med Cases. 13(5):244-8. 12. Barbara M, Dhingra S, Mindikoglu AL. (2020) Ligandrol (LGD-4033)-Induced Liver Injury. ACG Case Rep J. 7(6):e00370. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0272 Leogrande P , Jardines D , Martinez Brito D , de La Torre X , Botrè F , Luch A , Diel P , Parr MK Physiological and post-training effects of ecdysteroid supplements. A multivariate study of the human serum metabolome Laboratorio Antidoping FMSI, Federazione Medico sportiva Italiana, Rome, Italy ; Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Berlin, Germany ; Institute for Cardiovascular Research and Sports Medicine, German Sport University, Cologne, Germany ; Institute of Pharmacy, Freie Universität Berlin, Berlin, Germany Abstract Metabolites, known to be responsible for a variety of biological functions (signaling molecules, biomarkers, and modulators of physiological processes), constitute the metabolome, whose highly dynamic nature makes it an instantaneous indicator of system perturbations induced by different physiological and/or pathological conditions as well as by the intake of biologically active substances. Since specific metabolic phenotypes can result from years of training, discrimination of physiological effects from those caused by the administration of substances banned by the World Anti-Doping Agency can be a valuable analytical strategy for interpreting adverse analytical results. This work aims to characterize the athlete serum profile after the administration of ecdysteroids, natural steroid hormones recently proved to enhance physical performance. The combination of mass spectrometry and chemometric tools may allow the differentiation of physiological effects from post- training and intake-driven effects. Metabolic profiles of 46 healthy male volunteers were measured by coupling an LC-MS/MS system with the Biocrates Absolute-IDQ p180 kit. In this way, a large panel of metabolites was quantified, which was then subjected to multivariate analysis. Unsupervised analysis of the data found no significant differences between the placebo group and the group taking the suggested dose of the ecdysteroids supplement. Considering a unique group consisting of both volunteers taking the recommended dose and those taking the supplement overdose, a clear discrimination between the control and placebo groups was observed. Phosphatidylcholines were the most significant characteristics of ecdysteroid administration to discriminate between the placebo and control groups, showing a significant influence only in the control samples and a dose-dependent effect in the others. 1 1 1 1 1 2 3 4 1 2 3 4 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0273 Saad K , Vonaparti A , Al-Mohannadi I , Khelifi S , Salma S , Mahieddine S , Saleh A , Lommen A , Horvatovich P , Beotra A , Abushareeda W , Al Maadheed M , Georgakopoulos C Preliminary study of MetAlign software automatic LC/MS screening data evaluation Doping Analysis Lab, Antidoping Lab Qatar, Doha, Qatar ; Institute of Doping Analysis and Sports Biochemistry Dresden (IDAS), Kreischa, Germany ; RIKILT Wageningen University and Research, Wageningen, The Netherlands ; University of Groningen, Groningen, The Netherlands ; Faculty of Pharmacy, University of Athens, Athens, Greece Abstract The World Anti-Doping Agency (WADA) annually updates the List of prohibited substances and methods [1]. WADA Accredited Laboratories analyze urine samples for a wide range of Prohibited and Monitored substances and report their findings to WADA. MetAlign is a freeware software designed to reduce the size of the raw datafiles acquired in full scan (FS) mode by liquid chromatography (LC) high resolution (HR) – Orbitrap mass spectrometry (MS) and to perform a fast search for substances in thousands of the reduced datafiles [2-4]. The purpose of this study was to assess the automatic data evaluation of LC/HRMS screening for beta blockers and diuretics using a combination of MetAlign reducing and searching software and the manual evaluation method. The searching and detection parameters of MetAlign are the combination of accurate ion mass to charge values (m/z), retention times and ion abundances. The MetAlign search validation criteria were based on the reliable peak detection, correct identifications and on the number of false positive (FP) and false negative (FN) findings, compared to the optical evaluation of the chromatographic peaks (generated by Xcalibur and LCQUAN software) performed by the analyst. For each substance, an abundance threshold was calculated as the mean-3 standard deviation from MetAlign's reduced quality control sample data files. These data were originated from the validation data of ten (10) different urine samples that were negative for the analytes covering the normal range of matrix differences, e.g., SG, pH, sex, turbidity, in the current validation study of the initial testing procedure (ITP). The 10 samples were evaluated before and after the addition of the substances (beta-blockers and diuretics) at 5 different concentration levels: MRL/MRPL, 75% MRL/MRPL, 50% MRL/MRPL, 25% MRL/MRPL, 10% MRL/MRPL [5,6]. An additional software module has been in-house developed with the capability to cycle over all outcomes and summarize the information in a spreadsheet format, ready to be used additionally in quality assurance applications showing the substance specific detection over long time periods. As shown in Table 1, the applied intensity thresholds provide a reliable tool for identifying positive and negative cases in the data, improving the accuracy and efficiency of the analysis. MetAlign's search capability was validated using 577 ADLQ screening (ITP) LC-HR-FS-MS reduced datafiles originating from original antidoping samples analyzed by the Laboratory during 2022 for WADA Prohibited List beta blockers and diuretics substances. The study will be continued for : 1) the incorporation of all the LC-screening analytes, 2) the reduction of the time and effort of LC screening data processing of the one out of two analysts by examining manually only the MetAlign searching results and not the full volume of the 1 1,2 1 1 1 1 1 3 4 1 1 1 5 1 2 3 4 5 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0274 thousands of the ion chromatograms of a routine batch, 3) the creation of quality control charts of the routine LC screening analytes, 4) the creation of the database of the reduced LC-screening datafiles ready to be used for reprocessing in the framework of the preventive reanalysis and long-term sample storage. Table 1. Summary for 5 of the 25 beta blockers (MRL 50 ng/mL) and 5 of the 41 diuretics studied References 1. World Anti-Doping Agency. The 2023 WADA Prohibited List. https://www.wada-ama.org/en/resources/world- anti-doping-program/2023-prohibited-list, (access date 10.01.2023) 2. Lommen A. (2009) MetAlign: interface-driven, versatile metabolomics tool for hyphenated full-scan mass spectrometry data preprocessing. Anal Chem. 81(8),3079-86. 3. Lommen A, Elaradi A, Vonaparti A, Blokland M, Nielen MW, Saad KA, Abushreeda, WM, Horvatovich P, Al- Muraikhi AE, Al-Maadheed M, Georgakopoulos C. (2019) Ultra-fast retroactive processing of liquid chromatography high-resolution full-scan Orbitrap mass spectrometry data in anti-doping screening of human urine. Rapid Commun Mass Spectrom. 33,1578–1588. 4. Khelifi S, Saad K, Vonaparti A, Mahieddine S, Salama S, Saleh A, Al-Mohannadi M, Al-Thaiban H, Lommen A, Horvatovich P, Beotra A, Abushareeda W, Al Maadheed M, Georgakopoulos C. (2021) Ultra-fast retroactive processing by MetAlign of liquid chromatography/high-resolution full-scan Orbitrap mass spectrometry data in WADA Human Urine Sample Monitoring Program. Rapid Commun Mass Spectrom. 35(17):e9141. 5. World Anti-Doping Agency. The World Anti-Doping Code International Standard for Laboratories (ISL). https://www.wada-ama.org/en/resources/laboratories/international-standard-for-laboratories-isl (access date 10.01.2023) 6. World Anti-Doping Agency. WADA Technical Document-TD2022MRPL. https://www.wada- ama.org/sites/default/files/2022-01/td2022mrpl_v1.1_eng_0.pdf. (Access date 12.11.2022) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0275 RECENT ADVANCES IN DOPING ANALYSIS 2023(31) Table of contents Lectures Poster Presentations Presentations Abstracts - Lectures Presentations Abstracts - Posters Scientific Board Contributions 1_Lectures 23_Fedoruk 2_Poster Presentations 01_Athanasiadou 02_Soni 03_Suominen 04_Wu 07_Fußhöller 09_Guddat 10_Montes de Oca Porto 15_Kwon 16_Cutler 18_Loungyot 20_Grucza 21_Grucza 22_Görgens 24_Sahu 27_Okano 28_Saavedra 30_McPherson 39_da Costa 41_Stojanovic 46_Yan 47_Chang 49_Thomas 51_Görgens 56_Radu 57_Wicka 58_Sobolevsky 66_Liu 67_Grainger 68_Yan 69_Wicka 70_Wicka 3_Presentation Abstracts - Lectures 01_Ahi 02_Al-Qahtan 03_Al-Mohannadi 04_Brooker 05_Torres 06_Rubio 07_Kwon 08_Colpaert 09_Krug 10_Santos 11_Paßreiter 12_Wagener 13_Naumann 14_Euler 15_Krombholz 16_Möller 17_Coll 18_Alechaga 19_Ventura 20_Langer 21_Thilmany 22_Breuer 24_Aguilera 25_Piper 26_Nair 27_Piper 28_de la Torre 29_Piper 30_Goodrum 31_Marchand 32_Loria 33_Dørum 34_Leuenberger 35_Heiland 36_Requena-Tutusaus 37_Reichel 38_Miller 39_Garzinsky 4_Presentation Abstracts - Posters 05_Martinez 06_Martinez 08_Martinez 11_Sobolevsky 12_Andersson 13_Euler 14_Wen 17_Peters 19_Keiler 23_Sahu 25_Walpurgis 26_Walpurgis 29_Fiallo 31_He 32_Joonyeop 33_Mongongu 34_Somay Selbes 35_Donati 36_Donati 37_Ponzetto 38_Guo 40_Buisson 42_Alechaga 43_Musenga 44_Miyamoto 45_Sletten 48_Heiland 50_Brockbals 52_Kim 53_González-Rubio 54_Keiler 55_Cristea 59_Upadhyay 60_Naumann 61_Akiyama 62_Son 63_Son 64_Aldubayyan 65_Al-Naimi 71_Petterson-Bohlin 72_Leogrande 73_Saad

M. THEVIS H. GEYER U. MARECK (EDITORS) RECENT ADVANCES IN DOPING ANALYSIS (31) Proceedings of the Manfred Donike Workshop 41st Cologne Workshop on Dope Analysis 27th February to 3rd March 2023 SPORTVERLAG Strauß - Hellenthal 2023 RECENT ADVANCES IN DOPING ANALYSIS (31) 2 ISBN 978-3-86884-049-0 Bibliografische Information Der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über abrufbar. Thevis, Mario; Geyer, Hans; Mareck, Ute (Eds.) Recent Advances in Doping Analysis (31). Proceedings of the Manfred Donike Workshop, 41st Cologne Workshop on Dope Analysis, 27th February to 3rd March 2023/ [MDI e.V.] – 2023 Sportverlag Strauß. ISBN 978-3-86884-049-0 ©SPORTVERLAG Strauß Neuhaus 12 – 53940 Hellenthal Tel. +49 (0)2448/2470040 – Fax +49 (0)2448/9195610 E-mail: info@sportverlag-strauss.de www.sportverlag-strauss.de Satz: Autorensatz RECENT ADVANCES IN DOPING ANALYSIS (31) 3 ISBN 978-3-86884-049-0 MANFRED DONIKE WORKSHOP 2023 TABLE OF CONTENTS PAGE LECTURES Fedoruk M, Breuer J, Thevis M, Silk N: Investigating claims of sexual contamination by analysis for the presence of semeno- gelin in athlete urine samples ...................................................................................................... 14-15 POSTER PRESENTATIONS Athanasiadou I, Tsivou M, Geisendorfer T, Gmeiner G: Occurrence of ecdysteroids in doping control urine samples analysed by the Seibersdorf Laboratory ................................................................................................................ 16-21 Soni A, Rani S, Sahu PL: Effect of mefenamic acid on the urinary endogenous anabolic androgenic steroid profile ............................................................................................................................................ 22-26 Suominen T, Piirainen M, Vanne H: Analysis of steroid esters and quantitation of endogenous steroids in blood as Girard P derivatives .................................................................................................................................... 27-31 Wu Q, Yan K, Dong T, Liu L, Wang Y, Wang Z: Quantification of testosterone and androstenedione in human serum by HRMS ..................... 32-36 Fußhöller G, Geyer H, Görgens C, Guddat S, Jübner M, Lucuta L, Thevis M: Excretion pattern of urinary metabolites of dehydrochloromethyltestosterone after transdermal application ................................................................................................................ 37-40 Guddat S, Fußhöller G, Geyer H, Görgens C, Jübner M, Lucuta L, Thevis M: Excretion pattern of urinary metabolites of metandienone after transdermal application ..... 41-44 Montes de Oca Porto R, Martinez Brito D, Lopez Castillo D, Rodriguez Fernandez A, Laffita Jorrin D: Behaviour of 19-norandrosterone levels in negative samples ........................................................ 45-49 RECENT ADVANCES IN DOPING ANALYSIS (31) 4 ISBN 978-3-86884-049-0 PAGE Kwon OS, Muresan AR, Rahaman KA, Xu Y, Jeon MJ, Kim MY, Jung S, Chin AL, Min H, Kim HJ, Sung C, Kim KH, Lee KM, Kang MJ, Lee J, Son J: The proposed mass fragmentation pathways for bolasterone metabolite identification in rats by high resolution LC-HRMS and GC-MS/MS .................................................................... 50-56 Cutler C, Viljanto M, Hincks P, Habershon-Butcher J, Scarth J, van Eenoo P: Comparison of the human and equine metabolism of the SARM S-23 in vitro and in urine .............................................................................................................................................. 57-60 Loungyot T, Thangboonjit W, Prompittayayut P, Yaikwawong M, Songsaeng R, Panan S, Thitamadee S, Kongpatanakul S: Alterations of muscular atrophy gene expressions in clenbuterol induced human myotubes ....................................................................................................................................... 61-65 Grucza K, Wicka M, Drapala A, Konarski P, Stanczyk D, Kaliszewski P, Kwiatkowska D: Furosemide glucuronide as a valuable biomarker for the interpretation of results for furosemide in doping control analysis s ....................................................................................... 66-70 Grucza K, Wicka M, Drapala A, Konarski P, Stanczyk D, Kaliszewski P, Kwiatkowska D: Occurrence of higenamine and its sulfate conjugate in routine doping control analysis - interpretation of results ............................................................................................................. 71-75 Görgens C, Guddat S, Bosse C, Thevis M: Enantiomeric separation of various stimulants in urine after chiral derivatization using Marfey’s reagent for doping control purposes ............................................................................ 76-80 Sahu PL, Handa T, Sahu K, Soni A, Kori RK, Mahajan S, Padder AH, Nandi U, Ahmed QN, Reddy DS: Synthesis and characterization of 17α-hydroxymethyl-17β-methyl-18-nor-2-oxa-5α- androst-13-en-3-one (Oxandrolone M2), a long-term metabolite via a Kratena’s reaction approach ........................................................................................................................................ 81-85 Okano M, Akiyama K, Sato M, Mombayashi A: Can current WADA-approved direct detection methods identify the use of GH analogue Somatrogon in doping control? .................................................................................................... 86-90 Saavedra MJ, Sánchez-Wandelmer J, Polo ML, Rodríguez-de Francisco G, Muñoz G: Bandmate™ as a good alternative to BlotCycler™ for erythropoietin detection by Western blot .................................................................................................................................. 91-96 Mc Pherson Medina A, Fiallo Fernandez T, Martinez Brito D, Montes de Oca Porto R: Stability of the biotinylated monoclonal antibody BAM2871 in the 1% non-fat milk solution ........................................................................................................................................ 97-101 da Costa S, Ruivo J, Casimiro T, Pereira L, Salema B: Long-term stability study of erythropoietin and erythropoietin receptor agonists detection in dried blood spots and comparison of different absorbent sample supports .... 102-106 RECENT ADVANCES IN DOPING ANALYSIS (31) 5 ISBN 978-3-86884-049-0 PAGE Stojanovic B, Pecher D, Geisendorfer T, Tsivou M, Gmeiner G: Dried blood spot microsampling as a suitable tool to monitor diuretics misuse in sport ...... 107-111 Yan X, Yan S, Chang W, Zhang L, Wang Z, Yang S: A comparative study of DFT calculated and experimental UV/Visible spectra for the E/Z testosterone ester oximes at trace levels ....................................................................................... 112-116 Chang W, Yan X, Yan S, Liu Y, Yang S: Pilot study of small peptide analysis in dried blood spots using automated SPE and UPLC-HRMS without DMSO ............................................................................................................... 117-121 Thomas A, Garzinsky A, Görgens C, Guddat S, Geyer H, Thevis M: Carbonic anhydrase inhibitors brinzolamide and dorzolamide in dried blood spots .............. 122-125 Görgens C, Geyer H, Guddat S, Machnik M, Schenk I, van de Bovenkamp A, Wagener F, Thevis M: Trimetazidine: pilot study data illustrating approaches to evaluate a potential contamination scenario ...................................................................................................................... 126-128 Radu M, Catana D, Berghes B, Cristea C, Toboc A, Stan C: Development and validation of a chromatographic separation method for betametha- sone and dexamethasone .................................................................................................................. 129-133 Wicka M, Konarski P, Drapala A, Kaliszewski P, Grucza K, Stanczyk D, Kwiatkowska D: LC-MS/MS method for the detection of synthetic cathinones in urine ..................................... 134-138 Sobolevsky T, Ahrens B: Simplified method for detection of urinary cobalt by LCMS ....................................................... 139-142 Liu L, Wang Z, Yan K, He G, Xing Y, Wu Q: Doping control analysis of morphine in human urine by ultraperformance liquid chromatographic-electrospray ionization tandem mass spectrometry? .................................. 143-148 Grainger J, Agon V, Croft M, Goebel C: Stability assessment of two small peptide ( < 2kDa) mixed standard ........................................ 149-153 Yan X, Yan S, Chang W, Yang S: A warning list to athletes of musk as ingredients from Traditional Chinese Medicine .......... 154-157 Wicka M, Drapala A, Konarski P, Zyfka-Zagrodzinska E, Golaszewska A, Marusza M, Gorczyca D, Kwiatkowska D: Hemp oils and potential for unintentional anti-doping rule violations ..................................... 158-162 Wicka M, Drapala A, Konarski P, Grucza K, Siek P, Kopczynska M, Kwiatkowska D: BPC-157 – case study ........................................................................................................................... 163-168 RECENT ADVANCES IN DOPING ANALYSIS (31) 6 ISBN 978-3-86884-049-0 PRESENTATIONS ABSTRACTS - LECTURES Ahi S, Wolff K: An overview of doping control analysis at the Birmingham 2022 Commonwealth Games ............................................................................................................................................... 169 Al-Qahtani A, Abushareeda W, Vonaparti A, Kraiem S, Elsaftawy W, Saad K, Al-Wahaibi A, Hilal N, Dbes N, Nandlal V, Beotra A, Al Maadheed M: Dope Testing: Overview of FIFA World Cup 2022 .................................................................... 170-171 Al-Mohannadi I, Samsam W, Al-Darwish S, Cherif A, Al-Jaber M, Abushreeda W, Al-Qahtani A, Kraiem S, Al-Mohammed H, Farooq A, Nimker V, Stuart M, Robinson N, Zangenfeind G, Rovira D, Beotra A, Al-Maadheed M, Mohamed-Ali V: First drone delivery of anti-doping samples: safe, secure, rapid, reliable and robust ........... 172-173 Brooker L, Shimko K, Speers N, Goebel C, Thomas K: Summary and evaluation of the Australian PIED wastewater-based epidemiology collaboration ............................................................................................................................. 174-175 Torres C, Oliveira F, Jooris LF, Padilha M, Pereira H: The presence of doping agents in dietary supplements: A glimpse into the Brazilian situation ..................................................................................................................................... 176-177 Rubio A, Thomas A, Euler L, Geyer H, Krug O, Reis G, Costa Padilha M, Pereira HMG, MunizSantos R, Cameron LC, Stojanovic B, Kuehne D, Lagojda A, Mc Leod MD, Thevis M: Investigations into Annona fruit consumption as a potential source of dietary higenamine intake in the context of sports drug testing ........................................................ 178-179 Kwon OS, Rahaman KA, Muresan AR, Xu Y, Jeon MJ, Kim MY, Jung S, Chin AL, Min H, Kim HJ, Sung C, Kim KH, Lee KM, Kang MJ, Lee J, Son J: The urinary metabolite Ac-Tβ1-14 in huvec cells and rats treated with thymosin β4: A potential biomarker for doping tests ........................................................................................... 180 Colpaert T, de Hon O, Duiven E, Olijhoek M, T'sjoen G, van Eenoo P, Deventer K, Coppieters G, van Uytfanghe K: Detection of thyroid hormones T3 and T4 in serum samples after T4 administration in healthy volunteers .................................................................................................................... 181-182 Krug O, Guddat S, Möller T, Piper T, Görgens C, Thevis M: Determination of urinary deanol-N-oxide excretion profiles after ingestion of nutritional supplements containing deanol ............................................................................. 183-184 Santos V, Carneiro G, Braz B, Santelli R, Machado S, Pereira H Coordination chemistry applied to antidoping science: cobalt quantification as diethyldithiocarbamate complex by LC-HRMS ............................................................................... 185-186 RECENT ADVANCES IN DOPING ANALYSIS (31) 7 ISBN 978-3-86884-049-0 PAGE Paßreiter A, Naumann N, Thomas A, Grogna N, Delahaut P, Thevis M: How to detect CRISPR with CRISPR - employing SHERLOCK for doping control purposes ........... 187 Wagener F, Naumann N, Leciejewska N, Görgens C, Guddat S, Thevis M: Using organ-on-a-chip for predicting the metabolism of the selective androgen receptor modulator RAD140 in comparison to established in vitro approaches .......................... 188 Naumann N, Wapurgis K, Rubio A, Thomas A, Paßreiter A, Thevis M: Detection of doping control sample substitutions via SNP-based ID typing ................................. 189 Euler L, Deinert K, Thomas A, Thevis M: Mass spectrometric characterization and development of detection methods for in vitro synthesized metabolites of the troponin activators reldesemtiv and tirasemtiv for doping control purposes ................................................................................................................. 190 Krombholz S, Thomas A, Delahaut P, Miller GD, Bidlingmaier M, Thevis M: A combined top-down and bottom-up LC-HRMS/MS method for the quantification of human growth hormone in plasma ................................................................................................. 191 Möller T, Naumann N, Krug O, Thevis M: Identification and synthesis of selected in vitro generated metabolites of the novel selective androgen receptor modulator (SARM) 2f ........................................................................ 192 Coll S, Bressan C, Alechaga E, Monfort N, Carbó M, Ventura R: Elimination profile of methylprednisolone in urine and plasma after oral administration: Evaluation of the reporting level and washout period ........................................ 193 Alechaga E, Bressan C, Coll S, Carbó M, Monfort N, Ventura R: Glucocorticoids and DBS I. Concentrations of glucocorticoids in DBS after oral administrations ................................................................................................................................ 194 Ventura R, Bressan C, Coll S, Alechaga E, Carbó M, Monfort N: Glucocorticoids and DBS II. Correlation of glucocorticoid concentrations in DBS and plasma samples ................................................................................................................................ 195 Langer T, Nicoli R, Kuuranne T, Musenga A: Comparison of serum and urine analysis for the confirmation of oral testosterone undecanoate administration .................................................................................................... 196-198 Thilmany S, Hardy T, Thomas A, Vogel M, Scholl C, Thevis M: Transcriptomic analysis of the effects of steroid hormones on neuronal cells ...................... 199-200 Breuer J, Thomas A, Delahaut P, Schänzer W, Geyer H, Thevis M: Investigations into the concentration and metabolite profiles of stanozolol and LGD- 4033 in blood plasma and seminal fluid using liquid chromatography high-resolution mass spectrometry ........................................................................................................................... 201 RECENT ADVANCES IN DOPING ANALYSIS (31) 8 ISBN 978-3-86884-049-0 PAGE Aguilera R: Athlete isotopic signature of δ C values. Complementary data from testosterone gel application (males and females) ..................................................................................................... 202 Piper T, Krombholz S, Thevis M: Carbon isotope ratios of phenethylamine and phenylacetylglutamine and their applicability to doping control ........................................................................................................ 203 Nair M, Howa J, Kucherova Y, Ahrens B, Pereira HMG, Nicoli R, Kuuranne T, Fedoruk M: Applicability of longitudinal monitoring of IRMS values - A case study with unusual values ................................................................................................................................................ 204 Piper T, Thevis M: Investigations into the human metabolism of ecdysterone ................................................... 205-206 de La Torre X, Colamonici C, Curcio D, Botrè F: 19-Norandrosterone confirmation by GC-C-IRMS: 10 years of experience ................................... 207 Piper T, Thevis M: Employing 11-ketotestosterone as target analyte for adrenosterone (11OXO) administrations in doping control ................................................................................................... 208 Goodrum J, Nair V, Moore C, Eichner D, Miller G: Effects of heavy biotin supplementation on detection of hCG after Ovidrel® administration to males ................................................................................................................... 209 Marchand A, Roulland I, Semence F, Jaffredo F, Dehainault C, Mekacher LR, Ericsson M: Detection of the homologous transfusion of a red blood cell concentrate in vivo: evaluation of flow cytometry and DNA analysis methods ...................................................... 210-211 Loria F, Breenfeldt Andersen A, Bejder J, Bonne T, Grabherr S, Kuuranne T, Leuenberger N, Baastrup Nordsborg N: Erythropoiesis-related RNAs biomarkers are sensitive and specific to low-dose erythropoietin treatment at sea level and altitude ........................................................................ 212 Dørum S, Sletten C, Oll Ø, Dehnes Y: EPO screening with K9 ..................................................................................................................... 213 Leuenberger N, Kuuranne T, Castella V: Sequencing c.577del in EPO gene in urine and dried blood samples...................................... 214-215 Heiland CE, Leuenberger N, Ericsson M, Marchand A, Martin L: DBS in practice for EPO doping control analysis: in vivo evaluation ...................................... 216-218 RECENT ADVANCES IN DOPING ANALYSIS (31) 9 ISBN 978-3-86884-049-0 PAGE Requena-Tutusaus L, Anselmo I, Alechaga E, Bergés R, Ventura R: Detection of ERAs in a single-spot DBS sample at MRPL level: improving applicability of DBS in doping control .................................................................................................................. 219 Reichel C, Gmeiner G, Thevis M: Cost minimized immunoaffinity purification of EPO and its analogs in doping control – A step-by-step protocol for human blood and urine ............................................................... 220-221 Miller G, Goodrum J, Campbell T, Eichner D: Assessing the stability of recombinant EPO in urine ...................................................................... 222 Garzinsky A, Harth J, Reihlen P, Leipp F, Thomas A, Thevis M: Can the detection of EPO and its derivatives be manipulated by the addition of oral fluid to doping control urine samples? ........................................................................................... 223 PRESENTATIONS ABSTRACTS - POSTERS Martinez Brito D, de La Torre X, Botrè F: Analysis of testosterone esters by gas chromatography-highresolution mass spectrometry with low-energy electron ionization ................................................................. 224-225 Martinez Brito D, de La Torre X, Botrè F: Correlation of androstenedione and testosterone measurements by LC-MS/MS and GC-LE-TOF .................................................................................................................................. 226-227 Martinez Brito D, de La Torre X, Botrè F: Effect of thyroid hormones administration on the urinary endogenous steroid profile of the athlete biological passport ............................................................................................. 228-229 Sobolevsky T, Walpurgis K, Görgens C, Fedoruk M, Lewis L, Ahrens B, Thevis M: Detection of capromorelin in urine following different routes of administration ........................ 230 Andersson A, Pohanka A, Ekström L: 18-Methyl-19-noretiocholanolone detected after use of emergency contraceptive ................... 231 Euler L, Mürdter T, Heinkele G, Schwab M, Miller GD, Eichner D, Thomas A, Thevis M: Identification of (Z )-3‘-hydroxy clomiphene as a new potential dopingrelevant metabolite of clomiphene ............................................................................................................... 232 Wen H, Wagener F, Neudörfl J, Thevis M, Schäfer M: Investigation of the phase I metabolism of a new 20-keto-steroid S42 by C-6 oxidation and GC-MS analysis ................................................................................................................... 233-234 RECENT ADVANCES IN DOPING ANALYSIS (31) 10 ISBN 978-3-86884-049-0 PAGE Peters I, Naumann N, Görgens C, Guddat S, Thevis M: 3D cultivation of HepaRG cells for metabolite prediction of the calstabin-ryanodine receptor complex stabilizer S107 in preventive doping research .................................................. 235 Keiler AM, Rzeppa S, Voss SC, Thieme D: In vitro biotransformation of voxelotor .......................................................................................... 236 Sahu PL, Matsa R, Pawar S, Seth K, Radhakrishnanand P, Murty U, Handa T, Sahu K, Kori RK, Kumar P: Synthesis and characterization of ostarine metabolite: applicable for TL 12 ............................... 237 Walpurgis K, Agricola J, Thomas A, Thevis M: Myostatin inhibitory peptides in sports drug testing ..................................................................... 238 Walpurgis K, Gäde A, Thomas A, Gochard S, Delahaut P, Thevis M: Detection of extracellular hemoglobin from Arenicola marina in doping control serum and plasma samples by means of LC-HRMS/MS ............................................................................. 239 Fiallo Fernandez T, Mc Pherson Medina A, Martinez Brito D, Montes de Oca Porto R: Evaluation of the Cuban CBSSEPO monoclonal antibody for the detection of erythropoiesis-stimulating agents in doping control ..................................................................... 240 He S, Liu X, Zhou X: Administration study of rEPO on c.577del carriers ......................................................................... 241 Joonyeop Y, Minyoung K, Mijin J, Hophil M, Byung-Gee K, Junghyun S, Changmin S: Direct multiplex-PCR based gene doping detection for human growth hormone and its isoform using CRISPR/Cas12a-assisted HOLMES ............................................................................ 242 Mongongu C, Madi Moussa E, Marchand A, Buisson C, Ericsson M: Cubosomic supramolecular solvents: synthesis, characterization and potential for high throughput multiclass testing of banned substances in urine ....................................................... 243 Somay Selbes Y, Demirel AH, Elaldi K: Comparison of Insulin-like growth factor 1 measured between Siemens Immulite and IDS-iSYS ............................................................................................................................................. 244 Donati F, de La Torre X, Botrè F: Screening of exogenous hemoglobin with Sysmex XN analyzer .................................................... 245 Donati F, de La Torre X, Botrè F: Real-Time PCR SNP genotyping for the detection of homologous blood transfusion in dried blood spots .............................................................................................................................. 246 RECENT ADVANCES IN DOPING ANALYSIS (31) 11 ISBN 978-3-86884-049-0 PAGE Ponzetto F, Settanni F, Nonnato A, Nicoli R, Ghigo E, Kuuranne T, Mengozzi G: Volumetric absorptive microsampling (VAMS) for targeted LC-MS/MS measurement of novel blood markers of EAAS doping ....................................................................................... 247-248 Guo J, Goucher E, Patterson C, Gibson R, van Natta K, Samra S: Direct quantitation of 77 drugs of abuse in dried blood spots using the fully automated transcend DSX-1 system ................................................................................................................... 249 Buisson C, Smires S, Ericsson M: Dried blood spots for doping controls – Validation of a quantification procedure for corticosteroids ........................................................................................................................... 250-251 Alechaga E, Bressan C, Coll S, Monfort N, Ventura R: Multiclass method for the screening of doping agents in dried blood spot .................................. 252 Musenga A, Langer T, Salamin O, Nicoli R, Kuuranne T: A fast screening of doping agents in DBS by LC-MS/MS ................................................................. 253 Miyamoto A, Ota M, Sato M, Okano M: Simultaneous detection method of 26 testosterone, nandrolone and boldenone esters in dried blood spots by LC-MS/MS .................................................................................................. 254 Sletten C, Solheim SA, Bækken LV, Dehnes Y: Effect of Sustanon® administration on ALAS2 expression ............................................................. 255 Heiland CE, Mongongu C, Semence F, Pohanka A, Ericsson M, Marchand A, Ekström L: IGF-I variation in DBS and serum .............................................................................................. 256-257 Brockbals L, Thomas A, Schneider TD, Kraemer T, Steuer AE, Thevis M: LC-MS/MS-based peptide analysis for dried blood spot sampling time point estimation ........... 258 Kim KH, Jeong W, Eun Byul C, Min H, Son J: Development of an online immunopurification assay for urinary insulin and insulin mimetics using AF4-ESI-MS/MS ....................................................................................................... 259 González-Rubio S, Caballero-Casero N, Ballesteros-Gómez A, Cuervo D, Muñoz G, Rubio S: A general screening method for testing of banned substances in urine based on cubosomic supramolecular solvents and liquid chromatography/time-of-flight mass spectrometry .................................................................................................................................... 260 Keiler AM, Derwand D, Rzeppa S, Voss SC, Thieme D: QuEChERS as alternative extraction procedure in doping analyses .............................................. 261 RECENT ADVANCES IN DOPING ANALYSIS (31) 12 ISBN 978-3-86884-049-0 PAGE Cristea C, Radu M, Catana D, Berghes B, Toboc A, Stan C: Triple quadrupole UHPLC-MS/MS method as an alternative of orbitrap UHPLC-HRMS method used for detection of growth hormone-releasing hormones ........................................... 262 Upadhyay AK, Bhardwaj A, Sahu P: Comprehensive identification method for hypoxia-inducible factor (HIF) activating agents................................................................................................................................................ 263 Naumann N, Vollmert C, Krajina M, Thevis M: Evaluation of a novel gene doping detection approach via high multiplex MALDI-TOF MassARRAY analysis ........................................................................................................................ 264 Akiyama K, Momobayashi A, Okano M: Individual identification method using samples related to doping tests: a comparison of mitochondrial and nuclear genetic data ..................................................................................... 265 Son J, Park H, Cho Y, Sung C, Min H, Lee KM, Bahn Y: Enhancement of anti-doping strategy using artificial intelligence (AI): A study on improving the accuracy and efficiency of detecting performance-enhancing drugs in athletics as a proposed nextgeneration doping diagnosis strategy ............................................... 266 Son J, Cho Y, Park H, Jeon S, Sung DJ, Jeong JH, Sung C, Min H, Lee KM, Kim H, Yoon SS: Clinical study of brain doping diagnosis (bD ): Confirmation of sports performance enhancement and neurochemical alterations via brain stimulation ............................................. 267 Aldubayyan A, Castrignano' E, Abbate V: Short- and long-term stability of synthetic cathinones and dihydrometabolites in human urine samples: considerations for antidoping and forensic toxicology applications ...................................................................................................................................... 268 Al-Naimi N, Kraiem S, Samsam W, Beotra A, Orie N, Mohamed-Ali V, Almaadheed M: Turkesterone a noval ecdysteroid: High resolution orbitrap mass spectrometry identification of turkesterone in supplements and urine samples ......................................... 269-270 Pettersson-Bohlin K, Pohanka A, Ekström L: Detection of SARMs in the Swedish society ............................................................................. 271-272 Leogrande P, Jardines D, Martinez Brito D, de La Torre X, Botrè F, Luch A, Diel P, Parr MK: Physiological and post-training effects of ecdysteroid supplements. A multivariate study of the human serum metabolome ........................................................................................ 273 Saad K, Vonaparti A, Al-Mohannadi I, Khelifi S, Salma S, Mahieddine S, Saleh A, Lommen A, Horvatovich P, Beotra A, Abushareeda W, Al Maadheed M, Georgakopoulos C: Preliminary study of MetAlign software automatic LC/MS screening data evaluation ......... 274-275 RECENT ADVANCES IN DOPING ANALYSIS (31) 13 ISBN 978-3-86884-049-0 SCIENTIFIC BOARD F. Botrè, Rome (Italy) D. Cowan, London (UK) X. de la Torre, Rome (Italy) H. Geyer, Cologne (Germany) G. Gmeiner, Seibersdorf (Austria) A. Keiler, Dresden (Germany) U. Mareck, Cologne (Germany) M. Okano, Tokyo (Japan) H. Pereira, Rio de Janeiro (Brazil) T. Piper, Cologne (Germany) C. Reichel, Seibersdorf (Austria) W. Schänzer, Cologne (Germany) M. Thevis, Cologne (Germany) A. Thomas, Cologne (Germany) P. van Eenoo, Ghent (Belgium) R. Ventura, Barcelona (Spain) S. Rzeppa, Dresden (Germany) Fedoruk M , Breuer J , Thevis M , Silk N Investigating claims of sexual contamination by analysis for the presence of semenogelin in athlete urine samples Science & Research, U.S. Anti-Doping Agency (USADA), Colorado Springs, United States of America ; Centre for Preventive Doping Research, Institute of Biochemistry, German Sport University Cologne, Cologne, Germany Abstract In cases where athletes allege sexual contamination has resulted in a positive test, semenogelin has emerged as an important potential marker of semen exposure from sexual contact that could help to corroborate an athlete's claim. Methods to identify semenogelin I, a major and specific protein constiuent of semen, in urine samples collected during doping control have been published using two analytical approaches, a laminar flow immunological test strip (initial testing procedure) and a bottom-up LC- HRMS/MS-based test (confirmation) method, specific for semenogelin I. Four previous cases were examined where positive samples were kept in long-term storage to determine whether the presence or absence of semenogelin I could corroborate the athlete's version of events. Based on these further analyses, it was determined that the presence or absence of semenogelin in the urine does not provide unequivocal evidence as to how a drug or its metabolites are introduced into an athlete's urine sample, but could provide helpful supporting evidence. Specific factors surrounding seminal exposure and intimate contact may influence the ability to detect semenogelin in an athlete's urine sample. Introduction The athlete exposome has recently become an important focus of anti-doping research due to the likelihood that athletes are exposed to prohibited substances through circumstances that may not be related to doping. Contamination scenarios based on the ingestion of food, cosmetics, dietary supplements, medications, uncommon metabolic drug interactions, and physical contact are all proven avenues for adverse analytical findings (AAFs)[1]. The differentiation between intentional drug use and all the various contamination scenarios is critical in order to ensure that athletes are able to effectively defend themselves, as the burden remains on the athlete to demonstrate the source of a positive doping test. Using urine in long-term storage, urine samples were analyzed for the presence of sperm residue marker, semenogelin, to examine the possibility of a prohibited substance being present in a female athlete’s urine sample due to residual ejaculate being introduced by recent sexual intercourse into the vaginal area of an athlete and contaminating the urine sample collected during doping control. Trace amounts of semenogelin could be used to support an athlete’s claim of contamination due to their doping male partner transferring a prohibited substance to their body. Experimental Athlete samples were tested using a protocol of a commercially available lateral flow immuno‐ 1 2 2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-014 chromatographic test directed against semenogelin. Secondly, a liquid chromatography/tandem mass spectrometry (LC-MS/MS)-based method was used, employing solid-phase extraction of urine, followed by trypsinization of the retained protein content and subsequent detection of semenogelin I-specific peptides [2]. Results and Discussion Signals were not observed for semenogelin in an athlete’s urine sample that reported sexual intercourse with her male partner shortly before sample collection. Prior further investigation of this case demonstrated that the male partner was doping with letrozole and GW-1516 at the time of multiple unprotected sexual encounters with the athlete. Similiarly, no semenogelin was detected in an athlete’s sample that reported unprotected sexual intercourse two days prior to sample collection; however, the athlete tested positive for LGD-4033 metabolite dihydroxy-LGD-4033. An investigation in this case demonstrated her male partner was using therapeutic doses of LGD-4033 at the time of intimate contact. Two further samples contained semenogelin, however the athletes in these cases admitted to using oral dehydroepiandrosterone (DHEA), or sexual contamination was not communicated as an explanation for the positive test. Two other samples tested negative for the presence of semenogelin where athletes claimed sexual contamination as a possible source of both stanozolol and ostarine positive tests. During the results management of these doping cases, sexual contamination ruled out as a plausible route of exposure based on a comprehensive investigation of the facts. Importantly, it appears additional consideration must be given to the specific circumstances and timing of sexual contact relative to sample collection, the viracity of alleged use by the partner of prohibited substances and the estimated concentrations of prohibited substances and/or their metabolites observed in the athlete's urine sample. Conclusions Semenogelin presence can be used as strong supporting evidence for prohibited substance exposure via the introduction of male ejaculate ‘dripping’ into a female athlete’s doping control urine sample, however there are some important limitations, and the presence (or absence) cannot be used as unequivocal evidence of prohibited substance exposure via sexual transmission. References 1. Thevis M, Kuuranne T, Fedoruk M, Geyer H. Sports drug testing and the athletes' exposome. Drug Test Anal. 2021 Nov;13(11-12):1814-1821 2. Breuer, J, Thomas, A, Geyer, H, Thevis, M. Probing for the presence of semenogelin in human urine by immunological and chromatographic-mass spectrometric methods in the context of sports drug testing. Anal Sci Adv. 2022; 3: 21– 28 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-015 Athanasiadou I, Tsivou M, Geisendorfer T, Gmeiner G Occurrence of ecdysteroids in doping control urine samples analysed by the Seibersdorf Laboratory Doping Control Laboratory, Seibersdorf Labor GmbH, Seibersdorf, Austria Abstract In 2020, ecdysterone was included in the monitoring program of the World Anti-Doping Agency (WADA) to monitor its prevalence of misuse in professional sports, in and out of competition. Ecdysterone has been suggested to be included in WADA’s Prohibited List in class S.1.2 Other Anabolic Agents with a reporting cut-off limit [1].The screening of 20525 doping control samples' “digital matrix” data was used to extract information regarding ecdysterone and turkesterone use in relation to the athlete's gender, type of competition and sport. The occurrence of ecdysteroids in the athlete population was estimated to be approx. 1% with a higher prevalence in OOC samples from strength and endurance sports originating from Balkan and Eastern European countries. Furthermore, a controlled administration study with a single oral dose of approx. 50 mg of a beta-ecdysone supplement was conducted to monitor the excretion profiles of ecdysterone and its main metabolite 14-deoxy-ecdysterone and to investigate any effects on steroid profile parameters (testosterone, epitestosterone, androsterone, etiocholanolone, 5α- androstane-3α,17β-diol (5αAdiol), 5β-androstane-3α,17β-diol (5βAdiol) and ratios. All the steroid profile markers followed the normal urinary circadian rhythm. No effect was observed on steroid profile markers or the relevant respective ratios in the presence of ecdysterone. Introduction In sports, ecdysteroids are widely marketed to athletes as dietary supplements advertised to increase strength and muscle mass during resistance training, to reduce fatigue and to ease recovery. Several studies have reported a wide range of pharmacological effects of ecdysteroids in mammals, most of them beneficial to the organism [2]. The most active phytoecdysteroid, ecdysone (also known as “Russian secret”), was suspected to be used by Olympic athletes back in the 1980s. Recently, ecdysterone was reported to enhance performance in sports [3] and to promote an anabolic effect even stronger than that of the anabolic steroid metandienone [4-9]. In addition, a global interest in turkesterone supplements distributed online was observed in the last couple of years, especially in countries such as Brazil, Australia, Norway, the United States, and Canada. “Digital matrix” of the analysed doping control samples containing a significant amount of information (e.g. gender, type of competition, sport discipline, etc) adds a tremendous value for retrospective data mining purposes [10,11]. The screening of the “digital matrix” of doping control data could lead to useful conclusion regarding the prevalence and patterns of the use of substances based on different aspects of the athlete population. In this context, the current project exploited the “digital matrix” of 20525 doping control urine samples for the retrospective data mining of ecdysterone and turkesterone presence to obtain a snapshot of the athlete population for the period between June 2020 and September 2021. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-016 Experimental Oral administration of ecdysterone and urine collection A controlled excretion study was performed after the administration of a single oral dose of approx. 50 mg (20-beta-hydroxy-ecdysone, Westpharmaceuticals LLC, Indiana, USA) to one male volunteer (55 yrs, weight of 95 kg, and height of 193 cm). Supplement analysis The content of one capsule (100 mg as referred in the product label) was diluted with 10 mL MeOH and then incubated in the ultrasound bath for 10 min. 10 µL of the diluted capsule were transferred in a conical tube, spiked with 50 µL internal standard and diluted up to 1 mL with MeOH/Water (10/90). 100 µL of the prepared mixture were transferred into an autosampler vial and 10 μL was injected to the LC-MS system for analysis. Table 1. Mass spectrometric information for the analytes under investigation Evaluation of excretion profile in urine In this study, the excretion profiles of ecdysterone and its main metabolite after a single-dose administration of 50 mg of pure ecdysterone were evaluated. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-017 Evaluation of the steroid profile markers Steroid profile markers including testosterone, epitestosterone, androsterone, etiocholanolone, 5α- androstane-3α,17β-diol (5αAdiol), 5β-androstane-3α,17β-diol (5βAdiol) and the respective ratios were evaluated in the excreted urine samples as mentioned in the WADA technical document [12]. Urine concentrations of the steroid profile markers were adjusted based on the specific gravity (SG) measurements of the excretion samples. Retrospective analysis using TraceFinder software Retrospective analysis was performed on all urine samples analysed by the Seibersdorf Doping Control Laboratory between June 2020 and September 2021 (in total 20525 data files evaluated). Using the Tracefinder software (TraceFinderTM 5.1), a Compound Database and a Master Method created based on the mass spectrometric info presented for the target analytes. A statistical evaluation based on the athlete's gender, the type of competition and the sport was conducted to obtain a snapshot of ecdysterone and turkesterone use in the athlete population. Results and Discussion Supplement analysis Supplement analysis showed ~ 50 mg ecdysterone per capsule (exact amount 48 mg/capsule). However, the amount of ecdysterone on the product label was equal to 100 mg/capsule. Evaluation of excretion profile of ecdysterone and its main metabolite Following a single-dose administration of 50 mg ecdysterone, the parent compound resulted to be the most abundant analyte in all post-administration urine samples. C (2221 ng/mL) was detected 3.67 h after administration (TAD), whereas the main metabolite (14-deoxy-ecdysterone) was detectable from 9.83 h up to 3 days with C (743 ng/mL) observed at 43 h TAD. Excretion profiles of ecdysterone and its metabolite are presented in Figure 1. Figure 1. Urinary excretion profile (concentration-time curve) of ecdysterone (left) and 14-deoxy-ecdysterone (right), following a single-dose administration of 50 mg of pure ecdysterone in a male volunteer max max MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-018 Evaluation of the steroid profile markers No effect was observed on steroid profile markers or the relevant respective ratios. The testosterone excretion profile and the T/E ratio are depicted as examples in Fiure 2. All the steroid profile markers followed the normal urinary circadian rhythm. Figure 2. Urinary excretion profile (concentration-time curve) of endogenous testosterone (left) and testosterone/ epitestosterone (T/E) (right) Retrospective and statistical analysis The occurrence of ecdysteroids in the athlete population tested was equal to be approx. 1%. Declared nutritional supplements containing ecdysterone and/or turkesterone include but are not limited to: Retibol Eiselt Research, Mega Ecdysterone, Ecdysterone-S, Beta Ecdyx Pure, Eksumid (Exumid), EcDyBol, etc. Russia, Estonia, Ukraine, Greece, Hungary, Lithuania, Moldova and Serbia were among the countries with the highest number of declared nutritional supplements containing ecdysteroids. As shown in Figure 3a, findings were distributed as 61% out of competition (OC) and 39% in-competition (IC) samples. Ecdysterone findings were distributed as 62% in males and 38% in females. Weightlifting, canoe/kayak, athletics and aquatics were the sports with the highest prevalence of ecdysterone and turkesterone. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-019 Figure 3. Graphic distribution of ecdysterone and turkesterone findings based on the type of distribution (a), the gender (b) and sport (c) Proof of concept The 2020 WAADS Educational Samples (pooled urine from an excretion study after ingestion of a supplement containing ecdysterone and separately turkesterone – according to the provider), were used to prove the fit for purpose of the developed retrospective method. Samples were processed with the retrospective method and all analytes were successfully detected. Conclusions The current study underlines the high occurrence of ecdysteroids in the athlete population (approx. 1%). In addition, the hereby presented retrospective approach can be a useful tool [11] - despite the limitation of the raw data files (approx. 1 Terabyte), when investigating the abuse of substances, that are not yet included in the prohibited menu of accredited laboratories but are available on the black market. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-020 References 1. WADA The 2021 Monitoring Program https://www.wada-ama.org/sites/default/files/resources/files/2021list_monitoring_program_en_0.pdf 2. M. Mazzarino, G. Ambrosio, A. Grieco, et al. Ecdysterone: a snapshot of use in the athlete’s population. Poster presented at the 38 Manfred Donike Workshop on Doping Analysis, Cologne, Germany, 2020. 3. E. Isenmann, G. Ambrosio, JF. Joseph, et al. Ecdysteroids as non-conventional anabolic agent: performance enhancement by ecdysterone supplementation in humans. Archives of Toxicology (2019), 93:7, 1807- 1816. 4. M. Parr, G. Ambrosio, B. Wuest, et al. Targeting the administration of ecdysterone in doping control samples. Forensic Toxicology (2020), 38:1, 172-184. 5. MK. Parr, F. Botre, A. Nass, et al. (2015) Ecdysteroids: a novel class of anabolic agents? Biol Sport 32(2):169–173. 6. NN. Orie, A. Raees, MY. Aljaber, et al. 20-Hydoxyecdysone dilates muscle arterioles in a nitrc oxide- dependent, estrogen ER-and beta receptor-independent manner. Poster presented at the 40 Manfred Donike Workshop on Doping Analysis, Cologne, Germany, 2022. 7. D. Kwiatkowska, K. Grucza, K. Chajewska, et al. Ecdysterone - possible sources of origin in urine. Poster presented at the 40 Manfred Donike Workshop on Doping Analysis, Cologne, Germany, 2022. 8. S. Kraiem, S. Bouabdallah, S. Touil, et al. Elimination profile of 20-hydroxyecdysone (20-OHE) in urine - Liquid-Liquid extraction and dilute and inject methodology using UHPLC/HRMS. A comparative study. Poster presented at the 40 Manfred Donike Workshop on Doping Analysis, Cologne, Germany, 2022. 9. D. Kwiatkowska, K. Grucza, K. Kowalczyk, et al. Ecdysterone - excretion study after ingestion of spinach. Poster presented at the 39 Manfred Donike Workshop on Doping Analysis, Cologne, Germany, 2021. 10. S. Khelifi, K. Saad, A. Vonaparti, et al. Ultra-Fast retroactive processing by MetAlign of LC-Chromatography High-resolution Mass Spectrometry Data in WADA Human Urine Sample Monitoring Program. Poster presented at the 39 Manfred Donike Workshop on Doping Analysis, Cologne, Germany, 2021. 11. M. Thevis, K. Walpurgis, A. Thomas. Analytical Approaches in Human Sports Drug Testing: RecentAdvances, Challenges, and Solutions. Anal.Chem. (2020), 92, 506−523. 12. WADA Measurement and Reporting of Endogenous Anabolic Androgenic Steroid (EAAS) Markers of the Urinary Steroid Profile; www.wada-ama.org/sites/default/files/2022-01/td2021eaas_final_eng_v_2.0.pdf Acknowledgements The current work was supported by the Austrian Ministry of Sports. The authors would like to acknowledge the Cologne Laboratory and Dr. Geyer for the ecdysterone supplement. th th th th th th MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-021 Soni A, Rani S, Sahu PL Effect of mefenamic acid on the urinary endogenous anabolic androgenic steroid profile National Dope Testing Laboratory, New Delhi, India Abstract Endogenous anabolic androgenic steroids (EAAS) are prohibited in sports when administered exogenously. The World Anti-Doping Agency (WADA) accredited laboratories monitor the steroid profile markers (concentrations and concentration ratios of “pseudo-endogenous” steroid, especially testosterone, its precursors and metabolites) in urine samples collected from athletes in and out of competition. The Athlete Biological Passport (ABP) has been used for long-term monitoring of alterations of the intra-individual steroid concentrations and ratios in anti-doping analysis. As steroid metabolism is very complex and certain non-prohibited drugs may influence the concentrations of the urinary steroid profile or its interpretation. Aldo-keto-reductases (AKRs), particularly AKR1C3, are a significant group of enzymes that are crucial for steroidogenesis. It was discovered that NSAIDs are powerful inhibitors of AKR1C3 enzymes. Thus, the aim of this work was to investigate the effect of mefenamic acid on the urinary steroid profile. A urinary excretion study after drug administration for two consecutive days (twice a day) was carried out using a validated GC-MS/MS method of the laboratory. Data analysis was performed by using Agilent Mass-Hunter Quantitative software. As expectetd, significant changes in a few steroid concentrations and their ratios were observed in some volunteers after the intake of mefenamic acid. The significant changes in the steroid profile were detected especially the concentration of androsterone and the Andro/T ratio. Introduction Endogenous anabolic androgenic steroids (EAAS) abuse in sports is indicated by changes in steroid profile markers [1]. The steroidal module of the Athlete Biological Passport has been used to monitor steroid profiles and it compares individual reference ranges established from several test results of the same athlete [2]. But recent studies have shown, that some other (neither prohibited nor monitored) drugs, so called confounding factors, can alter the individual's steroid profile. Mefenamic acid (trade name in India, e.g., Meftal) is a nonsteroidal analgesic (NSAID) approved by the FDA for the treatment of mild to moderate acute pain. A previous study reports that mefenamic acid inhibits the aldo-keto reductase 1C2 (AKR1C2) enzyme in vitro [4]. This enzyme plays a key role in endogenous steroid metabolism (e.g., conversion of potent 5a-dihydrotestosterone (5aDHT) to inactive 5a-androstane- 3a,17b-diol (ADIOL)) [3]. This inhibition may cause changes in the steroid profile and a misleading interpretation by the ABP module could lead to unnecessary but very costly confirmative analyses by GC/C/IRMS. This work shows an in vivo excretion study on two volunteers followed by analysis of urinary steroids included in the steroid profile using GC-MS/MS. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-022 Experimental Materials Reference standards of endogenous steroids and deuterated internal standards were procured from Sigma-Aldrich and National Measurement Institute, Australia. Inert-sep HLB sample preparation cartridges were procured from GLSciences, the enzyme β-glucuronidase from E. coli was purchased from Roche, USA. MSTFA and all other solvents and chemicals were of high-performance liquid chromatography (HPLC) grade and analytical grade, respectively, from Merck and Sigma-Aldrich mainly. Selection of volunteers and in vivo administration trial Two volunteers (1 female and 1 male) were selected by screening of their urinary steroid profile. The age range of selected volunteers was 18-30 years. A 500 mg mefenamic acid tablet (manufactured by Blue Cross Laboratories Pvt. Limited) was provided twice a day for two consecutive days to each volunteer. Urine samples were collected in the following manner: - 4 urine samples before drug administration (sampling points: 07:00 h, 12:00 h, 17:00 h and 21:00 h) - 4 urine samples for 2 days during the administration (same time-points as before) - 2 urine samples for next 4 days - 1 sample for further 4 days after drug administration. All samples were anonymized, collected in sterile plastic tubes and stored at -20 °C until analysis. The study was approved by the ethics committee of the National Dope Testing Laboratory (NDTL), India and all volunteers signed the informed consent. Urine sample preparation Extraction of endogenous steroids (as free and glucuronide fraction) was performed using an already established protocol for steroid profiling of endogenous steroids as per TD2021EAAS which is routinely used in the dope testing laboratory at New Delhi, India. In brief, 2 mL of urine, 50 µL of internal standard mixture 1 mL of 0.2 M phosphate buffer (pH 7.0) and 50 µL of β-glucuronidase enzyme (E. coli) were added, followed by incubation of the sample at 60 °C for 60 minutes. These pre-treated samples were placed on Gilson ASPEC, eluted with 2 mL of 5% methanol in ethyl acetate into the labelled tubes. We dried the elute(s) using a nitrogen evaporator at 60 °C and dissolved the dried elute with 50 µL of MSTFA/ IODO –TMS/DTE mixture (1000/2/2: v/v/w).The tubes were incubated at 60 °C for 30 minutes. The derivatized extract was injected directly into the GC-MS/MS. Steroid profile measurements on GC-MS/MS A previously described method as per TD2021EAAS, which is validated, accredited under ISO17025, and currently used in routine samples analysis for the detection and quantification of endogenous steroids was used. In brief, analysis was performed on an Agilent GC–MS/MS 7890 A/7000C with an Agilent HP Ultra-1 column (length: 17 m; diameter: 0.2 mm; film-thickness: 0.11 µm) and 7683B Automatic Liquid Sampler. Carrier gas was helium, flow rate 2.25 mL/min, injection and transfer line temperature were set to 280 °C and injection was performed in split mode with a ratio of 11:1. The oven program was as follows: initial temparature of 180 °C hold for 1 min, ramp 1: 3 °C/min to 229 °C, ramp 2: 40 °C/min to 300 °C; final temparature was hold for 4 min. The ionization energy was set to 70 eV. The acquisition was carried out in multiple-reaction monitoring (MRM) mode. Quantitation of the urinary steroids was MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-023 performed based on the peak area ratio of the analyte to the corresponding internal standard. Calibration and quality control samples were prepared in steroid free urine. Results and Discussion Steroid profile parameters including markers and their ratios were calculated as per method characterization given in TDEAAS and validated excel sheets being used for routine sample reporting [1]. Mefenamic acid metabolites and endogenous steroids under current study are depicted in Table 1. The measured concentrations of the markers of the steroid profile were reported versus the collection time to evaluate their physiological fluctuation. All data were normalized for the specific gravity applying the following formula: (concentration ng/mL) x (1:020-1)/ (specific gravity sample-1). Table 1. List of Endogenous Steroids under investigation As mefenamic acid is an inhibitor of AKR1C3, the most logical assumption was that the concentrations and ratios of the steroid profile that contains 3-hydroxy-steroids should increase. For both volunteers, changes in the steroid profile were observed and these changes were evaluated statistically using one- way analysis of variance (ANOVA) using metaboanalyst software. In the male volunteer significant changes in the steroid profile were detected for three steroid-profile markers: the concentration of androsterone and the ratios Andro/T and Andro/Etio (boxplots with significance levels represented in Figure 1, A-C). For Andro, a significant decrease from difference in mean values; P = 3×10 was observed, similarly, the ratio Andro/T decreased from difference in mean values; P = 0.014 in the concentration was detected during the administration of mefenamic acid compared to pre- administration, whereas no significant difference was detectable between the samples collected during and after the application of mefenamic acid. For the female volunteer significant changes were detected for Andro/T (P = 6×10 ), 5A-diol/5B-diol (P = 1.6×10 ), 5A-diol/E (P = 9.7×10 ) and E (P = 0.01). Boxplots with the changes in steroid concentrations are displayed in Figure 1, D-H. Since the steroid concentrations investigated in the steroid profile are much lower in females compared to males, interpretation is more challenging and a lot of factors should be taken in to account when interpreting the results [5]. As assumed, concentrations and concentration ratios of the steroid profile involving one - 3 -7 -4 -4 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-024 3-hydroxy-steroid (Andro, 5A-diol, Etio, 5B-diol, Andro/T and Andro/E) are reduced. Unfortunately, the picture is not so clear from the two volunteers in this study. Figure 1. Boxplots of urinary steroid concentrations and ratios included in the steroid profile from the male and female volunteer with significant changes MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-025 Furthermore, the metabolite of mefenamic acid (3-OH methylmefenamic acid) was reported significantly till 10 days after stopping drug intake in both of the volunteers (Figure 2. A-B). Figure 2. Excretion profile of 3-hydroxy methyl mefenamic acid in male (A) and Female (B) volunteers Conclusions An inhibition of aldo-keto-reductases (AKRs), especifically AKR1C3, by non-steroidal anti-inflammatory drugs (NSAIDs), which are neither prohibited nor monitored, but frequently used drugs in sports, should be demonstrated. Mefenamic acid (NSAID) is an easy-over the counter accessible pain killer. This study shows the relevance of AKR1C3 inhibition by mefenamic acid (NSAIDs). Changes in the steroidal profile caused by the intake of therapeutic doses of mefenamic acid over two consecutive days (twice a day) were shown by anaylzing the urine samples using a validated method of the laboratory. In the male volunteer significant changes in the steroid profile were detected for the concentration of androsterone and the ratios Andro/T and Andro/Etio and in the female volunteer significant changes were detected for Andro/T, 5A-diol/5B-diol, 5A-diol/E and E. Since the inhibition by mefenamic acid is significanntly causing changes in some steroid profile markers, unnecessary GC/C/IRMS may be avoided during interpreting atypical steroid profiles in the ABP’s steroidal module. Furthermore, this study proposes an administration trail with a larger number of volunteers to elucidate the role of mefenamic acid and NSAIDs as confounding substances for steroid profiling. References 1. 2021 WADA Technical Document – TD2021EAAS. 2. 2021 WADA Technical Document – TD2021APMU. 3. Gobec S, Brozic P, Rizner TL. Nonsteroidal anti-inflammatory drugs and their analogues as inhibitors of aldo-keto reductase AKR1C3: new lead compounds for the development of anticancer agents. Bioorg Med Chem Lett. 2005 Dec 1;15(23):5170-5. doi: 10.1016/j.bmcl.2005.08.063.. 4. Shiiba M, Yamagami H, Yamamoto A, Minakawa Y, Okamoto A, Kasamatsu A, Sakamoto Y, Uzawa K, Takiguchi Y, Tanzawa H. Mefenamic acid enhances anticancer drug sensitivity via inhibition of aldo-keto reductase 1C enzyme activity. Oncol Rep. 2017 Apr;37(4):2025-2032. doi: 10.3892/or.2017.5480 5. Mareck U, Geyer H, Opfermann G, Thevis M, Schänzer W. Factors influencing the steroid profile in doping control analysis. J Mass Spectrom. 2008 Jul;43(7):877-91. doi: 10.1002/jms.1457 Acknowledgements The authors acknowledge financial support from Government of India. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-026 Suominen T, Piirainen M, Vanne H Analysis of steroid esters and quantitation of endogenous steroids in blood as Girard P derivatives Doping Control Laboratory Helsinki, Finnish Institute for Health and Welfare (THL), Helsinki, Finland Abstract A wide range of testosterone as well as other steroid formulations (e.g. nandrolone and trenbolone) with different structures and thus differing pharmacokinetic parameters are available. As most steroids are not well absorbed as oral formulations, the most widely used forms of administration are intramuscular injections of steroid ester preparations. Detection of intact steroid esters in blood provides indisputable proof of steroid misuse. Steroid esters are present at low concentrations in blood, and therefore the method used for their analysis shall be able to detect the compounds at low pg/ml concentrations. In this work, an LC-MS/MS method suitable for the quantitative analysis of testosterone and androstenedione and for the quantitative analysis of steroid esters was developed and validated. Introduction Detection of steroid misuse in sport is mainly based on the urinary steroid profile, followed by possible GC-C-IRMS confirmation of samples with atypical profiles. An alternative approach is to detect steroid esters in blood, requiring less time-consuming sample preparation compared to IRMS analysis. Steroid esters have been analyzed with different methods, such as GC-MS and more commonly by LC-MS, usually as oxime or Girard derivatives. For extraction of the steroid esters from the sample matrix, solvents such as MTBE, cyclohexane, toluene, mixtures of hexane and ethyl acetate have been successfully used. The aim of this work was to develop a LC-MS/MS method suitable for the quantitative analysis of testosterone and androstenedione, and for the quantitative analysis of steroid esters. Experimental Sample preparation ISTDs d -T (Cerilliant), d -A4 (TRC) and d -testosterone undecanoate (Alsachim), were added to 200 µL of serum or plasma, followed by extraction with 3 mL of TBME. After evaporation to dryness, 50 µL of 10 % acetic acid in methanol and 20 µL of Girard P reagent (4 mg/mL in H O) were added, followed by incubation at 60 °C for 10 min and transfer to autosampler vials. For oxime derivatization, 75 µL of hydroxylamine (100 mM in meOH/H O) was added, followed by incubation at 60 °C for 30 min. LC-MS/MS Analysis was performed using Agilent 1290 Infinity autosampler and 6460 Triple Quadrupole MS with Agilent Zorbax Eclipse Plus C18 (2.1 x 50 mm, 1.8 µm) column operated at 55 °C. 2.5 mM ammonium formate in 0.1 % formic acid (A) and 2.5 mM ammonium formate in 0.1 % formic acid in 95 % methanol 3 3 4 2 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-027 (B) were used as mobile phases. The flow rate was 0.3 mL/min and injection volume 5 µL. All compounds were monitored in positive ionization (SRM mode). The capillary voltage, drying gas flow, drying gas temperature, nebulizer, nozzle voltage, sheath gas flow and sheath gas temperature were set at 3500 V, 8 L/min, 200 °C, 35 psi, 500 V, 12 L/min, 350 °C. To wash out remaining Girard P reagent, the eluent was directed to waste for the first 3 minutes. Validation The aim of the method was to a) quantitate T and A4 in serum samples and b) detect exogenous steroid esters in serum and plasma samples according to the WADA Guideline Quantification of Endogenous Steroids in Blood for the Athlete Biological Passport and the WADA TN on Analytical Method validation. The validation included establishing repeatability, selectivity, LOD, carryover and extraction recovery for all analytes, and additionally LOQ, MU and linear range for the quantitation of T and A4. For the quantitation of T and A4, commercial calibrators (Chromsystems Multilevel Serum Calibration Set Steroid Panel 2), as well as in-house calibrators, were used. The in house-calibrators were prepared by spiking T and A4 into a buffer (0.1 BSA/PBS). Results and Discussion Method development: The intensities of the protonated molecules were compared for non-derivatized, oxime derivatization and Girard P derivatization. For most analytes, the Girard P derivatization provided the best intensity of the protonated ion. For some steroid esters the oxime derivative was more intensive, but the S/N were similar, and the Girard P derivatization was selected for use in the validation. The two most prominent SRM transitions for the compounds were chosen for the ITP method. The protonated compounds have a target ion consisting of M+N C H . Figure 1. Comparison of the ionization of selected steroid esters with no derivatisation, oxime derivatisation and Girard P derivatisation 3 7 8 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-028 Table 1. Compounds and SRM transitions used for ITP Different HPLC columns were tried during method development, but the best separation of the analytes was with a Agilent Zorbax Eclipse Plus C18 column. Linearity was evaluated using two independent sets of calibrators; the Chromsystems Multilevel Serum Calibrator Set, and in-house calibrators spiked into PBS-buffer. The linearity was evaluated according to the WADA Guideline “Quantification of Endogenous Steroids in Blood for the Athlete Biological Passport” from 0.05-15 ng/mL for both T and A4. With both calibrators, the linearity was excellent for both T and A4 (r > 0.999). The extraction recoveries were between 70-100%. 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-029 Figure 2. Linearity of T and A4 using in-house calibrators For T and A4, the LOQ was defined as the lowest concentration with a S/N of 6 and acceptable bias ( < 30%). For both T and A4, the LOQ was at 0.05 ng/mL. For the steroid esters, the LODs were established by spiking the compounds into six different serum and plasma samples, serially diluted with the same plasma/serum, and set at the concentration with a s/n of ≥ 3. Based on the analysis of parallel serum and plasma samples, either blank samples or samples to which the exogenous steroid esters had been spiked, the method was proven to be repeatable and selective. The MU was evaluated based on the analysis of EQAS samples; for T uc = 7.2% and for A4 uc = 7.7 %. Table 2. LODs, LOQs and extraction recoveries Conclusions With the developed and validated method, the quantitation of testosterone and androstenedione as well as the qualitative analysis of exogenous steroid esters can be done. The method was developed and validated according to the Guideline (WADA) criteria. The method consists of a fairly simple extraction protocol, followed by derivatization, and the chromatographic conditions have been optimized to wash out the derivatization reagent in the beginning of the chromatographic run, thus making any extra clean- up steps unnecessary. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-030 References 1. Forsdahl, G et al (2015). Detection of testosterone esters in blood. Drug Test Anal, 7, 983-989 2. Van Renterghem, P et al (2020). Validation of an ultra-sensitive detection method for steroid esters in plasma for doping analysis using positive chemical ionization GC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci 15(1141:122026) 3. Keski-Rahkonen, P, Huhtinen, K, Poutanen, M and Auriola, S (2011). Fast and sensitive liquid chromatography–mass spectrometry assay for seven androgenic and progestagenic steroids in human serum. Journal of Steroid Biochemistry & Molecular Biology 127, 396–404 4. Häkkinen, M R et al (2019). Simultaneous analysis by LC–MS/MS of 22 ketosteroids with hydroxylamine derivatization and underivatized estradiol from human plasma, serum and prostate tissue. Journal of Pharmaceutical and Biomedical Analysis 164, 642–652 5. de la Torre, X, Iannone, M, Botré, F (2021). Improving the detection of anabolic steroid esters in human serum by LC–MS. Journal of Pharmaceutical and Biomedical Analysis 194 (2021) 113807. 6. Frey, A J et al (2016). Validation of highly sensitive simultaneous targeted and untargeted analysis of keto- steroids by Girard P derivatization and stable isotope dilution-liquid chromatography-high resolution mass spectrometry. Steroids 116, 60–66 7. Langer, T et al (2022). A comprehensive UHPLC–MS/MS method for the analysis of endogenous and exogenous steroids in serum for anti-doping purposes. Drug Test Anal. 14, 1904–1919 8. World Anti-Doping Agency, Laboratory Guidelines (2023). Quantification of Endogenous Steroids in Blood for the Athlete Biological Passport Acknowledgements The authors would like to acknowledge Dr. Günther Gmeiner for valuable advice with the method development. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-031 Wu Q , Yan K , Dong T , Liu L , Wang Y , Wang Z Quantification of testosterone and androstenedione in human serum by HRMS Beijing Sport University, Beijing, People's Republic of China ; Beijing Anti-Doping Laboratory, Beijing Sport University, Beijing, People's Republic of China Abstract The identification and judgement of whether the atypical change of the concentrations of endogenous steroids originates from exogenous steroid drug abuse by athletes is a complicated and difficult topic in doping-control. Through the ‘steroidal module’ of the Athlete Biological Passport by GC-MS (/MS), the quantification of selected endogenous anabolic androgenic steroids (EAAS) from athletes’ urine is the major monitoring method. However, many confounding factors such as ethnicity, individual difference, micro-biological degradation, gene polymorphism, intaking of non-prohibit substances or alcohol cause some limitations of the urinary steroid profile. Quantification of endogenous steroids, especially testosterone and androstenedione from human serum was considered as a possible and effective complementory program. In this study we developed an ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) method for quantifying testosterone and androstenedione in human serum. The method involves precipitating proteins (PPT) from a 50 µL serum sample and optimizing the concentration steps for micro aliquots to meet the LOQ requirement of 0.1 ng/mL. All measurements were conducted under UPLC-HRMS routine conditions. Method validation included recovery at various concentrations, specificity, intra-day and inter-day precision, accuracy, and uncertainty, with a linear range of 0.1 to 10 ng/mL. This method allows the quantification of testosterone and androstenedione in human serum at low cost and microsample volume, and results also shown high accuracy and stability. Introduction Testosterone and androstenedione are endogenous steroids. Especially testosterone plays a key role in regulating effects for human body muscle content, aggression, physical strength and resilience. Due to the identification and judgement of whether the atypical change of endogenous steroid originates from exogenous steroid drug abuse by athlete is a complicated and difficult topic in doping-control. An UPLC- HRMS method for quantifying the concentration of testosterone and androstenedione in human serum was developed and validated in this study. The method demonstrats to quantify testosterone and androstenedione levels in human serum accurately and it may become the indirect proof to monitor athlete’s behaviour in the future 1 2 2 2 2 2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-032 Experimental Charcoal stripped human serum In this work, charcoal stripped human serum (CSHS) was used to evaluate linearity, specificity, recovery and precision. It was made by dextran coated charcoal (Merck, Sigma-Aldrich, C6241), followed by at least two rounds of centrifugation to collect the supernatant. Each batch of CSHS was evaluated regarding its purity before it was employed in the experiment process. Calibrators 1 mL of Cal 1 to Cal 5 and controls were prepared by CSHS which was finished evaluation of purification and reference materials in each batch. Then 50 µL of single serum was separated into 1.5 mL centrifuge tubes and stored at -20 °C before use. The concentrations of the calibrators were 0.1, 0.5, 1.0, 5.0 and 10.0 ng/mL of testosterone (T) and androstenedione (AD), respectively. Sample preparation All serum samples were prepared as follows: 50 µL of serum was transferred into 1.5 mL centrifuge tube, 5 µL of d -T (0.01 ng/µL in methanol) and 5 µL of d -AD (0.01 ng/µL in methanol), which were internal standard, were added into the tube. 140 µL of acetonitrile, used for protein precipitation, was added to the tube and the solution was thoroughly mixed by vortex mixing. All samples were centrifuged for 10 minutes at 12000 rpm, 170 µL of supernatant was transferred and evaporated to dryness by vacuum concentrator (Eppendorf concentrator plus, Germany) at 45 °C. The samples were reconstituted with 100 µL of initial mobile phase and centrifuged again for 10 minutes. Finally, 90 µL of supernatant was transferred to a vial prior to analysis. UPLC-HRMS Thermo Q-Exactive Plus with Dionex UltiMate 3000 was used for sample analysis.The LC conditions did not require switching between this method and the routine urine analysis method. The instrument conditions are shown in Table 1. Table 1. UPLC-HRMS instrument conditions for the quantification method 3 7 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-033 Results and Discussion In this work, solid-phase extraction (SPE), solid-liquid extraction (SLE) and protein precipitation (PPT) were initially compared. The responses show that SPE and SLE are much higher than PPT, but the response of PPT prepared at LOQ of 0.1 ng/mL also met the quantitative requirements. Compared to other options, PPT is the least expensive and the method is simple and fast to operate. Therefore, the PPT pre-processing method was selected for development and optimization. Selectivity, LOQ, recovery, precision, accuracy, and uncertainty were evaluated. The results were favourable and are shown in Figure 1 and Table 2. Figure 1. Selectivity and specificity of testosterone and androstenedione CSHS+LOQ: Spike 0.1 ng/mL to charcoal stripped human serum; HS: Human serum; CSHS: Charcoal stripped human serum Table 2. Validation of the quantification method 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-034 As shown in Figure 2, the R of T and AD linearity ranges was over 0.995. The response of the LOQ at 0.1 ng/mL was consistent with the quantification requirements. The recovery at different concentrations was exceeding 60%. The coefficient of variation was less than 15% at each concentration from inter- and intra-precision. The bias of accuracy was less than 12%. The uncertainty was less than 30% the LOQ level, and less than 20% for the medium and high concentrations. This method was capable of quantifying testosterone and androstenedione in human serum under the condition of low cost and low sample volume, and the result showed high accuracy and good stability. Figure 2. Calibration curves of testosterone and androstenedione. A: Testosterone; B: Androstenedione Testosterone: Y = 0.9932 X + 0.01924; R : 0.9989; Origin: Ignore; W: 1/X; Area Androstenedione: Y = 1.24 X – 0.008512; R : 0.9988; Origin: Ignore; W: 1/X; Area In this method validation experiment, recovery, linearity, and precision density focus on the stability of the quantitative method, so CSHS was selected for evaluation with RM (Figure 1 and Table 2). Accuracy and uncertainty are closely related to the matrix, CSHS no longer has the ability to access the deviation caused by different matrices due to its purification process, therefore, the different normal human serum (HS) was selected (Table 2). The synthetic uncertainty was calculated by Sw from HS and Bias (Ext) from “Multilevel Serum Calibrator Set” (Chromsystem, Munich, Germany). 2 2 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-035 Conclusions In this study, we developed and validated a UPLC-HRMS method for the quantification of testosterone and androstenedione from human serum in accordance with the World Anti-Doping Agency (WADA) guidelines. Based on the optimized protein precipitation technology, the quantitative results show high accuracy and good stability at low cost and microsample volume. By providing additional proof beyond the urine steroid profile, this method has the potential to improve the monitoring of exogenous steroid drug abuse in athletes and contribute to the fairness and integrity of sports competitions. References 1. Salamin O, Ponzetto F, Cauderay M, Boccard J, Rudaz S, Saugy M, Kuuranne T, Nicoli R. Development and validation of an UHPLC-MS/MS method for extended serum steroid profiling in female populations. Bioanalysis. 2020 Jun;12(11):753-768. doi: 10.4155/bio-2020-0046. 2. Sobolevsky T, Samara V, Ahrens B. Detection of testosterone and androstenedione in serum by LC-HRMS. In: Thevis M, Geyer H, Mareck U (eds.) Recent advances in doping analysis (30). Sportverlag Strauß, Hellenthal (2022).63-66 3. Ponzetto F, Boccard J, Baume N, Kuuranne T, Rudaz S, Saugy M, Nicoli R. High-resolution mass spectrometry as an alternative detection method to tandem mass spectrometry for the analysis of endogenous steroids in serum. J Chromatogr B Analyt Technol Biomed Life Sci. 2017 May 1;1052:34-42. doi: 10.1016/j.jchromb.2017.03.016 4. Ponzetto F, Parasiliti-Caprino M, Gesmundo I, Marinelli L, Nonnato A, Nicoli R, Kuuranne T, Mengozzi G, Ghigo E, Settanni F. Single-run UHPLC-MS/MS method for simultaneous quantification of endogenous steroids and their phase II metabolites in serum for anti-doping purposes. Talanta. 2023 Apr 1;255:124218. doi: 10.1016/j.talanta.2022.124218 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-036 Fußhöller G , Geyer H , Görgens C , Guddat S , Jübner M , Lucuta L , Thevis M Excretion pattern of urinary metabolites of dehydrochloromethyl‐ testosterone after transdermal application Institute of Biochemistry, German Sport University, Cologne, Germany ; Center of Preventive Doping Research, German Sport University, Cologne, Germany ; Department of Toxicology, Institute of Legal Medicine, Faculty of Medicine and University Hospital, University of Cologne, Cologne, Germany Abstract The aim of the present study was to investigate whether the transdermal application of dehydrochloromethyltestosterone (DHCMT) leads to a characteristic excretion pattern of urinary metabolites, differing from those known to exist after oral administration of the drug. Six volunteers received transdermal administrations of 10 mg of DHCMT dissolved in 100 μL DMSO on different skin regions. Urine samples were collected before and till 330 hours after the application and were analysed with GC-MS/MS and LC-HRMS for 5 different DHCMT metabolites. Compared with literature data obtained after oral administration of DHCMT, it could be shown that, in case of transdermal application, DHCMT glucuronide constitutes a long-term metabolite, whereas after oral application it is commonly categorized under the group of short-term metabolites. Introduction Several studies have shown that the transdermal administration of testosterone leads to excretion patterns of urinary testosterone metabolites that differ from those observed after oral administration or injection of testosterone due to an increased 5a-reductase activity of the skin [1,2]. In a recent study with clostebol, such distinct differences between the transdermal and oral administration of clostebol were not corroborated [3]. Based on data obtained from investigations of Gessner et al. [4], the present study aimed at assessing whether the transdermal application of dehydrochloromethyltestoterone (DHCMT) leads to a characteristic excretion pattern of urinary metabolites differing from those known to exist after oral administration of the drug. Experimental The experimental details are described in the publication of Gessner et al. [4]. In short, 6 volunteers received transdermal administrations of 10 mg DHCMT dissolved in 100 µL DMSO. Two volunteers each received the substance/DMSO mixture on the back of the hand, on the top of the lower arm and on the neck. Urine samples were collected before and till 330 hours after the application. The urine samples were analysed with GC-MS/MS and LC-HRMS for the DHCMT metabolites DHCMT glucuronide, 6-OH- DHCMT and M1, M2 and M3 (nomenclature of M1-M3 according to Sobolevsky and Rodchenkov [5]). 1,2 1,2 1,2 1,2 3 3 1,2 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-037 Results and Discussion The detection windows for the different DHCMT metabolites obtained with LC-MS/MS and GC-MS/MS after transdermal administration of DHCMT are presented in Figure 1a-c (two volunteers for each skin region). The data originate from initial testing procedures, i.e. for some presumptive findings it is not guaranteed that a confirmation of the target analyte in further procedures would be successful. For comparison, data of one volunteer (volunteer 5) after an oral administration of 5 mg DHCMT from an investigation of Loke et al. [6] are presented in Figure 2. After transdermal application, the longest detection window for all volunteers and all skin regions could be observed for the DHCMT glucuronide (Figure 1a-c). This is different to the oral application, where one of the metabolites M1-M3 has the longest detection window and DHCMT glucuronide is only a short term metabolite (Figure 2 [6]). In none of the samples after transdermal administration, the metabolites M1 and M2 were detected. Figure 1a-b. Detection windows for the different DHCMT metabolites after transdermal administration of DHCMT on the hand (a) and lower arm (b) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-038 Figure 1c. Detection windows for the different DHCMT metabolites after transdermal administration of DHCMT on the neck Figure 2. Detection windows for the different DHCMT metabolites after oral administration of DHCMT [6] Conclusions The metabolic pattern after the transdermal application of DHCMT is different from the pattern after oral application. In case of transdermal application the DHCMT glucuronide is a long-term metabolite. Therefore the sole presence of DHCMT glucuronide in a doping control sample may be possible and may be an indication of a transdermal application of DHCMT. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-039 References 1. Geyer H, Flenker U, Haenelt N, Mareck U, Piper T, Schmechel A, Schrader Y, Platen P, Schänzer W. Steroid profiling and GC/C/IRMS for the detection of the misuse of testosterone gel. Schänzer W, Geyer H, Gotzmann A, Mareck U (eds.) Recent advances in doping analysis (15). Sportverlag Strauß, Köln (2007) 133-142 2. Mullen J, Börjesson A, Hopcraft O, Schulze JJ, Ericsson M, Rane A, Lehtihet M, Ekström L. Sensitivity of doping biomarkers after administration of a single dose testosterone gel. Drug Test Anal. 2018 May;10(5):839-848.Piper T, Emery C, Saugy M. Norandrosterone analysis by GC/C/IRMS. In: Schänzer W, Thevis M, Geyer H, Mareck U (eds.) Recent advances in doping analysis (20). Sportverlag Strauß, Köln (2012) 201-204 3. de la Torre X, Colamonici C, Iannone M, Jardines D, Molaioni F, Botrè F. Detection of clostebol in sports: Accidental doping? Drug Test Anal. 2020 Nov;12(11-12):1561-1569. 4. Gessner L, Thevis M, Rothschild MA, Jübner M. Detectability of oxandrolone, metandienone, clostebol and dehydrochloromethyltestosterone in urine after transdermal application. Drug Test Anal. 2022 Oct;14(10):1744-1761 5. Sobolevsky T, Rodchenkov G. Detection and mass spectrometric characterization of novel long-term dehydrochloromethyltestosterone metabolites in human urine. J Steroid Biochem Mol Biol. 2012 Feb;128(3- 5):121-7. 6. Loke S, de la Torre X, Iannone M, La Piana G, Schlörer N, Botrè F, Bureik M, Parr MK. Controlled administration of dehydrochloromethyltestosterone in humans: Urinary excretion and long-term detection of metabolites for anti-doping purpose. J Steroid Biochem Mol Biol. 2021 Nov;214:105978. Acknowledgements The authors acknowledge support from Sport Ireland (Dublin, Ireland), the Federal Ministry of the Interior, Community and Building of the Federal Republic of Germany (Berlin, Germany) and the Manfred Donike Institute for Doping Analysis (Cologne, Germany). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-040 Guddat S , Fußhöller G , Geyer H , Görgens C , Jübner M , Lucuta L , Thevis M Excretion pattern of urinary metabolites of metandienone after transdermal application Institute of Biochemistry, German Sport University, Cologne, Germany ; Department of Toxicology, Institute of Legal Medicine, Faculty of Medicine and University Hospital, University of Cologne, Cologne, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract In the present study, the urinary excretion pattern of metandienone metabolites after transdermal application of the drug was investigated and the presence of potentially characteristic excretion profiles differing from those known to exist after oral administration of the drug was assessed. Six volunteers received transdermal administrations of 10 mg of metandienone dissolved in 100 µL DMSO on different skin regions. Urine samples were collected before and till 330 hours after the application and were analysed with GC-MS/MS and LC-HRMS for 8 different metandienone metabolites. Compared with literature data after oral administration it could be shown that, in case of transdermal application, 6-OH- metandienone constitutes a long-term metabolite, whereas after oral application it is commonly categorized under the group of short-term metabolites. Introduction Several studies have shown that the transdermal administration of testosterone leads to excretion patterns of urinary testosterone metabolites that differ from those observed after oral administration or injection of testosterone due to an increased 5a-reductase activity of the skin [1,2]. In a recent study with clostebol, such distinct differences between the transdermal and oral administration of clostebol were not corroborated [3]. Based on data obtained from investigations of Gessner et al. [4], the present study aimed at assessing whether the transdermal application of metandienone leads to a characteristic excretion pattern of urinary metabolites differing from those known to exist after oral administration of the drug. Experimental The experimental details are described in the publication of Gessner et al. [4]. In short, 6 volunteers received transdermal administrations of 10 mg of metandienone dissolved in 100 µL DMSO. Two volunteers each received the substance/DMSO mixture on the back of the hand, on the top of the lower arm and on the neck. Urine samples were collected before and till 330 hours after the application. The urine samples were analysed with GC-MS/MS and LC-HRMS for the metandienone metabolites 6-OH- metandienone (6OH-Metand), 17α-methyl-5α-androstane-3α,17β-diol (tetrahydromethyltestosterone, THMT), 18-norepimetendiol (NorEMD), epimetendiol (EMD), epi-LTM (epi-long-term metabolite [5]), LTM (long-term metabolite [5]), epimetandienone (epiMD) and metandienone (MD). 1 1 1 1 2 2 1,3 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-041 Results and Discussion The detection windows for the different metandienone metabolites obtained with LC-MS/MS and GC- MS/MS after transdermal administration of metandienone are presented in Figures 1a-c (two volunteers for each skin region). The data originate from initial testing procedures, i.e. for some presumptive findings it is not guaranteed that a confirmation of the target analyte in further procedures would be successful. For comparison, data gathered after oral administration of 5-10 mg metandienone from Polet et al. [6] are presented in Figure 2. After transdermal application, the longest detection windows for all volunteers and all skin regions could be observed for the metandienone LTM and 6OH-metandienone (Figure1a-c). This is different to the oral application, where 6OH-metandienone is considered as a short-term metabolite (Figure 2 [6]). Figure 1a-b. Detection windows for the different DHCMT metabolites after transdermal administration of metandienone on the hand (a) and lower arm (b) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-042 Figure 1c. Detection windows for the different Metandienone metabolites after transdermal administration of metandienone on the neck Figure 2. Detection windows for the different metandienone metabolites after oral administration of metandienone [6] Conclusions The metabolic pattern after the transdermal application of metandienone is different from the pattern after oral application. In case of transdermal application, 6OH-metandienone constitutes a long-term metabolite, whereas after oral application it is commonly categorized under the group of short-term metabolites. The presence of 6OH-metandienone together with the long-term metabolite of metandienone (LTM) may be an indication of a transdermal application of metandienone. Similar to MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-043 scenarios where the LTM of metandienone is exclusively detected after oral administration, the sole presence of 6OH-metandienone after transdermal application of metandienone cannot be excluded. References 1. Geyer H, Flenker U, Haenelt N, Mareck U, Piper T, Schmechel A, Schrader Y, Platen P, Schänzer W. Steroid profiling and GC/C/IRMS for the detection of the misuse of testosterone gel. Schänzer W, Geyer H, Gotzmann A, Mareck U (eds.) Recent advances in doping analysis (15). Sportverlag Strauß, Köln (2007) 133-142 2. Mullen J, Börjesson A, Hopcraft O, Schulze JJ, Ericsson M, Rane A, Lehtihet M, Ekström L. Sensitivity of doping biomarkers after administration of a single dose testosterone gel. Drug Test Anal. 2018 May;10(5):839-848.Piper T, Emery C, Saugy M. Norandrosterone analysis by GC/C/IRMS. In: Schänzer W, Thevis M, Geyer H, Mareck U (eds.) Recent advances in doping analysis (20). Sportverlag Strauß, Köln (2012) 201-204 3. de la Torre X, Colamonici C, Iannone M, Jardines D, Molaioni F, Botrè F. Detection of clostebol in sports: Accidental doping? Drug Test Anal. 2020 Nov;12(11-12):1561-1569. 4. Gessner L, Thevis M, Rothschild MA, Jübner M. Detectability of oxandrolone, metandienone, clostebol and dehydrochloromethyltestosterone in urine after transdermal application. Drug Test Anal. 2022 Oct;14(10):1744-1761 5. Schänzer W, Geyer H, Fußhöller G. Mass spectrometric identification and characterization of a new long- term metabolite of metandienone in human urine. Rapid Commun Mass Spectrom. 2006;20(15):2252- 2258.. 6. Polet M, Van Gansbeke W, Van Eenoo P, Deventer K. Efficient approach for the detection and identification of new androgenic metabolites by applying SRM GC-CI-MS/MS: a methandienone case study. J Mass Spectrom. 2016 Jul;51(7):524-34. Acknowledgements The authors acknowledge support from Sport Ireland (Dublin, Ireland), the Federal Ministry of the Interior, Community and Building of the Federal Republic of Germany (Berlin, Germany) and the Manfred Donike Institute for Doping Analysis (Cologne, Germany). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-044 Montes de Oca Porto R, Martinez Brito D, Lopez Castillo D, Rodriguez Fernandez A, Laffita Jorrin D Behaviour of 19-norandrosterone levels in negative samples Antidoping Laboratory, Havana, Cuba Abstract Background: One of the pathways for the formation of 19-norandrosterone (19-NA) in the human organism is through the transformation of testosterone to estradiol with the participation of the aromatase enzyme. Several authors have described the inhibition or increase of the activity of this enzyme due to physiological processes or interactions with other drugs, even in different metabolic pathways. This study describes the behavior of 19-norandrosterone levels in samples analyzed from 2017 to 2022. Experimental: The study included samples with the presence of 19-NA in female (n = 217) and male (n = 239) samples below 2.5 ng/mL (adjusted for specific gravity). The samples were classified by sports groups (n = 4) according to control of sports training literature. Statistical analysis of the data distribution was performed (Shapiro-Wilk and Anderson-Darling tests, α = 0.05) and statistical differences between groups were evaluated (Kruskal-Wallis test, α = 0.05). Results: Non-parametric distributions were observed for all the groups evaluated except for the male group in competitive arts sports, probably influenced by the number of samples. Significant differences were observed between the female and male groups for the concentration of 19-NA (p = 5.3 x 10 ) with higher values for the male group. Likewise, significant differences were found between the sexes for the group of sports with balls (p = 0.00037). Specifically, within the male group, significant differences were observed in the ball sports group compared to the rest. While in the female group there are no differences between the sport groups studied. Introduction One of the pathways for the formation of 19-norandrosterone (19-NA) in the human organism is through the transformation of testosterone to estradiol with the participation of the aromatase enzyme. Several authors have described the inhibition or increase of the activity of this enzyme due to physiological processes or interactions with other drugs, even in different metabolic pathways [1-3]. To maintain an observation on this affirmation, the goal of this work was to describe the behavior of 19-norandrosterone levels in female and male samples analyzed from 2017 to 2022. Experimental Urine samples of athletes received in the Havana Antidoping Laboratory from 2017 to 2022 that presented 19-norandrosterone below 2.5 ng/mL (after the concentration was adjusted by SG) were classified, and statistically compared. All samples included in the study met the NA identification criteria according to the TD-IDCR technical document. The group was divided into females (F, n = 217) and -6 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-045 males (M, n = 239). Then, they were classified by sports groups (n = 4) according to control of sports training literature into ball sports (F: n = 50, M: n = 154), endurance sports (F: n = 68, M: n = 42), combat sports (F: n = 56, M: n = 32) and competitive art sports (F: n = 33, M: n = 7). Sample preparation under routine conditions were done following a solid phase extraction on C18 cartridges, enzymatic hydrolysis (50 µL β-glucuronidase Roche, pH 6.8) and then liquid-liquid extraction (TBME, 5 mL, pH 10). Trimethylsylil derivatives (MSTFA: NH I: 2-ME, 100:2:6, m:V:m) were analysed on an Agilent 7890A gas chromatograph coupled to an Agilent 7000B triple quadrupole. The column used was a HP1MS methyl fused-silica capillary column (17 m x 0.2 mm, 0.11 μm film thickness). The data acquisition was performed in multiple reaction monitoring (MRM). Statistical analyses were done by using MedCalc Statistical Software version 20.2 (MedCalc Software Ltd, Ostend, Belgium; https://www.medcalc.org; 2022). Data distribution was checked by Shapiro-Wilk and Anderson-Darling tests (p < 0.05). Statistical differences between groups were checked by applying Kruskal-Wallis test, p < 0.05. Results and Discussion Except males in competitive art sports, no data showed normal distribution (Shapiro-Wilk and Anderson- Darling tests, p 20 ng/mL) and furosemide glucuronide (est. conc. > 20 ng/mL) This is the most frequent situation in routine doping control analysis so far. The exemplary chromatograms from a CP analysis are shown in Figure 1. Figure 1. Chromatograms of athlete's sample from confirmatory analysis performed after sample preparation involving double LLE SCENARIO 2: Furosemide (est. conc. > 20 ng/mL) and furosemide glucuronide (est. conc. < 20 ng/mL) According to the TL-24, this situation constitutes an AAF, as well. This situation was observed e.g. in December 2022. Two samples with Specific Gravities of 1.003 and 1.004 were collected from one Athlete competing in Powerlifting. The confirmation procedure of the first sample showed furosemide and its metabolite at concentrations of 24 ng/mL and 6 ng/mL, respectively. In turn, in the second sample, the estimated concentrations of furosemide and its metabolite were 25 ng/mL and 6 ng/mL, respectively. SCENARIO 3: Furosemide (est. conc. < 20 ng/mL) and furosemide glucuronide (est. conc. < 20 ng/mL) To date, for the correct interpretation and reporting of results from the furosemide analysis, the WADA TL-24 shall be applied. Should a modification of this TL consider the inclusion of phase-II metabolites such as the furosemide glucuronide in the future, the second-phase metabolite of furosemide could be MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-068 used as a target compound for the Initial Testing Procedure (ITP) and Confirmation Procedures (CP) for furosemide analysis, as well. This would then also be compatible with WADA TD 2022 MRPL (point "c" in the MRL table) [5] stating "when the Analytical Method used includes also the determination of phase-II Metabolites (e.g., glucuronides, sulfates) of the specific target substance, the MRL is applied to the total concentration (i.e., free and conjugated fractions) of the substance. This estimation is obtained either by separate determination of the molecular species (e.g., by LC-MS analysis) or following the de-conjugation of the phase-II Metabolite(s) […]” SCENARIO 4: Only Furosemide presence in a sample (est. conc. > 20 ng/mL or < 20 ng/mL) According to TL-24, the presence of furosemide in each case at an estimated concentration greater than (> ) 20 ng/mL, shall be reported as an AAF, in turn, ≤ 20 ng/mL, shall be reported as a Negative. As the sole exception to this new MRL, the presence of this compound in a sport or discipline which are listed in Annex 1. SCENARIO 5: Only metabolite presence in a sample (est. conc. > 20 ng/mL or < 20 ng/mL) The situation is similar to Scenario 4. Due to the limited number of papers concerned with the elimination process of furosemide, the exclusive presence of the furosemide metabolite in a sample might necessitate consideration for doping control samples collected from defined sport disciplines. Conclusions The WADA TL-24 harmonized the scheme for the reporting of furosemide as AAF, ATF, or Negative finding. According to this document, the presence of furosemide (parent compound only), in each case at an estimated concentration greater than (> ) 20 ng/mL, shall be reported as an AAF. The presented data show that the second-phase metabolite of furosemide is a valuable biomarker in furosemide detection (especially as it confirms the administration, biotransformation, and elimination of the drug). Including this metabolite in routine operations could be valuable for the interpretation of results for furosemide analysis purposes also, e.g. with furosemide glucuronide potentially being an indicator of the time point of application of furosemide. In addition, targeting furosemide glucuronide could be of added value when screening doping control samples of sport disciplines listed in Annex 1 WADA TL-24 (sport/disciplines with relevant weight classes). In order to assess the extent of this added value, the monitoring of furosemide glucuronide could be initialized by the volunteer laboratories for some time to evaluate a scale of the presence of this metabolite in athlete samples. Undeniably, the identification of new metabolites of prohibited substances is a crucial aspect of developing the anti-doping policy, as well as research concerning the elimination process of furosemide seems to be necessary. References 1. M. Espinosa Bosch, A.J. Ruiz Sanchez, F. Sanchez Rojas, C. Bosch Ojeda. Recent developments in analytical determination of furosemide. J.Pharm. Biomed. Anal. (2008) 48, 519–532. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-069 2. World Anti-Doping Agency. Prohibited List 2023. Available at https://www.wada- ama.org/sites/default/files/2022-09/2023list_en_final_9_september_2022.pdf [26 January 2023]. 3. World Anti-Doping Agency. WADA Technical Letter – TL24. Available at https://www.wada- ama.org/sites/default/files/2022-01/tl24_diuretics_v2.0.pdf [26 January 2023]. 4. Grucza K., Wicka M., Kowalczyk K., StaÅ„czyk D., Kaliszewski P., Michalak D., Szutowski M., Kwiatkowska D.: Identification of glucuronide conjugate of furosemide in routine doping control analysis. Poster presented at the 37 Manfred Donike Workshop on Doping Analysis, Cologne, Germany, 17.02. - 22.02. 2019 5. World Anti-Doping Agency. TD2022MRPL. Available at https://www.wada-ama.org/sites/default/files/2022- 01/td2022mrpl_v1.1_eng_0.pdf [31 January 2022]. Acknowledgements Financial support from the Ministry of Health of the Republic of Poland under the project number 1/85195/PLAD/2022/4186/406 is gratefully acknowledged. th MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-070 Grucza K , Wicka M , Drapala A , Konarski P , Stanczyk D , Kaliszewski P , Kwiatkowska D Occurrence of higenamine and its sulfate conjugate in routine doping control analysis - interpretation of results Polish Anti-Doping Laboratory, Warsaw, Poland ; Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland Abstract Higenamine (Norcoclaurine) is a very popular substance in Chinese medicine present in many plants and is one of the substances occurring in dietary supplements. Moreover, it is included in Class S3 (β-2- agonists) of the WADA 2023 Prohibited List. This poster describes instances where performing hydrolysis would cause an AAF status and indicates that change in the current principles of interpretations of results should be considered. Introduction Higenamine (Norcoclaurine) is a very popular substance in Chinese medicine present in many plants and is one of the substances commonly occurring in dietary supplements [1]. Although extracts of these plants are listed as ingredients in many pre-workout and fat-burner products, several products were contaminated, without notice on the label [2]. Higenamine is prohibited in sport at all times and included in Class S3 (β-2-agonists) of the WADA 2023 Prohibited List [3]. The presence of higenamine in urine samples at concentrations greater than 10 ng/mL constitutes an Adverse Analytical Finding (AAF) [4]. This poster shows instances where performing hydrolysis would cause an AAF status and indicates that change of the current principles of interpretations of results should be considered. Experimental Urine samples were prepared by 2 protocols: Dilute-and-Shoot (DaS) approach (200 µL of urine and 800 µL of water) and a method involving acid hydrolysis and double extraction as described previously [5]. Analysis was performed on a UPLC™ Acquity chromatograph (Waters, Milford Massachusetts, USA) equipped with an HSST3 column (1.8 μm, 2.1 × 100 mm). The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B) and the LC gradient was employed at a constant flow rate of 300 μL/min at 45 °C. The concentration of acetonitrile was gradually increased in a linear manner: from 0% to 15% within the first 6 minutes, and from 15% to 100% in 1 minute. Then it was kept constant for additional 2 minutes. Finally, the column was re-equilibrated for 2 minutes with the mobile phase of the initial composition. Samples were stored at 5 °C in the autosampler prior to analysis and the injection volume was fixed at 5 μL. Multiple reaction monitoring (MRMs) of higenamine and metabolite were traced with Xevo TQ-S (Waters, USA) mass spectrometer equipped with a new atmospheric pressure ionization source commercialized as UniSpray™. Analytes were investigated in the positive (+) mode. The 1 1 1 1 1 1 1,2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-071 desolvation gas flow was set at 800 L/h at 500 °C and the source temperature was 150 °C. The impactor voltage applied was 3.0 kV. The cone and collision gas flows were set at 150 L/h and 0.20 mL/min, respectively. Traced MRMs and their corresponding MS settings are listed in Table 1. Table 1. MS settings (MRMs traced, Cone [v], Collision [eV]) of the substances tested Results and Discussion At the WADA-accredited laboratory in Warsaw, higenamine as well as higenamine sulfate are detected in routine anti-doping analysis by the DaS approach. In August 2016, WADA obliged all accredited laboratories to detect higenamine as soon as possible. In turn, afterward, the metabolite of higenamine was included in the ITP in 2017 [5]. Since then, cases where only the metabolite was present in a sample were observed 18 times: 4 times in 2017, 4 times in 2018, 4 times in 2019, 1 time in 2020, 1 time in 2021, 3 times in 2022, and 1 time in 2023 (so far) (Figure 1). For these cases, the ratios for higenamine metabolite to meldonium-D3 were calculated as well. As a result, samples prepared according to the second protocol showed the presence of the higenamine at estimated concentrations ranging between 1 ng/mL and 50 ng/mL as the total concentration (sum of free form, glucuronide conjugation, and sulfate conjugation)(Figure 2). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-072 Figure 1. Chromatograms of the Initial Testing Procedure (DaS approach) for higenamine sulfate (only detection window): (a) blank urine, (b) athlete`s sample. The presence of higenamine (parent compound) was not observed in the athlete`s sample Moreover, higenamine was identified at the estimated concentration above MRL in seven cases. Undoubtedly, the above concentration range does not allow us to indicate whether an athlete consumed higenamine for doping purposes or whether it was an unintentional doping case by an athlete, as well. The first information about reporting only the free form of Higenamine was included in WADA TD2018MRPL as: „The reporting limits specified for salmeterol and higenamine apply to the determination of the free parent compound only” and is present in WADA TD2022MRPL as well [4]. This clearly indicates that hydrolysis shall not be performed. As a consequence of this principle of interpretation of results, such an approach may cause an underestimation of the concentration of higenamine in urine samples. In turn, the WADA threshold concentration is based on the sum of free form and the glucuronide conjugate for salbutamol and formoterol. As it is well known, higenamine is excreted in unchanged form, and as second-phase metabolites(both glucuronide and sulfate conjugates), and consequently hydrolysis of a sample should be considered for the proper estimation of the higenamine. Looking more closely, the principle of higenamine detection as „the determination of the free (non- conjugated) parent compound" [4] does not eliminate the risk of reporting unintentional doping by athletes. Conversely, not carrying out sample hydrolysis may affect that laboratories may not report „intentional doping” (an AAF for Higenamine), especially, in cases where the estimated concentration of higenamine after performed sample hydrolysis will exceed MRL. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-073 Figure 2. Chromatograms of analysis performed after sample preparation involving acid hydrolysis and double LLE: (a) blank urine, (b) athlete's sample, and (c) quality control sample (10 ng/mL) Conclusions Undeniably, monitoring of second-phase metabolites of prohibited substances is a crucial activity for doping control purposes. It seems to be undoubted that point "c" in the MRL table of the WADA TD2022MRPL should be applied for all β-2-agonist without sole exceptions to avoid reporting false negative findings for higenamine. Therefore, a change of the current principles of interpretation of the results for higenamine should be considered as soon as possible. Moreover, research concerning the presence of higenamine in nutritional supplements should be conducted to set a new MRL for higenamine metabolites or verify general MRL for higenamine. References 1. Cohen PA, Travis JC, Keizers PHJ, Boyer FE, Venhuis BJ. The stimulant higenamine in weight loss and sports supplements. Clin Toxicol. 2019;57(2):125-130. 2. Comunità F, de la Torre X, Mazzarino M, Botrè F. Higenamine and coclaurine positive doping case associated massage cream use. In: Schänzer W, Thevis M, Geyer H, Mareck U, eds. Recent Advances in Doping Analysis. Köln: Sportverlag Strauss; 2017:47-50. 3. World Anti-Doping Agency. Prohibited List 2023. Available at https://www.wada- ama.org/sites/default/files/2022-09/2023list_en_final_9_september_2022.pdf [03 February 2023] 4. World Anti-Doping Agency. TD2022MRPL. Available at https://www.wada-ama.org/sites/default/files/2022- 01/td2022mrpl_v1.1_eng_0.pdf [03 February 2023] MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-074 5. Grucza K., Kwiatkowska D., Kowalczyk K., Wicka M., Szutowski M., Cholbinski P.: Analysis for higenamine in urine by means of UHPLC/MS/MS: Interpretation of results. Drug Test Anal. 2017;10(6):1017-1024. Acknowledgements Financial support from the Ministry of Health of the Republic of Poland under the project number 1/85195/PLAD/2022/4186/406 is gratefully acknowledged. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-075 Görgens C , Guddat S , Bosse C , Thevis M Enantiomeric separation of various stimulants in urine after chiral derivatization using Marfey’s reagent for doping control purposes Institute of Biochemistry, German Sport University, Cologne, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract Enantiomers of chiral drugs often provide different pharmacological and pharmacodynamic properties due to their stereoselectivity. In many instances, the desired pharmacological activity is primarily attributed to one specific enantiomer, whereas the other enantiomer may exhibit reduced activity or even possesses toxic properties. For metamfetamine, the detected enantiomer determines the sub- classification by the World Anti-Doping Agency (WADA) as a “specified” or a “non-specified” stimulant, which is essential for the sanctioning process of the athlete. Therefore, the use of confirmation assays, which are able to distinguish between different enantiomers are essential for the compound’s unequivocal identification. Besides different LC-MS/MS-based approaches using chiral chromatographic analysis, derivatization strategies with chiral derivatizing agents followed by GC-MS/MS or LC-MS/MS detection also proved capable. The aim of the study was to develop a simple and fast LC-MS/MS-based assay for the enantioselective identification of some of the most frequently detected stimulants in sports drug testing after derivatization using Marfey’s reagent. Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide) is used to separate and to determine enantiomeric, amino-group containing compounds. The reagent reacts stoichiometrically with primary or secondary amino groups to produce stable diastereomeric derivatives, which can readily be separated by conventional reversed-phase HPLC without the need of a special and cost-intensive chiral column. The developed enantioselective assay is highly specific and reproducible, providing limits of identification (LOI) fulfilling WADA requirements for stimulants assigned with a minimum reporting level (MRL) of 50 ng/mL. As proof-of-concept, authentic urine samples containing the target compounds were analyzed and their enantiomeric composition was assessed. The approach proved suitable for the chiral separation of a total of six selected enantiomeric stimulants prohibited in sports. Introduction In recent years, a trend towards more efficient initial testing procedures (ITP), using LC-MS/MS methods covering hundreds of prohibited compounds as well as their metabolites in one single chromatographic run has been observed in sports drug testing. Using these high-throughput analytical assays, some crucial characteristics of specific analytes, e.g. chirality or isomerism of small polar compounds (m/z 100 – m/z 200) often remain undetected in the ITP, according to a non-enantioselective separation on common reversed-phase analytical columns and/or a limited number of product ions using electrospray ionization mass spectrometry. However, for some stimulants, e.g. metamfetamine, the detected enantiomer determines the sub-classification by the World Anti-Doping Agency (WADA) as “specified” or 1 1 1 1,2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-076 “non-specified”, which is essential for the sanctioning process of the athlete [1]. Moreover, the enantiomers of chiral drugs often provide different pharmacological and pharmacodynamic properties due to their stereoselectivity. In many instances, the desired pharmacological activity is primarily attributed to one specific enantiomer, whereas the other enantiomer may exhibit reduced activity or even possesses toxic properties. Therefore, the use of confirmation assays, which are able to distinguish between different enantiomers are essential for the compound’s unequivocal identification. Besides different LC-MS/MS-based approaches using chiral chromatographic analysis, derivatization strategies with chiral derivatizing agents followed by GC-MS/MS or LC-MS/MS detection proved valuable [2-4]. The aim of the study was to develop a simple and fast LC-MS/MS-based assay for the enantioselective identification of some of the most frequently detected stimulants in sports drug testing after derivatization using Marfey’s reagent. Figure 1. Nucleophilic substitution reaction of l-amfetamine (I) and Marfey’s reagent (II) to yield the corresponding l,l-diastereomer derivative (III) with elimination of hydrogen fluoride Experimental Materials and methods The reference materials of d-amfetamine, l-amfetamine, d-metamfetamine, l-metamfetamine and 4- methylhexan-2-amine were obtained from from Cerilliant (Round Rock, TX, USA). 5-methylhexan-2-amine and 1,3-dimethylbutylamine were purchased from Sigma-Aldrich (Deisendorf, Germany), while 3-methyl- hexan-2-amine was bought from Curpys Chemicals (Kyiv, Ukraine). Experiments were carried out using an Exploris 480 orbitrap mass spectrometer operating in negative-ionization full scan and parallel reaction monitoring (PRM) acquisition mode coupled to a Vanquish UHPLC (Thermo Scientific, Bremen, Germany), equipped with a Nucleoshell RP 18 (100 x 2 mm, 2.7 µm) analytical column (Macherey Nagel, Düren, Germany). The mobile phase composed of 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B). Initial conditions were 60% of solvent B at a flow rate of 250 µL/min. After 5 min of isocratic elution, the content of solvent B was increased linear from 60% to 80% within 10 min. After another one-minute of isocratic elution at 98% B, re-equilibration at 60% B started for 4 min, resulting in an overall runtime of 20 min. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-077 Samlpe preparation To 50 µL of urine sample, 10 µL of the racemic internal standard amfetamine-D11 and 20 µL of 1 M NaHCO was added. After mixing, 100 µL of a 0.1% ( w/v) solution of Marfey's reagent (1-fluoro-2-4- dinitrophenyl-5-L-alanine amide) dissolved in acetone, was added to the mixture, vortexed and heated at 45 °C for 1 hour. The reaction was stopped by adding 40 µL of 1 M HCl in water and evaporation to dryness under nitrogen at 60°C. The samples were reconstituted in 100 µL of 40:60 water:methanol (v/v) before injection into the instrument. Results and Discussion As demonstrated in Figure 2, analysis of the aforementioned stimulants after derivatization using Marfey’s reagent and LC-HRMS/MS detection provides excellent enantioselective separation with almost zero biological noise. As demonstrated in Table 1, the assay was found the be highly specific and reproducible, providing limits of identification (LOI) fulfilling WADA requirements for stimulants assigned with a minimum reporting level (MRL) of 50 ng/mL. As a proof-of-concept, authentic urine samples containing the target compounds were analyzed and their enantiomeric composition was assessed. The approach proved suitable for the chiral separation of a total of six selected enantiomeric stimulants prohibited in sports. Table 1. Method validation results 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-078 Figure 2. Extracted ion chromatogram of spiked urine samples with (A) 3-methylhexan-2-amine, (B) 4- methylhexan-2-amine, (C) 5-methylhexan-2-amine, (D) metamfetamine and amfetamine, and (E) 1,3- dimethylbutylamine (in each case 50 ng/mL) after derivatization using Marfey‘s reagent MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-079 Conclusions Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide) is used to separate and to determine enantiomeric, amino-group containing compounds. The reagent reacts stoichiometrically with primary or secondary amino groups to produce stable diastereomeric derivatives, which can readily be separated by conventional reversed-phase HPLC without the need of a special and cost-intensive chiral column (Figure 1). The developed sample preparation is simple and fast and fulfills WADA requirements for MRL compounds. References 1. World Anti-Doping Agency. The 2023 Prohibited List. 2023, https://www.wada- ama.org/sites/default/files/2022-09/2023list_en_final_9_september_2022.pdf (24-01-2023) 2. Thomas A, Thevis M. Stereoisomers in sports drug testing: Analytical strategies and applications. J Chromatogr A. 2022; 1674:463154. doi: 10.1016/j.chroma.2022.463154. Epub 2022 May 17. 3. Newmeyer MN, Concheiro M, Huestis MA, Rapid quantitative chiral amphetamines liquid chromatography- tandem mass spectrometry method in plasma and oral fluid with a cost-effective chiral derivatizing reagent, J Chromatogr A. 2014; 1358 (5): 68–74. https://doi.org/10.1016/j.chroma.2014.06.096 4. Foster BS, Gilbert DD, Hutchaleelaha A, Mayersohn M, Enantiomeric determination of amphetamine and methamphetamine in urine by precolumn derivatization with Marfey's reagent and HPLC, J Analytical Toxicology. 1998; 22 (4): 265-269. https://doi.org/10.1093/jat/22.4.265 Acknowledgements The presented work was conducted with support of the Federal Ministry of the Interior, Building and Community, of the Federal Republic of Germany, and the Manfred-Donike Institute for Doping Analysis (Cologne, Germany). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-080 Sahu PL , Handa T , Sahu K , Soni A , Kori RK , Mahajan S , Padder AH , Nandi U , Ahmed QN , Reddy DS Synthesis and characterization of 17α-hydroxymethyl-17β-methyl-18- nor-2-oxa-5α-androst-13-en-3-one (Oxandrolone M2), a long-term meta​bolite via a Kratena’s reaction approach National Dope Testing Laboratory, New Delhi, India ; CSIR-Indian Institute of Integrative Medicine, Jammu, India, Jammu, India Abstract The oxandrolone long-term urinary metabolite (OxM2) belongs to S1.1 Anabolic Androgenic Steroids under S1 Anabolic Agents class listed in the WADA Prohibited List. Despite its potent anabolic activity and less side effects, anabolic steroids like oxandrolone have limited positive findings, making it a popular choice for athletes who cheat. Oxandrolone marketed under the brand name Oxandrin and Anavar is an Androgenic Anabolic Steroid (AAS). Despite of its therapeutic usage, it is associated with one or more side effects such as liver cell tumors, peliosishepatis, intra-abdominal hemorrhage, liver failure, fatal and blood lipid changes associated with increased risk of atherosclerosis, cholestasis hepatitis, increased risk for the development of prostatic hypertrophy and prostatic carcinoma in geriatric patients and hypercalcemia in patients with breast cancer. So, FDA announced the final decision on June 28, 2023 to withdraw approval of brand and generic Oxandrin and decided that the drug products should be removed from the market [1]. For the synthesis of OxM2, literature report by Kratena et al. was followed and due to various bottle neck challenges in the reported method, series of optimizations were performed to successfully achieve the formation of OxM2. So, in the present report, an optimized procedure for the synthesis of OxM2 along with its spectral analysis is reported. Keywords : oxandrolone, long-term metabolite, OxM2 Introduction Anabolic Androgenic Steroids are still popular prohibited substances among athletes and are the most frequently found drug in tests, as stated by the statistics provided by WADA [2]. Oxandrolone has two long-term urinary metabolites which are C-17-epimeric substances, i.e 17α-hydroxymethyl-17β-methyl- 18-nor-2-oxa-5α-androst-13-en-3-one (Ox M2) and its epimer 17β-hydroxymethyl-17α-methyl-18-nor-2- oxa-5α-androst-13-en-3-one (Ox M1). The merit of these metabolites for the anti-doping community is the prolonged detection period, which significantly increases the effectiveness of sports drug testing. In the present work, some optimizations of the approach of Kratena et al. are carried out to access the OxM2 metabolite [3], which is required as reference material in anti-doping laboratories for the detection of Oxandrolone misuse by athletes. 1 1 1 1 1 2 2 2 2 2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-081 Experimental Procedure for the synthesis of OxM2 Oxandrolone 1 (0.900 g) was dissolved in dry pyridine (20 mL) and POCl (2 mL) was added at -78 °C and stirred overnight for 48 h at room temperature. After cooling the mixture to room temperature, it was carefully poured into ice cold water. The suspension thus obtained was extracted with dichloromethane (DCM), the organic phases were dried over Na SO and evaporated in vacuo to obtain the crude product which was recrystallized in DCM and hexane. Solid thus obtained was filtered and characterized as mixture of isomers 2. Further 2 (0.889 g) was dissolved in 10 mL dry DCM, 1.2 equiv. m-CPBA (0.639 g) was added portion wise at 0 °C and then stirred at room temperature for 3 h. The solution was then quenched with 40 mL saturated sodium thiosulfate and 40 mL NaHCO solutions and extracted with DCM. The crude product thus obtained was purified by column chromatography (230-400 mesh silica gel, neutralized with triethylamine) using 15% ethyl acetate:hexane as eluent to give 0.443 g mixture of epoxides 3 and 4. The mixture of 3 and 4 (0.436 g) was further dissolved in glacial acetic acid (9.6 mL) and stirred overnight at room temperature for 24 hours. The reaction mixture was then treated with 20 mL water, neutralized with saturated solution of NaHCO and extracted with DCM. The combined organic extracts were washed with brine and dried over Na SO . The crude product obtained by evaporation of the combined organic extracts was purified using column chromatography (230-400 mesh silica gel, neutralized with triethylamine) with 17% EtOAc:hexane as eluent. For achieving 95% pure desired OxM2 metabolite, it was further purified using column chromatography (100-200 mesh silica gel, neutralized with triethylamine) with 24% EtOAc:hexane as eluent to obtain the desired OxM2 metabolite (30 mg) as a white waxy solid. Results and Discussion Initially, Oxandrolone 1 was dehydrated as reported by Kratena to obtain a crude mixture of regioisomers 2. As reported, it was used without purification for epoxidation but some undesired products were obtained, when the rearrangement of isolated mixture of epoxides was performed using Kratena’s approach. So we modulated the reaction conditions and performed the dehydration by adding POCl at - 78 °C followed by overnight stirring at room temperature for 48 h to obtain 2 after recrystallization in DCM with hexane (Scheme 1, 50% yield). 2 was further subjected to epoxidation with portion wise addition of 1.2 equiv. of m-CPBA at 0 °C and then at room temperature stirring for 3 h in the absence of Na HPO /KH PO buffer. After 3 h, the formation of the desired mixture of epoxides 3 and 4 was observed along with unreacted reactant (Scheme 2) which was isolated using column chromatography. 3 2 4 3 3 2 4 3 2 4 2 4 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-082 Figure 1. Scheme 1 and Scheme 2 Mixture of 3 and 4 was then stirred for 24 h in glacial acetic acid ( Scheme 3) which resulted in the rearrangement of 3 to yield OxM2 which was purified using column chromatography. Figure 2. Scheme 3 1 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-083 H NMR (400 MHz, CDCl ): δ 4.24 (d, J = 10.7 Hz, 1H), 3.90 (d, J =10.7 Hz, 1H), 3.29 (dd, J =35.1, 10.5 Hz, 2H), 2.47 (dd, J =18.7, 5.8 Hz, 1H), 2.21-2.13 (m, 2H), 2.09-1.96 (m, 3H), 1.95-1.84 (m, 3H), 1.82-1.73 (m, 1H), 1.68-1.62 (m, 1H), 1.56-1.46 (m, 3H), 1.30-1.23 (m, 1H), 1.18-1.14 (m, 1H), 1.05 -0.96 (m, 2H), 0.91 (s, 3H), 0.89 (s, 3H). C NMR (101 MHz, CDCl ): δ 170.57, 139.98, 136.51, 81.00, 68.96, 51.60, 47.28, 40.21, 36.07, 34.68, 33.92, 33.72, 30.56, 30.41, 27.51, 22.85, 22.17, 21.68, 9.84. IR (KBr) ν cm : 3441.91, 2926.41, 2858.69, 1734.03, 1447.24, 1409.10, 1384.19, 1265.3, 1208.65, 1037.66, 917.9, 800.57, 665.84. Figure 3. H (CDCl , 400 MHz) and C NMR (CDCl , 101 MHz) spectrum of OxM2 Figure 4. HRMS analysis of OxM2 1 3 13 3 -1 1 3 13 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-084 Conclusions The isomeric metabolite of oxandrolone i.e. OxM2 has been successfully synthesized via revisiting Kratena's approach and fully characterized using H, C NMR and mass spectrometry to improve sports drug testing in respect to develop comprehensive analytical strategies for the effective detection of oxandrolone abuse. References 1. Federal Register / Vol. 88, No. 123 / Wednesday, June 28, 2023 / Notices https://www.regulations.gov/document/FDA-2023-N-2226-0001. 2. List_en_final_9_september_2022.pdf; https://www.wada-ama.org/sites/default/files/2022-09/2023 3. Kratena N, Stöger B, Weil M, Enev VS, Gärtner P. Synthesis of two epimeric long-term metabolites of oxandrolone. Tetrahedron Letters, 2017, 58;13:1316-1318. doi.org/10.1016/j.tetlet.2017.02.048. Acknowledgements Authors are thankful to CSIR-Indian Institute of Integrative Medicine, Canal Road Jammu for providing all the necessary research facilities and National Dope Testing Laboratory (NDTL), New Delhi for providing the funding. 1 13 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-085 Okano M, Akiyama K, Sato M, Mombayashi A Can current WADA-approved direct detection methods identify the use of GH analogue Somatrogon in doping control? Anti-Doping Laboratory LSI Medience Corporation, Tokyo, Japan Abstract Somatrogon is a long-acting human growth hormone (GH) with a C-terminal peptide domain derived from human chorionic gonadotropin (hCG). In 2022, somatrogon was added as an example of GH analogues on the S2.2.3 of the World Anti-Doping Agency (WADA) Prohibited List. The study purpose is to verify whether the detection of somatrogon use is possible using the current WADA–approved direct methods (i.e. GH isoform test and urinary hCG tests). An injectable solution of somatrogon (0.66 mg/kg) was subcutaneously injected to six healthy subjects (three males and three females). The detectability of somatrogon use was dependent on each GH isoform kit (Kit1 and Kit2). The Pit_kit1 showed a dose response, but the Rec_kit1 did not show that, resulting in a profile in which the Rec/Pit1 ratio decreased from the baseline. In contrast to Kit1, the Rec_kit2 showed post-dose response. The Rec/Pit2 ratio exceeded the WADA decision limit in all subjects. It was suggested that antigen-antibody interaction at the epitope of Rec_kit1 was disrupted by hCG β-subunit. Since Kit2 can effectively detect the use of somatrogon, it is recommended to perform the further GH biomarkers test in cases where Kit1 shows an abnormally low Rec/Pit1 ratio. Total hCG above 5 IU/L was detected in two of three male subjects after administration. On the other hand, the result of the intact hCG analysis was negative in all subjects as expected. This suggests that hCG β-subunit is excreted into urine as a metabolite/degradation product of somatrogon. Introduction The illicit use of human growth hormone (hGH) has been prohibited by the World Anti-Doping Agency (WADA)[1]. Somatrogon is a recombinant fusion glycoprotein composed of C-terminal peptides of the β- subunit of human chorionic gonadotropin (hCG) at positions 1-28, 220-247 and 248-275 and hGH at positions 29-219, which was designed/developed to have a long action compared with the conventional recombinant hGH [2]. In 2022, somatrogon was added as an example of GH analogues on the S2.2.3 of the WADA Prohibited List [1]. The study purpose is to verify whether the detection of somatrogon use is possible using the current WADA–approved direct methods (i.e. GH isoform test and urinary hCG tests). Experimental Materials Somatrogon (Ngenla ) was purchased from Pfizer Inc. (Tokyo, Japan). ® MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-086 GH isoform differential immunoassay The detection method was a quantitative analysis of recombinant GH (Rec) and pituitary GH (Pit) using immunoassay kits provided by CMZ-Assays GmbH (Berlin, Germany). The kits consist of two parts: Kit1 and Kit2. The assay was fully validated and performed according to the WADA technical document [3]. Determination of urinary hCG The initial testing procedure (ITP) was performed using Immulite 2000 (Siemens, IL, USA ), which is specific for total hCG. The limit-of-quantification (LOQ) was set at 1.00 IU/L. The confirmation analysis (CP) using an assay-specific α /β heterodimeric hCG was performed. A Roche Cobas e411 (Roche Diagnostics, In, USA) using the Elecsys hCG STAT assay. The LOQ for each test was 1.00 IU/L. Both assays were fully validated in accordance with WADA technical document – TD2021CGLH [4]. Cross-reactivity test The somatrogon formulation was prepared at 3 ng/mL and 30 ng/mL in sheep serum and analyzed to confirm cross-reactivity with each assay system. Human subjects An injectable solution of somatrogon (0.66 mg/kg) was subcutaneously injected to six healthy volunteers (three males: BB01-03, three females: BB04-06) at 9:00 am. The administration study was reviewed and approved by the Ethical Review Board of LSI Medience Corporation. The time courses of sample collection were -24, -20, -16, -12, 0 (pre-administration), 2, 4, 6, 8, 10, 12, 24, 28, 32, 36, 48, 52, 56, 60 and 96 h. Serum and urine samples collected were stored at -20°C until analysis. Results and Discussion Cross-reactivity As shown in Table 1, the Rec_kit2 showed a strong reactivity, but the Rec_kit1 did not. It was suggested that steric hindrance disrupts antigen-antibody interaction at the epitope of Rec_kit1. Neither the intact hCG kit nor the total hCG kit showed cross-reactivity with intact somatotrogon. GH isoform test (Kit1) As shown in Figure 1, the Pit_kit1 showed a dose response, but the Rec_kit1 did not show that, resulting in a profile in which the Rec/Pit1 ratio decreased from the baseline. It was consistent with the results of cross-reactivity test (Table 1). GH isoform test (Kit2) In contrast to Kit1, the Rec_kit2 showed post-dose response (Figure 2). The cross-reactivity test shown in Table 1 also indicated the full reactivity with the Rec_kit2. The Rec/Pit2 ratio exceeded the WADA decision limit (DL) in all 6 subjects (Table 2). In all female cases, it did not return to baseline even 96 h after administration, indicating a long-acting profile. In the future, it may be possible to detect even male by setting individual reference values based on athlete biological passport instead of DL judgment. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-087 Table 1. Cross-reactivity test; Rate (%) = (Measured value / Target conc.) x 100 Figure 1. GH isoform test result after administration of somatrogon (Kit1) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-088 hCG tests As shown in Table 2, total hCG above 5 IU/L was detected in 2 of 3 male subjects after administration (Table 2). On the other hand, the result of intact hCG measurement was negative in all subjects as expected. Cross-reactivity testing of the formulation showed no reactivity with either the Immulite2000 kit or the intact hCG kit (Table 1). This suggests that hCG β-subunit is excreted into urine as a metabolite/degradation product of somatrogon. Figure 2. GH isoform test result after administration of somatrogon (Kit2) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-089 Table 2. Detectability of GH isoform test (Kit2) and total hCG (urine) Conclusions The detectability of somatrogon use was dependent on each GH isoform kit. Since Kit2 can effectively detect the use of somatrogon, it is recommended to perform the further GH biomarkers test in cases where kit1 shows an abnormally low Rec/Pit ratio. As expected, an administration of somatrogon did not affect the CP for urinary intact hCG (no false positive of hCG), whereas the ITP for total hCG might detect some somatrogon fragments/metabolites having β-subunit of hCG. If ITP with Immulite2000 results in suspected total hCG in urine, it may be beneficial to recommend additional GH testing, although limited to male athletes, due to the possibility of using somatrogon. References 1. World Anti-Doping Agency. The 2023 Prohibited list. International Standard, Montreal (2023) https://www.wada-ama.org/sites/default/files/2023-05/2023list_en_final_9_september_2022.pdf (access date 21.08.2023) 2. Hershkovitz O, Bar-Ilan A, Guy R, Felikman Y, Moschcovich L, Hwa V, Rosenfeld RG, Fima E, Hart G. (2016) In vitro and in vivo characterization of MOD-4023, a long-acting carboxy-terminal peptide (CTP)-Modified human growth hormone. Mol Pharm. 13(2), 631-9 3. World Anti-Doping Agency. WADA Technical Document – TD2021GH, Montreal (2020) https://www.wada- ama.org/sites/default/files/2022-01/td2021gh_final_eng_0.pdf (access date 21.08.2023) 4. World Anti-Doping Agency. WADA Technical Document – TD2021CG/LH, Montreal (2020) https://www.wada- ama.org/sites/default/files/2022-01/td2021cg-lh_final_eng_0.pdf (access date 21.08.2023) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-090 Saavedra MJ , Sánchez-Wandelmer J , Polo ML , Rodríguez-de Francisco G , Muñoz G Bandmate™ as a good alternative to BlotCycler™ for erythropoietin detection by Western blot Madrid Anti Doping Laboratory, Health Institute Carlos III, Madrid, Spain ; Madrid Antidoping Laboratory, Tragsatec, Madrid, Spain Abstract Analysis of erythropoietin receptor agonists (ERAs) is crucial in antidoping control, being the Western blot analysis the most extended method for their detection [1,2]. The robustness in the performance of this kind of analytical technique is key to warrant fiability of results. Manual methods are often inconsistent and show high variability. Due to that, and also with the aim to increase productivity, most of the antidoping laboratories decided to use an automatized system for the analysis of erythropoietins by Western blot with BlotCycler as the system of choice [3,4]. This system allows the simultaneous incubation (first with the primary antibody and then with the secondary antibody or streptavidin-POD) of up to 4-midi gels, making possible to process up to 56 samples. However, and probably due to the need of working under cold conditions, this instrument does not seem to be robust enough for the requirements and use in antidoping laboratories, suffering an important deteriorating in very short time. In this work, the alternative system called Bandmate Western blot Processor (from Invitrogen) has been tested for automatization of ERAs analysis. The system has been tested in Madrid Doping Control Laboratory in the analysis of several urine samples spiked at 50% of MRPL of the more relevant erythropoietins (recombinant EPO, NESP, CERA and EPO-Fc). All the species were properly detected according TD2022EPO requirements [5]. Introduction Erythropoietin (EPO) is a glycoprotein hormone that promotes the production of red blood cells in response to cellular hypoxia. This biological function has made erythropoietin a good candidate for the development of therapeutic analogues. Both recombinant erythropoietins (EPOs) and erythropoietin receptor agonists (ERAs) have been extensively used for the treatment of several clinical conditions such as anemia or renal insufficiency. The stimulation of erythropoiesis and hence, of oxygen transport to tissues by these drugs has also made them attractive for athletes participating in endurance sports. EPOs and agents affecting erythropoiesis are therefore listed in section S2 of the World Anti-Doping Agency (WADA) Prohibited List and the number of adverse analytical findings (AFF) for these substances is always the highest in S2 Prohibited List. Exogenous EPO and ERAs differ from endogenous erythropoietin in molecular weight. Therefore, current methods to detect them are based on these variations, using polyacrylamide gel electrophoresis (SAR- PAGE) followed by immunoblotting, which is the only method accepted in the current WADA Technical Document for the analysis and reporting of EPOs and ERAs (TD2022EPO). Although both SDS and SAR- PAGE can be used for EPO detection, SAR-PAGE showed better sensitivity for PEGylated EPOs than SDS- 1 2 1 1 1 1 2 TM TM MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-091 PAGE [6] and it is currently the method of choice for analysis of EPOs in urine samples. Selectivity and sensitivity of this analysis are not only affected by the electrophoresis conditions, but also immunodetection is an important factor to be considered as well. Automatization of this process would certainly contribute to decrease variability due to manual intervention and contributes to intra and interlaboratory standardization of methodology to ensure reliable results. In this study, the Bandamate Western blot processor (Invitrogen) has been tested to further automate of the analysis of ERAs in urine samples as an alternative to using the BlotCycler , which was already reported to decrease membrane background and increase sensitivity [3] compared to manual processing. Here, we confirmed that by performing Western blot on the Bandmate . We were able to detect recombinant EPO, NESP, CERA and EPO-Fc in positive urine QCs, and in urine contaminated at both MRPL and 50% MRPL. In addition, the use of Bandmate offered several advantages such as a significant reduction in antibody volume without affecting detection sensitivity and low maintenance demands. Experimental Materials EPO-BRP (European Pharmacopoeia), NESP (Darbepoein alfa, Amgen), Mircera (CERA, Roche) Dynepo (DYN, Shire Pharmaceutical Ibérica S.L), and EPO-Fc (Prospec), ELISA Kit (Stemcell Technologies), EPO NuPAGE 10% Bis-Tris Midi gels (ThermoFisher), EPO clone AE7A5 (R&D Systems BAM2871), Streptavidin- POD Conjugate (Roche), SuperSignal West Femto (ThermoFisher). Samples In all cases two sets of three different urine samples from healthy volunteers were spiked with different types of ERAs at MRPL or below: BRP-EPO, Aranesp (NESP), Mircera (CERA) or EPO-Fc (ProSpec). The ERA- Mix standards are DYN-NESP-CERA in 0.1% casein in PBS. Urine ultrafiltration The samples were concentrated by ultrafiltration [2]. 15 mL of urine, 1.5 mL Tris- HCl (3.75 mM), and 200 µL complete solution (1 tablet / 2 mL water) were mixed and centrifuged (3900 rpm, 20 min). The supernatant was transferred to an Amicon Ultra 15 filter (30 kDa MWCO) and centrifuged (3900 rpm, 20 min). The retentate was washed (15 mL aprox. 50 mM Tris-HCl buffer and 200 µL complete and centrifuged (3900 rpm, 20 min). Finally, the retentate was transferred to an Amicon Ultra 0.5 filter (14000 rpm, 15 min) and recovered by reserved spinning at 1000 RCF for 2 min. Immunopurification All urine retentates were incubated on the ELISA plate overnight at 4 °C, next washed five times with 300 µL PBS, eluted with 15 µL of sample elution buffer and heated for 5 min at 95 ºC under shaking (650 rpm; ThermoMixer). Cool down, and centrifuge (3900 rpm, 1 min). SAR-PAGE, single-blotting, and detection Samples were loaded onto the electrophoretic gel, followed by SAR-PAGE electrophoresis at 125V / 25W / constant voltage for 3 h in a cold bath. Transfer was performed at 200 mA / 25 V for 45 min, the PVDF TM TM TM TM ® ® TM MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-092 membrane was blocked with 5% milk and incubations were performed overnight in a BlotCycler and Bandmate . Chemiluminescent signals were detected using a camera (Amersham Imager 680) and analyzed with GASepo software [7,8]. Results and Discussion Figure 1 and Figure 2 show the immunoblot of the two sets of samples performed in BlotCycler or Bandmate . In both cases, membranes were blocked offline and subsequently incubated with 20 mL of primary antibody (1:1000 dilution in 1% milk in PBS) for 12 hours and with 20 mL of Streptavidin (1:2000 dilution in 1% milk in PBS) for 5 hours in the automated Western blot systems evaluated. Every set of samples was composed of a set of positive quality controls (negative urine samples spiked with every type of the ERAs at MRPL). Two different stock solutions of EPO-Fc were used (1 and 2) in the positive controls and two different urine samples spiked with a mix of ERAs at MRPL and 50% MRPL. The comparison of the images processed using GASepo software showed that the results obtained in both systems were comparable in terms of sensitivity and background. Figure 1. Immunoblot of the indicated samples performed in BlotCycler TM TM TM TM TM MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-093 Figure 2. Immunoblot of the indicated samples performed in Bandmate In addition, in the case of Bandmate , it was also tested the online blocking of the membrane and the possibility of incubation using half the volume of the primary antibody (10 mL instead of 20 mL of 1:1000 in dilution in 1% milk in PBS). Figure 3 shows that the results for the positive quality control samples were similar to the ones obtained in the previous case. TM TM MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-094 Figure 3. Immunoblot of the indicated samples performed in Bandmate . The membrane was blocked in the instrument and subsequently incubated with 10 mL of primary antibody (1:1000 dilution in 1% milk in PBS) and with 10 mL of streptavidin (1:2000 dilution in 1% milk in PBS) Conclusions Bandmate™ acts as the BlotCycler™ for the detection of ERAs by immnunodetection. Its productivity is reduced compared to BlotCyclerrTM, as only 2 midi gels can be processed in the same run. However, it increases cost-effectiveness, as the volume of antibody solutions can be reduced, leading to significant savings in both primary and secondary antibodies or streptavidin. In addition, Bandmate™ easily allows the recovery of antibodies for reuse and, in contrast to BlotCycler™, Bandmate™ only requires a quick flush with water after use and very low maintenance operations. In summary, we conclude that Bandmate™ is a good alternative to BlotCycler™ for immunodetection of ERAs. References 1. Reichel C Detection of peptidic erythropoiesis-stimulating agents in sport. Br J Sports Med. 2014 May;48(10):842-7. doi: 10.1136/bjsports-2014-093555. Epub 2014 Mar 27. 2. Masquelier M, Heiland C, Bhuiyan H, Erlandsson G, Molin A, Magnus E. Optimization of ESAs detection in urine and blood: comparison of different immunopurification techniques. In: Thevis M, Geyer H, Mareck U (eds.) Recent Advances In Doping Analysis (26). Sport und Buch Strauß - Köln 2018, pp 58-65 3. Reichel C, Gmeiner G, Reihlen P, Thevis M, Schänzer W. SARCOSYL-PAGE: Optimized Protocols for the Separation and Immunological Detection of PEGylated Proteins. Methods Mol Biol. 2019;1855:131-149. doi: 10.1007/978-1-4939-8793-1_14 TM MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-095 4. Reichel C, Erceg D, Lorenc B, Scheiblhofer V, Farmer L, Zanitzer K, Geisendorfer T, Gmeiner G, Thevis M. Data from a microdosed recombinant human erythropoietin administration study applying the new biotinylated clone AE7A5 antibody and a further optimized sarcosyl polyacrylamide gel electrophoresis protocol. Drug Test Anal. 2023 Feb;15(2):163-172. doi: 10.1002/dta.2989. Epub 2021 Jan 21. PMID: 33450134. 5. WADA Technical Document TD EPO: Harmonization of Analysis and Reporting of EPO and other Erythropoietin Receptor Agonists (ERAs) by Polyacrylamide Gel Electrophoretic (PAGE) Analytical Methods. 6. Reihlen P, Blobel M, Kempkes R, Reichel C, Völker Schänzer E, Majer B, Schänzer W. Optimizing SAR-PAGE. Drug Test Anal. 2015 Nov-Dec;7(11-12):1014-6. doi: 10.1002/dta.1915. 7. Bajla I, Holländer I, Minichmayr M, Gmeiner G, Reichel Ch. GASepo-a software solution for quantitative analysis of digital images in Epo doping control. Comput Methods Programs Biomed. 2005 Dec;80(3):246- 70. doi: 10.1016/j.cmpb.2005.09.005 8. Bajla I, Holländer I, Gmeiner G, Reichel Ch. Quantitative analysis of images in erythropoietin doping control. Med Biol Eng Comput. 2005 May;43(3):403-9. doi: 10.1007/BF02345819. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-096 Mc Pherson Medina A, Fiallo Fernandez T, Martinez Brito D, Montes de Oca Porto R Stability of the biotinylated monoclonal antibody BAM2871 in the 1% non-fat milk solution Antidoping Laboratory, Havana, Cuba Abstract Clone AE7A5 anti-EPO antibody recognizes an epitope within the first 26 amino acids of the amino terminal chain of human and recombinant erythropoietin. Its biotinylated version (BAM2871) improves the detection with respect to its non-biotinylated version (MAB2871). This work evaluates the stability of BAM2871 incubation solution considering the conductivity of water and the origin of the non-fat milk. Three BAM2871 solutions were prepared with Millipore non-fat milk for microbiological use prepared with water MQ (conductivity values of 0.1 [S1] and 0.5 μS/cm [S2]) and Nestlé commercial non-fat milk prepared with water MQ (conductivity of 0.1 μS/cm [S3]). Gels with quality negative and positive controls in urine, at 50% MRPL, were analyzed simultaneously. Analyses were carried out from 0 to 35 days and the 1% non-fat milk solutions were conserved at 4 °C during this time. Values of pH were measured, and EPO-Fc, rEPO, CERA, NESP and Dynepo were tested. From day 15, turbidity and sediment were observed i n S3. Also, the pH started to increase from day 5 and, an excessive background on the membrane difficulted the interpretation of the bands. From day 20, a decrease in the intensity of the bands was observed when S2 was used and a stench was detected at day 25 after the preparation and the pH value started to increase before day 15. On the contrary, results obtained with S1 were repeatable up to day 30. Its pH value was stable until day 30. Introduction Clone AE7A5 anti-EPO antibody recognizes an epitope within the first 26 amino acids at the N-terminus of human urinary and human recombinant EPO [1]. Its biotinylated version (BAM2871, R&D Systems) has allowed compliance with the MRPL described in the TD2022EPO [2]. Usually, the antibody is added in amounts that exceed those necessary to guarantee a quantitative reaction [3]. Furthermore, it binds only to regions of the membrane where a related protein is found, therefore, after incubation, the remanent of the solution containing the antibody should be usable again. This work aimed to evaluate the stability of the BAM2871 monoclonal antibody incubation solution taking into consideration the conductivity of MQ water and the origin of the non-fat dry milk. Experimental BAM2871 was purchased from R&D System; Milk powder fat-free suitable for microbiology (1.15363, Millipore) was from SIGMA and Nestlé Carnation was from Nestle. The quality of all reagents and materials meets the criteria for immunoassays and are described in the SOP to detect and to confirm ESAs accredited by the requirements of ISO/IEC 17025 [4]. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-097 BAM2871 was prepared in three different non-fat milk solutions at a concentration of 1%. Non-fat milk were dissolved in PBS solution and the water used was microfiltrated to exclude the presence of bacteria. The solutions were identified as: Millipore 1.15363, water conductivity: 0.1 μS/cm Millipore 1.15363, water conductivity: 0.5 μS/cm Nestlé Carnation, water conductivity: 0.1 μS/cm Three gels were analyzed simultaneously every 5 days (from to 0 to 35 days) using the solutions S1, S2 and S3 with the monoclonal antibody BAM2871 to detect EPO-Fc, rEPO, CERA, NESP and Dynepo at 50% of the MRPL reference material and urine. The volume absolute of the bands obtained by the GASepo software [5] was plotted for each ESA and for each solution S1, S2 and S3. After the incubation was finished, solutions S1, S2 and S3 were conserved at 4 °C until the next analysis. Results and Discussion The results for each solution S1, S2 and S3 are represented in Figure 1. Figure1. Results of the band densities for EPO-Fc, rEPO, CERA, NESP and Dynepo during the experiment from day 0 – 35 for Millipore and water conductivity 0.1 μS/cm (S1); Millipore and water conductivity 0.5 μS/cm (S2); Nestlé Carnation and water conductivity 0.1 μS/cm (S3) and behavior of the pH values for S1, S2 and S3 (pH) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-098 The solution S3, prepared in the non-fat milk from Nestlé, started to increase values of pH from day fifth after the preparation. The maximum pH value of 8.5 was reached on day 20 and it was stable until the end of the experiment. After fifteen days, the solution showed turbidity, stench, and sediments, which provoked the presence of spots and unspecific bands that increased the background, making interpretation difficult and decreased the quality of the image. The most affected ESAs were NESP and EPO-Fc. Figure 2 shows an example of gels on days 5 and 25. Figure 2. GASepo image for the gels after 5 and 25 days incubating BAM2871 in a solution of non-fat milk Nestlé Carnation prepared with a water conductivity of 0.1 μS/cm (S3) The solution S2, containing BAM2871 dissolved in the non-fat milk from Millipore and water conductivity of 0.5 μS/cm, showed a decrease in the intensity of the bands from day 20 after preparation. A stench was detected at day 25 after the preparation and the pH value started to increase before day 15, showing a maximum value of pH of 8.3 after day 25. Although to a lesser extent than for S3, the presence of spots and unspecific bands that increased the background was also observed (Figure 3). The solution S1, containing BAM2871 solved in the non-fat milk from Millipore and water conductivity of 0.1 μS/cm, showed repeatable results from day 0 to the end of the experiments on day 35. Only the last measurement showed a decrease on the band intensities except for EPO-Fc, that showed a decreased intensity after day 25. The pH of the solution was also stable and until day 35 when the value increased up to 8, and no sediments were observed. As Figure 4 shows, also the quality of the image was stable during all the experiment. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-099 Figure 3. GASepo image for the gels after 5 and 30 days incubating BAM2871 in a solution of non-fat milk Millipore prepared with a water conductivity of 0.5 μS/cm (S2) Figure 4. GASepo image for the gels after 5 and 35 days incubating BAM2871 in a solution of non-fat milk Millipore prepared with a water conductivity of 0.1 μS/cm (S1) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0100 Conclusions The stability of the solutions of non-fat milk used in the incubations with BAM2871 to detect ESAs is not the same depending on the water quality. The preparations of solutions of Millipore milk in water with a conductivity of 0.1 μS/cm showed no alterations of the image background. It seemed to be stable at least for 30 days conserved at 4 °C. On the contrary, preparations of non-fat milk with water showing higher levels of conductivity favoured the presence of sediments, stench, elevation of pH value, and presence of spots and/or unspecific, that can make image interpretation difficult. References 1. W. Jelkmann. Erythropoiesis Stimulating Agents and Techniques: A Challenge for Doping Analysts. Curr. Med. Chem., 2009, 16, 1236–1247. 2. World Anti-Doping Agency. WADA Technical Document – TD2022EPO. Harmonization of analysis and reporting of erythropoietin (EPO) and other EPO-receptor agonists (ERAs) by Polyacrylamide gel electrophoretic (PAGE) Analytical Methods. Version 1.0, January 2022. 3. R. F. Ochoa-Azze. Bases Metodológicas para la evaluación de Anticuerpos en Ensayos Clínicos de Vacunas Mediante Técnicas Inmunoenzimáticas, Finlay Ediciones, Ciudad de La Habana, 2008. 4. ISO/IEC 17025:2017 General requirements for the competence of testing and calibration laboratories. 5. Bajlaa, I., Holländer, I., Minichmayr, M., Gmeiner, G., Reichel, Ch. GASepo - a software solution for quantitative analysis of digital images in Epo doping control. Computer Methods and Programs in Biomedicine 80, 246-270, 2005. doi:10.1016/j.cmpb.2005.09. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0101 da Costa S, Ruivo J, Casimiro T, Pereira L, Salema B Long-term stability study of erythropoietin and erythropoietin receptor agonists detection in dried blood spots and comparison of different absorbent sample supports Laboratório de Análises de Dopagem, INSA, Lisbon, Portugal Abstract The use of DBS for the analysis of doping substances has been increasing in anti-doping field. For that reason, the present study aims to understand whether capillary blood in DBS is a viable complement [1] for the detection of EPO and ERAs. This study involved 30 volunteers, from whom capillary blood and venous blood samples were taken. Urine and blood samples were collected from 4 volunteers. DBS samples were immunopurified (Gel Purification Kit – MAIIA nº 1430) prior to sodium N-lauroysarcosinate polyacrylamide gel electrophoresis (SAR-PAGE) and Western Blot using the biotinylated monoclonal mouse anti-human EPO clone AE7A5 (R&D Systems BAM2871). Limits of detection (LODs) were determined for the main ERAs: BRP (3.15 mUI/mL), NESP (3.15 pg/mL), EPO-Fc (31 pg/mL) and CERA (31.3 pg/mL). The comparison study of 6 different absorbent sample supports was also carried out to assess EPO and ERAs recovery. DBS Collection Card for erythropoietin analysis from MAIIA proved to be the support that shows best results for both endogenous EPO and spiked ERAs. Stability tests performed on DBS, serum and urine samples, stored at different temperatures (room temperature (RT), 4 °C and ‑20 °C showed promising results for DBS. DBS samples were re-analyzed 1 year after collection and all ERAs were present. This study reinforces the utility of DBS as a possible complementary biological matrix for the detection of EPO and ERAs especially when long transport times and limited storage capacities are expected. Introduction Requirements for the analysis of EPO and ERAs are described, in TD2022EPO, for urine and serum/plasma [2]. WADA published, in 2021, a specific document for the analysis of DBS, as a result of its increasing applicability, outlined in the most recent Testing Figures Report [3]. Since it is currently possible to perform capillary blood analysis by DBS for ERAs detection, it has become essential to carry out more studies to understand if DBS is a viable complement matrix [1] for the detection of EPO and ERAs. Several parameters were tested, being the stability study and comparison of different absorbent sample supports the most prominent. Useful information was gathered for collection, storage and stability time and temperatures. Experimental Subjects Capillary finger blood and venous blood samples (EDTA tube) were collected from 30 healthy subjects MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0102 and applied on the 903 Protein Saver card (air dried for at least 1h). From 4 of the 30 volunteers, urine and serum were also collected (samples used on the stability test). Questionnaire 3 questions related to pain level and collection preference among the 3 matrices: urine, venous or capillary blood were asked after the collections. Procedure 60 µL of capillary blood on DBS were immunopurified using Gel Purification Kit – MAIIA nº1430. Retentate was concentrated in Ultracell YM-30 tubes prior to sodium N-lauroysarcosinate polyacrylamide gel electrophoresis (SAR-PAGE) and Western Blot using the biotinylated monoclonal mouse anti-human EPO clone AE7A5 (R&D Systems BAM2871) and Streptavidin-HRP (Biospa) using Blotcycler during about 19 hours. Detection was carried out using the LAS-4000 camera (Fujifilm) and the images were processed using the Gasepo software. For urine, 10 mL was immunopurified using StemCell ELISA and the retentate was concentrated in Amicon Ultra-0.5 (30 K). For serum, 500 µL were immunopurified using EPO Purification Kit - MAIIA nº1390 and for both matrices the procedure was the same as described above for DBS. Selectivity and Limits of Detection (LODs) 60 µL of capillary blood from 30 volunteers were analyzed for selectivity and seven different concentrations of the 4 main ERAs (BRP, NESP, EPO-Fc and CERA) were tested to establish the LOD. Comparison of different absorbent sample supports Comparison of 6 brands of filter paper/DBS/Volumetric Absorptive Microsampling (VAMS) to understand the impact on recovery of bEPO and main ERAs. Filter paper: Whatman #1 and Macherey-Nagel DBS: 903 Protein Saver Card (Whatman), DBS Collection Card for EPO analysis (MAIIA) and Microsampling HemaXis DB10 Kits (DBS Systems SA) VAMS: Neoteryx Long-term stability study Carried out with capillary blood in DBS, serum and urine samples, stored at different temperatures: room temperature (RT), 4 °C and -20 °C and analyzed after 1, 2 and 3 months. DBS were also re-analyzed 1 year after collection. Results and Discussion Questionnaire Urine is the 1 option by 73.3% volunteers and capillary blood is the 2 most chosen option by 63.3%. On a pain scale of 1 to 10, the average pain rating for venous blood collection was 3.8 (mild-to-moderate pain), while for capillary blood was 2.3 (no pain-mild). Selectivity and limits of detection (LODs) Currently, the requirements for the analysis of EPO and ERAs in anti-doping laboratories are only st nd MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0103 described for urine and serum/plasma. It was possible to observe all bEPO bands with adequate intensities in the 30 samples analyzed with 60 µL. Compared to the MRPL for serum/plasma defined in TD2022EPO, values below 50% MRPL were achieved for rEPO (BRP) and NESP, and values around 125% MRPL were detected for CERA and EPO-Fc, resulting in limits higher than those defined for serum/plasma (Table 1). Table 1. Estimated limits of detection (LODs) for main ERAs in DBS Comparison of different absorbent sample supports Figure 1 shows visible bEPO on all supports with minimal differences in relative band volumes. The results showed that for filter paper #1 (Whatman) and DBS Collection Card for EPO (MAIIA) more intense bands were obtained when compared to other supports (Figure 1). Figure 1. Comparison of 6 different absorbent sample supports Long-term stability study After 1 month, degradation can be observed in serum and urine samples at all temperatures, although in the DBS (Figure 2) promising results were obtained as it is the only one that allows the preservation of all ERAs after 2 months at all storage temperatures analyzed. Similar results can be observed after 3 months of storage. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0104 Figure 3 shows DBS samples analyzed after 12 months demonstrating that some steps allowed to optimize the extraction of ERAs. After this period, for the 3 temperatures studied, samples showed greater intensity when stored at -20 °C. Figure 2. Stability study after 2 months storage at room temperature, 4 °C and ?20 °C of capillary blood in DBS, serum and urine spiked with the main ERAs at the same concentration MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0105 Figure 3. Stability study after 12 months storage at room temperature, 4 °C and -20 °C of capillary blood in DBS spiked with the main ERAs Conclusions According to the LODs obtained, an optimization of the method should be made to decrease the values for CERA and EPO-Fc, upon a possible scenario of the establishment os specific MRPL for capillary blood on DBS in TDEPO. The analysis of the three matrices stored at different temperatures (RT, 4 °C, ‑20 °C) after 3 months, allowed to demonstrate the ability of preservation of all ERAs in DBS, even after 12 months, when compared to serum and urine. The DBS Collection Card for EPO analysis (MAIIA) proved to be the one with the best results. This study confirms the usefulness of DBS as a possible complementary biological matrix for the detection of EPO and ERAs in doping control analysis. References 1. Heiland CE, Ericsson M, Pohanka A, Ekström L, Marchand A. (2022) Optimizing detection of erythropoietin receptor agonists from dried blood spots for anti-doping application. Drug Test Anal. Aug;14(8):1377-1386. 2. World Anti-Doping Agency. Technical Document. TD2022EPO - Harmonization of analysis and reporting of Erythropoietin (EPO) and other EPO-Receptor Agonists (ERAs) by polyacrylamide gel electrophoretic (PAGE) analytical methods (2022); https://www.wada-ama.org/sites/default/files/2023- 02/td2022epo_v1.0_final_eng.pdf (acess date 31.08.2023) 3. World Anti-Doping Agency. Testing Figures Report (2021); https://www.wada- ama.org/sites/default/files/2023-01/2021_anti-doping_testing_figures_en.pdf (acess date 31.08.2023) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0106 Stojanovic B, Pecher D, Geisendorfer T, Tsivou M, Gmeiner G Dried blood spot microsampling as a suitable tool to monitor diuretics misuse in sport Doping Control Laboratory, Seibersdorf Labor GmbH, Seibersdorf, Austria Abstract In recent years, the implementation of dried blood spot (DBS) microsampling increased significantly in the field of antidoping. This is supported by the World Antidoping Agency (WADA) TD2023DBS [1] as well as numerous publications [2,3]. However, the high number of substances in the WADA Prohibited List [4], their different physico-chemical properties, the small sampling volume of available devices as well as the complex matrix represent a challenge when developing comprehensive analytical methods for DBS samples. In the present study, the suitability of non-volumetric (DBS cards) and volumetric dried blood spotting devices (TASSO and volumetric absorptive microsampling (VAMS)) for selected substances from S5. class of the WADA Prohibited List was investigated. For this purpose, a sensitive liquid chromatography high resolution mass spectrometry (LC-HRMS) method was developed, validated in accordance with the current International Standard for Laboratories (ISL) [5] and applied to real sample analysis. As proof-of- concept, samples obtained from 2 healthy volunteers after administration of a single dose of a two- component medication (amiloride 2.5 mg/chlortalidone 25 mg) were used. Amiloride was identified in samples collected up to 24 h after administration, while its presence in the 48 h samples was confirmed in TASSO and VAMS devices only. Chlortalidone was identified in all collected samples. Consequently, dried blood microsampling is proved to be a suitable tool for the monitoring of diuretic misuse in sport. Introduction DBS microsampling is a complementary technique to traditional sample collection methods, which is recently gaining popularity and significance in the doping community [2,3,6]. The well-known benefits of a small amount of blood dried on a collection device forces the development of new methods. However, to perform the two-step protocol in doping analysis – initial testing procedure (ITP) and confirmation procedure (CP) – using one device can be challenging. Therefore, multi-component CP for substance groups such as diuretics may be a suitable strategy to cope with this challenge. In the current study, a protocol for multi-component LC-HRMS analysis for selected diuretics in 3 types of DBS collection devices was developed and applied to samples collected after administration of selected diuretics (amiloride and chlortalidone). Experimental Sample preparation for ITP and CP Fortified blood (20 µL) was spotted onto the DBS cards (FTA DMPK cards, Merck, Germany), VAMS® MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0107 (20 mL Mitra Clamshell, Neoteryx, The Netherlands) and TASSO-M20 (Tasso, Inc. USA) and allowed to dry for minimum 2 h. A 6 mm punch from DBS cards was taken from the center of the spot. For ITP: Substances were extracted from the spot using the extraction solvent - mixture of methanol- acetonitrile (4:1 v/v; 2 mL) containing ISTD. The samples were shaken in a water bath (50 °C) for 30 minutes and centrifuged at 2100 rpm for 5 minutes. Afterwards, 5 µL of 3 M HCl was added. The solvent was transferred into a glass test tube and evaporated. Samples were reconstituted in 100 µL of a mixture of methanol-water (3:7 v/v) and injected into the LC/MS/MS system. For CP: Methanol-acetonitrile (1:1 v/v;1 mL) containing ISTD was used as extraction solvent. The extraction protocol was the same as for ITP but only without adding 3 M HCl. Finally, after reconstitution in a mixture of methanol-water (3:7 v/v), samples were injected into the LC/HRMS system. Chromatographic conditions are presented in Table 1. Table 1. Chromatographic conditions Administration study As proof-of-concept, samples obtained from 2 healthy volunteers (1 male, 1 female) after oral administration of a single dose of a two-component medication (amiloride 2.5 mg/chlortalidone 25 mg) were used. Samples were collected at 0, 2, 4, 6, 8, 10, 12, 24, and 48 h after administration, dried for a minimum of 2 h at room temperature, and stored at 4 °C until analysis. Samples were initially analyzed using the ITP for DBS analysis. Subsequently, only the samples in which the administered diuretics were detected, were subjected to the CP analysis. Results and Discussion In the current study, an LC-HRMS method suitable for the confirmation of 20 substances from substance group S5 of the WADA Prohibited List was developed. The method was successfully validated for all 3 tested microsampling devices (DBS, VAMS, and TASSO). The method proved to be selective, robust, and no carry over was observed. The obtained limits of identification (LOI) of this method for each device are presented in Table 2. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0108 Table 2. Performance parameters of the developed LC/HRMS confirmation method In this administration study, amiloride was detected in samples collected up to 24 h post administration, while its presence in the 48 h sample was confirmed in the TASSO and VAMS devices only. Chlortalidone was identified in all collected samples. The estimated concentrations of amiloride and chlortalidone obtained when using the TASSO device are depicted in Table 3. The c was obtained 4 h post administration for amiloride and 4-10 h post administration for chlortalidone. m a x MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0109 Table 3. Estimated concentrations 0-48 h after administration for amiloride (top) and chlortalidone (bottom) The results obtained for amiloride are comparable to the already published data [7]. Chlorthalidone is concentrated in erythrocytes which resulted in a concentration in blood 7-10 times higher compared to plasma as reported in a previous study [8]. Data gathered in the present study could assist in definition of the minimal required performance level (MRPL) for diuretics in DBS, but an administration study with more volunteers should also be considered. Conclusions Dried blood microsampling proved to be a suitable tool for the monitoring of diuretics misuse in sport. The capability of non-volumetric and volumetric devices for selected diuretic analysis was comparable and the obtained results have shown good compliance. Results obtained for samples collected in an administration study confirmed the applicability of the tested devices for the analysis of diuretics, but also confirmed that the developed and validated multi-component LC-HRMS confirmation method is fit for purpose. References 1. World Antidoping Agency. DRIED BLOOD SPOTS (DBS) FOR DOPING CONTROL TD2023DBS | World Anti- Doping Agency (wada-ama.org) (access date 27.01.2023) 2. Thevis M, Walpurgis K, Thomas A. (2023) DropWise: current role and future perspectives of dried blood spots (DBS), blood microsampling, and their analysis in sports drug testing. Crit Rev Clin Lab Sci. 60 (1) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0110 https://doi.org/10.1080/10408363.2022.2103085 3. Mazzarino M, Di Costanyo L, Comunita F, Stacchini C, Torre X, Botre F. (2022) UHPLC−HRMS Method for the Simultaneous Screening of 235 Drugs in Capillary Blood for Doping Control Purpose: Comparative Evaluation of Volumetric and Non-volumetric Dried Blood Spotting Devices. ACS Omega 7, 31845-31868. 4. World Antidoping Agency. The 2023 Prohibited list. International Standard, 2023 Prohibited List | World Anti-Doping Agency (wada-ama.org) (access date 27.01.2023) 5. World Antidoping Agency. International Standard for Laboratories 2021, https://www.wada- ama.org/sites/default/files/resurces/files/isl_2021.pdf (access date 14.07.2022) 6. Garzinsky A-M, Thomas A, Guddat S, Görgens C, Dib J, Thevis M. (2023) Dried blood spots for doping controls – Development of a comprehensive initial testing procedure with fully automated sample preparation. Biomed Chrom. DOI: 10.1002/bmc.5633 7. Sun Q, Sever P. (2020) Amiloride: a review Journal of the Renin-Angiotensin-Aldosterone System (1-9) doi.org/10.1177/1470320320975893 8. Carter BL, Ernst ME, Cohen JD (2004) Hydrochlorothiazide Versus Chlorthalidone Hypertension. 43:4–9 DOI: 10.1161/01.HYP.0000103632.19915.0E MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0111 Yan X, Yan S, Chang W, Zhang L, Wang Z, Yang S A comparative study of DFT calculated and experimental UV/Visible spectra for the E/Z testosterone ester oximes at trace levels Beijing Anti-Doping Laboratory, Beijing Sport University, Beijing, People's Republic of China Abstract Dried blood spot (DBS) is a new alternative matrix used in 2022 Beijing Winter Olympics and Paralympics. It can realize the unequivocally distinguish the exogenous testosterone preparations without the aid of GC-C-IRMS confirmation. The detective method used methoxylamine reacting with the testosterone undecanoate, which formed the E and Z isomers. However, the absolute configurations of the oxime products have not been elucidated. This paper used a density function theory (DFT) calculation method to get the theoretical UV spectra data. The basis set was B3LYP, 6-31G (d,p) for the geometry optimizations. The Polarizable Continuum Model (PCM) was used. The actual UV spectra were determined by HPLC with DAD detector. The wavelength was set from 190 nm to 400 nm. By comparison the results, the E or Z configuration of testosterone undecanoate oxime can be deduced at nanogram levels (ng/mL). Introduction The prohibition history of testosterone is dated back to 1976 [1]. Dried blood spot (DBS) is recently a new alternative matrix used in anti-doping analysis [2-4]. It can distinguish exogenous testosterone preparations without the aid of GC-C-IRMS. Thus, it is recognized as a powerful technique introduced by WADA and played an important role in the 2020 Tokyo Olympics [5], 2022 Beijing Winter Olympics and Paralympics [6]. The method used methoxylamine reagent reacting with the eight testosterone esters, which all formed the E and Z isomers. However, the absolute configurations of the oxime products have not been elucidated. This paper focuses on using a density function theory (DFT) approach combined with experimental UV visible spectra to tentatively solve this problem at nanogram levels (ng/mL). Experimental Reagents and Materials Testosterone acetate (R1), testosterone propionate (R2), testosterone benzoate (R3), testosterone phenylpropionate (R4), testosterone cypionate (R5), testosterone enanthate (R6), testosterone decanoate (R7) and testosterone undecanoate (R8) are purchased from Sigma-Aldrich. The chemical structures are shown in Figure 1. Methanol, tert-butyl methyl ether and formic acid are HPLC grade, and the D3-testosterone is purchased from Sigma-Aldrich. Ammonium formate is purchased from Fluca Inc. Sodium bicarbonate, sodium carbonate and sodium hydroxide were purchased from Sinopharm Reagent Co., Ltd. Methoxylamine is purchased from Sigma-Aldrich. DMPK-C filter card was purchased from GE Healthcare company (USA). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0112 Figure 1. The chemical structures of the testosterone esters: testosterone acetate (R1), testosterone propionate (R2), testosterone benzoate (R3), testosterone phenylpropionate (R4), testosterone cypionate (R5), testosterone enanthate (R6), testosterone decanoate (R7) and testosterone undecanoate (R8) Sample preparation A mixture of the eight testosterone esters are diluted with methanol at a concentrations of 2.5 ng/μL. 50 μL of the standard solution was aliquoted, and 100 μL 10 mM methoxylamine methanol solution was added. The derivatization was conducted in capped glass tubes heated for 30 mins at 80 °C. After centrifugation at 4000 rpm for 3 mins, the residues were transferred to vials for injection. A volume of 10 μL was injected. Mass spectrometry and HPLC-UV spectrum For DBS samples, an ESI-LC-HRMS analysis was performed on a Vanquish Q Exactive HF orbitrap mass spectrometer (Thermofisher, Bremen, Germany). The UV-visible spectrum was determined on an Agilent 1200 HPLC (Agilent, USA). The conditions and parameters are listed in Table 1. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0113 Table 1. Instruments parameters used in Q Exactive HRMS and Agilent 1200 HPLC DFT theoretical calculation The theoretical calculation was performed using Gaussian 16 software (Gaussian Inc, Wallingford, CT, USA). The geometries of E/Z testosterone oxime isomers’ ground and excited states, excitation energy and UV absorption spectra were calculated by using the DFT functional, B3LYP, 6-31G (d,p) basis set [7- 8]. For the atoms in geometry optimizations, the Polarizable Continuum Model (PCM) was used [9]. Vertical excitation energy calculation was carried out by using time-dependent density functional theory (TD-DFT). Results and Discussion The testosterone oxime isomers have characterizations of double peaks in the extraction chromatograph by mass spectrometry. After the process of heating at 80 °C, one low abundance and one high abundance peak with 1:3 (peak area ratio) were observed. The E/Z isomers have the same mass spectra and ion fragments for each testosterone ester oxime. There is no difference observed from the mass spectra. Main fragment ions are m/z of 126.0913 and 138.0913, with mass accuracy error less than 3 ppm. The mass spectrum of testosterone undecanoate is shown in Figure 2. The UV-HPLC is improved by using acetonitrile as the B mobile phase, and a baseline separation is achieved, which is better than methanol as B phase. For testosterone undecanoate oxime isomers, the MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0114 retention times are 19.339 and 19.866 min, respectively. The UV spectrum of the peaks showed the absorption wavelength at 258 nm for the peak RT = 19.339, and 248 nm for the RT = 19.866, respectively (Figure 3). The theoretical calculation of testosterone undecanoate E isomer shows that the first excitation wavelength is 252.2 nm, while for the Z isomer it is 258 nm. The differences or errors are 4.2 nm and 0.4 nm. Therefore, the E and Z isomers are tentatively identified by comparing the theoretical calculations with the actual UV spectrum determinations. Figure 2. Mass spectra of testosterone undecanoate oxime: mass spectrum of the peak at RT = 12.66 min (top); mass spectrum of the peak at RT = 12.79 min (bottom) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0115 Figure 3. UV-HPLC analysis of the testosterone undecanoate oxime E/Z isomers. The upper chromatograph shows the UV spectra of the low abundance peak at RT = 19.33 min Conclusions The E or Z isomer of testosterone undecanoate oxime can be separated by HPLC and the UV spectrum determined. A DFT calculation using each previously determined isomer can give the theoretical results of the UV-Vis spectrum. And the E or Z isomer can be assigned based on these spectrometric data. Therefore, this method can tentatively distinguish betrween the E or Z isomers of the oxime products. References 1. World Anti-Doping Agency. The 2023 Prohibited List. International Standard, Montreal (2023) https://www.wada-ama.org/sites/default/files/2023-05/2023list_en_final_9_september_2022.pdf 2. Thomas A, Geyer H, Guddat S, et al. (2011) Dried blood spots (DBS) for doping control analysis. Drug Test Anal. 3(11-12), 806-813 3. Tretzel L, Thomas A, Geyer H, et al. (2014) Use of dried blood spots in doping control analysis of anabolic steroid esters. J Pharm Biomed Anal. 96(5), 21-30 4. Forsdahl G, Erceg D, Geisendorfer T, et al. (2015) Detection of testosterone esters in blood. Drug Test Anal. 7(11-12), 983-989 5. Okano M, Ikekita A, Sato M, et al.(2022) Doping control analyses during the Tokyo 2020 Olympic and Paralympic Games. Drug Test Anal. 14(11-12), 1836-1852 6. Wang Y, Zhang L, Xing Y. (2022) Operation of the anti-doping laboratory for the Beijing 2022 Olympic and Paralympic Winter Games. Drug Test Anal. 14(11-12), 1853-1863 7. Tirado-Rives J, Jorgensen W. (2008) Performance of B3LYP density functional methods for a large set of organic molecules. J Chem Theory Comput. 4(2), 297-306 8. Frisch M, Trucks G, Schlegel H, et al. Gaussian 16, Gaussian, Inc., Wallingford CT, 2016. 9. Cossi M, Barone V, Cammi R, Tomasi J. (1996) Ab initio study of solvated molecules: a new implementation of the polarizable continuum model. Chemical Physics Letters. 255(4-6), 327-335 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0116 Chang W, Yan X, Yan S, Liu Y, Yang S Pilot study of small peptide analysis in dried blood spots using automated SPE and UPLC-HRMS without DMSO Beijing Anti-Doping Laboratory, Beijing Sport University, Beijing, People's Republic of China Abstract Small peptides are banned by WADA in sports because they can either increase the performance or be used as masking agents. Compared to the mature methods used for small peptide analysis in human urine, the detection protocols for small peptides in dried blood spots (DBS) have not yet been carried out in many anti-doping laboratories. In this pilot study, an optimized automated SPE pretreatment and LC- HRMS method is presented to detect 48 types of small peptides in DBS without the assistance of DMSO. Validation was performed including selectivity, LOD, extraction stability and carryover. The data showed that the method herein is selective for small peptides without obvious interference at the retention times of target analytes. All peptide analytes could be detected at a concentration of 20 ng/mL. In addition, carryover was not observed at a concentration of 80 ng/mL, and all small peptide analytes were detectable at least for up to 72 h in the autosampler. It is believed that the method has the potential to reach LODs below 20 ng/mL after future formal evaluation. It could be deduced that, even without DMSO enhancing the ionization efficiency, this method can still be used to detect small peptides in DBS samples, which is helpful to further realize the simultaneous detection of small peptides and small molecular compounds in a single injection. Introduction While the detection methods for small peptides in human urine are well performed in routine analysis [1- 4], the mature protocols for the dried blood spots (DBS) detection have not carried out in many anti- doping laboratories. It is noted that several clinical trials have investigated the administration of small peptides, and these studies have reported peak concentrations of certain peptide analytes in blood samples exceeding 20 ng/mL [5-6]. Thus, Lange et. al reported a promising method to screen 46 small peptide analytes with the assistance of DMSO, and the LODs range between 0.5 and 20 ng/mL [7]. However, DMSO will cause adverse influence when small molecular compounds are detected together with peptide analytes. This pilot work thus presents an optimized automated SPE pretreatment and LC- HRMS method to detect 48 types of small peptides in DBS without the aid of DMSO, which is likely to provide prospective LOD levels below 20 ng/mL. Experimental Sample preparation Stock solutions of small peptide analytes and their mixed solution of 1 mg/mL were prepared in 1% formic acid in acetonitrile/water (v/v= 1/1) mixture in low-binding microtubes and stored in the freezer. A MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0117 bovine insulin solution of 50 μg/mL was prepared with the above-mentioned solvent mixture used to prepare the stock solutions, and a working solution of the peptide analytes at 0.1 ng/μL was prepared in the bovine insulin solution. A shell device developed by CHINADA was used to obtain triplicate 20 μL spots on filter paper once (Figure 1)[8]. To check LOD levels and extraction stability for the peptide analytes, the pre-extraction DBS samples were prepared by adding 4 μL of the working solution onto the center of a DBS. In addition, 16 μL of the working solution was added onto the center of the DBS to check carryover. Figure 1. DBS device developed by CHINADA (left); triplicate 20 μL spots are collected from a drop of fingertip blood (right) Sample extraction Samples were extracted by adding 0.4 mL of bovine insulin solution, then 0.8 mL water and 20 μL IS solution. After vortexing, 1.2 mL of phosphate buffer (0.2 M, pH 6) was added, followed by centrifuging at 10,000 rpm for 10 mins. Afterwards, 2 mL of the supernatant was loaded onto an Oasis WCX cartridge (30 mg, 1 cc) previously conditioned with 1 mL of methanol and 1 mL of water by Biotage Automated Sample Preparation System. The cartridge was washed with 1 mL water and 0.5 mL methanol and then analytes were eluted with 1 mL methanol containing 5% formic acid. After evaporation, the dry residues were dissolved with 100 μL of 2% acetic acid and injected into the UPLC-HRMS. UPLC-HRMS parameters are shown in Table 1, the ion transitions and the corresponding normalized collision energy (NCE) have been listed in Table 2. The injection volume was 10 μL. ® MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0118 Table 1. Dionex Ultimate 3000 LC/Q Exactive Plus mass spectrometer parameters for the detection of small peptide analytes Table 2. Retention times, monitored ion transitions and normalized collision energies for the detection of small peptide analytes Results and Discussion The analytical method of small peptides in DBS samples needs to eliminate most of the interferences in the matrix and get LODs at an appropriate level. As these are non-threshold substances, the developed method for small peptides in DBS was validated with regard to the following parameters: selectivity, limit MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0119 of detection (LOD), carryover and sample extract stability in the autosampler. For selectivity, ten blank DBS samples were analyzed to distinguish analytes from matrix compounds present in the DBS samples. There was no obvious interference at the retention times for each analyte, indicating that the presented method herein is selective for the small peptides. For the LODs, ten above-mentioned blank DBS samples, fortified with 4 μL of working solution of the small peptides at a level of 20 ng/mL, were analyzed. As shown in Figure 2, all of the peptide peaks at this level could exhibit a signal to noise ratio significantly higher than 3. The ion transitions and the corresponding normalized collision energies (NCE) for all analytes are listed in Table 2. Furthermore, carryover was evaluated with the consecutive analysis of a DBS sample fortified with peptide analytes at 80 ng/mL and two following blank DBS samples. No carryover was observed. In addition, ten above-mentioned urine samples for checking LODs were re-analyzed on the instrument autosampler after 72 h, and all small peptide analytes were still detectable. Conclusions This study presents a detection method for small peptides in DBS including sample extraction and UPLC- HRMS detection. All peptide analytes could be detected at concentrations of 20 ng/mL, and the method has the potential to reach LODs below 20 ng/mL after future formal evaluation. The performance of the presented method is comparable with that reported by Lange et.al [7], which is possibly caused by the optimized sample extraction to increase recoveries of the target analytes. The validation data show that our method can be used for the detection of small peptides in DBS samples without DMSO in the UPLC mobile phase, which is helpful to further realize the simultaneous detection of small peptides and small molecular compounds in one injection. Considering that DBS analysis is already a specific test, the com‐ bined analysis of small peptides and other low molecular substances is meaningful, as analyzing DBS for multiple prohibited substance categories would significantly increase the cost of testing. References 1. Görgens C, Guddat S, Thomas A, Thevis M. (2018) Recent improvements in sports drug testing concerning the initial testing for peptidic drugs ( < 2 kDa) - sample preparation, mass spectrometric detection, and data review. Drug Test Anal. 10(11-12), 1755-1760 2. Coppieters G, Deventer K, Van Eenoo P and Judák P. (2021) Combining direct urinary injection with automated filtration and nanoflow LC-MS for the confirmatory analysis of doping-relevant small peptide hormones. J Chromatogr B: Anal Technol Biomed Life Sci. 1179, 122842 3. Jing J, Tian T, Wang Y, Xu X, Shan Y. (2022) Multi-analyte screening of small peptides by alkaline pre- activated solid phase extraction coupled with liquid chromatography-high resolution mass spectrometry in doping controls. J Chromatogr A. 1676, 463272 4. Chang W, He G, Yan K, Wang Z, Zhang Y, Dong T, Liu Y, Zhang L, Hong L. (2021) Doping control analysis of small peptides in human urine using LC-HRMS with parallel reaction monitoring mode: screening and confirmation. Anal Methods.13(48), 5838-5850 5. Roumi M, Lenaerts V, Boutignon F, Wuthrich P, Deghenghi R, Bellemare M, Adam A, Ong H. (1995) Radioimmunoassay for hexarelin, a peptidic growth hormone secretagogue, and its pharmacokinetic studies. Peptides. 16(7), 1301-1306 6. Zdravkovic M, Olsen A, Christiansen T, Schulz R, Taub M, Thomsen M, Rasmussen M, Ilondo M. (2003) A clinical study investigating the pharmacokinetic interaction between NN703 (tabimorelin), a potential inhibitor of CYP3A4 activity, and midazolam, a CYP3A4 substrate. Eur J Clin Pharmacol. 58(10), 683-688. 7. Lange T, Thomas A, Walpurgis K, Thevis M. (2020) Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide and non-peptide doping agents by means of LC- HRMS. Anal Bioanal Chem. 412(15), 3765-3777 8. Wen H, Gao J, Wang X, Yu L. (2021) Application of dried blood spot sampling equipment and method in doping control. Chin. Sport Sci. 41(11), 30-37. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0120 Figure 2. Extracted ion chromatogram (EIC) of small peptide analytes in the DBS sample at a concentration of 20 ng/mL MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0121 Thomas A, Garzinsky A, Görgens C, Guddat S, Geyer H, Thevis M Carbonic anhydrase inhibitors brinzolamide and dorzolamide in dried blood spots Institute of Biochemistry, German Sport University, Cologne, Germany Abstract Dried blood spots as a possible sample matrix in doping control has been approved since 2022 and is becoming increasingly popular. DBS are currently applicable to the analysis of prohibited substances that are banned at all times and are not subject to any threshold or MRL. This is the case, for example, for diuretics and masking agents (S5 of the WADA Prohibited List), where the two carbonic anhydrase inhibitors brinzolamide (BA) and dorzolamide (DA) are also listed. With the exception of a local ophthalmic application, BA and DA are prohibited in sports. The retrospectivity achievable with DBS is usually lower compared to urine samples, because urine represents a quasi-integral of plasma concentrations. Thus, the detection window for drugs of low molecular mass is usually higher than in the respective blood samples. However, in the case of the two carbonic anhydrase inhibitors brinzolamide (BA) and dorzolamide (DA), this is exactly the opposite. Due to the rapid incorporation of these substances into erythrocytes (with an average lifetime of 120 days), BA and DA are detectable for much longer in whole blood samples (such as DBS) than in urine. A half-life of 24 weeks in whole blood has been described, so that a detectability of months, if not years, can be assumed. DA and BA are carbonic anhydrase inhibitors and often used by topical administration to treat glaucoma. Although locally applied, BA and DA will also enter the circulation via the blood-retinal-barrier and are enriched in the erythrocytes. Two post administration samples (DBS and urine, 2 and 8 hours after one drop of BA 10 mg/mL) yielded BA concentrations of approx. 10 resp. 100 ng/mL in urine and 19 resp. 17 µg/mL in the corresponding DBS samples. This phenomenon might explain a comparably high percentage of BA and DA observations in DBS. After one year of DBS doping control sampling BA and DA showed a significant prevalence most likely due to the prevalence of glaucoma in athletes. Introduction Dried blood spots as a possible sample matrix in doping control has been approved since 2022 and is becoming increasingly popular. DBS are currently applicable to the analysis of prohibited substances that are banned at all times and are not subject to any threshold or MRL [1]. This is the case, for example, for diuretics and masking agents (S5 of the WADA Prohibited List), where the two carbonic anhydrase inhibitors brinzolamide (BA) and dorzolamide (DA) are also listed (see Figure 1). With the exception of a local ophthalmic application, BA and DA are prohibited in sports. The retrospectivity achievable with DBS is usually lower compared to urine samples, because urine represents a quasi-integral of plasma concentrations. Thus, urine concentrations (and detcetion windows) for drugs of low molecular mass are usually higher than in the respective blood samples. However, in the case of the two carbonic anhydrase inhibitors brinzolamide (BA) and dorzolamide (DA), this is exactly the opposite. Due to the rapid MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0122 incorporation of these substances into erythrocytes (with an average lifetime of 120 days), BA and DA are detectable for much longer in whole blood samples (such as DBS) than in urine. A half-life of 24 weeks in whole blood has been described, so that a detectability of months, if not years, can be assumed [2]. DA and BA are carbonic anhydrase inhibitors and often used by topical administration to treat glaucoma (see Figure 2). Although locally applied, BA and DA will also enter the circulation via the blood-retinal-barrier and are enriched in the erythrocytes [3]. Figure 1. Structure of a) brinzolamide (BA) and b) dorzolamide (DA) Figure 2. Ocular drug administration following a) topical and b) intracameral administration Experimental Sample preparation For DBS sampling DMPK cellulose cards as well as TASSO devices were used and found to be appropriate. Both were loaded with 20 µL of capillary whole blood (venous EDTA blood for validation). The cards were extracted fully automated by means of the Gerstel DBS autosampler (Mülheim, Germany) following the protocol described elsewhere [4]. The TASSO samples were extracted following an adapted manual protocol with the nearly identical conditions. In brief, samples were fortified with ISTD mixture (stable isotope labelled clenbuterol, dexamethasone, hydrochlorothiazide, amphetamine) solution and extracted with a mixture of methanol/water (80/20). The extract is acidified with 5 µL of hydrochloric acid (0.3 N) prior to evaporation and reconstituted in acetonitrile/water (80/20) before injection into the LC-MS system. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0123 Post administration samples Two paired post administation samples (one male, one female) were collected after topical ophthalmic administration of one drop of BA (10 mg/mL, twice a day, as permanent glaucom treatment > 2 weeks). DBS and urine samples were collected 2 resp. 8 hours after the last administration. The urine samples were analysed according to the established direct urine injection with the validated LC-HRMS initial testing procedure. Here the urinary concentrations were estimated at approx. 13 ng/mL (after 2 h) resp. 107 ng/mL (after 8h). The corresponding DBS samples showed concentrations of 19 µg/mL (after 8 h) resp. 17 µg/mL (after 2 h). These findings strongly confirm the very long detection window of BA in DBS samples. Especially, considering the described half-life of approx. 24 weeks [2]. Results and Discussion The prevalence of glaucoma increases with age from 0% for babies to approximately 2% for patients with 45 years of age or older. Because DA and BA are common therapeutic drugs used to treat glaucoma, a comparatively high prevalence is also expected in samples from athletes accordingly. This is especially given in consideration of the expected long detectability in DBS samples. Direct differentiation between permitted topical- and prohibited systemic administration, is not enabled with the present method, because both will yield high whole blood levels with low (undetectable) urine concentrations. For BA it is described, that topic administration will produce lower levels of the known metabolite N-Desethyl-BA in the erythrocytes, but this phenomenon has to be confirmed with controlled administration samples yet [2]. Table 1 shows the validation results for the confirmation of BA and DA in dried blood spots according to the current requirements of the WADA. Table 1. Validation results (ITP/CP non-threshold) Conclusions In 2022, the Cologne laboratory had two cases of confirmed BA samples with DBS. The estimated concentrations of BA were 5 resp. 9 ng/mL and the corresponding urine samples were negativ (no BA detectable). In addition, several other samples showed traces of BA and DA, which were not reported due to the low concentration of BA and DA (below the LOI of 5 ng/mL). Figure 3 shows the extracted ion chromatograms of the product ion experiments used for confirmatory analysis. Due to the prevalence of glaucoma in the population as well as in competing athletes and the long detection window, the analysis of DBS for BA and DA will yield a considerable high number of analytical findings. In comparison, the 2021 anti-doping testing figure report of the WADA shows 28 DA and 23 BA cases considering > 200,000 urine samples [5]. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0124 Figure 3. Extracted ion chromatograms from a) a blank DBS sample, b) a DBS from an athlete (AAF) and c) a DBS fortified at 5 ng/mL with BA References 1. WADA, TD2023 DBS, https://www.wada-ama.org/sites/default/files/2022-11/td2023dbs_cleanversion.pdf, 2023 2. Fachinformation Brinzolamide, https://www.ema.europa.eu/en/documents/product-information/azopt-epar- product-information_de.pdf, 2023 3. Suzuki G, Kunikane E, Shigemi W, Shinno K, Kozai S, Kurata M, Kawamura A. Ocular and systemic pharmacokinetics of brimonidine and brinzolamide after topical administration in rabbits: comparison between fixed-combination and single-drug formulations. Curr Eye Res. 2021 Mar;46(3):380-386. doi: 10.1080/02713683.2020.1800046. 4. Garzinsky AM, Thomas A, Guddat S, Görgens C, Dib J, Thevis M. Dried blood spots for doping controls- Development of a comprehensive initial testing procedure with fully automated sample preparation. Biomed Chromatogr. 2023 Aug;37(8):e5633. doi: 10.1002/bmc.5633. 5. WADA, Anti-Doping Testing Figure Report 2021, https://www.wada-ama.org/sites/default/files/2023- 01/2021_anti-doping_testing_figures_en.pdf Acknowledgements The study was carried out with support of the Manfred Donike Institute for Doping Analysis (Cologne, Germany) and the Federal Ministry of the Interior, Community and Building of the Federal Republic of Germany (Bonn, Germany). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0125 Görgens C , Geyer H , Guddat S , Machnik M , Schenk I , van de Bovenkamp A , Wagener F , Thevis M Trimetazidine: pilot study data illustrating approaches to evaluate a potential contamination scenario Institute of Biochemistry, Center of Preventive Doping Research, German Sport University, Cologne, Germany ; Amsterdam Universitair Medische Centra, Amsterdam, Netherlands ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract Triggered by an AAF for trimetazidine, allegedly caused by a contamination scenario, several pilot studies were conducted to provide data for RMAs to evaluate such scenarios in the future. The pilot studies consisted of the LC/HRMS analyses of hair and oral fluid samples collected from patients on a therapeutic trimetazidine regimen as well as urine samples of a volunteer, who orally administered a microdose (5 µg) of trimetazidine. The obtained data of the pilot studies provide first insights regarding concentrations of trimetazidine in oral fluid and hair samples of patients as well as urinary concentrations and detection windows after microdosing of trimetazidine. Introduction In connection with an AAF for trimetazidine in 2022, it was published that the AAF was presumably attributable to a contamination scenario. Trace amounts of the substance may have been unknowingly ingested by the athlete, sharing a glass of water with a person who used trimetazidine therapeutically. The approximate concentration of trimetazidine in the doping control urine sample, resulting in the AAF, was reported to be 2.1 ng/mL [1]. Analytical data supporting the evaluation of such scenarios are scarce, and pilot studies were conducted to answer the following questions: 1. At which magnitude do concentrations of trimetazidine exist in the oral fluid (OF) of patients receiving (repeated) therapeutic doses of trimetazidine? 2. Which urinary concentrations of trimetazidine are obtained after the administration of a microdose (simulating a contamination) of trimetazidine (5 µg)? 3. Is it possible to detect trimetazidine in hair of patients receiving therapeutic doses of trimetazidine? Experimental Hair samples (strand of hair, with a diameter of ca. 0.5 cm from the back of head) and OF samples (2 mL) were collected from two patients (54 and 52 years old) who had been taking trimetazidine at a daily dose of 2 x 35 mg/day of slow-release trimetazidine dihydrochloride for 11 months. The samples were collected 2 hours after the last trimetazidine application. Urine samples were obtained from a male volunteer (49 years) who orally administered 5 µg of (free) trimetazidine. Therefore, trimetazidine dihydrochloride was dissolved in methanol and an aliquot corresponding to 5 µg of dissociated 1 1 1 1 1 2 1 1,3 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0126 trimetazidine was evaporated and consumed after solution in vodka. Urine samples were collected before and till 24 hours after the drug administration. The urine and OF samples were analyzed with LC/HRMS for trimetazidine using an established routine doping control analytical method reported elsewhere [2]. The validated LOD was 2.1 ng/mL. The trimetazidine concentration in the urine samples were not corrected for specific gravity. The hair samples were pulverized, 50 mg of each sample were extracted according to a method of Nguyen [3], and the extracts were analyzed by LC/HRMS with the same method as the urine and OF samples. Results and Discussion Oral fluid samples: in the OF samples of the two patients, trimetazidine was detected at roughly estimated levels of 540 ng/mL and 1130 ng/mL, respectively. Hair samples: in the hair samples of both patients trimetazidine was identified. The roughly estimated concentrations of trimetazidine were 880 pg/mg and 350 pg/mg, respectively. It is not known, if a single application of trimetazidine in doses in the low microgram range can be detected in hair. Urine samples: after the oral application of 5 µg of trimetazidine, a maximum value of trimetazidine of ca. 4.7 ng/mL was obtained after 13.5 h (concentrations not corrected for specific gravity (SG)). After 19.5 h trimetazidine could no longer be detected (Figure 1). Figure 1. Urinary concentrations after oral administration of 5 µg trimetazidine (conc. not corrected for SG) Conclusions These pilot study data provide first insights into drug deposition and elimination profiles, which might assist RMAs in future case evaluations concerning trimetazidine findings (if hair samples are available and collected in a timely manner). Follow-up investigations considering e.g. hair segment analyses and/or additional microdosings simulating exposure appear warranted. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0127 References 1. CAS award OG 22/08 -CAS OG 22/09 -CAS OG 22/10. https://www.tas- cas.org/fileadmin/user_upload/OG_22_08-09-10_Arbitral_Award__publication_.pdf; access: 02.02.2023 2. Görgens, C, Guddat, S, Bosse, C, Knoop, A, Geyer, H, Thevis, M. Implementation of the HIF activator IOX-2 in routine doping controls – Pilot study data. Drug Test Anal. 2020; 12: 1614– 1619. 3. Nguyen V.M.C. (2020): Entwicklung und Validierung eines Nachweisverfahrens für ausgewählte Dopingsubstanzen im Pferdehaar unter Berücksichtigung verschiedener Einflussfaktoren wie Farbe, Morphologie und Vorbehandlung der Haare. Köln, Deutsche Sporthochschule Köln, Institut für Biochemie, 1. Staatsexamen Lebensmittelchemie Acknowledgements The authors acknowledge support from the Manfred Donike Institute for Doping Analysis (Cologne, Germany) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0128 Radu M , Catana D , Berghes B , Cristea C , Toboc A , Stan C Development and validation of a chromatographic separation method for betamethasone and dexamethasone LC-MS, Romanian Doping Control Laboratory, Bucharest, Romania ; Romanian Doping Control Laboratory, Bucharest, Romania Abstract Betamethasone and dexamethasone are prohibited in sports, in competition only, and are included in the World Anti-Doping Agency (WADA) Prohibited List, class S.9 Glucocorticoids. The two compounds are epimers, the only difference between them being the orientation of the methyl group at carbon 16. This results in similar spectra and a very difficult chromatographic separation. Our study proposes a method for the identification of betamethasone and dexamethasone by liquid chromatography in tandem with mass spectrometry (LC-MS/MS) technique after investigating several chromatographic conditions and using a C8 reversed phase chromatographic column for separation. The method was validated for specificity, limit of identification, carry-over and robustness. The results indicat, that this method is specific and ensures reliable analytical results without interferences. Therefore, it is suitable for the identification of betamethasone and dexamethasone in accordance with the current WADA Technical Documents TD2023IDCR and TD2022MRPL. Introduction Betamethasone and dexamethasone are included in the World Anti-Doping Agency (WADA)’s Prohibited List in class S9. Glucocorticoids and are prohibited in competition only [1]. Due to their structural similarity, their MS-MS spectra have the same fragments that differ only by abundance [2], and are difficult to separate by chromatography. Various studies regarding analysis of betamethasone and dexamethasone in pharmaceutical products [3,4] and biological samples [5,6] were performed, the best results being obtained using chiral columns [7]. This study proposes a method for the separation and identification of betamethasone and dexamethasone in urine samples using a C8 reversed phase chromatographic column and detection by tandem mass spectrometry. Experimental Betamethasone and dexamethasone were purchased from LGC Standards (Wesel, Germany). Tert-butyl methyl ether, acetonitrile potassium bicarbonate and potassium carbonate were purchased from Merck (Redox Romania). Ultrapure water was obtain using a Milli-Q Q-POD equipment from Merck. Sample preparation method is presented in Figure 1. 1 2 2 2 2 2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0129 Figure 1. Sample preparation scheme Equipment used was AB SCIEX 6500 / EXION LC AD. The chromatographic column was Zorbax Eclipse XDB-C8 (4.6 x 150 mm, 5 µm). The A solvent was 5 mM amonium formate, 1‰ formic acid in water, B solvent 5 mM amonium formate, 1‰ formic acid in 90% acetonitrile and 10% water. LC flow was 450 µL/min, injection volume 2 µL and the column was thermostated at 30 °C. The MS parameters were the following: acquisition mode MRM, polarity positive, curtain gas 35 psi, ionization voltage 5500 V, source temperature 550 °C, nebulizer gas 60 psi, auxiliary gas 60 psi, collision cell gas (CAD) 12. B gradient and MRM transitions are presented in Table 1. Table 1. LC program and MRM transitions The methods were investigated for matrix effects, limit of identification, carry-over, and robustness: Matrix effects: 10 negative control samples were injected and then the presence of interferences at the retention times of the analyte of interest was checked. + MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0130 Limit of identification (LOI): scalar dilutions in urine to obtain the lowest concentration for substances identification (S/N> 3). 10 negative urine samples from different sources were fortified with the standard betamethasone and dexamethasone, establishing that the identification limit has the value of 15 ng/mL and 30 ng/mL. Carry-over was evaluated with the consecutive injection of a sample fortified at 300 ng/mL and blank samples to investigate if there are any contamination. Robustness was evaluated by modifying column temperature, source temperature, flow, injection volume, TBME volume, reconstitution volume, hydrolysis time. Results and Discussion The following columns and gradients were tested (data not shown): Zorbax SB-C18 (2.1 x 50 mm, 5 µm) with the following gradient: flow 0.3 mL/min, 0 - 20 min 22% B, flow 0.50 mL: 21 - 26 min 70% B, and four minutes reequilibration at 22% B Poroshell 120 EC-C18 (3 x 50 mm, 2.7 µm) with the following gradient flow 0.3 mL/min, 0 - 2 min 0% B, 2 - 22 min 45% B, 22 - 25 min 45% - 100% B, 25 - 28 min 100% B, 28.1 - 31 min 0% B Zorbax Eclipse XDB-C18 (4.6 x 150 mm, 5 µm) and Zorbax Eclipse XDB-C8 (4.6 x 150 mm, 5 µm) with the following gradients: flow 0.5 mL/min gradient 1: 0 - 5 min 25% B, 5.5 - 12 min 32% B, 12.5 - 17 min 48% B, 17.5 - 25 min 70% B then reequilibration for 5 min at 25% B flow 0.45 mL/min gradient 2: 0 - 5 min 25% B, 5.5 - 12 min 32% B, 12.5 - 17 min 48% B, 17.5 - 25 min 70% B then reequilibration for 5 min at 25% B flow 0.45 mL/min gradient 3: 0 - 5 min 20% B, 5.5 - 12 min 25% B, 12.5 - 17 min 40% B, 17.5 - 25 min 65% B then reequilibration for 5 min at 20% B flow 0.40 mL/min gradient 4: 0 - 23 min 0% B, 23.1 - 27 min 90% B, 27.1 - 32 min 0% B flow 0.35 mL/min gradient 5: 0 - 1 min 0% B, 1 - 23 min 0-70% B, 23 - 27 min 70-100% B, 27 - 29 min 100% B, then reequilibration for 5 min at 0% B (0.5 mL/min on reequilibration step) flow 0.40 mL/min gradient 6: 0 - 5 min 20% B, 5.5 - 12 min 30% B, 12.5 - 17 min 45% B, 17.5 - 25 65% B, then reequilibration for 5 min at 20% B (0.5 mL/min on reequilibration step) The best separation was achieved under the conditions presented in Table 1. Although baseline separation was not achieved, the method is capable to differentiate between beta‐ methasone and dexamethasone when are present in the urine sample simultaneously. The separation between betamethasone and dexamethasone proved to be sufficient to meet the WADA chromatographic identification criteria only for betamethasone when it is tested against a dexamethasone individual standard and vice versa. Chromatograms of a urine fortified with betamethasone and dexa​methasone and individual standards of betamethasone and dexa​methasone are presented in Figure 2. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0131 Figure 2. Chromatograms for A: negative urine sample; B: urine sample spiked with betamethasone and dexamethasone at 15 ng/mL; C: individual standards of betamethasone and dexamethasone in H O spiked at 30 ng/mL The results obtained during validation showed that the method is specific for identification of betamethasone and dexamethasone and no interference signals were present in blank samples. The limit of identification was established at 15 ng/mL and it is compliant with the WADA TD2022MRPL document. The method presents no significant carry-over and is robust for investigated parameters for both compounds. 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0132 Conclusions This study presents a method for the separation and identification of betamethasone and dexamethasone in urine samples using a reversed phase C8 chromatographic column and detection by tandem mass spectrometry. Even though baseline separation was not achieved, the retention time difference between the two compounds is enough to meet the WADA identification criteria, so that betamethasone can’t be misidentified as dexamethasone or vice versa. The method has a limit of identification of 15 ng/mL and is suitable for the identification of betamethasone and dexamethasone in doping control urine samples in compliance with the WADA technical documents TD2023IDCR and TD2022MRPL. References 1. World Anti-Doping Agency. The 2023 Prohibited List. https://www.wada-ama.org/en/resources/world-anti- doping-program/2023-prohibited-list#resource-download (access date 23.01.2023). 2. De Wasch K, De Brabander HF, Van de Wiele M, Vercammen J, Courtheyn D, (2001) Differentiation between dexamethasone and betamethasone in a mixture using multiple mass spectrometry. J. Chromatogr. A. 926, 79–86. 3. Xiao, KP, Xiong, Y, & Rustum, AM (2008) Quantitation of trace betamethasone or dexamethasone in dexamethasone or betamethasone active pharmaceutical ingredients by reversed-phase high-performance liquid chromatography. J. Chromatogr. Sci. 46(1), 15-22. 4. Arthur, KE, Wolff, JC, Carrier, DJ (2004) Analysis of betamethasone, dexamethasone and related compounds by liquid chromatography/electrospray mass spectrometry. RCM 18(6), 678-684. 5. Cirimele, V, Kintz, P, Dumestre, V, Goulle, JP, Ludes, B (2000) Identification of ten corticosteroids in human hair by liquid chromatography–ionspray mass spectrometry. Forensic Sci. Int. 107(1-3), 381-388. 6. Deventer, K, Delbeke, FT (2003) Validation of a screening method for corticosteroids in doping analysis by liquid chromatography/tandem mass spectrometry. RCM. 17(18), 2107-2114. 7. Karatt, TK., Sayed, R, Nalakath, J, Perwad, Z, Albert, PH, & Khader, KA (2018) Separation and identification of the epimeric doping agents–Dexamethasone and betamethasone in equine urine and plasma: A reversed phase chiral chromatographic approach. Steroids 140, 77-82. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0133 Wicka M , Konarski P , Drapala A , Kaliszewski P , Grucza K , Stanczyk D , Kwiatkowska D LC-MS/MS method for the detection of synthetic cathinones in urine Polish Anti-Doping Laboratory, Warsaw, Poland ; Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland Abstract Synthetic cathinones (SCs) are a broad group of chemicals, which have a very similar structure. Cathinones are derivatives of the parent compound cathinone, which is one of the active compounds found in the khat plant (Catha edulis). The cathinone structure is strictly related to amphetamine. However, synthetic cathinones can be much more potent than the drugs that they are intended to mimic, which increases the risk of overdose and death. In the WADA Prohibited List, SCs are classified in section S6 (stimulants) and are prohibited in competition only. The aim of the study was to develop an initial testing analytical method that would allow the detection of 25 cathinones in urine to be used for routine testing of athletes by means of ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The method was verified for selectivity, the limit of detection (LOD), recovery (RE), matrix effects (ME), and process efficiency (PE). The presented method allows the simultaneous detection and identification of 25 cathinones in urine by means of the LC-MS/MS approach and is fit-for-purpose for routine analysis of doping samples. Introduction Synthetic cathinones (SCs) are β-keto phenethylamine derivatives, which have the same core structure [1,2]. The cathinone structure is strictly related to amphetamine, with the difference being the presence of a keto group [2-5]. Cathinone derivatives can be synthesized by the addition of several substituents at different sites of the cathinone scaffold as represented in Figure 1. Synthetic cathinones are widely abused due to their hallucinogenic and stimulant effects. SCs are replacing cocaine and MDMA (3,4- methylenedioxymethamphetamine), which are much more expensive. However, synthetic cathinones can be much more potent than the drugs that they are intended to mimic, which increases the risk of overdose and death [2]. In the WADA Prohibited List, SCs are classified in section S6 (stimulants) and are prohibited in competition only [6]. The aim of the presented work was to develop and validate an LC- MS/MS method for the detection of 25 cathinones in urine to be used for routine testing of athletes. The described method is suitable for use on complex biological matrices. 1 1 1 1 1 1 1,2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0134 Figure 1. The general structure of cathinone derivatives Experimental Sample pre-treatment The sample preparation is a two-step procedure involving enzymatic deconjugation of glucuronides and then liquid-liquid extraction with 6 mL of methyl tert-butyl ether. The residue was reconstituted in 100 µL of mobile phase (acetonitrile/water, 1/1, v/v), transferred in a vial and 2 µL was injected into the LC- MS/MS system. Instrumental analysis Chromatographic separation was conducted by means of a Waters Acquity I-Class UPLC System liquid chromatography with BEH C18 column (1.7 µm, 100 mm x 2.1 mm, Waters). The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B), and the LC gradient was employed at a constant flow rate of 300 µL/min at 45 °C. MRMs of the studied substances were traced with a Xevo TQ-XS mass spectrometer equipped with an electrospray source. All analytes were investigated in the ESI mode. Desolvation gas flow was set at 800 L/h at 600 °C with ion source temperature at 150 °C. The capillary voltage was 1.0 kV (Table 1). Method validation The validation process was performed in accordance with the WADA technical document TD2023IDCR [7]. The method was verified for selectivity, limit of detection (LOD), retention time (RT), extraction recovery (RE), process efficiency (PE), and matrix effects (ME). + MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0135 Table 1. Selected tandem mass spectrometry transitions for analysis Results and Discussion The extraction recovery of the method showed that, with two exceptions (15.3% for 4-CEC and 28.3% for cathinone), the percentages of recovery ranged from 31.6% to 95.3%. The process efficiency was 5.3 % to 82.9%. The matrix effect for the analyzed compounds ranged from 18.2% to 96.5% (Table 2). Selectivity was assessed by analyzing 6 blank samples from different individuals (3 males and 3 females). Figure 2 shows an example chromatogram for the strongest MRM for each cathinone (for the final concentration of 10 ng/mL). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0136 Table 2. The value obtained from LOD, RT, RE, ME, and PE of cathinones in urine Figure 2. Typical MRM chromatograms of urine samples – blank urine and analyzed cathinone at a concentration of 10 ng/mL MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0137 Conclusions All compounds were successfully fragmented into ions . All the selected ionic transitions have proved to be specific. No interfering signals from the matrix were observed for any of the MRMs - high selectivity of the method. The method is suitable for use with complex biological matrices (% RSD 408.0 (CE 75 V) for Co-5-Br-PADAP (target, blue) and 665.1 > 362.1 (CE 65 V) for Co-5-Cl-PADAP (ISTD, yellow) Lastly, as sediment is often present in urine samples, it is important to know whether analyte losses can occur due to coprecipitation. This may be critical in cases where secondary aliquot is taken after urine had sufficient time to settle, or when sediment is intentionally removed prior to SPE. Here, 22 randomized urine samples containing visually different amounts of sediment were spiked with cobalt ions, mixed thoroughly, and refrigerated. After 72 h the samples (both spiked and non-spiked) were carefully resuspended and secondary aliquots were immediately taken for analysis. The remaining MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0141 volume was centrifuged and an aliquot of supernatant was processed as well. Using a single-point calibration, concentration of cobalt was found to be not significantly different between aliquots taken with and without sediment (Table 2). Table 2. Negative and spiked urine analyzed before and after centrifugation (n = 22) Conclusions The method presented herein can be suggested for use as an ITP in those laboratories which do not have the ICP-MS capability. Given the short LC run time ( < 3 min per sample), this method can easily be adapted to a 96-well plate format, if high productivity is desired. Although a reporting threshold has yet to be established by WADA, broader testing for cobalt is vital to the effectiveness of the global anti- doping program. References 1. Sobolevsky T, Ahrens B. (2021) Measurement of urinary cobalt as its complex with 2-(5-chloro-2- pyridylazo)-5-diethylaminophenol by liquid chromatography-tandem mass spectrometry for the purpose of anti-doping control. Drug Test Anal. 13(6), 1145-1157 2. Dos Santos VF, Carneiro GRA, Braz BF, Santelli RE, de Paula Machado S, Gualberto Pereira HM. (2023) Coordination chemistry applied to anti-doping analysis: Cobalt quantification as its diethyldithiocarbamate complex by liquid chromatography coupled to high resolution tandem mass spectrometry. Drug Test Anal. doi: 10.1002/dta.3571. Epub ahead of print MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0142 Liu L, Wang Z, Yan K, He G, Xing Y, Wu Q Doping control analysis of morphine in human urine by ultra- performance liquid chromatographic-electrospray ionization tandem mass spectrometry Beijing Sport University, Beijing Anti-Doping Laboratory, Beijing, People´s Republic of China Abstract Morphine is a narcotic analgesic commonly used in clinics, while prohibited in the time of sports competition. Morphine could be metabolized in the urine from codeine and ethylmorphine. Nowadays, the use of codeine and ethylmorphine is allowed, while the threshold for total morphine is 1 μg/mL, which is regulated by the latest valid WADA technical document (TD) on decision limits (DL). Based upon these conditions, an ultra-performance liquid chromatographic-electrospray ionization tandem mass spectrometry (UPLC–ESI-MS/MS) method for three prohibited morphine substances was developed and validated, including morphine, morphine-3β-D-glucuronide and morphine-6β-D-glucuronide in human urine in the concentration ranges between 100 and 1000 ng/mL. The quantitative bias caused by the pretreatment conditions and the different matrix was reduced by a straightforward sample preparation of the dilute and shoot method. The compounds were analyzed directly without the need for hydrolysis, solvent transfer, evaporation or reconstitution. Since codeine is not included in the prohibited list by WADA, it is important to determine whether a positive morphine finding is a result of codeine metabolism. Hence, the codeine and codeine-6β-D-glucuronide were monitored simultaneously in the single injection. The method proved to be specific, accurate and precise for linearity; the limit of quantification, intermediate precision, repeatability, matrix effects and uncertainty have been evaluated. The Beijing anti-doping laboratory has applied this method in routine tests and reported adverse analytical finding for many years. The relative comparison experiment results from WADA-external quality assessment scheme have been further discussed. Introduction Morphine is a narcotic analgesic commonly used in clinics, while prohibited in the time of sports competition. Morphine could be metabolize from codeine and ethylmorphine in urine [1]. Codeine is widely used as a milder analgesic and antitussive, whereas ethylmorphine is used to treat scleritis [2]. Nowadays, the use of codeine and ethylmorphine is permitted, and a concentration of morphine only above the relevant threshold (or decision limit) results in an AAF [3], if the criteria involving the consideration of codeine or ethylmorphine and corresponding metabolites are met [4]. Based upon these conditions, it is necessary to establish a simple and accurate quantitative method fot the simultaneous determination of the concentration of total morphine (M) including the parent and glucuronide metabolites (M3G & M6G) in human urine, which has been applied in routine testing and reported adverse analytical findings. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0143 Experimental 2.1 Instrumental analysis The UPLC-MS/MS system consisted of an Agilent 1295 LC system, and a 6470 triple-quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source. The MS/MS parameters including the MRM transitions, collision energy (CE) and retention times are presented in Table 1. Other mass spectrometry parameters were as follows: capillary voltage, 3.5 kV; gas temperature, 300 °C; sheath gas temperature, 350 °C and nebulizer pressure, 45 psi. High purity nitrogen was employed as drying and heating gas with a flow rate of 11 L/min. High purity argon was employed as nebulizing gas with a flow rate of 6.0 L/min. The chromatographic separation was achieved on an ACE Excel 3 Super C column (150 x 4.6 mm) with the temperature maintained at 30 °C. The mobile phase using a flow rate maintained 0.8 mL/mL, was a mix of 0.1% ammonia and 10 mmol/L ammonium formate solution (A) and MeOH (B). The eluting conditions were optimized as follows: isocratic at 20% B (0–3 min), 20% to 40% B (3–4 min), isocratic at 40% B (4–15 min), 40-100% B (15–20 min), 100-20% B (20–20.01 min), isocratic at 20% B (20–25 min). Table 1. method validation parameters 2.2 Sample pretreatment procedure The LC/MS method has the ability to determine the parent and glucuronide metabolites simultaneously. However, it has been reported that the optimization of hydrolysis conditions is difficult and the hydrolysis rates vary according to the type of enzyme, temperature, incubation time, etc. [5-6]. The dilute and shoot method was applied in this quantitative method, while hydrolysis and extraction steps of the normal screening procedure have been replaced by a single centrifugation and filtering. More specifically, for the quality control samples including the calibrations, standard solutions are spiked separately and dried under nitrogen; then 1 mL of blank urine is spiked with 50 μL of D -mophine at 10 μg/mL. After fully mixing and centrifugation, the supernatant is injected or diluted with water if needed. For the blank, negative control and actual urine samples, the pretreatment procedure is similar without the dried step. 2.3 Method validation The limit of quantification (LOQ) for 100 ng/mL, linearity ranging from 100 ng/mL to 1000 ng/mL, the intermediate precision and repeatability preformed at 500 ng/mL of M, M3G and M6G were fully validated and evaluated. The uncertainty (Uc) evaluation was performed using the methods B and D 18 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0144 recommended in WADA-TD-DL [3]. The intermediate precision and Uc data could validate the method stability comprehensively. Results and Discussion 3.1 Method validation The established method demonstrated satisfactory linearity, intermediate precision, LOQ, and repeatability within the measurement range of 100 to 1000 ng/mL. The relevant validation parameters are presented in Table 2. Table 2. MRM transitions and ESI-MS/MS parameters 3.2 Experimental results The structures of morphine substances are similar, so to obtain the suitable product ions, the parameters for the fragmentor and the collision energies were optimized. The representative mass spectra and fracture manners of these components are listed in Figure 1a. Typical chromatograms of QCP added with the 3 analytes and IS and the actual urine sample are illustrated in Figure 1b. No coeluting peaks or interfering signals were detected at the retention times of the targets operated at their specific mass transition. Therefore, it can be concluded that the present method is selective for the determination of morphine substances. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0145 Figure 1. (a) MS/MS spectra; (b) chromatograms of QCP and actual urine sample 3.3 Comparison results The measurement of uncertainty is evaluated according to the results from relevant rounds of the EQAS, and partial results are listed in Figure 2a and 2b. In the practical application process, except for the quantitative process of morphine, the accurate assessment of codeine concentration is equally necessary. Codeine could undergo metabolism to morphine in the liver via CYP2D6, and affect the metabolite profiling in a certain extent. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0146 Figure 2. (a) Application results of morphine; (b) comparison results; (c) QC chart of morphine 3.4. Discussion It is worth noting, that during the method validation process the peak shape and ionization efficiency of M3G and M6G could be easily influenced by the concentration. To avoid this, the linearity was designed to range from 100 ng/mL to 1000 ng/mL when the concentration of morphine substances larger than 1000 ng/mL; the dilution operation is necessary to ensure the accuracy. Beyond that, 0.1% ammonia and 10 mmol/L ammonium formate solution was used to improve peak shape and enhance ESI. The results showed that the uncertainties of the morphine substances were all below the maximum uncertainties specified by WADA, as shown in Figure 2c. Due to the Olympic requirement of fast turn-around analysis time, a convenient and reliable quantitative method is essential. And there was one positive case for morphine reported during the XIII Paralympic Winter Games. Conclusions The measurement of morphine in human urine is influenced by many factors, such as individuals with ultra-rapid CYP2D6 metabolism [7], the parallel and efficiency of pretreatment procedures, or taking several non-prohibited drugs simultaneously. Determining the origin of morphine found in a doping control urine sample of athletes is the basic requirement for anti-doping laboratories. In this report, urine samples were diluted and directly injected into the UHPLC-MS/MS system without any sample pretreatment, also the use of deuterated internal standard was necessary to reduce the matrix effects. The QC chart indicates that the method allows retrospective data analysis, which is strong in operability and convenient for the application in routine testing and reporting of adverse analytical findings. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0147 References 1. Kolmonen M, Leinonen A, Kuuranne T, Pelander A, Ojanper I. (2010). Hydrophilic interaction liquid chromatography and accurate mass measurement for quantification and confirmation of morphine, codeine and their glucuronide conjugates in human urine. J Chromatogr B Analyt Technol Biomed Life Sci, 878(29), 2959-2966. 2. Vree T B, Wissen V V.(2010). Pharmacokinetics and metabolism of codeine in humans. Biopharm. Drug Dispos., 13(6), 445-460. 3. World Anti-Doping Agency. Decision Limits for the Confirmatory Quantification of Threshold Substances. (2022). https://www.wada-ama.org/en/resources/ lab-documents /td2022dl#resourcedownload 4. World Anti-Doping Agency. WADA Technical Letter - TL22 Ethylmorphine. (2020). https://www.wadaama.org/en/resources/tl22#resource-download 5. Hackett L P, Dusci L J, Ilett K F, Chiswell G M. (2002). Optimizing the hydrolysis of codeine and morphine glucuronides in urine. Ther Drug Monit. 24(5), 652. 6. Wang P, Stone J A, Chen K H, Gross S F, Haller C A, Wu A H. (2006). Incomplete recovery of prescription opioids in urine using enzymatic hydrolysis of glucuronide metabolites. J Anal Toxicol, 30(8):570-5. 7. He YJ, Brockmöller J, Schmidt H, Roots I, Kirchheiner J. (2008). CYP2D6 ultrarapid metabolism and morphine/codeine ratios in blood: was it codeine or heroin? J Anal Toxicol. Mar;32(2):178-82. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0148 Grainger J, Agon V, Croft M, Goebel C Stability assessment of two small peptide ( < 2kDa) mixed standard Australian Sports Drug Testing Laboratory (ASDTL), Sydney, Australia Abstract ASDTL commissioned Auspep Pty Ltd to synthesise and prepare two small peptide mixed standards. SPMix-1 and SPMix-2 contained the same 41 small peptides and metabolites with the addition of bovine insulin to SPMix-2 to act as a carrier peptide. The homogeneity and stability of the two mixes were evaluated. Three peptides for which isotopically labelled internal standards were available – Leuprolide, GHRP-2 and GHRP-2(1-3)-OH were used to evaluate homogeneity as they provided the most precise data sets. Analysis of Variance (ANOVA) of the three data sets obtained for the duplicate analysis of 14 vials determined the between vial variability to be ≤ 2.50% for SPMIX-1 and ≤ 2.16% for SPMix-2. Peptide stability was assessed over a 12-month period under four storage conditions: in solution or dried at either -20 °C or -80 °C. Most peptides in both SPMixes were found to be best stored at -80 °C whether dried or in solution. Drying caused losses of hexarelin and its metabolites in both SPMixes, with decreases of 10-30% observed when stored at -20 °C. Losses of around 15% were observed for AOD-9604(7-16) when stored in solution at -20 °C. Both SPMixes were distributed to all WADA laboratories for assessment. Of the laboratories that responded, all found the quality of the SPMixes to be comparable to in-house standard mixes but would prefer additional peptides to be included. Introduction The peptides and metabolites in WADA’s 2020 Prohibited List [1] cover a range of sub-classes including LH releasing hormones, GH secretagogues, GH releasing peptides, GH fragments, masking peptides and Tβ4 derivatives. Reliable reference standards are an essential component of multi-residue anti-doping testing procedures. Over 50 peptides/metabolites custom synthesised for ASDTL are available to WADA laboratories. This study aimed to produce a multi-peptide mixed standard to aid the harmonisation of peptide testing across WADA laboratories. Including all peptides/metabolites in the mix would have been cost prohibitive, so peptide metabolism [2-4] was considered. GHRP-1 and alexamorelin, for example, were excluded because they are completely metabolised and not found in urine [3,4], while metabolites detected in urine for longer periods, were included. Experimental Auspep Pty Ltd (Melbourne, Australia) was commissioned to synthesise and prepare two SPMixes, each containing 41 peptides (Table1) with SPMix-2 also containing bovine insulin to act as carrier. Two hundred vials of each mix were produced. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0149 Table 1. List of small peptides included in the SPMixes prepared by Auspep Pty Ltd (25 µg net of each peptide) Homogeneity and stability assessment was performed using 14 randomly selected vials of each SPMix. After reconstitution, 200 μL aliquots from each vial were transferred into five tubes. One tube was used for homogeneity assessment. The remaining tubes were placed into four different long-term storage (LTS) conditions – in solution at -20 °C; in solution at -80 °C; dried at -20 °C; and dried at -80 °C. After 1, 2, 3, 4, 5, 6 and 12 months tubes were removed and stored at -80 °C until tested. Sample analysis was performed by LC-HRMS. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0150 The long-term stability of the peptides after reconstitution was assessed using the isochronous approach described in ISO Guide 35 [5]. Aliquots stored under the same storage conditions were analysed by LC- HRMS in a single batch under repeatability conditions at the completion of the stability period to minimise analytical variance. Assessment of the homogeneity of the mass of peptides across individual vials was based on the recommendations of ISO Guide 35 [5]. Isotopically labelled analogues of GHRP-2, GHRP-2(1-3)-OH and leuprolide were used in the measurement procedure. This resulted in more precise data sets for these analytes, which were then used to estimate between vial homogeneity by ANOVA. Results and Discussion Homogeneity ANOVA of the concentrations of the three representative peptides measured from 14 vials was used to indicate if, at a level of confidence of 95%, the variance observed between vials was significantly different to the variance observed within vials (duplicate analysis). The between vial variability for leuprolide, GHRP-2(1-3)-OH and GHRP-2 was 1.80%, 1.67% and 2.5% respectively in SPMix-1 and 2.05%, 2.16% and 2.14% respectively in SPMix-2. Long Term Stability In-Solution (1 mL 0.2% FA) Assessment of both SPMixes stored in solution at -20 °C showed an overall decrease in concentration of all peptides over 12 months. AOD-9604(7-16) and LHRH(2-10) decreased by 10-15% in both SPMixes (Figure 1), while hexarelin and hexarelin(1-3)-OH had losses of approximately 8% (Figure 2). When stored in solution at -80 °C the decrease over the same period was less. AOD-9604(7-16), hexarelin and hexarelin(1-3)-OH decreased by approximately 5%, while LHRH(2-10) had negligible losses. Figure 1. Stability of LHRH(2-10) and AOD-9604(7-16) in SPMIX-1 and SPMIX-2 when stored in solution at -20 °C MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0151 Dried (vacuum concentrator, 55 °C) With the exception of hexarelin and hexarelin(1-3)-OH, the majority of peptides in both SPMixes were slightly more stable dried and stored at -20 °C or -80°C, than stored in solution at -20 °C or -80 °C. Hexarelin and hexarelin(1-3)-OH were severely affected when dried with losses of 10-30% in both SPMixes stored at -20 °C (Figure 2) and losses of approximately 6% at -80 °C, over the study period. Figure 2. Comparison of stability of hexarelin and metabolite in SPMIX-1 over 12-month period when stored in solution or dried at -20 °C Assessment by WADA Laboratories All WADA laboratories (~30) were given vials of both SPMixes for assessment. The 11 labs that responded found the quality of the SPMixes compared well with in-house mixes but most would prefer the mixes include additional peptides. Only two laboratories preferred the addition of bovine insulin. The majority of laboratories were prepared to purchase the SPMixes noting the advantages of time-saving and harmonisation across laboratories for peptide testing. Conclusions The production and supply of suitable mixed peptide standards offers an efficient and cost-effective means by which laboratories can satisfy WADA requirements for peptide reference materials. This study demonstrated the viability of producing a suitable material. Two mixed peptide standards were prepared by Auspep Pty Ltd, each containing 41 peptides. Homogeneity testing determined between bottle variability was ≤ 2.5% for SPMix-1 and ≤ 2.2% for SPMix-2 over the 200 vial production batch. Stability testing over 12 months determined the best storage conditions for the SPMixes after reconstitution to be in solution at -80 °C. The majority of WADA laboratories responding to the assessment survey of these materials noted the time-saving and harmonisation advantages provided and were interested in using the SPMixes. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0152 References 1. World Anti-Doping Agency. The 2020 Prohibited List. International Standard, Montreal (2020). https://www.wada-ama.org/sites/default/files/wada_2020_english_prohibited_list_0.pdf (access date 1/07/2020) 2. Esposito S, Deventer K, Geldof L, Van Eenoo P. In vitro models for metabolic studies of small peptide hormones in sport drug testing. J Pept Sci. 2015;21(1):1-9. 3. Semenistaya E, Zvereva I, Thomas A, Thevis M, Krotov G, Rodchenkov G. Determination of growth hormone releasing peptides metabolites in human urine after nasal administration of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin. Drug Test Anal. 2015;7(10):919-25. 4. Thomas A, Delahaut P, Krug O, Schänzer W, Thevis M. Metabolism of Growth Hormone Releasing Peptides. Anal Chem. 2012;84(23):10252-9. 5. International Organisation of Standardisation (2017). Reference materials - Guidance for characterisation and assessment of homogeneity and stability (ISO Guide 35:2017). Acknowledgements This study was carried out with the support of the National Measurement Institute (Sydney, Australia) and the World Anti-Doping Agency (WADA, 17A11CG). MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0153 Yan X, Yan S, Chang W, Yang S A warning list to athletes of musk as ingredients from Traditional Chinese Medicine Beijing Anti-Doping Laboratory, Beijing Sport University, Beijing, People's Republic of China Abstract Musk, as a traditional Chinese medicine, can be used to treat traumatic injuries and cardiovascular disease. Numerous steroid compounds have been found in musk. Anabolic androgenic steroids feature prominently in the doping analysis field. Here we report an adverse analytical finding resulting from a previous administration of musk-containing drugs. This article sorts a list of medicines containing musk as an ingredient by the Pharmacopoeia of the People’s Republic of China (Chinese Pharmacopoeia). In summary, there are 74 traditional Chinese medicines containing musk extracts in the Chinese Pharmacopoeia. We suggest that athletes do not administrate those drugs to avoid triggering Adverse Analytical Findings (AAFs). Introduction Steroids, due to their anabolic effects, have been banned by WADA and IOC for many years. In recent years, various steroidal components including endogenous steroids were detected in musk, one of traditional Chinese medicines (TCMs), indicating a potential risk of triggering AAFs in doping tests, if athletes use a drug containing musk unintentionally [1]. Therefore, this article summarizes 74 traditional Chinese medicines containing musk from the Chinese Pharmacopoeia as a checklist for athletes and their team staff. Occurence of musk deer and composition of musk Musk deer are widespread in China, which is one of the original distribution areas around the world (Figure 1). However, the distribution area in China has been greatly reduced compared with ancient times, mainly distributed in Sichuan, Tibet, Qinghai, and Gansu provinces, and other places like Heilongjiang and Jilin also have small amounts. In addition to the wild, musk deer are widely domestically raised in recent years. As in 2020, there are about 30,000 domestic musk deer, mainly Moschus berezovskii Flerov, distributed in Shaanxi, Sichuan, Gansu, and Hubei provinces. Reports were also made in Shanxi, Fujian, and Shanghai. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0154 Figure 1. The distribution area of musk deer in China The chemical composition of musk is very complex. At present, the quality of traditional Chinese medicines containing musk is determined by the concentration of muscone. Amino acids, peptides, proteins, and steroidal compounds are also considered as main components of musk. Anabolic steroids found in musk ingredients were testosterone (T), epitestosterone (ET), dehydroepiandrosterone (DHEA), androsterone (An), etiocholanolone (Etio), and epiandrosterone (Epi-A). In addition, musk grains also contain small amounts of cholesterol, fatty acids, and esters (Table 1)[1]. Table 1. Steroids have been identified in Musk MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0155 Musk containing medicaments Musk extract has been used as a TCM for more than 2,000 years. Its medical application was first published in the “Shennong Ben Cao Jing”. This ancient Pharmacopoeia was created in the Qin-Han dynasty (about 200 years B.C.) in China [2]. Musk grains could be obtained from the pod of mature male musk deer (Moschus berezovskii Flerov, Moschus sifanicus Przewalski, Moschus Moschiferus Linnaeus ). In terms of the Chinese Pharmacopoeia, musk can be used for the treatment of cardiovascular diseases and traumatic injuries [3]. A list of traditional Chinese medicines containing musk is shown in Table 2. In the Chinese Pharmacopoeia 2020 edition, the number of traditional Chinese medicines containing musk has increased to 74, with up to 12 preparations, including 13 varieties of natural musk. Table 2. List of Traditional Chinese Medicines containing musk (from Chinese Pharmacopoeia, 2020 edition) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0156 Results and Discussion There have been approximately 20 steroidal compounds identified in musk extract so far, and nine steroids among them are listed in the WADA Prohibited List [1,4,5]. The use of musk extract has contributed to the triggering of AAFs at the 2011 FIFA Women World Cup [1]. It was reported that musk- containing drugs can alter steroid profile in human urine samples which could make athletes be tested positive in anti-doping analysis [6,7]. According to Gong Xiaoli et al., there are 469 kinds of traditional Chinese medicines containing musk in China [8]. The majority are used to affect the nervous system, and there are 101 drugs among them that are applied to the musculoskeletal system [9]. There are crucial differences between natural musk and artificial musk, especially the variety and content of macrocyclic ketones and androgens [10]. The relative content of natural musk components in different varieties and habitats also variy greatly. All kinds of TCM containing musk are complicated, and quality control is an essential task in the pharmaceutical factories in China nowadays. Conclusions A portion of anabolic androgenic steroids found in musk deer extracts are of the same kind as those in the human body, the main differences are that the concentrations may vary. Many more anabolic androgenic steroids have been identified in musk (Table 1)[1]. The prohibited endogenous steroids in musk can be abused as doping. This poster provides a list of traditional Chinese medicines containing musk. It is suggested that athletes should check the ingredients and keep alert to avoid Adverse Analytical Findings. References 1. Thevis M, Schänzer W, Geyer H, Thieme D, Grosse J, Rautenberg C, Flenker U, Beuck S, Thomas A, Holland R, Dvorak J. (2012) Traditional Chinese medicine and sports drug testing: identification of natural steroid administration in doping control urine samples resulting from musk (pod) extracts. Br J Sports Med. 47, 109–114 2. Ren L, Gao H, Zheng L, Peng C, Hou X. (2022) History and literature review of Moschus. Mod Chin Med. 25(3), 608-615 3. Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China. The medicine science and technology press of China, Beijing (2020), 402-403 4. World Anti-Doping Agency. The 2023 Prohibited List. International Standard, Montreal 5. (2023). https://www.wada-ama.org/sites/default/files/2023-05/2023list_en_final_9_september_2022.pdf (access date 05.08.2023) 6. Zhou J, Li N, Xie W, Song M, Han F, Li F, Li G. (2022) Advance in study on chemical constituents of natural musk and their pharmacological effects. Lishizhen Med Mater Med Res. 33(1), 185-188 7. He Y, Wang J, Liu X, Xu Y, He Z. (2013) Influences of musk administration on the doping test. Steroids. 78, 1047–1052 8. Wang J, He Y, Liu X, Yang Z, Yang W. (2017) Steroid profile and IRMS analysis of musk administration for doping control. Drug Test Anal. 9, 1779–1787 9. Gong L, Wang B, Luo S, You Z, Wang X, Li L, Zhao N. (2022) Research Status of Quality of Chinese Patent Medicines Containing Moschus. Mod Chin Med. 25(5), 1155-1162 10. Zhang Z, Huang H, Luo Y, Tan T, Yang M. (2019) Chemical Constituents from Musk of Moschus berezovskii. J Chin Med Mater. 42(2), 331-332 11. Jiang Y, Luo Y, Tan T, Yang M, Liao G. (2018) Comparison of Volatile Chemical Compositions of Moschus from Different Species and Producing Areas by GC-MS and Chemometric Analysis. Chin J Exp Tradit Med Formulae. 24(3), 49-55 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0157 Wicka M , Drapala A , Konarski P , Zyfka-Zagrodzinska E , Golaszewska A , Marusza M , Gorczyca D , Kwiatkowska D Hemp oils and potential for unintentional anti-doping rule violations Polish Anti-Doping Laboratory, Warsaw, Poland ; Lazarski University, Warsaw, Poland ; LymeLab Pharma, Warsaw, Poland ; Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland Abstract All natural and synthetic cannabinoids with one exception – cannabidiol (CBD) - are prohibited in competition and are included in section S8 in the WADA Prohibited List. CBD products are widely available on the commercial market. It is highly probable that the use of this group of preparations could cause unintentional violations of anti-doping regulations. Moreover, CBD preparations from cannabis plants may contain additional minor cannabinoids. The aim of the study was to analyze products containing CBD (hemp teas and oil) and urine samples collected after taking these preparations. Afterwards, the laboratory tested 15 oils from dried hemp available on the commercial market. Furthermore, urine samples from routine doping control have been analyzed for the presence of CBD. The presented study was conducted by means of two different LC- MS/MS systems. Introduction Cannabidiol is popularly known as CBD, a substance that is part of the cannabinoids, chemical components extracted from the cannabis or hemp plant [1]. All natural and synthetic cannabinoids are prohibited in sport in-competition with one exception - cannabidiol - and are classified under section S8 of the WADA Prohibited List [2]. CBD can be consumed in multiple products, in drops of oil, processed foods, drinks, and other products that athletes can find in a supermarket or specialized sports store, and that is why the consumption of CBD has increased significantly among athletes. It is highly probable that the use of this group of preparations could cause unintentional violations of anti-doping regulations [3]. Moreover, CBD preparations from cannabis plants may contain additional minor cannabinoids [1,4,5]. CBD could become an alternative to non-steroidal anti-inflammatory drugs, opioids or corticosteroids [6]. The aim of the study was to analyze products containing CBD (hemp teas and oil) and urine samples after taking these preparations. Moreover, the laboratory tested 15 oils from dried hemp available on the commercial market. In addition, urine samples from routine doping control have been analyzed for the presence of CBD. The presented study was conducted by means of two different LC-MS/MS systems. 1 1 1 2,3 3 2 2,3 1,4 1 2 3 4 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0158 Experimental The samples were prepared using two different methods described below. Method 1 (for urine) The sample preparation is a two-step procedure involving enzymatic deconjugation of glucuronides and then liquid-liquid extraction with 6 mL of methyl tert-butyl ether. The residue was reconstituted in 100 µL of mobile phase (acetonitrile/water, 1/1, v/v), transferred in a vial, and then 2 µL was injected into the LC-MS/MS system. Method 2 (for tested oils) 10 µL of the test sample was weighed into a centrifuge tube. Next, 400 µL of isopropanol was added and the sample was vortexed for 30 seconds. After the dissolution of the sample, 400 µL methanol was added and the sample was vortexed again for 30 seconds. Finally, the sample was filtered through a 0.2 µm PTFE syringe filter into a brown glass HPLC vial. Instrumental analysis Figure 1. Chromatographic separation and MS settings MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0159 Results and Discussion Fifteen oils from dried hemp, available on the commercial market were tested. For the analysis of the cannabinoids, aliquots of the stock solutions were diluted with ethanol 10 and 100 times and injected in duplicates in randomized orders onto the LC-MS for analysis. Some of the 15 oils had a full spectrum of cannabinoids, as the producer declared, and some of them contained only isolates of selected cannabinoids (mainly CBD, and CBG). Olive oil was used as a blank sample. The results of the analysis are shown in Table 1. Table 1. Results of analysis of 15 oils – Method 2 and Instrumental Analysis II Three different products - two teas and one oil - were purchased (Figure 2). The studies were performed on three healthy volunteers (1 female and 2 males) who consumed the hemp products as recommended by the manufacturer for 3 consecutive days (Table 2). Two spontaneous urine samples were collected per day i.e. in the morning and evening (the first one 8 hours after the first administration). Urine samples were collected for 3 consecutive days after the first administration. What’s more, urine samples from routine doping control (male and female, in- and out- of- competition) were analyzed for the presence of CBD. Concentrations of cannabinoids in urine samples were not adjusted for specific gravity. Results of the analysis are shown in Table 3A and Table 3B. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0160 Table 3 . A – Results of analysis of urine samples from routine doping control – Method 1 and Instrumental Analysis 1, B – Results of analysis of urine samples after administration of hemp products (excretion study) and analysis of taken hemp products for cannabinoids – Method 1 and Instrumental Analysis 2 Conclusions The consumption of hemp products could lead to the detection of prohibited cannabinoids in urine. According to WADA, CBD is not banned, but commercial products often contain banned substances such as THC and other cannabinoids. All CBD and hemp products should be avoided to prevent accidental doping from these often contaminated products. References 1. Rojas-Valverde D, Fallas-Campos A. Cannabidiol in sports: insights on how CBD could improve performance and recovery. Front Pharmacol. 2023; 14: 1210202, doi: 10.3389/fphar.2023.1210202 2. World Anti-Doping Agency. The 2023 Prohibited List, https://www.wada-ama.org/en/resources/world-anti- doping-program/2023-prohibited-list 3. Mareck U, Fusshöller G, Schertel T, Petring S, Huestis MA, Thevis M. Risk of unintentional antidoping rule violations by consumption of hemp products. Drug Test Anal. 2023; 15:27-41. DOI: 10.1002/dta.3327 4. Mareck U., Fussholler G., Geyer H., Huestis M., Scheiff A. B., Thevis M. Preliminary data on the potential for unintentional antidoping rule violations by permitted cannabidiol (CBD) use. Drug Test Anal. 2021; 13:539- 549. DOI: 10.1002/dta.2959 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0161 5. Ramos-Guerrero L., Montalvo G., Cosmi M., Garcia-Ruiz C., Ortega-Ojeda F.E. Classification of Various Marijuana Varieties by Raman Microscopy and Chemometrics. Toxics 2022, 10, 115, https://doi.org/10.3390/toxics10030115 6. Gamelin F-X et al. Cannabidiol in sport: Ergogenic or else? Pharmacol Res. 2020 Jun:156:104764. doi: 10.1016/j.phrs.2020.104764. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0162 Wicka M , Drapala A , Konarski P , Grucza K , Siek P , Kopczynska M , Kwiatkowska D BPC-157 – case study Polish Anti-Doping Laboratory, Warsaw, Poland ; Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland Abstract BPC-157 is a stable gastric pentadecapeptide composed of 15 amino acids – Gly-Glu-Pro-Pro-Pro-Gly-Lys- Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val. It is an experimental compound that has been tested for the treatment of ulcerative colitis, multiple sclerosis, and acute liver lesions. According to the World Anti-Doping Agency (WADA) Prohibited List from 2022, BPC-157 is included in section S0 (non-approved substances). Nowadays, BPC-157 has become an easily available supplement that brings beneficial effects in sports, for example accelerated healing of muscle tissue and joints (e.g. after injuries), and improved functioning of the brain. The presented case study resulted from the investigation following the detection of GHRP-2 and its metabolite in a urine sample undergoing routine doping control analysis. The athlete declared the administration of only one supplement labeled BPC-157, which he delivered for further analysis. Analysis was conducted by means of ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Introduction BPC-157, or body-protective compound 157, is a peptide isolated from human gastric juice, composed of 15 amino acids – Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val [1,2]. It is an experimental compound, that has been tested for the treatment of ulcerative colitis, multiple sclerosis, and acute liver lesions [1,3]. BPC-157 is stable in human gastric juice, and thus it has good oral bioavailability (always given alone) [3]. It can also be administered intramuscularly and subcutaneously. Moreover, BPC157 has demonstrated remarkable potential as a therapeutic agent for severe trauma and stress damage and can promote the healing of wounds, ligament injuries, tendon injuries, and fractures [4]. The beneficial effects of its use may cause athletes more often to decide to use BPC-157. Hitherto, there is not enough information from clinical trials to assess the possible harmful effects of the use of BPC-157. Therefore, BPC-157 is included in section S0 (non-approved substances) of the WADA Prohibited List from 2022 [5]. The presented case study indicates possible consequences after the administration of the supplement containing BPC-157 (as of 2021, BPC-157 was not namely including on the list). In this case, it was the detection of substances from the WADA prohibited list - GHRP-2 and its metabolite. This paper describes also the validation of an LC-MS/MS method for the detection of BPC-157 in human urine. 1 1 1 1 1 1 1,2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0163 Experimental The samples were prepared using three different methods described below. Method A 200 µL of urine sample was transferred to 1.5 mL tube (Eppendorf), spiked with mildronate-d (internal standard, ISTD), and diluted with 800 µL of water. Samples were strongly centrifuged and 200 µL of supernatant was transferred to a vial. 10 µL was injected into the LC-MS/MS system. Method B 10 µL of ISTD ([Lys-8]-vasopressin, d -GRP 2 (1-3), FA) and 200 µL of phosphate buffer (1 M, pH 6.5) were added to 2 mL urine transferred to Protein LoBindTubes (Eppendorf), and centrifuged. Samples were loaded onto an Oasis WCX – microelution plate (2 mg, Waters), previously conditioned with 1 mL of methanol and 1 mL of water, and washed with 1 mL of water. Afterwards, the target base fraction analytes were eluted with 1 mL of 25% ammonium hydroxide in water/methanol (1:9, v/v), and the target acid fraction analytes were eluted with 1 mL of 10% formic acid in water/methanol (1:9, v/v) into a round 96-well collection plate (Waters). Both fractions have been evaporated (SpeedVac) and reconstituted in 200 µL of mobile phase (acetonitrile/water, 1/1, v/v). 10 µL was injected into the LC- MS/MS system. Method C 10 µL of ISTD ([Lys-8]-vasopressin, d -GRP 2 (1-3), FA) and 1 mL 2% of acetic acid in water were added to 1 mL urine transferred to Protein LoBindTubes (Eppendorf), and centrifuged. Samples were loaded onto an Oasis WCX – microelution plate (2 mg, Waters), previously conditioned with 1 mL of methanol and 1 mL of water, and washed with 2% carbonate buffer (0.5 mL, pH ca.11), 1 mL of water, and 1 mL of methanol. Afterwards, the target base fraction analytes were eluted with 1 mL of 10% formic acid in water/methanol (1:9, v/v) into a round 96-well collection plate (Waters). The fraction has been evaporated (SpeedVac) and reconstituted in 150 µL of mobile phase (acetonitrile/water, 1/1, v/v). 10 µL was injected into the LC-MS/MS system. Instrumental analysis Chromatographic separation was conducted by means of a Waters Acquity I-Class UPLC System liquid chromatograph with HSS T3 column (1.8 µm, 100 mm x 2.1 mm, Waters). The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B), and the LC gradient was employed at the constant flow rate of 300 µL/min at 45 °C. MRMs of the studied substances were traced with a Xevo TQ-S mass spectrometer equipped with a UniSpray source. All analytes were investigated in the US mode. Desolvation gas flow was set at 300 L/h at 500 °C with ion source temperature at 150 °C. The capillary voltage was 3.0 kV. Method validation The validation process was performed in accordance with the WADA technical document TD2023IDCR [6]. The method was verified for selectivity, linearity, limit of detection (LOD), limit of identification (LOI), intra-day precision, and carryover (Figure 1). 3 4 4 + MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0164 Figure 1. Characteristics of the method validation performed and mass spectrometry parameters for BPC-157 Results and Discussion The validation was conducted for three different methods and for two of them (A and C) correlation coefficients were > 0.997 (covering a concentration range from 0.1 ng/mL to 100 ng/mL), with LOD at 0.5 ng/mL (Figure 1). The results and parameters of the initial test procedure for the athlete's urine sample in which GHRP-2 and its metabolite were detected and the results and parameters for the confirmation procedure are shown in Figure 2. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0165 Figure 2. Mass spectrometry parameters for ITP and CP detection of GHRP-2 and its metabolite The laboratory received a vial of white lyophilized powder called BPC-157 for analysis. The concentration declared by the producer was 10 mg per vial and was intended for intramuscular injections. The actual concentration of BPC 157 in the received vial was not estimated in. The analysis of the supplement labeled BPC-157, which indicated the presence of not declared peptide GHRP-2, is shown in Figure 3. Figure 3. Supplement analysis for BPC-157 and GHRP-2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0166 A reanalysis of the athlete’s urine sample has not confirmed the presence of BPC-157 (Figure 4). Figure 4. Reanalysis of the urine sample after investigation of administration of the contaminated supplement named BPC-157 Conclusions According to the published data [7,8], it is unlikely to detect BPC-157 (parent compound) in urine. Therefore, for detecting the presence of BPC 157 or its metabolites, it is important to specify a period of time from peptide administration to sample collection. Based on pharmacokinetics, it would be worth to consider monitoring BPC-157 metabolites. The above case is another example that indicates the problem of contamination of permitted supplements with prohibited compounds. References 1. Sikiric P., Seiwerth S., Grabarevic Z., Rucman R., Petek P., Rotkvic I. et.al. Hepatoprotective effect of BPC 157, A 15-aminoacid peptide, on liver lesions induced by either restraint stress or bile duct and hepatic artery ligation or CCl4 administration. A comparative study with dopamine agonists and somatostatin. Life Sciences, v.53 (18), 1993, pages: PL291-PL296. 2. Jelovac N., Sikiric P., Rucman R., Petek M., Marovic A., Perovic D., Prkacin, I. Pentadecapeptide BPC 157 attenuates disturbances induced by neuroleptics: the effect on catalepsy and gastric ulcers in mice and rats. Eur. J. Pharmacol. 1999; 379(1), 19-31. 3. Vukojevic J., Milavic M., Perovic D., IIic S., Cillic A. Z., Duran N., Strbe S., Zoricic Z., Filipcic I., Brecic P., Siverth S., Sikiric P. Pentadecapeptide BPC 157 and the central nervous system. Neural Regen Res. 2022 Mar; 17(3): 482-487. 4. Lei He et. al. Pharmacokinetics, distribution, metabolism, and excretion of body-protective compound 157, a potential drug for treating various wounds, in rats and dogs. Front Phrmacol 2022; 13: 1026182. doi: 10.3389/fphar.2022.1026182 5. World Anti-Doping Agency. The 2023 Prohibited List, https://www.wada-ama.org/en/resources/world-anti- MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0167 doping-program/2023-prohibited-list 6. The World Anti-Doping Agency (WADA). Minimum Criteria for Chromatographic-Mass Spectrometric Confirmation of The Identity of Analytes for Doping Control Purposes. Available online: https://www.wada- ama.org/en/resources/lab-documents/td2023idcr 7. Cox HD, Miller GD, Eichner D. Detection and in vitro metabolism of the confiscated peptides BPC 157 and MGF R23H. Drug Test Anal. 2017 Oct;9(10):1490-1498. 8. Lei He, et. al. Pharmacokinetics, distribution, metabolism, and excretion of body-protective compound 157, a potential drug for treating various wounds, in rats and dogs. Front. Pharmacol., 2022; doi: https://doi.org/10.3389/fphar.2022.1026182 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0168 Ahi S, Wolff K An overview of doping control analysis at the Birmingham 2022 Commonwealth Games King's College London, Drug Control Centre, London, United Kingdom Abstract Doping control analysis is an integral part of any sporting event. The XXII Commonwealth Games took place in Birmingham, United Kingdom between 28 July and 8 August 2022. The collaborative work and effective communication between Commonwealth Games Federation, UK Anti Doping (UKAD) and the Drug Control Center (DCC) made the event a success. This presentation would summarize the doping control analysis performed by the DCC, King’s College London as per the guidelines stipulated in the World Anti Doping Agency (WADA) International Standard for Laboratories (ISL, 2021) incuding details of laboratory infrastructure, staffing, training, methods and result management. The DCC operated continuously during the games period to provide services as per an agreed turn-around-time (TAT). Of all the samples tested during the event the presence of some prohibited substances was confirmed, resulting in 1 atypical finding (ATF) and 4 adverse analytical findings (AAFs) which would also be discussed. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0169 Al-Qahtani A , Abushareeda W , Vonaparti A , Kraiem S , Elsaftawy W , Saad K , Al-Wahaibi A , Hilal N , Dbes N , Nandlal V , Beotra A , Al Maadheed M Dope Testing: Overview of FIFA World Cup 2022 Doping Analysis Lab, Antidoping Lab Qatar, Doha, Qatar ; Institute of Doping Analysis and Sports Biochemistry (IDAS) - Dresden, Kreischa, Germany Abstract The FIFA World Cup, one of the major football events in the world was played in the State of Qatar in 2022 (November 18 to December 19 ). For the first time in history, the games were hosted in one of the GCC countries, the State of Qatar. A stringent Test Distribution Plan (TDP) was implemented by the FIFA Medical Committee in agreement with the requirements of the World Anti-Doping Agency (WADA). A total of 1612 samples (623 urine and 622 blood) were collected during in and out of competition. In addition, a total of 367 blood samples were collected for the hematological passport (ABP) during out of competition only and were analyzed within 24 hrs. Additional analysis for erythropoiesis stimulating agents (ESAs), small peptides and IRMS were carried out as per the requests received. The Test Distribution Plan (TDP) for the FIFA World Cup 2022 was challenging as there was an increased number of tests for human growth hormone (hGH) and the Athlete Biological Passport (ABP). The reporting of results was done within 24 to 72 hours for all the samples including ESAs analysis. A strict chain of custody was followed by the FIFA medical doctors/DCOs to hand over the samples to ADLQ to analyze athlete samples and safeguard the integrity of the samples. The FIFA Medical Committee was prompt in responding to any irregularity or clarification requested by the laboratory related to the samples. Figure 1 shows the distribution of samples received each day (1a) and the comparison of hGH isoform tests performed in various Olympic games with FIFA 2022 (1b). A thorough risk assessment and its mitigation was done prior to the event to ensure effective controls for a high number of hGH isoform test (622) and the overall conduct of the testing for the event. However, proper planning and collaboration together with the FIFA Medical Committee led to successful completion of testing. In this paper, an overview of the logistics, challenges, and statistics of FIFA World Cup 2022 testing was presented. The laboratory handled the event as per the scope of accredited methods by ISO/IEC 17025:2017 [1] and in accordance with the WADA International Standard for Laboratories (ISL [2]). The testing of the event was supported by 27 permanent scientists, 9 temporary scientific staff, 2 secretaries and 2 laboratory assistants. The Laboratory was supported by 6 international scientific experts from other WADA-accredited laboratories. The international experts were chosen to complement the existing staff and allow high-level scientific expertise during the event. 1 1 2 1 1 1 1 1 1 1 1 1 1 2 th th MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0170 Figure 1. a) Distribution of samples and analysis performed during in an out of competition, b) comparison of hGH isoform tests performed in various Olympic games with FIFA 2022 References 1. SO/IEC 17025:2017. General requirements for the competence of testing and calibration laboratories 2. WADA International Standard for Laboratories (ISL) https://www.wada-ama.org/en/resources/laboratories/international-standard-for-laboratories-isl) MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0171 Al-Mohannadi I , Samsam W , Al-Darwish S , Cherif A , Al-Jaber M , Abushreeda W , Al-Qahtani A , Kraiem S , Al-Mohammed H , Farooq A , Nimker V , Stuart M , Robinson N , Zangenfeind G , Rovira D , Beotra A , Al-Maadheed M , Mohamed-Ali V First drone delivery of anti-doping samples: safe, secure, rapid, reliable and robust Doping Analysis Lab, Antidoping Lab Qatar, Doha, Qatar ; Aspetar - Qatar Orthopaedic and Sports Medicine Hospital, Doha, Qatar ; International Testing Authority, Lausanne, Switzerland ; Rigi Technologies, Prilly, Switzerland ; Centre for Metabolism and Inflammation, Division of Medicine, University College London, London, UK Abstract As drone technology advances, its diverse applications, from medical logistics to parcel delivery, have rapidly expanded. However, transporting anti-doping samples during major sports events still needs to be explored. Although drones promise speed and efficiency, concerns regarding the stability of biochemical and haematological parameters post-transport, especially under adverse conditions, remain. The potential and challenges of drone applications in medical logistics are evident from contemporary research, including studies by Zailani et al. (2021) and Johannessen (2022). In this collaborative project between the International Testing Authority (ITA), Anti-Doping Lab Qatar (ADLQ), and Rigitech, a Swiss start-up, the study investigated the efficacy and compliance of drone delivery with the World Anti-Doping Agency (WADA) Code & WADA International Standard for Testing and Investigations (ISTI) requirements. The central objective of this research was to validate the hypothesis that drone transport does not compromise the integrity of blood, urine, and DBS samples, in alignment with Article 9.3 of the WADA ISTI. The study lasted four days, during which Eiger VTOL drones from Rigitech were used to transport samples in temperature-regulated compartments over distances ranging from 5 km to 20 km. Samples obtained from 47 healthy volunteers (including three females) before and after ingesting caffeine or placebo tablets were transported by either car or drone. The study was authorized by a nationally registered ethical committee in Doha, Qatar (IRB-AOSM-2020-007). In this research, the evaluation of blood parameters was conducted using Sysmex. Gas and liquid chromatography, along with mass spectrometry, were employed to detect steroid profiles and caffeine levels. Additionally, Metabolon's hardware and software aided in the analysis of metabolites through Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). Key results indicated that the drone was twice as fast as the car for short distances, and three times faster for a longer distance. Most haematological and clinical chemistry profiles retained consistency post-transport, irrespective of mode. The haematological factors and urinary steroid profile were stable, independent of mode of transport, over both distances (5 and 20 km). For example, when investigating differences between car and drone in testosterone, the p-values for the 5 km and 20 km groups were 0.11 and 0.13, respectively. For epitestosterone, the values were 0.48 and 0.65; for HGB, the values were 0.78 and 0.52. Following metabolomics analysis, concentrations of most metabolites (drone: 92%, 1 1 1 1 1 1 1 1 1 2 1 3 3 4 4 1 1,5 1,5 1 2 3 4 5 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0172 car: 83%) were stable regardless of the means of transport. Thus, for the current testing compounds, drone delivery seems a secure, faster and environmentally safer mode of transport. Multi-omics data, metabolites in this study, also seem largely stable but needs further validation. In conclusion, integrating drone transport with both the current and advanced analytical techniques offers promising avenues for future anti-doping measures. However, comprehensive research, accounting for diverse conditions and sample types, remains crucial. References 1. Zailani, M.A., Azma, R.Z., Aniza, I. et al. Drone versus ambulance for blood products transportation: an economic evaluation study. BMC Health Serv Res 21, 1308 (2021). 2. Johannessen KA. A Conceptual Approach to Time Savings and Cost Competitiveness Assessments for Drone Transport of Biologic Samples with Unmanned Aerial Systems (Drones). Drones. 2022; 6(3):62. 3. Perlee D, van der Steege KH, den Besten G. The effect of drone transport on the stability of biochemical, coagulation and hematological parameters in healthy individuals. Clin Chem Lab Med. 2021;59(11):1772- 1776. 4. Amukele TK, Hernandez J, Snozek CLH, et al. Drone Transport of Chemistry and Hematology Samples Over Long Distances. Am J Clin Pathol. 2017;148(5):427-435. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0173 Brooker L , Shimko K , Speers N , Goebel C , Thomas K Summary and evaluation of the Australian PIED wastewater-based epidemiology collaboration Australian Sports Drug Testing Laboratory (ASDTL), National Measurement Institute, North Ryde, Australia ; Queensland Alliance for Environmental Health Sciences, University of Queensland, Brisbane, Australia ; Science and Medicine, Sport Integrity Australia, Canberra, Australia Abstract Wastewater-based epidemiology (WBE) is a proven methodology that can provide spatial and temporal information on the use of and exposure to chemicals within the general population. It has been suggested that the illegal use of performance and image enhancing drugs (PIEDs) such as anabolic steroids and SARMs is increasing in general society. In this collaboration between the Queensland Alliance for Environmental Health Sciences (QAEHS, University of Queensland), Sport Integrity Australia (SIA) and the Australian Sports Drug Testing Laboratory (ASDTL), WBE methodology was developed and validated for AAS and SARMs, and applied to authentic archived wastewater samples to provide an insight into the emergence and occurrence of PIED use in Australia. To evaluate the results from this comprehensive project and to reflect on the effectiveness of modifications to initial testing procedures (ITP) over time at the ASDTL, reported findings from non- athletes (so-called workplace samples) over the period 2011-2022 were collated and tabulated graphically. For traditional S1.1 class substances such as testosterone, nandrolone, metandienone, trenbolone, stanozolol and clenbuterol, overall trends to fewer reported AAFs in recent years could be seen. This was potentially due to reduced sample numbers submitted for analysis through the COVID pandemic period or possibly users were moving to other forms of PIED use. When substances such as SARMs and also related PIEDs like ibutamoren (MK677) and cardarine (GW1516) were inspected, distinct trends could be observed. The first detections occurred from 2015 onwards, peaking through 2017 and 2019, with a decline through the years 2022-2022 (Figure 1). These trends from the findings of non-athletes urine samples analysed at ASDTL correlate remarkably well with the obtained WBE results. It can be noted that ASDTL did not report any enobosarm (ostarine) through the years 2011-2015 though WBE has shown that this substance was in use in the Australian community (Figure 1, yellow box). However, for more recent substances such ligandrol (LGD4033) it seems as if the laboratory was able to implement the appropriate target compound into the ITP prior to widespread use in the community starting. Lastly, it can be remarked how trends in analytical findings at anti-doping laboratories, especially for non-athlete samples, is useful information for others in the academic field of the study of the illegal use of performance and image enhancing drugs. 1 2 3 1 2 1 2 3 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0174 Figure 1. A side-by-side comparison of ASDTL non-athlete findings (2011-2022) and wastewater-based epidemiology results (2009-2021) for SARMs and related PIEDs This work has been published in the following articles: K. Shimko, T. Piatkowski, K. Thomas, N. Speers, L. Brooker, B. Tscharke, J. O’Brien. (2021) Performance and image enhancing drug use in the community: use prevalence, user demographics and the potential role of wastewater-based epidemiology. Journal of Hazardous Materials, 419, 126340 (1-12). K. Shimko, J. O’Brien, J. Li, B. Tscharke, L. Brooker, P.K. Thai, P.M. Choi, S. Samanipour and K. Thomas. (2022) In-sewer stability assessment of anabolic steroids and selective androgen receptor modulators. Environmental Science and Technology, 56, 3, 1627-1638. K. Shimko, J. O’Brien, X. Lin, B. Tscharke and K. Thomas. (2023) In-Sample Stability of 52 Performance- and Image-Enhancing Drug Biomarkers in Wastewater, ACS EST Water, 3, 3, 669–678. K. Shimko, J. O’Brien, B. Tscharke, L. Brooker, C. Goebel, R. Shiels, N. Speers, J, Mueller, K. Thomas. (2023) Emergence and Occurrence of Performance-Enhancing Substance Use in Australia Determined by Wastewater Analysis. Nature Water, in press. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0175 Torres C , Oliveira F , Jooris LF , Padilha M , Pereira H The presence of doping agents in dietary supplements: A glimpse into the Brazilian situation Chemistry Institute - UFRJ, Rio de Janeiro, Brazil ; Bazilian Doping Control Laboratory - LBCD, Rio de Janeiro, Brazil Abstract Dietary supplements (DS) are intended for healthy people to maintain or improve their overall health. According to the Brazilian Health Regulatory Agency (ANVISA), DS are products that are orally taken to provide nutrients, substances, enzymes, or probiotics missing in diets. Its consumption is widespread in a large part of the general population and at all levels of athletes aiming to optimize physical performance. Nevertheless, the DS intake can also pose health risks and may lead to Adverse Analytical Findings (AAF) due to the possibility of its contamination or adulteration with doping agents. Recent studies have shown an increasing number of doping cases associated with, a priori, the ingestion of contaminated DS with prohibited substances [1–3]. The detection of such substances in athletes' samples is considered a violation of the World Anti-Doping Code [4]. In this context, to raise awareness regarding the risks associated with DS consumption by athletes, this work aims to present the descriptive data concerning the presence of prohibited substances [5] in DS products reported by the Brazilian Doping Control Laboratory (LBCD) after Testing Authorities (TAs) analysis requests. All analyzed DS samples were related to AAF cases, and product contaminations were claimed during the results management or in the sports tribunal to justify the adverse findings. All DS samples (n=140) received by the LBCD between 2017 and 2022 (up to May) were included in the study. Most of the samples were presented as solid contents (capsules, tablets, and powder). For liquid samples, 1 mL was transferred and diluted in 9 mL of the same solvent. The materials were extracted with 10 mL of methanol. An aliquot of 1 ml of the methanolic solution was used for the analyses. The sample extracts were screened by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high resolution mass spectrometry (LC-HRMS), using similar method conditions applied to the anti-doping samples routine analysis. The findings of DS analyzed by the LBCD showed an alarming number of tainted samples. Diuretics were the most common adulterants found in all supplement types. However, the profile of prohibited substances in manufactured and compounded dietary supplements (MDS and CDS, respectively) were distinct, with stimulants being most prevalent in MDS, mainly due to the slimming product profiles and anabolic agents in CDS products. Additionally, MDS samples generally presented higher estimated concentrations of banned substances (mg/g), while CDS samples revealed lower estimated concentrations of prohibited substances (µg/g). The common practice of DS intake by athletes continues to be of great concern for a doping-free sport, given the high prevalence of prohibited substances detected in the analyzed samples by the LBCD. The current outlook regarding detecting doping agents in supplement samples reinforces the importance of raising awareness for the sports community of the possible consequences of an inadvertent doping case linked to DS use. 1 2 2 2 2 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0176 The topic of this workshop presentation was published as: Torres CL, de Oliveira FAG, Jooris LF, Padilha MC, Pereira HMG. The presence of doping agents in dietary supplements: A glimpse into the Brazilian situation. Drug Test Anal. 2024 Jan;16(1):38-48. doi: 10.1002/dta.3517. References 1. V. R. Kozhuharov, K. Ivanov, S. Ivanova. Dietary Supplements as Source of Unintentional Doping. Biomed Res. Int., 2022, 2022, 8387271. 2. U. Mareck, H. Geyer, T. Schertel, S. Petring, O. Krug, M. Thevis. Detection of undeclared doping substances in nutritional supplements in the context of follow-up investigations concerning adverse analytical findings. Drug Test. Anal., 2021, 13, 1911–1914. 3. N. M. Mathews. Prohibited Contaminants in Dietary Supplements. Sports Health, 2018, 10, 19–30. 4. World Anti-Doping Agency - WADA. “World Anti-Doping Code,” available at: https://www.wada-ama.org/sites/default/files/resources/files/2021_wada_code.pdf, 2021. 5. World Anti-Doping Agency. “The 2022 prohibited list,” available at: https://www.wada-ama.org/en/news/wada-publishes-2022-prohibited-list, 2022. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0177 Rubio A , Thomas A , Euler L , Geyer H , Krug O , Reis G , Costa Padilha M , Pereira HMG , Muniz- Santos R , Cameron LC , Stojanovic B , Kuehne D , Lagojda A , Mc Leod MD , Thevis M Investigations into Annona fruit consumption as a potential source of dietary higenamine intake in the context of sports drug testing Laboratory Medicine, Hospital Universitario Son Espases, Palma, Spain ; Center for Preventive Doping Research - Institute of Biochemistry, German Sport University, Cologne, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne / Bonn, Germany ; Brazilian Doping Control Laboratory (LBCD - LADETEC / IQ - UFRJ) - Chemistry Institute, Rio de Janeiro, Brazil ; Laboratory of Protein Biochemistry, The Federal University of State of Rio de Janeiro, Rio de Janeiro, Brazil; Seibersdorf Labor GmbH, Seibersdorf, Austria ; Bayer AG, Crop Science Division, Bayer AG, Monheim, Germany ; Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory, Australia Abstract Higenamine is a non-selective β -agonist prohibited in sports at all times by the World Anti-Doping Agency (WADA). Since it has been described as a key component of a great variety of natural plants, including the Annonaceae family, one aim of this research project was to evaluate whether the ingestion o f Annona fruit could lead to higenamine adverse analytical findings (AAF) in sports. Single-dose administration studies including three of the most widely consumed Annona species (i.e. A. muricata, A. cherimola, and A. squamosa) were conducted, leading to higenamine findings below the established minimum reporting level (MRL) of 10 ng/mL in urine, and supporting the position that single-dose administrations, considering the ingested amount, are rather unlikely to lead to an AAF in sports. In consideration of C values (7.8 ng/mL) and t values (up to 24 h) observed for higenamine after single-dose administration of Annona fruit, a multidose administration study was also conducted, indicating cumulative effects under the chosen conditions, and thus increasing the possibility of an AAF for higenamine by Annona fruit ingestion. However, the MRL was also not exceeded at any time point. Further, as both administration studies showed a major urinary excretion of higenamine in its conjugated forms, especially at the expense of the sulfo-conjugates, and the MRL refers to the free compound only, the estimation of contributions of higenamine phase-II metabolite to the substance´s elimination was assessed by enzymatic hydrolysis. The contribution of the glucuronide conjugates represented only 1.7– 2.5% of the sulfo-conjugates, and the unconjugated form represented 1.1–1.9% of the sulfo-conjugates, corroborating the major urinary excretion of higenamine in its sulfo-conjugated form. In the light of the herein presented results, a stability study concerning higenamine and its intact major phase-II metabolite (higenamine sulfate) in urine was performed. Higenamine was found to be stable under all storage conditions for up to 90 days, except for one volunteer at room temperature, and +4 °C. Higenamine sulfo-conjugate was found to be stable under all storage conditions at all different studied times, indicating that it does not liberate higenamine over time. Therefore, higenamine sulfo-conjugates were also considered optimal biomarkers for indicating higenamine intake. In the absence of reference material, higenamine sulfo-conjugates were synthesized and comprehensively characterized by means of LC-HRMS(/MS) and H-NMR, suggesting the predominant presence of higenamine 7-sulfate. 1 2 2 2,3 2,3 4 4 4 5 5 6 7 7 8 2,3 1 2 3 4 5 6 7 8 2 max max 1 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0178 In addition, due to the reported potential of higenamine-containing sports supplements to result in anti- doping rule violations, the option to include complementary biomarkers of diet-related higenamine intake into routine doping controls was also investigated. This research was based on the monitoring of intermediate compounds of the benzylisoquinoline alkaloids (BIAs) biosynthetic pathway. For that purpose, urine samples collected after single- and multi-dose administration of three different higenamine-containing supplements were also evaluated. A characteristic urinary pattern attributed to isococlaurine, reticuline, and yet not fully characterized bismethylated higenamine glucuronide was observed after Annona fruit ingestion but not after supplement use (Figure 1), providing a promising dataset of a total of five urinary biomarkers, which supports the discrimination between different sources of urinary higenamine detected in sports drug testing programs. Published as: Rubio A, Thomas A, Euler L, Geyer H, Krug O, Reis G, Padilha MC, Pereira HMG, Muniz-Santos R, Cameron LC, Stojanovic B, Kuehne D, Lagojda A, McLeod MD, Thevis M. Investigations into Annona fruit consumption as a potential source of dietary higenamine intake in the context of sports drug testing. Drug Test Anal. 2023 Nov-Dec;15(11-12):1488-1502. doi: 10.1002/dta.3558. Figure 1. Extracted ion chromatograms with diagnostic precursor-product ion pairs for the target analytes (MS/MS experiment) are shown as an example for one volunteer. The data originated from the analyses of urine samples collected before application (A) and 8 h following single-administration of supplement 1 (B), supplement 2 (C), A. muricata (D), or A. cherimola (E). Noteworthy differences in the urinary metabolite pattern were observed after Annona fruit ingestion when monitoring specific glucuronide conjugates such as the tentatively identified isococlaurine-, reticuline-, and N,X-bismethylhigenamine glucuronides MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0179 Kwon OS , Rahaman KA , Muresan AR , Xu Y , Jeon MJ , Kim MY , Jung S , Chin AL , Min H , Kim HJ , Sung C , Kim KH , Lee KM , Kang MJ , Lee J , Son J The urinary metabolite Ac-Tβ in huvec cells and rats treated with thymosin β4: A potential biomarker for doping tests Doping Control Center, Korea Institute of Science and Technology (KIST), Seoul, South Korea ; Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul, South Korea ; Center for Advanced Molecular Recognition, KIST, Seoul, South Korea Abstract Thymosin β4 (Tβ4) was reported to exert various beneficial bioactivites such as tissue repair and regeneration, anti-inflmmation, and reduced scar formation, and it is listed on the prohibited substnaces by the World Anti-Doping Agency. No metabolism studies of Tβ4 were not reported yet. Previously, our lab reported that total 13 metabolites were found by using multiple enzymes, and 6 metabolites (Ac-Tβ31-43, Ac-Tβ 1-11, Ac-Tβ 17-43, Ac-Tβ 1-14, Ac-Tβ 1-15, and β1-17) were confirmed by comparing with the synthetic standards. The study was aimed at identifying new metabolites of Tβ4 in biological systems such as leucine aminopeptidase, human kidney microsomes, huvec cells and rats to develope biomarkers for detecting doping in sports. A method for detecting and quantifying Ac-Tβ1-14 was developed and validated using Q-Exactive orbitrap mass spectrometry. LOD and LOQ of the Ac-Tβ 1-14 were 0.19 and 0.58 ng/mL and showed a good linearity (r =0.9998). This work was conducted in in vitro pure enzyme and microsome systems, cultured huvec cells, and rats after administration of parent protein. Among 6 metabolites above, Ac-Tβ was found in huvec cells exposed to 10 μg/mL and in urine of rats intraperitoneally treated with 20 mg/kg Tβ4. As a result, Ac-Tβ , the metabolite of Tβ4, was detected in all in vitro biological systems including pure enzyme and microsome systems, huvec cells, and rat urine samples. In addition, the metabolite Ac-Tβ was quantitatively determined to 48 hr in rats, with the highest concentration occurred between 0- 6 hr. Ac-Tβ was not detected in non-treated control groups including human blank urine. This results suggest that Ac-Tβ in urine is a potential biomarker for screening the parent Tβ4 in doping tests. Published as: Rahaman KA, Muresan AR, Hasan ML, Joung YK, Min H, Son J, Kang MJ, Kwon OS. Detection and quantification of the metabolite Ac-Tβ in in vitro experiments and urine of rats treated with Ac-Tβ4: A potential biomarker of Ac-Tβ4 for doping tests. Drug Test Anal. 2023 Nov-Dec;15(11-12):1454-1467. doi: 10.1002/dta.3552. 1,2 1,2 1,2 1 1 1 1 1 1 1 1 1 1 2,3 1 1 1-14 1 2 3 2 1-14 1-14 1- 14 1-14 1-14 1-14 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0180 Colpaert T , de Hon O , Duiven E , Olijhoek M , T'sjoen G , van Eenoo P , Deventer K , Coppieters G , van Uytfanghe K Detection of thyroid hormones T3 and T4 in serum samples after T4 administration in healthy volunteers DoCoLab Universiteit Gent-UGent, Ghent, Belgium ; Doping Authority Netherlands, Capelle aan den IJssel, Netherlands ; Ghent University Hospital, Ghent, Belgium ; Laboratory of Toxicology - UGent, Ghent, Belgium Abstract Thyroid hormones triiodothyronine (T3) and thyroxine (T4) are iodine-containing hormones involved in a wide range of metabolic processes. Protein synthesis/catabolism, lipid catabolism, and cardiac output are all influenced by T4 and the more active, yet less abundant, T3. There are rumours that some athletes started supplementing these hormones to lose weight, due to their catabolic effect on fat mass. Currently, thyroid hormones are not mentioned on the World Anti-Doping Agency (WADA) Prohibited List, even though multiple anti-doping organizations have lobbied for their inclusion. To further broaden the knowledge on thyroid hormone supplementation in a doping-related context, an administration study was performed where 1 mg Levothyroxine was administered to six healthy volunteers. Urine, serum, and dried blood spot (DBS) excretion samples were collected up to seven days prior to T4 administration to evaluate baseline T3 and T4 concentration levels. Post-administration samples were collected to investigate the intensity and duration of potential changes in these levels. To analyse these samples, a quantitative LC-HRMS/MS-based method was developed and validated for serum. The same method, with slight changes to the extraction protocol, was validated for urine. Figure 1 provides an overview of all serum post-administration samples. Samples are marked in yellow if their value exceeded the 95% confidence interval (CI) upper limit (average + 2 * standard deviation of pre-administration samples) or in green if their value exceeded the 99.7% CI upper limit (average + 3 * standard deviation of pre-administration samples). On the left side of the table, CI values were calculated for each volunteer individually (like a biological passport), whereas on the right side, CI values were calculated for all six volunteers together (like a population). Not shown in Table 1 are the baseline (pre- administration) samples that were marked in yellow. Using an individual 95% CI, 1 serum T4 and 2 serum T3 values would be incorrectly marked as significantly elevated. Using a group-based 95% CI, 2 serum T3, and 2 serum T4/T3 ratio values would be incorrectly marked as significantly elevated. It is clear that T4 supplementation only affects serum T4 levels, with serum T3 levels remaining constant throughout the duration of the study. Since T4 is converted to T3 by enzymes (deiodinases) within target cells, T4 supplementation would only raise intracellular T3 concentrations, not seen in the bloodstream. Our findings support this claim. Both T4 and T4/T3 ratio appear to be acceptable metrics for detecting the administration of T4, showing significant elevations up to one day post-administration. Unfortunately, no later samples were collected, making it unknown how long the elevations last. Since a 95% CI threshold results in some false positives, a more strict 99.7% CI threshold must be used. Comparing the 1 2 2 2 3 1 1 1 4 1 2 3 4 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0181 findings from individually calculated thresholds (biological passport) and one single group-based threshold, the former results in a more sensitive method. Nonetheless, all volunteers had multiple true positives using the single population threshold, something which is more easily implemented in routine doping control. Figure 1. Results for post-T4 administration serum samples from six volunteers. Yellow and green bars respectively indicate samples exceeding the 95% and 99.7% confidence interval upper limit, calculated using individual (left) or group (right) baseline values Full paper citation: TBA MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0182 Krug O , Guddat S , Möller T , Piper T , Görgens C , Thevis M Determination of urinary deanol-N-oxide excretion profiles after ingestion of nutritional supplements containing deanol Center for Preventive Doping Research - Institute of Biochemistry, German Sport University, Cologne, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne / Bonn, Germany Abstract 2-(Dimethylamino)ethan-1-ol (Deanol) is a widely produced chemical used by both industry and consumers in a variety of applications. Meclofenoxate, a stimulant classified on the World Anti-Doping Agency Prohibited List, metabolizes into deanol and, presumably, its main metabolite deanol-N-oxide. Hence, using liquid chromatography-tandem mass spectrometry, a quantitative detection method for deanol-N-oxide in urine was developed. Subsequently, the urinary excretion of deanol-N-oxide after oral application of 130 mg of deanol was determined in 6 volunteers, and urine samples of a cohort of 180 male and female athletes from different sports were analyzed. In addition, urinary deanol-N-oxide was determined in an exploratory study with one volunteer ingesting 250 mg of meclofenoxate. The developed test method allowed for limits of detection and quantification for deanol-N-oxide at 0.05 and 0.15 µg/mL, respectively. Urinary deanol-N-oxide c levels were found between 100 and 250 µg/mL 2-5 h post-administration of 130 mg of deanol. Similarly, urine samples collected after the administration of 250 mg of meclofenoxate exhibited c levels of 115 µg/mL (Figure 1). In contrast, deanol-N-oxide urine concentrations of pre-administration specimens and 180 routine doping control urine sample were found between 0.3 and 1.3 µg/mL, and below LOQ and 1.8 µg/mL, respectively. The study suggests that the use of deanol and meclofenoxate result in significantly elevated urinary deanol-N-oxide levels. Whether or not monitoring deanol-N-oxide in doping controls can support decision- making processes concerning the detection of meclofenoxate use necessitates further investigations taking into consideration the elimination kinetics of 4-chlorophenoxyacetic acid, the main metabolite of meclofenoxate, and deanol-N-oxide. Figure 1. Urinary elimination profiles of deanol-N-oxide in 6 volunteers after ingestion of 130 mg deanol and one volunteer after ingestion of 250 mg meclofenoxate (red line) 1,2 1 1 1 1 1,2 1 2 max max MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0183 Published as: Krug O, Guddat S, Görgens C, Piper T, Möller T, Thevis M. Determination of urinary deanol-N-oxide excretion profiles after ingestion of nutritional supplements containing deanol. Drug Test Anal. 2023 Nov- Dec;15(11-12):1312-1318. doi: 10.1002/dta.3580. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0184 Santos V , Carneiro G , Braz B , Santelli R , Machado S , Pereira H Coordination chemistry applied to antidoping science: cobalt quantification as diethyldithiocarbamate complex by LC-HRMS Brazilian Doping Control Laboratory - LBCD, Rio de Janeiro, Brazil ; Chemistry Institute - UFRJ, Rio de Janeiro, Brazil Abstract Cobalt, known as a producer of a chemical or metal-induced hypoxia, was included in the WADA’s Prohibited List in 2015 due to its effect on stimulus of erythropoiesis via stabilization of hypoxia-inducible factor. Although its proven benefits as a performance enhancement agent [1,2], especially in endurance sports, relatively low attention has been provided to the cobalt issue on last years. One factor that discourage research and improvements on this topic is the unavailability of inductively coupled plasma– mass spectrometry (ICP-MS) in most of the WADA-accredited laboratories. Therefore, the main objective of the study was to develop and validate, based on the WADA guidelines, an analytical method for cobalt quantification as its diethyldithiocarbamate (C H NCS , DDC) complex by liquid-chromatography coupled to high resolution tandem mass spectrometry (LC-HRMS/MS). As cobalt is a naturally occurring trace element in the human body and its typical urinary concentrations are reported in ranges between 0.1 and 2 ng/mL [3], a study with ICP-MS was conducted to access possible matrix influences. Aliquots of homogenized urine samples from fifteen volunteers returned a cobalt concentration in the range of (0.23 – 2.72) ng/mL in the total fraction (i.e., including both sediments and its supernatant) and a range of (0.23 – 2.88) ng/mL only in the supernatant fraction. However, for one volunteer, approximately 52.5% (i.e., 1.58 ng/mL) of the measured cobalt were aggregated to sediments. Therefore, a sample digestion is highly recommended for cobalt quantification by LC-HRMS/MS to avoid possible matrix effects. In the LC-HRMS/MS analysis, palladium was proposed as internal standard and was included in the first step of sample preparation. A microwave-assisted digestion with purified concentrated HNO was essential to liberate cobalt from possible matrix components. After the samples’ mineralization, a neutralization and pH adjustment to pH 6, a complexation with DDC and an oxidation with citric acid were conducted. Then, the samples were subjected to the liquid-liquid extraction (LLE) with tert-butyl methyl ether, and, after centrifugation and separation of the organic phases, rhodium was included as a complexation control to guarantee that there was excess of ligand even after the complexation reaction. The reconstituted samples were injected in a Dionex Ultimate 3000 ultra-high performance liquid chromatography system coupled to a QExactive Plus hybrid quadrupole-Orbitrap mass spectrometer. As validation, the method showed adequate selectivity in differentiating typical urinary cobalt concentrations found in population in relation to supraphysiological levels. The limit of identification (LOI) was estimated in 0.21 ng/mL, the samples were stable in autosampler at 20 °C for at least 48 hours and the method was not prone to carryover with spiked cobalt concentration at 100 ng/mL. Also, the method presented equivalent performance for small changes in complexation, time for LLE and time for evaporation to dryness. For the quantitative confirmation, the limit of quantification (LOQ) was estimated 1 1 2 2 2 1 1 2 4 10 2 3 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0185 at 2.5 ng/mL, a coefficient of variation (CV%) of 7.76% was obtained on day 1 and 12.8% on day 2, resulting in an intermediate precision of 10.2%. Finally, a combined standard uncertainty (u ) of 11.4% was obtained for the developed quantification method. Therefore, the method for cobalt quantification in urine showed an adequate precision and accuracy and represents a feasible way to encourage more anti-doping laboratories to start monitoring this analyte in routine analysis. Also, a risk assessment from the international anti-doping system regarding the use of cobalt as doping agent becomes necessary, as well as the evaluation of a threshold value. For more information, please refer to: dos Santo, VF, Carneiro GRA, Braz BF, Santelli RE, de Paula Machado S, Gualberto Pereira HM. Coordination chemistry applied to anti-doping analysis: Cobalt quantification as its diethyldithio‐ carbamate complex by liquid chromatography coupled to high resolution tandem mass spectrometry. Drug Test Anal. 2023; 1-11. doi:10.1002/dta.3571 References 1. Hoffmeister T, Schwenke D, Wachsmuth N, Krug O, Thevis M, Byrnes WC, Schmidt WFJ. Erythropoietic effects of low-dose cobalt application. Drug Test Anal. 2019 Feb;11(2):200-207. doi: 10.1002/dta.2478 2. Hoffmeister T, Schwenke D, Krug O, Wachsmuth N, Geyer H, Thevis, M, Byrnes WC, Schmidt WFJ. Effects of 3 Weeks of Oral Low-Dose Cobalt on Hemoglobin Mass and Aerobic Performance. Front Physiol. 2018 Sep 19;9:1289. doi: 10.3389/fphys.2018.01289. 3. Unice KM, Monnot AD, Gaffney SH, Tvermoes BE, Thuett KA, Paustenbach DJ, Finley BL. Inorganic cobalt supplementation: prediction of cobalt levels in whole blood and urine using a biokinetic model. Food Chem Toxicol. 2012 Jul;50(7):2456-61. doi: 10.1016/j.fct.2012.04.009 Acknowledgements The authors thank CNPq (PhD scholarship: 162996/2018-7) and Autoridade Brasileira de Controle de Dopagem for financial support, and Juliana Scofano Barrabin for the statistical support. c MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0186 Paßreiter A , Naumann N , Thomas A , Grogna N , Delahaut P , Thevis M How to detect CRISPR with CRISPR - employing SHERLOCK for doping control purposes Institute of Biochemistry, German Sport University, Cologne, Germany ; Département Santé, CER Groupe, Marloie, Belgium Abstract The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) tool kit constitutes one of today’s most frequently used gene editing techniques. Editing of virtually any DNA sequence can be realised, due to the quickly progressing research into different Cas effectors and their ever-expanding range of targets. Moreover, the simplicity and cost-effectiveness of those CRISPR tools can, unfortunately, also facilitate the illicit utilisation of CRISPR/Cas in order to achieve performance enhancements amongst athletes. Consequently, there is an urgent need for the direct detection of illegally applied CRISPR/Cas methods in doping control samples, for which a promising strategy is presented herein employing Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) for targeted nucleic acid detection. An analytical method was developed that enables the detection of sgRNA associated with Cas9 from Streptococcus pyogenes (SpCas9) in serum samples by means of reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection via SHERLOCK in combination with a complementary gel-based screening procedure in order to uncover doping attempts with lipid mediated CRISPR ribonucleoprotein (RNP) complexes. Initial qualitative method characterisation confirmed the specificity of both procedures and established a detection sensitivity of 10 nM uncomplexed target sequence and 100 pM sgRNA in the form of RNP complexes. Furthermore, a proof-of-concept in vivo administration study simulating a hypothetical gene doping scenario employing a mouse model revealed a detection window of 8 h after intravenous injection, supporting the principal applicability of the test strategy to authentic doping control samples in the future and encouraging progressing method optimisation. Published as: Paßreiter A, Naumann N, Thomas A, Grogna N, Delahaut P, Thevis M. How to detect CRISPR with CRISPR - employing SHERLOCK for doping control purposes. Analyst. 2022 Nov 21;147(23):5528-5536. doi: 10.1039/d2an01318e 1 1 1 2 2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0187 Wagener F , Naumann N , Leciejewska N , Görgens C , Guddat S , Thevis M Using organ-on-a-chip for predicting the metabolism of the selective androgen receptor modulator RAD140 in comparison to established in vitro approaches Institute of Biochemistry, German Sport University, Cologne, Germany ; University of Life Sciences, Poznan, Poland ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne, Germany Abstract The identification of metabolites for the expansion of possible targets for anti-doping analysis is an important field of research to ensure the fairness of sporting competitions. Especially for novel substances such as selective androgen receptor modulators (SARMs), information on metabolic fate is scarce. Human in vivo experiments yield samples with the highest applicability for anti-doping analysis but require administration studies, which can be difficult to conduct with pharmacologically relevant doses. Animal models provide information about the metabolism of substances but can be of limited use due to inter-species variability in metabolism and should be reduced due to animal-welfare concerns. In vitro models using liver enzymes are accessible and fast ways to produce metabolites, yet lack the full complexity of human biology. Novel approaches such as the organ-on-a-chip technology may provide a metabolic profile that resembles human in vivo samples more closely than approaches that rely on human liver enzymes only. Liver spheroids formed using HepaRG cells and human stellate cells were cultivated in an organ-on-a-chip platform and subjected to the SARM RAD140. Samples were collected over a three-week period and analyzed using HPLC-HRMS/MS. The detected metabolites resulting from the organ-on-a-chip experiment were compared to a human urine sample containing RAD140 as well as in vitro samples after incubation with human liver enzymes. A total of 15 metabolites was detected during this study, including five formerly unknown biotransformation products. Both organ-on-a-chip and established in vitro approaches yielded metabolites also found in human urine. Because some metabolites were exclusively detected in samples of one of the employed techniques, the approaches can be viewed as complementary to one another. Published as: Wagener F, Naumann N, Göldner V, Görgens C, Guddat S, Karst U, Thevis M. Comparison of in vitro approaches for predicting the metabolism of the selective androgen receptor modulator RAD140. Anal Bioanal Chem. 2023 Sep;415(23):5657-5669. doi: 10.1007/s00216-023-04835-z. 1 1 2 1 1 1,3 1 2 3 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0188 Naumann N , Wapurgis K , Rubio A , Thomas A , Paßreiter A , Thevis M Detection of doping control sample substitutions via SNP-based ID typing Center for Preventive Doping Research / Institute of Biochemistry, German Sport University Cologne, Cologne, Germany ; Laboratory Medicine, Hospital Universitario Son Espases, Palma de Mallorca, Spain Abstract The authenticity of a doping control sample is a key element of sports drug testing programs. Doping control sample manipulation by providing another individual’s urine or blood (instead of the tested athlete’s sample) has been observed in the past and is an unequivocal violation of the World Anti-Doping Agency (WADA) anti-doping rules. To determine attempts of manipulations by sample swapping, the utility of a single nucleotide polymorphism (SNP)-based sample authentication with a multi-target SNP panel was assessed. The panel comprises detection assays for 44 different SNPs, three gender markers, and five quality control markers for DNA-profile determination. Sample analysis is based on a multiplex polymerase chain reaction (PCR)-step followed by a multiplex single base extension (SBE)-reaction and subsequent SBE-product detection by MALDI-TOF MS. Panel performance was evaluated for urine and dried blood spot (DBS) samples. Urine (8 mL) and DBS (20 µL) test samples were reliably typed and matched to whole blood reference samples, while efficient typing of urine samples correlated with sample quality and input amounts. Robust profiling of urine doping control specimens was confirmed with an assay input of 12 mL. Samples can be processed in a high-throughput format with an overall assay turnaround time of approximately 11 hours. SNP-based DNA typing via MALDI-TOF MS thus represents a high throughput-capable possibility for doping control sample authentication. SNP-profiling of samples could offer the opportunity to complement existing steroid profile analytics to substantiate sample manipulations and to support quality control processes in high throughput routine settings. Published as: Naumann N, Walpurgis K, Rubio A, Thomas A, Paßreiter A, Thevis M. Detection of doping control sample substitutions via single nucleotide polymorphism-based ID typing. Drug Test Ana l. 2023 Nov-Dec;15(11- 12):1521-1533. doi: 10.1002/dta.3597. 1 1 2 1 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0189 Euler L, Deinert K, Thomas A, Thevis M Mass spectrometric characterization and development of detection methods for in vitro synthesized metabolites of the troponin activators reldesemtiv and tirasemtiv for doping control purposes Institute of Biochemistry, German Sport University, Cologne, Germany Abstract The fast skeletal muscle troponin activators (FSTAs) reldesemtiv and tirasemtiv were developed for patients suffering from a non-curable degenerative disease of the motor nervous system (e.g. ALS). The drug candidates are able to increase the sensitivity of troponin C to calcium by selectively activating the troponin complex. This slows down the rate of dissociation of calcium from the troponin complex and sensitizes the sarcomere to calcium, resulting in increased skeletal muscle contraction. This effect of FSTAs on the human body could be abused by athletes to enhance performance. The aim of the present study was to identify and characterize metabolites of reldesemtiv and tirasemtiv in order to ensure their reliable identification in doping control analysis, if the class of substances is considered as relevant by the World Anti-Doping Agency (WADA). The biotransformation was studied in vitro using pooled human liver microsomes. The metabolites were analyzed by means of HPLC-HRMS. In total, six phase I and three phase II for reldesemtiv and three phase I and two phase II metabolites for tirasemtiv were formed by the liver microsomes. To obtain further information on the metabolic fate of reldesemtiv and tirasemtiv, in vitro experiments were carried out using the human hepatic cell line HepaRG. For a potential implementation in doping control analysis, a HPLC-MS/MS method was developed to detect reldesemtiv and tirasemtiv in urine. As a proof of concept, urine samples were spiked using in vitro-generated phase II metabolites and subsequently processed and analyzed according to the developed method. The details of this study will be published elsewhere. * The 2023 Manfred Donike Award for the best oral presentation went to Luisa Euler, graduate student at the Institute of Biochemistry of the German Sport University, Cologne. Mrs. Euler provided first information on the human metabolism of the troponin activators reldesemtiv and tirasemtiv, which belong to a new class of substances featured on the 2024 WADA Prohibited List. Knowledge about the metabolism and, hence, suitable target analytes for routine doping controls is vital for effective anti- doping work concerning new drugs and drug candidates. This information was produced thoroughly and presented comprehensively regarding phase-I and phase-II metabolism, allowing the implementation of these compounds into initial testing procedures in sports drug testing laboratories. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0190 Krombholz S , Thomas A , Delahaut P , Miller GD , Bidlingmaier M , Thevis M A combined top-down and bottom-up LC-HRMS/MS method for the quantification of human growth hormone in plasma Institute of Biochemistry, German Sport University, Cologne, Germany ; Departement Sante, CER Groupe, Marloie, Belgium ; Sports Medicine Research and Testing Laboratory, Salt Lake City, Utah, USA ; Ludwig-Maximilians-University, Medizinische Klinik?Innenstadt, Munich, Germany Abstract The precise determination of human growth hormone (GH) levels in serum/ plasma is crucial for the diagnosis and control of diseases like GH-deficiency or acromegaly, as well as in the field of sports drug testing. This can, however, be challenging due to the pulsatile character and the heterogeneous isoform composition of the protein, in addition to inter-assay variations in the ligand-binding assays (LBA) currently used for routine tests. Quantitative determination of proteins by top-down liquid chromatography-mass spectrometry (LC-MS) can provide a valuable alternative to LBA, offering improved specificity, selectivity and precision, as well as the opportunity to determine several analytes simultaneously. Here, we present a combined top-down and bottom-up LC-HRMS/MS approach for the quantification of human growth hormone in plasma. Extraction from 200 µL of plasma was achieved by protein precipitation, followed by an immunocapture step using Protein-A-coupled magnetic beads and a polyclonal anti-GH antibody. Analysis by 2D-LC-HRMS/MS on an orbitrap system enabled the selective quantification of the intact monomeric 22 kDa-Isoform of GH in a representative working range of 0.5 - 10 ng/mL. Subsequent bottom-up analysis of the eluate involving trypsin digestion provided additional data on total plasma GH and the second most abundant isoform 20 kDa GH. Growth hormone concentrations in plasma samples of healthy adults, as well as acromegaly patients and in samples obtained after administration of recombinant human growth hormone were determined using the presented approach, demonstrating the proof-of-principle and how the selectivity of this method can offer further information in case results obtained from routine immunoassays are contradictory or ambiguous. Published as: Krombholz S, Thomas A, Delahaut P, Bidlingmaier M, Schilbach K, Miller G, Thevis M. A combined top- down and bottom-up LC-HRMS/MS method for the quantification of human growth hormone in plasma and serum. Growth Horm IGF Res. 2023 Oct-Dec;72-73:101560. doi: 10.1016/j.ghir.2023.101560. 1 1 2 3 4 1 1 2 3 4 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0191 Möller T , Naumann N , Krug O , Thevis M Identification and synthesis of selected in vitro generated metabolites of the novel selective androgen receptor modulator (SARM) 2f Institute of Biochemistry, German Sport University, Cologne, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract Among anabolic agents, selective androgen receptor modulators (SARMs) represent a new class of potential drugs that can exhibit anabolic effects on muscle and bone with reduced side effects due to a tissue-selective mode of action. Besides possible medical applications, SARMs are used as performance- enhancing agents in sports. Therefore, they are prohibited by the World Anti-Doping Agency (WADA) in and out of competition. Since their inclusion into the WADA Prohibited List in 2008, there has been an increase in not only the number of adverse analytical findings, but also the total number of SARMs, making continuous research into SARMs an ongoing topic in the field of doping controls. 4-((2R,3R)-2- Ethyl-3-hydroxy-5-oxopyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile (SARM 2f) is a novel SARM candidate and is therefore of particular interest for sports drug testing. This study describes the synthesis of SARM 2f using a multi-step approach, followed by full characterization using liquid chromatography–high- resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR). To provide the first insights into its biotransformation in humans, SARM 2f was metabolized using human liver microsomes and the microsomal S9 fraction. A total of seven metabolites, including phase I and phase II metabolites, were found, of which three metabolites were chemically synthesized in order to confirm their structure. Those can be employed in testing procedures for routine doping controls, further improving anti-doping efforts. Published as: Möller T, Wen H.-C, Naumann N, Krug O, Thevis M. Identification and Synthesis of Selected In Vitro Generated Metabolites of the Novel Selective Androgen Receptor Modulator (SARM) 2f. Molecules. 2023 Jul; 28(14):5541. https://doi.org/10.3390/molecules28145541 1 1 1,2 1,2 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0192 Coll S , Bressan C , Alechaga E , Monfort N , Carbó M , Ventura R Elimination profile of methylprednisolone in urine and plasma after oral administration: Evaluation of the reporting level and washout period Catalonian Antidoping Laboratory Fundació Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain ; Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain Abstract Methylprednisolone (MP) is prohibited in sports competitions when administered by all injectable, oral or rectal routes, and is permitted at all times when dermatologically administered. A general minimum reporting level (MRL) of 30 ng/mL was initially established to discriminate between allowed and prohibited administrations of all glucocorticoids. Since 2022, new compound-specific MRLs have been established for some GC. For MP, although low concentrations were reported after dermatological applications, the MRL was maintained at 30 ng/mL because the sensitivities after oral and injectable administrations were considered adequate. The aim of the present work is to study the urinary and plasma profiles of MP after single and multiple oral administrations in order to provide additional data to evaluate the current regulations to detect MP misuse in sports. MP was administered to healthy volunteers using a single-dose oral treatment (12 mg, n=8 males) and multiple-dose oral treatment (12 mg/day for 3 days, n=8 males). Urine and plasma samples collected before and after administration were analysed using a liquid chromatography-tandem mass spectrometry method. The current reporting level and wash-out period used to detect MP misuse in sports will be evaluated by assessing the urinary profile of MP obtained after different oral administrations. Furthermore, MP and cortisol concentrations obtained in plasma will be presented and discussed. 1 1 1,2 1 2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0193 Alechaga E , Bressan C , Coll S , Carbó M , Monfort N , Ventura R Glucocorticoids and DBS I. Concentrations of glucocorticoids in DBS after oral administrations Catalonian Antidoping Laboratory Fundació Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain ; Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain Abstract There is an interest in the evaluation of alternative sampling techniques for anti-doping purposes. Dried Blood Spots (DBS) present several advantages compared to conventional doping control matrices such as urine and blood or serum, as they facilitate sample collection, storage and transport. Conversely, the reduced sample volume available represents a limitation to reach the necessary detection limits and requires the use of highly sensitive instrumentation. Another limitation is the difficulty for quantitation of drugs due various factors such as the heterogeneous spreading of blood on the support or the necessity of controlling spot volume. In this work, a quantitative method for the determination of dexamethasone (DEX) and methyl- prednisolone (MP) in DBS samples has been developed and validated. Sample preparation consisted of a two-step extraction procedure with methanol:methyl tert-butyl ether 1:4 and acetone. The developed method was applied to samples collected in clinical studies after oral administrations of these glucocorticoids to healthy volunteers. For DEX, single dose (4 mg, n = 8 males) or multiple dose (2x 2 mg/day, 5 days, n = 8 males) treatments were applied, while for MP, a single dose administration of 12 mg (n=8 males) was studied. In both cases, DBS samples from capillary blood were collected at different time points covering up to 3 days after administration and submitted to analysis. Results from quantitative validation were satisfactory in terms of linearity, accuracy and precision. The relevance of the different factors that might affect quantitation will be assessed. Elimination profiles of DEX and MP in DBS samples will be presented. In a following presentation, correlations of plasma and DBS concentrations will be discussed. 1,2 1 1 2 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0194 Ventura R , Bressan C , Coll S , Alechaga E , Carbó M , Monfort N Glucocorticoids and DBS II. Correlation of glucocorticoid concentrations in DBS and plasma samples Catalonian Antidoping Laboratory Fundació Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain ; Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain Abstract Among the different advantages that the use of dried blood sports (DBS) has for anti-doping analyses, the possibility to correlate DBS concentrations with plasma concentrations is one of the most relevant, specially when minimum reporting levels (MRL) have to be defined for specific groups for forbidden substances prohibited only in competition. There are several known factors that might explain differences between plasma and DBS concentrations for a particular compound, e.g., blood-to-plasma ratios of the analytes, hematocrit level, differences in composition of capillary and venous blood, etc. This work is a follow-up of previous studies presenting concentrations of dexamethasone (DEX) and methylprednisolone (MP) in plasma, DBS and urine samples obtained after oral administrations. Here, the correlation of the concentrations of the aforementioned glucocorticoids in plasma and DBS has been evaluated in samples collected in two clinical studies. DEX and MP were administered orally to healthy volunteers in single-dose (4 mg, n = 8 males, and 12 mg, n=8 males, respectively) and multiple-dose (2x2 mg/day, 5 days, n = 8 males, only for DEX) treatments. Plasma and DBS samples were collected at the same time points and analyzed by liquid chromatography coupled to tandem mass spectrometry. Correlation between the concentrations in both matrices will be evaluated. Statistical comparison of the results will be performed by paired t-test, Passing-Bablok regression and Bland-Altman plots. Finally, the comparability of plasma and DBS results according to different factors will be discussed. Based on the results obtained, MRLs for DEX and MP in DBS will be proposed. Results from the analysis of urine samples also collected during the same studies will be also evaluated to assess the correspondence of all matrices and the agreement with the defined MRLs. 1 1 1 1,2 2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0195 Langer T, Nicoli R, Kuuranne T, Musenga A Comparison of serum and urine analysis for the confirmation of oral testosterone undecanoate administration Laboratoire Suisse d'Analyse du Dopage Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Epalinges, Switzerland Abstract Uncovering doping practices with pseudo-endogenous substances is one of the most important challenges in the fight for clean competitions. The Athlete Biological Passport (ABP) has proven to be an invaluable tool for the indirect detection of this kind of doping practices. However, for the detection of testosterone doping, the longitudinal monitoring of the urinary steroid profile is not always specific enough to report adverse analytical findings (AAF). To increase the likelihood of a doping scenario, urine samples that are considered suspicious are most often confirmed with a method that is capable of directly and unequivocally identify exogenous origin of testosterone and its metabolites. For urine samples, this method is isotope ratio mass spectrometry (IRMS). However, despite its specificity, this process is tedious, resource- and time-consuming. Similar to the urinary steroid profile, endogenous steroids can be measured in serum samples, allowing longitudinal monitoring [1]. While it has been shown that the determination of the origin of endogenous steroids in serum is possible [2], this approach is very recent and not yet available for all laboratories. However, when testosterone is administered in the form of esters, the direct detection of the intact esters in serum samples by LC-MS/MS is currently a much simpler approach compared to IRMS analysis. We developed a method that is capable of analysing a simplified steroid profile containing androstenedione (A4), testosterone (T), and dihydrotestosterone (DHT) in serum as well as detecting steroid esters in the same sample aliquot [3]. Serum samples from a testosterone undecanoate (TU) administration study including 19 male volunteers [4] were analysed, highlighting the influence on the steroid profile and demonstrating the detectability of intact TU after the administration. During this administration study, urine samples were collected as well, so urinary steroid profiles could be established, and the urine samples of nine volunteers were analysed by IRMS according to the routine laboratory procedure [5]. All results were compared to propose an efficient process for the detection of oral TU administration. Steroid profiles in urine and serum were influenced depending on the amount of detected TU. While the urinary ratio of testosterone to epitestosterone (T/E) was generally a good indicator of the administration, the ratio of 5α-androstane-3α,17β-diol to epitestosterone (5αAdiol/E) was more indicative for the volunteers with the UGT2B17 del/del polymorphism. Serum steroid profiles were less affected than urinary steroid profiles with DHT being a better marker than T or T/A4. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0196 Figure 1. Comparison of average urinary IRMS results with the average TU concentration in serum after the administration. Serum TU is shown as black line, the Δ C-values of T, androsterone (A), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5αAdiol) and 5β-androstane-3α,17β-diol (5βAdiol) are indicated as bar-graphs and were calculated using pregnanediol (PD) as endogenous reference. The standard deviations are indicated in the table below the average values. The colour and pattern of the bars correspond to the background colour of the steroids in the table. Full-grey cells indicate that the average value was above the threshold, hatched grey cells indicate, that the average is below the threshold, but individual values were above. Results showed that, by analysing serum samples, we were able to detect intact TU up to 24 h post- administration, although with high inter-individual variability. For half of the volunteers, TU was detectable 12 h after the administration or even longer. In comparison, using IRMS on the corresponding urine samples, it was also possible to determine the exogenous origin of testosterone and its metabolites, leading to AAF up to 24 h after the administration and to atypical findings (ATF) up to 48 h after the administration. In conclusion, the detection window in IRMS is slightly longer than the detection 13 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0197 window of TU in serum, but directly proportional to the detected TU concentration. However, the workload needed for the two methods is very different. Steroid esters in up to 80 serum samples can be analysed within 1-2 days, while the IRMS analysis of just 8 samples takes a full week, giving a slightly extended detection window. In the case that the urinary steroid profile is flagged as atypical and both urine and serum samples are available, it could be beneficial to perform the fast and simple steroid esters analysis in serum by LC-MS/MS before considering IRMS analysis of steroids in urine. Published as: Langer T, Nicoli R, Schweizer-Grundisch C, Grabherr S, Kuuranne T, Musenga A. Comparison of analytical approaches for the detection of oral testosterone undecanoate administration in men. Drug Test Anal. 2024 Jan 11. doi: 10.1002/dta.3638. References 1. Ponzetto F, Mehl F, Boccard J, et al. Longitudinal monitoring of endogenous steroids in human serum by UHPLC-MS/MS as a tool to detect testosterone abuse in sports. Anal Bioanal Chem. 2016;408(3):705-719. 2. Piper T, Geyer H, Nieschlag E, Bally L, Thevis M. Carbon isotope ratios of endogenous steroids found in human serum-method development, validation, and reference population-derived thresholds. Anal Bioanal Chem. 2021;413(22):5655-5667. 3. Langer T, Salamin O, Nicoli R, Grabherr S, Kuuranne T, Musenga A. A comprehensive UHPLC-MS/MS method for the analysis of endogenous and exogenous steroids in serum for anti-doping purposes. Drug Test Anal. 2022;14(11-12):1904-1919. 4. Badoud F, Boccard J, Schweizer C, Pralong F, Saugy M, Baume N. Profiling of steroid metabolites after transdermal and oral administration of testosterone by ultra-high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. J Steroid Biochem Mol Biol. 2013;138:222-235. 5. Piper T, Emery C, Thomas A, Saugy M, Thevis M. Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sports drug testing. Anal Bioanal Chem. 2013;405(16):5455-5466. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0198 Thilmany S , Hardy T , Thomas A , Vogel M , Scholl C , Thevis M Transcriptomic analysis of the effects of steroid hormones on neuronal cells Institute of Biochemistry, German Sport University Cologne, Cologne, Germany ; Research Department, Federal Institute for Drugs and Medical Devices, Bonn, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract Introduction Steroid hormone use is still popular among athletes; according to the 2021 testing figures of the World Anti-Doping Agency (WADA), almost 900 samples tested positive for steroids [1]. Steroids are also widely used for medical reasons, e.g., hormonal contraception [2], hormone replacement therapy [3], or cancer treatment [4,5]. In addition to the desired therapeutic effect, steroids can cause numerous undesirable side effects. For instance, an influence of hormonal contraceptives on the development of mental illnesses has been controversially discussed for years [6–12], and several reports are suggesting a similar effect with corticoids [13,14]. Consequently, the entire class of steroids and steroid receptor ligands is suspected of negatively influencing users’ mental health. So far, the findings are based on clinical and epidemiological studies; to the best of our knowledge, no evidence has been obtained under controlled laboratory conditions. This project aims to investigate the influence of steroids on the transcriptome of neuronal cells. Methods Human neuroblastoma cells were treated with different steroid hormone receptor ligands in cell culture. Samples were collected over the duration of the treatment, and transcriptome analysis was performed using DNA microarrays. Statistical analysis of gene expression changes over time identified genes affected by the treatment. Results Significant changes in gene expression were detected for all steroid hormone receptor ligands tested. These differentially expressed genes support the theory that steroid receptor ligands may have a negative impact on mental health as comparisons with published data from depression research show that there are numerous similarities to already known potential depression markers. The most significant changes involved pathways of proliferation, the neuronal system, neuronal development, neuronal degeneration, neurotransmission, neuroinflammation, and the immune system. Outlook Since the proteome is closer to biological function and thus allows more precise conclusions to be drawn about changes in the organism and the mechanism of action of steroids, the transcriptome analysis results are to be expanded in the further course of the project by analyses at the proteome level. For this purpose, a bottom-up proteomics platform is currently being established, with the help of which the neuronal proteome can be relatively quantified by means of isobaric labeling. 1,2 2 1 2 2 1,3 1 2 3 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0199 References 1. World Anti-Doping Agency. 2021 Anti-Doping Testing Figures. Report. 2023. 2. United Nations and Department of Economic and Social Affairs and Population Division. World Contraceptive Use 2022. Dataset. 2022. 3. Deutsche Gesellschaft für Gynäkologie und Geburtshilfe (DGGG), Östereichische Gesellschaft für Gynäkologie und Geburtshilfe (OEGGG) und Schweizerische Gesellschaft für Gynäkologie und Geburtshilfe (SGGG). S3-Leitlinie Peri- und Postmenopause - Diagnostik und Interventionen. Version 2.3. Guideline 015- 062. 2020. 4. Leitlinienprogramm Onkologie der Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften e.V. (AWMF), Deutschen Krebsgesellschaft e.V. (DKG) und Deutschen Krebshilfe (DKH). S3-Leitlinie Früherkennung, Diagnostik, Therapie und Nachsorge des Mammakarzinoms. Version 4.4. Guideline 032-045OL. 2021. 5. Leitlinienprogramm Onkologie der Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften e.V. (AWMF), Deutschen Krebsgesellschaft e.V. (DKG) und Deutschen Krebshilfe (DKH). S3-Leitlinie Prostatakarzinom. Version 6.2. Guideline 043-022OL. 2021. 6. Gingnell, M. et al. „Oral contraceptive use changes brain activity and mood in women with previous negative affect on the pill–a double-blinded, placebo-controlled randomized trial of a levonorgestrel- containing combined oral contraceptive“. In: Psychoneuroendocrinology 38 (7). 2013. DOI: 10.1016/j.psyneuen.2012.11.006. 7. Skovlund, C. W. et al. „Association of Hormonal Contraception With Suicide Attempts and Suicides“. In: Am J Psychiatry 175 (4). 2018. DOI: 10.1176/appi.ajp.2017.17060616. 8. Skovlund, C. W. et al. „Association of Hormonal Contraception With Depression“. In: JAMA Psychiatry 73 (11). 2016. DOI: 10.1001/jamapsychiatry.2016.2387. 9. Toffol, E. et al. „Hormonal contraception and mental health: results of a population-based study“. In: Hum Reprod 26 (11). 2011. DOI: 10.1093/humrep/der269. 10. Hamstra, D. A. et al. „Oral contraceptives positively affect mood in healthy PMS-free women: A longitudinal study“. In: J Psychosom Res 103. 2017. DOI: 10.1016/j.jpsychores.2017.10.011. 11. Scheuringer, A. et al. „Use of an estradiol-based combined oral contraceptives has no influence on attentional bias or depressive symptoms in healthy women“. In: Psychoneuroendocrinology 113. 2020. DOI: 10.1016/j.psyneuen.2019.104544. 12. Duke, J. M. et al. „Is there an association between the use of oral contraception and depressive symptoms in young Australian women?“ In: Contraception 75 (1). 2007. DOI: 10.1016/j.contraception.2006.08.002. 13. Brown, E. S. et al. „Mood and Cognitive Changes During Systemic Corticosteroid Therapy“. In: Prim Care Companion J Clin Psychiatry 3 (1). 2001. DOI: 10.4088/pcc.v03n0104. 14. Brown, E. S. „Effects of glucocorticoids on mood, memory, and the hippocampus. Treatment and preventive therapy“. In: Ann N Y Acad Sci 1179. 2009. DOI: 10.1111/j.1749-6632.2009.04981.x. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0200 Breuer J , Thomas A , Delahaut P , Schänzer W , Geyer H , Thevis M Investigations into the concentration and metabolite profiles of stanozolol and LGD-4033 in blood plasma and seminal fluid using liquid chromatography high-resolution mass spectrometry Institute of Biochemistry, German Sport University, Cologne, Germany ; CER Groupe, Marloie, Belgium ; European Monitoring Center for Emerging Doping Agents, Cologne/Bonn, Germany Abstract Potential scenarios as to the origin of minute amounts of banned substances detected in doping control samples have been a much-discussed problem in anti-doping analysis in recent years. One such debated scenario has been the contamination of female athletes’ urine with ejaculate containing doping agents and/or their metabolites. The aim of this work was to obtain complementary information on whether relevant concentration ranges of doping substances are excreted into the ejaculate and which metabolites can be detected in the seminal fluid (sf) and corresponding blood plasma (bp) samples. A method was established to study the concentration and metabolite profiles of stanozolol and LGD-4033 – substances listed under anabolic substances (S1) on the World Anti-Doping Agency’s Prohibited List – in bp and sf using liquid chromatography high-resolution mass spectrometry (LC-HRMS). For sf and bp, methods for detecting minute amounts of these substances were developed and tested for specificity, recovery, linearity, precision, and reliability. Subsequently, sf and bp samples from an animal administration study, where a boar orally received stanozolol at 0.33 mg/kg and LGD-4033 at 0.11 mg/kg, were measured. The developed assays proved appropriate for the detection of the target substances in both matrices with detection limits between 10 and 40 pg/mL for the unmetabolized drugs in sf and bp, allowing to estimate the concentration of stanozolol in bp (0.02–0.40 ng/mL) and in sf (0.01– 0.25 ng/mL) as well as of LGD-4033 in bp (0.21–2.00 ng/mL) and in sf (0.03–0.68 ng/mL) post- administration. In addition, metabolites resulting from different metabolic pathways were identified in sf and bp, with sf resembling a composite of the metabolic profile of bp and urine. Published as: Breuer, J., Thomas, A., Delahaut, P. Schänzer, W., Geyer, H., Thevis, M. Investigations into the concentration and metabolite profiles of stanozolol and LGD-4033 in blood plasma and seminal fluid using liquid chromatography high-resolution mass spectrometry. Anal Bioanal Chem 415, 669–681 (2023). https://doi.org/10.1007/s00216-022-04456-y 1 1 2 1 1,3 1,3 1 2 3 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0201 Aguilera R Athlete isotopic signature of δ C values. Complementary data from testosterone gel application (males and females) Drug Control Centre King's College, London, United Kingdom Abstract Isotope Ratio Mass Spectrometry continues to be the most powerful technique to confirm the use of synthetic endogenous steroid in the doping sport arena. The technique is based on the fact that pharmaceutical steroids have a lower C ratio than endogenous naturally produced steroids. Today these isotope ratios of C/ C can be measured with high accuracy and precision. Nonetheless, there is a significant natural variation of d C values within different populations and analytical issues related to calibration and sample preparation within laboratories. This final research project was focused on the variable d C values reported from different analytical laboratories and the micro doses used during recovery time by athletes. Some of these differences can be due to differences in an individual’s steroid metabolism variation. In order to avoid these inconsistencies in d C values between analytical groups and individual populations we have established the Athlete Internal Metabolic Reference by use of an isotopic signature or isotopic fingerprint including the detection of testosterone gel. 13 13 13 12 13 13 13 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0202 Piper T, Krombholz S, Thevis M Carbon isotope ratios of phenethylamine and phenylacetylglutamine and their applicability to doping control Institute of Biochemistry, German Sport University, Cologne, Germany Abstract Phenethylamine (PEA) is a naturally occurring trace amine that acts as a modulator in the central nervous system. It is widely sold as a dietary supplement and advertised for its mood enhancing effects and should support weight loss. It is prohibited in sports and itemized as a stimulant on the Prohibited List issued by the World Anti-Doping Agency (WADA). After oral administration of PEA, its urinary concentration is found only slightly elevated while metabolites of PEA show a significant increase [1,2]. Besides 2-(2-hydroxyphenyl)acetamide sulfate, especially phenylacetylglutamine (PAG) was found at significantly elevated urinary concentrations after the administration [2]. Due to large inter- and intra- individual variations in urinary concentrations of all metabolites, establishing a concentration or concentration ratio-based threshold remained complicated to unambiguously identify post-administration samples [2]. In accordance with the approach employed in detecting testosterone misuse, the applicability of isotope ratio mass spectrometry to differentiate between endogenously elevated concentrations and PEA administrations was investigated. A method encompassing solid-phase extraction combined with acetylation and high-performance liquid chromatography (HPLC)-based clean- up was developed and validated for PEA. The more abundant metabolite PAG was purified by a direct injection approach on the HPLC and could be analysed without the need for derivatization. Both methods were validated considering applicable WADA regulations. A reference population was investigated to establish population-based thresholds considering the carbon isotope ratios (CIR) found at natural abundance for PAG. The derived threshold was tested for its applicability by re-analysis of numerous post-administration samples encompassing single- and multi-dose trials [2]. Published as: Piper T, Krombholz S, Thevis M. Carbon isotope ratios of phenethylamine and its urinary metabolite phenylacetylglutamine. Drug Test Anal. 2023 Dec 4. doi: 10.1002/dta.3616 References 1. G. Sigmund, J. Dib, L. Tretzel, T. Piper, C. Bosse, W. Schänzer, M. Thevis. Monitoring 2-phenylethanamine and 2-(3-hydroxyphenyl)acetamide sulfate in doping controls. Drug Test Anal 2015, 7, 1057-1062. 2. S. Krombholz, A. Thomas, T. Piper, A. Lagojda, D. Kühne, M. Thevis. Urinary phenylethylamine metabolites as potential markers for sports drug testing purposes. Biomedical Chromatography 2021; e5274. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0203 Nair V , Howa J , Kucherova Y , Ahrens B , Pereira HMG , Nicoli R , Kuuranne T , Fedoruk M Applicability of longitudinal monitoring of IRMS values - A case study with unusual values The Sports Medicine Research and Testing Laboratory (SMRTL), South Jordan, United States ; UCLA Olympic Analytical Laboratory, Los Angeles, USA ; Brazilian Doping Control Laboratory, Rio de Janeiro, Brazil ; Laboratoire Suisse d?Analyse du Dopage Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland ; United States Anti-Doping Agency, Colorado Springs, United States Abstract Longitudinal monitoring of IRMS values has been previously suggested as a way to increase the sensitivity of the IRMS assay. Accordingly, data from athletes who received multiple IRMS tests over a period of three years were evaluated longitudinally. The overall variability of the Δδ C values was low. However, possible dietary changes caused noticeable fluctuation in the absolute δ C values for some athletes. While these changes occur across all assay analytes, it is possible that there could be transient perturbation in the subject's Δδ C profile. It is extremely unlikely that these changes will exceed the threshold set in the IRMS technical document but may flag a sample in a longitudinal model. A possible application of longitudinal monitoring may be for samples where measured isotope values do not meet criteria for positivity according to the IRMS technical document but are inconsistent with physiological variability. A case study with multiple unusual isotope measurements will be presented. No exogenous substances were detected in urine but analysis of serum and DBS samples from the same athlete for the presence of exogenous steroids and esters produced suspicious but as yet inconclusive results. A satisfactory explanation for these results has not yet been elucidated. 1 1 2 2 3 4 4 5 1 2 3 4 5 13 13 13 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0204 Piper T, Thevis M Investigations into the human metabolism of ecdysterone Institute of Biochemistry, German Sport University, Cologne, Germany Abstract The possible performance-enhancing effects and medical benefits of ecdysterone (ECDY) have been discussed several times throughout the last decades [1-4]. In 2020, WADA decided to include ECDY in their monitoring program and continued this prevalence study until now. Only little is known about the metabolism of ECDY. The first study performed on human subjects in the field of sports drug testing was already conducted in 2001 [5]. Besides the parent compound, 2-deoxyecdysterone and deoxyecdysone were identified in post-administration urine samples. These results were recently corroborated by two different studies, adding 14-deoxy-poststerone (14DP) as a potential urinary ECDY metabolite to the scope of investigations [6,7]. Aim of this study was the in-depth investigation on human ECDY metabolism to improve its detectability and to support the decision-making processes as to how ECDY can be implemented most effectively into sports drug testing regulations. In a first trial, one male volunteer was administered with deuterated ECDY to enable the detection and potential identification of all urinary metabolites still comprising the deuterium label by employing hydrogen isotope ratio mass spectrometry and high resolution/high accuracy mass spectrometry [8]. Samples were collected for up to 14 days and metabolites excreted unconjugated, glucuronidated and sulphated were investigated separately. The detected deuterated metabolites were confirmed in a second administration trial encompassing 3 male and female volunteers. After the administration of 50 mg ECDY, urine samples were collected for up to 7 days. Besides the already described urinary metabolites of ECDY, more than 20 new metabolites were detected encompassing all expected metabolic conversions including the side chain cleavage described for 14DP. A significant inter-individual variation in the amounts of excreted metabolites was noted. Published as: Piper T, Thevis M. Investigations into the human metabolism of ecdysterone. Drug Test Anal. 2023 Nov- Dec;15(11-12):1503-1520. doi: 10.1002/dta.3582. References 1. Bucci LR. Selected herbals and human exercise performance. Am. J. Clin. Nutr. 2000, 72(suppl), 624S– 636S. 2. Le Bizec B, et al. Ecdysteroids: one potential new anabolic family in breeding animals. Anal. Chim. Acta 2002, 473, 89-97. 3. Simnadis TG, et al. Physiological Effects Associated with Quinoa Consumption and Implications for Research Involving Humans: a Review. Plant Foods Hum. Nutr. 2015, 70, 238–249. 4. Isenmann E, et al. Ecdysteroids as non-conventional anabolic agent: performance enhancement by ecdysterone supplementation in humans. Arch. Toxicol. 2019, 93(7), 1807-1816. 5. Tsitsimpikou C, et al. Study of excretion of ecdysterone in human urine. Rapid Commun. Mass Spectrom. 2001, 15, 1796-1801. 6. Kraiem S, et al. Analytical strategy for the detection of ecdysterone and ist metabolites in vivo in uPA(+/+)- SCID mice with humanized liver, human urine samples, and estimation of prevalence of ist use in anti- doping samples. Drug Test Anal. 2021, 13, 1341–1353. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0205 7. Ambrosio G, et al. Urinary Elimination of Ecdysterone and Its Metabolites Following a Single-Dose Administrations in Human. Metabolites, 2021, 11, 366. 8. Thevis M, et al. Hydrogen isotope ratio mass spectrometry and high-resolution/ high-accuracy mass spectrometry in metabolite identification studies: Detecting target compounds for sports drug testing. Rapid Commun. Mass Spectrom. 2013, 27, 1904–1912. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0206 de La Torre X, Colamonici C, Curcio D, Botrè F 19-Norandrosterone confirmation by GC-C-IRMS: 10 years of experience Laboratorio Antidoping FMSI, Federazione Medico sportiva Italiana, Rome, Italy Abstract The use of GC-C-IRMS in antidoping analysis to determine the origin of urinary 19-norandrosterone was introduced at the beginning of 2000s and implemented in WADA Technical documents in 2010. The method should be able to distinguish between 19-norandrosterone produced in vivo in specific physiological conditions or ex-vivo in unstable urine samples (the so-called active urines) where some microbial activity is suspected. As with many other IRMS methods, this is based on the principle that the in vivo or ex vivo “endogenous” delta values can be distinguished from the values obtained from the pharmaceutical preparations or dietary supplements containing nandrolone precursors potentially used by athletes. The method is time- consuming and requires a large volume of urine (usually > 15-20 mL) since the amounts to be detected are low ( < 2-5 mL). This is not a mandatory procedure to be available in all WADA accredited laboratories and can be subcontracted to another WADA laboratory. The work dealt with the evolution of the confirmation procedure for 19-NA by IRMS, described the experience of more than 150 analyses in our laboratory and raised some difficulties that the antidoping community may encounter. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0207 Piper T, Thevis M Employing 11-ketotestosterone as target analyte for adrenosterone (11OXO) administrations in doping control Institute of Biochemistry, German Sport University, Cologne, Germany Abstract The human metabolism of 11-ketotestosterone (11KT) was recently investigated to enable the detection of 11KT administrations in doping control samples [1]. For both, the initial testing procedure (ITP) based on urinary concentrations and the confirmation procedure (CP) based on carbon isotope ratios (CIR), 11KT itself was the most promising candidate to detect 11KT administrations. Due to the structural correspondence between 11KT and 11OXO (adrenosterone, androst-4-ene-3,11,17-trione) and the described metabolism of 11OXO towards 11KT, the investigation on 11KT as potential target for 11OXO administrations was warranted [2]. In the context of sports drug testing, 11OXO was already investigated more than 10 years ago [3,4]. Urinary concentrations of 11-hydroxy-androsterone (11OHA) greater 10,000 ng/mL or a ratio of 11OHA divided by 11-hydroxy- etiocholanolone were suggested as potential markers during ITP. Samples found above these thresholds should be forwarded to isotope ratio mass spectrometry-based determinations of 11OHA. In the Cologne laboratory, the suggested ITP-thresholds were found to be suboptimal as samples with elevated 11OHA concentrations were extremely uncommon, while samples with elevated ratios are observed comparably often (approx. 10 % of all samples). Therefore, one male volunteer administered 100 mg 11OXO (2 capsules of a nutritional supplement) and collected urine sample before and up to 45 h after administration. All samples were subjected to the developed sample preparation, and both urinary concentrations and CIR were determined for relevant metabolites [1]. For both the ITP and the CP 11KT showed superior capabilities in detection of 11OXO administration compared to the already described target analytes. Employing 11KT as target analyte at a urinary concentration of 100 ng/mL may simplify and improve the detection for both, 11OXO and 11KT. Further investigations in a larger reference population encompassing n = 2000 routine doping control samples are ongoing to improve the statistical power of the threshold suggested for the urinary concentration of 11KT. Additionally, the CIR of urine samples found with elevated concentrations of 11KT will be investigated to establish a reliable Δ-value for this target analyte. As soon as these investigations are finished, the results will be published in detail elsewhere. References 1. Piper T, Thevis M. Development of mass spectrometry-based methods for the detection of 11- ketotestosterone and 11-keto​dihydro​testosterone. Drug Test. Analysis 2023, accepted 2. Pretorius E, Arlt W, Storbeck KH. A new dawn for androgens: Novel lessons from 11-oxygenated C19 steroids. Molecular and Cellular Endocrinology 2017, 441, 76-85. 3. Brooker L, Parr MK, Cawley A, Flenker U, Howe C, Kazlauskas R, Schänzer W, George A. Developement of criteria for the detection of adrenosterone administration by gas chromatography-mass spectrometry and GC/C/IRMS for doping control. Drug Test. Analysis 2009, 1, 587-595. 4. Brooker L, Cawley A, Kazlauskas R, Goebel C, George A. Carbon isotope ratio analysis of endogenous glucocorticoid urinary metabolites after cortisone acetate and adrenosterone administration for doping control. Drug Test. Analysis 2012, 4, 951–961. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0208 Goodrum J, Nair V, Moore C, Eichner D, Miller G Effects of heavy biotin supplementation on detection of hCG after Ovidrel administration to males The Sports Medicine Research and Testing Laboratory (SMRTL), Salt Lake City, United States Abstract Human chorionic gonadotropin (hCG) is a protein hormone that can be used as a performance enhancing substance by male athletes to re-establish or increase endogenous testosterone production. Doping control testing for hCG is conducted in urine, often on a variety of immunoanalyzer platforms, some of which utilize biotin-streptavidin dependent sandwich immunoassays where biotin is a well-known confounding factor. The extent of biotin interference in serum samples has been well studied, however biotin interference in urine samples has yet to be characterized. The goal of this study was to understand the extent of biotin interference in urine samples on biotin-dependent anti-doping hCG testing platforms to determine if biotin could act as a masking agent for hCG abuse. Ten recreationally active, male individuals self-administered hCG for 3 weeks concurrent with either heavy biotin or placebo supplementation. Urinary hCG detectability was evaluated using a biotin-dependent assay on the Roche Cobas e411 immunoanalyzer and a biotin-independent assay on the Siemens Advia Centaur XPT immunoanalyzer. Additionally, biotin concentration in urine samples from both groups was quantified. Finally, urinary steroid profiles were generated for each individual throughout the administration period. Data from these analyses will be presented. ® MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0209 Marchand A , Roulland I , Semence F , Jaffredo F , Dehainault C , Mekacher LR , Ericsson M Detection of the homologous transfusion of a red blood cell concentrate in vivo: evaluation of flow cytometry and DNA analysis methods Laboratoire AntiDopage Francais (LADF), Paris, France ; IGNA, Nantes, France ; CHU Tizi-Ouzou, Tizi-Ouzou, Algeria Abstract Two doping cases of homologous blood transfusion (HBT) during Tokyo 2020 Summer Olympics have shown that more controls are needed. The method of detection using flow cytometry to evaluate the expression of minor blood group antigens from red blood cells (RBC) and identify different RBC populations is efficient but still complex to perform with multiple antigens detection. Recently the interest of using forensic DNA analysis was also highlighted as a potential new method to detect HBT, with possibility to start from dried blood spots (DBS) instead of fresh blood. After a first phase of development a protocol was validated for HBT detection using DNA profiling analysis after extraction from DBS. Presence of a second DNA was clear down to 2% of donor blood in vitro. A flow cytometry protocol was also developed with preparation and analysis in 96-well plates and detection of 9 different antigens in total (C ,c, E, Jka, Jkb, Fya, Fyb, S,s) with two different primary antibodies per well whenever possible recognized by two different secondary antibodies (IgM coupled to a fluorescent 488 tag for detection of anti-C, anti-c, anti-E, anti -Jka and anti Jkb ; IgG coupled to a fluorescent RPE tag for detection of anti-S, anti-s, anti-Fya and anti-Fyb). The objective of the project was to evaluate the window of detection of an HBT performed in vivo with 150 mL of RBC concentrate prepared from whole blood without a deleukocytation step on two healthy volunteers. Blood samples obtained over 7 weeks post-transfusion were analyzed. While a positive control (mix of blood with 2% minor blood) was correctly identified with a DNA mix, DNA profiling from DBS was not sensitive enough to detect the presence of a second DNA in the sample obtained from the transfusion study even one day after transfusion, possibly due to the fast elimination of the donor white blood cells (which are the only blood cells containing DNA). On the contrary the flow cytometry protocol was very efficient and allowed identification of double populations (DP) of RBC (mix of expressing/ non expressing RBC). This was detected by the presence of a minor peak beside the major peak in one histogram of fluorescence (PE or AF-488 depending on the secondary antibody used for each antigen detection) and by the presence of a second cloud of points in the associated dot-plots PE versus AF-488 fluorescence.). Three to four different antigens with DP were detected in each sample post-transfusion until day 50 post-transfusion. The confirmation procedure (CP) was performed by using a new aliquot of the blood conserved at 4 °C: for each antigen with a suspected double population, three different dilutions of the primary antibody were tested (the same used for the screening analysis and 2-fold more and 2-fold less). Presence of a clear DP in at least 2 of the 3 antibody dilutions confirmed the DP for one antigen and if two antigens presented a DP in the same sample, HBT was confirmed. Reanalysis of the 1 1 1 2 2 3 1 1 2 3 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0210 blood samples stored at 4 °C for more than 30 days, despite hemolysis and a lower sensitivity for some antigens still allowed identification of double populations of RBC and identification of HBT. This protocol can be fully validated for a future application to doping control samples. For more details see publication: Marchand A, Roulland I, Semence F, et al. (2023) Evaluation of the detection of the homologous transfusion of a red blood cell concentrate in vivo for antidoping. Drug Test Anal . 2023; 1-13. doi:10.1002/dta.3448. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0211 Loria F , Breenfeldt Andersen A , Bejder J , Bonne T , Grabherr S , Kuuranne T , Leuenberger N , Baastrup Nordsborg N Erythropoiesis-related RNAs biomarkers are sensitive and specific to low-dose erythropoietin treatment at sea level and altitude Swiss Laboratory for Doping Analyses, Lausanne University Hospital, Lausanne, Switzerland ; Department of Nutrition, Exercise and Sports, University of Copenhagen, Copenhagen, Denmark ; University Center of Legal Medicine, Lausanne University Hospital, Lausanne, Suisse Abstract Background Indirect detection of recombinant human erythropoietin (rhEPO) abuse in Anti-Doping world is done by hematological module of athlete biological passport (ABP). However, actual markers (reticulocytes (%RET) and hemoglobin (HGB)) of this module are not enough specific and have difficulties to distinguish rhEPO administration from altitude sojourn especially if both condition are merged. Previous study have demonstrated that erythropoiesis-related RNAs, genes 5’-aminolevulinate synthase 2 (ALAS2) and carbonic anhydrase 1 (CA1) in capillary dried blood spots (DBS) could be potential biomarkers to increased specificity and sensitivity of the ABP in case of hypoxic conditions. Study design and method In the present study, we monitored ALAS2 and CA1 expression in DBS after injection of 20 IU kg bw epoetin alpha at sea level and the effect merged with altitude exposure. For this purpose, DBSs samples collected from 39 volunteers’ males and females were analyzed. Participants were divided in two groups (EPO and placebo) and subjects from both groups sojourned at sea level and at altitude. The purpose was to evaluate the performance of RNAs biomarkers for the detection of rHEPO injection in a longitudinal blinded setting. Results Our results illustrated a clear significant increase of ALAS2 until to 300% when treated at sea level and at altitude. Regarding CA1 the increase is up to 200% in both conditions. Interestingly, our results suggested no combined effect of altitude and rhEPO injection on ALAS2 and CA1 biomarkers as previously demonstrated with %RET values. In placebo group. Moreover, no effect of altitude on ALAS2 and CA1 was observed in contrast to %RET parameter. Results from blind setting test demonstrated a performance of interpretation of 92% regarding ALAS2 and CA1 monitoring. Conclusion Erythropoiesis-related RNAs in capillary DBS such as ALAS2 and CA1 were sensitive and specific biomarkers for low-dose rhEPO misuse at sea level and altitude. Interestingly, altitude seemed not to be a confounding factor for ALAS2 and CA1 biomarkers in contrast to %RET. Furthermore, this investigation provide evidence that low-dose of rhEPO injections can be detected in a longitudinal blinded setting with RNA biomarkers in DBS. 1 2 2 2 3 1 1 2 1 2 3 -1 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0212 Dørum S , Sletten C , Oll Ø , Dehnes Y EPO screening with K9 Norwegian Doping Control Laboratory, Oslo University Hospital, Oslo, Norway ; Asker Dog Centre, Oslo, Norway Abstract Doping with recombinant EPO (rEPO) has a long history of abuse in endurance sports. rEPO is considered to be a doping agent with a potentially high performance enhancing effect, making it an attractive doping agent for cheating athletes. To date, there is no fast and sensitive screening method for detection of rEPO. The method implemented in the WADA laboratories to detect misuse of rEPO is both sensitive and specific, but it is time-consuming and expensive, and only a small percentage of all samples are selected for EPO analysis. The aim of this project was to develop a rapid pre-screening method for detection of rEPO in urine from doped athletes, by taking advantage of the exceptional nose of the dogs. With such a method, a far higher amount of urine samples could be pre-screened for rEPO to select suspicious samples for further analysis in the laboratory. This would save both time and money, and it could potentially increase the number of positive rEPO samples identified. Together with Asker Dog Centre, we trained dogs to recognize low amounts of rEPO in urine. Here we will show the training strategy and present results from this highly challenging project. 1 1 2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0213 Leuenberger N , Kuuranne T , Castella V Sequencing c.577del in EPO gene in urine and dried blood samples Laboratoire Suisse d'Analyse du Dopage Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Epalinges, Suisse ; Forenic genetic unit, Epalinges, Suisse Abstract Human erythropoietin (hEPO), which increases the human red blood cell count, has been banned in sports since the 1990s. To detect recombinant EPO (rEPO), an analytical method has been set up using an electrophoretic approach, such as sarcosyl-polyacrylamide gel electrophoresis (SAR-PAGE) or sodium dodecyl sulfate-PAGE (SDS-PAGE), which separates the proteins by size in consideration of the molecular weight difference between the rEPO and the endogenous hEPO protein. Recently, a single nucleotide polymorphism (SNP) for EPO was detected in the East Asian population c.577del in the EPO gene, which can produce EPO with different molecular weights. This single mutation results in size increase of approximately 2.9 kDa. Therefore, the current confirmation method for rEPO with electrophoretic separation can lead to misinterpretation of the protein pattern of the EPO variant (VAR-EPO) for rEPO. The prevalence of the mutation is low (an allele frequency of approximately 0.5 – 1 %) and limited to East Asian populations. Nevertheless, to strengthen the analytical method, in October 2021, the WADA published a new version of the technical document for EPO analysis, including gene sequencing, in Annex B of TD2022EPO. According to this document, EPO sequencing shall be performed as a further investigation if double-band (heterozygous of EPO c.577del variant) or single band (homozygous of EPO c.577del variant) at a position higher than wild-type EPO is observed in blood samples with SDS-/SAR- PAGE analytical results. However, the detection of rEPO abuse for an athlete carrying the VAR-EPO is currently not possible with this approach. Collecting and transporting blood samples may be logistically difficult from certain areas, slowing down the overall process and delaying the reporting of the results. Different approaches, still based on the collection of blood samples, have already been proposed for the analyses, using either SDS-PAGE or CRISPR/dCas9 analyses. Urines are currently used as a source of DNA for genetic analyses. Urine contains about 1-20 ng/mL DNA from epithelial cells (shed from the urinary tract for both sexes plus the vagina for the females), leukocytes, and cell-free DNA. Blood contains about 20,000-40,000 ng/mL DNA from white blood cells and cell-free DNA. Considering equal volumes, urine contains about 1,000 to 40,000 times less DNA than blood. However, contrary to blood, urine can be collected using non-invasive methods. Therefore, urine can be used as an alternative source of DNA for the detection of the EPO c.577 del variant, provided the success rate is high. In recent years, considerable anti-doping research and development have focused on the dried blood samples (DBS). Moreover, DBS has been used for years in forensic genetics. DBS testing exhibits advantages in collection, transportation, and storage compared to traditional anti-doping testing matrices such as venous blood. For the practicalities of sample collection, an important benefit with DBS is the minimally invasive collection of capillary blood by puncture from a finger-prick or upper arms. In this study, we first analyzed 20 fresh urine samples from volunteers (10 females and 10 males) to define the amount of DNA needed to obtain a conclusive sequence of the EPO exon 5. Then, using this threshold, we 1 1 2 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0214 determined the proportions of conclusive EPO sequences that could be expected from urine and DBS samples by considering 191 urine samples with varying storage duration that were analyzed for anti- doping cases and 20 cellulose paper and polymer resin DBS (10 Whatman 903 cards and 10 Tasso-M20, respectively) from female and male volunteers. The amount of DNA recovered and the analytical success rate of urine samples were compared with those from published studies. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0215 Heiland CE , Leuenberger N , Ericsson M , Marchand A , Martin L DBS in practice for EPO doping control analysis: in vivo evaluation Stockholm Doping Control Laboratory, Karolinska Univeristy Hospital, Stockholm, Sweden ; Swiss Laboratory for Doping Analyses, Lausanne University Hospital, Lausanne, Switzerland ; Laboratoire AntiDopage Français, Université Paris-Saclay, Paris, France Abstract Background The applicability of dried blood spots (DBS) to erythropoiesis stimulating agent (ESA) detection has been evaluated previously using 20 to 60 µL sample volumes and various immunopurification methods [1,2], but it is still debated, particularly due to the risk of reduced sensitivity. Here, we demonstrate the suitability of DBS for ESA analysis via athlete samples and administration studies using Tasso-M20, Whatman 903 filter paper, and Mitra VAMS. Samples Detection limit samples: Venous blood (K EDTA, BD Vacutainer, NJ, USA) was collected from healthy volunteers with approval from the Bioethics Department of the French Ministère de l’Education de l’Enseignement Supérieur et de la Recherche (DC-2019-3644). Samples were stored at 4 °C. In-competition samples: DBS samples were collected from 111 athletes (with written consent) during two competitions organized by the Professional Triathletes Organization (PTO) in 2022. One Tasso-M20 (Tasso Inc., WA, USA) was used for blood collection from the upper arm skin and two of the four blood spots were used for analysis (about 35 µL capillary blood in total). Also, 34 urine samples were collected from some of these athletes simultaneously with the DBS samples. DBS samples were stored at room temperature and urine was stored at -20 °C until analysis. Aranesp administration study: Venous blood was collected from a patient (with verbal consent) in Switzerland undergoing Aranesp (NESP) treatment (30 µg/kg) every two weeks. The blood was pipetted onto Whatman 903 (Cytiva, MA, USA) filter paper (20 µL/spot), dried, sent to LADF, and stored at room temperature until analysis. Samples from pre-injection, days 4, 7, 14, and 28 were analysed (two spots/analysis). Eprex administration study: At University Hospital Tizi-Ouzou, Algeria, two healthy volunteers (one male, one female) received a micro-dose of Eprex (10 IU/kg) and venous blood was collected pre- injection, and 24-, 48-, and 72-hours post-injection (Ethical permit 006/CAN/Int/TECH/2021). Samples were sent to LADF, where the blood was applied to Mitra VAMS (Neoteryx, CA, USA), dried, and stored at room temperature until analysis. Methodology Spiking: To determine the detection limits of recombinant EPO (rEPO; NeoRecormon ), NESP, CERA, and EPO-Fc from analysis of two Tasso spots, venous blood was spiked at various concentrations (rEPO: 5-40 IU/mL; NESP: 5-80 pg/mL; CERA: 50-400 pg/mL; EPO-Fc: 25-400 pg/mL) and pipetted into the Tasso device. 1 2 3 3 3 1 2 3 2 ® ® ® ® ® MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0216 Immunopurification: Starting from two spots of 17.5-20 µL each collected on Tasso devices, Whatman filter paper, or Mitra VAMS, EPO was extracted using the EPO Purification Gel Kit (EPGK; MAIIA Diagnostics, Uppsala, Sweden). Eluates of 200 µL were concentrated down to ~15 µL using Amicon with 30kDa cut-off, and then analysed with polyacrylamide gel electrophoresis (PAGE). The other two immunopurification methods tested were extractions with buffer from EPGK combined with anti-EPO antibody coupled magnetic beads, and 1% tween solution combined with antibody coupled magnetic beads, based on a previously published method [3] For urine samples, EPO was quantified, and samples were immunopurified by previously published methods [3]. PAGE and Western blot: DBS samples were analysed with SAR-PAGE (and SDS-PAGE CAPS discontinuous transfer for detection limit tests) with Western blot, based on previously described methods [1,3]. Urine samples were analysed with SDS-PAGE according to a previously published method [3]. Results and conclusions The detection limits determined for the ESAs were: 10 IU/L rEPO, 10 pg/mL NESP, 250 pg/mL CERA, and 100 pg/mL EPO-Fc. High sensitivity and selectivity were achieved with samples immunopurified with EPGK and analysed with SAR-PAGE. Therefore, this method was applied to the in-competition and administration study samples. For the in-competition samples, no urine or DBS samples showed the presence of any prohibited ESAs. Endogenous EPO signals were identified in all DBS samples, whereas in 38% of the corresponding urine samples, EPO was undetectable. DBS samples on Whatman filter paper collected from a patient treated with Aranesp demonstrated that a detection window of at least two weeks can be obtained after use of therapeutic doses. A longer detection window is likely at such a dose since NESP signals were still strong two weeks after Aranesp® administration. Study subjects who received one Eprex micro-dose showed that this rEPO was detectable in Mitra VAMS for at least three days (Figure 1). However, differences between the two individuals were seen in signal strength, where the female subject (Subject 2) had generally fainter rEPO signals than the male subject (Subject 1). All these data are in favour of implementing DBS (polymer and filter paper supports) for EPO analysis from 35-40 µL dried blood as a supplemental routine analysis. Figure 1. Detection of Eprex micro-dose from DBS by SAR-PAGE: A single administration of 10 IU/kg Eprex was performed in 2 volunteers. One baseline (D0) and three post-administration (24, 48, and 72 h later, D1-3) samples were analysed for each subject: a male (Subject 1) and female (Subject 2). DBS samples were spotted from venous blood applied to Mitra VAMS. Two spots of 20 µL were used for analysis. Reference standard mixes (Ref), a negative control (Neg), and Eprex reference standard (Eprex ref) were also loaded. ® ® ® ® ® ® MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0217 Details published in: Heiland CE, Martin L, Zhou X, Zhang L, Ericsson M, Marchand A. Dried blood spots for erythropoietin analysis: Detection of micro-doses, EPO c.577del variant and comparison with in-competition matching urine samples. Drug Test Anal. 2023 Nov 9. doi: 10.1002/dta.3596. References 1. Heiland CE, Ericsson M, Pohanka A, Ekstrom L, Marchand A. (2022) Optimizing detection of erythropoietin receptor agonists from dried blood spots for anti-doping application. Drug Test Anal. 14(8):1377-86 2. Reverter-Branchat G, Ventura R, Din ME, Mateus J, Pedro C, Segura J. (2018) Detection of erythropoiesis- stimulating agents in a single dried blood spot. Drug Test Anal. 10(10):1496-507 3. Martin L, Martin JA, Audran M, Marchand A. (2022) An optimized SDS-PAGE protocol with a new blotting system for the initial testing procedure of ESAs in doping control. Drug Test Anal. 14(1):181-7 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0218 Requena-Tutusaus L , Anselmo I , Alechaga E , Bergés R , Ventura R Detection of ERAs in a single-spot DBS sample at MRPL level: improving applicability of DBS in doping control Catalonian Antidoping Laboratory Fundació Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain ; Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain ; Catalonian Antidoping Laboratory Fundació Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain Abstract Erythropoietin (EPO) receptor agonists (ERAs) are a group of substances prohibited by the World Antidoping Agency (WADA), that includes rEPO, NESP, EPO-Fc and CERA. ERAs are generally monitored in urine and blood samples (either serum or plasma), for which sampling, transportation and storage may be costly. For this reason, there is a great interest in the development of analytical procedures for doping control in new matrices such as dried blood spots (DBS). In this study a direct method for the detection of ERAs in DBS samples using one single spot of 25 µL has been evaluated. Two different immunopurification methods, MAIIA EPGK and StemCell EPO ELISA kit, were adapted to achieve maximum sensitivity and minimize background. Several steps of the immunopurification process were optimized, and the MAIIA kit was selected as it presented superior performance overall in terms of sensitivity and recovery. After immunopurification, automated SAR-PAGE western blot using primary biotinylated antibody anti-EPO (BAM2871) was applied to achieve excellent sensitivity and lower background. A complete validation of selectivity, limit of detection, recovery, stability, robustness and carryover was conducted for the optimized procedure. Results of the validation protocol will be presented. Stability of the four ERAs in this matrix was evaluated at different storage temperatures for up to 5 months, showing promising results regarding long-term storage of DBS samples. Performance of the developed method will be compared with the requirements established for conventional liquid matrices such as urine, serum or plasma. Particularly, the capability of detecting ERAs in a single low volume spot at the Minimum Required Performance Level (MRPL) established in blood will be assessed. 1,2 3 2,3 3 3 1 2 3 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0219 Reichel C , Gmeiner G , Thevis M Cost minimized immunoaffinity purification of EPO and its analogs in doping control – A step-by-step protocol for human blood and urine Seibersdorf Labor GmbH Doping Control Laboratory, Seibersdorf, Austria ; Institute of Biochemistry/Center for Preventive Doping Research, German Sport University Cologne, Cologne, Germany Abstract During the COVID-19 pandemic, many doping control laboratories were facing difficulties in obtaining consumables required for EPO analysis (e.g. ultrafilters, materials for immunoaffinity purification, sample tubes). While these shortages have been largely overcome, costs for materials and reagents drastically increased between 2020 and 2023 (e.g. +42% for the biotinylated clone AE7A5 EPO-antibody, +27% for clone 9C21D11 EPO capture antibody, + 21% for the EPO immunoaffinity isolation plate). Sample preparation of urine and blood specimens for the detection of EPO and its analogs is quite costly due to the requirement of immunoaffinity purification (IP) before electrophoretic separation (TD2022EPO, chapter 2.2).[1] Additionally, the “CP should differ, where necessary, from the ITP” (chapter 2.2.1.2 TD2022EPO), e.g. by application of an IP method using a different capture antibody. While for urine samples sufficient commercial kits fulfilling this requirement are available (i.e. EPO-ELISA plate from Stemcell Technologies [2], EPO purification products from MAIIA Diagnostics [3-5]), for blood only the kits from MAIIA Diagnostics exist.[3, 5] However, they use the same capture antibody (clone 3F6). We focused on a protocol requiring no covalent immobilization procedure and significantly modified it in order to increase its sensitivity and specificity. Furthermore, it is now universally applicable for blood and urine samples, performs with the same sensitivity as the commercial products but at a fraction of their costs. Figure 1 shows the protocol for urine. For more details, please refer to the full article published in Drug Testing and Analysis [6]. References 1. World Anti-Doping Agency (WADA). Technical Document TD2022EPO. Harmonization of analysis and reporting of erythropoietin (EPO) and other EPO-receptor agonists (ERAs) by polyacrylamide gel electrophoretic (PAGE) analytical methods. https://www.wada- ama.org/sites/default/files/resources/files/td2022epo_v1.0_final_eng.pdf. Accessed 1 September 2023. 2. Stemcell Technologies. EPO Immunoaffinity Isolation Plate. https://www.stemcell.com/products/epo- immunoaffinity-isolation-plate.html. Accessed 17 March 2023. 3. MAIIA Diagnostics. EPO Purification Kit (Art. 1390). https://maiiadiagnostics.com/wp- content/uploads/2022/06/DfU-EPK.pdf. Accessed 17 March 2023. 4. MAIIA Diagnostics. EPO Purification Gel Kit for Urine (Art. 1410). https://maiiadiagnostics.com/wp- content/uploads/2022/06/DfU-EPGKU-2022.pdf. Accessed 17 March 2023. 5. MAIIA Diagnostics. EPO Purification Gel Kit for Blood (Art. 1430). https://maiiadiagnostics.com/wp- content/uploads/2022/06/DfU-EPGKB-2022.pdf. Accessed 17 March 2023. 6. Reichel C, Gmeiner G, Thevis M. Cost minimized immunoaffinity purification of EPO and its analogs in doping control – A step-by-step protocol for human urine and blood. Drug Test Anal. (in press) 1 1 2 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0220 Figure 1. Protocol for immunopurification of EPO and its analogs from urine Acknowledgements The project was carried out with support of the Austrian Ministry of Sports and the Federal Ministry of the Interior of the Federal Republic of Germany. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0221 Miller G, Goodrum J, Campbell T, Eichner D Assessing the stability of recombinant EPO in urine The Sports Medicine Research and Testing Laboratory (SMRTL), Salt Lake City, United States Abstract The WADA Technical Document for EPO analysis includes a section on the reporting of samples that show no electrophoretic band for either endogenous or recombinant EPO. Although these samples are reported negative, we cannot be certain that the urine sample was provided by an athlete clean from EPO. And while the TD lists specifically samples with no endogenous or exogenous EPO signal, Western blot interpretation also becomes difficult with samples showing signs of degradation and in samples with very low total EPO content. It is unclear exactly what causes this EPO instability, so we designed a three-part study to further understand the stability of endogenous, and, more importantly, recombinant EPO in human urine. First, samples showing no EPO bands or degraded EPO bands analyzed at SMRTL in 2022 were examined to understand trends or similarities amongst the samples. Analysis variables included sport/discipline, sample pH, sample SG, presence of microbial activity according to the steroid profile, shipping time/distance, time of year, and geographic location of sample collections. Second, anti-doping urine samples showing either no endogenous EPO or degraded endogenous EPO after screening were spiked with a combination of recombinant EPO, darbepoetin, and Mircera. Western blotting results from the spiking of 100 samples will be presented. The third portion of the study included a single-dose administration of EPO to twelve male and female volunteers. Each volunteer was administered 40 IU/kg of EPOGEN (epoetin alfa) by subcutaneous injection. A bulk urine collection was performed at an assigned, specific time point following the injection (up to 48 hours). Aliquots from the bulk collection were stored under various time and temperature conditions, designed to mimic shipping conditions at various times in the year. Samples were then analyzed for the presence of endogenous and recombinant EPO, and any potential degradation products. Data from the administration study will be presented, in addition to suggested guidance on the collection, handling and EPO analysis of urine samples. MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0222 Garzinsky A , Harth J , Reihlen P , Leipp F , Thomas A , Thevis M Can the detection of EPO and its derivatives be manipulated by the addition of oral fluid to doping control urine samples? Institute of Biochemistry, German Sport University, Cologne, Germany ; Ecole Nationale Superieure de Chimie de Paris, Universite Paris Sciences et Lettres, Paris, France Abstract In the field of sports drug testing, so called “no-shows” represent a well-known problem when it comes to the analysis of erythropoiesis-stimulating agents (ESAs) by means of SAR-PAGE and Western blotting. In these specimens, endogenous EPO is not identifiable. Past studies already demonstrated, that proteases were found to contribute to this observation by leading to the degradation of ESAs. Since oral fluid (OF) comprises proteases, among others, it was assumed that OF could also lead to the degradation of ESAs and thus to impaired EPO analyses, resulting in a readily accessible method for urine sample tampering. The aim of this study was to investigate this assumption and to develop a detection assay in order to identify present OF in urine doping control samples. For this purpose, a total of 1080 urine samples were subjected to EPO analysis and evaluated with regard to variations due to the subject, sex, volume of OF, time point of OF-sampling, and storage conditions. The results showed, that OF can indeed lead to masking of ESA abuse. In particular, interindividual differences as well as the sex and the timing of OF-sampling (pre- and post-prandial) were observed to have an impact on the analysis. In addition, the volume of OF in urine is of major relevance, but realistic amounts, which can be achieved e.g. by spitting once or twice, were found to impair the EPO analysis in a significant number of cases. In order to identify contaminations or urine samples with OF, detection methods targeting human salivary α-amylase (sAA) were developed, as it was found to be a specific and most abundant protein in OF. For this purpose, both a lateral flow strip test (rapid test) and a bottom-up proteomic assay involving tryptic digestion followed by LC-HRMS/MS analysis were evaluated in terms of selectivity, sensitivity, and stability. Carry-over effect as well as linearity were additionally assessed for the bottom-up proteomic approach. Both approaches successfully identified sAA in urine, and the negative controls and OF- enriched samples could be clearly distinguished from each other. However, the naturally excreted level of sAA in urine presented a major challenge. A proof-of-concept study revealed an intersection between individuals with naturally occurring high levels of sAA in urine and those with low levels despite contaminations with OF, e.g. due to degradation processes caused by high concentrations of proteases in OF. First follow-up experiments demonstrated that peptides of the protein “salivary acidic proline-rich phosphoprotein ½” could be used as complementary biomarkers, but further research is required to confirm and subsequently optimize this approach in terms of its applicability. The details of this study will be published elsewhere. 1 1 1 2 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Lecture RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0223 Martinez Brito D, de La Torre X, Botrè F Analysis of testosterone esters by gas chromatography-high- resolution mass spectrometry with low-energy electron ionization Laboratorio Antidoping FMSI, Federazione Medico sportiva Italiana, Rome, Italy Abstract Rationale High-resolution mass spectrometry (HRMS) has demonstrated to be an alternative platform for quantitative analyses, identifying unknown compounds and gathering information for elucidation of chemical structures. This work describes a method to detect thirteen esters of testosterone (T) in 0.1 mL of human serum by gas chromatography coupled to HRMS. Method Analytes were extracted from serum after deproteinization and liquid-liquid extraction. The trimethylsilyl derivatives were analyzed using a gas chromatograph coupled to HRMS at low electron energy to minimize molecule fragmentation. The acquisition in profiling full scan mode was applied with a resolving power of 30,000 at m/z 400. The method was evaluated for linearity, lower limit of quantitation (LOQ) and measurement uncertainty. Mass accuracy (MA) and mass extraction window (MEW) were also assessed. Results The T esters showed a linear response between 0.25-10 ng/mL (except for undecanoate, enanthate and propionate that showed linear responses between 0.5-10 ng/mL and isocaproate between 2-10 ng/mL). Detection limits remained between 0.1-0.5 ng/mL and accuracy between 81-119%. The MA (MEW = 10 ppm) was maintained between -2.4 and 4.8 ppm. Conclusions The method to detect T esters in serum using gas chromatography coupled to HRMS showed linear responses up to 10 ng/mL with adequate linearity, precision, and accuracy. It was possible to distinguish cholesterol from T-isocaproate based on the MEW of 10 ppm preventing false positives. The difference between the mass 458.32176 of T-isocaproate and the mass 458.39415 of cholesterol is higher than 100 ppm (Figure 1). Besides, this method in full scan acquisition allows searching for other biomarkers and/or unknown metabolites (and other ester forms not included here) but at a later stage if necessary. Since the results were encouraging, the need to look for criteria to identify the analytes when HRMS is used, is imposed. Published as: Martínez Brito D, Leogrande P, Jardines D, de la Torre X, Botrè F. Simultaneous quantification of endogenous biomarkers and detection of testosterone esters in human serum using gas chromatography-high-resolution mass spectrometry. Rapid Commun Mass Spectrom. 2024 Mar 30;38(6): e9685. doi: 10.1002/rcm.9685. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0224 Figure 1. Extracted ion chromatogram m/z 458.32 ± 0.5 Da in a positive control (A); extracted ion chromatogram m/z 458.3944 ± 10 ppm for cholesterol (B); reduced mass spectra for cholesterol trimethylsilyl derivative (B1); extracted ion chromatogram m/z 458.3216 ± 10 ppm for T-isocaproate and -caproate (C); reduced mass spectra for T-isocaproate trimethylsilyl derivative (C1); overlapping chromatograms B and C and mass spectra at the RT at which the two compounds coelute (A1). Also, chemical structures, formula and accurate mass are presented MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0225 Martinez Brito D, de La Torre X, Botrè F Correlation of androstenedione and testosterone measurements by LC-MS/MS and GC-LE-TOF Laboratorio Antidoping FMSI, Federazione Medico sportiva Italiana, Rome, Italy Abstract In the frame of the implementation of the steroidal module of the athlete biological passport (ABP) in serum, the World Anti-Doping Agency (WADA) has established the minimum requirements to quantify testosterone (T) and androstenedione (A4) in serum. The method should be based on liquid chromatography combined with tandem Mass Spectrometry (LC-MS ). This work evaluated an alternative method to quantify T and A4 in 0.1 mL of human serum using gas chromatography coupled to high- resolution mass spectrometry (HRMS) taking advantage of an assay designed to detect T-esters. Both procedures using LC-MS/MS and GC-HRMS instruments were validated using the 6PLUS1 Multilevel Serum Calibrator Set MassChrom Steroid Panel 2. Positive and negative quality controls and human serum samples were used to check the correlation between these two approaches. Correlation, regression analysis, and Bland-Altman tests showed a good correlation between these two procedures. Only the T limit of quantification did not meet the requirement established for this parameter. LOD of T was 0.2 ng/mL instead of 0.1 ng/mL as required. Figure 1. Scatter diagram of the regression analysis for A4 and T (upper panel) and Bland-Altman plots for A4 and T (lower panel) n ® ® MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0226 As required by WADA technical document TD-IDCR, the identification criteria are based on the fact that each measured mass used for identification shall be within ± 0.5 Da of the corresponding mass of the same diagnostic ion acquired from a reference analyzed in the same analytical batch. Also, when using single-stage MS, at least three diagnostic ions shall be acquired. The identification of analytes using HRMS instrumentation is not yet regulated in the technical document but given the increase in the use of this type of platforms in recent years in anti-doping laboratories, it is a situation that should be addressed. The data evaluation of T and A4 by GC-TOF was done with a MEW of ± 3 ppm from the accurate mass of A4 (m/z 430.2723) and T (m/z 432.2879), which represents a range of ± 0.000697 Da from the accurate mass of the substance. The second evaluated fragment was the [M -CH ] characteristic of the trimethylsilyl derivatives. In this case, given the characteristics of the mass spectra of both compounds (intense molecular ion and low fragmentation), the error of the second fragment was ± 8 ppm (± 0.00186 Da), which is more than 250 times lower than the value of 0.5 Da established in the technical document TD-IDCR. The TD-IDCR does not include the acceptance criteria when HRMS platforms are used, but it should be considered because of the expansion of its use in antidoping laboratories. + 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0227 Martinez Brito D, de La Torre X, Botrè F Effect of thyroid hormones administration on the urinary endogenous steroid profile of the athlete biological passport Laboratorio Antidoping FMSI, Federazione Medico sportiva Italiana, Rome, Italy Abstract This work focused on the possible alterations of the markers of the steroidal module of the Athlete Biological Passport, considering samples of athletes declaring and not-declaring the supplementation with thyroid hormones (TH) in the Doping Control Form (DCF). Concentrations of 5α-androstane-3α,17β-diol (5α-Adiol), 5β-androstane-3α,17β-diol (5β-Adiol), testosterone (T), androsterone (A), etiocholanolone (Etio), epitestosterone (E), pregnanediol (PD), dehydroepiandrosterone (DHEA) and 11β-hydroxy-androsterone (OHA) were established by gas chromatography-tandem mass spectrometry. Metabolic ratios between the above biomarkers were also estimated. The data set was composed of samples from females and males declaring (FD and MD) and not-declaring (FND and MND) TH supplementation in the DCF. To corroborate these observations, a controlled urinary excretion study was carried out with multiple doses of sodium liothyronine (T3) in a healthy female volunteer. Data from females showed significant differences for the concentrations of 5α-Adiol, A, DHEA, E, OHA, and T and the ratio A/Etio between FD and FND groups (Figure 1), while the male groups only showed significant differences in OHA concentration. In both cases, males and females declaring the consumption of levothyroxine showed a narrower data distribution and diminished percentiles from 17 to 67% with respect to the non-declaring corresponding groups. Concentrations of 5α-metabolites showed a higher depression in the FND, and both FD and MD groups showed an odd behavior for the PD concentrations. The controlled urinary excretion study agreed with the observations, mainly for the female group. Significant differences were observed for concentrations of E, Etio, 5α-Adiol, 5β-Adiol. The interpretation of the steroid markers of the ABP steroidal module should consider TH administrations, due to the actions and metabolism of TH that involve androgen and estrogen metabolism. Published as: Martínez Brito D, de la Torre X, Botrè F. Effect of thyroid hormones administration on urinary endogenous steroid profile of the athlete biological passport. Drug Test Anal. 2023 Nov-Dec;15 (11-12):1361-1370. doi: 10.1002/dta.3534. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0228 Figure 1. Box-and-whisker plot showing the minimum, maximum and median value, and the first and third quartiles of the data for the female groups. Data identified as white boxes correspond to the group not declaring consumption of TH and orange boxes correspond to the group declaring the consumption of TH for testosterone, epitestosterone, androsterone and etiocholanolone concentrations, and T/E and And/Etio ratios. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0229 Sobolevsky T , Walpurgis K , Görgens C , Fedoruk M , Lewis L , Ahrens B , Thevis M Detection of capromorelin in urine following different routes of administration UCLA Olympic Analytical Laboratory, Los Angeles, United States ; Institute of Biochemistry, German Sport University Cologne, Cologne, Germany ; U.S. Anti-Doping Agency, Colorado Springs, United States Abstract Capromorelin (CP-424,391) is a non-peptidic ghrelin receptor agonist that has been shown to stimulate production of growth hormone (GH) and insulin growth factor-1 in humans and other animal species. It was originally developed by Pfizer as a potential treatment for cachexia and frailty in the elderly, but its development was eventually discontinued. Subsequent studies demonstrated capromorelin’s ability to increase food intake in animals, leading to approval in the U.S. and Europe as an appetite stimulant for cats (Elura) and dogs (Entyce). Capromorelin is prohibited in sports due to its ability to stimulate GH production and enhance performance, with any amount detected in an athlete’s sample to be reported as an adverse analytical findings (AAF).Given that its veterinary preparation is formulated as a highly concentrated solution (20 or 30 mg/mL) delivered orally, incidental ingestion or dermal absorption may result in an AAF by way of direct exposure during oral administration to a pet. An administration study was conducted by either oral or transdermal application of capromorelin solution to mimic the scenario of inadvertent exposure to the drug. Ingestion of 30 µg of capromorelin orally (equivalent to 1 µL of Entyce) resulted in detectable amounts of capromorelin in urine for up to 48 hours after administration with a maximum urinary concentration of 7 ng/mL. Importantly, when applied directly to the skin on the hands in larger quantities mimicking a pet administration exposure scenario (30 mg, or 1 mL of Entyce), capromorelin was also detected reaching a maximum urinary concentration of 0.7 ng/mL. Athletes and testing authorities should be aware of the risk of an AAF arising due to incidental exposure to veterinary preparations of capromorelin. To our knowledge, before 2022, no positive test for capromorelin had ever been reported. Published as: Sobolevsky T, Walpurgis K, Goergens C, Fedoruk M, Lewis L, Ahrens B, Thevis M. Detection of capromorelin in urine following oral and dermal routes of administration. Drug Test Anal. 2023 Nov-Dec; 15(11-12):1449-1453. doi: 10.1002/dta.3573. 1 2 2 3 3 1 2 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0230 Andersson A , Pohanka A , Ekström L 18-Methyl-19-noretiocholanolone detected after use of emergency contraceptive Department of Laboratory Medicine, Karolinska Institut, Stockholm, Sweden ; Doping Control Laboratory, Karolinska University Hospital, Stockholm, Sweden Abstract The world anti-doping agency puts forth a list each year which prohibits substances to keep sports clean. Athletes are inherently responsible for everything they consume, which becomes an issue when the metabolic pathways of prohibited and non-prohibited compound intersect. This was the case when an 18- methylnandrolone metabolite was detected in a routine sample of an athlete that had recently ingested an emergency contraceptive pill. Our aim was to study the metabolism and excretion of levonorgestrel in urine. Five women were recruited into a study to elucidate the link between 18-methyl-19- noretiocholanolone and levonorgestrel. After providing a pre-treatment sample, NorLevo was ingested and six additional samples were collected. The sample were analysed with GC-MS/MS after extraction and derivatisation. This study demonstrates a metabolic link between 18-methyl-19-noretiocholanolone and levonorgestrel. Confirming the need to exclude a levonorgestrel intake before reporting an adverse analytical finding. If not, women might be penalised for the use of hormonal contraceptives. 1,2 1,2 1,2 1 2 ® MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0231 Euler L , Mürdter T , Heinkele G , Schwab M , Miller GD , Eichner D , Thomas A , Thevis M Identification of (Z)-3‘-hydroxy clomiphene as a new potential doping- relevant metabolite of clomiphene Institute of Biochemistry, German Sport University, Cologne, Germany ; Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany ; Sport Medicine Research & Testing Laboratory, Salt Lake City, USA Abstract The anti-estrogen clomiphene is used therapeutically to induce ovulation in women. This effect has also been observed in animals. For instance, studies with laying hens that received orally administered clomiphene have shown a significantly increased egg production rate but, as a consequence, eggs were found to incorporate residues of clomiphene. A recent study has shown that consumption of just two eggs containing clomiphene residues could result in an AAF for clomiphene in a doping control. To protect athletes from sanctions due to anti-doping rule violations arising from an unintentional ingestion of the doping agent, a method was developed to distinguish whether clomiphene was ingested in its pharmaceutical form or as residues of the substance in contaminated eggs. After consumption of clomiphene-containing eggs, (Z)-4-hydroxy clomiphene accounted for over 90% of the hydroxy metabolites. In contrast, in urine from 8 out of 9 participants who had received a microdose of (E/Z)-clomiphene citrate, (Z)-3-hydroxy clomiphene, a metabolite of (E)- clomiphene, accounted for > 80% of the mono-hydroxylated metabolites. Re-analyses of doping control samples with clomiphene AAFs demonstrated that the major hydroxy clomiphene isomer could not be assigned to any of the reference compounds available in this study. Comparison with the microdose excretion study samples suggested that this hydroxy metabolite may be a long-term metabolite. Ion mobility experiments were performed to obtain preliminary information on the structure of this metabolite. Based on this data, new reference compounds were synthesized to be included in the differentiation method. Consequently, (Z)-3'-hydroxy clomiphene was identified as the potential long- term metabolite. Using this updated method, samples from a previous study were re-analyzed in which male study participants had received therapeutic doses for 30 days. The excretion profiles of two volunteers were measured. In both cases, (Z)-3'-hydroxy clomiphene was excreted and the relative concentration increased only after peak concentrations. Published as: Euler L, Mürdter T, Heinkele G, Schwab M, Miller GD, Eichner D, Thomas A, Thevis M. Identification and synthesis of (Z)-3'-hydroxy clomiphene as a new potential doping-relevant metabolite of clomiphene. Rapid Commun Mass Spectrom. 2023 Sep 15;37(17):e9599. doi: 10.1002/rcm.9599. 1 2 2 2 3 3 1 1 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0232 Wen H , Wagener F , Neudörfl J , Thevis M , Schäfer M Investigation of the phase I metabolism of a new 20-keto-steroid S42 by C-6 oxidation and GC-MS analysis Institute of Organic Chemistry, University of Cologne, Cologne, Germany ; Institute of Biochemistry, German Sport University Cologne, Cologne, Germany Abstract Introduction Selective androgen receptor modulators (SARMs) are relevant as illicit drugs for performance enhancement in the sports doping context due to their anabolic properties combined with less side effects based on their tissue selectivity [1]. In 2009, a new synthetic 20-keto-steroid S42 was introduced as a new SARM candidate [2]. Since S42 showed anabolic and anti-catabolic effects on cultured myotubes, it was proposed to be applied as a cure for muscle-related diseases [3], triggering also use in sports doping [4]. However, fundamental understanding of the GC-MS behavior of S42 and its metabolism is indispensable for reliable qualitative and quantitative analysis of this pharmaceutical and of its metabolites in body fluid matrices. Preliminary Data S42-mono-OH, S42-bis-OH and S42-tris-OH were found by GC-MS analysis after S42 phase I metabolism. The structure of S42-mono-OH was closely analyzed, and we assign the hydroxylation position to be in ring A or B based on 3 experimental findings. Firstly, the mass difference of S42-TMS isomers and S42- d -TMS isomers decreased from 6 Da to 5 Da after phase I metabolism. This implies that the possible hydrolysis position is at C-1 to C-3, C-6 or C-7. Secondly, the dominating signals in the GC-Mass spectrum of TMS derivatized S42-mono-OH (1) are found at m/z 219.1200, at m/z 245.1359 and at m/z 297.1671 suggesting that the TMSO functional group is attached to either rings A+B or A+B+C (Figure 1b). This assumption rests on the significantly mass shifted fragment ions of the respective TMS-S42 plus 88 Da ([M-H+TMSO]) molecular ions. Furthermore, [TMSOH] and [TMSOD] peaks were found of the ions noted above, indicating the TMS moiety in tandem MS experiments. However, the TMSO group may not be at the A ring since this elimination reaction is not favorable at an aromatic ring. Thirdly, an indicator ion at m/z 143.0887 which originated from the underivatized D-ring of TMS-S42, was observed. Therefore, a hydroxylation at the D-ring is ruled out. In addition, a S42-C6-ketone was synthesized and the TMS derivatized substance (2) showed similar fragment ion formation with a mass difference of 2 Da to TMS- S42-mono-OH (1) (Figure 1). These observations indicate that the B-ring hydroxylation in the positions C-6 or C-7. 1 2 1 2 1 1 2 6 • + • + MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0233 Figure 1. GC-MS spectra of TMS derivatized S42-C6-ketone (2) and S42-mono-OH (1) References 1. Solomon ZJ, Mirabal JR, Mazur DJ, Kohn TP, Lipshultz LI, Pastuszak AW. Selective Androgen Receptor Modulators: Current Knowledge and Clinical Applications. Sex Med Rev. 2019 Jan;7(1):84-94. doi: 10.1016/j.sxmr.2018.09.006. 2. Min L, Yanase T, Tanaka T, Fan W, Nomura M, Kawate H, Okabe T, Takayanagi R, Nawata H. A novel synthetic androgen receptor ligand, S42, works as a selective androgen receptor modulator and possesses metabolic effects with little impact on the prostate. Endocrinology. 2009 Dec;150(12):5606-16. doi: 10.1210/en.2009-0405. 3. Muta Y, Tanaka T, Hamaguchi Y, Hamanoue N, Motonaga R, Tanabe M, Nomiyama T, Nawata H, Yanase T. Selective androgen receptor modulator, S42 has anabolic and anti-catabolic effects on cultured myotubes. Biochem Biophys Rep. 2019 Jan 15;17:177-181. doi: 10.1016/j.bbrep.2019.01.006. 4. Thevis M, Walpurgis K, Thomas A. Analytical Approaches in Human Sports Drug Testing: Recent Advances, Challenges, and Solutions. Anal Chem. 2020 Jan 7;92(1):506-523. doi: 10.1021/acs.analchem.9b04639. 5. Uyanik C, Malay A, Hanson JR, Hitchcock PB, Tiryakioglu S. Ring a Aromatic Steroids in the Pregnane Series. Journal of Chemical Research. 2006;(7):417-419. doi:10.3184/030823406777980619 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0234 Peters I , Naumann N , Görgens C , Guddat S , Thevis M 3D cultivation of HepaRG cells for metabolite prediction of the calstabin-ryanodine receptor complex stabilizer S107 in preventive doping research Center for Preventive Doping Research - Institute of Biochemistry, German Sport University, Cologne, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne, Germany Abstract In preventive doping research, the prediction of potential metabolites of substances banned in sport is essential. This applies to non-approved substances that are assigned to the category S0 of the World Anti-Doping Agency (WADA) Prohibited List, such as the calstabin-ryanodine receptor complex stabilizer S107. In general, various in vivo and in vitro approaches can be applied to predict metabolite formation and secretion of these substances into urine. Human excretion studies provide the most accurate prediction of metabolites; however, these in vivo studies are often difficult to perform with non-approved substances. Another in vivo option is to perform animal studies, where transferability from animal model systems to humans is usually limited. 2D in vitro cell culture models with human cell lines or experiments with human liver microsomes and S9 liver fractions can provide first indications of metabolites, but are not complex enough to mimic human physiological conditions. Therefore, in the present study, a 3D-liver model was applied for metabolite prediction of the drug candidate S107 by using HepaRG liver spheroids cultured in an ultra-low attachment multi-well plate. The metabolic transformation of S107 was analyzed during a three-week experimental period with repetitive substance application. Supernatants at the experimental time points d3 to d21 were analyzed by HPLC-HRMS/MS to identify the metabolites formed. It can be shown that the metabolic profile found is comparable to that one detected in previous in vitro studies. This leads to the conclusion that HepaRG liver spheroids are an appropriate model system to further investigate the metabolic biotransformation of relevant substances in preventive doping research. In addition, the 3D cultivation of HepaRG cells is planned to be used in the so-called organ-on-a-chip technology to even better mimic physiological conditions in humans. The details of this study will be published elsewhere. 1 1 1 1 1,2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0235 Keiler AM , Rzeppa S , Voss SC , Thieme D In vitro biotransformation of voxelotor Institute of Doping Analysis and Sports Biochemistry (IDAS) - Dresden, Kreischa, Germany ; Environmental Monitoring & Endocrinology, TU Dresden University of Technology, Faculty of Biology, Dresden, Germany Abstract Voxelotor (GBT440) has been integrated in the List of Prohibited Substances and Methods under category M 1 Manipulation of Blood and Blood components only on January 1 2023 [1]. This haemoglobin S polymerisation inhibitor is a therapeutic treatment of patients with sickle cell disease. Voxelotor increases haemoglobin’s oxygen binding affinity and thereby enhances arterial oxygen saturation. In addition, first-in-human studies showed increased serum erythropoietin levels and haemoglobin concentrations in healthy adults as side effects [2]. The present study aimed at the identification and characterization of voxelotor metabolites for doping control purposes. In vitro biotransformation was performed using human HepG2 cells and human pooled liver microsomes. For analysis, we performed high-performance liquid chromatography coupled to tandem mass spectrometry or to high-resolution mass spectrometry. The HepG2 cells generated three phase I metabolites resulting from mono- hydroxylation and reduction, and six phase II metabolites resulting from mono-hydroxylation, reduction and O-methylation or glucuro-conjugation in different combinations. Moreover, the glucuro-conjugate of voxelotor was also formed as a minor metabolite. The pooled human liver microsomal incubation only yielded two phase I metabolites, one also formed by the HepG2 cells. With the spectrometric behaviour of voxelotor and its in vitro metabolites described herein, an implementation in doping control screening and consequently a detection of an abuse in an athlete urine sample might be possible. The presence of the metabolites identified in vitro in human urine has to be proven in future studies. Published as: Rzeppa S, Voss SC, Thieme D, Keiler AM. Identification of human in vitro metabolites of the haemoglobin S polymerization inhibitor voxelotor for doping control purposes. Drug Test Anal. 2023 Nov-Dec;15(11- 12): 1403-1409. doi: 10.1002/dta.3489. References 1. World Anti-Doping Agency. List of Prohibited Substances and Methods. World Anti-Doping Code Int Stand. 2023 2. Hutchaleelaha A, Patel M, Washington C, Siu V, Allen E, Oksenberg D, Gretler DD, Mant T, Lehrer-Graiwer J. Pharmacokinetics and pharmacodynamics of voxelotor (GBT440) in healthy adults and patients with sickle cell disease. Br J Clin Pharmacol. 2019 Jun;85(6):1290-1302. doi: 10.1111/bcp.13896. 1,2 1 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0236 Sahu PL , Matsa R , Pawar S , Seth K , Radhakrishnanand P , Murty U , Handa T , Sahu K , Kori RK , Kumar P Synthesis and characterization of ostarine metabolite: applicable for TL 12 National Dope Testing Laboratory, New Delhi, India ; National Institute of Pharmaceutical Education and Research, Guwahati, India, Guwahati, India Abstract The World Anti-Doping Agency (WADA) publishes and updates a prohibited substance list annually. It has a variety of substances and metabolites that are rarely found. Ostarine, which would further metabolize to O-dephenyl ostarine, is one of the substances in the “Other Anabolic Steroid” category. Furthermore, since O-dephenyl ostarine could also be present in urine samples as a contaminant/impurity and/or minor metabolite of the permitted drug bicalutamide, this metabolite shall not be considered as the sole criterion for the reporting of an Adverse Analytical Finding (AAF) for ostarine (TL 12). Hence, this metabolite plays a significant role for the reporting of AAF. Subsequently, it was planned to synthesize reference material (RM) of the ostarine metabolite. The present poster describes the synthetic route of O-dephenyl ostarine and its characterization studies. Keywords: O-dephenyl Ostarine, Synthesis, Technical Letter (TL 12), The prohibited list. 1 2 2 2 2 2 1 1 1 2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0237 Walpurgis K , Agricola J , Thomas A , Thevis M Myostatin inhibitory peptides in sports drug testing Institute of Biochemistry/Center for Preventive Doping Research, German Sport University Cologne, Cologne, Germany ; Division of BioAnalytical Chemistry, Faculty of Science, Vrije Universiteit Amsterdam, Amsterdam, Netherlands ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract Across species, skeletal muscle mass is negatively regulated by the TGF-β cytokine myostatin (MSTN). Inhibitors of this growth factor and its signaling pathways are therefore not only promising therapeutics for muscular diseases but also potential performance-enhancing agents in sports. Within this study, protein precipitation and liquid chromatography high-resolution tandem mass spectrometry (LC- HRMS/MS) were employed to develop a detection method for six novel MSTN inhibitory peptides derived from the regulatory MSTN propeptide and the natural MSTN inhibitor follistatin (FST) from doping control serum samples. The approach was comprehensively characterized and found to allow for a specific detection down to concentrations of 3–9 ng/mL. Moreover, several potential metabolites of the drug candidates referred to as DF-3, DF-25, and Peptide 7 were identified as valuable complementary analytical targets for doping control analytical assays. Overall, the acquired data pave the way for an implementation of MSTN inhibitory peptides into routine sports drug testing. Even though no drug candidate has obtained clinical approval yet, a proactive development of detection assays is of utmost importance to deter athletes from misusing such compounds, which are readily available for research purposes and on the black market. Published as: Walpurgis K, Agricola J, Thomas A, Thevis M. Myostatin inhibitory peptides in sports drug testing. Drug Test Anal. 2023 Nov-Dec;15(11-12):1477-1487. doi: 10.1002/dta.3473. 1 2 1 1,3 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0238 Walpurgis K , Gäde A , Thomas A , Gochard S , Delahaut P , Thevis M Detection of extracellular hemoglobin from Arenicola marina in doping control serum and plasma samples by means of LC-HRMS/MS Institute of Biochemistry/Center for Preventive Doping Research, German Sport University Cologne, Cologne, Germany ; Pig for Life SA, Marche-en-Famenne, Belgium ; CER Groupe, Marche-en-Famenne, Belgium ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract The manipulation of blood and blood components in sports is prohibited at all times and besides blood transfusions, also hemoglobin-based oxygen carriers (HBOCs) can be employed to artificially improve the oxygen transport capacity of the blood. As cell-free hemoglobin (Hb) is not only very ineffective in reversible oxygen binding and release, but further exhibits toxic effects, different strategies for Hb stabilization were employed during HBOCs development. Unfortunately, the in vivo use of these products was still found to be associated with serious side effects such as vasoconstriction and hypertension. However, the natural giant extracellular Hb from the marine invertebrate Arenicola marina (lugworm) could be a promising alternative for both transfusion medicine and cheating athletes, as it was found to be well tolerated in pre-clinical animal studies. Within this research project, lugworm Hb was implemented into the existing doping control detection method for bovine HBOCs based on ultrafiltration (cut-off: 100 kDa), tryptic digestion, and LC-HRMS/MS. For the mass spectrometric detection of lugworm Hb, two precursor-product ion pairs for a total of four tryptic peptides originating from subunits hbA2 (T ), hbB1 (T & T ) and the linker chain (T ) were employed. The modified approach was comprehensively characterized and found to allow for a both specific and sensitive detection of lugworm Hb down to concentrations of 10 µg/mL from 50 µL of serum or plasma. Therefore, it can serve as confirmation procedure for Lugworm Hb following visual or electrophoretic screening. Moreover, a proof-of-concept rat administration study was conducted, and the observed detection windows of at least 4 (dose: 200 mg/kg) and 8 hours (dose: 600 mg/kg) demonstrated, that the presented approach can be readily employed to efficiently test in-competition doping control samples for the presence of the drug. Published as: Walpurgis K, Gäde A, Thomas A, Gochard S, Delahaut P, Thevis M. Detection of extracellular hemoglobin from Arenicola marina in doping control serum samples by means of liquid chromatography and high- resolution tandem mass spectrometry. Drug Test Anal . 2023 Nov-Dec;15(11-12):1430-1438. doi: 10.1002/dta.3591. 1 1 1 2 3 1,4 1 2 3 4 7 3 6 17 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0239 Fiallo Fernandez T, Mc Pherson Medina A, Martinez Brito D, Montes de Oca Porto R Evaluation of the Cuban CBSSEPO monoclonal antibody for the detection of erythropoiesis-stimulating agents in doping control Antidoping Laboratory, Havana, Cuba Abstract Background Erythropoietin levels are one of the essential factors for the homeostasis of the human organism. Since its cloning and commercialization as recombinant human erythropoietin in its different glycosylation variants (ESAs), its use for doping purposes has been increasing. After their inclusion in the Prohibited List, WADA issues and periodically updates a technical document (TD-EPO) that establishes the analytical conditions for the detection of these compounds in urine and blood. Looking for an alternative to the primary monoclonal antibody established in TD-EPO, the Cuban CBSSEPO monoclonal antibody, initially designed for quality control activities in the industry, was evaluated. Experimental Different concentrations were used for CBSSEPO (200, 500, 1000 ng/mL) and the analytical signals for each of the ESAs were compared taking into consideration those obtained with the monoclonal antibody BAM2871. Time for the lecture on the camera Image Quant LAS 4000 mini and the quality of images were assessed by using the GASepo software quantitative analysis of digital images. EPO-Fc, rEPO, CERA and NESP were tested at 0.25(MRPL), 0.5(MRPL), MRPL, 2(MRPL) and 3(MRPL) levels. Results The optimal working concentration of the Cuban CBSSEPO was 1 µg/mL, five times more than the concentration used for the BAM2871. The exposure time varied from 60 to 300 seconds depending on the epoetin tested. The minimum concentration observed in urine matrix for rEPO and EPO-Fc was the MRPL level, while for CERA was 3(MRPL) level. It was not possible to detect NESP in any of the concentrations studied since an analytical signal that corresponds to the current technical document was not obtained. For that reason, Cuban CBSSEPO monoclonal antibody does not meet the requirements to detect ESAs with doping purposes at least in his current design. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0240 He S, Liu X, Zhou X Administration study of rEPO on c.577del carriers Beijing Anti-Doping Laboratory, Beijing Sport University, Beijing, People's Republic of China Abstract The issue of the variant c.577del in the human EPO gene has been found in recent years. Although WADA has published an annex to the TD2022EPO (Annex B) to direct how to deal with the rEPO positive samples, complicated and time-consuming procedure made further analysis inefficiently, on the other hand, the abuse of rEPO by the variant c.577del carriers is difficult to detect. In order to solve this issue, the method of analyzing de-N-glycosylated EPO in blood samples was developed, both VAR-EPO and rEPO can be determined using this method, however, the method cannot be applied until it is evaluated with rEPO excreted samples from variant carriers. For this purpose, five heterozygous carriers of the variant c.577del were recruited in an administration study of rEPO, urine and blood samples were collected at different time before and after subcutaneous injection with single-dose, and the samples were conserved appropriately until analysis. A series of experiments were designed, the urine samples collected from this administration study, including blank samples and washed-out samples, were analyzed for intact EPO with SAR-PAGE, while the serum samples were analyzed for both intact EPO and de-N-glycosylated EPO with SDS-PAGE. The result of urine samples showed that the typical smear band was detected in both blank samples and washed- out samples, which were difficult to distinguish. The result of serum samples showed that, for the analysis of both intact EPO and de-N-glycosylated EPO, double bands with similar intensity were detected in blank samples. While, for the washed-out serum samples, the intact EPO in samples collected in the first few days after injection showed a typical smear band , then two separated bands were detected vaguely several days after injection. The de-N-glycosylated EPO in all washed-out samples showed two well-separated bands with different intensity. The intensity ratio of lower band to upper band decreased with wash-out periods. Due to this result, we can not only determine whether the blood sample is collected from the variant c.577del carrier, detect rEPO according to the ratio of lower band to upper band, so the de-N-glycosylated EPO could be an effective complementary method for confirmation of rEPO in blood samples. Published as: He S, Liu X, Song H, Zhou X. Administration study of recombinant erythropoietin on the carriers of variant c.577del in EPO gene. Drug Test Anal. 2023 Nov-Dec;15(11-12):1410-1416. doi: 10.1002/dta.3566. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0241 Joonyeop Y , Minyoung K , Mijin J , Hophil M , Byung-Gee K , Junghyun S , Changmin S Direct multiplex-PCR based gene doping detection for human growth hormone and its isoform using CRISPR/Cas12a-assisted HOLMES Korea Institute of Science and Technology, Doping Control Centre, Seoul, Korea ; Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul, Korea ; Bio-MAX/N-Bio Institute, Seoul National University, Seoul, Korea Abstract With the recent development of gene therapy technology, the unethical genetic attempt to improve athletic performance became an immediate threat to the field of anti-doping. This kind of genetic approach is called gene doping and is prohibited by the World Anti-Doping Agency. Various DNA analysis techniques such as quantitative PCR, digital PCR, real-time PCR, loop-mediated isothermal amplification (LAMP), and targeted next-generation sequencing (NGS) have been developed, and research on the development of exogenous gene detection methods using these technologies is also attempted. One of these techniques, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based detection methods such as Specific High Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR), and one-HOur Low-cost Multipurpose highly Efficient System (HOLMES) has demonstrated its high sensitivity in nucleic acid detection. These methods utilize the target-dependent trans-cleavage activity of the CRISPR-associated proteins (e.g., Cas12a, Cas13, etc.) along with fluorescence-labeled single-stranded DNA reporter (ssDNA), allowing for highly sensitive and versatile applications in genetic diagnosis. In this study, we aimed to develop a direct multiplex-PCR based gene doping detection assay using CRISPR/Cas12a-assisted HOLMES for human growth hormone (hGH) and its isoform. The gene of hGH is named 22K-GH and lacks the first 45 nucleotides in exon 3 of 22 kDa- G H is named 20K-GH, were selected as potential targets due to their short half-life in serum and well-known metabolic effects such as muscle mass increasing. During the direct multiplex-PCR, each 22K-GH and 20K-GH could be selectively amplified from 5 μL of whole blood sample using an exon-exon junction spanning primers and the Locus Control Region (LCR) is also amplified as an internal control of PCR. In HOLMES, if amplified exogenous 22K-GH and 20K-GH genes exist in the reaction, the Cas12a/crRNA binary complex forms a ternary complex with PCR products, which in non-target ssDNA reporter (5’FAM-3’BHQ1) illuminates fluorescence by trans-cleavage. To confirm the exogenous GH gene and isoform, all PCR products were separated and visualized on a 2.5% agarose gel. As a result, 2.5~5 copies of the exogenous GH gene could be detected through our assay, and there were no cross-reactions and non-specific amplifications found. Our proposed method is highly convenient as it does not require pretreatment of blood and purification after PCR, making it a feasible gene doping detection assay with high sensitivity and specificity. 1,2 1 1 1 3 1 1 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0242 Mongongu C, Madi Moussa E, Marchand A, Buisson C, Ericsson M Top-down IGF-I quantification in serum using LC-HRMS analysis for longitudinal follow-up: impact of calibration on measurement variability Laboratoire AntiDopage Francais (LADF), Paris, France Abstract The determination of Insulin Growth factor-I (IGF-I) concentration is used for the diagnosis of Growth Hormone-related disorders. This growth factor is synthesized by the liver under the biological effect of GH (growth hormone). IGF-I circulates in the blood as a complex bound to specific high affinity proteins (IGFBPs), regulating its excretion and tissue availability. The quantification of IGF-I is widely performed using immunoassays for clinical purposes but some concerns about the reproducibility between assays have emerged. In recent years, IGF-I analysis using mass spectrometry (MS) has been developed (bottom-up and top-down analysis), to benefit from MS advantages in terms of accuracy, precision and the possibility to differentiate IGF-I from its variants (endogenous and exogenous). In the anti-doping field, IGF-I serum concentration is used as a biomarker of GH doping by athletes. In order to overcome interindividual variability, longitudinal monitoring of GH biomarkers will soon be implemented in the "Athlete's Biological Passport (ABP)" by the World Anti-Doping Agency (WADA). In this work, we evaluated different quantification strategies to determine IGF-I concentration based on a top-down analysis of the intact protein in serum. The sample preparation consists of a microelution solid phase extraction after IGF-I release from its binding proteins (protein precipitation). After dilution, the eluate is then directly analysed by LC-HRMS. Using 20 µL of serum, the developed method was successfully validated according to WADA requirements. The impact of different calibration strategies on the variability of IGF-I concentrations determined in intermediate precision tests for a low and a high quality control (QC) was evaluated. We tested either a calibration curve or a single point calibrator using IGF-I NIST SRM 2926 standard prepared freshly, or alternatively an endogenous human serum used as a single point calibrator. This latest was validated against IGF-I NIST SRM 2926 standard, aliquoted and stored frozen at -80 °C until analysis. Our results supports the use of a single point human serum- based calibrator as it allows the lowest variability in QCs IGF-I concentrations over time, which would be an asset for effective longitudinal monitoring. In addition, a second batch of endogenous human serum calibrator was prepared, calibrated against the first single point human serum-based calibrator and used to quantify IGF-I in the two QCs. The measured IGF-I values were similar to the ones obtained with the first single point human serum-based calibrator showing a very low variability over time. That validated the possibility of easily implementing this approach in anti-doping laboratories. A good practice would be that all laboratories use the same WADA-approved human serum-based calibrator to attribute the value of their own endogenous human serum used thereafter as calibrator. ® ® ® ® MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0243 Somay Selbes Y, Demirel AH, Elaldi K Comparison of Insulin-like growth factor 1 measured between Siemens Immulite and IDS-iSYS Turkish Doping Control Center (TDKM), Ankara, Turkey Abstract Insulin-like growth factor-1 (IGF-1) is a known key hormone that mediates the physiological response to both endogenous and exogenous growth hormone (GH). IGF-1, along with the N-terminal pro-peptide of type III collagen (PIIINP), has been used to detect the abuse of hGH, GH secretagogues, and IGF-I in sports. There are two WADA-defined immunoassays for the detection of IGF-1 levels in anti-doping laboratories. The dependence of these tests on a specific device and occasional problems in kit procurement raises the question of whether other devices and IGF-1 test kits can be considered as acceptable testing techniques in anti-doping laboratories. Therefore, in this study, IGF-1 results obtained with IDS iSYS were compared with Immulite 2000 IGF-I test results. Spearman's rank correlation coefficient showed a moderate correlation between the two measurements (ρ =0,473; p 0.98 are achieved using a weighting factor of 1/x, and the limit of quantification (LOQ) values are established with % RSD and % CV < 15, │% Diff│ < 20, and relative ion ratio < %20. The LOQ values are largely in the low ng/mL levels, which meet the screening and confirmation sensitivity needs of analytical methodologies in labs that routinely monitor sports anti-doping. Conclusion Transcend DSX-1 combines a dried spot autosampler and TurboFlow LC-MS/MS and provides a complete workflow for fast and robust quantification of 77 drug-of-abuse analytes in dried blood spots. 1 2 1 1 1 1 1 2 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0249 Buisson C, Smires S, Ericsson M Dried blood spots for doping controls – Validation of a quantification procedure for corticosteroids Université Paris-Saclay, Laboratoire AntiDopage Français, Chatenay-Malabry, France Abstract Introduction To improve doping control in sport, analytical procedures must constantly evolve to get ahead of new doping techniques and to meet unique challenges of sensitivity. Lately, a particular interest has been shown in dried blood spots (DBS) which simply consists of a drop of blood deposited on an appropriate paper for further analysis. Indeed, compared to conventional samples, like venous blood and urine, a DBS sampling can be less invasive, and reduce storage and shipping costs. Moreover, regarding doping control, DBS could be useful as a complementary matrix to urine for the detection and quantification of doping substances, enabling the correlation of pharmacological effects to urinary concentration. According to the World Anti-Doping Agency (WADA), glucocorticoids, prohibited only in competition by all routes of administration, are one of the most commonly used classes of prohibited substances by athletes. Therefore, the objective of this work is to develop and validate a DBS assay for glucocorticoids such as Triamcinolone acetonide (TA), 6βOH-Triamcinolone acetonide (6βOH-TA), and Betamethasone by LC-MS/MS. Methodology In order to develop a DBS assay, Capitainer® devices were spotted with simulated drops of fortified blood samples and the following parameters were evaluated in accordance with WADA technical documents: recovery, LOQ and dynamic ranges, repeatability and intermediate precision, measurement uncertainty (MU) and selectivity. The extraction method developed is divided into three steps. Firstly, 10 µL of the internal standard are put in a tube with the 10 µL spot, and the spot is diluted twice in a mix of 250 µL H O/CH CN (30:70). Secondly, a liquid-liquid extraction is realized with 400 µL of H O and 800 µL of TBME. Finally, the dry residue is taken up in 50 µL of mobile phase. The doping agents are separated along a gradient within 9.5 min on a Kinetex XB-C18 column. The mobile phase is constituted of H O 10 mM HCOONH /CH CN (initially 70:30), the flow is set up at 0.4 ml/min, and the volume of injection at 10 µL. Results For TA the dynamic range has been validated from 1 ng/mL to 20 ng/mL, for 6bOH-TA from 1.5 ng/mL to 20 ng/mL, and for Betamethasone from 2.5 ng/mL to 25 ng/mL. Performed tests showed a relative standard deviation (RSD) of repeatability lower than 10%, a RSD of intermediate precision lower than 15%, and measurement uncertainty lower than 15% for the three molecules for quality controls (QC) at low, middle and high level of concentration. The extraction yield has been rated greater than 75% for the three molecules. The study of blank samples made it possible to establish the specificity of the method. 2 3 2 2 4 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0250 At least two precursor-product ion transitions (SRM transitions) were validated to guarantee the identification capacity of the method. Conclusion The DBS assay developed is validated for the three glucocorticoids studied with LOQs < 2.5 ng/mL (10 µL of blood). The extraction method had a recovery higher than 85% with CV for repeatability < 10% for the three glucocorticoids, reinforcing the usefulness of volumetric DBS devices for quantification methods. The proposed protocol can thus be used in clinical studies required to establish MRL in blood. However, the need to prepare DBS devices spotted with fortified blood as calibrant may pose challenges for the implementation in routine. Therefore, in parallel of this validation, tests have been done on DBS cards spotted with fortified serum in order to simplify the practical application. However, the need to prepare DBS devices spotted with fortified blood as calibrant may pose challenges for the implementation in routine. Therefore, in parallel of this validation, tests have been done on DBS cards spotted with fortified serum in order to simplify the practical application. The results of these tests show a bias < 20% for the three glucocorticoids with a systematic underestimation for two of them (6βOH-TA and Betamethasone). The use of a correction factor could be considered to enable the use of serum as a matrix for calibration curves. Further tests are thus needed to validate this approach. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0251 Alechaga E , Bressan C , Coll S , Monfort N , Ventura R Multiclass method for the screening of doping agents in dried blood spots Catalonian Antidoping Laboratory Fundació Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain ; Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain Abstract Dried Blood Spots (DBS) are a sampling technique routinely used for newborn screening, and recently evaluated as an alternative to blood matrices such as serum or plasma. DBS possess several advantages with respect to conventional liquid biological samples: they simplify sample collection, storage and transport, and they generally present increased analyte stability. For these reasons, there is an increasing interest in adapting analytical methodologies for doping control to this new matrix. However, they require the use of highly sensitive methodology, due to the very low volume of sample (usually < 30 µL) collected in each spot. This work presents the development of a multi-class screening method for compounds belonging to different groups of the World Anti Doping Agency (WADA) prohibited list, including anabolic agents, growth hormone secretagogues, beta-2 agonists, aromatase inhibitors, anti-estrogenic substances and hypoxia-inducible factor activating agents. DBS samples for method development were obtained depositing 20 µL of venous blood and letting it to dry on pure cellulose cards. The whole DBS spot was then punched out and extracted prior to the analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. The chromatographic separation was carried out with a C18 column and a binary mobile phase of acetonitrile:water, both with 0.01% formic acid. The methodology was validated for qualitative purposes, and different parameters were evaluated, such as limit of detection, selectivity, recovery, matrix effects and intra-assay precision. The vast majority of the compounds could be reliably detected at sub-ng/mL level. Satisfactory results were obtained in terms of recovery and precision, considering only non-threshold compounds without Miminum Reporting Level (MRL). Also, matrix effects were negligible for most compounds, as expected considering the low amount of sample analyzed. Additionally, samples after the administration of boldenone, oxandrolone and tamoxifene to healthy volunteers were analyzed. The method showed good performance and robust results, making it fit-for-purpose for its application to doping control. 1,2 1 1 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0252 Musenga A, Langer T, Salamin O, Nicoli R, Kuuranne T A fast screening of doping agents in DBS by LC-MS/MS Swiss Laboratory for Doping Analyses, University Center of Legal Medicine Lausanne-Geneva, Lausanne University Hospital and University of Lausanne, Switzerland, Epalinges, Switzerland Abstract Doping control is mostly based on the analysis of urine samples, thanks to the relatively low-invasiveness of sample collection. However, this analysis mainly targets the metabolites of the prohibited substances. Blood samples, on the other hand, can provide a more representative information on the presence of substances at the time of collection as well as the possibility to target the parent compound for detection, though sample collection requires trained personnel (e.g. a phlebotomist), is more invasive and the shipment and storage requirements are more demanding (e.g. temperature control). Widely used for the screening of new-borns, the analysis of DBS has recently gained interest in the anti-doping community, since it can overcome some of the difficulties associated with blood samples [1]. This collection method is minimally invasive and the process of drying stops the degradation processes so that samples can be transported at room temperatures as long as the humidity is kept to a minimal level [2]. To regulate the use of this matrix for doping control, the World Anti-Doping Agency (WADA) has issued a technical document [3], which commands that the analysis can only be conducted on substances without threshold or reporting limits. Therefore, DBS qualitative analysis can be applied to the classes of substances that are prohibited in sports at all time, like anabolic agents, b2-agonists, diuretics and hormone modulators. Nevertheless, the DBS analysis of substances belonging to other classes such as stimulants or narcotics is feasible [4], even though the small sample volumes require sensitive techniques. We have developed a fast and comprehensive method for the detection in DBS of over 100 substances across all WADA classes S1-S9. Compounds are quickly extracted from either a cellulose or a polymer based support material using methanol. After the addition of hydrochloric acid to limit the evaporation of basic volatile compounds, the sample extract is dried under a flow of nitrogen and finally reconstituted with mobile phase. The analysis is performed with a 10 min gradient by UHPLC- MS/MS using a mobile phase composed of water, acetonitrile and formic acid. The method has been validated according to ISO 17025 and the International Standard for Laboratories and is currently used in routine for the analysis of non-threshold substances without MRL in agreement with WADA requirements. However, our validation also included substances belonging to the WADA classes S6-S9, demonstrating the capability to expand the analysis to these compounds in case new guidelines are released. References 1. Thevis M, Geyer H, Tretzel L, Schänzer W. Sports drug testing using complementary matrices: Advantages and limitations. J Pharm Biomed Anal. 2016 Oct 25;130:220-230. doi: 10.1016/j.jpba.2016.03.055. 2. World Anti-Doping Agency: International Standard for Testing and Investigations, 2023 3. World Anti-Doping Agency: Technical document TD2023DBS, 2023 4. Salamin O, Garcia A, González-Ruiz V, Rossi F, Bigard X, Déglon J, Daali Y, Faiss R, Saugy M, Rudaz S. Is pain temporary and glory forever? Detection of tramadol using dried blood spot in cycling competitions. Drug Test Anal. 2020 Nov;12(11-12):1649-1657. doi: 10.1002/dta.2923. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0253 Miyamoto A, Ota M, Sato M, Okano M Simultaneous detection method of 26 testosterone, nandrolone and boldenone esters in dried blood spots by LC-MS/MS Anti-Doping Laboratory, LSI Medience Corporation, Tokyo, Japan Abstract WADA approved the use of dried blood spots (DBS) as a sample collection method in doping control in 2021. Since testosterone esters are not directly excreted into urine, the IRMS analysis is essential for detecting the testosterone doping. On the other hand, testosterone esters are stable in DBS, and if they can be detected, IRMS analysis for their metabolites in urine is not required. It has been reported that direct detection of steroid esters in DBS is useful for detecting doping. In this study, a simultaneous detection method was developed for 11 testosterone esters, 9 nandrolone esters and 6 boldenone esters in DBS. Absorption devices for DBS were evaluated with DMPK-C (fingertip sampling) and Tasso M20 (brachial capillary sampling). The method allowed the detection of concentration levels at 0.5 ng/mL for all 26 steroid esters. In addition, the method allowed for confirmation analysis that all evaluated testosterone esters met the TDIDCR at 0.8 ng/mL. For applicability test, the capillary blood samples were collected using both devices after administration of testosterone enanthate (100 mg, i.m.) and testosterone undecanoate (80 mg, p.o.). Testosterone enanthate was detectable up to 96 h and testosterone undecanoate was detectable up to 6 h at least in DBS after each administration. If the MRPL is set at 1 ng/mL for testosterone esters, the method can be applied to doping control sampling using Tasso M20 and DMPK-C card. The full paper will be published elsewhere. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0254 Sletten C , Solheim SA , Bækken LV , Dehnes Y Effect of Sustanon administration on ALAS2 expression Norwegian Doping Control Laboratory, Oslo University Hospital, Oslo, Norway ; Anti Doping Denmark, Brøndby, Denmark Abstract ALAS2 mRNA has previously been proposed as a biomarker for blood doping-stimulated erythropoiesis. In this project we wanted to investigate the intra-individual variation and whether injection of testosterone esters (Sustanon ) affects ALAS2 expression. Capillary blood from fingerprick was collected on Whatman™ DMPK-C cards (GE Healthcare) 14 days before the injection (baseline sample) and 1, 3, 5, 10 and 14 days post injection. DBS cards were stored for 4-5 years at -80 °C before analysis. Quantitative PCR was performed on a an StepOne Plus Real-Time PCR System (Applied Biosystems) using Comparative C analysis with baseline samples as calibrators. Our preliminary results show that RNA can successfully be isolated from DBS after several years in storage. A marked intra-individual variation in the relative expression of ALAS2 was observed in both the treatment group and the placebo group. The CV variance in the relative expression of ALAS2 was higher after use of testosterone ester, although we could not find a significant difference in mean between the two groups, probably due to too few subjects. 1 1,2 1 1 ® 1 2 ® T MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0255 Heiland CE , Mongongu C , Semence F , Pohanka A , Ericsson M , Marchand A , Ekström L IGF-I variation in DBS and serum Doping Control Laboratory Karolinska University Hospital, Stockholm, Sweden ; Laboratoire AntiDopage Français, Université Paris-Saclay, Paris, France ; Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Abstract Background The athlete biological passport (ABP) now includes an endocrine module to longitudinally follow the endogenous growth hormone marker insulin-like growth factor 1 (IGF-I). However, DBS are not established for monitoring this biomarker. Here, we examine the correlation and inter-individual variability of the IGF-I concentration in DBS and serum samples for six weeks. Experimental Samples: DBS Mitra VAMS (Neoteryx, CA, USA) and serum (Becton Dickson, NJ, USA) were collected from 14 subjects (10 males, 4 females; median age 28 years old) over a six-week collection period (Swedish Ethical Review Committee permit Dnr 2020-04258). DBS samples were stored for about six months at room temperature before analysis took place and serum samples at -20 °C. Serum samples (n = 41) were analyzed in duplicate in the LC analysis. DBS samples were analyzed once with LC (n=63) and serum samples were analyzed once with the IDS-iSYS Multi-disciplined Automated System (n = 40). Reference standards, quality controls, calibrators: IGF-I SRM 2926 (NIST, Gaithersburg, MD, USA) stock solution (305 µg/µL IGF-I) was diluted to make an IGF-I reference stand solution (10 ng/µL), also containing 37 ng/µL IGFBP-3 (mole-to-mole; Sigma-Aldrich, Saint-Quentin Fallavier, France). The internal standard N-IGF-I working solution (5 ng/µL) was diluted from SRM 2927 (285 ng/µL N-IGF-I; NIST, Gaithersburg, MD, USA). IGF-II was used at a concentration of 50 ng/µL. Rat serum was used to prepare the working solutions. Methodology: DBS samples were sonicated for 2 hours and both DBS and serum underwent protein precipitation before extraction of IGF-I using Biotage Extrahera LV based on a previously described method (1). A calibration curve was prepared in the same way. LC analysis for DBS used a Zorbax SC18 column, 25-µL injection volume, and a flow rate of 500 µL/min (mobile phase A: 0.1% formic acid/H O, B: 0.1% formic acid/acetonitrile). LC analysis for serum used a Triart C18 column, 5-µL injection volume, and a flow rate of 400 µL/min (same mobile phases). IDS-iSYS was used as previously described (2). Results and discussion There was a strong correlation in IGF-I concentration between serum and DBS (rs = 0.86, p < 0.0001), particularly at lower concentrations in DBS (Figure 1A). The concentration of IGF-I in DBS was about 5x lower than that in serum (Figure 1B). The CV% of IGF-I in DBS was 14-34% (n = 63), while in serum it was 4-12% (n = 24). Serum IGF-I concentrations measured with LC were similar to those measured with IDS- iSYS. After storing the samples for several months at room temperature, it is possible that IGF-I degraded 1 2 2 1 2 2 3 1 2 3 15 15 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0256 slightly and that the desorption was inefficient, thus contributing to lower detection levels in DBS. Overall, DBS may be used for longitudinal IGF-I monitoring when an inter-laboratory harmonization of sample storage, calibrator preparation, and methodology is performed. Figure 1. IGF-I concentrations in blood matrices. (A) Correlation in endogenous IGF-I concentration between DBS and serum from 9 subjects. (B) Intra-individual variation of IGF-I concentration in one participant over a six-week period References 1. Mongongu C, Moussa EM, Semence F, Roulland I, Ericsson M, Coudoré F, et al. (2020) Use of capillary dried blood for quantification of intact IGF-I by LC-HRMS for antidoping analysis. Bioanalysis. 12(11):737- 52. 2. Marchand A, Martin JA, Collot D, Hoang O, Roulland I, Semence F, et al. (2019) Combined administration of microdoses of growth hormone and erythropoietin: Effects on performance and evaluation of GH detection capability using anti-doping methods. Drug Test Anal. 11(11- 12):1698-713. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0257 Brockbals L , Thomas A , Schneider TD , Kraemer T , Steuer AE , Thevis M LC-MS/MS-based peptide analysis for dried blood spot sampling time point estimation Institute of Biochemistry, German Sport University, Cologne, Germany ; Department of Forensic Pharmacology, University of Zurich, Zurich, Switzerland Abstract Along with the recent acknowledgement of the World Anti-Doping Agency to use dried blood spot (DBS) samples for routine doping control purposes, there have been propositions to use DBS as a matrix that allows regular proactive remotely supervised self-sampling, providing potential longitudinal monitoring of an athlete’s exposure to doping agents. However, several organisational aspects have to be considered before implementation, such as the verification of the sample collections time-point. Based on a previous untargeted proteomics workflow utilizing liquid chromatography-high resolution mass spectrometry (LC- HRMS) to identify protein/peptide markers to define the time since deposition of a bloodstain, the aim of the current study was to develop a targeted LC-HRMS/MS analytical method for promising peptidic target analytes. A long-term DBS storage experiment was carried out over a three-month period (sample collection time-points: 0, 2, 4, 7, 14, 21, 28, 42, 56, 70, 84 and 91 days) with DBS samples of 10 volunteers for longitudinal investigation of signal abundance changes of targeted peptide sequences at different storage temperatures (room temperature (RT), 4 °C and -20 °C). Prior to experimental analysis, LC-HRMS/MS method characteristics were successfully assessed, including intra-day precision, carryover and sample extract stability. For estimation of DBS sample collection time points, ratios of two peptides that originate from the same protein prior to tryptic digestion were created. Two targeted peptide area ratios were found to significantly increase after stored at RT for 28 days, representing potential markers for future use in routine doping controls that contribute to advancing complementary avenues in anti- doping. Published as: Brockbals L, Thomas A, Schneider TD, Kraemer T, Steuer AE, Thevis M. Do dried blood spots have the potential to support result management processes in routine sports drug testing? – Part 3: LC-MS/MS- based peptide analysis for dried blood spot sampling time point estimation. Drug Test Anal. 2023 Feb 24. doi: 10.1002/dta.3463. 1 1 2 2 2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0258 Kim KH , Jeong W , Eun Byul C , Min H , Son J Development of an online immunopurification assay for urinary insulin and insulin mimetics using AF4-ESI-MS/MS Korea Institute of Science and Technology, Doping Control Centre, Seoul, Korea ; Chemistry, Sogang University, Seoul, Korea Abstract Insulins and its mimetics could perform a wide range of roles in the body such as a hormone that regulates blood sugar levels. Among these, the anabolic and anticatabolic effects of insulin drugs, related to muscle strength and endurance, may influence improving elite athletes’ performance. Therefore, they were prohibited by WADA as metabolic modulators, and it has been shown to be detected in urine metabolism, so the analysis of insulin in urine is receiving a great deal of attention. In this study, we demonstrated a new online immunopurification analysis method based on asymmetrical flow field-flow fractionation (AF4-ESI-MS/MS) for urinary insulin and six insulin mimetics: Lispro, Glulisine, Aspart, Degludec, Detemir, and Glargine. AF4 is a separation technique based on analytes size by the flow of a carrier solution, and the analytes are focused in a narrow lane on the device then small matrix could be removed through the membrane pores. In order to achieve immunopurification in AF4, the PVDF membrane and RC membrane were used for protein immobilization and filtration, and the pH of the mobile phase was adjusted to control the antigen-antibody reaction. As a result, the mass spectrum of analytes through immunopurification was confirmed. An increase in sensitivity was observed when protein A, which improves antibody orientation, was added to the immunopurification step. Moreover, the optimization was completed within 5 and 10 minutes, which is considerably shorter than the general method. The developed assay was validated for limit of detection (LOD), linearity, matrix effect, intra- day precision, and inter-day precision. Furthermore, studies were conducted on the enrichment performance of AF4 and its potential was confirmed. The AF4-ESI-MS/MS is able to perform the three steps including concentration, immunopurification, and mass spectrometry in a single run, and the immunopurification hyphenated mass spectrometry platform will be useful in the analysis of proteins from a wide variety of biological samples. 1 1,2 1,2 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0259 González-Rubio S , Caballero-Casero N , Ballesteros-Gómez A , Cuervo D , Muñoz G , Rubio S A general screening method for testing of banned substances in urine based on cubosomic supramolecular solvents and liquid chromato- graphy/time-of-flight mass spectrometry Department of Analytical Chemistry, Institute of Chemistry for Energy and the Environment, University of Córdoba, Córdoba, Spain ; Madrid Anti Doping Laboratory, Health Institute Carlos III, Madrid, Spain Abstract Multiclass screening methods in doping control should be comprehensive, allow high throughput sample processing and be cost-effective and green. In this respect, one of the greatest challenges of screening methods is how to extract efficiently the target compounds and reduce matrix effects while preventing the loss of chemicals with very different physicochemical properties during sample extraction and purification. Here, cubosomic supramolecular solvents (SUPRASs), able to extract efficiently multiclass substances while removing matrix interferences were firstly investigated for the multiclass extraction of banned substances in urine prior to their screening analysis by LC-QTOF/MS. For this purpose, eighty prohibited substances and/or their metabolites, selected from the 10 categories (S1-S9, P1) of the WADA list were tested. These substances included a wide range of polarity (log P from -2.4 to 9.2) and chemical structures (e.g. compounds with alcohol, amine, carboxyl, ether, ester, ketone, sulfonyl, etc. functional groups). The SUPRAS selected was synthesized directly in the urine by the spontaneous self-assembly and coacervation of 1,2-hexanediol, induced by sodium sulfate. Method validation was carried out according to WADA guidelines. No interfering peaks were observed for any of the 80 substances investigated. Around 84-93% of drugs were efficiently extracted (recoveries 70-120%) and 83-94% of the analytes did not show matrix effects (±20 %) in the ten urines tested. The only substance with recoveries below 30% for some urine samples was ecgonine methyl ester. It is worth noting that ritalinic acid (log P -2.4) was extracted with recoveries within the range 72.6-93.3%. Method detection and quantification limits for the drugs selected were in the intervals 0.002-12.9 ng mL and 0.007-43.2 ng mL , respectively. The applicability of the method was evaluated by the screening of thirty-six blinded and anonymized urine samples previously analyzed by conventional methods by the Doping Control Laboratory of the Health Institute Carlos III in Madrid. 1 1 1 2 2 1 1 2 -1 -1 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0260 Keiler AM , Derwand D , Rzeppa S , Voss SC , Thieme D QuEChERS as alternative extraction procedure in doping analyses Institute of Doping Analysis and Sports Biochemistry (IDAS) - Dresden, Kreischa, Germany ; Environmental Monitoring & Endocrinology, TU Dresden, Faculty of Biology, Dresden, Germany Abstract QuEChERS is a well-known extraction method in food analysis used for residue analyses, such as pesticides and mycotoxins [1]. The name reflects the advantages of this extraction procedure: Quick, Easy , Cheap, Efficient, Rugged, Safe. The procedure is based on a liquid-liquid extraction with acetonitrile in the presence of a buffer/salt-mixture, which leads to a phase separation. Aim of the present study was to investigate the extractability of the small molecules from groups S1 to S9 of the WADA Prohibited List. As a starting point, the parameters for the extraction were first optimized with regard to pH and buffer/salt ratio. With the optimized parameters, we were able to extract approximately 90 % of the small molecules targeted in our routine GC-MS/MS and LC-MS/MS screening procedure using the QuEChERS method. Furthermore, we observed significant improvements for individual substances and substance groups compared to our established methods. As example, the extractability of stanozolol- N-glucuronides and HIF modulators was significantly improved. Overall, this pilot study demonstrates the potential of QuEChERS as alternative extraction procedure for screening and confirmatory methods in doping analysis. Published as: Derwand D, Rzeppa S, Voss SC, Zschiesche A, Keiler AM. QuEChERS as alternative extraction procedure in doping analyses. Drug Test Anal. 2023 Dec 3. doi: 10.1002/dta.3610. References 1. Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and „dispersive solid-phase extraction“ for the determination of pesticide residues in produce. J AOAC Int. 2003;86(2):412–31. 1,2 1,2 1 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0261 Cristea C , Radu M , Catana D , Berghes B , Toboc A , Stan C Triple quadrupole UHPLC-MS/MS method as an alternative of orbitrap UHPLC-HRMS method used for detection of growth hormone-releasing hormones Romanian Doping Control Laboratory, Bucharest, Romania ; University of Bucharest, Faculty of Chemistry, Bucharest, Romania Abstract The use of growth hormone-releasing hormone (GHRHs) is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). Considering the complexity of urine samples and the low concentrations at which these analytes should be detected, analysis of GHRHs is a challenging task. In most of the studies, GHRHs are analyzed using UHPLC-HRMS with orbitrap. The aim of the current study was to develop and validate a method for the detection of growth hormone-releasing hormones (Tesamorelin, CJC-1295, CJC-1295 DAC, Sermorelin (1-29), Sermorelin (3-29)-NH2, Somatorelin) using triple quadrupole UHPLC/MS-MS method with solid phase extraction (SPE), based on weak cation exchange, which is able to detect concentrations as low as 0.2 ng/mL (Figure 1). This is due to the high extraction selectivity of the sorbent’s surface with carboxylic acid groups, which enables to target the analyte of interest removing interferences that can lead to misidentification of GHRH compounds and further false positive results, as well as due to the specificity of the UHPLC/MS-MS system, which ensure reliable analytical results, without interferences. The method developed by our laboratory was validated according to WADA technical documents for specificity, limit of detection, recovery, carry-over and robustness. The results show that the method has adequate recoveries and sensitivity, hence, it can be employed for routine screening in anti-doping laboratories. Published as: Cristea CD, Radu M, Toboc A, Stan C, David V. Cationic exchange SPE combined with triple quadrupole UHPLC-MS/MS for detection of GHRHs in urine samples. Anal Biochem. 2023 Dec 1;682:115336. Figure 1. UHPLC-MS/MS chromatograms recorded for: A. Urine samples spiked at 0.2 ng/mL with all compounds, B. Negative urine. 1,2 1 1 1 1 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0262 Upadhyay AK, Bhardwaj A, Sahu P Comprehensive identification method for hypoxia-inducible factor (HIF) activating agents National Dope Testing Laboratory, New Delhi, India Abstract Hypoxia-inducible factors (HIFs) activating agents are included in the Prohibited List of the World Anti- Doping Agency (WADA) because they can induce hypoxia, that results in increased red blood cells and erythropoietin (EPO) production. The detection of HIFs in doping control urines using a single method poses a challenge for anti-doping laboratories due to the required detection limit and structural variability in prohibited HIFs. The aim of this study is to develop a comprehensive identification method for the detection of HIFs in doping control urine samples. The sample preparation includes enzymatic hydrolysis followed by liquid-liquid extraction in alkaline and acidic phase using TBME and ethyl acetate. The method was developed on Liquid chromatography- tandem mass spectrometry (LC-MS/MS, 5500 Qtrap) coupled with Waters UPLC. The results of the method were found fit for the purpose of the identification of all prohibited HIFs in a single method. The limit of identification of the developed method is found from half MRPL to below 10 of MRPL for targeted HIFs as per the prohibited list. The LOI and specificity of the method demonstrate that it can identify targeted HIF-activating agents below half of MRPL in doping control samples. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0263 Naumann N , Vollmert C , Krajina M , Thevis M Evaluation of a novel gene doping detection approach via high multiplex MALDI-TOF MassARRAY analysis Center for Preventive Doping Research / Institute of Biochemistry, German Sport University Cologne, Cologne, Germany ; Agena Bioscience GmbH, Hamburg, Germany Abstract Since 2003, gene doping has been prohibited under the regulations of the World Anti-Doping Agency (WADA). In this project, a new approach for high multiplex detection of seven potential human gene doping targets in one reaction and analysis is presented. The approach is based on a multiplex PCR amplification step and subsequent single-base extension (SBE)-procedure with a final detection step via Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) on a MassARRAY System. In a proof-of-principle study, a gene doping panel prototype has been developed, which demonstrates the capability of simultaneously detecting transgenic DNA encoding for human EPO, FST, GH-1, IGF-1, MSTN (Propeptide), VEGF-A, and VEGF-D via at least two exon-exon junctions per target transcripts. First tests of the panel prototype provided promising results with high specificity and sensitivity comparable to qPCR approaches, underlining the opportunity to establish a novel time- and cost-effective approach for gene doping detection. Next steps and focus of the project is the in depth- validation of the panel prototype and test-runs concerning the method’s applicability in routine screening procedures. In addition, suitable reference material via a SNP-based detection approach will be generated and evaluated in this project. The topic of this poster will be published elsewhere. 1 2 2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0264 Akiyama K, Momobayashi A, Okano M Individual identification method using samples related to doping tests: a comparison of mitochondrial and nuclear genetic data Anti-Doping Laboratory, LSI Medience Corporation, Tokyo, Japan Abstract Doping offences include sample substitution. Therefore, it has been recommended that identification by STR (short tandem repeat) analysis should confirm that the doping control samples collected belong to the same athlete. However, it may be difficult to obtain full STR analysis using degraded or a very small amount of samples. Mitochondrial DNA (mtDNA) is characterized molecular stability and high cellular copy number. Therefore, the use of mtDNA in urine might be suitable for individual identification in doping controls. In this study, we conducted sensitivity, concordance, and case-type studies in order to compare mtDNA testing with STR analysis. In sensitivity studies, mtDNA testing was a highly sensitive analytical method compared to STR analysis. Concordance studies confirmed that all samples were concordant with mtDNA sequences and STR profiles. However, in some urine samples examined by STR analysis, allelic drop-out occurred as expected. In the case-type samples study, assuming actual testing, we found that mtDNA testing could be used to obtain DNA profiles of test samples such as urine, needle and blood stains, similar to STR analysis. We conclude that mtDNA testing would be suitable for analysis of low-concentration and highly degraded DNA samples, such as urine samples, compared to STR analysis. Since mtDNA is inherited maternally, we propose that mtDNA testing using urine samples is conducted for the initial testing procedure. Further STR analysis in blood may be required in cases where the DNA match is detrimental to the athlete. The full paper will be published elsewhere. *This year's Manfred Donike Award for the best poster presentation went to Kentaro Akiyama of the Tokyo Anti-Doping Laboratory. His work on analyzing mitochondrial DNA from urine and blood offered a complementary and robust option to individualize doping control urine samples, especially when low concentration and/or extensive DNA degradation (e.g. in urine samples) is observed. Comparing urinary mtDNA profiles with DBS-derived profiles was shown to be fit for purpose, adding a new tool to uncover sample manipulation attempts. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0265 Son J , Park H , Cho Y , Sung C , Min H , Lee KM , Bahn Y Enhancement of anti-doping strategy using artificial intelligence (AI): A study on improving the accuracy and efficiency of detecting performance-enhancing drugs in athletics as a proposed next- generation doping diagnosis strategy Doping Control Center, Korea Institute of Science and Technology, Seoul, Korea ; Department of Biotechnology, Yonsei University College of Life Science and Biotechnology, Seoul, Korea ; Department of Microbiology and Immunology, Yonsei University College of Medicine, Seoul, Korea Abstract The list of prohibited substances in anti-doping is constantly evolving as new performance-enhancing drugs and methods are developed, making it a challenging task to keep up with the latest developments. Additionally, the manual evaluation of large amounts of biological sample data is time-consuming and prone to human error, making it necessary to explore alternative methods of analysis. We propose that the use of artificial intelligence (AI) technologies in anti-doping efforts can significantly improve the accuracy, efficiency and speed of the testing process. A total of 145,544 data collected over 4 years at the Doping Control Center (South Korea) were used for supervised machine learning, and the integrated dataset consisted of a total of 4 datasets including retention times and MRM pairs for 153 doping agents. The accuracy of doping diagnosis was confirmed with 10 classification algorithm models, and the optimal algorithm model for each dataset was determined by naive Bayes and SMOTE-Tomek for the whole group and beta group, and naive Bayes for alpha and gamma groups. The results of the study, with an accuracy of 93% for the whole group and 99% for the alpha, beta, and gamma groups, demonstrate the potential of AI to accurately diagnose doping in athletes. The high accuracy of the model suggests that this new approach could be an effective next-generation anti-doping strategy. 1 1,2 1,3 1 1 1 2 1 2 3 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0266 Son J , Cho Y , Park H , Jeon S , Sung DJ , Jeong JH , Sung C , Min H , Lee KM , Kim H , Yoon SS Clinical study of brain doping diagnosis (bD ): Confirmation of sports performance enhancement and neurochemical alterations via brain stimulation Doping Control Center, Korea Institute of Science and Technology, Seoul, Korea ; Department of Microbiology and Immunology, Yonsei University College of Medicine, Seoul, Korea ; Department of Biotechnology, Yonsei University College of Life Science and Biotechnology, Seoul, Korea ; Bionics Research Center, Korea Institute of Science and Technology, Seoul, Korea Abstract A novel form of doping, known as “brain doping”, involves temporarily activating certain brain regions to enhance sports performance. However, there is no clear regulation or process for detecting it. We have continued our research to establish a brain doping diagnosis based on the concept that neurochemicals mediate brain stimulation to improve sports performance. In this study, a clinical study was conducted that focused on the effects of transcranial direct current stimulation and tested whether receiving stimulation prior to exercise enhanced sports performance and produced neurochemical alterations. Fifteen volunteers participated in a single-blind crossover test and performed a maximal incremental exercise test until volitional exhaustion on a treadmill after stimulation. In order to identify changes in neurochemicals, urine was collected at a certain time point from participants and analyzed using an ultra-sensitive analysis method developed in a previous study. Compared to sham conditions, brain stimulation led to changes in movement-related cortical potentials and increases in sports performance. As a result of analyzing the urine of the subjects, neurotransmitters having a significant difference were identified according to the sports performance, and six substance ratios were suggested as diagnostic criteria for brain doping. In this study, by detecting a whole shift in patterns for neurochemicals, we carried out research to establish a diagnosis strategy for brain doping for the first time. In addition, the analysis method developed in this research is effective for the simultaneous measurement of neurochemicals in biological samples and is anticipated to be applicable in several sectors, including doping control. 1 1,2 1,3 1 4 4 1 1 1 4 2 2 1 2 3 4 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0267 Aldubayyan A , Castrignano' E , Abbate V Short- and long-term stability of synthetic cathinones and dihydro- metabolites in human urine samples: considerations for antidoping and forensic toxicology applications Department of Analytical, Environmental & Forensic Sciences, School of Cancer and Pharmaceutical Sciences, King's College London, London, United Kingdom ; Drug Control Centre, Department of Analytical, Environmental & Forensic Sciences, School of Cancer and Pharmaceutical Sciences, King's College London, London, United Kingdom Abstract Synthetic cathinones, among the most prevalent classes of new psychoactive substances, pose a large analytical and interpretative challenge for forensic toxicology and antidoping laboratories. Cathinone and its analogues, e.g. mephedrone, methedrone, and α-pyrrolidinovalerophenone, along with other substances having a similar chemical structure or biological effect(s), are prohibited specified stimulants in-competition from class S6 of the World Anti-Doping Code International Standard Prohibited List 2023. Various factors may influence the stability of synthetic cathinones in diverse degrees between sampling and analysis, and therefore stability studies are of great value to bring insight into the best storage conditions and extend period of detection. The study involved 16 synthetic cathinones and 10 dihydro- metabolites spiked in human urine to evaluate the stability under common storage conditions to imitate real forensic toxicology/antidoping samples submitted for synthetic cathinone’s analysis. The samples were stored at either room temperature (22–23 °C) up to 3 days, refrigerated (4 °C) up to 14 days or frozen (–40 °C) up to 12 months and analysed in triplicated using a validated liquid chromatography– tandem mass spectrometry method. Analytes’ concentrations decreased over time, although slower when stored frozen. All analytes remained stable (> 80%) for 1 month when stored frozen before losses in content were more apparent for some analytes, depending on their chemical structure. Under all storage conditions, the highest instability was observed for analytes containing halogens (i.e., chlorine or fluorine). Irrespective of parent analytes, dihydro-metabolites had improved stability at each tested temperature, which highlights their importance as potential biomarkers when retesting is required after a long period of storage. It is therefore suggested that biological evidence containing synthetic cathinones should be analysed immediately, and when storage is required then caution must be taken after extended period of time as it may directly impact the accurate determination of analyte’s concentration. 1 1,2 1 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0268 Al-Naimi N , Kraiem S , Samsam W , Beotra A , Orie N , Mohamed-Ali V , Almaadheed M Turkesterone a noval ecdysteroid: High resolution orbitrap mass spectrometry identification of turkesterone in supplements and urine samples Doping Analysis Lab, Antidoping Lab Qatar, Doha, Qatar ; Centre for Metabolism and Inflammation, Division of Medicine, University College London, London, UK Abstract Ecdysteroids are polyhydroxylated ketosteroids, which were initially isolated from arthropods, and thought to play a role in the molting process. They are now known to be abundant in plants and can be detected in the tissues and body fluids of mammals after dietary consumption [1]. This family of phytosteroids, especially 20-hydroxyecdysone, have gained research interest in recent years because of their anabolic potential in mammals and growing popularity among athletes, with prevalence of use by elite athletes estimated at 0.4–5% [2,3]. Unlike typical anabolic androgenic steroids (AASs), ecdysteroids do not bind to the androgenic receptor (AR), which makes them perfect anabolic agents for use by athletes without androgenic distraction. 20-hydroxyecdysone, the most studied of these compounds is now on the Monitoring List of the World Anti-Doping Agency (WADA). Since the listing, the method of detection and biological effects of 20-hydroxyecdysone have been established and reported [3,4]. However, Turkesterone, an analogue of 20-hydroxyecdysone with an additional hydroxyl group at position C 11 of its structure, is thought to be more potent and is not on the WADA list, therefore more likely to be abused by athletes. There is, as yet no validated method to detect the steroid, and little is known about its pharmacokinetic properties. Therefore, this project aimed to develop a method for the identification and quantification of Turkesterone and its metabolites in different biological matrices, including samples collected with micro-sampling devices. The developed method was applied to detect and quantify the presence of Turkesterone in commercially available dietary supplements, as well as in urine of volunteers after its ingestion. The method used was based on UHPLC high resolution mass spectrometry. A method was developed which was fit for the purpose of detecting Turkesterone. Optimization of various parameters, such as mobile phase gradient, flow rate, run time, injection time, the orbitrap resolution, the automatic gain control (AGC), injection time (ms), and collision energy, resulted in the method development. Mass accuracy less than 2 ppm was obtained with UHPLC/HRMS. LOD of 3 ng/mL was obtained when analyzed on UHPLC/ HRMS, applying the dilute-and-shoot procedure. Out of the four dietary supplements obtained through online suppliers and tested for the presence of Turkesterone, only one showed the presence of the ecdysteroid, but a concentration (33.8 mg/g) far less than claimed on the label (500 mg/g). However, all the four supplements tested showed the presence of 20- hydroxyecdysone (see Figure 1). 1,2 1 1 1 2 1,2 1,2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0269 Figure 1. A) Chromatograms of turkesterone and B) 20HE in each supplement; Suppl. 1: pro force- super-bol, Suppl. 2: pro force l-citrulline turkesterone, Suppl. 3: pro force T40 turkesterone, Suppl. 4: s5 supplement labs turkesterone. Turkesterone was eluted at RT 5.67 and 20HE was eluted at RT 6.05 Urine from 2 volunteers (one male and one female) given Turkesterone (5 mg each), showed peak levels of the compound in the urine within 8 hours of administration in both the volunteers.In conclusion, a method was successfully developed for the detection of Turkesterone. As for other ecdysteroids, while dietary supplements do contain this phytoestrogen its level are significantly lower than the claims made by the manufacturers There may be a gender specific difference in the metabolism of this ecdysteroid, that is currently being investigated. Finally, the prevalence of Turkesterone in stored urine samples, from elite athletes, previously analyzed for 20-hydroxyecdystrone (20HE), will be reanalyzed and compared with the use of 20HE. References 1. Lafont R, Dinan L. Practical uses for ecdysteroids in mammals including humans: an update. J Insect Sci. 2003;3:7. doi: 10.1093/jis/3.1.7. 2. Parr, M.K., Ambrosio, G., Wuest, B. et al. Targeting the administration of ecdysterone in doping control samples. Forensic Toxicol 38, 172–184 (2020). doi.org/10.1007/s11419-019-00504-y 3. Kraiem S, Al-Jaber MY, Al-Mohammed H, Al-Menhali AS, Al-Thani N, Helaleh M, Samsam W, Touil S, Beotra A, Georgakopoulas C, Bouabdallah S, Mohamed-Ali V, Al Maadheed M. Analytical strategy for the detection of ecdysterone and its metabolites in vivo in uPA(+/+)-SCID mice with humanized liver, human urine samples, and estimation of prevalence of its use in anti-doping samples. Drug Test Anal. 2021 Jul;13(7):1341-1353. doi: 10.1002/dta.3032. 4. Nelson N Orie, Asmaa Raees, Maneera Y. Aljaber, Nada Mohamed-Ali, Halima Bensmail, Mostafa M. Hamza, Nasser Al-Ansari, Alka Beotra, Vidya Mohamed-Ali, Mohammed Almaadheed. 20-Hydroxyecdysone dilates muscle arterioles in a nitric oxide-dependent, estrogen ER-β receptor-independent manner. Phytomedicine Plus, 1(3), 100078. doi.org/10.1016/j.phyplu.2021.100078 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0270 Pettersson-Bohlin K , Pohanka A , Ekström L Detection of SARMs in the Swedish society Laboratory Medicine, Karolinska Institute, Doping Control Laboratory Karolinka University Hospital, Stockholm, Sweden ; Doping Control Laboratory Karolinka University Hospital, Stockholm, Sweden Abstract Selective Androgen Receptor Modulators (SARMs) are a heterogenous group of substances that all binds to the androgen receptor. SARMs are marketed as having less androgenic effect and more potent anabolic effect compared to AAS [1]. SARMS are since 2008 forbidden by WADA. According to the Swedish doping law (1991:1969) all anabolic androgenic steroids (AAS) and substances increasing the testosterone levels are juridically forbidden [2]. Subsequently, SARMS are not tested for outside WADA community and the prevalence of SARMs in society is unknown. The aim of this study was to assess the prevalence of SARMs in Swedish health care samples originally subjected to AAS testing. The samples analyzed in this study were urine leftovers from AAS testing done at Drug Abuse Laboratory, Medical Unit of Clinical Pharmacology, Karolinska University Hospital collected between 1 September 2021 and 29 August 2022. In total 651 samples were analyzed, the only information following the samples were gender. The analysis was done using our accredited WADA LC-HRMS methods for detecting the prevalence of SARMs included in the prohibited list [3]. The SARMs found in this study are shown in (Figure 1.), LGD-4033 was in top followed by RAD140 and ostarine. These findings match the findings in sports testing, i.e., according to WADA statistics (2019- 2021) these three SARMs were the most detected. None of the female samples (n=100) contained any SARM even though one might speculate that SARMs may be favorable in females due to minimizing the risk of having irreversible masculinizing effects that are associated with AAS use [4]. In 47% of the samples screened for SARMs, LGD-4033, and/or only the LGD-4033 long lasting metabolite (LGD-4033 LL met) [5,6] were detected, highlighting the importance of including metabolites prolonging the detection window. In the SARM positive samples, two or more SARMs were found in 45% of the cases. There could be several reasons for this, stacking of several SARMs to increase performance outcome can be one reason [7]. Another reason can be contamination problems with the bought products, since studies has shown that labeling and found concentration can vary a lot [8-10]. The variability in concentration of SARM products increases the risk of consuming a toxic dose. SARMs can give acute liver damage when consumed in high doses which has been reported in case reports [11,12]. A limitation herein is that the concentrations of SARMs could not be evaluated, and hence it is not possible to estimate if toxic doses have been used by the study subjects. Another limitation of the study design is that some individuals might have been tested several times during the collection time. The tested individuals are mainly people from abuse treatment programs or having a suspicious AAS use, which makes this group unique. When collecting the next round of samples, the same individual will only be collected once and the concentration of the found SARMs will be evaluated. 1 2 2 1 2 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0271 To conclude, SARMs were found in samples taken outside WADA community. Therefore, there is a need of educating healthcare and sport organizations not associated to WADA about their potential liver toxicity and to inform that patients using SARMs in Sweden will not be positive if conducted to a AAS test. Figure 1. Top three SARMs found in the healthcare samples, LGD-4033 followed by RAD140 and ostarine. Presented in % with (the number of detects) References 1. Narayanan R, Coss CC, Dalton JT. (2018) Development of selective androgen receptor modulators (SARMs). Mol Cell Endocrinol. 465:134-42. 2. Riksdagen. Lag (1991:1969) om förbud mot vissa dopningsmedel https://www.riksdagen.se/sv/dokument- lagar/dokument/svensk-forfattningssamling/lag-19911969-om-forbud-mot-vissa-dopningsmedel_sfs-1991- 1969. (access date 09.12.2022) 3. Pettersson-Bohlin K, Pohanka A, Andersson A, Ekström L. Detection of Anabolic Agents including Selective Androgen Receptor Modulators in samples outside of sport. DTA-23-0203. 4. Havnes IA, Jorstad ML, Innerdal I, Bjornebekk A. (2021) Anabolic-androgenic steroid use among women - A qualitative study on experiences of masculinizing, gonadal and sexual effects. Int J Drug Policy. 95:102876. 5. Thevis M, Lagojda A, Kuehne D, Thomas A, Dib J, Hansson A, et al. (2015) Characterization of a non- approved selective androgen receptor modulator drug candidate sold via the Internet and identification of in vitro generated phase-I metabolites for human sports drug testing. Rapid Commun Mass Spectrom. 29(11):991-9. 6. Pitsinos EN, Angelis YS, Petrou M. (2022) Structure revision and chemical synthesis of ligandrol's main bishydroxylated long-term metabolic marker. Org Biomol Chem. 20(46):9112-6. 7. Bonnecaze AK, O’Connor T, Burns CA. (2021) Harm Reduction in Male Patients Actively Using Anabolic Androgenic Steroids (AAS) and Performance-Enhancing Drugs (PEDs): a Review. Journal of General Internal Medicine. 36(7):2055-64. 8. Chakrabarty R, Grainger J, Goebel C, Brooker L, George A. (2021) “For research use only”: A comprehensive analysis of SARMs and related IPEDs purchased on local Australian websites between 2017 and 2018. Performance Enhancement & Health. 9(3-4). 9. Van Wagoner RM, Eichner A, Bhasin S, Deuster PA, Eichner D. (2017) Chemical Composition and Labeling of Substances Marketed as Selective Androgen Receptor Modulators and Sold via the Internet. JAMA. 318(20):2004-10. 10. Leaney AE, Beck P, Biddle S, Brown P, Grace PB, Hudson SC, et al. (2021) Analysis of supplements available to UK consumers purporting to contain selective androgen receptor modulators. Drug Test Anal. 13(1):122-7. 11. Weinblatt D, Roy S. (2022) Drug-Induced Liver Injury Secondary to Enobosarm: A Selective Androgen Receptor Modulator. J Med Cases. 13(5):244-8. 12. Barbara M, Dhingra S, Mindikoglu AL. (2020) Ligandrol (LGD-4033)-Induced Liver Injury. ACG Case Rep J. 7(6):e00370. MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0272 Leogrande P , Jardines D , Martinez Brito D , de La Torre X , Botrè F , Luch A , Diel P , Parr MK Physiological and post-training effects of ecdysteroid supplements. A multivariate study of the human serum metabolome Laboratorio Antidoping FMSI, Federazione Medico sportiva Italiana, Rome, Italy ; Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Berlin, Germany ; Institute for Cardiovascular Research and Sports Medicine, German Sport University, Cologne, Germany ; Institute of Pharmacy, Freie Universität Berlin, Berlin, Germany Abstract Metabolites, known to be responsible for a variety of biological functions (signaling molecules, biomarkers, and modulators of physiological processes), constitute the metabolome, whose highly dynamic nature makes it an instantaneous indicator of system perturbations induced by different physiological and/or pathological conditions as well as by the intake of biologically active substances. Since specific metabolic phenotypes can result from years of training, discrimination of physiological effects from those caused by the administration of substances banned by the World Anti-Doping Agency can be a valuable analytical strategy for interpreting adverse analytical results. This work aims to characterize the athlete serum profile after the administration of ecdysteroids, natural steroid hormones recently proved to enhance physical performance. The combination of mass spectrometry and chemometric tools may allow the differentiation of physiological effects from post- training and intake-driven effects. Metabolic profiles of 46 healthy male volunteers were measured by coupling an LC-MS/MS system with the Biocrates Absolute-IDQ p180 kit. In this way, a large panel of metabolites was quantified, which was then subjected to multivariate analysis. Unsupervised analysis of the data found no significant differences between the placebo group and the group taking the suggested dose of the ecdysteroids supplement. Considering a unique group consisting of both volunteers taking the recommended dose and those taking the supplement overdose, a clear discrimination between the control and placebo groups was observed. Phosphatidylcholines were the most significant characteristics of ecdysteroid administration to discriminate between the placebo and control groups, showing a significant influence only in the control samples and a dose-dependent effect in the others. 1 1 1 1 1 2 3 4 1 2 3 4 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0273 Saad K , Vonaparti A , Al-Mohannadi I , Khelifi S , Salma S , Mahieddine S , Saleh A , Lommen A , Horvatovich P , Beotra A , Abushareeda W , Al Maadheed M , Georgakopoulos C Preliminary study of MetAlign software automatic LC/MS screening data evaluation Doping Analysis Lab, Antidoping Lab Qatar, Doha, Qatar ; Institute of Doping Analysis and Sports Biochemistry Dresden (IDAS), Kreischa, Germany ; RIKILT Wageningen University and Research, Wageningen, The Netherlands ; University of Groningen, Groningen, The Netherlands ; Faculty of Pharmacy, University of Athens, Athens, Greece Abstract The World Anti-Doping Agency (WADA) annually updates the List of prohibited substances and methods [1]. WADA Accredited Laboratories analyze urine samples for a wide range of Prohibited and Monitored substances and report their findings to WADA. MetAlign is a freeware software designed to reduce the size of the raw datafiles acquired in full scan (FS) mode by liquid chromatography (LC) high resolution (HR) – Orbitrap mass spectrometry (MS) and to perform a fast search for substances in thousands of the reduced datafiles [2-4]. The purpose of this study was to assess the automatic data evaluation of LC/HRMS screening for beta blockers and diuretics using a combination of MetAlign reducing and searching software and the manual evaluation method. The searching and detection parameters of MetAlign are the combination of accurate ion mass to charge values (m/z), retention times and ion abundances. The MetAlign search validation criteria were based on the reliable peak detection, correct identifications and on the number of false positive (FP) and false negative (FN) findings, compared to the optical evaluation of the chromatographic peaks (generated by Xcalibur and LCQUAN software) performed by the analyst. For each substance, an abundance threshold was calculated as the mean-3 standard deviation from MetAlign's reduced quality control sample data files. These data were originated from the validation data of ten (10) different urine samples that were negative for the analytes covering the normal range of matrix differences, e.g., SG, pH, sex, turbidity, in the current validation study of the initial testing procedure (ITP). The 10 samples were evaluated before and after the addition of the substances (beta-blockers and diuretics) at 5 different concentration levels: MRL/MRPL, 75% MRL/MRPL, 50% MRL/MRPL, 25% MRL/MRPL, 10% MRL/MRPL [5,6]. An additional software module has been in-house developed with the capability to cycle over all outcomes and summarize the information in a spreadsheet format, ready to be used additionally in quality assurance applications showing the substance specific detection over long time periods. As shown in Table 1, the applied intensity thresholds provide a reliable tool for identifying positive and negative cases in the data, improving the accuracy and efficiency of the analysis. MetAlign's search capability was validated using 577 ADLQ screening (ITP) LC-HR-FS-MS reduced datafiles originating from original antidoping samples analyzed by the Laboratory during 2022 for WADA Prohibited List beta blockers and diuretics substances. The study will be continued for : 1) the incorporation of all the LC-screening analytes, 2) the reduction of the time and effort of LC screening data processing of the one out of two analysts by examining manually only the MetAlign searching results and not the full volume of the 1 1,2 1 1 1 1 1 3 4 1 1 1 5 1 2 3 4 5 MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0274 thousands of the ion chromatograms of a routine batch, 3) the creation of quality control charts of the routine LC screening analytes, 4) the creation of the database of the reduced LC-screening datafiles ready to be used for reprocessing in the framework of the preventive reanalysis and long-term sample storage. Table 1. Summary for 5 of the 25 beta blockers (MRL 50 ng/mL) and 5 of the 41 diuretics studied References 1. World Anti-Doping Agency. The 2023 WADA Prohibited List. https://www.wada-ama.org/en/resources/world- anti-doping-program/2023-prohibited-list, (access date 10.01.2023) 2. Lommen A. (2009) MetAlign: interface-driven, versatile metabolomics tool for hyphenated full-scan mass spectrometry data preprocessing. Anal Chem. 81(8),3079-86. 3. Lommen A, Elaradi A, Vonaparti A, Blokland M, Nielen MW, Saad KA, Abushreeda, WM, Horvatovich P, Al- Muraikhi AE, Al-Maadheed M, Georgakopoulos C. (2019) Ultra-fast retroactive processing of liquid chromatography high-resolution full-scan Orbitrap mass spectrometry data in anti-doping screening of human urine. Rapid Commun Mass Spectrom. 33,1578–1588. 4. Khelifi S, Saad K, Vonaparti A, Mahieddine S, Salama S, Saleh A, Al-Mohannadi M, Al-Thaiban H, Lommen A, Horvatovich P, Beotra A, Abushareeda W, Al Maadheed M, Georgakopoulos C. (2021) Ultra-fast retroactive processing by MetAlign of liquid chromatography/high-resolution full-scan Orbitrap mass spectrometry data in WADA Human Urine Sample Monitoring Program. Rapid Commun Mass Spectrom. 35(17):e9141. 5. World Anti-Doping Agency. The World Anti-Doping Code International Standard for Laboratories (ISL). https://www.wada-ama.org/en/resources/laboratories/international-standard-for-laboratories-isl (access date 10.01.2023) 6. World Anti-Doping Agency. WADA Technical Document-TD2022MRPL. https://www.wada- ama.org/sites/default/files/2022-01/td2022mrpl_v1.1_eng_0.pdf. (Access date 12.11.2022) MANFRED DONIKE WORKSHOP 2023 Poster RECENT ADVANCES IN DOPING ANALYSIS (31) ISBN 978-3-86884-049-0275 RECENT ADVANCES IN DOPING ANALYSIS 2023(31) Table of contents Lectures Poster Presentations Presentations Abstracts - Lectures Presentations Abstracts - Posters Scientific Board Contributions 1_Lectures 23_Fedoruk 2_Poster Presentations 01_Athanasiadou 02_Soni 03_Suominen 04_Wu 07_Fußhöller 09_Guddat 10_Montes de Oca Porto 15_Kwon 16_Cutler 18_Loungyot 20_Grucza 21_Grucza 22_Görgens 24_Sahu 27_Okano 28_Saavedra 30_McPherson 39_da Costa 41_Stojanovic 46_Yan 47_Chang 49_Thomas 51_Görgens 56_Radu 57_Wicka 58_Sobolevsky 66_Liu 67_Grainger 68_Yan 69_Wicka 70_Wicka 3_Presentation Abstracts - Lectures 01_Ahi 02_Al-Qahtan 03_Al-Mohannadi 04_Brooker 05_Torres 06_Rubio 07_Kwon 08_Colpaert 09_Krug 10_Santos 11_Paßreiter 12_Wagener 13_Naumann 14_Euler 15_Krombholz 16_Möller 17_Coll 18_Alechaga 19_Ventura 20_Langer 21_Thilmany 22_Breuer 24_Aguilera 25_Piper 26_Nair 27_Piper 28_de la Torre 29_Piper 30_Goodrum 31_Marchand 32_Loria 33_Dørum 34_Leuenberger 35_Heiland 36_Requena-Tutusaus 37_Reichel 38_Miller 39_Garzinsky 4_Presentation Abstracts - Posters 05_Martinez 06_Martinez 08_Martinez 11_Sobolevsky 12_Andersson 13_Euler 14_Wen 17_Peters 19_Keiler 23_Sahu 25_Walpurgis 26_Walpurgis 29_Fiallo 31_He 32_Joonyeop 33_Mongongu 34_Somay Selbes 35_Donati 36_Donati 37_Ponzetto 38_Guo 40_Buisson 42_Alechaga 43_Musenga 44_Miyamoto 45_Sletten 48_Heiland 50_Brockbals 52_Kim 53_González-Rubio 54_Keiler 55_Cristea 59_Upadhyay 60_Naumann 61_Akiyama 62_Son 63_Son 64_Aldubayyan 65_Al-Naimi 71_Petterson-Bohlin 72_Leogrande 73_Saad

M. THEVIS H. GEYER U. MARECK (EDITORS) RECENT ADVANCES IN DOPING ANALYSIS (30) Proceedings of the Manfred Donike Workshop 40th Cologne Workshop on Dope Analysis 28th March to 1st April 2022 SPORTVERLAG Strauß - Hellenthal 2022 RECENT ADVANCES IN DOPING ANALYSIS (30) 2 ISBN 978-3-86884-048-3 Bibliografische Information Der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über abrufbar. Thevis, Mario; Geyer, Hans; Mareck, Ute (Eds.) Recent Advances in Doping Analysis (30). Proceedings of the Manfred Donike Workshop, 40th Cologne Workshop on Dope Analysis, 28th March to 1st April 2022 / [MDI e.V.] – 2022 Sportverlag Strauß. ISBN 978-3-86884-048-3 ©SPORTVERLAG Strauß Neuhaus 12 – 53940 Hellenthal Tel. +49 (0)2448/2470040 – Fax +49 (0)2448/9195610 E-mail: info@sportverlag-strauss.de www.sportverlag-strauss.de Satz: Autorensatz RECENT ADVANCES IN DOPING ANALYSIS (30) 3 ISBN 978-3-86884-048-3 MANFRED DONIKE WORKSHOP 2022 TABLE OF CONTENTS PAGE LECTURES Matos RR, Aquino RM, Lopez NP, Anselmo CDS, Magalhães A, Sardela VF, Pereira HMG: Diving into the study of metabolism of SARMs through Zebrafish Water Tank (ZWT) - A proof-of-concept comparing water tank and blood analysis .................................................. 12-14 Rzeppa S, Mußhoff F, Thieme D, Keiler AM: Case report for cocaine with unusual results .............................................................................. 15-18 Rubio A, Thomas A, Geyer H, Euler L, Reis G, Costa-Padilha M, Pereira HMG, Cameron LC, Thevis M: Can the ingestion of fruits from the Annonaceae family lead to the detection of higenamine in doping control urine samples? ............................................................................ 19-26 Gomes G, Santos V, Carneiro G, Trajano C, Pereira H: An old issue under a new perspective: interpretation of a coca tea excretion study in the light of the WADA criteria ..................................................................................................... 27-29 POSTER PRESENTATIONS Piper T, Thevis M: A brief summary of different isotope ratio mass spectrometry-based approaches to detect low-dose testosterone applications................................................................................. 30-34 Kwiatkowska D, Kaplinski A, Urbaniak-Zbyszynska A, Jarek A, Tarka M, Chajewska K, Siek P, Pasik M, Szczepanska Z, Wojtowicz-Zawadka M: Pregnancy as a factor influencing the change of the steroid profile in terms of assessment of the athlete's biological passport .......................................................................... 35-39 Kwiatkowska D, Wicka M, Wojtowicz-Zawadka M, Urbaniak-Zbyszynska A, Tarka M, Chajewska K, Grucza K, Szczepanska Z, Pasik M, Zalewska Z, Jarek A, Kaplinski A: Gender change in the aspect of assessing the athlete’s biological passport .............................. 40-43 Mareck U, Fußhöller G, Geyer H, Görgens C, Guddat S, Haenelt N, Romberg S, Thevis M: Evaluation of urinary ethyl glucuronide levels in doping control samples 2018-2020 .............. 44-46 RECENT ADVANCES IN DOPING ANALYSIS (30) 4 ISBN 978-3-86884-048-3 PAGE Mareck U, Fußhöller G, Hülsemann F, Geyer H, Thevis M: Results of confirmation analyses for boldenone in the Cologne laboratory from 2017- 2021 ............................................................................................................................................... 47-49 Mareck U, Geyer H, Fußhöller G, Thevis M: Results of confirmation analysis for 19-norandosterone in the Cologne laboratory from 2017-2021 ...................................................................................................................................... 50-53 Hülsemann F, Mareck U, Fußhöller G, Geyer H, Thevis M: GC/C/IRMS results for 19-norandosterone findings in the Cologne laboratory from 2017-2021 ...................................................................................................................................... 54-58 Gmeiner G, Schlögl T, Göschl L: Profile analysis of phase II stanozolol metabolites to evaluate the time of last application ................................................................................................................................................ 59-62 Sobolevsky T, Samara V, Ahrens B: Detection of testosterone and androstenedione in serum by LC-HRMS .................................... 63-66 de Souza B, Gomes M, Nunes I, Padilha M, Pereira H: Zebrafish Water Tank model as a tool to evaluate phase I metabolites of methyl- testosterone .................................................................................................................................. 67-69 Dos Santos L, Rodrigues Matos R, M. G. Pereira H: Metabolism study of the selective estrogen receptor modulator tamoxifen through the Zebrafish Water Tank model ........................................................................................................ 70-72 Desharnais P, Naud J: A modified Towbin transfer buffer for a simple, versatile and sensitive detection of ESAs ................................................................................................................................................ 73-78 Desharnais P, Hamelin C, Douangpanya R, Stinfil CJ, Hébert N, Ferrand P, Ayotte C, Naud J: Evaluation of a single-point calibrator for the adjustment of IGF-1 concentrations measured by Immulite immunoassay .......................................................................................... 79-83 Cuervo D, Fernández-Alvarez M, Muñoz G: Determination of small peptides in doping control: HPLC-HRMS (QTOF) vs HPLC-MSMS (QQQ) analysis following SPE on microplates .............................................................................. 84-88 Stojanovic B, Geisendorfer T, Jeitler V, Illedits S, Gmeiner G: Capability and stability of dried blood spots for doping analysis ............................................... 89-91 Blázquez I, Espinosa P, de La Cal A, García L, Fernández B, Muñoz G: Solid phase extraction by anion exchange for hypoxia inducible factor (HIF) activating agents............................................................................................................................................. 92-96 RECENT ADVANCES IN DOPING ANALYSIS (30) 5 ISBN 978-3-86884-048-3 PAGE Almeida CVP, Alves I, Ruivo J: Potentialities of adsorptive micro-extraction in doping control – a new perspective in sample preparation targeting green chemistry ......................................................................... 97-102 Berghes B, Cristea CD, Radu M, Catana D, Toboc A, Stan C: The development of an identification method for heptaminol by liquid chromatography-tandem mass spectrometry (LC-MS/MS) .................................................... 103-107 Pereira D, Mourato M, Rocha Gomes T, Salema B, Ruivo J: HIF: Validation of confirmation procedure by LC-MS/MS ....................................................... 108-113 Sobolevsky T, Ahrens B: Metal-sensitive analytes in LC-MS: implications for doping control .......................................... 114-117 Wicka M, Grucza K, Stanczyk D, Drapala A, Konarski P, Wojtkowiak K, Kaliszewski P, Kwiatkowska D: Comparison of the use of the ESI ion source and UniSpray for the analysis of compounds prohibited in sport in urine samples .......................................................................... 118-122 Wicka M, Grucza K, Stanczyk D, Drapala A, Konarski P, Wojtkowiak K, Burstein K, Kwiatkowska D: Study of doping substance isomers – the possibility of their separation in human urine using LC-MS/MS ................................................................................................................................... 123-127 Aquino R, Anselmo C, Sardela V, Pereira H: Development of an LC-HRMS/MS approach for the detection of ethylmorphine and its metabolites. Comparison of different acquisition methods aiming untargeted analysis ...... 128-130 Grucza K, Drapala A, Konarski P, Kaliszewski P, Stanczyk D, Wojtkowiak K, Wicka M, Kwiatkowska D: Identification of the glucuronide conjugate of diphenhydramine during doping control analysis .................................................................................................................................................. 131-136 Constantinescu G, Pop A, Berghes B, Radu M, Penu R, Cristea C, Catana D, Toboc A, Stan C: Three months screening for chlorphenesin .................................................................................... 137-141 Athanasiadou I, Tsivou M, Gmeiner G: Data mining of HIF activating agents ............................................................................................... 142-146 Görgens C, Guddat S, Thevis M: Gas-phase water adduct formation of IOX-2 in ion trap mass spectrometers ......................... 147-151 Guddat S, Görgens C, Delahaut P, Sobolevsky T, Thevis M: Follow-up: meldonium contamination in milk – a possible scenario for inadvertent doping in sports? ................................................................................................................................. 152-156 Krug O, Geyer H, Thomas A, Piper T, Thevis M: Black market products suspected to contain doping relevant ingredients – report for 2021 ........................................................................................................................................................ 157-161 RECENT ADVANCES IN DOPING ANALYSIS (30) 6 ISBN 978-3-86884-048-3 PAGE PRESENTATIONS ABSTRACTS - LECTURES Piper T, Thevis M: Addressing recent challenges in isotope ratio mass spectrometry – Development of a method applicable to 1-androstene-steroids, 6αhydroxy-androstenedione, and androstatrienedione ................................................................................................................. 162-163 Piper T, Thevis M: Development of mass spectrometry-based methods for the detection of 11-keto- testosterone and 11-ketodihydrotestosterone ....................................................................... 164-165 Albertsdóttir AD, van Gansbeke W, van Eenoo P, Polet M: Sulfated steroids: Bridging the LC vs. GC divide Comparison of non-hydrolysed sulfated metabolites of metenolone and mesterolone analysed by 4 different techniques .......................................................................................... 166-167 Keiler AM, Gronert A, Liu L, Bredendiek F, Hobohm L, Froschauer A, Zierau O, Parr MK, Thieme D: New models for human-like steroid metabolism – shown for metandienone ............................. 168 Gmeiner G, Glatt A: Adverse analytical findings for testosterone esters in blood samples .......................................... 169 Langer T, Nicoli R, Kuuranne T, Musenga A: A holistic approach to serum steroid profiling by LC-MS/MS ................................................. 170-171 Wagener F, Guddat S, Görgens C, Petrou M, Angelis Y, Lagojda A, Kühne D, Thevis M: Investigations into the elimination profiles and metabolite ratios of micro-dosed selective androgen receptor modulator LGD-4033 for doping control purposes ......................... 172 Coll S, Bressan C, Alechaga &, Monfort N, Ventura R: Elimination profile of dexamethasone after oral administrations: Evaluation of the reporting level and washout periods .............................................................................................. 173 Euler L, Wagener F, Thomas A, Thevis M: Elimination profile of microdosed zilpaterol mimicking consumption of contaminated cattle meat ........................................................................................................................................ 174 Coppieters G, Deventer K, van Eenoo P, Judák P: Application of dilute-and-shoot on a nanoflow LC-MS setup for the confirmatory analysis of small peptide hormones ................................................................................................. 175-176 Reichel C, Gmeiner G, Thevis M: Detection of black market myostatin propeptide ................................................................... 177-178 RECENT ADVANCES IN DOPING ANALYSIS (30) 7 ISBN 978-3-86884-048-3 PAGE Marchand A, Miller G, Martin L, Gobbo C, Crouch AK, Eichner D, Ericsson M: Detection of erythropoiesis stimulating agent luspatercept after administration to healthy volunteers for antidoping purposes ............................................................................ 179-180 Naud J, Desharnais P: Detection of activin receptor type IIA and IIB-Fc fusion proteins by automated capillary immunoassay ............................................................................................................................. 181-182 Krombholz S, Thomas A, Thevis M: Investigations into the in vitro metabolism of hGH and IGF-I employing a stable- isotope-labelled reporter ion screening approach .................................................................. 183-184 Paßreiter A, Naumann N, Thomas A, Thevis M: How to detect CRISPR with CRISPR for doping control purposes employing SHERLOCK .............. 185 Ponzetto F, Settanni F, Nonnato A, Nicoli R, Mengozzi G, Ghigo E, Kuuranne T: Investigating physical exercise and circadian rhythm as possible confounding factors of the blood steroid profile ........................................................................................................... 186-187 Goodrum J, Lewis L, Fedoruk M, Eichner D, Miller G: Feasibility of microcapillary whole blood collections for usage in athlete biological passport analysis ....................................................................................................................... 188-189 Al-Jaber M, Al-Nesf A, Mohamed-Ali N, Acquaah V, Al-Nesf M, Mohamed Y, Orie NN, Voss SC, Georgakopoulos C, Bhatt R, Al-Ansari N, Beotra A, Al-Maadheed M, Mohamed-Ali V Untargeted metabolomics identifies a novel panel of markers for autologous blood transfusion ................................................................................................................................. 190-191 Buisson C, Touzani Z, Tekla E, Ericsson M: Usefulness of artificial intelligence to enhance the Athlete Biological Passport – a pilot study on the steroidal module.................................................................................................. 192-193 Gotzmann A, Trinks S: Application of the dried blood spot technique under different perspectives of a National Anti-Doping Organization ................................................................................................. 194 Loria F, Stutz A, Rocca A, Grabherr S, Kuuranne T, Pruijm M, Leuenberger N: Monitoring of hemoglobin and erythropoiesis-related mRNA in athletes and patients dried blood spots .............................................................................................................................. 195 Salamin O, Nicoli R, Saugy M, Pitteloud N, Kuuranne T: Dried blood spot as alternative matrix for the blood steroid profile and the detection of testosterone doping in women ............................................................................................ 196-197 RECENT ADVANCES IN DOPING ANALYSIS (30) 8 ISBN 978-3-86884-048-3 PAGE Garzinsky A, Thomas A, Guddat S, Görgens C, Dib J, Thevis M: Dried blood spots for doping controls – development of a comprehensive initial testing procedure with fully automated sample preparation .................................................................... 198 Okano M, Ikekita A, Sato M, Kageyama S, Inoue T, Akiyama K, Aoi A, Miyamoto A, Momobayashi A, Ota M, Saito M, Sakurai H, Shiomura S, Takemine M, Watanabe Y, Hikota T: Doping control analyses during the Tokyo 2020 Olympic and Paralympic Games ....................... 199 Zhang L, Wang Y, Xing Y, Zhang Y, Wang S, Shen L, Wang Z: Operation of the doping analysis laboratory for Beijing 2022 Winter Olympic and Paralympic Games under COVID-19 pandemic ............................................................................... 200 Mareck U, Fußhöller G, Schertel T, Petring S, Thevis M: Risk of unintentional antidoping rule violations by consumption of hemp products .................. 201 PRESENTATIONS ABSTRACTS - POSTERS Keiler A, König S, Rzeppa S, Thieme D: Agreement of steroid profiles in Athlete Biological Passport residues and corres- ponding serum samples ................................................................................................................... 202 Stojanovic B, Rasic J, Andjelkovic M, Dikic N, Forsdahl G, Gmeiner G: Characterization of the urinary excretion profile of higenamine after multiple dose oral administration utilizing on-line SPE LC with HRMS detection ........................................................ 203 Göschl L, Gmeiner G, Gärtner P, Steinacher M, Forsdahl G: Detection of DHCMT long-term metabolite glucuronides with LC-MSMS as an alternative approach to conventional GC-MSMS analysis ...................................................... 204-205 Thieme D, Anielski P, Keiler AM: Unusual steroid findings: steroid design or synthetic accidents? .................................................. 206 Toscano Bayona L, Chaves DC, Martinez Ramirez JA, Cárdenas Cuadros PA: Biotransformation of metandienone with an in-vitro model with the fungus Cunninghamella elegans .................................................................................................................. 207 Sahu PL, Rani S, Sahu K, Soni A, Mahajan S, Teja Illa G, Nandi U, Prakash Gupta A, Ahmed QN, Reddy DS Synthesis and characterization of etamivan sulfate: PK study of etamivan to decipher its metabolites ........................................................................................................................... 208-209 RECENT ADVANCES IN DOPING ANALYSIS (30) 9 ISBN 978-3-86884-048-3 PAGE Kraiem S, Bouabdallah S, Touil S, Beotra A, Mohamed Ali V, Al-Maadheed M: Elimination profile of 20-hydroxyecdysone (20-OHE) in urine: Liquidliquid extraction and dilute and inject methodology using UHPLC/HRMS. A comparative study ........................... 210 Min H, Son J, Seo Y, Park J: Analysis of intact glycopeptide in erythropoietin for doping control using liquid chromatography-mass spectrometry .............................................................................................. 211 Joon-Yeop Y, Minyoung K, Byung-Gee K, Junghyun S, Changmin S: CRISPR/dCas9 based erythropoietin variant detection assay: Simple visualization method for single nucleotide polymorphism of erythropoietin .................................................... 212 Joon-Yeop Y, Minyoung K, Byung-Gee K, Junghyun S, Changmin S: CRISPR/dCas9-based high-throughput gene doping analysis (HiGDA) for exogenous human erythropoietin ...................................................................................................................... 213 Zhou X, He S, Liu X, Wu D: Detection of de-N-glycosylated EPO with SDS-PAGE: A complementary confirmation procedure for recombinant EPO in blood samples ......................................................................... 214 Thomas A, Thilmany S, Hofmann A, Thevis M: Probing for peptidic drugs (2-10 kDa) in doping control blood samples ....................................... 215 Rahaman KA, Muresan AR, Kim KH, Lee KM, Min H, Kim HJ, Sung C, Kang M, Lee J, Son J, Kwon O: Increased 5-oxoproline and 5-oxoprolinase level in stored RBCs: Biomarkers for homologous blood doping ............................................................................................................... 216 Ota M, Miyamoto A, Sato M, Kageyama S, Okano M: Doping control analysis of trimetazidine in DBS ............................................................................. 217 Dos Santos L, Anselmo C, Pereira H, Carneiro M, Carneiro AC: Development and validation of a dried blood spot assay for the analysis of stimulants and glucocorticoids .......................................................................................................................... 218 González-Rubio S, Ballesteros-Gómez A, Muñoz G, Rubio S: Cubosomic supramolecular solvents: synthesis, characterization and potential for high throughput multiclass testing of banned substances in urine ....................................................... 219 Rubio A, Görgens C, Guddat S, Piper T, Garzinsky AM, Krug O, Thevis M: Chiral analysis of selected enantiomeric drugs relevant in doping controls ................................. 220 Lee J, Jeong TY, Kang M, Jang H, Kim M: Mobile-Phase Composition Map (MPC Map) for ionization efficiency and chromatographic behavior of 311 prohibited substances in LC-ESI/MS analysis ......................... 222 RECENT ADVANCES IN DOPING ANALYSIS (30) 10 ISBN 978-3-86884-048-3 PAGE Kim KH, Kim SH, Jeong W: Qualitative analysis applied with derivatization for formoterol and salbutamol in human urine by liquid chromatography-mass spectrometry ..................................................................... 223 Ayotte C, Charlebois A, Couture M, Desjardins M, Lalonde K: Presence of β -agonists growth promoters in human urine samples. GC-MS/MS evaluation of the excretion profiles of ractopamine administered in microdoses ....................... 224 Breuer J, Thomas A, Geyer H, Thevis M: Probing for the presence of semenogelin in human urine by immunological and chromatographic-mass spectrometric methods in the context of sports drug testing................. 225 Sahu PL, Kalita S, Kumar J, Pawar S, Sethi K, Radhakrishnanand P, Murty USN, Dubey S, Sahu K, Upadhyay A, Kori RK, Kumar P: Synthesis and characterization of reference materials of certain drugs and their metabolites ....................................................................................................................................... 226 Orie NN, Raees A, Alijaber MY, Mohamed-Ali N, Bensmail H, Hamza MM, Al-Ansari N, Beotra A, Mohamed-Ali V, Al-Maadheed M: 20-Hydoxyecdysone dilates muscle arterioles in a nitric oxide-dependent, estrogen ER-β receptor-independent manner . ....................................................................................... 227-228 Möller T, Wagener F, Thevis M: Synthesis of metabolites of the selective androgen receptor modulator LGD-4033 for doping control purposes .................................................................................................................. 229 Aljaber MY, Orie NN, Raees A, Kraiem S, Al-Jaber M, Samsam W, Hamza MM, Abraham D, Kneteman NM, Al-Ansari N, Beotra A, Mohamed Ali V, Al-Maadheed M: Downregulation of CYP17A1 by 20-hydroxyecdysone: Plasma progesterone and its vasodilatory properties ............................................................................................................. 230-231 Kwiatkowska D, Grucza K, Chajewska K, Konarski P, Wojtkowiak K, Drapala A, Wicka M: Ecdysterone - possible sources of origin in urine............................................................................ 232 Sobolevsky T, Piper T, Ahrens B, Thevis M: AICAr to SAICAr ratio can serve as additional marker of AICAr use............................................... 233 RECENT ADVANCES IN DOPING ANALYSIS (30) 11 ISBN 978-3-86884-048-3 SCIENTIFIC BOARD C. Buisson, Paris (France) X. de la Torre, Rome (Italy) H. Geyer, Cologne (Germany) G. Gmeiner, Seibersdorf (Austria) U. Mareck, Cologne (Germany) M. Mazzarino, Rome (Italy) R. Montes de Oca Porto, Havana (Cuba) M. Okano, Tokyo (Japan) T. Piper, Cologne (Germany) C. Reichel, Seibersdorf (Austria) W. Schänzer, Cologne (Germany) M. Thevis, Cologne (Germany) D. Thieme, Dresden (Germany) A. Thomas, Cologne (Germany) P. van Eenoo, Ghent (Belgium) R. Ventura, Barcelona (Spain) S. Rzeppa, Dresden (Germany) Matos RR, Aquino RM, Lopez NP, Anselmo CDS, Magalhães A, Sardela VF, Pereira HMG Diving into the study of metabolism of SARMs through Zebrafish Water Tank (ZWT) — A proof-of-concept comparing water tank and blood analysis Chemistry Institute, Brazilian Doping Control Laboratory - LBCD, Rio de Janeiro, Brazil Abstract Andarine, S-4 (AND), and ostarine, S-22 (OST) are selective androgen receptor Mmodulators (SARMs) that share the arylpropionamide nucleus and differ only in the ring substituents, presenting similar physicochemical characteristics. The World Anti-Doping Agency (WADA) has included SARMs in the list of banned substances in sports due to their muscle- and bone-anabolic properties and, consequently, potential misuse to enhance performance in sports. In addition, SARMs are still under clinical investigation. However, these substances are easily found in illegal markets around the world. The metabolism of both doping agents was investigated in zebrafish (Danio rerio) using zebrafish water tank (ZWT) and fish blood analysis. Therefore, this work combines (1) NMR and liquid chromatography with high-resolution mass spectrometry (LC-HRMS/MS) analysis to characterize the AND and OST supplements obtained via the Internet, (2) the investigation of the metabolism of the two SARMs using the ZWT model, and (3) the comparison of the ZWT with fish blood analysis. The ZWT settings consisted of adding each SARM to a 200 mL recipient of eight fish at 32 ± 1 °C, resulting in a final concentration of 1 µg/mL during 12 h of experiment. Aliquots from tank water for each SARM were analyzed in three different sample pretreatments using LC-HRMS / MS in negative ionization mode. At the end of the experiment, zebrafish blood was collected from wounds made on the anal fin and extracted by low centrifugal force after euthanasia of the fish. As a result, in the aquarium water, hydroxylation, O-dephenylation, glucuronidation, sulfate conjugate, and combinations of these reactions were observed for OST and AND. In addition, amide hydrolysis, nitroreduction, and acetylation reaction products were detected as AND in vivo biotransformation products. All metabolites found in the water were also found in zebrafish blood. However, metabolites of OST O-dephenylated glucuronide have only been detected in zebrafish blood. These results provide new insights into xenobiotic metabolism by zebrafish and establish it as a valuable tool for SARM doping control analysis. Introduction Andarine, S-4 (AND), and ostarine, S-22 (OST) are selective androgen receptor modulators (SARMs) that share the arylpropionamide nucleus, differing only in the ring substituents. Hence, these SARMs show similar chemical and pharmacological characteristics. Both are banned substances in sports, but easily found in illegal markets around the world. One of the reasons to study the metabolism of AND and OST is that improving the analytical window of all time forbidden drugs depends on knowledge of the metabolic behavior of doping agents. The zebrafish water tank (ZWT) uses the water tank instead of the body fluids MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-312 or tissues from the animal itself, and has proven useful to screen for doping agents’ metabolites. The use of the water tank is possible because the fish excrete xenobiotics and hormones into the water either through the gills via passive diffusion or through urine and feces. However, doping agents’ metabolites have not yet been studied in the blood of hte fish. Therefore, the metabolism of both SARMs was investigated using zebrafish (Danio rerio) water tank (ZWT) model and fish blood analysis. Experimental This study combined NMR and LC-HRMS/MS to characterize AND and OST supplements obtained by online purchase. NMR experiments were carried out at room temperature (25 °C) in a Bruker AVANCE III- HD, 400.13 MHz (9.4 Tesla) equipped with PABBO probehead. H, at the resonance’s frequencies 400.13 was collected at traditional mode. ZWT model details were described in a previous study of the group. Briefly, each SARM was added to a 200 mL recipient of eight fish at 32 ± 1 °C, resulting in a final concentration of 1 µg/mL. The total time of the experiment was 12 hours. Aliquots from ZWT were pretreated by dilute-and-shot (DS) method: 90 µL of the ZWT in natura samples and 10 µL of MeOH, 0.1 % formic acid (mobile phase B) and analyzed in triplicate applying LC-HRMS/MS in negative ionization mode. At the ending of the ZWT experiment, each fish was euthanized and its caudal region was cut off at the end of the anal fin. The blood was withdrawn by low centrifugal force from the wound and collected as a pool of each tank. Results and Discussion The NMR H confirmed that the illegally bought supplements were composed of AND and OST as expected. In ZWT, metabolites resulting from hydroxylation, O-dephenylation, glucuronidation, sulfate conjugate, and combinations of these reactions were observed for OST and AND. In addition, amide hydrolysis, nitro reduction, and acetylation reaction products were detected as AND in vivo transformation products. All metabolites found in the water tank were also found in zebrafish blood. Moreover, the OST O-dephenylated glucuronide have only been detected in zebrafish`s blood. This could be explained by the increase in sensitivity after the pre-concentrations step adopted in blood analysis. These results provide new insights into xenobiotic metabolism studies by zebrafish and highlight the blood matrix as an analytical option for investigating biotransformation reactions. Such results also indicate that the ZWT model is a valuable tool for studying the metabolism of SARMs. Conclusions Metabolism of AND and OST were investigated using the ZWT model. The main metabolites already observed in humans were produced, opening the perspective of use the ZWT for other SARMs poorly studied. A modification of the original experimental design using the zebrafish blood seems also promising, especially for metabolites with low concentrations in the tank. These promising results, shows the potential of using zebrafish a useful tool for doping control laboratories to biosynthesize SARMs analytical targets when the study of their metabolism in humans is unfeasible, or when metabolites are not yet commercially available to be analytical targets. 1 1 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-313 References 1. Matos RR, Anselmo CS, Sardela VF, Pereira HMG (2021) Phase II stanozolol metabolism study using the zebrafish water tank (ZWT) model. J Pharm Biomed Anal. 195, (20), 113886. 2. Matos RR, Martucci MEP, Anselmo CS, Alquino Neto FR, Pereira HMG, Sardela VF. (2019) Pharmacokinetic study of xylazine in a zebrafish water tank, a human-like surrogate, by liquid chromatography Q-Orbitrap mass spectrometry. Forensic Toxicol. (38), 108–121. 3. Babaei F, Ramalingam R, Tavendale A, Liang Y, Yan LSK, Ajuh P, Cheng SH, Lam YW. 2013. Novel blood collection method allows plasma proteome analysis from single zebrafish. J Proteome Res. 12(4):1580– 1590. https://doi.org/10.1021/pr3009226 4. Thevis M, Gerace E, Thomas A, Beuck S, Geyer H, Schlörer N, Kearbey JD, Dalton JT, Schänzer W. 2010. Characterization of in vitro generated metabolites of the selective androgen receptor modulators S-22 and S-23 and in vivo comparison to post-administration canine urine specimens. Drug Test Anal. 2(11–12):589– 598. https://doi.org/10.1002/dta.211 5. Thevis M. 2009. Detection of the arylpropionamide-derived selective androgen receptor modulator (SARM) S-4 (Andarine) in a black-market product. Drug Test Anal. 1(8):387–392. https://doi.org/10.1002/dta.91 Acknowledgements The authors would like to thank the Brazilian Anti-Doping Agency (ABCD) for the financial support. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-314 Rzeppa S , Mußhoff F , Thieme D , Keiler AM Case report for cocaine with unusual results Institute of Doping Analysis and Sports Biochemistry, Kreischa, Germany ; Forensic Toxicological Center, Munich, Germany ; Enviromental Monitoring and Endocrinology, Facultaty of Biology, Technische Universität Dresden, Dresden, Germany Abstract At the Institute of Doping Analysis and Sports Biochemistry (IDAS) an unusual doping case related to cocaine was handled in 2021. In an urine doping control sample, cocaine was confirmed with a concentration of about 16 ng/mL and benzoylecgonine as main metabolite with about 47 ng/mL. In the majority of doping controls associated with cocaine intake, very low, if any, cocaine concentrations are detectable, while detectable benzoylegconine concentrations are significantly higher. In accordance with the requirements given by WADA at this date, the present case was reported as an AAF due to a detectable concentration of cocaine. During result management by the responsible national anti-doping agency, the athlete initially completely denied the intake of cocaine. In view of the results, a single contact with cocaine was first granted. Based on the confirmed concentrations, the athlete admitted a five-time intake of cocaine under further case treatment. Last consumption of cocaine should have been be six days before doping control sample collection. The simultaneous consumption of cocaine and alcohol was also discussed as a possible reason for the prolonged excretion of cocaine at such high concentrations. In order to achieve a better insight into the present case, the Forensic Toxicological Center Munich analyzed a hair sample of the athlete. Cocaine has been detected in concentrations of 39 ng/mg up to 75 ng/mg depending on the hair section. Cocaethylene in unusual high concentrations and markers for increased alcohol consumption were also detected in the hair sample. However, re-investigation of the original urine sample showed no relevant amounts of cocaethylene and ethyl glucuronide. Thus, in the overall evaluation of literature review and the results from hair and urine analyses, it can be assumed that a single intake, initially claimed by the athlete, doesn’t result in the observed marker concentration. Cocaine was rather consumed intensively over a longer period, obviously in combination with alcohol. The results also show a long-lasting cocaine elimination at unusually high concentrations compared to benzoylecgonine after intensive long-term cocaine consumption. Introduction Cocaine is metabolized rapidly and almost completely to its main metabolite benzoylecgonine. In addition, metabolism of benzoylecgonine to corresponding hydroxy metabolites and other metabolites like benzoylnorecgonine is also known. Cocaine itself can be also metabolized to norcocaine, ecgonine methyl ester and hydroxy metabolites. Simultaneous consumption of cocaine and ethanol can lead to a so-called transesterification yielding cocaethylene. Cocoaethylene can also be further metabolized to metabolites comparable to those of cocaine, but with the ethyl-function instead of the methyl-function 1 2 1 1,3 1 2 3 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-315 [1]. For the doping analysis the most relevant analytes are cocaine itself and the main metabolite benzoylecgonine. For both analytes, reporting limits are given by WADA [2]. Experimental For initial testing, the urine sample was analyzed according to the procedure used in routine analysis. 50 µL of urine sample were mixed with 200 µL buffer containing internal standards. Analysis was carried out on a LC-MS/MS system (Agilent 1290, AB Sciex TripleQuad 5500) in positive MRM mode. For benzoylecgonine the transition m/z 290> 168 and for cocaine the transition m/z 304> 182 were used. Ethyltheophylline with the transition m/z 209> 69 was used as internal standard. Chromatographic separation was carried out using an Eclipse XDB-C8 column (2.1 x 100 mm, 3.5 µm; Agilent) with the following gradient: 0 min 0% B, 5 min 90% B, 6.5 min and 90% B, A (ACN/H O, 5/95 v/v containing 2 mmol/L ammonium acetate and 0.1% acetic acid) and B (ACN/H O, 95/5 v/v containing 2 mmol/L ammonium acetate and 0.1% acetic acid). Flow rate was set at 200 µL/min. Injection volume was 5 μL. The dilute-and-shoot approach was also chosen for confirmation procedure. 200 µL urine were diluted with 800 µL buffer containing benzoylecgonine-d3 as internal standard. Analysis was carried out on a LC- MS/MS system (Agilent 1290, AB Sciex TripleQuad 6500) in positive MRM mode. For benzoylecgonine the transitions m/z 290> 168, m/z 290> 77 and m/z 290> 82, for cocaine the transitions m/z 304> 182, m/z 304> 82 and m/z 304> 72, and for cocaethylene the transitions m/z 318> 196 and m/z 318> 82 were used. Benzoylecgonine-d3 with the transition m/z 293> 171 was used as internal standard. Chromatographic separation was carried out with the same setup as for the initial testing procedure. Results and Discussion The object of this case report is a urine sample, in which cocaine and benzoylecgonine were detected during initial testing procedure. Identity of cocaine and benzoylecgonine in the urine samples were confirmed during confirmation procedure at estimated concentrations of about 14 ng/mL for cocaine and of about 47 ng/mL for benzoylecgonine. According to the recommendations given by WADA, which were valid at that time in 2021, samples with a detectable concentration of cocaine shall be reported as Adverse Analytical Finding even if the benzoylecgonine concentration is below the reporting limit (50 ng/mL) [3]. During result management by the responsible National Anti-Doping agency, the athlete initially completely denied the intake of cocaine. After communicating the results, the athlete granted a single, very low dose contact with cocaine. Under further case treatment, the athlete admitted a five-time intake of cocaine as confirmed concentrations are too high according to literature and therefore in contrast to the first statement of the athlete [4]. Last consumption of cocaine should have been six days before doping control sample collection. However, this is in contrast to the low ratio of cocaine to benzoylecgonine of about 3, suggesting consumption and sample collection have been taken place chronologically close to each other. Therefore, an attempt was made to explain the present results through the simultaneous consumption of cocaine and alcohol. This mixed consumption could account for these unusual results and the prolonged excretion time due to a possible delayed elimination of cocaine. A reanalysis for cocaethylene as typical marker for a possible mixed consumption of alcohol and cocaine 2 2 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-316 was made. In the urine sample, only traces of cocaethylene could be detected. In order to contribute a clarification in the present case, a hair sample was collected from the athlete. The analysis was carried out by the Forensic Toxicological Center Munich [5]. For the analysis, the hair sample was divided into two segments, each 1.5 cm. Ethyl glucuronide as marker of alcohol consumption as well as cocaine and typical metabolites were detected. Estimated concentration of cocaine ranged from 39 to 75 ng/mg depending on the segment. In addition, also cocaetehylene was detected at a concentration of 2.5 ng/mg. Table 1. Results of the hair analysis. The analyses were carried out by the Forensic Toxicological Center Munich. The hair segment of 3 cm represents a time period of approx. 3 months. Since the hair sample was taken six months after the positive urine sample, the hair analysis covers a time period after the positive urine sample. Nevertheless, this hair sample provides valuable insight into the consumption patterns of the athlete regarding cocaine and alcohol. Even an exact correlation between concentration in hair and aplied amount is not possible, the results indicate a chronic excessive consumption of both, alcohol and cocaine. The high concentration of cocaethylene indicates, that several consumptions of cocaine had taken place in combination with alcohol. Other studies showed that chronic cocaine use can result in comparable urinary cocaine and benzoylecgonine concentrations. In addition, the excretion time can be significantly prolonged in the case of chronic use of cocaine compared to a single use [6]. Conclusions Chronic and extensive consumption of cocaine can result in unusual results for cocaine and its metabolite benzoylecgonine in urine samples. Relatively high concentration of cocaine compared to benzoylecgonine can indicate such a chronic consumption in urine samples. In addition, a prolonged excretion of cocaine can be explained by the chronic use of cocaine. In addition, the results of this case report show that a single positive urine sample can only be a snapshot. In some cases, it can therefore be useful to have a look at additional sample materials covering longer period of time. Hair samples can be such a biological sample material, which can cover a period of several months. In the present case, the results of the hair samples indicate a chronic and extensive consumption of cocaine. In addition, results suggest a mixed consumption of cocaine and alcohol. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-317 References 1. Cami et al., Cocaine Metabolism in Humans after Use of Alcohol Clinical and Research Implications, Recent Developments in Alcoholism, 1998 2. WADA, TD2022 MRPL, January 2022 3. WADA, Analysis and Reporting of Cocaine Findings, January 2021 4. Ambre, The urinary excretion of cocaine and metabolites in humans: a kinetic analysis of published data, Journal of Analytical Toxicology, 9, 1985 5. Musshoff et al., Determination of hydroxy metabolites of cocaine in hair samples for proof of consumption, Drug Testing and Analysis, 10, 2018 6. Preston et al., Occurrence of Cocaine in Urine of Substance-Abuse Treatment Patients, Journal of Analytical Toxicology, 22, 1998 Acknowledgements The financial support by the German Ministry of the Interior and Community is gratefully acknowledged. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-318 Rubio A , Thomas A , Geyer H , Euler L , Reis G , Costa-Padilha M , Pereira HMG , Cameron LC , Thevis M Can the ingestion of fruits from the Annonaceae family lead to the detection of higenamine in doping control urine samples? Institute of Biochemistry, German Sport University, Cologne, Germany ; Brazilian Doping Control Laboratory (LBCD ? LADETEC / IQ - UFRJ), Rio de Janeiro, Brazil ; Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, Brazil ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract The presence of higenamine in a great variety of plant-based remedies and different weight loss and sports supplements has been shown to possess the potential to result in unintentional anti-doping rule violations during the last years. The aim of this study is to investigate whether the ingestion of fruits from the Annonaceae family can lead to adverse analytical findings (AAFs) in sports. For that purpose, single-dose administration studies were conducted with three Annona species: A. cherimola, A. muricata, and A. squamosa. Higenamine was detected in post-ingestion urine samples but, under the chosen conditions, at concentrations ranging exclusively below the established MRL. Higher urinary higenamine concentrations were observed in case of A. muricata and A. squamosa, with C values of 0.6 – 4.1, and 1.0 – 7.8 ng/mL, respectively. A substantial interindividual variability of higenamine urinary concentrations, and t values was observed, especially in case of A. muricata and A. squamosa. The high t values observed in some volunteers suggest that multidose administrations might necessitate consideration in future studies. The herein obtained data showed a major urinary excretion of higenamine in its conjugated form, especially at the expense of its sulfo-conjugates. The outcome supports the position that single-dose administrations of these fruit species are rather unlikely to lead to AAFs in sports. However, substantial variability of the natural higenamine content in fruits exists; hence, whilst less likely, it cannot be excluded that, under specific circumstances, the current MRL is exceeded. Introduction Higenamine is a non-selective β -agonist prohibited in sports at all times due to its bronchodilating and cardiac stimulating effects [1]. A reporting level of 10 ng/mL (50% of the Minimum Required Performance Level (MRPL)) applies for the detection of β -agonists in doping control urine samples, and in case of higenamine, the reporting level refers to the determination of the unconjugated compound only [2]. From 2016 till 2020, up to 227 samples were reported as an AAF for higenamine [3-7]. Higenamine has been described as a key component of a great variety of natural plants such as Nandina domestica, Tinospora crispa, and different species of Annona genus [8-13], and has been found to be an ingredient – often unlabeled – of different weight loss and sports supplements [9-12]. The content of higenamine in natural plants is variable, and different studies have investigated whether the ingestion of different plant extracts or commercially available plant-based products can lead to an AAF, corroborating 1 1 1 1 2 2 2 3 1,4 1 2 3 4 max max max 2 2 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-319 the risk originating from diets that include certain higenamine sources such as Nelumbo nucifera [8,11]. Some tropical fruit plants of the Annonaceae family have been reported to contain higenamine, and a pilot study from L.C. Cameron´s Laboratory (Protein Biochemistry Laboratory, Federal University of Rio de Janeiro, Brazil) showed that single-ingestion of Annona fruit led to detectable higenamine in post- administration urine samples. Hence, the aim of this study as to investigate whether the ingestion of such fruits can lead to AAFs in sports considering the currently applicable WADA regulations. Experimental Three Annona species were included in the herein project, and single-dose administration studies were conducted: Consumption and elimination studies with A. cherimola and A. muricata were performed by the Cologne Laboratory, and accordingly A. squamosa administration studies were done by the Brazilian Laboratory of Doping Control (LBCD – LADETEC / IQ – UFRJ, Rio de Janeiro, Brazil). For each administration study, a pulp fruit puree was prepared as homogeneous mixture of different fruits from the same specie in order to ensure an identical higenamine intake by each participant. A. cherimola (harvested in Spain) fruit puree was prepared in Cologne, and A. muricata and A. squamosa (Brazil) fruit purees were prepared by LBCD. Preparations were stored at -20°C until consumption. For the estimation of higenamine content, 1 g of fruit puree homogenate was diluted into 4 mL of water. After ultrasonication for 30 min and centrifugation for 5 min, the higenamine content was determined by means of standard addition, i.e. a sequence of supernatant aliquots were enriched with 0.5, 1, 2, and 4 µg of higenamine (Toronto Research Chemicals / TRC, Toronto, Canada). Isoxsuprine-d (0.2 µg; TRC) was used as ISTD. Two consecutive TBME (KMF, St. Augustin, Germany) extractions at pH 9.6 were performed, the combined organic phases were evaporated to dryness, and reconstituted in ACN/H O (1:9). A. cherimola, A. muricata, and A. squamosa administration studies included considerable numbers of volunteers, i.e. 12 (6 x male, 6 x female), 24 (12 x male, 12 x female), and 33 (16 x male, 17 x female), respectively, and comparable single servings of fruit puree were administered i.e. 330 g of the first two species, and 173 - 528 g of A. squamosa. Within the 24 h following ingestion, every urine sample was collected; on the consecutive days and up to 72 h, only two samples were required. All urine specimens were stored at -20°C until analysis. The studies were performed with ethical approval, and all participants provided written informed consent. Urine samples were prepared for analysis following direct injection, and SPE approaches employing isoxsuprine-d (50 ng/mL in urine), and 7-propyl-theophilline as ISTDs by the Cologne Laboratory and LBCD, respectively. Strata-X-C cartridges (Phenomenex) were used for SPE, equilibrated with water and methanol, and loaded with 2.5 mL of sample. After washing with water and methanol, elution was performed with methanol/formic acid (95:5), and the eluate was evaporated to dryness, and reconstituted in water. Both assays were comprehensively characterized. Measurements were performed by means of LC-HRMS(/MS) on a Vanquish UHPLC system coupled to an Orbitrap Exploris 480 mass spectrometer, and Dionex Ultimate 3,000 coupled to a Q Exactive-Plus Orbitrap (Thermo Fisher Scientific, Dreieich, Germany), by the Cologne Laboratory and LBCD, respectively (Table 1). 5 2 5 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-320 Table 1. Summary of LC-HRMS(/MS) analytical conditions LC-HRMS(/MS) data were evaluated by using TraceFinder 4.0 software (Thermo Fisher Scientific). For all target analytes, the protonated molecules were used as precursor ions, and two precursor/product ion pairs were used for identification. Higenamine sulfates and glucuronides were also monitored by the Cologne Laboratory (Table 2). ISTD-normalized peak areas were used for higenamine quantification and, further, metabolite abundance ratios (constructed with the ISTD) were evaluated. Higenamine concentrations were specific gravity-adjusted. Table 2. Target analytes included in the study MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-321 Results and Discussion The method employed for the quantitative determination of higenamine following direct injection was fully characterized according to WADA guidelines [14], considering criteria for non-threshold substances with a minimum reporting level (MRL). The approach was found to be highly specific and linear from 5 to 100 ng/mL (R > 0.99) with an estimated LOQ of 0.6 ng/mL ( i.e. 6% MRL), and carryover effects were not observed. Ion suppression effects ranged from 33.2% to 69.8% and the method´s intra- and interday imprecision, and accuracy were determined at three different concentration levels and varied from 5.6% to 11.0%, 16.4% to 18.2%, -12.4% to 12.1%, respectively. Higenamine stability in the autosampler was shown to be fulfilled for at least 24 h at 4°C with a detection rate of 100 % for all fortified samples at the MRL. The method used for the quantitative determination of higenamine following SPE approach was also found to be highly specific, showed an LOQ of 1 ng/mL (i.e. 10% of MRL), recovery of 106%, and imprecision of 15%. The analysis of the pooled batches of extracted fruit puree confirmed higenamine contents at estimated concentrations of ca. 491 ng/g for A. cherimola, 733 as well as 969 ng/g (two separately pooled batches; 6 x male, 6 x female each) for A. muricata, and 340 – 1,090 ng/g for A. squamosa (fruit servings were adjusted in this case to ensure equal higenamine intake). This higenamine content would lead to an estimated ingestion of 162 µg, 244 - 320 µg, and 186 µg of higenamine per serving in the herein conducted elimination studies of A. cherimola, A. muricata, and A. squamosa, respectively. A summary of the results of the three elimination studies is shown in Table 3. Urinary higenamine was detected at all times (except for one volunteer after A. squamosa ingestion), however only minute amounts below the assay’s formally validated LOQ were observed in the A. cherimola elimination study, excluding quantification of higenamine in most samples. Higher concentrations were observed after ingestion of A. muricata and A. squamosa, with urinary C levels of higenamine of 0.6 – 4.1 ng/mL and 1.0 – 7.8 ng/mL, respectively, compared to 0.6 – 0.9 ng/mL for A. cherimola. The applicable WADA MRL of 10 ng/mL was not exceeded at any sampling time point. These results support the findings described by Okano et al. in 2017 after the administration of a throat lozenge containing Nandina domestica fruit. A substantial inter-individual variability of higenamine urinary concentrations and t values was observed in all elimination studies, especially in case of A. muricata and A. squamosa, and higenamine was detected up to 72 hours for some volunteers in minute amounts. Table 3 . Summary of single-dose elimination studies results after ingestion of three different Annona fruit species 2 max max MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-322 Urinary higenamine elimination profiles following single application of A. muricata, and A. squamosa are depicted in Figure 1. The values shown are the average concentrations from 24 and 33 samples, respectively per time point and error bars indicate the respective minimum / maximum at each time point. Since the herein obtained data showed a major urinary excretion of higenamine in its conjugated form, higenamine sulfates and glucuronides were also monitored after ingestion of A. cherimola and A. muricata, showing a major contribution from the sulfo-conjugates compared to the glucuronide conjugates, which were not detectable in most cases, especially in case of A. cherimola. Hence, only excretion profiles from the major sulfo-conjugate are plotted for both fruit species in Figure 2. T values were again subject of high inter-individual variability, especially in case of A. muricata, where peak urinary amounts of higenamine and its sulfo-conjugate were reached after 2-24 h and 4-24 h, respectively. Figure 1. Urinary higenamine elimination profiles following single application of A. muricata (top) and A. squamosa (bottom) max MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-323 Figure 2. Urinary higenamine sulfo-conjugate elimination profiles following single application of A. cherimola (top) and A. muricata (bottom). Area ratio was found to increase proportionally to the administered dose. Higenamine sulfo-conjugate was also detected up to 72 h post-administration ( i.e. end of sample collection period) for both fruit species, as it is shown in Figure 3, showing wider urinary detection window than higenamine for some volunteers, supporting previous preliminary data from Grucza et al. 2018 [15]. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-324 Figure 3. Maximum detection times of urinary target analytes after single-dose administration of A. muricata (orange) and A. cherimola (blue). Detection times were also found to increase proportionally to the administered dose. Conclusions The present investigation represents a single-dose ingestion of tropical fruit plants of the Annonaceae family, leading to higenamine findings in post-ingestion urine samples but, under the chosen conditions, at urinary concentrations ranging exclusively below the established MRL. Nevertheless, the high higenamine t values observed in some volunteers suggest that multidose administrations might necessitate consideration in future studies, and stability studies concerning both higenamine and its sulfo-conjugates in human urine appear sensible. The outcome supports the position that single-dose administrations of these fruit species are rather unlikely to lead to AAFs in sports. However, substantial variability of the natural higenamine content in fruits exists; hence, whilst less likely, it cannot be excluded that under specific circumstances the current MRL is exceeded. References 1. World Anti-Doping Agency. The 2022 Prohibited List. International Standard, Montreal (2022) https://www.wada-ama.org/sites/default/files/2022-01/2022list_final_en_0.pdf (access date 29.08.2022). 2. World Anti-Doping Agency. WADA Technical Document – TD2022MRPL: Minimum required performance levels for detection and identification of non-threshold substances (2022) https://www.wada- ama.org/sites/default/files/resources/files/2022-01/ td2022mrpl _ v1.1_eng_0.pdf (access date 12.10.2022). 3. World Anti-Doping Agency, 2016 Anti-Doping Testing Figures Report (2017) https://www.wada- ama.org/sites/default/files/resources/files/2016_anti-doping_testing_figures.pdf (access date 29.08.2022). 4. World Anti-Doping Agency, 2017 Anti-Doping Testing Figures Report (2018) https://www.wada- ama.org/sites/default/files/resources/files/2017_anti-doping_testing_figures_en_0.pdf (access date 29.08.2022). max MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-325 5. World Anti-Doping Agency, 2018 Anti-Doping Testing Figures Report (2019) https://www.wada- ama.org/sites/default/files/resources/files/2018_testing_figures_report.pdf (access date 29.08.2022). 6. World Anti-Doping Agency, 2019 Anti-Doping Testing Figures Report (2020) https://www.wada- ama.org/sites/default/files/resources/files/2019_anti-doping_testing_figures_en.pdf (access date 29.08.2022). 7. World Anti-Doping Agency, 2020 Anti-Doping Testing Figures Report (2021) https://www.wada- ama.org/sites/default/files/2022-01/2020_anti-doping_testing_figures_en.pdf (access date 29.08.2022). 8. M. Okano, M. Sato, S. Kageyama. (2017) Determination of higenamine and coclaurine levels in human urine after the administration of a throat lozenge containing Nandina domestica fruit. Drug Test Anal. 9 (11- 12), 1788-1793. 9. Stajic A, Andelkovic M, Dikic N, Rasic J, Vukasinovic-Vesic M, Ivanovic D, Jancic-Stojanovic B. Determination of higenamine in dietary supplements by UHPLC/MS/MS method. J Pharm Biomed Anal. 2017 Nov 30;146:48-52. 10. K. Grucza, K. Kowalczyk, M. Wicka, M. Szutowski, E. Bulska, D. Kwiatkowska. (2019) The use of a valid and straightforward method for the identification of higenamine in dietary supplements in view of anti-doping rule violation cases. Drug Test Anal. 11 (6), 912-917. 11. K. Yan, X. Wang, Z. Wang, Y. Wang, Z. Luan, X. Gao, R. Wang. (2019) The risk of higenamine adverse analytical findings following oral administration of Plumula nelumbinis capsules. Drug Test Anal. 11 (11-12), 1731-1736. 12. H. Wagner, M. Reiter, W. Ferstl. New drugs with cardiotonic activity I Chemistry and pharmacology of the cardiotonic active principle of Annona squamosa L. (1980) Planta Med – J Med Plant Res. 40, 77-85. 13. V. R. Kozhuharov, K. Ivanov, S. Ivanova. (2022) Higenamine in Plants as a Source of Unintentional Doping. Plants. 11 (3), 354. 14. WADA Laboratory Technical Note on Analytical Method Validation for Doping Control. Version 01, 2020. 15. K. Grucza, D. Kwiatkowska, K. Kowalczyk, M. Wicka, M. Szutowski, P. Cholbinski. (2018) Analysis for higenamine in urine by means of ultra-highperformance liquid chromatography–tandem mass spectrometry: Interpretation of results. Drug Test Anal. 10, 1017-1024. Acknowledgements The authors thank the Manfred Donike Institute for Doping Analysis, and the World Anti-Doping Agency (Montreal, Canada, grant #T20M01MT) for supporting the presented study, and the Brazilian Laboratory of Doping Control (LBCD – LADETEC / IQ – UFRJ, Rio de Janeiro, Brazil), and the Universidade Federal do Estado do Rio de Janeiro (UNIRIO) for their collaboration in this project. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-326 Gomes G , Santos V , Carneiro G , Trajano C , Pereira H An old issue under a new perspective: interpretation of a coca tea excretion study in the light of the WADA criteria Chemistry Institute, Brazilian Doping Control Laboratory - LBCD, Rio de Janeiro, Brazil ; Brazilian Olympic Committee - COB, Rio de Janeiro, Brazil Abstract A coca tea excretion study was used in different hypothetical scenarios to evaluate the typical concentrations of cocaine (COC) and benzoylecgonine (BZE) under the WADA criteria in force. A huge interindividual variation was observed concerning both concentrations and time of elimination. Also, the samples fulfil the MRL criteria even when the administration occurs two days before the in- competition period. However, the addition of buffer in the samples just after the collection highlight the impact of pH in the spontaneous COC-BZE conversion. Other hydrolysis reactions could also be relevant in the results interpretation. To one specific volunteer, a positive sample became negative due to the inhibition of the COC hydrolysis, resulting in BZE level below 50 ng/mL. Thus, temperature and transport time become important factors to be considered for those analytes. Introduction The strategy of using Minimum Reporting Levels (MRLs) is incorporated in the anti-doping laboratories routine. Therefore, MRLs and estimation of concentrations reported by the laboratories have been used in court by the legal representatives under argumentations concerning pharmacokinetic illations. Recently, MRL strategy for cocaine cases was reenforced by the TD2022MRPL and, despite the availability of other metabolites, only benzoylecgonine (BZE, MRL 50 ng/mL) and/or cocaine (COC, MRL 10 ng/mL) are used in the results interpretation [1]. Notwithstanding, it is well known that COC is also converted into BZE by spontaneous hydrolysis, specially under alkaline pH and higher temperatures (normally achieved during samples transportation to the laboratories). This phenomenon was also mentioned in the WADA guidance note for substance abuse under the 2021 code [2]. Therefore, the aim of the project was to evaluate the results from an excretion study of a commercially available coca tea based on the WADA criteria in force. Experimental A commercially available coca tea bag (Coca Zagradha, Colombia) was infused for five minutes in approximately 100 mL of hot water. The coca tea excretion study involved twelve volunteers, which were equally divided into two groups with different times of administration (i.e., morning and afternoon). A negative control for each volunteer was collected before administration. All urine samples were collected since administration up to 48 hr, and only the first urine in the morning in 72, 96 and 120 hr. The proposed experimental design also englobed an aliquot of approximately 5 mL of the collected sample, 1 1 1 2 1 1 2 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-327 transferred to a falcon tube containing pH 4 ammonium formate/formic acid buffer 2M by the volunteer immediately after each urine collection. The study was approved by the local ethics committee of the Federal University of Rio de Janeiro (32815120.0.0000.5257). All urine samples were subjected to solid- phase extraction and injected in a QExactive quadrupole-Orbitrap mass spectrometer equipped with electrospray ionization. Separation was performed by ultra-high performance liquid chromatography with a reversed phase column, adapted from Sardela et al. [3]. A pool of the administered coca teas was used for the estimation of the COC content by isotopic dilution. Results and Discussion An average of 3 mg of COC was estimated in the administered coca teas, in agreement with the literature [4,5]. Huge interindividual variation was observed among the volunteers regarding urinary maximum concentration C (BZE from 630 ng/mL to 2432 ng/mL and COC from 2.4 ng/mL to 87 ng/mL) and time T (BZE from 2.8 hr to 8.5 hr and COC from 0.8 hr to 11.2 hr). For results interpretation, different hypothetic scenarios were built. Scenario #1 considered the administration within an in-competition (IC) period, which resulted in an AFF for all samples based on the BZE criterion. COC criterion was able to flag an AAF only in samples administrated near the hypothetical collection time (66%). Admitting the administration one day before the competition/sample collection (Scenario #2), almost 83% of the samples triggered AAFs based on the BZE criterion. A same interpretation considering the administration two days before the competition/administration turned 2 of the 12 samples (near 15%) positive for BZE (Scenario #3). The addition of pH 4 buffer in the samples just after the collection highlight the impact of pH in the spontaneous COC-BZE conversion. In one specific case, a positive sample became negative due to the inhibition of the COC hydrolysis, resulting in BZE level below 50 ng/mL. Such huge interindividual variation in the urinary excretion profiles is a challenge factor to infer whether the administration happened in- or out-of-competition. The potential of chemical (non-enzymatic) hydrolysis, not only of COC, but also of BZE, makes the interpretation of urinary results still more difficult. In this context, as previously reported by Thevis et al. (2020), dried blood spots (DBS) matrix can bring additional information. In this study, although urinary concentrations of BZE exceeded the MRL concentrations up to 3.5 hr post-administration, the DBS levels of COC never exceeded 5 ng/mL, and BZE ranged from 40 ng/mL to 70 ng/mL [6]. These results show the potentiality of using different matrices to complement doping control analysis. Conclusions As a conclusion, the results showed an important interindividual variation, which hamper any pharmacokinetic extrapolations. Considering only the coca tea administration, samples fulfil the MRL criteria, even when the administration occurs 2 days before the IC period. On the other hand, the sample pH has the potential to impact in the analysis conclusion, considering the impact in the COC-BZE and other spontaneous hydrolysis reactions. Thus, temperature and transport time become important factors to be considered. It is noteworthy to mention that, in this study, only coca tea was administrated, which implicates that different administration routes with different doses and bioavailability can only be max max MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-328 speculated. Hence, more investigations are necessary to evaluate the diagnostic power of the current MRL criteria in COC cases. References 1. World Anti-Doping Agency. WADA Technical Document – TD2022MRPL. Minimum Required Performance Levels and Applicable Minimum Reporting Levels for Non-Threshold Substances Analysed by Chromatographic-Mass Spectrometric Analytical Methods. Montreal (2021) https://www.wada- ama.org/sites/default/files/resources/files/td2022mrpl_v1.0_final_eng.pdf (access date 30.09.2022). 2. World Anti-Doping Agency. Substances of Abuse Under the 2021 World Anti-Doping Code. Montreal (2021) https://www.wada-ama.org/sites/default/files/resources/files/2020-01- 11_guidance_note_on_substances_of_abuse_en_0.pdf (access date 30.09.2022). 3. Sardela, V. F., Martucci, M. E. P., de Araújo, A. L. D., Leal, E. C., Oliveira, D. S., Carneiro, G. R. A., Deventer K., Van Eenoo, P., Pereira, H. M. G., Aquino Neto, F. R. (2018). Comprehensive analysis by liquid chromatography Q-Orbitrap mass spectrometry: Fast screening of peptides and organic molecules. Journal of Mass Spectrometry, 53(6), 476-503. 4. Jenkins, A. J., Llosa, T., Montoya, I., Cone, E. J. (1996). Identification and quantitation of alkaloids in coca tea. Forensic Science International, 77(3), 179-189. 5. Feisthauer, E., Ameline, A., Gheddar, L., Arbouche, N., Raul, J. S., Kintz, P. (2022). Analysis of Cocaine and Its Metabolites in Urine after Consumption of Coca Tea by Five Subjects and Subsequent Hair Testing. Journal of Analytical Toxicology, 46(1), 108-113. 6. Thevis, M., Kuuranne, T., Dib, J., Thomas, A., Geyer, H. (2020). Do dried blood spots (DBS) have the potential to support result management processes in routine sports drug testing?. Drug Testing and Analysis, 12(6), 704-710. Acknowledgements The authors thank CNPq and Autoridade Brasileira de Controle de Dopagem (ABCD) for financial support. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-329 Piper T, Thevis M A brief summary of different isotope ratio mass spectrometry-based approaches to detect low-dose testosterone applications Institute of Biochemistry, German Sport University, Cologne, Germany Abstract The detection of continuous low-dose testosterone (T) applications especially in females remains highly challenging in sports drug testing. Different studies demonstrated that urinary steroids are only slightly affected and that the common approaches relying on steroid profiling and isotope ratio mass spectrometry (IRMS) often fail to unambiguously prove T administrations. So far, only the serum T concentration seems to be significantly altered after administration and may be a suitable marker for the initial testing procedure. Within this project we evaluated the potential of different recently developed IRMS-based approaches as a confirmation tool to substantiate administrations. Regarding urinary metabolites, we first focussed on 5α- and 5β-androstane-3α,17α-diol as these are metabolites of epitestosterone, and during prolonged administrations of T even epitestosterone may be affected in its carbon isotope ratios (CIR) resulting in a significant depletion of these androstanediols. In a second experiment, we investigated the potential of combining urinary concentrations and CIR resulting in a single marker named differences from weighted means (DWM) pointing towards an administration or not. Furthermore, the potential of the CIR of serum steroids was investigated based on our recently developed and validated method. Both approaches based on urinary steroids did not show promising results. The DWM-values followed the expected trend but never fell beyond individual thresholds. Especially 5β-androstane-3α,17α-diol was found depleted after the continuous T-administration, but in accordance with other urinary metabolites did not fall beyond the population-based threshold. Only IRMS-based analysis of serum steroids was able to demonstrate the exogenous origin of T-metabolites in approximately one third of the investigated samples (n = 21), mainly based on androsterone-sulfate as target metabolite. A direct comparison of CIR before and after the administration showed depleted values after administration in most of the individuals, but obviously the sensitivity was not sufficient for all samples under investigation. Nevertheless, these preliminary results are promising and further investigations in the CIR of serum steroids seem to be indicated. Introduction The detection of continuous low-dose testosterone (T) applications especially in females remains highly challenging in sports drug testing. Different studies demonstrated that urinary steroids are only slightly affected and that the common approaches relying on steroid profiling and isotope ratio mass spectrometry (IRMS) can fail in unambiguously proving T administrations. So far, only the serum T concentration seems to be significantly altered after administration and may be a suitable marker for the initial testing procedure [1-4]. Therefore, 3 different recently developed IRMS-based approaches were investigated regarding their potential to detect low-dose testosterone applications. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-330 Experimental Approach 1: Epidiols A total of 33 urine samples derived from 3 different volunteers were analysed within this study employing a further development of an already published method [5,6]. These aliquots represent a small subset of samples collected in the context of a larger investigation [3]. Three urine samples were collected before, 3 during the administration of T-gel (10 mg per day for 28 days) and 5 samples were collected 1, 2, 3, 4 and 7 days after cessation. Approach 2: DWM (ETIO) A new approach to simultaneously evaluate urinary concentrations and CIR of endogenous steroids has recently been introduced and was named difference from weighted mean (DWM)[7]. Based on the same urine samples employed in the first described approach, the suitability of DWM was tested for low-dose T- gel administrations. Approach 3: Serum-CIR Based on the described shortcomings of urinary T/Es in the detection of T-administrations to female athletes and considering the benefits of serum T-levels [1], a method was developed and validated to investigate the CIR of endogenous serum steroids [8]. This method was applied to serum samples collected from 21 female volunteers before and after T-treatment applying 10 mg/day for 10 weeks [2]. Results and Discussion Approach 1: Epidiols Urinary testosterone showed depleted carbon isotope ratios (CIR) during and directly after administration in all volunteers if concentrations above the LOQ were found (Figure 1). 5αAdiol and 5βAdiol were also found depleted, but only in 1 volunteer (V1), the CIR were found beyond established thresholds resulting in an AAF. The Epidiols (5α- and 5β-androstane-3α,17α-diol) have been investigated as potential long- term metabolites of T administrations and are mainly derived from epitestosterone (E) [5,6]. Due to the prolonged administration of T it was expected that E may also be influenced resulting in depleted CIR for both Epidiols (Figure 1). Unfortunately, only in 1 volunteer (V3) a depletion of the 5β-Epidiol was detected, demonstrating that in general the Epidiols are no suitable markers for low-dose T administrations. Figure 1. CIR found in 3 female volunteers before, during and after the administration of low-dose T-gel MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-331 Approach 2: DWM (ETIO) As depicted in Figure 2, the approach does not provide the necessary sensitivity to detect the low-dose administrations. The values for DWM (ETIO) show the expected trend towards lower values during and directly after the administration. This is due to the fact that the exogenous T is preferentially metabolized to A disturbing the endogenous equilibrium between ETIO and A. Neither population based nor individual thresholds were affected. Figure 2. Values calculated for DWM (ETIO) in 3 female volunteers before, during and after the administration of low-dose T-gel Approach 3: Serum-CIR Significant differences (t-test, p < 0.01) were found for the Δ-values measured before and after the treatment as depicted in Figure 3. The target compounds (TCs) showed consistently more depleted values after the administration. Considering the preliminarily established thresholds for serum steroids [8], at least 6 out of 20 post administration samples (1 sample fell below the LOQ) were found with CIR not in accordance with endogenous steroid production. Mainly the Δ-values encompassing androsterone were responsible for these findings. Figure 3. Boxplots comparing the Δ-values between the ERCs cholesterol (CHOL) and dehydroepiandrosterone (DHEA) and the TCs androsterone (A) and epiandrosterone (EpiA) Unfortunately, the CIR of the T-gel used for the administration trial was not determined. Considering the relatively depleted values found for the endogenous reference compounds (ERC) within this study (Figure 4), it may be hypothesized that the approach will show a better sensitivity in individuals with more enriched values in the ERCs. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-332 Figure 4. Absolute CIR found for both ERCs Conclusions Three different IRMS-based approaches have been investigated regarding their potential to detect the misuse of low-dose T-applications. For both the Epidiols and the DWM (ETIO) the sensitivity was not sufficient for sports drug testing. Interestingly, when urinary T itself was measurable, its CIR showed clearly the exogenous origin. Applying CIR to serum steroids allowed for the detection of T-misuse in 6 out of 20 investigated cases. In accordance with serum concentrations, serum CIR seem to be the most promising approach to detect low-dose T-administrations in female athletes. Further research may enable improvements in the sensitivity of this approach. References 1. Handelsman DJ, Bermon S. Detection of testosterone doping in female athletes. Drug Test Anal 2019; 11: 1566-1571. 2. Knutsson JE, Andersson A, Baekken LV, Pohanka A, Ekström L, Hirschberg AL. Disposition of urinary and serum steroid metabolites in response to testosterone administration in healthy women. J Clin Endocrinol Metab 2021; 106: 697-707. 3. Salamin O, Nicoli R, Langer T, Boccard J, Schweizer Grundisch C, Xu C, Rudaz S Kuuranne T, Pitteloud N, Saugy M. Longitudinal evaluation of multiple biomarkers for the detection of testosterone gel administration in women with normal menstrual cycle. Drug Test Anal 2021; 1–18. https://doi.org/10.1002/dta.3040. 4. Savkovic S, Ly LP, Desai R, Howa J, Nair V, Eichner D, Handelsman DJ. Detection of testosterone microdosing in healthy females. Drug Test Anal 2022; 1–14. https://doi.org/10.1002/dta.3202. 5. Piper T, Riemann P, Opfermann G, Mareck U, Geyer H, Vajiala G, Flenker U, Schänzer W. Determination of C/ C ratios of urinary epitestosterone and its main metabolites 5α- and 5β-androstane-3α,17α-diol. Drug Test Anal 2009;1:576–586. 6. Piper T, Rubio A, Thevis M. Investigations on carbon isotope ratios of potential novel long-term metabolites of testosterone and testosterone prohormone misuse - 5α- and 5β-androstane-3α,17α-diol. Manfred Donike Workshop - Proceedings of the 38 Cologne Workshop on Dope Analysis 2020. In: Thevis M, Geyer H, Mareck U (eds.) Recent Advances In Doping Analysis; 28. Sport und Buch Strauß - Köln 2020, 182-183. 7. Piper T, Haenelt N, Fusshöller G, Geyer H, Thevis M. Sensitive detection of testosterone and testosterone prohormone administrations based on urinary concentrations and carbon isotope ratios of androsterone and etiocholanolone. Drug Test Anal. 2021;13:1835–1851. 8. Piper T, Geyer H, Nieschlag E, Bally L, Thevis M. Carbon isotope ratios of endogenous steroids found in human serum—method development, validation, and reference population-derived thresholds. Anal Bioanal Chem 2021;413:5655-5667. 13 12 th MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-333 Acknowledgements We are grateful to O. Salamin and co-workers for providing urine samples of female volunteers to investigate both the Epidiols and the DWM (ETIO). The authors are indebted to L. Ekström and co-workers for providing serum samples after low-dose testosterone applications. We thank the Manfred Donike Institute for Doping Analysis e.V. (Cologne, Germany) and the Federal Ministry of the Interior, Building and Community (Berlin, Germany) for financial support. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-334 Kwiatkowska D , Kaplinski A , Urbaniak-Zbyszynska A , Jarek A , Tarka M , Chajewska K , Siek P , Pasik M , Szczepanska Z , Wojtowicz-Zawadka M Pregnancy as a factor influencing the change of the steroid profile in terms of assessment of the athlete's biological passport Polish Anti-Doping Laboratory, Warsaw, Poland ; Specialist Clinic - Arkadiusz K. Kaplinski, Warsaw, Poland Abstract The study was to investigate the changes in the hormonal balance in terms of factors changing the steroid profile of the athlete during pregnancy. For this purpose, a collaboration with a gynaecological clinic and pregnant women was established. The collected results gathered for tested samples were analyzed in terms of the steroid parameters observed when assessing the athlete's passport. Additionally, the concentrations of hCG were estimated. The observations confirmed that pregnancy, even the early one which the athlete may not know about, can affect the concentrations of the tested hormones as well as their ratios/indicators. Introduction The concept of an Athlete Biological Passport is based on detecting doping-induced changes in selected biomarkers by monitoring them in long term and comparing them with the values already obtained in the previous anti-doping analysis. It may indirectly allow the detection of the effects of doping, which may be an alternative to the detection of the substance itself or the use of a prohibited method to prove an athlete's anti-doping rule violation. Unfortunately, there are several factors that might influence steroid profiles such as e.g. inter-individual variability of steroid synthesis and metabolism (UGT2B17 polymorphism), pregnancy, contraceptive pills, alcohol, the administration of ketoconazole, human chorionic gonadotrophin in males, inhibitors of 5α-reductase and the influence of microorganisms existing in urine samples [1]. Experimental The carried out research was to broaden the knowledge about changes in the steroid profile in terms of assessing the athlete’s biological passport. Six steroid parameters such as androsterone (A), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5α-Adiol), 5β-androstane-3α,17β-diol (5β-Adiol), testosterone (T) and epitestosterone (E), and five their derivatives such as T/E ratio, A/Etio ratio, A/T ratio, 5α-Adiol/5β-Adiol ratio and 5α-adiol/E ratio were analyzed. An attempt was made to assess the steroids profile changes in the case of pregnancy. This study with the ethics approval nr KEBN-20-55-DK was carried out in cooperation with an expert from the Warsaw Athlete’s Passport Management Unit, Dr. Arkadiusz Kaplinski. 1 2 1 1 1 1 1 1 1 1 1 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-335 The analyzed urine samples were collected from seven healthy pregnant volunteers in the range of at least from seventy-third to one hundred and sixth day of pregnancy. The volunteers were in the age range of 27-47 years and they did not take any medicaments or alcohol during the urine sample collection. After delivery to the Polish Anti-Doping Laboratory (PLAD) from the gynecological clinic, all urine samples were being measured for specific gravity and stored at -20 °C until further analysis. Two mL of each collected urine sample was fortified with a mixture of internal standards (d -Etio for A, Etio, 5α-Adiol, 5β-Adiol and T, and d -T for E) followed by a sample preparation consisting of solid phase extraction, enzymatic hydrolysis of glucuronide conjugates, liquid-liquid extraction, trimethyl- silylation, and analysis using GC-MS system: 7890A Agilent Technologies system equipped with a J&W 190911z-008 HP1 column (17 m x 200 μm x 0.11 μm, Agilent Technologies), 7693 Agilent Technology autosampler and 5975C Agilent Technology system equipped with a quadrupole analyzer and an EI ion source. Moreover, the concentration of hCG was estimated using the immunochemical analyzer (Roche, Hitachi / Cobas e411). Results and Discussion Steroid concentrations for A, Etio, 5α-Adiol, 5β-Adiol, T and E were adjusted to a urine specific gravity (SG) of 1.020 based on the following equation: Conc = Conc * (1.020 -1) / (SG - 1). Three representative volunteers were selected for whom the tested time range of pregnancy was the longest. The results for the steroid profile of the analyzed urine samples from these volunteers are shown in Figure 1. 5 3 corr measured MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-336 Figure 1. The concentration profiles of T, E, 5α-Adiol, 5β-Adiol, A and Etio during pregnancy Moreover, the received profiles of steroid parameter derivatives such as T/E, A/Etio, A/T, 5α-Adiol/5β- Adiol and 5α-adiol/E are shown in Figure 2. Additionally, the obtained concentrations of hCG are shown in Figure 3. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-337 Figure 2. The ratio profiles of T/ E, 5α-Adiol /E, A/T, A/Etio and 5α-Adiol/5β-Adiol during pregnancy Figure 3. The concentration of hCG during pregnancy MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-338 The obtained results for the steroid profile of the analyzed urine samples from pregnant women have shown that pregnancy changes the steroid profile, which may be reflected in the ABP steroid module. The differences in the shape of the presented steroid profile curves for the volunteers were related to the inter-individual variability of steroid synthesis and metabolism. However, in some cases, the significant changes of T/E and 5α-adiol/E have been noticed at the beginning of the first trimester of pregnancy. In most cases, an increase in the concentration of E (in the third trimester of pregnancy) and a decrease in the ratio of T/E and 5α-diol/E (after the first trimester of pregnancy) have been observed. In some cases, an increase in the value of the ratio of A/Etio and 5α-diol/5β-diol has been also observed in the second and third trimesters of pregnancy. The fluctuations in the values of the steroid profile parameters were correlated with the hCG values. Conclusions The obtained results might to help interpret the steroid profile results in terms of assessment the athlete's biological passport and in terms of commissioning additional tests, including e.g. hCG testing in women to confirm pregnancy associated with changes in the steroid profile. References 1. Mareck U, Geyer H, Opfermann G, Thevis M, Schänzer W (2008) Factors influencing the steroid profile in doping control analysis. J Mass Spectrom 43, 877– 891. Acknowledgements Financial support from the Ministry of Sport and Tourism of the Republic of Poland under the project number 2020.0145/1575/UDOT/BM and the Ministry of Culture, National Heritage and Sport of the Republic of Poland under the project number 2021.0416/1575/Udot/DS./14/AM are gratefully acknowledged. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-339 Kwiatkowska D , Wicka M , Wojtowicz-Zawadka M , Urbaniak-Zbyszynska A , Tarka M , Chajewska K , Grucza K , Szczepanska Z , Pasik M , Zalewska Z , Jarek A , Kaplinski A Gender change in the aspect of assessing the athlete’s biological passport Polish Anti-Doping Laboratory, Warsaw, Poland ; Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland ; Specialist Clinic - Arkadiusz K. Kaplinski, Warsaw, Poland Abstract Athlete Biological Passport (ABP) is one of the tools to identify the use of WADA prohibited substances that affect an athlete's steroid profile (SP). It also provides longitudinal monitoring of steroidal markers and their variations over a period of time. The study was to monitor and investigate changes in concentrations and ratios of defined endogenous anabolic androgenic steroids (EAAS) in terms of gender change. For this purpose, the Polish Anti-Doping Laboratory (PLAD) collaborated with a gynecological wedge and a person undergoing the process of gender change. Collected urine samples were tested for EAAS. The results showed, that the process of gender change has a significant impact on the values of steroidal markers. Different body responses were observed depending on the treatment cycle. In order to find similarities, the obtained results were compared with the passport of an athlete who had a Therapeutic Use Exemption (TUE) for testosterone use, which could be a significant clue to correct interpretation of passport data in the future. Introduction The concept of the ABP is based on detecting doping-induced changes in selected biomarkers by their long-time monitoring and comparing with values obtained during previous analyses. To determine whether the change in the ABP is due to the body's physiological adaptation to the environment or not, each result is mathematically compared with values reported from former analyses of samples. Several factors might influence the SP: inter-individual variability of steroid synthesis and metabolism, pregnancy, contraceptive pills, a large intake of alcohol, the administration of ketoconazole, hCG in males, inhibitors of 5α-reductase, the influence of microorganisms existing in urine samples, the use of masking agents and diuretics. The SP values may also be influenced by mental stress or changes related to the age [1]. Experimental The proposed research was to broaden the knowledge about changes in the steroid profile in terms of assessing the athlete's biological passport. An attempt was made to assess the steroid profile changes in the case of a person undergoing the process of gender change and using a hormone treatment for this purpose. This study was carried out in cooperation with an expert from the Warsaw APMU (Athlete’s Passport Management Unit), Dr. Arkadiusz Kaplinski. The ethics approval nr is KEBN-20-55-DK. 1,2 1 1 1 1 1 1 1 1 1 1 3 1 2 3 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-340 The person undergoing the process of gender-changing from female to male has been taking a drug called “Omnadren 250”, cointaining four different testosterone esters: 30 mg testosterone propionate, 60 mg testosterone phenylopropionate, 60 mg testosterone isocaproate and 100 mg testosterone decanoate. 0.25 g/mL in the amount of 1 amp. has been administered by intramuscular injection every 21 days for the duration of the ongoing treatment. Urine samples were collected from June 22, 2019, to September 28, 2021. The reference urine samples were collected twice before starting the treatment. Following the administration, all spontaneous samples were collected for two days. Afterwards, one sample a day was collected till the end of December 2019. Then, the samples were collected weekly for the remaining time of the study. After the submission to the Polish Anti-Doping Laboratory (PLAD), the samples were stored at -20° C until analysis. The collected urine samples were prepared according to the routine procedures in PLAD for steroid profile determination. 2 mL of urine was fortified with a mixture of deuterated internal standards (d - etiocholanolone, d -testosterone, d -androsterone), then was purified and concentrated on a C18 cartridge (BAKERBOND spe 7020-06; J.T. Baker). The columns were activated and conditioned using 4 mL of methanol and 4 mL of water, respectively, prior to loading the samples. Subsequently, the columns were rinsed with 3 mL of water. Afterwards, steroids were eluted with 3 mL of methanol and an extract was collected for further analysis. After evaporation at 50 °C, 1 mL of 0.2 M of phosphate buffer (pH 7.0) and 50 µL of β-glucuronidase (E. coli) were added to the dry residues. Enzymatic hydrolysis of glucuronides was carried out at 50°C for 60 min. Next, the samples were cooled to ambient temperature and following the addition of phosphate buffer (pH 8-9), extracted with 6 mL of diethyl ether by shaking (20 min). Samples were then centrifuged for 6 min and frozen. The organic phase was collected and the solvent evaporated at 45°C under a nitrogen flow. Dry residues were reconstituted in 400 µL of diethyl ether, transferred to vials and submitted to derivatisation with 50 µL of a mixture containing MSTFA, ammonium iodide and ethanethiol (1 L/1 mg/4 mL) at (80 ± 5) °C for 20 min. All analyses were performed using GC/MS system: 7890A Agilent Technologies system equipped with a J&W 190911z-008 HP1 column (17 m x 200 μm x 0.11 μm, Agilent Technologies), 7693 Agilent Technology autosampler and 5975C Agilent Technology system equipped with a quadrupole analyzer and an EI ion source. Steroid concentrations for A, Etio, 5α-Adiol, 5β-Adiol, T and E were adjusted to a urine specific gravity (SG) of 1.020 based on the following equation: Conc = Conc * (1.020 – 1)/(SG-1). Additionally, the collected urine samples have been analyzed for the presence of alcohol and other confounding factors. Results and Discussion The results for the steroid profile of the analyzed urine sample were compiled using statistical programs to study the trend. The significant changes in the concentration of the selected steroids including testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5α-Adiol), 5β-androstane-3α,17β-diol (5β-Adiol) have been observed at the beginning of the treatment, especially immediately after the testosterone injection. More stable changes were observed over time, along with the visible change of the person’s gender in the direction of male (Fig. 1-3). 5 3 4 TM corr measured MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-341 The results of the samples with biological degradation haven’t been included in the charts. The samples in which ethyl glucuronide has been detected were included in the charts. Figure 1. The change of the person’s gender in the direction of male – T/E and 5α-Adiol/E Figure 2. The change of the person’s gender in the direction of male – A/T Figure 3.. The change of the person’s gender in the direction of male – A/Etio and 5α-Adiol/5β-Adiol We could make the comparison of the steroid profiles after gender-changing treatment with the steroid profile of an athlete with TUE for testosterone use. We can observe similar behaviour of the levels of steroids included in the steroid profile after the administration of testosterone in both cases. The concentration of T in both profiles increased with a simultaneous decrease of E concentration at the same time (data not shown) resulting in an increased value for the T/E ratio. A similar situation can be observed in the case of the 5α-Adiol/E ratio, next to a significant increase of the A/Etio ratio (Fig. 4). MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-342 Figure 4. ABP steroid profile of an athlete with TUE for testosterone use Conclusions The results indicate that the administration of exogenous testosterone significantly increases the concentration of T, A, Etio, 5α-Adiol, 5β-Adiol, which translates into changes in index values, an increase in the T/E, a decrease in the A/T, a decrease in the value of 5α-diol/5β-diol and an increase in 5α-diol/E ratio. Thanks to these studies, it was possible to observe changes immediately after the administration of testosterone and long-term changes. Given the similarities between the steroid profile of the athlete with a TUE for testosterone use and a person undergoing gender change, the obtained results can be of great importance in view of the interpretation of an athlete's ABP. References 1. Mareck U, Geyer H, Opfermann G, Thevis M, Schänzer W (2008) Factors influencing the steroid profile in doping control analysis. J Mass Spectrom 43, 877-891 Acknowledgements Financial support from the Ministry of Sport and Tourism of the Republic of Poland (project number 2020.0145/1575/UDOT/BM) and the Ministry of Culture, National Heritage and Sport of the Republic of Poland (project number 2021.0416/1575/Udot/DS./14/AM) are gratefully acknowledged. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-343 Mareck U, Fußhöller G, Geyer H, Görgens C, Guddat S, Haenelt N, Romberg S, Thevis M Evaluation of urinary ethyl glucuronide levels in doping control samples 2018-2020 Center for Preventive Doping Research / Institute of Biochemistry, German Sport University Cologne, Cologne, Germany Abstract The “urinary steroid profile” may be altered following the intake of alcohol, which can trigger Confirmation Procedure (CP) requests entailing time-consuming and costly GC/C/IRMS analysis. A total of 66229 doping control urine samples, analyzed between 2018 and 2020 in the Cologne anti-doping laboratory for the presence of ethyl glucuronide (ETG) by means of liquid chromatography-tandem mass spectrometry, was evaluated. Overall, 2584 (3.9%) specimens showed an estimated ETG concentration higher than 5 µg/mL, 89% were collected out-of-competition, and 78% originated from male athletes. 255 of the ETG-containing samples (~ 10%) led to Atypical Passport Findings (ATPFs) resulting in CPs and GC/C/IRMS analyses. All CPs returned negative IRMS results. Based on the evaluation results it appears warranted to discuss, if GC/C/IRMS analyses are applicable only for samples yielding ETG at levels under 20 µg/mL. Introduction The urinary “steroid profile” may be altered following the intake of alcohol. The determination of the confounding factor ethanol is performed via the identification and quantification of ethyl glucuronide (ETG) [1]. Alterations of the steroid profile may lead to Atypical Passport Findings (ATPF) or Suspicious Steroid Profiles (SSP), which can trigger Confirmation Procedure (CP) requests [1]. In 2018, an evaluation of the frequency and distribution of ETG in genders, sports and control types (in-competition, out-of- competition) for doping control urine samples analyzed in the Cologne doping control laboratory in 2016 and 2017 was already performed [2]. In order to examine the data precision, the same evaluation was performed for the period 2018 – 2020 with an additional evaluation of ETG in samples, which underwent CPs. Experimental A total of 66229 doping control urine samples from national and international federations, analyzed between 2018 and 2020 in the Cologne anti-doping laboratory for the presence of ETG by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS) [3], was evaluated. Results and Discussion 2584 (3.9%) doping control urine samples contained ETG between 5 and 750 µg/mL. In total, 89% of these urine specimens were collected out-of-competition (OOC) and 78% originated from male athletes MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-344 (Tab. 1a). Similar results were obtained in 2018 for the evaluation of 46785 doping control urine samples, analyzed in 2016 and 2017 [2]. For 1218 urine specimens (1.8%) analyzed between 2018 and 2020, ABP-CP was requested, 255 of them (21%) yielding ETG higher than 5 µg/mL, 94% collected OOC and 73% originated from male athletes (Tab. 1b). All samples returned negative GC/C/IRMS results. While the parameters gender and control types are similar, the frequency and distribution of ETG between ordinary doping control urine samples and ABP-CP specimens showed significant differences. In ordinary doping control urine samples, low ETG concentrations are more represented, vice versa in ABP-CP specimens, most probably triggering the CP request (Fig. 1a+b). When compared to the normal reference, the total of 66229 doping control urine samples showed 58% out of competition controls, 70% male specimens and 79% ATPFs without ETG. Conclusions ~ 10% of ETG containing specimens lead to ATPFs All CPs based on ETG findings returned negative IRMS results It should be discussed, whether the time-consuming and costly GC/C/IRMS can be waived for samples yielding more than 20 µg/mL ETG. References 1. World Anti-Doping Agency. Technical Document TD 2021EAAS, v.2.0., June 2021 https://www.wada- ama.org/sites/default/files/2022-01/td2021eaas_final_eng_v_2.0.pdf (access 14.07.2022) 2. U. Mareck, G. Fußhöller, H. Geyer, C. Görgens, S. Guddat, N. Haenelt, S. Romberg, W. Schänzer, M. Thevis, in Recent Advances in Doping Analysis (26), (Eds: M. Thevis, H. Geyer, U. Mareck), 2018, p. 167-169 3. C. Görgens, S. Guddat, A. Thomas, H. Geyer, W. Schänzer, in Recent Advances in Doping Analysis (19), (Eds: W. Schänzer, H. Geyer, A. Gotzmann, U. Mareck), 2011, p. 309-312 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-345 Acknowledgements The authors thank the Manfred Donike Institute for Doping Analysis e.V. (Cologne, Germany) and the Federal Ministry of the Interior, Building and Community (Berlin, Germany) for supporting the study. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-346 Mareck U, Fußhöller G, Hülsemann F, Geyer H, Thevis M Results of confirmation analyses for boldenone in the Cologne laboratory from 2017-2021 Center for Preventive Doping Research / Institute of Biochemistry, German Sport University Cologne, Cologne, Germany Abstract According to WADA regulations, a GC/C/IRMS analysis is mandatory for samples, in which the concen- tration of boldenone or the main boldenone metabolite is estimated between (≥) 2.5 and (≤) 30 ng/mL. A total of 72 doping control urine samples – analyzed in the Cologne anti-doping laboratory – for which a boldenone confirmation procedure enclosing GC/C/IRMS analysis was performed between 2017 and 2021, was evaluated. 49 specimens (68%) returned negative GC/C/IRMS results, 4 were reported as atypical (ATF), and 19 (26%) as adverse analytical finding (AAF). Based on GC/C/IRMS results, the estimated concentrations for boldenone and / or boldenone metabolite do not allow a prediction of the exogenous or endogenous origin. No additional criteria could be found for the decision-making process to reduce GC/C/IRMS analysis. Introduction According to WADA Technical Document TD2021IRMS, GC/C/IRMS analysis is mandatory on samples in which the specific gravity adjusted concentration of boldenone (B) or the main boldenone metabolite (BM1; 5β-androst-1-en-17β-ol-3-one) is estimated between (≥) 2.5 and (≤) 30 ng/mL [1]. The confirmation procedure includes the identification and estimation of the B and BM1 concentration as well as a time and costly GC/C/IRMS analysis. Also, several laboratories have not the analytical capacity to perform GC/C/IRMS analysis for B and BM1. They have (after consultation with the Testing Authority or the Results Management Authority) to transfer the samples to another laboratory that has such analytical capacity, further entailing time- and cost-intensive processes. In order to identify possible criteria that facilitate reducing the need for IRMS analyses and, thus, saving resources, further evaluations of the doping control urine samples with B and BM1 concentrations less than 30 ng/mL, which were analyzed between 2017 and 2021 with GC/C/IRMS, were performed. Experimental In total, 72 doping control urine samples with B and BM1 concentrations less than 30 ng/mL from national and international federations and subcontracted analyses, were analyzed between 2017 and 2021 in the Cologne anti-doping laboratory to confirm the estimated concentration of B and BM1 and to perform GC/C/IRMS analysis to establish the origin (endogenous or exogenous) of the detected substances. The initial testing procedure (ITP) and confirmation procedure (CP) of B and BM1 were conducted according to the method described by Thevis [2] with consideration of the rules described in MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-347 the TD2021IRMS [1]. The IRMS analyses were conducted according to the method described by Piper et al. [3]. Results and Discussion Of the 72 analyzed doping control samples, 49 (68%) returned negative GC/C/IRMS results, 4 were reported as atypical finding (ATF) and 19 (26%) were reported as adverse analytical finding (AAF); none of these exhibited signs of degradation, e.g. increased pH value, Δ -steroid-dehydrogenase or other bacterial activity. In 6 samples that produced AAFs, further prohibited substances were detected. Negative samples and samples reported as ATFs showed concentrations of B and/or BM1 equal or less than 14 ng/mL for all but one specimen. In samples reported as AAFs, the concentrations ranged between 1.8 and 27.3 ng/mL, with 11 samples equal or less than 10 ng/mL, i.e. B and/or BM1 concentrations alone do not allow to predict the result of a GC/C/IRMS analysis. Figures 1 and 2 presenting δ C-Boldenone and ΔERC-TC versus the Boldenone concentrations of the specimens support this assumption. Whereas B and BM1 concentrations of the negative samples are centered around 5 ng/mL with only a few samples exceeding 10 ng/mL, the B and BM1 concentrations of samples reported as AAFs are distributed over the whole concentration range. The obtained δ C-values for B allow a clear differentiation between negative/ATF samples with δ C-values in the usual endogenous range of -20 to ‑26 ‰ and samples reported as AAFs with δ C-values around -30 ‰. Calculations to find ratios as additional criteria for the decision making process were performed for several constellations between B or BM1 and endogenous steroids (testosterone, epitestosterone, androsterone (A), etiocholanolone). While for 19-norandrosterone (19-NA) findings, the ratios 19-NA/A and 19-NA/19-NE (19-noretiocholanolone) are most probably helpful criteria for the discrimination between exogenous and endogenous origin of 19-NA, similar criteria could not be identified for B or BM1. Figure 1. Boldenone concentration / δ C-Boldenone 1 13 13 13 13 13 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-348 Figure 2. Boldenone concentration / ΔERC-TC Conclusions Based on the evaluation of the available data, no further criteria can currently be proposed to support a reduction of GC/C/IRMS analysis. References 1. World Anti-Doping Agency. Technical Document TD 2021IRMS. https://www.wada- ama.org/sites/default/files/resources/files/td2021irms_final_eng_v_2.0.pdf (access 24.08.2021) 2. Thevis M. Mass Spectrometry in Sports Drug Testing – Characterization of Prohibited Substances and Doping Control Analytical Assays. Wiley, New Jersey, 201. 376 pages. ISBN: 978-0-470-41327-2. 3. Piper T, Geyer H, Gougoulidis V, Flenker U, Schänzer W. (2010) Determination of C/ C ratios of urinary excreted boldenone and its main metabolite 5β-androst-1-en-17β-ol-3-one. Drug Test Anal. 2, 217-224. Acknowledgements The authors thank the Manfred Donike Institute for Doping Analysis e.V. (Cologne, Germany) and the Federal Ministry of the Interior, Building and Community (Berlin, Germany) for supporting the study. 13 12 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-349 Mareck U, Geyer H, Fußhöller G, Thevis M Results of confirmation analysis for 19-norandosterone in the Cologne laboratory from 2017-2021 Center for Preventive Doping Research / Institute of Biochemistry, German Sport University Cologne, Cologne, Germany Abstract According to WADA regulations a GC/C/IRMS analysis is mandatory on samples with an estimated 19-NA concentration between 2.5 and 15 ng/mL. A total of 53 doping control urine samples – analyzed in the Cologne anti-doping laboratory – for which a 19-norandrosterone confirmation procedure enclosing GC/C/IRMS analysis was performed between 2017 and 2021, was evaluated. Overall, 34 of these urine specimens were reported negative, 91% showing an estimated 19-NA concentration less than 5 ng/mL, 76% less than 0.1% 19-NA/A and 53% were delivered with a time span between collection and reception of more than 21 days. 19 urine specimens were reported as Adverse Analytical Finding (AAF) or Atypical Finding (ATF). Based on the evaluation results it can be concluded that revisiting the criteria triggering GC/C/IRMS analysis is warranted. Introduction According to the WADA Technical Document TD2021 NA v2.0, a GC/C/IRMS analysis is mandatory on samples in which the concentration of 19-NA is estimated between (≥) 2.5 and (≤) 15 ng/mL, except in cases of pregnancy or in the presence of 3,5-tetrahydronorethisterone [1]. The confirmation procedure includes the identification and estimation of the 19-NA concentration as well as a time-consuming and costly GC/C/IRMS analysis. Also, several laboratories have not yet the analytical capacity to perform GC/C/IRMS analysis for 19-NA. They have (after consultation with the Testing Authority or the Results Management Authority) to transfer the samples to another laboratory that has such analytical capacity, further entailing time- and cost-intensive processes. In order to identify possible criteria that facilitate reducing the need for IRMS analyses and, thus, saving resources, further evaluations of the doping control urine samples with NA concentrations less than 15 ng/mL, which were analyzed between 2017 and 2021 with GC/C/IRMS, were performed. Experimental In total, 53 doping control urine samples with NA concentrations < 15 ng/mL from national and international federations were analyzed between 2017 and 2021 to confirm the estimated concentration of 19-NA and to perform GC/C/IRMS analysis to establish the origin (endogenous or exogenous) of the detected19-NA. The initial testing procedure (ITP) and confirmation procedure (CP) of 19-NA were conducted according to the method described by Hülsemann et al. [2] with consideration of the TD2021NA [1]. The IRMS analyses were conducted according to the method described by Piper et al. [3]. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-350 Results and Discussion Of the 53 analyzed doping control samples, 34 (64%) returned negative GC/C/IRMS results, and none of these exhibited an increased pH value or signs of bacterial activity. As shown in Figure 1, 31 out of the 34 negative samples showed NA concentrations below 5 ng/mL. Amongst the 19 adverse analytical (AAF) and atypical (ATF) findings, 12 samples presented urinary 19-NA concentrations < 5 ng/mL, i.e. the 19-NA concentration alone does not allow to predict the result of a GC/C/IRMS analysis (Table 1 and Figure 1). Consequently, the added value of further criteria was assessed: 19-NA/19-NE > 3: According to TD2021NA, the laboratory has to consider the ratio 19-NA/19-NE as a possible indicator of the administration of 19-norsteroids. Negative GC/C/IRMS results and a 19-NA/19-NE ratio > 3 have to be reported as atypical finding. From the 34 samples with negative GC/C/IRMS results, only 5 samples showed 19-NA/19-NE ratio > 3. All but one sample with AAFs or ATFs showed 19-NA/19-NE ratios > 3 (see Table 1). 19-NA/A < 0.1%: The 19-NA/A ratio < 0.1% was proposed as additional criterion for the decision-making process in 1999 [4] and further presented in a case study [5]. As a natural conversion from androsterone (A) to 19-NA cannot be excluded, this parameter may be a valuable tool. 26 (76%) out of the 34 negative samples showed 19-NA/A values < 0.1% whereas only 3 (15%) out of the 19 AAFs and ATFs showed values < 0.1% (see Table 1). Time span (collection-reception): Many of the specimens included in this study originated from a testing authority with unusually long delivery times. Although without detectable signs of urine alteration (e.g. increased pH value or bacterial activity) it is conceivable that long storage times under uncontrolled conditions may influence the conversion from endogenous steroids to 19-NA. Out of the 34 negative samples, 18 samples (53%) showed time spans of more than 20 days between collection and reception in the laboratory. Out of the 19 samples with AAFs and ATFs, 5 (26%) showed time spans > 20 days (see Table 1). Table 1. Summary of evaluation MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-351 Figure 1. 19-NA concentrations in negative, AAF and ATF urine samples Conclusions Based on the obtained results, the following proceeding is suggested. Samples can be reported negative without any confirmation and / or GC/C/IRMS analysis, when all of the following criteria are met: concentration of 19-norandrosterone < 5 ng/mL (without adjustment for the urine specific gravity) 19-NA/19-NE < 3 19-NA/A < 1:1000 ( < 0.1%) time span sample collection/sample reception > 21 days Applying the suggestion to the evaluated data would have resulted in omitting confirmation procedures in 9 cases. None of the AAF or ATF reported samples matched the proposal. Consequently, there would have been no false negative results. References 1. World Anti-Doping Agency. Technical Document TD 2021NA v.2.0. https://www.wada- ama.org/sites/default/files/resources/files/td2021na_final_eng_v2.0_m.pdf (access 27.07.2021) 2. Hülsemann F, Gougoulidis V, Schertel T, Fusshöller G, Flenker U, Piper T, Thevis M. (2018) Case Study: Atypical δ C values of urinary norandrosterone. Drug Test Anal. 10, 1728-1733. 3. Piper T, Emery C, Saugy M: Norandrosterone analysis by GC/C/IRMS. W Schänzer, M Thevis, H Geyer, U Mareck (eds.) Recent advances in doping analysis (20) Sport und Buch Strauß, Köln (2012) 4. Mareck-Engelke U, Geyer H, Schänzer W: 19-Norandrosterone – Criteria for the Decision Making Process. M Donike, H Geyer, A Gotzmann, U Mareck-Engelke (eds.) Recent advances in doping analysis (6). Sport und Buch Strauß, Köln (1999) 119-129 5. Fußhöller G, Geyer H, Haenelt N, Hülsemann F, Gougoulidis V, Blatt C, Thevis M: Additional investigations in connection with atypical findings for 19-norandrosterone – a case study. M Thevis, H Geyer, U Mareck (eds.) Recent advances in doping analysis (29) Sport und Buch Strauß, Köln (2021) 38-40 13 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-352 Acknowledgements The authors thank the Manfred Donike Institute for Doping Analysis e.V. (Cologne, Germany) and the Federal Ministry of the Interior, Building and Community (Berlin, Germany) for supporting the study. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-353 Hülsemann F, Mareck U, Fußhöller G, Geyer H, Thevis M GC/C/IRMS results for 19-norandosterone findings in the Cologne laboratory from 2017-2021 Institute of Biochemistry, German Sport University, Cologne, Germany Abstract An evaluation of 98 doping control urine samples with 19-NA concentrations < 22 ng/mL from national and international federations and subcontracted analyses, analyzed between 2017 and 2021 in the Cologne anti-doping laboratory via GC/C/IRMS to establish the origin (endogenous or exogenous) of the detected 19-NA, was performed. 66 specimens returned negative GC/C/IRMS results, 10 were reported atypical and 22 as adverse analytical finding (AAF). The results are supplemental data to the poster “Results of confirmation analysis for 19-norandrosterone (19-NA) in the Cologne laboratory from 2017 – 2021” and allow a review of the previous conclusions with an increased data set. In the case of samples with 19-NA concentrations < 15 ng/mL but with a prolonged time span between collection and reception greater than 21 days, it would be useful to check additional criteria such as 19- NA/19-NE > 3, 19-NA/A > 0.1%, and 19-NA concentration > 5 ng/mL before performing a GC/C/IRMS analysis. Samples not meeting the presented criteria turned out to be GC/C/IRMS negative. Using these criteria, the number of samples analyzed by GC/C/IRMS could be reduced by at least 12%, saving laboratory resources and avoiding unnecessary analytical procedures. Introduction The presented IRMS results are supplemental to the poster “Results of confirmation analysis for 19- norandrosterone in the Cologne laboratory from 2017 – 2021” [1]. The increased amount of data resulting from subcontracted analyses allows a review of the drawn conclusions of the initial investigation [1]. While the results of the initial investigation are based on 53 doping control urine samples from the Cologne anti-doping laboratory, the further evaluation encloses additional 45 subcontracted specimens. Experimental In total, 98 doping control urine samples with 19-norandrosterone (19-NA) concentrations < 22 ng/mL from national and international federations and subcontracted analyses, were analyzed between 2017 and 2021 in the Cologne anti-doping laboratory via GC/C/IRMS to establish the origin (endogenous or exogenous) of the detected 19-NA. The initial testing procedure (ITP) and confirmation procedure (CP) of 19-NA were conducted according to the method described by Hülsemann et al. [2] with consideration of the TD2021NA [3]. The GC/C/IRMS analyses were conducted according to the method described by Piper et al. [4]. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-354 For subcontracted specimens, concentrations for androsterone and 19-NE, as well as the time span between collection and reception in the initial laboratory are frequently not part of the provided information. Consequently, the evaluation is based on a limited amount of available data. Results and Discussion Of the 98 analyzed doping control samples, 66 (67%) returned negative GC/C/IRMS results, 10 (10%) were reported atypical (ATF), and 22 (22%) as adverse analytical finding (AAF); none of these exhibited an increased pH value or signs of bacterial activity based on androstanedione ratios [5]. 51 of 66 (77%) negative samples showed 19-NA concentrations below 5 ng/mL. However, 32 ATFs and AAFs exhibited also urinary 19-NA concentrations < 5 ng/mL; i.e. the 19-NA concentration solely does not allow the prediction of the GC/C/IRMS result (Figure 1). Figure 1. Comparison of 19-NA concentration of 98 doping control urine samples and corresponding Δ C - values Following the TD2021NA samples with a negative GC/C/IRMS result and a 19-NA/19-NE ratio > 3 have to be reported as atypical finding. In this study, due to limited information attached to subcontracted samples for GC/C/IRMS analyses, only for a subset of samples 19-NA/19-NE ratios were known. Out of these, for those 29 samples with negative GC/C/IRMS results, eight samples showed 19-NA/19-NE ratio > 3. All samples in this subset with AAFs showed 19-NA/19-NE ratios > 3 (Figure 2). The reported ATF results with an endogenous δ C signature may derive from an exogenous administration of a synthetic 19-NA precursor or ingestion of uncastrated boar offal/meat. 13 PD-NA 13 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-355 Figure 2. Subset of samples with known 19-NA/19-NE ratios in comparison to corresponding Δ C -values Accordingly, 35 (78%) out of the 45 negative samples with known 19-NA/A ratios were below 0.1% whereas 67 % of the 15 AAFs showed values > 0.1% (Figure 3). Regarding samples with a known time span between sampling and reception at the laboratory of more than 21 days, 17 (71%) returned negative GC/C/IRMS results, 7 specimens (29%) were AAF or ATF (Figure 4). 13 PD-NA MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-356 Figure 3. Subset of samples with known 19-NA/A ratios in comparison to corresponding Δ C -values Figure 4. Subset of samples with known time span between sampling and reception at the laboratiory in comparison to corresponding Δ C -values 13 PD-NA 13 PD-NA MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-357 If one of the criteria mentioned above is met, the sample should be analyzed by GC/C/IRMS as these criteria may indicate an adverse analytical finding. Samples with a time span greater than 21 days and none of the criteria mentioned above can be reported negative without need for GC/C/IRMS analysis. Conclusions The number of samples for GC/C/IRMS analyses with an expectable negative result can be reduced if additional information about the time span between sample collection and reception at the laboratory is considered as well as the additional criteria 19-NA/19-NE, 19-NA/A and 19-NA concentration. In our study, all samples with a prolonged time span of more than 21 days, a 19-NA/19-NE ratio < 3, a 19-NA/A ratio < 0.1% and a 19-NA concentration < 5 ng/mL turned out to be negative after GC/C/IRMS analysis. No false negative samples using this scheme are to be expected. The number of samples analyzed by GC/C/IRMS for 19-NA between 2017 and 2021 could have been reduced by at least 12% (12 out of 98 samples) following the presented scheme. References 1. Mareck U, Geyer H, Fußhöller G, Thevis M. (2022) Results of confirmation analysis for 19-norandrosterone in the Cologne laboratory from 2017 – 2021, Poster presented at the Manfred Donike Workshop, Köln 2. Hülsemann F, Gougoulidis V, Schertel T, Fusshöller G, Flenker U, Piper T, Thevis M. (2018) Case Study: Atypical δ C values of urinary norandrosterone. Drug Test Anal. 10, 1728-1733. 3. World Anti-Doping Agency. Technical Document TD2021NA v.2.0. https://www.wada- ama.org/sites/default/files/resources/files/td2021na_final_eng_v2.0_m.pdf (access 27.07.2021) 4. Piper T, Emery C, Saugy M: Norandrosterone analysis by GC/C/IRMS. W Schänzer, M Thevis, H Geyer, U Mareck (eds.) Recent advances in doping analysis (20) Sport und Buch Strauß, Köln (2012) 5. World Anti-Doping Agency. Technical Document TD2021EAAS v.2.0. https://www.wada- ama.org/sites/default/files/resources/files/td2021eaas_final_eng_v_2.0.pdf (access 11.10.2022) Acknowledgements The authors thank the Manfred Donike Institute for Doping Analysis e.V. (Cologne, Germany) and the Federal Ministry of the Interior, Building and Community (Berlin, Germany) for supporting the study. 13 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-358 Gmeiner G , Schlögl T , Göschl L Profile analysis of phase II stanozolol metabolites to evaluate the time of last application Doping Control Laboratory, Seibersdorf Labor GmbH, Seibersdorf, Austria ; Forensics, Seibersdorf Labor GmbH, Seibersdorf, Austria Abstract According to WADA statistics, stanozolol is one of the most commonly detected androgenic anabolic steroids in professional sports [1]. The WADA-accredited laboratory in Seibersdorf received three stanozolol-positive OOC urine samples. As a request of the RMA the time frame of application of the drug should be estimated. Excretion studies to analyze phase II glucuronides of stanozolol and statistical analysis were performed to estimate the timing of intake. Profile analysis of the samples together with the excretion studies were performed according to previously published literature [2,3]. A statistical analysis using linear regression models is presented including the highest probability of the last substance application. Introduction According to WADA statistics, stanozolol is one of the most commonly detected androgenic anabolic steroids in professional sports [1]. The WADA-accredited laboratory in Seibersdorf received three stanozolol-positive OOC urine samples. As a request of the RMA the time frame of application of the drug should be estimated. Excretion studies to analyze phase II glucuronides of stanozolol and statistical analysis were performed to estimate the timing of intake. Experimental Excretion studies The laboratory performed three excretion studies with a stanozolol tablet containing 9.6 mg of substance. All samples were analysed according to an online-SPE-LC-HRMS method described in a recent publication from the Seibersdorf laboratory in 2021 [2,3], based on previous work from Schänzer et al. in 2013 [4]. Peak areas of the signals of nine different glucuronide metabolites of stanozolol were used for statistical analysis (see Figure 1). In the course of time the pattern of the phase II metabolites of Stanozolol after oral application changed significantly. Stanozolol metabolite ratios decreased or increased significantly, leading to a change in the ratios of these metabolites. Linear regression model Metabolite data were analysed using a scatter plot matrix. Feasible predictive variables as well as possible metabolites for standardisation were determined. Two linear regression models were derived from the data using a different set of predictors. Their reliability was tested using cross validation. 1 2 1 1 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-359 Model Formula: y(x)=x(0)+ a*x(1)+b*x(2) In model 1, areas were normalized using Stanozolol metabolite 5 and metabolites 2 and 4 were used as predictors. Table 1: Statistical model 1 In model 2, areas were normalized using Stanozolol metabolite 2 and metabolites 4 and 1 were used as predictors. Table 2: Statistical model 2 Limitations of the model The applicability of the model is reduced by the facts that: The small sample size leads to a comparably large confidence interval The same dose of 9.5 mg was applied by all three volunteers, whereas the dose applied by the athletes is unknown. Consequently, the result is influenced by the means of data normalization. It was a single dose application, multiple dosing was not studied, but is commonly used by doped athletes. Only oral application was studied whereas injections would also be possible. Significant individual differences in metabolism cannot be excluded with such a small number of volunteers, resulting in a small sample size Consequently, the linear model yields a justified suspicion within the confidence interval but no final proof. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-360 Results and Discussion A simple visual interpretation of the phase II metabolite profiles (Figure 1) shows that the pattern of all 3 athletes samples correlates better with excretion afterca.30 h than after ca.200 h. Figure 1. Online-SPE-LC-HRMS results; Above, XICs of two exemplary samples from the elimination study and below, three athlete’s samples; For all samples the internal standard d3-testosterone glucuronide at ion transition 468.2671-> 109.0652 (CE = 35 eV), stanozolol glucuronides at ion transition 505.2908-> 329.2584 (CE = 60 eV) and hydroxystanozololglucuronides at ion transition 521.2858-> 345.2536 (CE = 60 eV) are shown MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-361 Statistical analysis According to model 1 the highest probability for the last application was – sample dependent – between 26 and 54 hours before sample collection. The standard deviation was calculated to be +/-11 h, comprising a 67% confidence interval. Table 3: Results according to statistical model 1 According to model 2 the highest probability for the last application was – sample dependent – between 27 and 56 hours before sample collection. The standard deviation was calculated to be +/-19 h, comprising a 67% confidence interval. The use of peak areas as basis for the statistical models is regarded as sufficient markers of the metabolic profiles. Even if reference substances for all of these nine markers would have been available, the corresponding concentrations are not expected to improve the precision of the model due to the uncertainty in the determination of the concentrations. Conclusions Taking into account the limitations of the model as indicated above, the highest likelihood for the last application is within approx. 20–60 hours before sample collection. In more detail for the athlete’s samples 1 and 2 it is likely that the last application was in the range of approx. 1 day before sample collection, whereas for athlete’s sample 3 two or more days are likely. However, due to the low number of volunteers in the model it cannot be completely excluded that the last application was even before. References 1. World Anti-Doping Agency (WADA). 2020 Anti-Doping Testing Figures. Montreal (2020) https://www.wada- ama.org/sites/default/files/2022-01/2020_antidoping_testing_figures_en.pdf (access date 02.02.2022) 2. L. Göschl, P. Gärtner, V. Enev, N. Kratena. (2020) Development and validation of a simple online-SPE method coupled to high-resolution mass spectrometry for the analysis of stanozolol-N-glucuronides in urine samples. Drug Test. Anal. 12 (8), 1–10 3. L. Göschl, G. Gmeiner, P. Gärtner, G. Stadler, V. Enev. (2021) Stanozolol-N -glucuronide metabolites in human urine samples as suitable targets in terms of routine anti-doping analysis. Drug Test. Anal., 13 (9), 1668–1677 4. W. Schänzer, S. Guddat, A. Thomas, G. Opfermann, H. Geyer, M. Thevis. (2013) Expanding Analytical Possibilities Concerning the Detection of Stanozolol Misuse by Means of High Resolution/High Accuracy Mass Spectrometric Detection of Stanozolol Glucuronides in Human Sports Drug Testing. Drug Test. Anal. 5 (11–12), 810–18. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-362 Sobolevsky T, Samara V, Ahrens B Detection of testosterone and androstenedione in serum by LC-HRMS UCLA Olympic Analytical Laboratory, Los Angeles, United States Abstract Measurement of testosterone and androstenedione concentrations in serum or plasma has long been an important diagnostic tool for clinical endocrinology. Recently, the steroidal module of the Athlete Biological Passport has incorporated serum steroids with the aim to improve detection of endogenous steroid abuse. The World Anti-Doping Agency (WADA) has now standardized method requirements to allow anti-doping laboratories to validate and implement this new method accordingly. Here we report a two-step solid phase extraction procedure for testosterone and androstenedione in serum that provides efficient clean-up and removal of major proteins and lipids followed by analysis by liquid chromatography - high resolution mass spectrometry (LC-HRMS). Unexpected yet important osbservation was that LC-HRMS system used for this assay should not be exposed to high concentrations of testosterone which is inevitable if used for other confirmatory work involving analysis of urinary total fraction. Introduction Several methods have been published for quantitation of testosterone and androstenedione in serum most of which are based on the use of liquid chromatography – mass spectrometry (LCMS) analysis following different sample preparation strategies such as liquid-liquid (LLE), solid phase (SPE), or supported liquid-liquid extraction [1,2]. Unlike urine, serum is a particularly challenging matrix due to high protein and lipid concentrations. The low concentration of steroids along with their strong protein binding also present challenges when selecting or designing an appropriate analytical protocol. Therefore, our goal was to develop a method that is capable of fulfilling WADA requirements including limited sample volume, sensitivity, and measurement uncertainty, while at the same time minimizing undesirable matrix effects and protecting LCMS instrument from contamination. Experimental Sample preparation Calibrators and controls obtained from ChromSystems were reconstituted in water per manufacturer instructions and aliquotted as 70-µL single use aliquots into 1.5-mL centrifuge tubes. As an additional control designated as LOQ, a calibrator from another vendor (RECIPE) was used with target concentrations of testosterone (T) and androstenedione (AD) at 0.10 and 0.11 ng/mL, respectively. One mL of 4% phosphoric acid was added to 70 µL of serum followed by brief mixing and further addition of 70 µL of internal standard (5 ng/mL each of C -T and C -AD in ethanol). Sample was loaded on a13 3 13 3 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-363 Strata X-A cartridge (30 mg/3 mL, Phenomenex). The cartridge was washed sequentially with 3 mL water and 40% methanol, and eluted with 0.6 mL methanol. The eluate was diluted with 2.4 mL water directly in the tube and loaded on a Strata C18-E cartridge (100 mg/3 mL, Phenomenex). The cartridge was eluted with 1 mL acetonitrile (no wash). The eluate was evaporated to dryness at 65 °C and reconstituted in 100 µL of 50% methanol before analysis. Instrumentation Thermo QExactive Plus with Dionex UltiMate 3000RS (S1) and QExactive with Vanquish Horizon (S2) were used. Separation was achieved using columns Waters Cortecs T3 (100 x 2.1, 2.7 µm) at 40 °C (S1) or XSelect Peptide CSH C18 (100 x 2.1 mm, 2.5 µm) at 60 °C (S2) with 0.3 mL/min of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). Gradient started from a 0.5 min at 60% B, increased to 85% B over 3.5 min, jumped to 98% B in 0.01 min, and after a 4.5-min hold switched back to 60% B followed by a 1.5-min equilibration before the next injection. Injection volume was 10 µL (S1) or 15 µL (S2). Mass spectrometer was operated in PRM mode at a resolution of 17500. Results and Discussion Initial experiments have shown that the use of LLE, in addition to being labor intensive, leads to introduction of a significant amount of neutral lipids onto the chromatographic column. These lipids cannot be eluted within a reasonable run time and would progressively accumulate in the column. Single- step SPE was also found to co-extract a lot of matrix interferences. Of note, these interferences did not directly affect T or AD, but could be seen in fullscan mode as massive peaks that kept eluting even after 5 minutes at 98% B. This was considered impractical for routine use due to concerns regarding method robustness. Therefore, we focused on a more elaborate sample clean-up. This was achieved with a two-step SPE first incorporating strong anion exchange to remove phospholipids (Strata X-A) and secondly silica-based octadecyl to trap the most hydrophobic neutral lipids (Strata C18-E). Linearity, repeatability, and intermediate precision were evaluated in a series of validation experiments (Table 1 & 2). Finally, a standard combined measurement uncertainty was calculated as shown below, which is within WADA requirements for this assay. Table 1. Validation summary of T MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-364 Table 2. Validation summary for AD A 1/x weighing was necessary to cover the linear range of the method (Figure 1). Figure 1. Calibration curves for testosterone and androstenedione An unexpected outcome was the observation of high T and AD backgrounds on the LC-HRMS system routinely used in our laboratory for confirmatory analyses (S1). We presume this background is due to persistent contamination of LC with endogenous steroids. On the contrary, S2 which is only used for ITP of peptides and does not see any T nor AD, did not show appreciable background (Figure 2). The high background affects the method linearity at low concentrations and must be avoided. Conclusions Quantitative method for testosterone and androstenedione in serum by LC-HRMS has been developed and validated. Method is based on a two-step SPE that helps remove the most hydrophobic matrix interferences, and can be easily adapted to a 96-well plate format for high throughput. Measurement uncertainty and sensitivity are within expectations set forth by WADA for this assay. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-365 Figure 2. Exemplary chromatograms on two different LC-HRMS systems (CAL1 is 0.05 ng/mL of T and 0.20 ng/mL AD). Transitions (normilized CE) used: AD, 287.2 > 97.0653 (45); C -AD, 290.2 > 100.0753 (45); T, 289.2 > 97.0653 (50); C -T, 292.2 > 100.0753 (50). References 1. Wang Y, Gay GD, Botelho JC, Caudill SP, Vesper HW. (2014) Total testosterone quantitative measurement in serum by LC-MS/MS. Clin Chim Acta. 436, 263-267 2. Ponzetto F, Boccard J, Baume N, Kuuranne T, Rudaz S, Saugy M, Nicoli R. (2017) High-resolution mass spectrometry as an alternative detection method to tandem mass spectrometry for the analysis of endogenous steroids in serum. J Chromatogr B Analyt Technol Biomed Life Sci. 1052, 34-42 13 313 3 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-366 de Souza B, Gomes M, Nunes I, Padilha M, Pereira H Zebrafish Water Tank model as a tool to evaluate phase I metabolites of methyltestosterone Chemistry Institute, Brazilian Doping Control Laboratory - LBCD, Rio de Janeiro, Brazil Abstract The Zebrafish Water Tank (ZWT) is a relative new model to research metabolic pathways of xenobiotics such as the prohibited substances class variety disclosed annually by WADA. The zebrafish (Danio rerio) has its genome already mapped, with several similarities in the enzymatic machinery when compared with humans, being widely used in pharmacological and toxicological investigations. Several classes of doping agents were already investigated using ZWT. However, steroids anabolic agents were barely studied by this model, mainly regarding the enzymatic redox reactions typically seen in steroid rings. In this work the ZWT was employed to investigate the metabolism of methyltestosterone (MT). The experimental design included three tanks containing 200 mL of ultrapure water each. First tank with 8 zebrafish; second tank with water fortified with MT; and a third tank with 8 zebrafish and MT. After 8 hours, 3 mL samples were retrieved from each tank and submitted to a sample preparation and analysis in a GC-MS-MS system following the procedure already used for anabolic steroids in LBCD. The ZWT model was able to yield several metabolites previously described in the literature in humans such as the metabolite 17α-methyl-5α-androstan-3α,17β-diol, and the minor metabolite of MT, 17α-methyl-5α- androstan-17β-ol-3-one (mestanolone). After a full-scan mode and mass spectra comparison, a putative metabolite isomer of 17α-methyl-5α-androstan-3α,17β-diol was displayed. According to these findings, the ZWT model proved to be an effective tool to investigate the enzymatic redox reactions seen in the steroid rings. The formation of phase II metabolites is under investigation using different analytical strategies and these results open perspective for new projects involving other steroids in the ZWT model aiming, for example, investigations about long-term metabolites. Introduction The Zebrafish Water Tank (ZWT) is a relatively new model to research metabolic pathways of xenobiotics such as prohibited substances classes disclosed annually by WADA. Among the strong sides of the model, it is possible to highlight the use of water as matrix of analysis, which is cleaner than biological fluids usually employed in other in vivo models. In addition, the zebrafish (Danio rerio) has its genome already mapped, with several similarities in the enzymatic machinery when compared with humans, being widely used in pharmacological and toxicological investigations [1]. However, steroidal anabolic agents were barely studied by the model, mainly regarding the enzymatic redox reactions typically seen in steroid rings. Stanozolol was the only AAS already studied by the ZWT model [2,3]. In this work the ZWT was employed to investigate the metabolism of methyltestosterone (MT). MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-367 Experimental The experimental design included three tanks (containing 200 mL of ultrapure water each): a negative control containing only 8 zebrafish; a positive control containing only water fortified with 200 µg MT; and a treatment tank containing pure water, 8 zebrafish and 200 µg MT. After 8 hours, 3 mL samples were collected from each tank and stored at -20°C until analysis [4]. The samples were submitted to a liquid- liquid extraction (LLE), derivatization and analysis in a GC-MS/MS system following the procedure already used for anabolic steroids in LBCD and a mass spectrometry data were obtained through full-scan acquisition [5]. Results and Discussion According to the chromatography and mass spectrometry data, ZWT was able to yield several metabolites previously described in the literature in humans. Reduction of α-β unsaturated ketone in ring A was observed, resulting in the metabolite 17α-methyl-5α-androstan-3α,17β-diol. As expected by the knowledge of the enzymatic homology of zebrafish with humans, 17α-methyl-5β-androstan-3α,17β-diol was not produced by the ZWT model [6]. The minor metabolite of MT, 17α-methyl-5α-androstan-17β-ol-3- one (mestanolone) was also identified using the relevant reference material (Figure 1A). A full-scan mode displayed a putative metabolite which was believed to be an isomer of 17α-methyl-5α- androstan-3α,17β-diol after comparison of both mass spectra based on the m/z 143 (characteristic for 17-hydroxy-17-methyl steroids after trimethylsilylation). The possibilities include 17β-methyl-5α- androstan-3α,17α-diol, 17β-methyl-5α-androstan-3β,17α-diol and 17α-methyl-5α-androstan-3β,17β-diol (Figure 1B). Due to the lack of reference material, it was not possible to state unequivocally about the structure. Figure 1. Scheme of the A-ring reductions (A); Structures of the putative metabolites (B) MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-368 Conclusions According to these findings, the solubility of MT in water, which could hamper the disponibility of the steroid to the fish, was not an issue and the ZWT model proved to be an effective tool to investigate phase I AAS metabolites. The metabolites produced by the ZWT model in this study were 17α-methyl-5α- androstan-3α,17β-diol, 17α-methyl-5α-androstan-17β-ol-3-one (mestanolone) confirmed with the reference materials. A putative metabolite which could be 17β-methyl-5α-androstan-3α,17α-diol, 17β- methyl-5α-androstan-3β,17α-diol or 17α-methyl-5α-androstan-3β,17β-diol was also observed. The formation of phase II metabolites is under investigation using different analytical strategies. These results open perspective for new projects involving other steroids in the ZWT model aiming, for example, investigations about long-term metabolites. Table 1. Identified metabolites References 1. Anselmo, C. de S., Sardela, V. F., de Souza, V. P. (2018) Zebrafish (Danio Rerio): A valuable tool for predicting the metabolism of xenobiotics in humans? Comparative Biochemistry and Physiology Part-C: Toxicology and Pharmacology. 212, 34-46. 2. Anselmo, C. de S., Sardela, V. F., Matias, B. F., de Carvalho, A. R., de Sousa, V. P., Pereira, H. M. G., Aquino Neto, F. R. (2017) Is zebrafish (Danio rerio) a tool for human-like metabolismo study? Drug testing and Analysis. 9, 1685-1694. 3. Matos, R. R., Anselmo, C. de S., Sardela, V. F., Pereira, H. M. G. (2021) Phase II stanozolol metabolism study using the zebrafish water tank (ZWT) model. Journal of Pharmaceutical and Biomedical Analysis. 195, 113886. 4. Prado, E., Matos, R. R., Gomes, G. M. L. (2020) Metabolism of synthetic cathinones through the zebrafish water tank model: a promising tool for forensic toxicology laboratories. Forensic Toxicology. 39, 73-88. 5. Cavalcanti, G. A., Garrido, B. C., Leal, F. D., Padilha, M. C., de la Torre, X., Aquino Neto, F. R. (2011) Detection of new urinary exemestane metabolites by gas chromatography coupled to mass spectrometry. Steroids. 76, 1010-1015. 6. Available at https://www.ncbi.nlm.nih.gov/gene/6718/ortholog/?scope=32523, on March 10 , 2022. Acknowledgements This work was supported by CAPES, the Rio de Janeiro Federal University, and the authors are grateful for the financial support from the Brazilian Authority of Doping Control. The authors thank Dr. Xavier de la Torre and Dr. Gustavo Cavalcanti by the fruitful discussions about steroid metabolism. th MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-369 Dos Santos L, Rodrigues Matos R, M. G. Pereira H Metabolism study of the selective estrogen receptor modulator tamoxifen through the Zebrafish Water Tank model Chemistry Institute, Brazilian Doping Control Laboratory - LBCD, Rio de Janeiro, Brazil Abstract The in vivo Zebrafish Water Tank (ZWT) model is an emerging model to assess the metabolism of xenobiotics. The class of selective estrogen receptor modulator (SERM) has not yet been evaluated by this model. This work aimed to evaluate the applicability of the ZWT model to study the tamoxifen metabolism, as a representative of the SERM. Tamoxifen was administered to a 200 mL tank containing water and 8 adult zebrafish at 32 ± 1°C for 8 hours. The water tank samples collected throughout the experiment were analyzed with and without pretreatment by LC-HRMS/MS. ZWT was able to reproduce the human metabolism of tamoxifen, with the main phase I metabolites being confirmed. Hence, ZWT could be applied to investigate other doping agents with similar structure in a very straightforward way. Introduction The use of selective estrogen receptor modulators (SERMs) has been banned by the World Anti-Doping Agency since 2005. Tamoxifen (TMX), a SERM, can be used as a doping agent to compensate for the adverse effects (gynecomastia) because of extensive use of anabolic androgenic steroids [1,2]. The enzymatic activity of CYP2D6 isoforms is reported to be one of the essential elements of TMX metabolism [3]. To study the metabolism of doping agents, zebrafish represents a viable alternative to the classical mammalian models, presenting the ability to perform phase I and phase II metabolism reactions, lower cost, and ease of handling compared to other rodent models, the substances of interest are absorbed by fish through water, among others [4]. The Zebrafish Water Tank (ZWT) model has shown success in studying the metabolism of different classes of doping agents [5]. However, among the doping agents previously studied in the ZWT model, SERMs have not yet been considered. Thus, taking into consideration that no zebrafish ortholog of human CYP2D6 has been reported in the literature yet, this study aimed to evaluate the applicability of ZWT to study Tamoxifen metabolism, as a representative of the SERM. Experimental The ZWT protocol used in previous studies was adopted [6]. TMX was administered to a 200 mL tank containing water and 8 adult zebrafish at 32 ± 1°C. Experiments were performed in triplicate and interrupted after 8 hrs. Additional tanks without fish with drug and without the drug with fish were used as controls (Figure 1). The water tank samples collected throughout the experiment were analyzed both with and without pretreatment (i.e., dilute-and-shoot, and Liquid-Liquid Extraction with and without hydrolysis) by Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC-HRMS/MS) in MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-370 positive ionization mode. Figure 1. ZWT’s experiment representation. Legend: R , R , and R – replicates with 8 fish + drug; PC – positive control: drug + water; NC – negative control: 8 fish without drug Results and Discussion By comparing the available reference material with the samples results according to WADA’s criteria (TD IDCR), 3 tamoxifen metabolites were identified (Table 1). The results obtained showed that these TMX’s metabolites were excreted mainly as free and gluco-conjugated, whereas only small amounts were excreted as sulphate. Table 1. Metabolites identified based on IDRC criteria A similar pattern was previously described for humans [1]. It was also noticed the presence of the main metabolites produced: 4-hydroxy-tamoxifen, N-desmethyl-tamoxifen, and endoxifen. Despite of the lack of reference material, the examination for others potential TMX metabolites was based on the search of typical biotransformation pathways, which have an associated change in the molecular formula and its corresponding mass shift compared to the TMX (Table 2). 1 2 3 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-371 Table 2. TMX’s metabolites found after 8h of experiment Thus, beside the 3 metabolites identified with reference material, 26 metabolites were found in the samples after 8h of experiment. This huge number is in alignment with what has been reported in the literature for other models such as humans (23 metabolites) and rats (38 metabolites) [1,7]. Some of the chemical reactions proposed for TMX’s biotransformation pathways include hydroxylation in different position(s), carboxylation, N-demethylation, N-oxidation, methoxylation and combinations of them. Primary standards would be required for further confirmation of the metabolite’s structure. Conclusions A total of 29 metabolites were detected in the ZWT model samples, including the main phase I metabolites well described in humans. These findings demonstrate the applicability of ZWT to investigate the metabolism of other doping agents with similar structure in a very straightforward way. References 1. MAZZARINO et al. Analytical and Bioanalytical Chemistry, 2013. doi: 10.1007/s00216-013-6961-7. 2. SALVADOR et al. Journal of Pharmaceutical and Biomedical Analysis, 2019. doi:10.1016/j.jpba.2018.04.027 3. SANCHEZ-SPITMAN et al. Expert Review of Clinical Pharmacology, 2019. doi: 10.1080/17512433. 2019.1610390. 4. ANSELMO et al. Comparative Biochemistry and Physiology Part - C: Toxicology and Pharmacology, 2018. doi: 10.1016/j.cbpc.2018.06.005. 5. ARAUJO et al. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 2021. doi: 10.1016/j.jchromb.2021.122826. 6. PRADO et al. Forensic Toxicology, 2020. doi: 10.1007/s11419-020-00543-w 7. DOMINGUEZ-ROMERO et al. Biomedical Chromatography, 2015. doi: 10.1002/bmc.3411 Acknowledgements The authors wish to express their gratitude to the UFRJ’s Institute of Chemistry and the financial support from the Brazilian research funding agency CNPq and the Brazilian Authority for Doping Control (ABCD). MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-372 Desharnais P, Naud J A modified Towbin transfer buffer for a simple, versatile and sensitive detection of ESAs Laboratoire de contrôle du dopage INRS - Centre AFSB, Laval, Canada Abstract Analytical procedures must be constantly evolving in order to improve their sensitivity regarding the detection of lower concentration of analytes, either for the detection of microdoses administration or for the detection from alternatives matrices, such as dried blood spots (DBS). The initial testing procedure (ITP) detection of ESAs is mainly performed by SAR-PAGE electrophoretic separation and western blotting. Since its introduction, the SAR-PAGE method uses Bjerrum buffer in protein transfer electroblotting process. Here, we show that not only reducing the percentage of methanol to 10% in the Bjerrum buffer improves the SAR-PAGE detection of high molecular weight ESA like Cera, but that using the classical Towbin buffer instead of the Bjerrum buffer improves the overall detection of all the ESAs. Our modified Towbin buffer was also compared to the newly suggested CAPS discontinuous buffer system on SDS-PAGE and shows that the sensitivity towards Cera reaches the one reported with CAPS buffer. New limit of detection (LOD) on SAR-PAGE were calculated for pure references standards, for urine and blood matrices as well as for DBS. Our results demonstrate that the modified Towbin buffer offers a simple, versatile and sensitive detection of ESAs for SAR-PAGE and SDS-PAGE analysis. Introduction The initial testing procedure (ITP) detection of ESAs is currently mostly performed by SAR-PAGE electrophoretic separation and western blotting immunodetection. Since first publication [1], SAR-PAGE method is accompanied with an electrophoretic protein transfer process in which Bjerrum buffer is used. In our laboratory’s standard operating procedure (SOP), methanol (MeOH) had previously been reduced in the Bjerrum buffer from 20% to 10% (v/v) to the increase in sensitivity for CERA. The main objective here is to demonstrate that the sensitivity towards all ESA’s could be further improved using Towbin buffer with reduced MeOH concentration. Results were also compared to a recently published discontinuous buffer system [2] on both SAR-PAGE and SDS-PAGE analysis. Experimental Electrophoresis SAR-PAGE and SDS-PAGE analysis were performed according to already described protocol at 150 V on 10% Nupage Novex Bis-Tris 1.0 mm Midi-gels. Semi-dry Western blotting After SDS- and SAR-PAGE, proteins were transferred on Immobilon-P membrane using four different MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-373 transfer buffers: 1) modified Towbin buffer (25 mM Tris, 192 mM glycine, pH 8.3) supplemented with 10% v/v MeOH 2) Bjerrum buffer (48 mM Tris, 39 mM glycine, 1.3 mM SDS) supplemented with 20% v/v MeOH 3) Bjerrum buffer supplemented with 10% v/v MeOH 4) CAPS discontinuous buffer All transfers were performed at 1.0 mA/cm for 45 minutes using the semi-dry blotter Transblot SD (BioRad). Eight layers of Electrode paper Novablot (GE Healthcare) were used on each side of the blotting sandwich except for the transfer with CAPS discontinuous buffer which was performed according to Martin et al. [2]. Immunoblotting Single blotting was performed using biotinylated MAb anti-EPO, clone AE7A5 (0.5 µg/mL in 1% NFM/PBS) as a primary antibody. LOD in urine A urine pool, adjusted to 2.5 mIU uEPO/15 mL, was spiked with 25 pg CERA, 25 pg EPO-Fc, 6.25 pg NESP and 5 mIU rhEPO BRP by 15 mL of urine and serially diluted in the non-spiked urine sample. Full ITP procedure was then performed on 15 mL of each dilutions using StemCell for immunoaffinity isolation. LOD in serum A serum pool, adjusted to 8 mIU/mL of endogenous EPO, was spiked with 100 pg CERA, 100 pg EPO-Fc, 6.25 pg NESP and 12.5 mIU rhEPO BRP/500 µL of serum and serially diluted in the non-spiked serum sample. Full ITP procedure was then performed on 500 µL of each dilutions using AF959-linked to Dynabeads M-270 Epoxy for immunoaffinity isolation. LOD in DBS K /EDTA blood sample was spiked with 640 pg CERA, 640 pg EPO-Fc, 160 pg NESP and 64 mIU rhEPO BRP/mL and serially diluted in the non-spiked sample. 20 µL of each dilution was spotted on individualized filter pads from Tasso-M20 units. Extraction was performed in PBS 1X/0.05% BSA and ESA isolated using AF959-linked to Dynabeads M-270 Epoxy immunomagnetic beads. 2 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-374 Figure 1. Immunoblotting results using different transfer buffers after SAR-PAGE. A. Serial dilutions of the ESA’s reference standards were prepared in loading buffer, analysed by SAR-PAGE, and transferred using the four different transfer buffers (starting in Lane 1 at 100 pg CERA, 100 pg EPO-Fc, 25 pg NESP, 10 mIU Dynepo). Std: ESA-standard mixture Dynepo/NESP/EPO-Fc/Cera. B. For 3 independent comparison assays, compilation of the densitometric intensities of each ESA bands from lane 1 to 7 was performed. The relative intensities for each ESA were compared to those obtained with the modified Towbin transfer buffer for which was assigned an arbitrary intensity of 100. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-375 Figure 2. Immunoblotting results using different transfer buffers after SDS-PAGE. A. Serial dilutions of the ESA’s reference standards were prepared in loading buffer, analysed by SDS-PAGE, and transferred using the four different transfer buffers (starting in Lane 1 at 100 pg CERA, 100 pg EPO-Fc, 25 pg NESP, 10 mIU Dynepo). Std: ESA-standard mixture Dynepo/NESP/EPO-Fc/Cera B. For 3 independent comparison assays, compilation of the densitometric intensities of each ESA bands from lane 1 to 7 was performed. The relative intensities for each ESA were compared to those obtained with the modified Towbin transfer buffer for which was assigned an arbitrary intensity of 100. Results and Discussion The classical formulation of the Towbin buffer contains an amount of 20% of methanol. Based on our previous observation, the reduction of methanol in Bjerrum buffer increased the sensitivity for CERA by MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-376 SAR-PAGE. In this study, the effect on the detection of all ERAs was evaluated with the same reduction of MeOH but in Towbin buffer. Results showed that a concentration 10% MeOH was optimal for the detection of CERA (data not shown). As observed in Figure 1, the reduction of methanol from 20% to 10% in Bjerrum buffer increase the sensitivity towards CERA. However, the usage of Towbin buffer results in a much greater increase. Densitometric analyses of bands obtained in Bjerrum 20% and 10% MeOH generate signal intensities for CERA that are only 17% and 39% of those obtained when modified Towbin buffer is used, respectively. For other ESAs, the effect is less pronounced, but the use of modified Towbin generally results in a 20% increase of the signals (Figures 1 and 2). The modified Towbin buffer was also compared to the newly suggested CAPS discontinuous buffer system and shows that the sensitivity towards CERA reaches levels close to those obtained with CAPS buffer on SDS-PAGE (Figure 2). In our hand, the performance of the CAPS discontinuous buffer was poor, apart for the detection of CERA by SDS-PAGE, particularly for the detection of rhEPO BRP and NESP in comparison to all other methods. Representative ITP results generated using the modified Towbin buffer are presented in Figure 3. They demonstrate that the modified Towbin buffer represents a versatile option for SAR-PAGE and SDS-PAGE analysis. New LOD’s were calculated for SAR-PAGE analysis on urine, serum, and DBS samples (Table 1). LODs obtained for serum/plasma samples are compliant with the Minimum Required Performance Levels (MRPL) of the technical document [3]. Interestingly, MRPL can be achieved for rEPO and NESP using DBS and can almost be reached for CERA and EPO-Fc (LOD of 40 pg/mL versus an MRPL of 25 pg/mL). Figure 3. Representative ITP results using Towbin buffer for SAR-PAGE and SDS-PAGE. Urine and serum samples were left untreated or spiked with the different ESAs and submitted to ITP using SAR-PAGE (left panel) and SDS-PAGE (right panel). MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-377 Table 1. New limit of detection (LOD) achievable by SAR-PAGE using the modified Towbin transfer buffer. *Based on ITP with 15 mL. **Based on ITP using 500 µL. The data presented are representative of at least two independent experiments. Conclusions The results presented herein show that not only reducing the percentage of methanol to 10% in the Bjerrum buffer improves the SAR-PAGE detection of high molecular weight ESA like CERA, but that using the classical Towbin buffer formulation instead of the Bjerrum formulation improves the overall detection of all ESAs. New limit of detection (LOD) on SAR-PAGE were calculated for pure references standards, for urine and blood matrices as well as for DBS. The results demonstrate that the modified Towbin buffer offers a simple, versatile, and sensitive detection of ESAs for SAR-PAGE and SDS-PAGE analysis References 1. Reichel C, Abzieher F, Geisendorfer T. SARCOSYL-PAGE: a new method for the detection of MIRCera- and EPO-doping in blood. Drug Test Anal. 2009, 1(11-12), 494-504. 2. Martin L, Martin JA, Audran M, Marchand A. An optimized SDS-PAGE protocol with a new blotting system for the initial testing procedure of ESAs in doping control. Drug Test Anal. 2022, 14(1), 181-18 3. WADA Technical Document – TD2022EPO. Harmonization of analysis and reporting of erythropoietin (EPO) and other EPO-receptor agonists (ERA) by polyacrylamide gel electrophoretic (PAGE) analytical methods. (Version 1.0. accessed August 26 , 2022) th MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-378 Desharnais P, Hamelin C, Douangpanya R, Stinfil CJ, Hébert N, Ferrand P, Ayotte C, Naud J Evaluation of a single-point calibrator for the adjustment of IGF-1 concentrations measured by Immulite immunoassay Laboratoire de contrôle du dopage INRS - Centre AFSB, Laval, Canada Abstract The measurement of insulin-like growth factor 1 (IGF-1) for the human growth hormone (hGH) biomarkers test is currently performed by mass spectrometry (LC-MS/MS or LC-HRMS) or immunoassays. However, there is a poor overall agreement between the results generated by these methods. To solve this, and to provide a better interlaboratory standardization, a single point serum-based calibrator was introduced for the bottom-up and top-down analysis of IGF-1 by mass spectrometry. Therefore, it can be proposed that the use of this calibrator could standardize the results obtained on the different immunoassay platforms with the results obtained by mass spectrometry. To verify this hypothesis, 103 serum sample were reanalysed by immunoassay (Immulite-2000, Siemens), including in each analytical run, a duplicate analysis of the single point calibrator. When compared to the bottom-up initial testing procedure (ITP) data obtained by LC-HRMS, it was shown that adjustment of the Immulite-2000 IGF-1 measurements with the calibrator reduces the mean difference concentrations from -37.2% to -3.8%, with only four outlier samples. Applied to the calculation of the GH-score, the mean difference for the GH-scores calculated with the IGF-1 immunoassay values went, after adjustment, from -1.00 to a minimal mean of -0.09 in comparison with scores calculated using the IGF-1 LC-HRMS concentrations. To further verify the possible effect of the adjustment using a calibrator, a retrospective simulation was performed on 117 serum samples formerly analysed using both methods. By adjusting the past IGF-1 immunoassay concentrations using the estimated correction factor determined from the single point calibrator analysis, a similar conclusion could be reached. Our results reveal that the addition of the calibrator to the IGF-1 testing by immunoassay allows the correction of the concentrations. The measured concentrations agree with those obtained by LC-HRMS and the effect on GH-Score is minimal. This could prove to be very useful for the large-scale implementation of an ABP module for the biomarkers test. Introduction The measurement of insulin-like growth factor 1 (IGF-1) for the human growth hormone (hGH) biomarkers test is currently performed by mass spectrometry (LC-MS/MS or LC-HRMS) or immunoassays. However, there is a poor overall agreement between the results generated by these methods which have led to the establishment of test-specific decision limits. To solve a part of this problem, and to provide a better interlaboratory standardization, a single point serum-based calibrator was introduced for the bottom-up and top-down analysis of IGF-1 by mass spectrometry. Our hypothesis was that adopting this single point calibrator for immunoassay testing could also help to generate measurements with these platforms that would then be in accordance with those obtained by mass spectrometry. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-379 Experimental Samples Dataset 1: 103 samples collected in SST-II Plus tubes, received for routine antidoping control testing, were initially analysed for IGF-1 by LC-HRMS using bottom-up approach as initial testing procedure (ITP). These samples were re-analysed using Immulite 2000 by including, in each independent analytical run (n=3), a duplicate analysis of the single point calibrator. Dataset 2: 117 serum samples were formerly analysed for IGF-1 using both LC-HRMS and Immulite 2000 immunoassay. In a retrospective simulation, the past IGF-1 immunoassay concentrations were adjusted using the mean estimated correction factor (1.55) determined from the single point calibrator analysis along the samples from the dataset 1. PIIINP Measurement by ADVIA Centaur CP Samples were analysed according to manufacturer’s instructions (Siemens, Material number:10994962, Lot 125022). IGF-1 measurement by Immulite -2000 Samples were analyzed according to manufacturer’s instructions (Siemens, Material number: 11128584, Lot #213). The test is a solid-phase enzyme-labelled chemiluminescent immunometric system calibrated against the WHO IS 02/254. IGF-1 measurement by LC-HRAM/MS Analysis was performed on Q-Exactive Plus (Thermofisher) according to an adapted version of the method published by Cox et al. [1]. Briefly: HPLC Ultimate3000 (Dionex), Halo peptide ES-C18 column, ESI HRMS positive mode, ACN/Formic gradient. Samples were quantified using the T2 peptide (22-36) for which the calculated peak area ratio (vs N15-labelled IGF-1) was compared with that of the single point calibrator assessed as 338 ng/mL. Results and Discussion When compared to the LC-HRMS data, adjusting the values obtained on Immulite-2000 with the single- point calibrator reduces their mean difference from -37.2% to -3.8% (Table 1), with only four outlier samples presenting a value exceeding a 20% difference after adjustment (Figure 1, bottom panel). Interestingly, all the outliers present LC-MS IGF-1 concentrations below 200 ng/mL. Applied to the GH- score, the mean difference for the GH-scores calculated with the adjusted IGF-1 immunoassay values went, from -1.00 to -0.09 in comparison with those calculated using the IGF-1 LC-MS concentrations (Table 1). This is well below our laboratory internal uncertainty measure of 0.312, associated to the GH- Score obtained with the assay pairing MS/Centaur. ® ® MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-380 Figure 1. Comparison of IGF-1 concentrations measured by Immulite versus those by LC-HRAM/MS. The IGF-1 concentrations from samples of the dataset 1 obtained by Immulite-2000 (IMM-2000, red triangles) were corrected with the single point calibrator 338 ng/mL (IMM-2000cal, blue squares) and compared to the IGF-1 concentrations obtained by LC-HRAM-MS (MS). Top panel: Orthogonal plot of data expressed in ng/mL. Bottom panel: Bland-Altman plot of percentage difference from MS values calculated for each sample. The thin lines depict an arbitrary +/-20% outlier zone from the MS values. Table 1. Summary of the mean IGF-1 concentration (ng/mL), percentage difference (%) and GH-Score for the 103 samples from Dataset 1 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-381 A similar conclusion was reached in a retrospective simulation of formerly analysed samples, by adjusting the past IGF-1 immunoassay concentrations with the estimated correction factor derived from 3 independent analysis of the single point calibrator along samples from dataset 1 (Figure 2, Table 2). The mean value measurement of the single-point calibrator by Immulite-2000 is 218 ng/mL, leading to an estimated correction factor of 1.55 to adjust it to the consensual target value of 338 ng/mL. Moreover, our results are comparable to those obtained in the inter-laboratory studies by mass spectrometry [1,2]. According to the most recent WADA Proficiency testing for the Biomarkers Test [3], only a few numbers of laboratories (5) have decided to apply LC-MS/MS or LC-HRMS for IGF-1 testing. This is in part due to the labor-intensive procedures associated with bottom-up analysis but also to the expertise and costly instruments required to implement this procedure in a laboratory. Figure 2. Comparison of GH-Scores from Immulite/Centaur pairing versus those from MS/Centaur pairing. GH- Scores, from the pairing of ADVIA Centaur PIIINP with Immulite-2000 (IMM-2000, red squares) and Immulite-2000 concentrations corrected with the single point calibrator (IMM-2000cal, blue squares), were compared to the MS- based scores for the dataset 1 samples. Top panel: Orthogonal plot of Immulite against MS GH-scores. Bottom panel: Bland-Altman plot of percentage difference from MS values calculated for each sample. The thin blue lines depict an arbitrary +/- 0.50 outlier zone from the MS GH-score values. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-382 Table 2. Summary of the mean IGF-1 concentration (ng/mL), percentage difference (%) and GH-Score for the 117 samples from Dataset 2 Conclusions Our results reveal that the addition of the single point calibrator to the IGF-1 testing by immunoassay generates concentrations in agreement with those obtained by bottom-up LC-HRMS. This could prove to be especially useful for the large-scale implementation of an ABP module for the biomarkers test. References 1. Cox HD, Lopes F, Woldemariam GA, Becker JO, Parkin MC, Thomas A, Butch AW, Cowan DA, Thevis M, Bowers LD, Hoofnagle AN. Interlaboratory agreement of insulin-like growth factor 1 concentrations measured by mass spectrometry. Clin Chem. 2014 Mar;60(3):541-8. doi: 10.1373/clinchem.2013.208538. 2. Moncrieffe D, Cox HD, Carletta S, Becker JO, Thomas A, Eichner D, Ahrens B, Thevis M, Bowers LD, Cowan DA, Hoofnagle AN. Inter-Laboratory Agreement of Insulin-like Growth Factor 1 Concentrations Measured Intact by Mass Spectrometry. Clin Chem. 2020 Apr 1;66(4):579-586. doi: 10.1093/clinchem/hvaa043 3. World Anti-Doping Agency. 2022 EQAS-01 Report - Part II (Serum samples), Version 2.0. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-383 Cuervo D, Fernández-Alvarez M, Muñoz G Determination of small peptides in doping control: HPLC-HRMS (QTOF) vs HPLC-MSMS (QQQ) analysis following SPE on microplates Madrid Doping Control Laboratory, CELAD, Madrid, Spain Abstract New recombinant peptidic therapeutics have strongly emerged in the doping field during the last twenty years, as proven by the number of doping-related peptides confiscated by customs or seized in operations against the doping drug trade. These species entered the WADA List of Prohibited Substances in 2015 distributed in a range of categories including Growth Hormone Releasing Factors (GHRFs), Gonadotrophin Releasing Hormones (GnRHs) or antidiuretic hormones. Accredited Doping Control Laboratories must control the presence of these ever-increasing peptidic-based drugs and/or their metabolites at the low MRPL (1-2 ng/mL) established for this class of compounds. Weak cation exchange solid phase extraction is typically the method of choice for sample preparation from urine specimen for the determination of the so-called small peptides (MW < 2 kDa), while instrumental analysis is mainly accomplished by HPLC-HRMS (Orbitrap or QTOF) or HPLC-MSMS (QQQ). A dilute-and-shoot approach has recently been reported for the determination of these small peptidic compounds, although state-of-the-art analytical instruments are required in order to achieve adequate results. In recent years our laboratory published a convenient, straightforward methodology for the analysis of 28 doping related small peptidic drugs by means of high-accuracy HPLC-QTOF analysis following SPE on microplates. In this work we present an update of this protocol covering 57 peptidic and mimetic substances by using this same sample preparation method and analysis by HPLC-HRMS (QTOF) or HPLC- MSMS (QQQ). A comparison between the results obtained with both instrumental analysis, together with their advantages and drawbacks, is also presented here. Introduction Doping-related small peptides are included in the WADA List of Prohibited Substances distributed in a range of categories, including Growth Hormone Releasing Peptides or Gonadotrophin Releasing Hormones. Highly sensitive analytical methods are required in order to fulfill the low MRPL [1] established for these compounds (1-2 ng/mL). Determination from urine samples is typically carried out by WCX-type SPE followed by HPLC-MS analysis. In recent years our laboratory published a methodology for the analysis of 28 peptidic drugs by HPLC-HRMS (QTOF) following SPE on microplates [2]. This same protocol has been expanded in our laboratory to 57 peptidic substances and also validated for analysis by HPLC- MSMS (QQQ). The comparison between the results obtained with both instrumental analysis is presented here. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-384 Experimental All solvents and reagents used were of analytical grade. Commercial standards were acquired from several sources: Auspep (Tullamarine, Australia), Sigma-Aldrich (Saint Louis, MO, USA), EP (Strasbourg, France), Bachem (Bubendorf, Switzerland) or synthesized by the Proteomics Unit, Spanish National Biotechnology Centre (CSIC, Madrid, Spain). From there, standard stock solutions were prepared in water/methanol/formic acid 76/19/5 (v/v) at concentrations of 1000 µg/mL, and working solutions were prepared in the same solvent mixture at concentrations of 10 and 1 µg/mL. Sample preparation was as follows: 15 µL of ISTD solution and 50 µL of phosphate buffer were added to 750 µL of centrifuged urine in polypropylene tubes. An Oasis 96 well plate manifold (Waters, Milford, MA, USA) was used for microextraction. Samples were loaded onto an Oasis WCX microelution 96-well sample plate (2 mg) from Waters, previously conditioned with 0.2 mL of methanol and 0.2 mL of water, and washed with 0.2 mL of water and 0.1 mL of methanol. Subsequently, the target analytes were eluted with 50 µL of 95/5 (75% acetonitrile in water/formic acid) into a round 96-well collector plate from Waters and diluted with 25 µL (for HPLC-QTOF) or 50 µL (for HPLC-QQQ) of water. The collector plate was placed in a linear shaker for 10 minutes (110 rpm) and finally placed into the HPLC autosampler. Samples were analyzed by HPLC-HRMS (QTOF) and HPLC-MS (QQQ) systems. Characteristics as well as chromatographic and spectrometric parameters of both systems are depicted in Figure 1. Results and Discussion 57 doping-related peptidic substances were validated for analysis by HPLC-HRMS (QTOF) and HPLC-MSMS (QQQ) (initial testing and confirmation) according to the validation guidelines proposed by WADA. SPE on microplates is adequate for the analysis of 57 out of the 58 peptidic substances studied in both QTOF and QQQ systems. Only the growth factor TB-500 showed too poor recoveries to reach the WADA MRPL required. In this regard, dilute-dilute-and-shoot and HPLC-QQQ analysis is the option of choice for the determination of TB-500 and forthcoming substances with similar behavior. This approach is not adequate for many of the analytes studied, due to matrix effects entailing lack of sensitivity in both QTOF and QQQ systems (Table 1). MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-385 Figure 1. Instrumental analysis by HPLC-HRMS (QTOF) and HPLC-MSMS (QQQ) Table 1. Sample preparation protocols tested for initial testing and confirmation, analysis by HPLC-HRMS (QTOF) and HPLC-MSMS (QQQ). MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-386 Table 2 shows the complete list of the substances included in this work and the 3 stable isotope-labelled peptides used as representative internal standards (ISTDs), together with the limits of detection (LOD) found. LOD were lower in QQQ analysis for 54 out of 57 substances and for 17 of them, LOD was at least one order of magnitude lower in QQQ against QTOF analysis. Table 2. Retention times and limits of detection of target compounds. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-387 Noteworthy, the microplate protocol showed lack of the sensitivity required for confirmatory purposes in QTOF analysis, since the MS/MS (Product Ion) mode required was found to be less sensible compared with the full scan mode used in the screening method. In contrast, SPE on microplates was adequate for confirmation via QQQ analysis. On the other hand, TOF full scan mode allows for retrospective data mining of untargeted analytes, while targeted QQQ analysis can solely detect the analytes included in the method. Furthermore, similar forthcoming related small peptides can be added in TOF analysis without any loss of performance. Instead, new analytes added to the method gradually reduce the cycles/sec of each transition in QQQ analysis, which entails progressive diminution of points-per-peak. Conclusions HPLC-HRMS (QTOF) or HPLC-MSMS (QQQ) analysis following SPE on microplates is a fast, cost-effective and high-throughput protocol for the determination of small doping-related peptides, by using conventional and affordable spectrometric instruments typically available in accredited doping control laboratories. Nearly all of the peptidic substances and their metabolites showed adequate recoveries as well as good chromatographic behavior and could be determined at the low concentration levels required by the technical document TD2022MRPL. Additionally, a complementary dilute-and-shoot protocol is necessary for the analysis of the small peptides that show too poor recoveries in the SPE protocol, in order to cover the whole range of small peptidic substances and mimetics banned in sports. References 1. World Anti-Doping Agency. Minimum required performance levels and applicable minimum reporting levels for non-threshold substances analyzed by chromatographic-mass spectrometric methods, Montreal (2022). www.wada-ama.org/sites/default/files/2022-01/td2022mrpl_v1.1_eng_0.pdf (access date 01.06.2022) 2. Cuervo D, Fernández-Álvarez M, Loli C, Muñoz G, Carreras D. (2017) Determination of doping peptides via solid-phase microelution and accurate-mass quadrupole time-of-flight LC-MS. J. Chromat. B. 1065-1066, 134-144. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-388 Stojanovic B, Geisendorfer T, Jeitler V, Illedits S, Gmeiner G Capability and stability of dried blood spots for doping analysis Doping Control Laboratory, Seibersdorf Labor GmbH, Seibersdorf, Austria Abstract To improve the fight against doping in sports new ways have to be paved every now and then to meet the challenges within this very unique field of chemical analysis. Examples of the past include the introduction of the athlete’s biological passport (ABP) both with blood and urine samples or the detection of growth hormone doping by the use of serum as sample matrix. Often such a strategy to introduce new ways of detection is dependent on developments or practices in comparable fields of analytical chemistry, like e.g. clinical analysis. Dried blood spots (DBS) are used for quite a long period especially an a clinical diagnostic setting due to its minimal invasion into the body to get a specimen for analysis. This is especially important for neonatal analysis. Besides the easiness and cost efficiency of sample transport and storage after analysis – e.g. cooling is not necessary – the use of DBS seems to have advantages in view of the analyte stability as well as with pharmacokinetic aspects in view of the most application of a prohibited substance. An analytical method suitable to detect about 200 substances and/or metabolites using liquid extraction followed by LC-MSMS detection is presented. Besides full validation, including extraction solvent selection, extraction time and extraction temperature, special emphasis focusses on analyte stability issues as well as potential cross contamination during the punching process. Finally, the method was applied for analysis of reference blood samples containing selected stimulants and narcotics. Introduction Since the TD2021DBS [1] document was released, popularity of DBS in sport drug testing significantly increased. Recognized advantages of DBS would be a simplification of sample collection, less invasive than current methods of urine or blood sample collection; the need for only a very small volume of blood, less expensive to collection and transportation of DBS samples compared to current methods, less space to store the samples and potential benefits with regards to sample stability. Potential advantages of DBS application in sport drug testing are numerous and provide enough reasons to develop and validate a method suitable for DBS analysis of substances banned in sports. Some recent publications shown great potential of DBS in doping field [2,3]. Experimental 20 µL of fortified blood was spotted onto a card and allowed to dry for a minimum of 2 hours. A 6 mm punch from Whatman FTA DMPK cards (Merck, Germany) was taken from the center of the spot and placed into a glass tube. A mixture of methanol-acetonitrile (4:1 V/V), containing internal standard (ISTD), was added, and the samples were shaken in a water bath for 30 minutes and centrifuged. TM ® MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-389 Afterwards, 5 µL of 3 M HCl was added. The solvent was evaporated, samples were reconstituted in mixture of methanol-water (3:7 V/V) and injected into an LC-MSMS system. Samples were analyzed using a Thermo Scientific LC system equipped with Vanquish Autosampler, Vanquish Column Compartment and Vanquish Pump (Thermo, Austin, TX, USA) interfaced to a TSQ Altis triple quadrupole (Thermo, Austin, TX, USA). As analytical pre-column, a 4 × 2 mm Security Guard C18 column was used; the analytical column was a 50 mm × 2.1 mm, 3.5 µm particle size Zorbax XBD C18 (Agilent, USA) column. Mobile phase A was water with 0.2% of formic acid (FA) and mobile phase B was methanol with 0.1% FA. A constant flow rate of 0.41 mL min was applied with the following gradient: 0% B (0–0.2 min), 0% →100% B (0.2 - 7 min), 100% B (7.0-8.0 min), 100% → 0% B (8.0-8.1 min), 0% B (8.1-10 min). The column temperature was maintained at 25 °C and the temperature in the autosampler was set to 4 °C. The mass spectrometer was equipped with a heated electrospray ionization (ESI) source and was operated in positive and negative ionization mode with a spray voltage set at 3500 V and 2500 V, respectively. The ion transfer tube temperature was adjusted to 325 °C while vaporized temperature was set at 350 °C. Sheath and auxiliary gas flow rates were 35 and 10 arbitrary units, respectively. The system was operated in selected ion monitoring (SRM) mode with argon as the collision gas at a pressure of 1.5 mTorr. Results and Discussion Sample preparation In order to establish an optimal sample preparation method suitable for the analysis of around 200 substances belonging to the groups S1, S3, S4, S5, S6, S7 and P1 of the WADA Prohibited List [4] in DBS a systematic approach was applied. Consequently, different extraction solvents and extraction solvent mixtures were tested. For all experiments an ISTD mixture was added to the extraction solvent. Ethyl acetate and MTBE as extraction solvents were excluded in early stages of the study, because the results obtained were not satisfactory for most of the substances. The best results were obtained using methanol and acetonitrile as extraction solvents in a ratio of 4:1 V/V. In addition, different mixing time (from 20 to 50 minutes), and different extraction temperature (room and 50 °C) were tested. Optimal results were obtained with 30 minutes mixing at 50 °C. Validation Method was validated in accordance with current International Standard for Laboratories (ISL) [5]. An overview of validation data is presented in Table 1. For the proof-of-concept Medidrug reference blood at low, medium and high concentrations containing different stimulants and narcotics was used. Additionally, a sample obtained from 1 female volunteer on therapy with nebivolol was used. All presented substances were successfully identified in DBS. Stability testing Additionally, a program for short-term and long-term stability was designed. For stability testing samples spiked at target concentrations and reference blood samples were used. The design of the stability study is presented in Table 2. Samples were stored in sealable plastic bags with silica gel as desiccant. Short- -1 ® MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-390 term stability shows that substances within the scope are stable in DBS. Long-term stability is still in progress but the results until now confirmed the stability of the tested analytes. Table 1. Validation data Table 2. Program for stability testing Conclusions The study shows that DBS in sport drug testing have great potential as complementary matrices and could bring improvement in routine doping control. Results so far show remarkable stability of the target substances, contributing to the importance of DBS in sport drug testing. References 1. World Antidoping Agency. DRIED BLOOD SPOTS (DBS) FOR DOPING CONTROL https://www.wada- ama.org/sites/default/files/item_6_3_3_attach_1_finalversion_td2021dbs_final.pdf (access date 14.07.2022) 2. Thevis M, Kuuranne T, Thomas A, Geyer H (2021) Do dried blood spots (DBS) have the potential to support result management processes in routine sports drug testing? – Part 2: Proactive sampling for follow-up investigations concerning atypical or adverse analytical finding Drug Test Anal 14 (6), 1040-1052 3. Jing J, Shan Y, Liu Z, Yan H, Xiang P, Chen P, Xu X (2021) Automated online dried blood spot sample preparation and detection of anabolic steroid esters for sports drug testing. Drug Test Anal 13, 505-509 4. World Antidoping Agency. The 2022 Prohibited list. International Standard, 2022 2022list_final_en_0.pdf (wada-ama.org) (access date 14.07.2022) 5. World Antidoping Agency. International Standard for Laboratories 2021, https://www.wada- ama.org/sites/default/files/resurces/files/isl_2021.pdf (access date 14.07.2022) MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-391 Blázquez I , Espinosa P , de La Cal A , García L , Fernández B , Muñoz G Solid phase extraction by anion exchange for hypoxia inducible factor (HIF) activating agents Laboratory LC-MS/MS, Madrid Anti Doping Laboratory, Madrid, Spain ; Madrid Anti Doping Laboratory, Madrid, Spain Abstract Hypoxia inducible factor (HIF) activating agents are being widely studied in the anti-doping context. HIF stabilizers increase the artificial erythropoiesis of athletes, which stimulates the production of red blood cells and enhances oxygen transport capacity. Therefore, World Anti-Doping Agency (WADA) prohibits the use of these compounds both in competition and out of competition. Consequently, the development of sensitive methods that comply with the identification criteria established by WADA (TD2021IDCR) are required. Most of the HIF activating agents have a carboxylic acid group and this structural characteristic has been used to develop a confirmation method for a set of HIF activating agents: vadadustat, desidustat, IOX-2, IOX-3 (FG-2216), JNJ-42041935, daprodustat (GSK1278863), daprodustat M2 (GSK2391220), and roxadustat (FG-4592). The initial testing procedure (ITP) of these prohibited substances, was performed by dilute and shoot after enzymatic hydrolysis. In order to reach greater selectivity and major sensitivity we propose a confirmation process based on an enzymatic hydrolysis followed by an anion exchange solid phase extraction (SPE). All experiments were performed by liquid chromatography on line with mass spectrometry (LC-MS/MS) as instrumental analysis. Preliminary results showed that all compounds could be identified at concentrations below 2 ng/mL. Introduction Hypoxia-inducible factor (HIF) activating agents are transcription factors that regulate the cellular response to hypoxia and act as regulators of oxygen homeostasis. The HIF activating agents are being widely studied in the anti-doping context, since there is a significant number of substances involved in the increase of artificial erythropoiesis. According to the World Anti-Doping Agency (WADA), these compounds are included in the list of prohibited substances in the category S2.1.2 Activating Agents of the Hypoxia Inducible Factor (HIF). The initial detection of substances with carboxylic acid groups was performed using the “dilute and shoot” technique, while an anion exchange solid phase extraction was suggested for its confirmation. The instrumental analysis performed in both procedures consists of a separation by reverse phase liquid chromatography in tandem with mass spectrometry (LC-MS-MS) and electrospray ionization in negative mode (ESI -). The objective of this work was to figure out if, employing MAX cartridges, the identification criteria established by WADA [4,5] are reachable at concentrations below the MRPL of the target analytes. 1 2 2 2 2 2 1 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-392 Figure 1. Chemical structures of the targeted compounds Experimental Sample treatment Samples from the initial treatment process were prepared according to a dilute and shoot approach. 500 µL of urine was spiked with 20 µL of pH 7 buffer, 10 µL of 1-dehydrocortesolone as internal standard and 20 µL of E. coli β-glucuronidase enzyme. The samples are heated for one hour at a temperature of 55 °C. A 1/5 dilution is applied to samples with 1 mM ammonium acetate/5% acetonitrile buffer. The samples are mixed and injected into the LC-MS/MS system. As a confirmation method, 2.500 µL of urine were enriched with 100 µL of pH 7 buffer, 7 µL of ISTD (2- benzyloxybenzoic acid) and 50 µL of E. coli β-glucuronidase enzyme. The samples were heated for one hour at a temperature of 55 °C. A solid phase extraction with anion exchange was performed using cartridges: Oasis MAX 30 mg, 1 cc (wash: 1 mL ammonium hydroxide 5% in distilled water; sample load: 2 mL sample; elution: 1 mL formic acid 2% in MeOH. Samples were dried under nitrogen and reconstituted with 400 μL of 1mM ammonium acetate/5% acetonitrile buffer. Samples were mixed and injected into the LC-MS/MS system. Instrumental Analysis All LC experiments were performed using an Agilent 1260 infinity Series HPLC pump with binary gradient system and automatic injector. Reverse-phase liquid chromatography was run on Poroshell 120 EC-C18 column (2.1 x 50 mm, 2.7 µm) from Agilent. The mobile phase consists of 1 mM ammonium acetate (A) and acetonitrile (B). A gradient program was set up starting at 2% B maintained for 0.2 min followed by a linear gradient up to 35% B in 3.3 min and 98% B during 2.6 min, returning to initial conditions and maintaining an equilibrium time of 3 min, which resulted in a total run time of 9.3 min. The flow rate was 350 µL/min. The column device was at 30 °C. The sample injection volume was 5 µL. Detection and confirmation data were acquired using SCIEX 5500 Qtrap mass spectrometer analyser equipped with an electrospray ionization source operating in negative polarity. The ion source temperature was 500 °C, the applied capillary voltage was -4500 V in negative mode. The mass spectrometer was run in multiple reaction monitoring (MRM) as acquisition mode with a detection window of 45 s. Three transition ions per analyte and one transition ion for the internal standard were used to identify each analyte. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-393 Table 1. Target compounds, MS parameters, retention times (RT), LODs and LOIs (for substances with * these are preliminary results based on one matrix, which not covering a range of pH, SG and different gender). Results and Discussion Using dilute and shoot, it was not possible to find three valid transitions according to TD2021IDCR. Therefore, the development of an alternative sample preparation for confirmation was necessary. Taking into account that the studied compounds have in common an acid group in their structure, an anion exchange solid phase extraction was chosen to increase sensitivity and selectivity and achieve a third transition that allows us to give a reliable result for the prohibited substance. The method proved to be specific (no significant interferences were detected at the retention times of the analytes under investigation), sensitive, and reproducible (retention times and relative abundances) (see Table 1). No carryover signals were detected in urine samples injected after spiked urine samples. With the "dilute and shoot" method (ITP), concentrations of 50% MRPL (1 ng/mL) are detected for vadadustat, desidustat, IOX-2, daprodustat M2 (GSK1391220) and roxadustat (FG-4592), concentrations of 35% MRPL (0.7 ng/mL) for IOX-3 (FG-2216) and concentrations of 25% MRPL (0.5 ng/mL) for JNJ- 42041935 and daprodustat (GSK1278863), while with solid phase extraction process concentrations of 50% MRPL (1 ng/mL) are identified for vadadustat, desidustat and IOX-3 (FG2216), concentrations of 25% MRPL (0.5 ng/mL) for IOX-2, JNJ-42041935 and daprodustat M2 (GSK1391220) and concentrations of 10% MRPL (0.2 ng/mL) for daprodustat ( GSK1278863) and roxadustat (FG-4592)(Figure 3). MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-394 Figure 3. Initial Testing Procedure vs Confirmation Process Figure 3. Initial Testing Procedure vs Confirmation Process These preliminary results indicate that the proposed SPE methodology, rather than dilute and shoot, could be employed for the confirmation of the target compounds since it allows their identification at concentrations below the MRPL with three diagnostic transitions fulfilling WADA requirements (Table 1). Conclusions With "dilute and shoot" at least two valid transitions are obtained, therefore is a reliable process for the initial detection of the HIF activating agents. Due to the presence of the acid group in the studied substances, anion exchange solid phase extraction has demonstrated to be an appropriate method to increase the signal of the target compounds and to improve selectivity, obtaining three transitions that MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-395 meet the WADA identification criteria (TD2021IDCR) [5] and reducing the noise usually derived from matrix and reagents interferences. Further experiments will be performed in order to fully validate the proposed confirmation methodology. References 1. Moses Philip, Binoy Mathew, Tajudheen K Karatt, Zubair Perwad, Michael Benedict Subhahar, Abdul Khader Karakka Kal (2021). Metabolic studies of hypoxia-inducible factor stabilisers IOX2, IOX3 and IOX4 (in vitro) for doping control. Drug Test Anal. 13(4):794-816. 2. De Wilde L, Roels K, Deventer K, Van Eenoo P. Automated sample preparation for the detection and confirmation of hypoxia-inducible factor stabilizers in urine. Biomed Chromatogr. 2021 Feb;35(2):e4970. doi: 10.1002/bmc.4970 3. World Anti-Doping Agency. The 2022 Prohibited List. International Standard, Montreal (2022). https://www.wada-ama.org/sites/default/files/prohibited_list_2022_en.pdf. 4. World Anti-Doping Agency. WADA Technical Document – TD2022MRPL. Minimum Required Performance Levels and Applicable Minimum Reporting Levels for Non-Threshold Substances Analysed By Chromatographic - Mass Spectrometric Analytical Methods. https://www.wada- ama.org/sites/default/files/resources/files/td2022mrpl_v1.0_final_en.pdf 5. WADA Technical Document – TD2021IDCR. Minimum Criteria for Chromatographic-Mass Spectrometric Confirmation of the Identity of Analytes for Doping Control Purposes. https://www.wada- ama.org/en/resources/lab-documents/td2021idcr MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-396 Almeida CVP , Alves I , Ruivo J Potentialities of adsorptive micro-extraction in doping control – a new perspective in sample preparation targeting green chemistry Laboratório de Análises de Dopagem, Lisbon, Portugal ; Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal Abstract During the development and implementation of analytical methodologies, the main goal consists in obtaining undoubtedly and irrefutable technical and scientific results, namely when working in doping control, as well as in any other area which follows strict and strong legal requirements. One of the first tasks throughout analytical method implementation is based on sample preparation. This continues to be a very relevant and important step, due to the high complexity of biological samples, such as urine. Therefore, the possibility of boosting analytical enrichment together with strong reduction or elimination of interferents are extremely important factors to achieve correct identification and quantification of the substances of interest. In the last decade, adsorptive micro-extraction emerges as a new perspective in sample preparation. The Bar Adsorptive Micro-Extraction (BAµE) device appears as one of the possibilities to be used. This device has shown to be a promising analytical strategy in several applications in order to overcome the limitations presented by other techniques such as the Stir Bar Sorptive Extraction (SBSE). BAµE operates in the static mode and with floating sample technology, using solid materials with suitable physical and chemical properties to achieve excellent efficiency for the polar and apolar target substances. Moreover, BAµE includes advantages such as high sensitivity, selectivity, efficiency, miniaturization, low cost, environmental friendliness using only low microliters of toxic solvents, and the option to select the most appropriate sorbent material for the substances under study. Overall, BAµE appears to be an excellent alternative sample preparation technique for enrichment purposes of target substances, and which at the same time allows highly effective elimination of interferents and the use of a low volume of urine. The present contribution aims to show the high analytical potentialities for the application of BAµE in doping analysis as unconventional sample preparation technique for enrichment purposes, while other sample preparation techniques may have analytical limitations. Introduction Introduced approximately 15 years ago, Bar Adsorptive MicroExtraction (BAµE) is an analytical device which operates under the floating sampling motion and in the static mode. This modern concept in analytical enrichment shows to be an alternative to other conventional µ-extraction techniques, in particular due to its high range of applicability to substances with polar and non-polar characteristics [1,2]. 1 2 1 1 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-397 During the µ-extraction enrichment process, the analytes migrate from the bulk sample into the sorbent material used. The transfer of the target analytes is promoted by magnetic stirring, which forces the device to stay vertically lined up under the vortex as a result of the centript force. This analytical alternative device presents several advantages with successful applications when compared to Stir Bar Adsorptive Extraction (SBSE) [3], such as the possibility to choose the most suitable sorbent phase, such as activated carbons, polymers, and ionic liquids, depending on the type of application. In addition, BAµE includes advantages such as high sensitivity, selectivity, efficiency, miniaturization, low cost and, in addition, it is an ecological approach. BAµE device can be combined with chromatographic systems and hyphenated ones, like HPLC, GC-MS, etc. [4-7]. The present contribution aims to show the high potentialities of BAµE in doping control to overcome the limitations shown by other analytical techniques. Preliminary assessments using stimulants (class S6) and β-blockers (class P1) as target model substances revealed good selectivity, efficiency, and linearity under non-optimized conditions using conventional GC-MS instrumentation. Experimental The present experiments were fully assessed with high purity reagents (≥ 98% and/or LC-MS quality). The BAμE device was lab-made prepared according to previous works, having approximately 7.5 mm in length and 3.0 mm in diameter [2,7]. Assays were performed in common glass vials (Ø 1.2× 3.2 cm length) for 1,4-dimethyl pentylamine and propranolol, and in flasks with 1.0 Ø and 5 cm length for cocaine. To each vial was pippeted 1 mL of urine sample for 1,4-dimethyl pentylamine and propranolol and 3 mL for cocaine. Suitable amount of buffer was added to each experiment, namely phosphate (pH 8) for 1,4-dimethyl pentylamine and cocaine, and acetate (pH 5) for propranolol. Appropriate amounts of standard and internal standard (dextrorphan) were used for spiking purposes, depending on the type of experiment. In the case of propranolol enzymatic hydrolysis was performed by adding 50 μL of ß-glucuronidase/arylsulfatase (Roche, Germany) for 1 hour at 55°C. Finally, the microextraction device, previously coated with powdered sorbent, was inserted into the sampling vial, as well as a conventional teflon stir bar. Furthermore, the sampling flasks were placed on a multipoint agitation plate at room temperature, where the microextraction took place under non optimized standard experimental conditions. Later on, the BAμE device was removed with clean tweezes, dried with a lint-free tissue and placed into vials containing conical shaped inserts with 150 μL of the stripping solvent, ensuring their total immersion prior to ultrasonic treatment (ELMA, Germany) at room temperature. Afterwards, the microextraction devices were removed, added 10 μL of MBTFA to the extract of 1,4-dimethyl pentylamine to prevent losses by evaporation, vortex-mixed and the solvent vaporized to dryness under vacuum at 45°C (Eppendorf Concentrator Plus, Germany). The last step was the derivatization with 50 μL of MSTFA at 80°C (Stuart oven block, UK) for 10 min and 15 μL of MBTFA at 80°C for 1,4-dimethyl pentylamine for 20 min and propranolol for 10 min, as well as 50 µL of MSTFA at 80 °C during 20 min for cocaine. Finally, after cooling down, the vials were placed on the auto-sampler for GC-MS analysis (Agilent 6890 Series gas chromatograph - Agilent 5973N Inert quadrupole mass selective detector). Figure 1 shows a schematic experimental procedure for the present assays. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-398 Figure 1. Experimental scheme of the present work Results and Discussion The results obtained from the preliminary assays showed several good perspectives in sample preparation for the selected compounds. In a first approach an efficiency of 42.1% was achieved for the propranolol test in urine sample under non-optimized experimental conditions. Observing the total ion chromatogram in Figure 2a for propranolol, excellent selectivity is observed when comparing to blank urine (Figure 2b) once the latter shows absence of any signal of interference. Figure 2. Total ion chromatogram for the spiked urines (a) and the blank urine (b) obtained for propranolol test MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-399 In the case of cocaine, good results were also obtained under non-optimized experimental conditions. By observing Figure 3, the test of spiking urine samples at the 10 ng/mL level (Figure 3b), shows good sensitivity and selectivity, once the blank urine shows absence of interferences for the m/z used at the cocaine retention time (Figure 3a). Figure 3. Chromatogram obtained for the extracted selected ions for cocaine in the negative urine (a) and for the spiked urine at 10 ng/mL (b) The third example was a linearity test assessed for 1,4-dimethyl pentylamine. By spiking urine samples in the range of 10 ng/mL to 200 ng/mL, including the negative sample, good linearity is observed with a determination coefficient (r ) of 0.9969, as depicted in Figure 4a. Moreover, the blank urine shows the absence of any signals for interferences, as well as for the 10 ng/mL spiked sample (Figure 4b), good and clean signals are obtained for the m/z used with a signal to noise > 3. Taking into consideration the fact that since the first appearance of the WADA Technical Document (TD) - Minimum Required Performance Level (MRPL) in 2004, to which many prohibited substances must comply, it was established a MRPL at the 500 ng/mL level. Actually, the TD-MRPL established a generic level of MRPL at 50 ng/mL for beta- blockers and stimulants. The MRPL constant decrease by 10-fold clearly suggest that the trend in the development of analytical methodologies must evidence high sensitivity, selectivity, specificity and robustness in order to fulfill the WADA analytical requirements present in the Technical Documents [8]. 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3100 Figure 4. Total ion chromatogram obtained for the linearity test (a) and extraction of the three ions selected at the level of 10 ng/mL (b), of 1,4-dimethyl pentylamine Conclusions The results obtained from preliminary assays using the modern bar adsorptive microextraction sample preparation approach showed to be an excellent alternative technique and different perspective in doping context. Advantages of BAµE, such as low-cost, miniaturized, fast and easy to work-up, as well as low volume of toxic solvents turn it to be environmentally friendly. Once results showed very good linearity and selectivity with absence of interferences, future work demands a complete optimization of the microextraction process and validation of the methodology, always with a vision for analytical improvements, such as lowering of volume sample. For a Fit-for-Purpose analytical methodology in regarding application for initial testing and/or confirmation procedures studies must include more compounds in number and/or several classes of compounds. References 1. Nogueira JMF, 2012 Novel sorption-based methodologies for static microextraction analysis: A review on SBSE and related techniques. Anal Chimica Acta 757 1-10. 2. Neng NR, Silva ARM, Nogueira JMF, Adsorptive micro-extraction techniques—Novel analytical tools for trace levels of polar solutes in aqueous media. 2010 J. Chromatogr. A 1217 7303-7310. 3. Baltussen E, Sandra P, David F, Cramers CA, 1999 Stir bar sorptive extraction (SBSE), a novel extraction MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3101 technique for aqueous samples: Theory and principles. J. Microcol. Sep. 11 737-747. 4. Almeida C, Nogueira JMF, 2012 Comparison of the selectivity of different sorbent phases for bar adsorptive microextraction—Application to trace level analysis of fungicides in real matrices. J. Chromatogr. A 1265 7- 16. 5. Neng NR, Ahmad SM, Gaspar H, Nogueira JMF, 2015 Determination of mitragynine in urine matrices by bar adsorptive microextraction and HPLC analysis. Talanta 144 105-109. 6. Almeida C, Nogueira JMF, 2015 Determination of steroid sex hormones in real matrices by bar adsorptive microextraction (BAμE). Talanta 136 145-154. 7. Ahmad SM, Almeida C, Neng NR, Nogueira JMF, 2016 Bar adsorptive microextraction (BAμE) coated with mixed sorbent phases-Enhanced selectivity for the determination of non-steroidal anti-inflammatory drugs in real matrices in combination with capillary electrophoresis. J. Chromatogr. B 1008 115-124. 8. World Anti-Doping Agency. The Minimum Required Performance Level. https://www.wada-ama.org/en - Access date 12-07-2022 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3102 Berghes B, Cristea CD, Radu M, Catana D, Toboc A, Stan C The development of an identification method for heptaminol by liquid chromatography-tandem mass spectrometry (LC-MS/MS) Romanian Doping Control Laboratory, Bucharest, Romania Abstract Heptaminol (6-amino-2-methyl-2-heptanol) is listed on the World Anti-Doping Agency 2022 Prohibited List as a specified stimulant. In the present study, we developed and validated a method based on solid phase extraction with sample pH ajusted to 5 using acetate buffer and elution with 5% NH in methanol followed by LC-MS/MS analysis. Method validation was performed at minimum required performance levels specified by WADA Technical Documents (50 ng/mL) for heptaminol, and the method was validated with regard to selectivity (no interference), limits of detection for confirmation 20 ng/mL and a 90.3% recovery. Introduction Heptaminol (6-amino-2-methyl-2-heptanol) is a stimulant with amino alcohol structure, which is classified as a cardiotonic and a vasodilator and it is listed on the World Anti-Doping Agency 2022 Prohibited List as a specified stimulant [1-4]. In doping control analysis the methods for identification of heptaminol are commonly based on the GC-MS technique [5-7]. In order to implement the method for the identification of heptaminol in our laboratory, different preparation methods were tested: liquid-liquid extraction (LLE) with different solvents and a solid-phase extraction (SPE) in which pH of the sample and the elution solution was varied. Analysis was performed using high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Experimental Materials and methods Materials: Heptaminol from LGC Standards, D -ephedrine from CERILLIANT, SPE cartridges PHENOMENEX STRATA X-CW 1 mL, ammonia 38%, methanol, acetonitrile, formic acid, ammonium formate form MERK. Liquid chromatography conditions: Liquid cromatograph HPLC Agilent 1290 Infinity, Mobile phase: Solvent A: H O (0.1% HCOOH and 5 mM amonium formate), Solvent B : 90% ACN (0.1% HCOOH and 5mM amonium formate) and 10% H O, Poroshell 120 EC-C18 3 x 50 mm, 2.7 microns, injection volume 1 µL, flow rate 0.25 mL/min. Sample preparation is described in Figure 1. 3 3 2 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3103 Figure 1. Sample preparation scheme Mass spectrometric conditions: Mass Spectomerter AB SCIEX 5500 Qtrap, Ionization source ESI positive, Source Temperature 600°C, Capillary voltage 5500 V, nebulizer gas (GS1) 40 psi, auxiliary gas (GS2) 60 psi, curtain gas (CUR) 25 psi, collision cell gas (CAD) High. Acquisition mode was multiple reaction monitoring (MRM) with transitions m/z 146.00> 128.00, 146.00> 69.00, 128.00> 69.00, 146.00> 111.00 (Figure 2). Figure 2. Validated fragment of Heptaminol MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3104 Validation Limit of identification (LOI): Scalar dilutions in urine to obtain the lowest concentration for substances identification (S/N> 3). Ten negative urine samples from different sources, 3 male and 7 female, with pH between 5-9 and sg 1.003-1.030, were fortified with the standard heptaminol, establishing that the identification limit has the value of 20 ng/mL. Specificity: 10 negative control samples are injected, and then the presence of interferences at the retention times of the analyte of interest is checked. Carry-over was evaluated with the consecutive injection of a sample fortified at 250 ng/mL and blank samples to investigate if there are any contamination. Robustness was evaluated by modifying column temperature, source temperature, flow, injection volume, elution volume, buffer volume, reconstitution volume. Results and Discussion In order to develop a method for identification of heptaminol, it was necessary to optimize the chromatographic and spectrometric parameters, but also the ones regarding sample preparation for meeting the requirements of the WADA technical documents (TDMRPL, TDIDCR, ISL). Using high purity heptaminol standard, the LC-MS method was developed. The parameters of the method are presented in material and methods section. For a specific extraction of urine samples several experiments were performed involving liquid-liquid extractions in an alkaline medium, with carbonate buffer pH 9, in which organic extraction solvents were varied (tert-methyl-butyl ether and ethyl acetate). This type of extraction was inefficient due to the interferences that were present at the retention time of the target compound (results not shown). Subsequently, solid phase extractions were performed and the adjusted parameters were: the pH of the sample (with formic acid for pH 2 and acetate buffer for pH 5) and the elution solution (5% formic acid and respectively 5% ammonia both in methanol). The optimal parameters for the sample preparation method involved solid phase extraction with sample pH ajusted to 5 with acetate buffer and elution with 5% NH in methanol. In order to demonstrate the fitness for purpose, the method was validated according to the ISL. The method is specific for heptaminol with no interfering signals on the target compound retention time. The identification limit has been established at a value of 20 ng/mL which is compliant with WADA TDMRPL (lower than MRPL value of 50 ng/mL). Negative control, positive control (negative urine spiked with heptaminol at 20 ng/mL) and H O spiked at 40 ng/mL are shown in Figure 3. Carry-over was not observed at a concentration of 250 ng/mL for heptaminol. The method is robust for investigated parameters. Recovery was evaluated at 20 ng/mL and the obtaining average recovery was 90.3% (Table 1). 3 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3105 Figure 3. Chromatograms for: A. Negative control, B. Positive control 20 ng/mL, C. Heptaminol standard in HO 40 ng/mL Table 1. Recovery results Conclusions According to our results, the developed methods, instrumental and preparation, have proved to be suitable for heptaminol identification and were validated according to WADA technical documents. The most appropiate method for sample preparation was solid phase extraction with sample pH adjusted to 5 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3106 with acetate buffer and elution with 5% NH in methanol, method which has 90.3% average recovery. The results from validation has shown that the method developed by our laboratory is “fit for purpose”. References 1. World Anti-Doping Agency. The 2022 Prohibited List. https://www.wada-ama.org/sites/default/files/ resources/files/2022list_final_en.pdf (access date 01.01.2022). 2. World Anti-Doping Agency Technical Document – TD2022MRPL. https://www.wada- ama.org/sites/default/files/2022-01/td2022mrpl_v1.1_eng_0.pdf (access date 01.01.2022). 3. World Anti-Doping Agency Technical Document – TD2021IDCR. https://www.wada- ama.org/sites/default/files/resources/files/td2021idcr_final_eng_0.pdf (access date 01.04.2022). 4. World Anti-Doping Agency International Standard for Laboratories. https://www.wada- ama.org/sites/default/files/resources/files/isl_2021.pdf (access date 01.01.2021). 5. Tseng Y L, Liu C H, Kuo F H, & Shieh M H. (2006) Solid-phase column chromatographic and gas chromatographic- mass spectrometric determination of heptaminol in human urine and related pharmacokinetic profiles. J. Anal. Toxicol. 30 (6), 365-369. 6. Dib J, Bosse C, Tsivou M, Glatt AM, Geisendorfer T, Geyer H, Gmeiner G, Sigmund G, Thevis M. Is heptaminol a (major) metabolite of octodrine? Drug Test Anal. 2019 Nov;11(11-12):1761-1763. doi: 10.1002/dta.2737 7. Sardela VF, Sardela PDO, Padilha MC, Pereira HMG, Neto FA. Analysis of adrenergic aliphatic amines in urine for doping control. Recent advances in doping analysis (19): Proceedings of the 29 Manfred-Donike- Workshop on Dope Analysis, Köln: Sportverlag Strauß, 2011, pp. 170-173 3 th MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3107 Pereira D, Mourato M, Rocha Gomes T, Salema B, Ruivo J HIF: Validation of confirmation procedure by LC-MS/MS Laboratório de Análises de Dopagem, Lisbon, Portugal Abstract Hypoxia-inducible factors (HIF) activating agents have a potential to enhance blood haemoglobin levels after oral administration, leading to an increase of the capacity for oxygen transport. Due to the increased number of HIF stabilizers available, their pharmacological potential and enhancing performance effects in athletes, these substances are included in the section S2.1.2 of WADA’s Prohibited List. The aim of this study was to develop and validate a procedure for HIF confirmation in urine samples by LC-MS/MS (ABSciex QTrap 5500). Sample preparation was performed by dilute-and-shoot for daprodustat (GSK1278863), daprodustat bishydroxylated metabolite (GSK2391220), desidustat (ZYAN1), FG-2216, IOX2, IOX4, JNJ-42041935, roxadustat (FG-4592) and vadadustat (AKB-6548) and by MCX solid- phase extraction for molidustat (BAY85-3934) and molidustat glucuronide (BAY1163348). The methods were validated for the parameters selectivity / specificity, limit of identification, robustness, carry over, matrix effect, stability and recovery (only for molidustat and molidustat glucuronide). The methods have shown to be fit-for-purpose, with limits of identification below or equal to 1 ng/mL, in compliance with WADA’s Technical Documents TD2022MRPL and TD2021IDCR. Introduction Hypoxia-inducible factors (HIF) activating agents are transcriptional activators of hypoxia inducible genes and have a potential to enhance blood haemoglobin levels after oral administration, leading to an increase of the capacity for oxygen transport. For this reasons and because of their pharmacological potential and enhancing performance effects, they are being used by athletes and were included in the section S2.1.2 of WADA’s Prohibited List [1-4]. Experimental Reference materials Molidustat and molidustat glucuronide were purchased from Bayer Pharma AG, desidustat, FG-2216, IOX2, IOX4, JNJ-42041935, roxadustat and vadadustat from Cayman Chemical Company and mefruside from LGC Standards. Daprodustat and daprodustat bishydroxylated metabolite were provided by GlaxoSmithKline. Sample preparation Sample preparation was performed by dilute-and-shoot for daprodustat, daprodustat bishydroxylated metabolite, desidustat, FG-2216, IOX2, IOX4, JNJ-42041935, roxadustat and vadadustat and by MCX solid-phase extraction for molidustat and molidustat glucuronide. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3108 Dilute-and-shoot: 20 µL of mefruside at 2 µg/mL (internal standard) were added to 480 µL of the samples, stirred and centrifuged and then transferred to a vial and analyzed in LC-MS/MS Solid-phase extraction (SPE): 40 µL of mefruside at 20 µg/mL (internal standard) and 400 µL of 0.6 M hydrochloric acid solution were added to 4 mL of the urine samples. Samples were centrifuged for 10 min at 2500 rpm. Then SPE was performed with an MCX cartridge, which was conditioned with 2 mL of ethanol, followed by 2 mL of 0.1 M hydrochloric acid solution. After sample elution, the cartridge was washed with 2 mL of 0.1 M hydrochloric acid solution and 2 mL acetonitrile. The analytes were eluted with 6 mL of acetonitrile fortified with 5% ammonia solution. The eluates were evaporated to dryness under a nitrogen stream at 60 °C and the residue was dissolved in 150 µL of a mixture of mobile phase A : mobile phase B (80:20%, v/v). Instrumental analysis ABSciex 5500 Qtrap LC-MS/MS was used for the analysis. The analytical column was Xbridge BEH C18 100 x 2.1 mm with 2.5 µm particle size and at 35 °C. Mobile phase A consisted of water with 0,1% formic acid: acetonitrile (95:5%, v/v) and mobile phase B of water with 0.1% formic acid: acetonitrile (5:95%, v/v) at 0.3 mL/min flow rate. Injection volume was 10 µL. Gradient elution for molidustat and metabolite and for the remaining analytes is shown in Figure 1. The mass spectrometry parameters are shown in Table 1. Figure 1. Chromatographic conditions – gradient elution Method validation The parameters studied in the validation of these methods were: seletivity/specificity (10 urines at MRPL in 2 different batches), limit of identification (LOI) (2 different batches, 6 urines at 3 levels: 50% MRPL, 25% MRPL and 10% MRPL), carry over (at MRPL and 10x MRPL), robustness, matrix effect (at MRPL and 10x MRPL), stability and recovery (only for SPE method and at MRPL and 10x MRPL). MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3109 Table 1. Mass spectrometry parameters MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3110 Results and Discussion Two methods were developed and validated for qualitative confirmation of HIF activating agents. Both methods showed to be specific, selective, robust, stable at least 36 hours after sample preparation, with LOI values below or equal to 1 ng/mL (50% MRPL), without matrix effect and carry over at concentrations levels of 10x MRPL. The extraction for molidustat and molidustat glucuronide showed acceptable recoveries. The results obtained for each analyte are presented in Table 2. Table 2. Validation results In Figure 2, an example of a blank urine and a positive control spiked with the HIF stabilizers at MRPL is shown. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3111 Figure 2. Chromatogram of a blank urine and a spiked urine at MRPL Conclusions This study presents a method validation for the confirmation of HIF stabilizers in human urine by LC- MS/MS. Due to the difference between analytes two different types of sample preparation were needed. The methods showed to be fit-for-purpose, with limits of identification below or equal to 1 ng/mL, which is in compliance with WADA Technical Documents TD2022MRPL and TD2021IDCR [5,6]. All analytes were included in the laboratory scope of accreditation under ISO 17025:2017. References 1. Dib, J., Mongongu, C., Buisson, C., Molina, A., Schänzer, W.,Thuss, U., Thevis, M. (2017), Mass Spectrometric characterization of the hypoxia-inducible factor (HIF) stabilizer drug candidate BAY 85-3934 (molidustat) and its glucuronidated metabolite BAY-348, and their implementation into routine doping controls, Drug Test. Analysis. 9 (1), 61-67 2. Thevis, M., Milosovich, S., Licea-Perez, H., Knecht, D., Cavalier, T., Schänzer, W. (2016), Mass spectrometric characterization of a prolyl hydroxylase inhibitor GSK1278863, its bishydroxylated metabolite, and its implementation into routine doping controls, Drug Test. Analysis. 8, 858-863 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3112 3. Berghes, B., Radu, M., Cristea, CD., Toboc, A. (2020), Validation of HIF activating agents confirmation methods. In Thevis, M., Geyer, H., Mareck, U. (eds.) Recent advances in doping analysis (28) Sportverlag Strauß, Köln, 124-128 4. World Anti-Doping Agency (WADA). The 2022 Prohibited List, International Standards, Montreal (2022) www.wada-ama.org/sites/default/files/resources/files/2022list_final_en.pdf (access date 03.03.2022) 5. World Anti-Doping Agency (WADA). WADA Technical Document – TD2022MRPL, v.1.1, Montreal (2022) www.wada-ama.org/sites/default/files/2022-01/td2022mrpl_v1.1_eng_0.pdf (access date 08.02.2022) 6. World Anti-Doping Agency (WADA), WADA Technical Document – TD2021IDCR, v.1.0, Montreal (2022) www.wada-ama.org/sites/default/files/resources/files/td2021idcr_final_eng_0.pdf (access date 08.02.2022) MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3113 Sobolevsky T, Ahrens B Metal-sensitive analytes in LC-MS: implications for doping control UCLA Olympic Analytical Laboratory, Los Angeles, United States Abstract The continually expanding WADA Prohibited List presents antidoping laboratories with an increasingly diverse range of target substances. As we incorporate more and more analytes into our initial testing procedures, we more frequently encounter analytes that just do not behave as expected under the existing conditions. It is has been established that secondary interactions with residual silanols on sorbent surface can lead to excessive peak tailing. In some cases, this can be mitigated by proper selection of a chromatographic column, increasing mobile phase ionic strength, or decreasing its pH. However, for compounds capable of chelation it can be very problematic to find a mass spectrometry friendly solution. Here we present our data on metal contamination of a typical liquid chromatography (LC) system and evidence of complex formation between IOX4 and iron which is responsible for poor peak shape. Further, it is demonstrated that the use of LC column with alkylsilane coating (Waters Premier brand) minimizes the adsorptive losses and greatly improves non-specific binding of metal sensitive analytes such as IOX4 and molidustat. Introduction There have been reports on adsorptive losses and poor chromatographic behavior of compounds capable of metal complex formation, in particular organophosphates (e.g. nucleotides) which have a propensity for iron or even titanium [1-3]. As desirable as it is to have a chromatographic system where metal ions from instrument hardware cannot leach into the mobile phase, it is not yet technically achievable. Major vendors of chromatography equipment offer so-called biocompatible systems with wetted parts made of alloys other than stainless steel, for instance, nickel-cobalt-chromium (MP35N) or titanium-aluminum alloys. Such systems may lend a false sense of “protection” against adverse effects related to metal contamination. Ironically, chromatographic columns are commonly made of stainless steel which largely negates the benefits of bioinert hardware. Experimental IOX4, molidustat and molidustat glucuronide were detected in a 96-well plate dilute-n-shoot LCMS assay following a 1:5 dilution of urine with 0.2 M formate buffer containing 2% methanol and isotopically labeled internal standards. Diluted samples were centrifuged directly in the plate at 1666 g for 5 min and analyzed on a Sciex 5500 TripleQuad instrument. Ten µL of diluted sample was injected onto an Acquity Premier HSS T3 column (Waters, 50 x 2.1 mm, 1.8 µm) at 40°C and 0.3 mL/min using 0.1% formic acid in MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3114 water and 0.1% formic acid in methanol as mobile phase. Gradient started from a 0.5-min hold at 2% B, increased linearly to 95% B over 7 min, and after a 1-min hold switched back to 2% B followed by a 1.5- min equilibration before the next injection. The experiments with volatile chelator acetylacetone (AcAc) were conducted on a Thermo QExactive Plus with Dionex UltiMate 3000RS. Separation was achieved using a variety of columns from different vendors at 40°C with a 0.3 mL/min flow of 0.1% formic acid in water and 0.1% formic acid in methanol or acetonitrile, each containing 2 mM AcAc. Gradient was adjusted as needed. Mass spectrometer was operated in fullscan mode, and positive ions were detected at a resolution of 17500. Results and Discussion It has been observed earlier that molidustat cannot be efficiently chromatographed using a low ionic strength water-methanol mobile phase (that is, unbuffered 0.1% formic acid). The only workaround was to detect molidustat glucuronide instead. A related compound IOX4 was also found to perform poorly on nearly every column we tested. With a certain compromise, IOX4 was incorporated into a dilute-n-shoot procedure using a Waters Cortecs T3 column. However, the peak shape of IOX4 was not consistent across different analytical batches and has always improved over repeated injections. Further, the replacement of a guard column always degraded the IOX4 peak shape, sometimes to the point that it was no longer detectable (Figure 1). Figure 1. Peak shape of IOX4 (2 ng/mL, 329 > 273, ESI+) on Cortecs T3 column, A-B: well-used precolumn & column, first injection (A), 96 injection (B), C-D: precolumn replaced, first injection (C) and 96 injection (D). We suspected that IOX4 and molidustat may form complexes with metal ions present either on silica surface of the sorbent or column hardware. On the contrary, in molidustat glucuronide, the nitrogen atom responsible for chelation is covalently bound to the glucuronide moiety, and this is why its detection does not pose any challenges. To understand the extent of metal contamination in the LC part we added acetylacetone into mobile phase (Figure 2). Under these conditions, the total ion current was found to be dominated by iron complex, which seemed counterintuitive for a biocompatible LC. Titanium and aluminum were also th th MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3115 present but this was not unexpected as pump heads are made of Ti-Al alloy. Further, a complex formation between iron and IOX4 was confirmed revealing the true reason behind poor chromatography. We postulate that acetonitrile, capable of donating its π-electrons to metal atoms, interferes with complex formation and this is why the acetonitrile-based mobile phase showed better peak shape for metal-sensitive analytes. Figure 2: Volatile metal complexes with acetyl acetone (A-D) and IOX4 adduct with Fe(AcAc) (E) An adequate performance of IOX4 and molidustat was observed on a specially coated column which recently become available from Waters (Figure 3). Figure 3: Peak shape of IOX4 (A-C) and molidustat (D) on Premier HSS T3 column (both at 2 ng/mL, 329 > 273 and 315 > 207, ESI+, respectively), A: brand new column, B-C: precolumn replaced, first injection (B) and 96 injection (C) 2+ th MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3116 Conclusions Utilizing IOX4 and molidustat as exemplary analytes, we have demonstrated that a column with alkylsilane coating (recently introduced by Waters under Premier brand) enables reliable detection of metal-sensitive analytes even with a low ionic strength water-methanol mobile phase. A nearly symmetrical peak shape is consistently obtained which greatly improves the detection limits. Needless to say that using a biocompatible LC system is critical for optimal chromatography, but it is essential that all wetted parts (solvent filters, tubing, and the column) are iron-free or properly passivated. Even clear borosilicate glass bottles can leach out metal ions (particularly, iron) when exposed to acidic mobile phase and contribute to the non-specific binding of sensitive analytes. References 1. De Pra M, Greco G, Krajewski MP, Martin MM, George E, Bartsch N, Steiner F. (2020) Effects of titanium contamination caused by iron-free high-performance liquid chromatography systems on peak shape and retention of drugs with chelating properties. J Chromatogr A. 1611, 460619 2. Siegel D, Permentier H, Bischoff R. (2013) Controlling detrimental effects of metal cations in the quantification of energy metabolites via ultrahigh pressure-liquid chromatography-electrospray-tandem mass spectrometry by employing acetylacetone as a volatile eluent modifier. J Chromatogr A. 1294, 87-97 3. Asakawa Y, Tokida N, Ozawa C, Ishiba M, Tagaya O, Asakawa N. (2008) Suppression effects of carbonate on the interaction between stainless steel and phosphate groups of phosphate compounds in high- performance liquid chromatography and electrospray ionization mass spectrometry. J Chromatogr A. 1198- 1199, 80-86 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3117 Wicka M , Grucza K , Stanczyk D , Drapala A , Konarski P , Wojtkowiak K , Kaliszewski P , Kwiatkowska D Comparison of the use of the ESI ion source and UniSpray for the analysis of compounds prohibited in sport in urine samples Polish Anti-Doping Laboratory, Warsaw, Polska ; Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland Abstract Most of the analysis by means of mass spectrometry is conducted using an electrospray ion source (ESI). The presented study aimed to compare a typical ESI ion source and the new ESI ion source produced by Waters company marked as UniSpray . Both sources were used for the identification of psychoactive compounds and glucocorticoids in urine by means of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS). Instrumental analyses were performed in the Multiple Reaction Monitoring (MRM) mode with positive ion detection. In the course of the research, the ion intensity ranges for tested ion sources were compared. The obtained results of the area under peak values of the analyzed compounds for the selected MRM transition indicated that the UniSpray ion source caused a significant increase in sensitivity. Introduction The presented study aimed to compare the use of different ion sources for the analysis of psychoactive compounds and glucocorticoids in urine. The evaluation covered the comparison of ionization sources in terms of the analysis of the identified substances and the influence of ionization on the signal responses for individual compounds. Experimental The comparative analysis was performed using 7 or 10 urine of different pH, specific gravity, and sex. In all cases, the final concentration for analyzed samples were the same for the individual compounds. Sample pre-treatment The sample preparation is a two-step procedure involving enzymatic deconjugation of glucuronides and then liquid-liquid extraction with 6 mL of methyl tert-butyl ether. The residue was reconstituted in 100 μL of mobile phase (acetonitrile/water, 1/1, V/V), transferred in a vial and 2 μL or 5 μL was injected into the LC-MS/MS system, respectively (Figure 1). Instrumental analysis Chromatographic separation was conducted using a Waters Acquity I-Class UPLC System liquid chromatography with BEH C18 (1.7 μm, 100 mm x 2.1 mm) column from Waters. The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B), and the LC gradient 1 1 1 1 1 1,2 1 1 1 2 TM MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3118 was employed at the constant flow rate of 300 μL/min at 45°C. Multiple Reaction Monitoring (MRM) of the studied substances were traced with a Xevo TQ-XS mass spectrometer equipped with an UniSpray (Figure 2) and Electrospray source. All analytes were investigated in the US and ESI modes. Desolvation gas flow was set at 1000 L/h at 600°C for US mode, and at 800 L/h at 600°C for ESI mode, with ion source temperature at 150°C. The capillary voltage was 3.0 kV (Figure 1). Figure 1. Samples were analyzed following the illustrated approach Figure 2. Unispray source shown on the Xevo TQ-XS [Source: Waters] MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3119 Results and Discussion In the course of the research, two ion sources - electrospray and UniSpray - incorporated in UPLC/ Xevo TQ-XS system - were tested. A batch combining a set of samples at the same concentration levels of selected compounds was analyzed using both systems. Recorded ion intensity ranges for the analyzed substances (Table 1) and values of the area under peaks for the analyzed substances (Table 2) were compared Table 1. Comparison of the ion intensity range depending on the type of ion source used (*) - 7 different urine samples; (**) - 10 different urine samples, with various pH and specific gravities, over a time interval of 3 months; for the selected MRM transition NR – no response for the standard MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3120 Table 2. Summary of responses for the substances depending on the type ion source - 7 different urine samples, with various pH and specific gravities, over a time interval of 3 months NR – no response for the standard The novel US ionization source (UniSpray) was evaluated for mass spectrometric analysis by direct comparison to electrospray ionization (ESI). The US is an ionization technique that produces results qualitatively similar to electrospray. On the other hand, an increased desolvation and subsequent ion sampling in the source enhanced the signal for most of the compounds in this study. The signal intensity observed for the US source was higher than the ESI signal for all compounds analyzed. The exceptions are compounds that were not ionized with a particular source (1,4-DMPA, octopamine, and CP 47,497 C8- homolog MT – in case of ESI and DMBA in case of the US). The comparison of the two sources showed that approximately 70% of the compounds had higher peak area responses in the US compared to ESI (defined as the ratio of peak area). On the other hand, about 30% obtained a comparable answer. The change of the ion source had a noteworthy influence in the case of psychoactive substances. For them, 1 1 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3121 an increase in the peak area was observed. In the case of compounds classified as glucocorticoids, for the majority of those compounds, the US source was not important, because no significant difference was observed compared to ESI sources. In this case, the observed increase in sensitivity also necessitates the use of high-purity LC/MS reagents (water and acetonitrile), which in practice means an increase in the costs of analyses. Conclusions The precision of the results (measured by RSD) for most compounds did not exceed 15%. The results of the area under peaks values of the analyzed compounds for the selected MRM transitions were compared proving that the UniSpray ion source ensured a significant increase in sensitivity. More significant differences were observed for compounds from the group of stimulants, narcotics, and cannabinoids, and less for glucocorticoids. References 1. Hammond J., Sanig R., Kirk J., Wrona M. A comparative study of electrospray and UniSpray sources Using ACQUITY UPC2 . Waters . Application Note.May.2018 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3122 Wicka M , Grucza K , Stanczyk D , Drapala A , Konarski P , Wojtkowiak K , Burstein K , Kwiatkowska D Study of doping substance isomers – the possibility of their separation in human urine using LC-MS/MS Polish Anti-Doping Laboratory, Warsaw, Polska ; Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland ; Medical Department, Medical University of Warsaw, Warsaw, Poland Abstract The WADA List of Prohibited Substances and Methods comprises substances with isomers [1]. These include, but are not limited to, formoterol and salbutamol - beta2-agonists. Both of these compounds are drugs that widen the respiratory tract. Thus, the use of formoterol and salbutamol is subject to WADA restrictions. Clenbuterol, on the other hand, belongs to a group of drugs called sympathomimetics that stimulate the sympathetic nervous system. Clenbuterol consists of a racemic mixture of the two R-(-) and S-(+) enantiomers. The conducted research aimed to separate chiral isomers utilizing a liquid chromatography system coupled with mass spectrometry. All the compounds were separated on an Astec Chirobiotic T2 column and detected by a tandem mass spectrometer in multiple reaction monitoring mode. The technique used for the research turned out to be effective in separating the isomers of the tested compounds. Both the enantiomers of formoterol, salbutamol, and clenbuterol were appropriately separated as intended. Introduction It is well known that more than half of the drugs available on the pharmaceutical market are chiral substances. Moreover, in the majority of the cases, one of the enantiomers is a pharmaceutically active ingredient, e.g. the (+)-enantiomer of ibuprofen, which is anti-inflammatory while its (-)-enantiomer is inactive. Another example is penicillamine, the (+)-enantiomer of which has an anti-rheumatic effect, while its (-)-enantiomer is highly toxic [1]. The WADA Prohibited List includes substances classified as isomers, i.a. formoterol and salbutamol [2]. Both compounds belong to a class of drugs known as bronchodilators. Formoterol consists of (R,R)- and (S,S)-enantiomers, most commonly administered as a 50:50 racemic mixture. Currently it is available in some markets as an enantiopure chiral product consisting of (R,R)-formoterol (arformoterol). Salbutamol enantiomers may have different activity profiles. The R-isomer carries most of the therapeutically broncho-dilating effect, while the S-isomer induces hypersensitivity in the airways and is metabolized more slowly than the R-isomer. Clenbuterol, on the other hand, belongs to a group of drugs called sympathomimetics, that stimulate the sympathetic nervous system. Clenbuterol is composed of a racemic mixture of the two enantiomers, R-(-) and S-(+). The R-(-) is responsible for stimulating β2-receptors, while the S-(+) blocks the effect of the β1-receptors [3]. The bronchodilator effect of clenbuterol is much more potent than the effect of salbutamol. Zhou et al. developed a chiral LC-MS/MS method for determination of salbutamol enantiomers in human plasma 1 1 1 1 1 1,2 3 1 1 2 3 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3123 and urine [4]. These substances were separated on an Astec Chirobiotic T column while others (Jacobson et al.) investigated enantiomers of formoterol using the same Astec Chirobiotic T column [5]. Experimental All the compounds were separated on a chiral column and detected by a tandem mass spectrometer in multiple reaction monitoring mode. Respective MRM transitions for formoterol, salbutamol, and clenbuterol are presented in Table 1. Chemical and reagents Reference materials of R-(-)-clenbuterol and S-(+)-clenbuterol, formoterol fumarate (mixture of diastereomers), arformoterol tartare, (R)-albuterol HCl, albuterol-d , and formoterol-d were purchased from Toronto Research Chemicals (TRC, Toronto, Canada). Formoterol fumarate and salbutamol were ordered from the National Measurement Institute (NMI, Camberra, Australia). Beta-glucuronidase from E.coli was purchased from Roche Diagnostic (Bazylea, Switzerland). Solvents were from Fisher Chemical (Hampton, USA). All reagents were of analytical grade. Sample pre-treatment The sample preparation is a two-step procedure involving enzymatic deconjugation of glucuronides and then liquid-liquid extraction with 6 mL of methyl tert-butyl ether. The residue was reconstituted in 100 μL of mobile phase (acetonitrile/water 1/1 V/V), transferred in a vial and 10 μL was injected into the LC- MS/MS system. Instrumental analysis Chromatographic separation was conducted using a Waters Acquity I-Class UPLC System liquid chromatography with Astec CHIROBIOTIC T2 chiral column (5 μm, 250 mm x 4.6 mm) from Sigma- Aldrich. The mobile phase consisted of 0.1% formic acid and 5 mM ammonium formate in 100% methanol. Elution was isocratic for 15 minutes. The flow rate was 0.6 mL/ min at 45 °C (Waters Column Heater Module). MRMs of the studied substances were traced with a Xevo TQ-XS mass spectrometer equipped with an Electrospray source. All analytes were investigated in the ESI+ mode. Desolvation gas flow was set at 950 L/h at 450 °C, with ion source temperature at 150 °C. The capillary voltage was 3.0 kV. Table 1. The MRM transitions for LC-MS/MS detection 3 6 ® MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3124 Results and Discussion The separations of tested substances were achieved with the chiral column using as the mobile phase of methanol containing 5 mM ammonium formate with 0.1% formic acid. Acetonitrile was also studied for elution, but the best separation was obtained using 100% methanol as the mobile phase. Formoterol enantiomers were satisfactorily separated (Figure 1.A). Figure 1.B demonstrates that the background sample matrix baseline from a blank urine was flow and flat. Figure 1. A Chromatogram of formoterol enantiomers in QC sample (10 ng/mL); B Exemplary UPLC-MS/MS chromatogram of real case sample of formoterol in urine overlaying a blank urine sample Figure 2.A shows the chromatogram obtained for a racemic mixture of R- and S-enantiomers of salbutamol. Different responses for enantiomers when analyzing urine spiked with the racemic solution of salbutamol were observed. This indicates that some features of the method can affect the reaction such as the matrix effect by ion suppression. When analyzed with the same racemic standard without matrix (urine) the responses for both enantiomers are equal (data not shown). Figure 2.B presents the absence of interferences for selected ionic transitions, which proves their specificity in the real case sample of salbutamol in urine. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3125 Figure 2. A.1 MRM chromatogram of urine spiked with salbutamol enantiomers (500 ng/mL); A.2 MRM chromatogram of urine spiked with R-salbutamol (500 ng/mL) - above, and salbutamol enantiomers (500 ng/mL) - below; B Exemplary UPLC-MS/MS chromatogram of real case sample of salbutamol in urine overlaying a blank urine sample The same was observed for the remaining analyzed compound (Figure 3.A and 3.B). Figure 3. A Chromatogram of clenbuterol enantiomers (0.2 ng/mL); B Exemplary UPLC-MS/MS chromatogram of real case sample of clenbuterol in urine overlaying a blank urine sample As preparations available on the market typically contain enantiomers, the developed method of separating isomers for doping purposes could be very useful. It is known that there is an ongoing debate about the use of beta 2-agonists under anti-doping laws concerning their performance-enhancing effects. The main concern is how to balance the need to treat asthma and exercise-induced bronchoconstriction (EIB) while minimizing the potential for doping. The presented research can be extended for other compounds, e.g. the possibility of separating clomiphene isomers can be checked. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3126 Conclusions 1. The applied technique with the selected chromatographic conditions used for the research proved to be effective in separating the isomers of the tested compounds. 2. The enantiomers of formoterol, salbutamol and clenbuterol were appropriately separated, as intended. 3. Both formoterol and salbutamol have been shown to exist in medicinal preparations as a mixture of enantiomers. 4. After testing the actual urine samples following the intake of preparations containing formoterol and salbutamol, neither albuterol nor levosalbutamol was detected. References 1. Reddy I., Mehvar R. Chirality in drug design and development. E-book, 2004 2. The Wada Prohibited List 2022, www.wada-ama.org 3. Velasco-Bejarano B., Bautista J., Noguez M.O., Camacho E., Rodriguez M.E., Rodriguez L. Resolution of R-(-) and S-(+) – enentiomers of clenbuterol in pharmaceutical preparations and black-market products using liquid chromatography-tandem mass spectrometry. Drug Test Anal. 2017, 9, 1738-1743. 4. Zhou T., Zeng J., Liu S., Zhao T., Wu J., Lai W., He M., Xu B., Qu S., Xu L., Tan W. Study on the determination and chiral inversion of R-salbutamol in human plasma and urine by liquid chromatography-tandem mass spectrometry. J Chrom B. 1002 (2015) 218-227. 5. Jacobson G.A, Hostrup M., Narkowicz Ch., Nichols D.S, Walters E. H. Enantioselective disposition of (R, R)- formoterol, (S, S)-formoterol and their respective glucuronides in urine following single inhaled dosing and application to doping control, Drug Test Anal. 2019; 11: 950-956. Acknowledgements Financial support from the Ministry of Culture, National Heritage,and Sport of the Republic of Poland under the project number 2021.0418/1575/UDOT/DS./14/AMW is gratefully acknowledged. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3127 Aquino R, Anselmo C, Sardela V, Pereira H Development of an LC-HRMS/MS approach for the detection of ethylmorphine and its metabolites. Comparison of different acquisition methods aiming untargeted analysis Chemistry Institute, Brazilian Doping Control Laboratory - LBCD, Rio de Janeiro, Brazil Abstract Narcotic opioids are included in the WADA’s List, being prohibited in-competition only. Morphine, unlike its 3-ethoxy derivative ethylmorphine, is one of the prohibited opioids. The classic metabolism of ethylmorphine has morphine and norethylmorphine as primary phase 1 metabolites. Zebrafish water tank (ZWT) is an in vivo experimental model used qualitatively for the study of doping agents of different classes. This work aimed to study the metabolism of ethylmorphine using ZWT. LC-HRMS (ESI+) was applied to investigate the ethylmorphine metabolites produced by ZWT. The experiment was performed in triplicate with negative control and stability control. Samples were aliquoted every hour during the experiment, which lasted 12 h. Collected aliquots were extracted in SPE, and dilute-and-shoot was used in parallel. At the end of the experiment, the fish were euthanized and the blood was collected for analysis by LC-HRMS. Three different acquisition methods were used to investigate the ZWT samples. Three classic ethylmorphine metabolites were identified using IDCR criteria, including morphine and norethylmorphine. The phase 2 metabolite found was morphine 3β-glucuronide. Morphine and morphine 3β-glucuronide were also observed in the blood. The comparison of the two untargeted acquisition methods (DDA and SWATH-type DIA) using PRM as a reference indicated that both are able to identify characteristic fragments of ethylmorphine and its metabolites, but the SWATH-type DIA has higher sensitivity. The results demonstrated that zebrafish are capable of producing ethylmorphine metabolites and that SWATH-type DIA has interesting potential to be used in metabolomics. Introduction Ethylmorphine is a 3-ethoxy derivative from morphine used as antitussive and analgesic. Differently from morphine, the administration of this opioid by athletes is allowed. In humans, ethylmorphine is metabolized to norethylmorphine and morphine via N-demethylation and O-deethylation pathways, respectively, and can be followed by O-glucuronidation of the parent and its metabolites. Different in vitro and in vivo models have been used to study the ethylmorphine metabolism. Zebrafish Water Tank (ZWT) is an in vivo experimental model that has already been successfully applied with different substance classes, such as anabolic steroids, cannabimimetics and stimulants. In this work, the metabolism of ethylmorphine was investigated applying ZWT model with the novelty of analyzing not only the tank water, but also the fish's blood. Experimental MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3128 This study applied liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) to investigate the ethylmorphine metabolites produced by ZWT. ZWT model details were described in a previous study of the group. Briefly, experiments were performed with eight fish placed in 200 mL glass tanks fitted with a heating device set to 32 ± 0.5 °C for 12 h, in triplicate. Samples were aliquoted every hour until the final of the experiment. Ethylmorphine standard in methanol was evaporated and resuspended into ultrapure water to a tank final concentration of 1 μ g/mL. The negative control was performed with the fish without ethylmorphine, and the stability control with the presence of only ethylmorphine in the tanks throughout the experiment. After 12 h, the fish were euthanized and blood was collected through a cut in the anal fin. ZWT samples were fortified with internal standard and treated by solid-phase extraction (SPE) using a weak cation exchange cartridge and in parallel the dilute and shoot technique, followed by analysis with LC-HRMS/MS (ESI+). Blood was extracted using methanol fortified with internal standard and centrifuged at high speed to separate the supernatant, followed by evaporation and resuspension in the mobile phase, which was then analyzed by LC-HRMS/MS (ESI+). Regarding MS/MS analysis three different strategies were applied: (i) Parallel Reaction Monitoring analysis (PRM), (ii) Data-dependent Acquisition (DDA), and (iii) Sequential Window Acquisition of All Theoretical Fragment Ions Data Independent Acquisition (SWATH-type DIA). Results and Discussion In the ZWT samples, three metabolites common to the human model were identified by IDCR and reference standards: norethylmorphine (N-demethylation), morphine (O-deethylation) and morphine 3β- glucuronide (O-deethylation + O-glucuronidation). Both morphine and its glucuronide metabolite were also observed in zebrafish blood. Due to the fact that zebrafish do not have an ortholog to the CYP2D family, which is responsible for carrying out mainly O-deethylation, the identification of morphine as an ethylmorphine zebrafish metabolite was interesting. This can be explained because, despite the lack of CYP2D, other enzymes from the vast enzymatic machinery of zebrafish can participate in this reaction, even if in smaller proportions. Glycoconjugate metabolites at position 6 were not detected. The literature already describes a lack of glycoconjugate at position 6 for the in vitro rat hepatocyte model, which signals a similarity between rat and zebrafish. DDA acquisition was compared to SWATH-type DIA based on the spectra obtained by PRM for ethylmorphine and its metabolites. Some characteristic fragments of ethylmorphine metabolites, such as m/z 165.06988, 183.08044 and 201.09101 (error 167.17, CE 15 eV. The desolvation gas flow was set at 800 L/h at 500 °C and the source temperature was 150 °C. The applied capillary voltage was 3.0 kV. The cone and collision gas flows were set at 150 L/h and 0.20 mL/min, respectively. All data were acquired and processed using the MassLynx™ software version 4.1 SCN905 (Waters, Milford, MA, USA). Results and Discussion The results obtained from the Initial Testing Procedure (ITP) of athletes and quality control (QC) samples are shown in Figure 1. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3132 Figure 1. Chromatograms of diphenhydramine (TIC) from Initial Testing Procedure. A. Blank urine; B. Athlete`s sample and C. QC sample of diphenhydramine (25 ng/mL) The retention time (RT) of DPH in both athlete`s and QC samples was 4.34 minutes. While monitoring MRM for DPH, an additional peak with shorter RT (4.04 minutes) was observed. Parent scan experiments revealed that the difference in m/z corresponded to a glucuronic acid conjugate. Experimentally, two MRMs for this conjugate were obtained, where the glucuronic acid conjugate was dissociated into its parent compound and a second ion, with a different collision energy setup (Figure 2). MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3133 Figure 2. Metabolite identification by determination of the m/z ratio of the compound of interest and its main fragments. MS Scan analysis showed that product ion 167.03 is about 2 times more intense than product ion 256.07. Cone voltage (CV) was set at 30 V with the following selected precursor ion-product ion transition at their respective collision energy (CE): m/z 432.17 > 167.03, CE 25 eV and m/z 432.17 > 256.07, CE 15 eV. DPH was observed occasionally in routine doping control analysis. However, every time both parent compound and metabolite were observed. An exemplary chromatogram of an athlete`s urine sample presented both compounds were shown in Figure 3. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3134 Figure 3. Chromatograms of an athlete urine from the Initial Testing Procedure prepared with the DaS method Conclusions The results from this study show that diphenhydramine is excreted in the unchanged form and as a glucuronide conjugate, so monitoring of this conjugate seems justified. Monitoring of DPH is necessary during the confirmatory analysis of modafinil and its metabolite modafinil acid. It should be noted, that both compounds fragment into the diphenyl carbinol group: m/z 274 > 167 and 275 > 167, respectively. This situation is also observed during diphenhydramine analysis. The presented poster draws the attention of the Laboratories, that diphenhydramine glucuronide should be monitored during doping control analysis to correct the interpretation of results. One should be wary, because the situation while the only metabolite will be present in the sample may be observed in routine analysis. As above, a detailed excretion study of diphenhydramine seems to be necessary for the future. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3135 References 1. Devrim B., Dinc E. and Bozkir A. Fast determination of diphenhydramine hydrochloride in reconstitutable syrups by CWT, PLS and PCR methods. Acta Poloniae Pharmaceutica Drug Research, Vol. 71 No. 5 pp. 721n729, 2014. 2. Gelotte CK, Brenda A. Zimmerman BA. and Thompson GA. Single-Dose Pharmacokinetic Study of Diphenhydramine HCl in Children and Adolescents. Clinical Pharmacology in Drug Development 2018, 7(4) 400–407. 3. World Anti-Doping Agency (WADA). WADA2022 Prohibited List. Available online: https://www.wada- ama.org/sites/default/files/2022-01/2022list_final_en_0.pdf (accessed on 9 March 2022). Acknowledgements Financial support from the Ministry of Culture, National Heritage and Sport of the Republic of Poland under project number 2021.0416/1575/Udot/DS./14/AM are gratefully acknowledged. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3136 Constantinescu G, Pop A, Berghes B, Radu M, Penu R, Cristea C, Catana D, Toboc A, Stan C Three months screening for chlorphenesin Romanian Doping Control Laboratory, Bucharest, Romania Abstract Meclofenoxate is a prohibited substance for athletes. The main product after administration of meclofenoxate is 4-chlorophenoxyacetic acid (4-CPA). 4-CPA may be also present in human urine after ingestion of food contaminated with chlorinated phenoxy acid herbicides or as a metabolite after oral or transdermal administration of other non-prohibited substances like chlorphenesin which is used as preservative with biocidal properties in a wide range of cosmetic products and chlorphenesin carbamate which is a muscle relaxant. The main metabolites of chlorphenesin are 3-(4-chlorophenoxy)-2- hydroxypropanoic acid (4-CPP), chlorphenesin glucuronide and chlorphenesin sulfate. Meclofenoxate, 4-CPA and 4-CPP are screened on LC-MS/MS. Chlorphenesin and chlorphenesin carbamate were added to the screening method on GC-MS/MS. It was proved that it is impossible to differentiate between them during screening. Chlorphenesin or chlorphenesin carbamate, 4-CPA and 4- CPP were identified in 33 out of 380 samples from the beginning of screening for CF. All these substances had concentrations below 2.1 µg/mL. Meclofenoxate was not detected in these samples. Introduction Meclofenoxate is a psychostimulant prohibited to be used by athletes during competition [1]. It is rapidly decomposed by spontaneous and enzyme-induced hydrolysis [2]. The main target metabolite for meclofenoxate is 4-chlorophenoxy acetic acid (4-CPA). Since 2006 it was known that 4-CPA is used as herbicide and growth regulator for plants and is considered to be absorbed and eliminated rapidly unchanged in urine [3]. Chlorphenesin is a preservative used in many cosmetic products at concentrations up to 0,32% in rinse-off products and at concentrations up to 0,3% in leave-on products [4]. Chlorphenesin carbamate is a muscle relaxant [5]. The purpose of this study was to offer a glance on CF presence and concentration level in anti-doping urine samples after 3 months of screeening. Experimental Chemical structures of meclofenoxate (1), 4-CPA (2), chlorphenesin (3), chlorphenesin carbamate (4) and 4-CPP (5) are presented in Figure 1. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3137 Figure 1. Chemical structures of meclofenoxate (1), 4-CPA (2), chlorphenesin (3), chlorphenesin carbamate (4) and 4-CPP (5) Reagents β-Glucuronidase from E.coli was purchased from Roche Diagnostics Division. Tert-butyl-methyl-ether, MSTFA, NH I, and ethanethiol from Merck. Chlorphenesin carbamate was purchased from LGC, chlorphenesin from Dr. Ehrenstorfer, 4-CPA from Sigma-Aldrich. 4-CPP was a gift from Cologne Laboratory. Instrumentation GC-MS/MS: The screening was performed on Thermo Scientific systems. The GC column was an HP-Ultra 1 from J&W Scientific. The transfer line temperature was set at 310 ºC. Helium 6.0 carrier gas had a flow rate of 1.18 mL/min. 2 µL of derivatized sample were injected in split mode (1:10) into GC. The acquisition was made in SRM mode. Transitions monitored: 346-200, 346-129, 346-103. LC-MS/MS: 6410 LC Agilent coupled with AB SCIEX 5500 QTrap. LC was equipped with a precolumn SecurityGuard ULTRA Cartridges UHPLC C18 and a Zorbax column SB-C18, 5 µm particle size. Solvent A: 5 mM HN -COOH with 1‰ H-COOH in water and solvent B: 5mM HN -COOH with 1‰ H-COOH in 90% acetonitrile + 10% water. The flow rate was 0.25 mL/min. Overall runtime was 14 min/injection. The mass spectrometer was operated in MRM ESI negative mode. Transitions monitored: 4-CPA: 185-127, 185-35; 4-CPP: 215-127, 215-35; meclofenoxate: 258-72, 258-213. Samples Chlorphenesin or chlorphenesin carbamate (CF) were identified in 33 out of 380 samples. In these 33 samples 4-CPA and 4-CPP were also detected. Sample preparation GC-MS/MS: 2 mL aliquot of urine sample, hydrolysis with β-glucuronidase from E.coli at 50 °C for 90 min, extraction in 5 mL tert-butyl-methyl-ether, evaporated under oxygen-free nitrogen at 40 °C. Derivatization: 100 µL of MSTFA/NH I/Ethanethiol (1000/2/3; v/w/v) [6]. LC-MS/MS: 1 mL aliquot of urine sample, centrifuged for 20 min at 6000 rpm. 40 µL from supernatant were transferred to a vial. 40 µL solvent A and 20 µL internal standard mefruside were added. 4 4 4 4 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3138 Results and Discussion In order to detect chlorphenesin and chlorphenesin carbamate in the samples they were added to the screening method developed on GC-MS/MS mainly for steroids. The spectrum of chlorphenesin (Figure 2) corresponds to the one in the literature [7]. Chlorphenesin and chlorphenesin carbamate give similar spectra (Figure 2-3) at the same retention time. This can be due to posible degradation of the carbamate in the injector. Figure 2. Chlorphenesin spectrum Figure 3. Chlorphenesin carbamate spectrum CF were identified in 33 out of these 380 samples during 3 months of screening, by GC-MS/MS. In these 33 samples 4-CPA and 4-CPP were also detected by LC-MS/MS. Meclofenoxate was not present in any of these samples. The estimation of concentrations was done against 1 calibration point and the values were not SG adjusted. For all 33 samples, the concentration for 4-CPA was below 5 µg/mL (Figure 4). In almost half of the samples which contained CF, 4-CPA and 4-CPP the concentrations were < 10 ng/mL for each of these compounds. Only one sample presented concentration of 4-CPA > 1 µg/mL. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3139 Figure 4. Obtained results for CF, 4-CPA and 4-CPP MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3140 There were only 8 samples where the concentration of 4-CPA exceeds the concentration of 4-CPP. In all of these cases the difference was less than three times the concentration of 4-CPP and the concentration of 4-CPP was greater than the concentration of CF for all these samples. This suggests that 4-CPA was formed only during the CF metabolism. Conclusions Chlorphenesin and chlorphenesin carbamate spectra are identical and they present the same retention time, so a rigurous distinction between these two compounds cannot be done. CF, 4-CPA and 4-CPP were observed together in close to 10% of the samples analysed during three months. In almost half of the samples which contained CF, 4-CPA and 4-CPP the concentrations were 1 µg/mL. The resulted concentrations for 4-CPA are well below than 5 µg/mL, concentration specified in TL01 [8]. References 1. World Anti-Doping Agency. WADA Technical Letter - TL01. (2021) https://www.wada- ama.org/sites/default/files/2022-01/tl01_meclofenoxate_v4.0.pdf (acces date 24.08.2021) 2. Yoshioka S, Aso Y, Uchiyama M. (1987) Kinetics of hydrolysis of meclofenoxate hydrochloride in human plasma. J. Pharm. Pharmacol. (39), 215-218 3. Guddat S, Sigmund G, Thomas A, Opfermann G, Thevis M, Schanzer W. (2006) Detection of Meclofenoxate and its degradation products dymethylaminoethanol and p-chloro-phenoxyacetic acid. In: Geyer H, Gotzamnn A, Mareck-Engelke (eds.) Recent Advances in Doping Analysis (14). Sport und Buch Strauß Schanzer W., Koln, 399-402. 4. Johnson W, Bergfeld W F, Belsito D V, Hill R A, Klaassen C D, Liebler D C, Marks J G, Shank R C, Slaga T J, Snyder P W, Andersen F A. (2014) Safety Assessment of Chlorphenesin. Int. J Toxicol. 33 (2), 5S-15S 5. Kurachi M, Aihara H. (1984) Effect of a Muscle Relaxant, Chlorphenesin Carbamate, on the Spinal Neurons of Rats. J. Pharmacol. Japan. 36, 7-11 6. Danila G M, Pop A M, Stan C, Toboc A. (2020) 7-keto-DHEA - a case study. In: Geyer H, Thevis M, Mareck U (eds.) Recent Advances in Doping Analysis (28). Sportverlag Strauß-Hellenthal, Koln, 85-89. 7. Rubio A, Görgens C, Krug O, Okano M, Fedoruk M, Ahrens B, Geyer H, Thevis M. (2021) Chromatographic- mass spectrometric analysis of the urinary metabolite profile of chlorphenesin observed after dermal application of chlorphenesin-containing sunscreen. Rapid Commun. Mass Spectrom. 35 (21). 8. World Anti-Doping Agency. The 2022 Prohibited List. International Standard, Montreal (2022) https://www.wada-ama.org/sites/default/files/2022-01/2022list_final_en_0.pdf (access date 10.01.2022) MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3141 Athanasiadou I, Tsivou M, Gmeiner G Data mining of HIF activating agents Doping Control Laboratory, Seibersdorf Labor GmbH, Seibersdorf, Austria Abstract Retrospective evaluation involves the re-processing of the results originally obtained on the samples (including those that may have originally been reported as negative), targeting the possible presence of prohibited substances that were unknown at the time of the original analysis without requiring additional sample preparation. The possibility of monitoring a huge amount of data opens the way to ‘omic’ approaches, where the alteration of specific biomarkers/biomarker profiles might be used as proof of a doping offense. In this context, a Compound Database and a Master Method were created, using the Tracefinder software, for retrospectively analysing data from routine doping control samples focusing on HIFs (class S2.1.2 of the WADA Prohibited List). A total of 18.844 data files were reprocessed corresponding to 54% in competition samples and 65% male samples. No suspicious findings indicating the misuse of HIFs were detected from the reprocessed samples analysed in the Seibersdorf Lab during the year 2018 and beginning 2019. Introduction Hypoxia-inducible factor (HIF) activating agents are prohibited in- and out-of-competition according to WADA Prohibited List (class S2.1.2) [1]. Since 2021, due to the high risk of doping with this category of substances, WADA accredited laboratories were advised to include in their testing menu six additional HIF activating agents, namely: IOX2, desidustat (ZYAN1), enarodustat (JTZ-951), IOX3 (FG-2216), IOX4, and JNJ-42041935. In 2019, as an outcome of the Operation Aderlass investigation, it was disclosed that during the preparation of endurance sports events, a powder mixture of HIFs was misused including molidustat (BAY 85-3934), roxadustat (FG-4592), daprodustat (GSK1278863) to get high reticulocyte numbers. In this context, retrospective analysis, targeting HIFs, was conducted from data originally obtained in the year 2018 and beginning of 2019 (including the 41 FIS Seefeld Nordic World Ski Championships’ period). Experimental Retrospective analysis using Trace-Finder software An extensive literature review was conducted for gathering analytical information available for HIFs (transitions, collision energy, etc.) [2-4] as shown in Table 1. As proof of concept for the Master Method created, samples from WADA blind (2021) and educational HIF rounds (2019, 2021) were reprocessed. st MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3142 Retrospective analysis was performed in 18.844 data files originally obtained from the initial routine doping control analysis of urine samples during the year 2018 including the first two months of 2019 (41 FIS Seefeld Nordic World Ski Championships’ period). Original urine samples from the above mentioned period were analyzed using the routine analytical method based on a full scan liquid chromatography-mass spectrometry method. A statistical evaluation based on the athlete`s gender, the type of competition and the sport was also conducted to obtain a snapshot of reprocessed data files in the athlete’s population. Table 1. Mass spectrometric info for the under-investigation analytes Results and Discussion As depicted in Figure 1, all the analytes present in WADA’s rounds were successfully detected. st MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3143 Figure 1. Chromatograms resulting from the reprocessing Master Method when applied to the WADA blind (2021) and educational samples (2019, 2020) The 18.844 reprocessed data files were distributed at 46% in out of competition (OC) samples and 54% in competition (IC) samples (Figure 2a). Regarding the gender distribution of the findings (Figure 2b), 65% in males and 35% in females were observed. Graphic distribution of samples based on the sport is presented at Figure 2c showing winter sports accounted for 14% of the reprocessed samples where HIFs analytes are most common to be detected during the preparation of endurance sports events. The reprocessed results from the samples analysed in the Seibersdorf Lab during the year 2018 and beginning 2019, showed no suspicious findings indicating the misuse of HIFs. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3144 Figure 2. Sample distribution according to type of competition (2a), gender (2b) and sport (2c) Conclusions No suspicious findings suggesting the misuse of HIFs during the year 2018 have been detected. The current retrospective approach can be extended in other classes of prohibited substances revealing useful information regarding the time of abuse of substances that are circulated in the black market while they are still unknown in the Anti-Doping community. The only limitation that can be referred is the large size of the raw data files usually reaching approximately 1 Terabyte. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3145 References 1. 2022list_final_en_0.pdf (wada-ama.org) 2. Dib J, Mongongu C, Buisson C, Molina A, Schänzer W, Thuss U, Thevis M. Mass spectrometric characterization of the hypoxia-inducible factor (HIF) stabilizer drug candidate BAY 85-3934 (molidustat) and its glucuronidated metabolite BAY-348, and their implementation into routine doping controls. Drug Test Anal. 2017 Jan;9(1):61-67. doi: 10.1002/dta.2011. 3. Görgens C, Guddat S, Bosse C, Knoop A, Geyer H, Thevis M. Implementation of the HIF activator IOX-2 in routine doping controls - Pilot study data. Drug Test Anal. 2020 Nov;12(11-12):1614-1619. doi: 10.1002/dta.2914. 4. Mazzarino M, Perretti I, Stacchini C, Comunità F, de la Torre X, Botrè F. UPLC-MS-Based Procedures to Detect Prolyl-Hydroxylase Inhibitors of HIF in Urine. J Anal Toxicol. 2021 Feb 13;45(2):184-194. doi: 10.1093/jat/bkaa055. Acknowledgements The current work was supported by the Austrian Ministry of Sports. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3146 Görgens C , Guddat S , Thevis M Gas-phase water adduct formation of IOX-2 in ion trap mass spectrometers Center for Preventive Doping Research/Institute of Biochemistry, German Sport University Cologne, Cologne, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract Since 2020, the HIF-activator IOX-2, bearing a quinoline-3-carboxamidic chemical structure, is implemented in routine doping control. Comparative analyses using tandem-in-time versus tandem-in- space mass spectrometry demonstrate significant differences in the product ion mass sepctra of the molecule, according to a potential gas-phase water adduct formation using ion trap mass spectro- metry. In accordance to previous investigations of structurally related HIF-activators, a dissociation pathway of the protonated IOX-2 molecule was proposed, including a loss of methylene-amine (-29 u) and a concomitant reversible water addition (+18 u) to the resulting acylium ion (m/z 278 ⇔ m/z 296). Moreover, the study demonstrates that the proportion of the water adduct increases if prolonged dwell times are set, while this penomenon was not observed using triple quadrupole mass spectrometry. Confirmation analysis of IOX-2 suspicious urine samples could therefore benefit from being performed using triple quadrupole mass spectrometry. Otherwise, using ion trap mass spectrometry, the product ions at m/z 278 and m/z 296 might complicate the substance’s identification using WADA TD2021IDCR. Introduction In 2020, the implementation of the HIF activator IOX-2 in routine doping controls using a combined mass spectrometric detection with roxadustat (FG-4592) and their hydroxylated metabolites was presented for the first time. As roxadustat and IOX-2 exhibit identical sum formulae of C H N O , and the fact that the structurally related pharmacophores both comprise a glycineamide side chain, the substances share similar collision-induced dissociation behaviors. [1] During method validation of the IOX-2 confirmation procedure using orbitrap mass spectrometry, ratios of the most characteristic product ions m/z 278 and m/z 296 demonstrated unexpected variabilities when analyzing different urine specimens. This unusual mass spectrometric dissociation behavior was not observed using triple quadrupole mass spectrometers, suggesting that the phenomenon occurs only in tandem-in-time and not in tandem-in-space mass spectrometry. The aim of the study was to further investigate the aforementioned observation and to propose a conclusive dissociation pathway of the protonated IOX-2 molecule in accordance with the well investigated dissociation behavior of structurally related protonated isoquinoline-3-carboxamides. [2-4] 1 1 1,2 1 2 19 16 2 5 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3147 Experimental Analytical parameters of IOX-2 (obtained from Sigma Aldrich, Deisendorf, Germany) were determined using established routine doping control methods reported elsewhere [1]. In brief, an aliquot of 90 µL of urine sample was fortified with 10 µL of an internal standard (IS) working solution (isoxsuprine-D ). 10 µL of the mixture was directly injected into the instrument, composed of a Vanquish UHPLC System (Thermo Scientific, Bremen, Germany) equipped with a Nucleodur C18 Pyramide analytical column (2 x 50 mm, 1.8 μm particle size; Macherey-Nagel, Düren, Germany) and an Exploris 480 orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) for mass spectrometric detection in Parallel Reaction Monitoring (PRM) acquisition mode. Since the filling time of the c-trap is a dynamic parameter, depending on the number of ions entering the ion trap, product ion mass spectra were investigated and compared after multiple injections of an IOX- 2 reference standard, varying the maximum injection time, while maintaining a constant automatic gain control (AGC) target of 5e , resulting in actual dwell times between 10 and 500 ms. Results and Discussion The investigations demonstrated a clear correlation between injection time and shift of the product ion ratio from 1/1 to 1/5 (m/z 278 / m/z 296), suggesting a gas-phase water adduct formation in the c-trap of the mass spectrometer. The proportion of the water adduct increases, the longer the actual dwell time in the c-trap is set (Figure 1). A potential dissociation pathway, based on investigations of structurally related protonated isoquinoline-3-carboxamides is demonstrated in Figure 2. After the elimination of carbon monoxide (- 28 u) and water (-18 u) from the protonated molecular ion, the nominal loss of 11 u is explained by a loss of methyleneamine (-29 u) and a concomitant and reversible water addition (+18 u) to the resulting acylium ion, yielding to the protonated 1-benzyl-3-carboxy-4-hydroxy-2-oxo-1,2- dihydroquinoline (m/z 296.0917)[2-4]. 5 TM TM TM TM 6 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3148 Figure 1. Comparison of IOX-2 (m/z 353.1132) product ion mass spectra using a constant AGC target set to 5e and a variable maximum injection time of a) 10 ms, b) 25 ms, c) 50 ms, d) 100 ms, e) 200 ms and f) 500 ms, demonstrating a significant correlation between the injection time and the shift of the product ion ratio between m/z 278 and m/z 296 towards the water adduct 6 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3149 Figure 2. Proposed dissociation pathway of the protonated IOX-2 molecule including a reversable gas-phase addition of water in ion trap mass spectrometers Conclusions Using tandem-in-time mass spectrometry, an unusual collision-induced dissociation behavior of IOX- 2 was observed, resulting from a gas-phase water adduct formation in the ion trap. It was demonstrated that the water adduct formation can be enforced by increasing the filling time of the trap. Therefore, confirmation analysis of IOX-2 suspicious urine samples could benefit from being performed using triple quadrupole mass spectrometry. Otherwise, using ion trap mass spectrometry, the product ions at m/z 278 and m/z 296 might complicate the substance’s identification using WADA TD2021IDCR. References 1. Görgens C, Guddat S, Bosse C, Knoop A, Geyer H, Thevis M, Implementation of the HIF activator IOX-2 in routine doping controls – Pilot study data. Drug Test. Anal. 2020;12(11-12): 1614-1619. 2. Thevis M, Kohler M, Schlörer N, Schänzer W. Gas Phase Reaction of Substituted Isoquinolines to Carboxylic Acids in Ion Trap and Triple Quadrupole Mass Spectrometers after Electrospray Ionization and Collision- Induced Dissociation. J. Am. Soc. Mass. Spectrom. 2008; 19: 151–158. 3. Beuck S, Schwabe T, Grimme S, Schlörer N, Kamber M, Schänzer W, Thevis M. Unusual Mass Spectrometric Dissociation Pathway of Protonated Isoquinoline-3-Carboxamides Due to Multiple Reversible Water Adduct Formation in the Gas Phase. J. Am. Soc. Mass. Spectrom. 2009; 20: 2034–2048. 4. Beuck S, Schänzer W, Thevis M, Hypoxia-inducible factor stabilizers and other small-molecule erythropoiesis-stimulating agents in current and preventive doping analysis. Drug Test. Anal. 2012;4(11): 830-845. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3150 Acknowledgements The presented work was conducted with support of the Manfred Donike Institute for Doping Analysis e.V. (Cologne, Germany) and the Federal Ministry of the Interior, Building and Community (Berlin, Germany). MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3151 Guddat S , Görgens C , Delahaut P , Sobolevsky T , Thevis M Follow-up: meldonium contamination in milk – a possible scenario for inadvertent doping in sports? Institute of Biochemistry and Center for Preventive Doping Research, German Sport University, Cologne, Germany ; CER Group, Marloie, Belgium ; Department of Pathology and Laboratory Medicine, Olympic Analytical Laboratory, UCLA David Geffen School of Medicine, Los Angeles, USA Abstract Recently, the possibility of the veterinary drug Emidonol as a potential origin for inadvertent doping in connection with the prohibited substance meldonium was described. Emidonol is approved in some eastern European countries for livestock breeding. As a follow-up, an exploratory Emidonol administration study was conducted to obtain first data on meldonium concentrations in milk. Therefore, one cow received 2 x 40 mL/day of Emidonol (10% solution) on 2 consecutive days. Milk was collected and analysed using an established protocol based on HILIC-HRMS/MS, slightly adapted to accommodate the milk matrix. Investigated Emidonol post-administration milk samples demonstrated meldonium concentrations in the µg/mL-range. Compared to earlier data, concentrations in milk were 10-fold higher and, thus, representing a realistic scenario for inadvertent doping in sports. Introduction The veterinary drug Emidonol (3-(2,2,2-trimethylhydrazinium)propionate-2-ethyl-6-methyl-3-hydroxy- pyridine disuccinate) has been debated in the context of inadvertent doping in connection with the prohibited substance meldonium [1]. Emidonol is approved in some eastern European countries for livestock breeding, e.g. for the treatment of cows. In animals, the drug rapidly dissociates into meldonium and emoxypine. Recently, Temerdashev et al. investigated raw milk of cows treated with Emidonol and determined maximum meldonium levels of up to approx. 700 ng/mL [2]. To investigate if such a contamination scenario may result in adverse analytical findings in sports drug testing, a pilot meldonium excretion study was conducted. Here, maximum urinary meldonium concentrations of 7.5 ng/mL after a single oral dose (50 μg) and 18.6 ng/mL after repeated doses (5 x 50 μg) were observed within 2-6 hours after spiked milk consumption. The applicable reporting level for meldonium of 100 ng/mL was not exceeded, nevertheless, the consumption of larger amounts of contaminated milk may create a possible risk for inadvertent doping in sports. As a follow-up, a pilot Emidonol administration study was conducted to obtain more data on meldonium concentrations and metabolic products in milk. Therefore, one cow (BW 720 kg) received 2 x 40 mL/day of Emidonol (10% solution) on 2 consecutive days (Fig. 1). Milk samples were analyzed using a well-established protocol based on HILIC-HRMS/MS, slightly adapted for the analysis of milk samples. 1 1 2 3 1 1 2 3 ® ® ® ® ® ® ® ® ® ® MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3152 Figure 1. The veterinary drug Emidonol (copied from https://avzvet.ru/en/product/emidonol-10-solution-for- injections/) Experimental Milk specimens: According to the manufacturer’s recommendations, a cow (BW 720 kg) received 2 x 40 mL/day of Emidonol i.m. (10% solution) on 2 consecutive days. One dose (40 mL) of the solution corresponded to 4 g Emidonol . A blank milk sample was collected 1 h before the first injection. Post- administration samples were collected 9, 23, 33, 47, 57, 71 and 81 h after the first injection. Each milk sample was divided in two aliquots of 100 mL, only one of which was pasteurized. Sample preparation: An aliquot of 270 µL of milk was fortified with 30 µL of the internal standard (meldonium-D , 1 μg/mL). The mixture was further diluted with 100 μL of a 100 mM ammonium acetate solution and 700 μL of acetonitrile. The sample was mixed for precipitation and the supernatant was transferred into a new vial. An aliquot of 10 μL was injected into the LC-MS/MS instrument. For semi- quantitative determination of meldonium, an 8-point calibration curve was used within a working range of 100-10000 ng/mL. LC-MS/MS: Milk samples were analyzed using a HILIC-HRMS/MS protocol published previously [3]. Briefly, the approach utilized an online sample clean-up using a dual pump setup equipped with Nucleodur HILIC columns (Macherey-Nagel, Düren, Germany) and a mobile phase that consisted of a 200 mM ammonium acetate buffer containing 0.15% acetic acid (pH 5.0), deionized water and acetonitrile. The Q Exactive mass spectrometer (Thermo Scientific, Bremen, Germany) was operated in t-HCD acquisition mode (precursor ion: m/z 147.1126, NCE: 50%, resolution: 35 000 FWHM). ® ® ® 3 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3153 Results and Discussion Representative extracted ion chromatograms using HILIC-HRMS/MS of a blank milk sample, a calibration point at a meldonium concentration of 6 µg/mL and a post-administration sample 47 h after the first injection (C : 6.8 µg/mL) are illustrated in Figure 2. For semi-quantification the relative area of precursor ion m/z 58.0651 (meldonium) and m/z 61.0848 (meldonium-D ) was used. Figure 2. Extracted ion chromatograms of meldonium (m/z 147.1128; resolution: 17,500 FWHM, NCE: 50%) of a blank milk sample, a spiked milk sample at 6 µg/mL and a post-administration milk sample (6.8 µg/mL, 47 h after the first injection) As depicted in Figure 3, meldonium concentrations in raw and pasteurized milk peaked at a concentration of 6.8 µg/mL 47 h after the first of 4 injections of 4 g Emidonol each. The applied dose corresponded to approx. 4.24 g meldonium (1.06 g/injection). The resulting concentrations in raw and pasteurized milk demonstrated no substantial difference. Compared to meldonium levels of approx. 700 ng/mL in milk presented by Temerdashev et al., observed levels in the present study were approx. 10- fold higher. Taking into account levels of 7.5 ng/mL observed after a single dose (50 μg) and 18.6 ng/mL after a multiple dose of meldonium (5 x 50 μg) presented earlier [4], consumption of milk containing meldonium at comparable levels as in the present study may lead to urinary meldonium concentrations greater than 100 ng/mL. max 3 ® MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3154 Figure 3. Meldonium concentrations in milk (µg/mL) following 4 injections of 40 mL of Emidonol solution i.m. (10%) to a cow, corresponding to 4 x 4 g Emidonol . First Injection (I 1) at 0 h, I 2 at 9 h, I 3 at 24 h and I 4 at 30 h. The graph in blue corresponds to raw milk and in red to pasteurized milk. Conclusions Since investigated Emidonol post-administration milk samples (i.m.) demonstrated a 10-fold increased meldonium concentrations (C : 6.8 µg/mL) compared to previously published data [2], a consumption of contaminated milk may lead to urinary meldonium concentrations exceeding the applicable reporting level for meldonium of 100 ng/mL. The investigated specimens demonstrated no significant difference between raw and pasteurized milk. To assist in the result managing process in sports drug testing, a differentiation between doping with meldonium or a possible contamination scenario would be of great interest. Here, information on emoxypine and metabolic products in milk may contribute to a more comprehensive analytical picture. As preliminary data, the investigation of the present post- administration samples provided evidence for the presence of emoxypine sulfate as a main metabolite found in milk. Nevertheless, more data is required to discriminate between both scenarios. References 1. World Anti-Doping Agency. Prohibited List 2021. https://www.wada-ama.org/sites/default/files/resources/files/2022list_en.pdf (accessed 2022/03/10). 2. Temerdashev A, Azaryan A, Dmitrieva E. Meldonium determination in milk and meat through UHPLC-HRMS. Heliyon. 2020 Aug 22;6(8):e04771. doi: 10.1016/j.heliyon.2020.e04771. 3. Görgens C, Guddat S, Dib J, Geyer H, Schänzer W, Thevis M. Mildronate (Meldonium) in professional sports - monitoring doping control urine samples using hydrophilic interaction liquid chromatography - high resolution/high accuracy mass spectrometry. Drug Test Anal. 2015 Nov-Dec;7(11-12):973-9. doi: 10.1002/dta.1788. ® ® ® max MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3155 4. Guddat S, Görgens C, Sobolevsky T, Thevis M. Meldonium residues in milk: A possible scenario for inadvertent doping in sports? Drug Test Anal. 2021 Nov;13(11-12):1906-1910. doi: 10.1002/dta.3145. Acknowledgements The authors thank P. Wachsmuth for his support with the practical work of the project. We would also like to acknowledge the financial support of the study by Sport Ireland (Dublin, Ireland), the Manfred Donike Institute for Doping Analysis e.V. (Cologne, Germany), and the Federal Ministry of the Interior, Building and Community (Berlin, Germany). MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3156 Krug O , Geyer H , Thomas A , Piper T , Thevis M Black market products suspected to contain doping relevant ingredients – report for 2021 Institute of Biochemistry / European Monitoring Center for Emerging Doping Agents, German Sport University, Cologne, Germany ; Institute of Biochemistry / Center for Preventive Doping Research, German Sport University, Cologne, Germany Abstract The European Monitoring Center for Emerging Doping Agents (EuMoCEDA) analyzed a total of 83 products qualitatively and quantitatively throughout 2021. Outstanding findings were the detection of ethylene glycol-oxymetholone and PEG-MGF (polyethylene glycol-conjugated mechano growth factor). Included substances were anabolic agents, stimulants, beta-2-agonists and narcotics. For the analysis of peptides and proteins, aliquots were separated by polyacrylamide gel electrophoresis and subsequently stained with coomassie blue. By bottom-up proteomic approaches including tryptic digestion and nano liquid chromatography / tandem high resolution mass spectrometry, proteinogenic ingredients were identified. Doping-relevant findings accounted in 101 cases for 46 different drugs (multi-findings included), from which 70% of the ingredients were not or falsely declared. Anabolic agents were determined in 63% of all identified doping relevant compounds (predominantly testosterone esters); 25% accounted for peptide hormones, growth factors, related substances and mimetics, 9% accounted for hormone and metabolic modulators, 1% related to non-approved substances, diuretics and masking agents, and stimulants, respectively. The finding of a (tentatively identified) ethylene glycol derivative of oxymetholone underlines the unpredictable composition of black market products due to manufacturing processes and/or intended manipulation, as well as the associated unknown health risks, corroborating the importance of continued monitoring of black market products. Introduction The black market for performance enhancing drugs is a common source for recreational / mass sport athletes [1-3]. The analysis of confiscated products, as well as products from test purchases is one part of monitoring the black market regarding emerging performance enhancing drugs. In 2021 the European Monitoring Center for Emerging Doping Agents (EuMoCEDA) analyzed again different products qualitatively and quantitatively. The 83 black market products were confiscated or purchased as test samples. Experimental Sample preparation Samples were solved / extracted with water, acetic acid (2% aq.), and/or acetonitrile (50:50 v/v), depending on their formulation (oily solution, lyophilized, etc.). Subsequently they were diluted to yield 1 2 2 2 1 1 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3157 an adequate concentration of labeled drug content. For gas chromatography, the extracted and subsequently dried samples were reconstituted in ethyl acetate, derivatized with N-methyl-N-(tri- methylsilyl)-trifluoroacetamide (MSTFA), or a mixture of MSTFA / ethanethiol and ammonium iodide, respectively [1]. PEG-MGF was subjected to a SDS-PAGE, tryptic digestion, and LC-(HR)MS/MS. Instrumentation The samples were screened by HPLC-ESI-MS using an Accela 1250 series HPLC interfaced via electrospray to a Thermo Scientific TSQ Vantage system. For HRMS experiments a Thermo Q-Exactive plus, a Thermo Exploris, and an Agilent 6550 iFunnel Q-TOF mass spectrometer were used. GC-MS experiments were performed on a Trace 1310 Gas Chromatograph in combination with a TSQ 8000 Evo Triple Quadrupole Mass Spectrometer from Thermo. Measurement To screen the most common target analytes in black market products, high performance liquid chromatography/mass spectrometry (HPLC-MS) experiments were conducted in single-reaction- monitoring (SRM) mode. Anabolic agents, stimulants, growth factors, natural and synthetic insulins, IGF-1 and synthetic analogs as well as growth hormone releasing factors could be determined by high performance liquid chromatography / high resolution mass spectrometry (HPLC-HRMS)-experiments in full-scan mode. Qualification and quantification of analytes were obtained by conducting product ion scans with substance specific fragmentation pathways. For gas chromatography / mass spectrometry (GC-MS) experiments, analytes were derivatized and measured in full-scan mode. Qualitative and quantitative analysis were accomplished by using reference substances and/or reference databases. Included substances were anabolic agents, stimulants, beta-2-agonists and narcotics [1]. For the analysis of peptides and proteins, aliquots were separated by polyacrylamide gel electrophoresis and subsequently stained with Coomassie blue. By bottom-up proteomic approaches including tryptic digestion and nano liquid chromatography/ tandem high resolution mass spectrometry, proteinogenic ingredients were identified. Analytes included, but were not limited to human growth hormone (hGH), growth factors (e.g.: FGF, MGF, etc.), various erythropoietins (EPO), and growth hormone releasing factors [4]. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3158 Table 1: Identified drugs in black market products MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3159 Results and Discussion In 2021, a total of 83 suspicious (illicit) black market products were analyzed at the Center for Preventive Doping Research. Doping-relevant findings accounted in 101 cases for 46 different drugs (multi-findings included), from which 70% of the ingredients were not or falsely declared (Tab. 1). As shown in Figure 1, 63% of the identified doping relevant compounds accounted for anabolic agents (predominantly testosterone esters); 25% accounted for Peptide hormones, growth factors, related substances and mimetics. Of these, 5 were EPO products, which were not further identified in detail. 9% accounted for hormone and metabolic modulators, 1% related to non-approved substances, diuretics and masking agents, and stimulants, respectively. The analytes, which were currently not doping relevant, were dermatologic and virilizing agents as well as sugars, amino acids, fatty acids, and vitamins. Outstanding findings were the detection of authentic PEG-MGF and an oxymetholone-derivative (Fig. 2 and 3). Products labelled to contain PEG-MGF were investigated in the past, but MGF conjugated to PEG was never observed in our analyses so far. The finding of a (tentatively identified) ethylene glycol derivative of oxymetholone underlines the unpredictable composition of black market products due to manufacturing processes and / or intended manipulation, as well as the associated unknown health risks, corroborating the importance of continued monitoring of black market products. Due to the absence of certified reference material, ethylene glycol oxymetholone was not quantified. Figure 1: Apportionment of identified doping relevant drugs in analyzed black market products 2021 Figure 2: Black market product containing oxymetholone-derivative (a), extracted ion chromatogram and MS²- product ion mass spectra @ ce 40 of oxymetholone (b), and C H O-oxymetholone-drivative (c)2 4 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3160 Figure 3: MS²-product ion mass spectrum @ ce 30 of trypsin-digested SDS-PAGE-spot (ca. 13 kDa, red frame). Precursor of tryptic MGF-fragment: m/z 934 Conclusions The use of faked and falsely labeled preparations from the black market by athletes of recreational sports induces risks for their health and undermines the spirit of sport. Anabolic agents and peptidic hormones are still the most popular products to improve performance and/or body shape. Remarkable is the recent finding of an ethylene glycol derivative of oxymetholone, which might be an artefact due to poor production processes. Nevertheless, as ingredient of the black market product, it enters the athletes body. This underlines that the risks for athletes are unpredictable and confirms once again the importance of continuous monitoring of the black market and the investigation of distributed products. The Cologne Anti-Doping Laboratory´s commitment under the umbrella of EuMoCEDA resulted again in the detection of emerging doping-relevant drugs. References 1. O. Krug, A. Thomas, K. Walpurgis, T. Piper, G. Sigmund, W. Schänzer, T. Laußmann, M. Thevis: Identification of black market products and potential doping agents in Germany 2010-2013 (2014) Eur J Clin Pharmacol, Vol. 70, 1303-1311 2. C. Weber, O. Krug, M. Kamber, M. Thevis: Qualitative and Semiquantitative Analysis of Doping Products Seized at the Swiss Border. (2017) Substance Use & Misuse , Vol 52, (6) 742-753 3. C. Weber, M. Kamber, V. Lentillon-Kaestner: Are doping substances imported into Switzerland mainly to increase athletic performance? (2016) Performance Enhancement & Health. Vol 5(2), 66–76. 4. M. Thevis, Y. Schrader, A. Thomas, G. Sigmund, H. Geyer, W. Schänzer: Analysis of Confiscated Black Market Drugs Using Chromatographic and Mass Spectrometric Approaches (2008) J Anal Toxicol, Vol. 32, 232 Acknowledgements The study was carried out with support of the Federal Ministry of the Interior, Building and Community (Berlin, Germany). MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3161 Piper T, Thevis M Addressing recent challenges in isotope ratio mass spectrometry – Development of a method applicable to 1-androstene-steroids, 6α- hydroxy-androstenedione, and androstatrienedione Institute of Biochemistry, German Sport University, Cologne, Germany Abstract In 2020 the World Anti-Doping Agency (WADA) issued 3 Technical Letters (TL) on several new pseudo- endogenous steroids that became relevant for sports drug testing. [1-3] In all cases the application of isotope ratio mass spectrometry (IRMS) has been recommended as a confirmation method but not for all of the mentioned steroids validated methods have already been developed. Regarding the anabolic androgenic 1-ene-steroids mentioned in TL 20 (5α-androst-1-ene-3β,17β-diol (1ADIOL), 5α-androst-1-ene- 3,17-dione (1AD), 17β-hydroxy-5α-androst-1-en-3-one (1T) and 3α-hydroxy-5α-androst-1-ene-17-one (1AND)) no IRMS method has been reported or developed so far. The same holds true for the hormone and metabolic modulators also mentioned in TL 20, i.e. Androsta-1,4,6-triene-3,17-dione (TRD) and its main metabolite 17β-hydroxy-androsta-1,4,6-triene-3-one (TR17OH). Regarding 6α-hydroxy-androst-4- ene-3,17-dione (6aOH) mentioned in TL 21 a method has already been reported but not fully validated considering all recommendations of the current Technical Document on IRMS. Aim of this research project was the development and validation of an IRMS method to determine the carbon isotope ratios (CIR) of all of the above-mentioned steroids plus 5α-androst-1-ene-3β,17β-diol (1EpiD) and 6β-hydroxy-androst-4-ene-3,17-dione (6bOH) to complement the approach. Due to numerous endogenous co-elutions a twofold high-performance liquid chromatography-based sample clean-up was found inevitably. All analytes were acetylated to further improve sample clean-up. The limits of quantification were validated at 10 ng/mL for a 20 mL urine aliquot for all analytes beside 1AND (20 ng/mL) and 1ADIOL, which was still affected by co-elutions. Found measurement uncertainties fall between 0.4 and 0.9 ‰. As a proof-of-concept samples collected after the single oral administration of a nutritional supplement containing 1AD and 1DHEA (5α-androst-1-ene-3β-ol-17-one) were analyzed. Urinary concentrations were significantly elevated for 45 h after administrations until the end of the study and found CIR could clearly substantiate the exogenous origin of urinary metabolites in all samples.‹ Published as: Piper T, Thevis M. Addressing recent challenges in isotope ratio mass spectrometry: Development of a method applicable to 1-androstene-steroids, 6α-hydroxy-androstenedione, and androstatrienedione. Drug Test Anal. 2022 Aug 24. doi: 10.1002/dta.3361. References 1. WADA Technical Letter – TL19. IN SITU FORMATION OF PREDNISONE AND PREDNISOLONE. www.wada- ama.org/sites/default/files/resources/files/tl19_prednisone_and_prednisolone_eng_2021_1.pdf, accessed 23.02.22 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3162 2. WADA Technical Letter – TL20. IN SITU FORMATION OF SPECIFIC SUBSTANCES WITH A STEROID STRUCTURE. www.wada- ama.org/sites/default/files/resources/files/tl20_in_situ_formation_of_specific_substances_eng_2021_1.pdf, accessed 23.02.22 3. WADA Technical Letter – TL21. IN SITU FORMATION OF 4-ANDROSTENE-3,6,17-TRIONE (6-OXO) AND METABOLITES. www.wada-ama.org/sites/default/files/resources/files/tl21_6oxo_eng_2021_1.pdf, accessed 23.02.22 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3163 Piper T, Thevis M Development of mass spectrometry-based methods for the detection of 11-ketotestosterone and 11-ketodihydrotestosterone Institute of Biochemistry, German Sport University, Cologne, Germany Abstract In recent years, several publications showed up dealing with the topic of the anabolic properties of 11- hydroxyandrostenedione (11OHA4) and its physiologically active metabolites 11-ketotestosterone (11KT) and 11-ketodihydrotestosterone (11KDHT).[1-4] Especially 11KT became easily available via internet based providers and its usefulness in the context of body-building is thoroughly discussed.[5] To the best of our knowledge no doping control methods for the detection of 11KT or 11KDHT exist, neither on the initial testing procedure level nor as confirmation method. The prohormone of 11OHA4 – adrenosterone (androst-4-ene-3,11,17-trione, A4TR) has already been addressed several years ago and the suggested urinary marker for its misuse was mainly the concentration of 11-hydroxyandrosterone (11OHA) to be greater than 10,000 ng/mL and for confirmation purposes the carbon isotope ratios (CIR) were taken into consideration.[6,7] Due to the relatively high urinary concentrations of 11OHA being a major metabolite of adrenal gland steroid production, the detection windows were rather short and most probably not sensitive enough to detect the potential misuse of 11KT. Therefore, we investigated the human metabolism of 11KT focusing on all urinary metabolites in order to enable the detection of 11KT and its prohormone A4TR. Two volunteers (one female and one male) orally administered 20 mg of 11KT each and urine samples were collected for 5 days. In a first step urinary concentrations of 11KT and its potential metabolites were investigated to enable implementation of reasonable metabolites into current screening procedures. A reference population encompassing 100 males and 100 females was investigated regarding baseline urinary concentrations of relevant metabolites in order to elucidate preliminary thresholds for identification of suspicious samples. As confirmation procedure an isotope ratio mass spectrometry-based method was developed in order to determine the carbon isotope ratios (CIR) of 11KT and relevant metabolites. After oral application of 20 mg of 11KT significantly elevated urinary concentrations were found for not more than 8 hours, depleted CIR were detected until 24 hours after administration pointing towards a fast metabolism of 11KT. Several new metabolites of 11KT have been detected, but they could not significantly prolong the detection times, neither during the screening procedure, nor considering the CIR. Published as: Piper, T, Thevis, M. Development of mass spectrometry-based methods for the detection of 11-keto- testosterone and 11-ketodihydrotestosterone. Drug Test Anal. 2023. doi.org/10.1002/dta.3442 References 1. Storbeck KH, Bloem LM, Africander D, Schloms L, Swart P, Swart AC. 11β-Hydroxydihydrotestosterone and 11-ketodihydrotestosterone, novel C19 steroids with androgenic activity: A putative role in castration MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3164 resistant prostate cancer? Molecular and Cellular Endocrinology 2013, 377, 135–146. 2. Bloem LM, Storbeck KH, Schloms L, Swart AC. 11β-Hydroxyandrostenedione Returns to the Steroid Arena: Biosynthesis, Metabolism and Function. Molecules 2013, 18, 13228-13244. 3. Swart AC, Storbeck KH. 11β-hydroxyandrostenedione: Downstream metabolism by 11βHSD, 17βHSD and SRD5A produces novel substrates in familiar pathways. Molecular and Cellular Endocrinology 2015, 408, 114–123. 4. Pretorius E, Arlt W, Storbeck KH. A new dawn for androgens: Novel lessons from 11-oxygenated C19 steroids. Molecular and Cellular Endocrinology 2017, 441, 76-85. 5. Introducing Olympus UK's SUP3R-11. The Ultimate 11-Ketotestosterone Guide. https://anabolicminds.com/community/threads/introducing-sup3r-11-the-ultimate-11-ketotestosterone- guide-all-you-need-to-know.280427/, accessed 24.02.2022 6. Brooker L, Parr MK, Cawley A, Flenker U, Howe C, Kazlauskas R, Schänzer W, George A. Developement of criteria for the detection of adrenosterone administration by gas chromatography-mass spectrometry and GC/C/IRMS for doping control. Drug Test. Analysis 2009, 1, 587-595. 7. Brooker L, Cawley A, Kazlauskas R, Goebel C, George A. Carbon isotope ratio analysis of endogenous glucocorticoid urinary metabolites after cortisone acetate and adrenosterone administration for doping control. Drug Test. Analysis 2012, 4, 951–961. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3165 Albertsdóttir AD, van Gansbeke W, van Eenoo P, Polet M Sulfated steroids: Bridging the LC vs. GC divide Comparison of non-hydrolysed sulfated metabolites of metenolone and mesterolone analysed by 4 different techniques DoCoLab Universiteit Gent-UGent, Ghent, Belgium Abstract In the re-analysis program from the 2004, 2008 and 2012 summer Olympic Games, 90% of the 191 counts of detected prohibited substances were due to the use of exogenous anabolic androgenic steroids (AAS). This has not only been credited to the improvement of instrumentation but also due to the discovery of new long-term AAS metabolites. As such, non-hydrolysed sulphated metabolites have gained renewed interest given that research demonstrated their extended detection time compared to the more conventional markers for clostebol, metenolone, mesterolone and methandienone. The investigations into their potential have been carried out using liquid and gas chromatography-mass spectrometry (LC- and GC-MS), both of which are used in the initial testing procedure (ITP) as these techniques are complementary. However, due to their complementary nature, it is probable that the most promising metabolite on one instrument does not necessarily exhibit the same behaviour on the other and vice versa. Therefore, a comprehensive comparison is needed where the most likely long-term metabolites are identified on both analytical platforms and compared on as much equal footing as possible. As a trial model, metenolone and mesterolone were selected as both are frequently detected. Using previous work, the most likely long-term sulphated metabolites were identified on the following instruments that also are generally used in the ITP in doping control laboratories: LC-QQQ-MS, GC-CI- QQQ-MS, GC-EI-QQQ-MS and GC-EI-QTOF-MS. Furthermore, our recent publication showed how, using a MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3166 modified sample preparation procedure, hydrolysed glucuronidated and non-hydrolysed sulphated metabolites could be analysed on GC-EI-QTOF-MS in a single run. Thus, additionally, this enabled detection time comparison between conventional markers and non-hydrolysed sulfated metabolites between different instruments. In the case of metenolone, the overall detection times were comparable across all 4 instruments where detection times ranged between 10 to 12 days. However, for mesterolone, the results varied where for individual 1 the detection times ranged from 15 to 22 days. For individual 2, the detection time was the same across all 4 instruments which was day 15. When comparing the detection time between different types of metabolites, the detection times were comparable for metenolone but for mesterolone, the sulfated metabolites provided 2 to 5 times longer detection times on all 4 instruments and both individuals. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3167 Keiler AM , Gronert A , Liu L , Bredendiek F , Hobohm L , Froschauer A , Zierau O , Parr MK , Thieme D New models for human-like steroid metabolism – shown for metandienone Institute of Doping Analysis & Sports Biochemistry, Kreischa, Germany ; Environmental Monitoring and Endocrinology, Technische Universität Dresden, Dresden, Germany ; Institute of Pharmacy, Pharmaceutical and Medicinal Chemistry, FU Berlin, Berlin, Germany ; Core Facility BiosupraMol, Department of Biology, Chemistry, Pharmacy, FU Berlin, Berlin, Germany Abstract Options to study biotransformation comprise, aside from human administration, recombinant enzymes, microsomal preparations, hepatic S9 fractions, primary hepatocytes, hepatocellular lines or cell lines of other origin. In addition to the classical 2D cultivation of mammalian cells, the generation of so-called spheroids by 3D culture is a way, which might lead to an improved liver enzyme expression and the possibility to investigate formation of long-term metabolites. We compared three different culture methods for generation of HepG2 spheroids with regard to morphology and differentiation by histological and immunohistochemical analyses as well as to the gene expression analysis of biotransformation enzymes. Moreover, we comparatively analyzed the HepG2 spheroid-mediated metandienone biotransformation by high performance liquid chromatography-high resolution mass spectrometry. In a second approach, we investigated whether medaka embryos (Oryzias latipes) might be a suitable in vivo test model for human-like metabolism (Liu et al., under revision). Beside the advantage that fish embryos are, according to the European Food Safety Authority (EFSA), excluded from animal testing regulations, an exposition with test substances allows for simultaneous assessment of developmental and toxicity parameters. We investigated toxicological and physiological effects as well as the biotransformation of medaka embryos exposed to different metandienone dosages at different developmental stages. One part has already been published as: Liu L, Hobohm L, Bredendiek F, Froschauer A, Zierau O, Parr MK, Keiler AM. Medaka embryos as a model for metabolism of anabolic steroids. Arch Toxicol . 2022 Jul;96(7):1963-1974. doi: 10.1007/s00204-022- 03284-4 Other parts will be published elsewere. 1,2 2 3 4 2 2 2 3 1 1 2 3 4 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3168 Gmeiner G, Glatt A Adverse analytical findings for testosterone esters in blood samples Doping Control Laboratory, Seibersdorf Labor GmbH, Seibersdorf, Austria Abstract The detection of a testosterone application is currently based on two principles: a) the steroidal module of the athlete biological passport (ABP) for monitoring of significant changes in the ratios of endogenous anabolic androgenic steroid (EAAS) markers of the urinary steroid profile; b) the steroid profile confirmation procedure including carbon isotope ratio mass spectrometry (IRMS) as final proof of the exogenous application of testosterone, mainly triggered by the ABP module. IRMS analysis is based on the difference in carbon isotope ratios of exogenously applied compared to endogenously produced testosterone. Several studies have indicated that quite a high percentage of testosterone-containing preparations are marketed with endogenous-like carbon isotope ratio [1]. Application of these preparations may not lead to analytical data sets fulfilling the positivity criteria of WADA TD IRMS in its current version. Due to the fact, that testosterone is available on the market as oily ester preparation for injection as possible route of administration, the detection as well as stability aspects of these prodrugs in blood and serum samples have been investigated in previous publications [2-4]. The detection of testosterone esters in blood or serum samples can serve as direct proof of its application, because unlike testosterone itself, the esters of these substances are not endogenously generated. Generally speaking, the detection of a prohibited substance in doping control samples is based on initial testing, followed by confirmation analysis in case of a presumptive finding in the initial screen. The detection strategy presented as a lecture at the Manfred Donike Workshop 2022 is based on derivatisation of the keto-moiety of the testosterone molecule, located at position 3. To improve the specificity of the detection assay testosterone esters are derivatized with hydroxylamine for initial testing, followed by derivatisation with Girard P reagent for the confirmation analysis. With this approach, four adverse analytical findings (AAF) for testosterone undecanoate or testosterone propionate were reported so far for 2021 and 2022. It is intended to publish the complete study elsewhere. References 1. G. Forsdahl, C. Östreicher, M. Koller, G. Gmeiner. Carbon isotope ratio determination and investigation of seized testosterone preparations. Drug Test. Anal. 3, 814–819 (2011). 2. Forsdahl, G., Vatne, H. K., Geisendorfer, T. & Gmeiner, G. Screening of testosterone esters in human plasma. Drug Test. Anal. 5, 826–833 (2013). 3. Forsdahl, G. et al. Detection of testosterone esters in blood. Drug Test. Anal. 7, 983–989 (2015). 4. Torre, X. de la, Iannonea, M. & Botrè, F. Improving the detection of anabolic steroid esters in human serum by LC–MS. J. Pharm. Biomed. Anal. (2020). Acknowledgements This project has been carried out with the financial support of WADA and the Austrian Sports Ministry. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3169 Langer T, Nicoli R, Kuuranne T, Musenga A A holistic approach to serum steroid profiling by LC-MS/MS Laboratoire Suisse d'Analyse du Dopage Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Epalinges, Suisse Abstract Traditionally, the detection of doping with anabolic steroids, exogenous as well as endogenous, is performed in urine. While the presence of exogenous steroids is an unambiguous proof of doping, demonstrating administration of endogenous steroids such as testosterone is more complicated, as they are naturally present in the body. Since 2014, longitudinal monitoring of urinary excreted steroid profiles in the Athlete Biological Passport (ABP) of an individual athlete has enhanced targeting of specific samples for confirmation with Isotope Ratio Mass Spectrometry (IRMS). However, urine as a matrix is not perfect: sample collection is prone to manipulation, steroids can be subject to microbial activity and steroid esters, which are used as pro-drugs, are not excreted in urine. In comparison with urine, blood is more difficult to tamper with, and allows for the analysis of steroid esters, while the lower sample volume poses some analytical challenges regarding sensitivity. Profiling of endogenous steroids in blood was proposed by Ponzetto et al. [1] for the detection of oral and transdermal testosterone administration in men. Later, Salamin et al.[2] could also show that steroid profiling in blood was an efficient tool to target the detection of testosterone gel application in women. In this work, we have further explored the potential of these methods by including the detection of exogenous steroids and steroid esters within the quantification of endogenous steroids. The sample preparation was based on the publication of Ponzetto [1], utilising supported liquid extraction but is adapted as shown in Figure 1 to accommodate the analysis of steroid esters. The first chromatographic analysis for the detection of several free exogenous steroids and the quantification of the endogenous steroids testosterone (T), androstenedione (A4) and dihydrotestosterone (DHT) was done on an Acquity BEH C18 column (2.1 x 100mm, 1.7 mm, Waters) with 0.1% formic acid in water and 0.1% formic acid in acetonitrile as mobile phases. After evaporation of the solvent, steroid esters were derivatised with Girard’s reagent T in acidic aqueous conditions. The derivatised species were then analysed on an Acquity BEH C8 column (2.1 x 100 mm, 1.7 mm, Waters) with 0.1% formic acid in water and 0.1% formic acid in methanol as mobile phases. All chromatographic conditions are more precisely described in the full article, submitted to Drug Testing and Analysis [3]. With this combined method, it was possible to analyse endogenous and exogenous steroids as well as steroid esters form one 200 μL aliquot of serum. The quantitative performance of T, A4 and DHT using the herein described method was verified by comparing quality control samples with the already published protocol [1]. Furthermore, the method was validated for the qualitative analysis of 18 exogenous steroids and 16 steroid esters. Limits of detection (LOD) in the range of 50 to 500 pg/mL were achieved. To demonstrate the applicability of the method, the samples of an oral testosterone undecanoate (TU) administration study with 19 male volunteers [4] were analysed. Steroid profiles were constructed for MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3170 each volunteer with individual tolerance limits based on the control phase of the study. No free exogenous steroids were detected in any of the samples. The samples were also analysed for steroid esters, detecting TU in all 19 volunteers up to 24 hours after oral administration. As the samples were collected in 2013 [3] and stored frozen at -20°C, the successful analysis of TU demonstrates the stability of this compound in serum under the chosen storage conditions. However, the TU concentrations found showed high inter-individual variabilities with maximum TU concentrations ranging from 0.37 to 171 ng/mL. From the three monitored endogenous steroids, DHT appeared to be the most promising marker for TU administration as a significant difference (p=0.025) was found between the control and administration phase. DHT concentrations were found to exceed the individual thresholds in 15 out of 19 volunteers after TU administration. Endogenous steroid concentrations in serum were more elevated in samples with higher TU concentrations. Since DHT was observed as the most influenced endogenous steroid, it was hypothesized that TU is also metabolised to DHT undecanoate (DHTU) prior to the hydrolysis of esters. This theory was reinforced further by the detection of DHTU in serum samples. Figure 1. Schematic illustration of the sample preparation and analytical workflow for the analysis of endogenous and exogenous steroids as well as steroid esters from one aliquot of 200 μL serum References 1. Ponzetto F, et al. (2016) Longitudinal monitoring of endogenous steroids in human serum by UHPLC-MS/MS as a tool to detect testosterone abuse in sports. Anal Bioanal Chem. 408 (3), 705-719 2. Salamin O, et al. (2022) Longitudinal evaluation of multiple biomarkers for the detection of testosterone gel administration in women with normal menstrual cycle. Drug Test Anal. 14 (5), 833-850 3. Langer T, et al. A comprehensive UHPLC-MS/MS method for the analysis of endogenous and exogenous steroids in serum for anti-doping purposes. Drug Test Anal. 2022 Oct 6. doi: 10.1002/dta.3379 4. Badoud F, et al. (2013) Profiling of steroid metabolites after transdermal and oral administration of testosterone by ultra-high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. J Steroid Biochem Mol Biol. 138, 222-235 Acknowledgements This project was carried out with financial support from the World Anti-Doping Agency (Grant 21A07RN). The authors wish to express their gratitude to Dr. Günter Gmeiner and Seibersdorf Laboratories for providing the DHTU standard. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3171 Wagener F , Guddat S , Görgens C , Petrou M , Angelis Y , Lagojda A , Kühne D , Thevis M Investigations into the elimination profiles and metabolite ratios of micro-dosed selective androgen receptor modulator LGD-4033 for doping control purposes Institute of Biochemistry, German Sport University, Cologne, Germany ; Cyprus Anti-Doping Authority, Nicosia, Cyprus ; Institute of Biosciences & Applications, National Center for Scientific Research "Demokritos", Doping Control Laboratory of Athens, Athens, Greece ; BayerCropScience AG, Monheim, Germany Abstract LGD-4033 (ligandrol) is a selective androgen receptor modulator (SARM), which is prohibited in sports by the World Anti-Doping Agency (WADA) in- and out-of-competition. But not only deliberate doping with LGD-4033 constitutes a problem. In the past years, some AAFs that concerned SARMs can be attributed to contaminated dietary supplements (DS). Thus, the urgency to develop methods to differentiate between inadvertent doping and abuse of SARMs to benefit from the performance-enhancing effect of the compound in sports is growing. To gain a better understanding of the metabolism and excretion patterns of LGD-4033, human micro-dose excretion studies at 1, 10 and 50 µg LGD-4033 were conducted. Collected urine samples were prepared for analysis using enzymatic hydrolysis followed by solid-phase extraction and analyzed via LC HRMS/MS. Including isomers, a total of 15 phase-I metabolites were detected in the urine samples. The LC HRMS/MS method was validated for qualitative detection of LGD-4033, allowing for a limit of detection (LOD) of 8 pg/mL. The metabolite M1, representing the epimer of LGD-4033 was synthesized and the structure elucidated by NMR spectroscopy. As the M1/LGD-4033 ratio changes over time, the ratio and the approximate LGD-4033 concentration can contribute to estimating the time point of drug intake and dose of LGD-4033 in doping control urine samples, which is particularly relevant in anti-doping result management. Published as: Wagener F, Guddat S, Görgens C, Angelis YS, Petrou M, Lagojda A, Kühne D, Thevis M. Investigations into the elimination profiles and metabolite ratios of micro-dosed selective androgen receptor modulator LGD-4033 for doping control purposes. Anal Bioanal Chem. 2022 Jan;414(2):1151-1162. doi: 10.1007/ s00216-021-03740-7 1 1 1 2 3 4 4 1 1 2 3 4 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3172 Coll S , Bressan C , Alechaga & , Monfort N , Ventura R Elimination profile of dexamethasone after oral administrations: Evaluation of the reporting level and washout periods Catalonian Antidoping Laboratory Fundació Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain ; Departament of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain Abstract Dexamethasone (DEX) is one of the most detected glucocorticoids in sports drug testing. Up to 2021, a minimum reporting level (MRL) of 30 ng/mL was used to discriminate between allowed and prohibited administrations of all glucocorticoids. Since 2022, new compound-specific MRLs have been established for some GC. For DEX, a MRL of 60 ng/mL was defined. Although very limited data on urinary concentrations after oral administration were available, the same criteria used for betamethasone was applied due to the similarities between both compounds. The aim of the present work was to study the urinary and plasma profiles of DEX after single and multiple oral administrations in order to evaluate the current reporting level and wash-out period. DEX was administered to healthy volunteers using a single-dose oral treatment (4 mg, n=8 males) and multiple- dose oral treatment (2x2mg/day for 5 days, n=8 males). Urine and plasma samples collected before and after administration were analysed using a liquid chromatography-tandem mass spectrometry method. The current reporting level and wash-out period used to detect DEX misuse in sports will be evaluated by assessing the urinary profiles of DEX and its metabolites obtained after different oral administrations. Furthermore, DEX and cortisol concentrations obtained in plasma samples will be presented. Both urinary and plasmatic profiles of DEX will be compared with the profiles obtained for betamethasone in a study previously reported by our group. 1 1 1,2 1 1 1 2 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN173 Euler L, Wagener F, Thomas A, Thevis M Elimination profile of microdosed zilpaterol mimicking consumption of contaminated cattle meat Institute of Biochemistry, German Sport University, Cologne, Germany Abstract Rationale: The synthetic β-adrenoreceptor agonist zilpaterol is legitimately used as an animal feed supplement in selected countries due to its known effects on lipolysis and protein biosynthesis. These pharmacological characteristics of zilpaterol have contributed to its classification as doping agent in sport by the World Anti-Doping Agency. However, the use as a feed supplement can lead to residues of the drug in edible tissues and, possibly, also in the urine of consumers. Methods: To provide urinary elimination profiles of microdosed zilpaterol and to determine whether the ingestion of zilpaterol below or at the acceptable daily intake level of 0.04 μg/kg bodyweight can result in an adverse analytical finding (AAF) in doping controls, healthy volunteers were administered single or multiple oral doses of 0.5 μg or 3 μg zilpaterol to mimic ingestion of contaminated cattle meat. Urine samples were collected and analyzed using a validated high-performance liquid chromatography- electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method and a newly developed chiral high-performance liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry (HPLC-APCI-MS/MS) method. Results: Urinary peak concentrations of zilpaterol were observed for all volunteers 1.5–12.5 h after ingestion, and maximum levels > 5 ng/mL, which would constitute an AAF in doping controls, were found after the intake of 3 μg of zilpaterol on five consecutive days in one out of five study participants. Noteworthy, the enantiomeric ratio of excreted zilpaterol remained constant over time. Conclusion: This study provides first insights into the urinary excretion of microdosed zilpaterol. Furthermore, a method was successfully developed and applied for the separation of the zilpaterol enantiomers with mass spectrometric detection. Published as: Euler L, Wagener F, Thomas A, Thevis M. Determination and enantioselective separation of zilpaterol in human urine after mimicking consumption of contaminated meat using high-performance liquid chromatography with tandem mass spectrometry techniques. Rapid Commun Mass Spectrom. 2022; 36(19):e9357. doi:10.1002/rcm.9357 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3174 Coppieters G, Deventer K, van Eenoo P, Judák P Application of dilute-and-shoot on a nanoflow LC-MS setup for the confirmatory analysis of small peptide hormones Diagnostic Sciences, DoCoLab Universiteit Gent-UGent, Ghent, België Abstract Nano-liquid chromatography (nanoLC) has proven itself as a powerful tool and its scope entails various applications in (bio)analytical fields. Operation at low (nL/min) flow rates in combination with reduced inner dimensions (ID < 100 μm), leads to significantly enhanced sensitivity when coupled with electrospray ionization-mass spectrometry (ESI-MS). Challenges that remain for the routine implementation of such miniaturized setups are related to clogging of the system and robustness in general, and thus the application of tedious sample preparation steps. To improve ruggedness, a filter placed upstream in the LC prevents particles from entering and clogging the system. This so-called online automatic filtration and filter back-flush (AFFL) system was combined with nanoLC and the direct injection principle for the sensitive confirmatory analysis of fifty different doping-relevant peptides in urine. Figure 1. Pressure monitoring of the online automatic filtration setup The presented assay was validated for routine purposes and was fully compliant with the most recent minimum required performance levels (MRPL) and chromatographic/mass spectrometric identification criteria (IDCR), as imposed by the World-Anti Doping Agency (WADA). In the absence of labour-intensive MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3175 sample preparation, the application of AFFL allowed for the injection of diluted urine samples without any noticeable pressure buildup in the nanoLC system. Contrary to earlier observations by our group and others, the addition of dimethylsulfoxide (DMSO) to the mobile phase did not enhance sensitivity in the presented nanoflow setup, yet was beneficial to reduce carryover. Although the robustness of the presented setup was evaluated only for the analysis of diluted urine samples, it is entirely conceivable that routine applications employing other matrices and currently running on analytical scale LC instruments could be transferred to micro/nanoLC scale systems to reach lower detection limits. Published as: Coppieters G, Deventer K, Van Eenoo P, Judak P. Combining direct urinary injection with automated filtration and nanoflow LC-MS for the confirmatory analysis of doping-relevant small peptide hormones. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences. 2021 Aug 1;1179:122842. doi: 10.1016/j.jchromb.2021.122842 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3176 Reichel C , Gmeiner G , Thevis M Detection of black market myostatin propeptide Chemical Analytics, Seibersdorf Labor GmbH Doping - Control Laboratory, Seibersdorf, Austria ; Institute of Biochemistry / Center for Preventive Doping Research / European Monitoring Center for Emerging Doping Agents, German Sport University Cologne, Cologne, Germany Abstract Myostatin propeptide it is prohibited according to chapter S4 of the “WADA 2022 List of Prohibited Substances and Methods” [1]. In the past, several attempts were made to develop therapies based on myostatin propeptide for curing muscular diseases including gene therapy, antisense oligonucleotide therapy and application of propeptide-Fc fusion proteins [2-8]. So far, no approved myostatin-propeptide pharmaceuticals are available. Nevertheless, myostatin-propeptides can be bought on the black market for “research purposes”. A study on black market myostatin propeptide products is presented as well as electrophoretic detection methods for serum and urine. Out of the twelve tested products, only nine actually contained the protein. Separation by SDS-PAGE (Figure 1) revealed that the nine products were relatively impure and that the main compound had a much higher mass (ca. 54-55 kDa) than expected (ca. 33 kDa). Further analyses by mass spectrometry showed, that the elevated molecular mass was due to the presence of a full length GST-tag on the propeptide. The developed detection method for serum is based on immunoprecipitation (IP) followed by SDS-PAGE and Western blotting. In total, three anti-myostatin propeptide antibodies were tested. All of them were well suited for either IP or immunoblotting. The final protocol applies a biotinylated polyclonal antibody, streptavidin-coated magnetic beads, and a monoclonal detection antibody. For a sample volume of 500 µL serum, the detection limit of the method is ca. 2.5 ng/mL. The urine method applies a commercial ELISA for IP and performs with an LOD of ca. 0.4 ng/mL. Furthermore, practically all currently available myostatin propeptide standards were also investigated. Due to the significant molecular mass difference of the black market products, an unambiguous differentiation from endogenous myostatin propeptide is possible. Published as: Reichel C, Gmeiner G, Thevis M. Electrophoretic detection of black market myostatin propeptide. Drug Test Anal. 2022 Nov;14(11-12):1812-1824. doi: 10.1002/dta.3398 References 1. World Anti-Doping Agency (WADA). The World Anti-Doping Code. International Standard. Prohibited List January 2022. https://www.wada-ama.org/sites/default/files/resources/files/2022list_final_en.pdf. Accessed 28 July 2022. 2. Bogdanovich S, Perkins KJ, Krag TO, Whittemore LA, Khurana TS. Myostatin propeptide-mediated amelioration of dystrophic pathophysiology. FASEB J. 2005;19(6):543-9. 3. Hamrick MW, Arounleut P, Kellum E, Cain M, Immel D, Liang LF. Recombinant myostatin (GDF-8) propeptide enhances the repair and regeneration of both muscle and bone in a model of deep penetrant musculoskeletal injury. J Trauma. 2010;69(3):579-83. 4. Cleasby ME, Jarmin S, Eilers W, Elashry M, Andersen DK, Dickson G, Foster K. Local overexpression of the myostatin propeptide increases glucose transporter expression and enhances skeletal muscle glucose 1 1 2 1 2 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3177 disposal. Am J Physiol Endocrinol Metab. 2014;306(7):E814-23 5. Jiang JG, Shen GF, Li J, Qiao C, Xiao B, Yan H, Wang DW, Xiao X. Adeno-associated virus-mediated expression of myostatin propeptide improves the growth of skeletal muscle and attenuates hyperglycemia in db/db mice. Gene Ther. 2017;24(3):167-175. 6. Ran N, Gao X, Dong X, Li J, Lin C, Geng M, Yin H. Effects of exosome-mediated delivery of myostatin propeptide on functional recovery of mdx mice. Biomaterials. 2020;236:119826. 7. Tsai SW, Tung YT, Chen HL, Yang SH, Liu CY, Lu M, Pai HJ, Lin CC, Chen CM. Myostatin propeptide gene delivery by gene gun ameliorates muscle atrophy in a rat model of botulinum toxin-induced nerve denervation. Life Sci. 2016;146:15-23 8. Arounleut P, Bialek P, Liang LF, Upadhyay S, Fulzele S, Johnson M, Elsalanty M, Isales CM, Hamrick MW. A myostatin inhibitor (propeptide-Fc) increases muscle mass and muscle fiber size in aged mice but does not increase bone density or bone strength. Exp Gerontol. 2013;48(9):898-904. Acknowledgements The project was carried out with support of the World Anti-Doping Agency (WADA), the Austrian Ministry of Sports and the Federal Ministry of the Interior of the Federal Republic of Germany. Figure 1. Separation of myostatin propeptide standards and two black market products (BM01, BM02) by SDS- PAGE (10% T, MOPS running buffer; 1 µg on gel, Coomassie R-250 stain). E. coli expressed proteins showed a narrow band at ca. 33 kDa while the mass of the HEK293 protein was ca. 39 kDa (blue arrows). Compared to that, the main band of the two BM products was also narrow but considerably higher (ca. 54 kDa; red arrows). MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3178 Marchand A , Miller G , Martin L , Gobbo C , Crouch AK , Eichner D , Ericsson M Detection of erythropoiesis stimulating agent luspatercept after administration to healthy volunteers for antidoping purposes Laboratoire AntiDopage Francais (LADF), Université Paris-Saclay, Châtenay-Malabry, France ; Sports Medicine Research and Testing Laboratory, Sports Drug Testing Laboratory, South Jordan, USA Abstract Luspatercept (Reblozyl ) is a newly approved anti-anemic drug composed of the extracellular domain of activin receptor IIB with an additional mutation (amino acid 79 L to D) coupled to the Fc part of immunoglobulin G. This protein drug acts more specifically on erythropoiesis by limiting apoptosis of immature erythroblasts, thus increasing differentiation and proliferation of erythroid progenitors. When tested in an healthy human population in a Phase I trial, 83.3% of the patients showed a prolonged increase in hemoglobin concentrations ([HGB] of > 1.0 g/dL) when administered a single dose of 0.25 mg/kg. Since 2020, Reblozyl (luspatercept-aamt) is now approved in the United States of America and European Union for treatment of several anemias as an alternative to other erythropoietin-derived drugs. The wider availability of this drug increases the risk of misuse of this product by athletes for doping and Luspatercept is prohibited by the World Anti-Doping Agency. Several detection methods for ActRIIB-Fc products have been described; they mostly use an initial immopurification step using antibodies recognizing the extracellular domain of ActRIIB followed by immunodetection after electrophoretic separation of the proteins by their molecular weight (SDS-PAGE or SAR-PAGE) or by their charge (IEF-PAGE), or identification of specific peptides by LC-HRMS or LC-MS/MS after tryptic digestion. Detection limits ranged between 50 ng/mL and 1 ng/mL considered appropriate to identify the product for several weeks after an administration of a therapeutic dose. However, all previous studies have been performed after spiking the drug in vitro into the sample matrix. The objective of this study was to perform the first administration of luspatercept in healthy volunteers for antidoping purpose and to evaluate the detectability in serum, dried capillary blood spots (DBS, collected using TASSO M20 device), and urine. Indirect detection was also evaluated by analyzing hematological parameters for the Athlete Biological Passport. Four volunteers (2 males, 2 females) received one subtherapeutic dose of luspatercept (0.25mg/kg) followed 3 weeks after by a second dose. Samples were collected from before administration until 7 weeks after the second dose. After immunopurification, electrophoretic separation (SDS-/SAR- or IEF-PAGE) and immunodetection, luspatercept was detected at high levels in serum until the end of the collection, sign of a very slow elimination and similarly detected unchanged at lower levels in urine from two days after the first administration until 7 weeks post-administration. DBS showed also the same long window of detection. Indirect detection of use of Luspatercept was more complex because the changes observed on hematological markers reticulocytes (RET%) and haemoglobin despite a clear trend to increase were however of limited amplitude and only two subjects presented atypical points outside the physiological limits during the study. In conclusion this in vivo study showed that luspatercept is readily detectable post-administration in serum (the most sensitive matrix) but also, urine and DBS for several weeks after a subtherapeutic dose 1 2 1 1 2 2 1 1 2 ® ® MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3179 using the electrophoretic methods SDS-, SAR- or IEF-PAGE. After a suspicion a changeof electrophoretic method and detection antibody can be used to confirm the presence of the drug in the sample. If luspatercept detection is introduced in the screening method for ESA by antidoping laboratories, the probability to catch any athlete using this product for doping will be very high. For more details refer to: Marchand A, Miller G, Martin L, Gobbo C, Crouch AK, Eichner D, Ericsson M. (2022) Detection of erythropoiesis stimulating agent Luspatercept after administration to healthy volunteers for antidoping purposes. Drug Test Anal. 2022 Nov;14(11-12):1952-1961. doi: 10.1002/dta.3341 Figure 1. Luspatercept detection in urine, serum and DBS post administration * The 2022 Manfred Donike Award for the best oral presentation went to Alexandre Marchand, Head of Biology at the French Anti-Doping Laboratory (AFLD), for his talk on the detection of luspatercept after administration in humans. He presented initial data on the detection window and applicable matrices including serum, DBS and urine for the detection of luspatercept. The information provided is critical in assessing if and how well the newly approved drug can be analysed using established anti-doping testing methods, especially in light of the fact that haematological parameters proved to be less sensitive than direct electrophoretic approaches in detecting manipulation with the recombinant fusion protein composed of human activin receptor type IIB and a fraction of immunoglobulin G. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3180 Naud J, Desharnais P Detection of activin receptor type IIA and IIB-Fc fusion proteins by automated capillary immunoassay Laboratoire de contrôle du dopage INRS - Centre AFSB, Laval, Canada Abstract Members of the transforming growth factor-β (TGFβ) superfamily, which include TGFβs, activins, growth differentiation factor 11 (GDF11), and bone morphogenetic proteins (BMPs) have been reported as essential regulators of erythropoiesis. Ligands in this large superfamily can limit RBC production by accelerating erythroid differentiation and inhibiting erythroid progenitor expansion. Sotatercept (ACE- 011) and luspatercept (ACE-536) are among the TGFβ ligand traps therapeutic fusion proteins in clinical trials. Sotatercept, originally developed to treat bone-loss disorders, revealed unexpected effects including increase in hematocrit and hemoglobin levels in human subjects. Luspatercept (ACE- 536), improves late-stage erythroid differentiation and help in the correction of anemia. Commercialized under the brand Reblozyl by Bristol Myers Squib, luspatercept has completed successfully phase 3 studies and has been recently approved by the US Food and Drug Administration (FDA) in 2019 and by the European Medecines Agency (EMA) in 2020. Because of their potential erythropoiesis stimulating potential, ActRIIA/B-Fc fusion proteins could be used in sports as performance-enhancement agents. Until now, several methods have been proposed for the detection of these molecules in human blood samples. Most of them are used for erythropoiesis stimulating agents (ESA) detection. Mass spectrometric strategies has also been suggested for their detection in blood and dried blood spots (DBS) Here, we report the detection of the ActRIIA-Fc and ActRIIB-Fc fusion proteins by automated capillary immunoassay (Simple Western). The lowest dose detected was 1.56 pg and 3.12 pg for sotatercept and lucpatercept, respectively (Figure 1). In serum samples, a LOD of 0.2 ng/mL was obtained for both molecules. Results obtained so far indicated that the method would be able to detect both molecules in dried blood spots. Finally, the method presented is suitable for the detection of luspatercept and sotatercepts and could be easily implemented by antidoping laboratories. Published as: Desharnais P, Naud JF. Detection of activin receptor type IIA and IIB-Fc fusion proteins by automated capillary immunoassay. Drug Test Anal. 2022 Nov;14(11-12):1938-1951. doi: 10.1002/dta.3378. Acknowledgements This work was funded by Partnership for Clean Competition (252557 R120). ® MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3181 Figure 1. Detection limits for ActRIIA-Fc and ActRIIB-Fc reference preparations. Samples were initially prepared to contain 50 pg of each molecule in 4 µL of PBS 1X/BSA 0.0025 %. Serial dilutions were performed down to 1.56 pg for each molecule, and samples were heat denatured following the addition of 1 µL of PS 5X buffer. Amounts are based on the absolute quantity in 5 µL of sample loaded on the wells of the pre-filled plates and analyzed by Wes automated capillary electrophoresis immunoassay. Detection limit, presented on the right panel is based on the lowest amount for which a peak with a ratio S/N > 10 is obtained. A) Detection limit for ActRIIA-Fc. B) Detection limit for ActRIIB-Fc. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3182 Krombholz S, Thomas A, Thevis M Investigations into the in vitro metabolism of hGH and IGF-I employ- ing a stable-isotope-labelled reporter ion screening approach Institute of Biochemistry, German Sport University, Cologne, Germany Abstract Studying the metabolism of prohibited substances is an essential element in anti-doping research in order to facilitate and improve detectability. Whilst pharmacokinetic studies on healthy volunteers are valuable, they are often difficult, not least due to safety reasons and ethical constraints, especially concerning peptidic substances, which must be administered parenterally. Hence, there is a growing need for suitable in vitro models and sophisticated analytical strategies to investigate the metabolism of protein- and peptide-derived drugs. These include human growth hormone (hGH) and its main mediator insulin-like growth factor-I (IGF-I), both prohibited in professional sports for their anabolic and lipolytic effects, while challenging in their detection, as they occur naturally in the human body. Within this study, the in vitro metabolism of hGH and IGF-I in various body fluids was investigated using a stable-isotope- labelled reporter ion screening strategy (IRIS). The experiments were performed with the uniformly- N- labelled peptides in human skin and liver S9 mix, as well as urine and serum. A combination of liquid chromatography, high-resolution mass spectrometry, and characteristic immonium ions generated by internal dissociation of the stable-isotope-labelled peptidic metabolites enabled the detection of specific fragments. Several degradation products for hGH and IGF-I were identified. Additionally, their stability in human serum was assessed, as these metabolites, potentially even indicative for subcutaneous administration of the drugs, could serve as promising targets for the detection of hGH and IGF-I misuse in future anti-doping applications. Figure 1. AIF chromatogram of the extracted ion traces corresponding to the most abundant N-labelled immonium ions, showing the metabolites obtained after incubation of N-hGH in skin S9 mix (a) in comparison to the respective substrate blank (b) 15 15 15 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3183 Published as: Krombholz S, Thomas A, Thevis M. Investigations into the In Vitro Metabolism of hGH and IGF-I Employing Stable-Isotope-Labelled Drugs and Monitoring Diagnostic Immonium Ions by High-Resolution/High- Accuracy Mass Spectrometry. Metabolites. 2022 Feb 4;12(2):146. doi: 10.3390/metabo12020146. PMID: 35208220; PMCID: PMC8877552. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3184 Paßreiter A, Naumann N, Thomas A, Thevis M How to detect CRISPR with CRISPR for doping control purposes employing SHERLOCK Institute of Biochemistry, German Sport University, Cologne, Deutschland Abstract The CRISPR/Cas systems have rapidly evolved as one of the most frequently utilized gene editing tools of these days. Due to an ever-expanding targeting range of different Cas effectors, the CRISPR/Cas systems enable highly specific gene editing of virtually any desired DNA sequence. Moreover, the application of those CRISPR tools is very simple and cost-effective compared to other gene editing techniques, which unfortunately also promotes the illicit utilization of CRISPR/Cas in order to achieve performance enhancing effects in elite sports. Consequently, there is an urgent need for direct detection of illegally applied CRISPR/Cas methods in doping control samples, which demonstrates the focus of the here presented study by employing Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) for targeted nucleic acid detection. SHERLOCK serves as an in vitro CRISPR-based diagnostic (CRISPR-Dx) platform, which leverages the target-dependent promiscuous ribonuclease activity, also referred to as “collateral activity”, of the CRISPR effector Cas13a in combination with isothermal amplification. Hence, the aim of this study was the development of an analytical method that enables the detection of sgRNA associated with Cas9 from Streptococcus pyogenes (SpCas9) in serum samples by means of reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection via SHERLOCK in combination with a complementary gel-based screening procedure in order to uncover illegal doping attempts with lipid mediated CRISPR RNP complexes. Initial qualitative method characterization confirmed the selectivity of both procedures and established a detection sensitivity of 10 nM target sequence. Furthermore, an in vitro study simulating a hypothetical gene doping scenario revealed a detection window extending 24 h, supporting the proposal to apply these test strategies for authentic doping control samples in the future. Published as: Paßreiter A, Naumann N, Thomas A, Grogna N, Delahaut P, Thevis M. How to detect CRISPR with CRISPR - employing SHERLOCK for doping control purposes. Analyst. 2022 Nov 21;147(23):5528-5536. doi: 10.1039/d2an01318e MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3185 Ponzetto F , Settanni F , Nonnato A , Nicoli R , Mengozzi G , Ghigo E , Kuuranne T Investigating physical exercise and circadian rhythm as possible confounding factors of the blood steroid profile Department of Medical Sciences, University of Turin, Turin, Italy ; Clinical Biochemistry Laboratory, City of Health and Science University Hospital, Turin, Italy ; Centre Hospitalier Universitaire Vaudois and University of Lausanne, Swiss Laboratory for Doping Analyses, Epalinges, Switzerland Abstract Anti-doping rule violations related to the abuse of endogenous anabolic androgenic steroids (EAAS) are currently discovered thanks to the urinary steroidal module of Athlete Biological Passport. Nevertheless, in the last few years, blood testing has emerged as a promising complementary strategy for the detection of EAAS doping [1], with various research works that have investigated the performance of longitudinal monitoring of both free and conjugated steroids serum concentrations, highlighting several markers of testosterone (T) doping [2-5]. Although this innovative approach is currently taking hold in anti-doping community, there is still lack of information regarding the possible confounding factors that could affect steroids concentrations in serum, making the interpretation of the novel “blood steroid profile” more complex and eventually giving rise to false negative as well as false positive findings. In this research project we investigated physical exercise and circadian rhythm as possible confounding factors of “blood steroid profile”. To obtain information about the effects of such factors, we analyzed biological samples collected during two different clinical studies using a recently developed UHPLC- MS/MS method for the simultaneous quantification of major circulating steroid hormones together with an extended panel of androgens’ glucuro- and sulpho-conjugated phase 2 metabolites. For investigating the effect of physical exercise, serum samples collected before and after a training session from 30 professional football players up to three times across a football season were used, while for evaluating the impact of circadian rhythm, serum samples collected from 19 healthy males at six different time points across 24 hours were analyzed. The comparison between steroids’ serum concentrations before and after monitored training sessions highlighted a significant increase in all ATCH-stimulated circulating hormones, a significant decrease in most glucuro-conjugated androgens and not significant variations in T, Dihydrotestosterone (DHT) and most of sulpho-conjugated androgens. The monitoring of circadian rhythmicity of target steroids pointed out that unlike all other free steroid hormones, which showed the highest concentrations at 8 a.m. then decreasing until 8 p.m., T, DHT and sulpho-conjugated androgens were not showing any fluctuations, while glucuro-conjugated androgens owned a shifted zenith at 10 a.m. The outcomes of these studies suggest that T and DHT, the two markers closest to the implementation in the future “blood steroid profile”, are not significantly affected by both physical exercise and circadian rhythm. Furthermore, androgens phase II metabolites and in particular sulpho- conjugated forms proved to have satisfactory performance in terms of stability related to the investigated confounding factors and to the intra-individual variability, being therefore highlighted as valuable markers of EAAS doping. 1 2 2 3 2 1 3 1 2 3 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3186 References 1. D.J. Handelsman, S. Bermon, Detection of testosterone doping in female athletes, Drug Test Anal, 2019, 11(10):1566-1571. 2. F. Ponzetto, J. Boccard, R. Nicoli, T. Kuuranne, M. Saugy, S. Rudaz, Steroidomics for highlighting novel serum biomarkers of testosterone doping, Bioanalysis, 2019, 11(12):1171-1187. 3. V.S. Nair, K. Sharpe, J. Husk, G.D. Miller, P. Van Eenoo, A. Crouch, D. Eichner, Evaluation of blood parameters by linear discriminant models for the detection of testosterone administration, Drug Test Anal, 2021, 13(7):1270-1281. 4. O. Salamin, R. Nicoli, T. Langer, J. Boccard, C. Schweizer, C. Xu, S. Rudaz, T. Kuuranne, N. Pitteloud, M. Saugy, Longitudinal evaluation of multiple biomarkers for the detection of testosterone gel administration in women with normal menstrual cycle, Drug Test Anal, 2021, doi: 10.1002/dta.3040. 5. T. Piper, H. Geyer, E. Nieschlag, L. Bally, M. Thevis, Carbon isotope ratios of endogenous steroids found in human serum – method development, validation, and reference population-derived thresholds, Anal Bioanal Chem, 2021, 413(22):5655-5667. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3187 Goodrum J , Lewis L , Fedoruk M , Eichner D , Miller G Feasibility of microcapillary whole blood collections for usage in athlete biological passport analysis The Sports Medicine Research and Testing Laboratory (SMRTL), Salt Lake City, United States ; United States Anti-Doping Agency, Colorado Springs, USA Abstract The hematological module of the Athlete Biological Passport (ABP) is an important tool in the pursuit to identify blood doping in athletes. This module is based on a complete blood count of a whole blood sample performed using a Sysmex XN hematology analyzer, and tracks parameters known to be affected by blood doping, specifically hemoglobin (HGB), reticulocyte percentage (RET%), and the combined stimulation index OFF-Score (10 x HGB x 60 x sqrt(RET%)). Currently, collecting blood samples for ABP analysis is cumbersome, invasive, and expensive; involving a venous blood draw performed by a trained phlebotomist followed by cold chain monitored shipping to the analysis laboratory. Developing innovative methods to collect and transport ABP blood samples while adhering to strict pre-analytical and analytical requirements has the potential to greatly increase testing frequency, and consequently, the effectiveness of the ABP program globally. One such method utilizes micro volumetric capillary blood collection via an upper arm site. This collection method does not require a trained phlebotomist and would allow for increased collection frequency, decreased transport costs associated with a smaller sample volume (and, smaller sample tube), and decreased athlete discomfort. The focus of this study was to compare venous and capillary blood collections to determine if capillary samples would be suitable for ABP analysis without sacrificing the analytical integrity required for anti-doping testing procedures. In this study, capillary blood was collected using the Tasso+ EDTA device, a novel micro-volumetric device that collects liquid, whole blood from skin capillaries on the upper arm. First, agreement between venous and capillary samples collected in tandem from 29 participants was assessed for all complete blood count parameters. Excellent agreement was observed between venous and capillary blood samples for most of the parameters considered as part of the ABP, including the three main ABP parameters: HGB, RET%, and OFF-Score. Plots of capillary values against venous values as well as Bland-Altman plots with calculated biases (calculated as capillary values minus venous values) and 95% limits of agreement for HGB, RET%, and OFF-Score can be seen in Figure 1. For the other parameters considered as part of the ABP, the only parameter to show a substantial difference between venous and capillary samples was platelets, which exhibited a 93.6 x 10 µL negative bias in capillary samples. Next, the stability of capillary samples collected from 10 participants after storage at 4°C, similar to what would be required during transport, was assessed. The stability was acceptable for up to 72 hours with only small, but statistically significant, changes observed in the three main ABP parameters. Specifically, we observed a 0.2 g/dL increase in HGB at 48 hours, a 0.184% increase in RET% at 72 hours, and a 3.27 increase in OFF-Score at 24 hours. These changes are small in the context of the ABP and would be unlikely to have a functional impact on passport interpretation. 1 2 2 1 1 1 2 6 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3188 Finally, we generated individual ABP profiles using the adaptive model for 10 participants over the course of 6 weeks. We observed excellent agreement between profiles generated with venous samples and profiles generated with capillary samples. No values from either sample type exceeded the adaptive model upper and lower limits, and any small variations apparent would not change the outcome of profile interpretation. The stability of the profiles indicate that values from one sample type could be interchanged with values obtained from the other sample type with minimal to no impact on the overall ABP profile. One limitation to micro volumetric capillary blood collections is the limited sample volume. For the Tasso+ EDTA devices used in this study, approximately 0.5 mL of blood is collected compared to the 3mL of blood collected in a typical venous draw. This volume only allows for about 4 runs on the Sysmex analyzer, which may create problems for sample acceptance during analysis. Additionally, this limited volume complicates later stage testing for erythropoiesis stimulating agents (ESAs) in plasma. In conclusion, these results indicate capillary blood collection with the Tasso+ EDTA device is a viable alternative to venous blood collections for ABP analysis and represents a groundbreaking shift in ABP collections, especially for large events where samples are analyzed on site, or in geographically challenging locations where a phlebotomist may not be available. Published as: Goodrum JM, Lewis LA, Fedoruk MN, Eichner D, Miller GD. (2022) Feasibility of micro- volumetric capillary whole blood collections for usage in Athlete Biological Passport analysis. Drug Test Anal. 2022; 14(7): 1291- 1299. doi:10.1002/dta.3254 Figure 1. Graphs plotting venous blood values against capillary blood values and Bland-Altman plots for HGB, RET%, and OFF-Score MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3189 Al-Jaber M , Al-Nesf A , Mohamed-Ali N , Acquaah V , Al-Nesf M , Mohamed Y , Orie NN , Voss SC , Georgakopoulos C , Bhatt R , Al-Ansari N , Beotra A , Al-Maadheed M , Mohamed-Ali V Untargeted metabolomics identifies a novel panel of markers for autologous blood transfusion Antidoping Lab Qatar, Doha, Qatar ; Centre for Metabolism and Inflammation, Division of Medicine, University College London, London, United Kingdom ; Hamad Medical Corporation, Doha, Qatar ; Department of Applied Health Research, University College London, London, United Kingdom Abstract Early conflicting reports on whether blood transfusion effects are too transient to be beneficial, are recently overshadowed by growing evidence on its aerobic and endurance enhancing effects. In the fight against doping, Homologous Blood Transfusion (HBT) can be identified by 1.) differences in expression of minor blood group antigens or 2.) short tandem repeats (STR) DNA analysis. On the other hand, autologous blood transfusion (ABT) resemble a greater challenge as blood is reinfused to the same donor. Current indirect method of ABT detection include Athletes Biological Passport. In this study a direct approach was taken to identify biomarkers to detect ABT by Metabolomics Profiling. After ethical approval, healthy male volunteers were recruited and 1 unit of blood (450 mL) was collected and stored for reinfusion. Blood and urine samples were collected at baseline (D0), post-donation (D2-D32) and 1- 168 hours post-transfusion. Total of 132 samples (blood and urine) were investigated using metabolomics analysis. More than a thousand metabolites were found in urine of which 912 were with known structural identities. In serum, 990 were found and 781 were of known identities. Data was analyzed and metabolites with p < 0.05 and a fold change of > 2 were identified. Data analysis revealed Seventeen known metabolites that were significantly altered throughout the experiment. Eight were not sustained in the post-transfusion stage. Four of the these 17 were found in serum (glycocholate, glycochenodeoxycholate, 12-HETE and lactosyl-N-palmitolspingosine). The other four were urinary (cystathionine, glucuronide of C H O (10*), glucuronide of C H O (1*) and enterolactone). Interestingly, upregulation of serum 12-HETE was a particularly robust marker for blood donation, which returned to baseline levels within 96 h post-transfusion. The remaining nine metabolites were significantly changed up to day 7 (+168 h) post-transfusion hence selected for the panel of biomarkers. These comprised of 7 metabolites identified in serum and only 2 in urine. The serum metabolites (glycoursodeoxycholate, downregulated; guanosine and inosine, upregulated) were significantly altered both post-donation and post-transfusion, compared to baseline (Figure 1 I–III). The other four serum metabolites in this panel (S-allcysteine, 17-alphahydroxypregnenalone 3, Glutamine conjugate of C H O (2)* and Sphinganine) were only altered post-transfusion (Figure-1 IV–VII). Though two plasticizers were transiently elevated in urine post-donation and posttransfusion, the selected panel did not include plasticizers and components of storage preservatives as these have been proven unreliable as markers for autologous blood transfusion. The seventh day post-transfusion time point allowed for the analysis of changes, which were independent of storage duration, concentration or dilution effects. 1 1 1 2 3 3 1 1 1 4 1 1 1,2 1 1 2 3 4 10 22 2 12 22 3 6 10 2 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3190 This study introduced two additional urinary metabolites as biomarkers, which are not plasticizers. This may be through utilizing different models of metabolomic analysis adopted by this study in comparison to previous reports. Our model showed good concordance in the data generated by reanalysis of samples in both our laboratory (HD4 ADLQ) and Metabolon Inc (HD4 Durham). Both ADLQ and the Durham HD4 platforms showed good recovery of metabolites from samples stored for 6–7 years. Thus, our model was stringently tested. Published as: Al-Nesf A, Mohamed-Ali N, Acquaah V, Al-Jaber M, Al-Nesf M, Yassin MA, Orie NN, Voss SC, Georgakopoulos C, Bhatt R, Beotra A, Mohamed-Ali V, Al-Maadheed M. Untargeted Metabolomics Identifies a Novel Panel of Markers for Autologous Blood Transfusion. Metabolites. 2022 May 10;12(5):425. doi: 10.3390/metabo12050425. Figure 1. Serum concentrations of 7 metabolites altered at different time points during the experi- ments. The log concentrations at different time-points are plotted for each metabolite as labelled in I to VII. * p < 0.05, ** p < 0.01, compared with baseline. References 1. Atkinson, T.; Kahn, M. Blood doping: Then and now. A narrative review of the history, science and efficacy of blood doping in elite sport. Blood Rev. 2020, 39, 100632. [CrossRef] [PubMed] 2. Lippi, G.; Banfi, G. Blood transfusions in athletes. Old dogmas, new tricks. Clin. Chem. Lab. Med. 2006, 44, 1395–1402. 3. D’Alessandro, A.; Reisz, J.; Zhang, Y.; Gehrke, S.; Alexander, K.; Kanias, T.; Triulzi, D.J.; Donadee, C.; Barge, S.; Badlam, J.; et al. Effects of aged stored autologous red blood cells on human plasma metabolome. Blood Adv. 2019, 3, 884–896. 4. Bejder, J.; Gürdeniz, G.; Cuparencu, C.; Hall, F.; Gybel-Brask, M.; Andersen, A.B.; Dragsted, L.O.; Secher, N.H.; Johansson, P.I.;Nordsborg, N.B. An Untargeted Urine Metabolomics Approach for Autologous Blood Transfusion Detection. Med. Sci. Sports Exerc. 2020, 53, 236–243. 5. Meeker, J.D.; Sathyanarayana, S.; Swan, S.H. Phthalates and other additives in plastics: Human exposure and associated health outcomes. Philos. Trans. R Soc. Lond B Biol. Sci. 2009, 364, 2097–2113. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3191 Buisson C , Touzani Z , Tekla E , Ericsson M Usefulness of artificial intelligence to enhance the Athlete Biological Passport – a pilot study on the steroidal module Laboratoire AntiDopage Francais (LADF), Chatenay-Malabry, France ; Deloitte Canada, Montreal, Canada Abstract The progress both in terms of accessibility and performance of machine learning (ML) in the last few years made it possible to be generalized to specific fields like anti-doping. During the evaluation of data from the ABP steroidal module, an important number of samples are wrongly flagged by ADAMS as suspicious or atypical. The reviewing of all these suspicious or atypical passports is time-consuming and could require several experts. This study proposes to develop a machine learning model to complement and refine ADAMS conclusions on passports by reducing the number of false-suspicious or false-atypical passports based on supervised learning. The problem we propose to tackle is a good candidate for machine learning for several reasons: There is one source of standardized data, the ABP steroidal module. The data is standardized, having 6 values and 5 ratios. There is an important quantity of data for training machine learning, more than 10,000 data are generated each year by anti-doping laboratories such as Paris lab. Moreover, those data are of high quality since standardized procedures are applied. Finally, the current way of analysis by experts is methodological and the activity is time- consuming for APMU's personnel. We used two different datasets for this study. The first one was with Paris APMU data only and the second set was with Paris laboratory and other longitudinal studies such as clinical studies. The main and most promising results were obtained on the first dataset. For the supervised learning approach, we have divided the data set into two parts (70% for machine learning and 30% for validation). The learning models used in this project are the Logistic Regression, the Random Forest, the XGBoost, the K-Nearest Neighbor, the Naïve Bayes, the Support Vector Machine (SVM) and the Multilayer Perceptron. For the unsupervised learning approach, we used a Principal Component Analysis (PCA) and an Auto-encoder. The results of this project validated our hypothesis that machine learning models have the capacity to learn from the data of the steroidal module. We have identified that the best individual machine learning models for that purpose ranked by performance are the SVM, the Naïve Bayes and the logistic regression. An ensemble voting model with the previous models including XGBoost had a similar performance in identifying the true-positive as the most performing machine learning model while reducing the number of false-positive. When compared to ADAMS, the performance of the ensemble voting model in identifying true-positive matched ADAMS performance, yet flagging significantly less false-positive than ADAMS. The use of anomaly detection technique with a PCA or an Auto-encoder shows that they could be useful to detect positive IRMS, although they performed less than the supervised learning models. An advantage of this approach is that the data don’t need to be labeled, this could remove possible human bias in the initial decision to do an IRMS to confirm a suspicious result. Unsupervised learning is a venue to be explored. 1 2 2 1 1 2 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3192 Different possibilities could be used to further improve the models. First, by adding more features. Some variables like the sport were not included when training these models. Also, using confounding factors could further improve the models. Next, to take into consideration the longitudinal aspect of the observations some learning algorithms could be tried like the LSTM. Finally, increasing the number of IRMS observations in general and positive ones, in particular, could also help enhance and improve the models. The results of this experiment have to be scaled with more data to confirm those preliminary findings, especially those related to the performance of the models. This work will be published in a peer-reviewed journal. Acknowledgements We thank the World Anti-Doping Agency and Fond de recherche du Quebec for funding this work. We would also like to thank the French Antidoping Agency and the Latvia Antidoping Agency for their permission to use the data from their athletes. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3193 Gotzmann A, Trinks S Application of the dried blood spot technique under different perspectives of a National Anti-Doping Organization National Anti-Doping Agency, Bonn, Germany Abstract The COVID-19 pandemic has shown that a testing system based on direct contact between control personnel and athletes can reach its limits. Lockdown phases and quarantine measures imposed by state authorities are likely to cause a considerable imbalance in the national and international testing system. In early 2020, NADA Germany has initiated two research projects to conduct non-contact tests by using the dried blood spot (DBS) method. The main goal of these two projects is to offer an alternative in testing. It is also important for NADOs and athletes to counteract the "general suspicion of unlimited doping" during this time. Basic experiences in handling and using the DBS in cooperation with German athletes had already been gained by NADA Germany in three previous projects since 2015. The latest research project launched by NADA Germany will further develop the online testing approach. For the first time, a remote testing solution is now being proofed. A newly developed application (App) has been optimized in order to completely map the administrative part of the whole control process. New test kits are also being tested in order to check their handling in practice. At the same time, the collected DBS samples provide a valid basis for protecting clean athletes from unjustified doping allegations, such as manipulation or sabotage acts by using analytical measures. The athletes themselves can take those tests independent and autonomous. Experts from NADA Germany accompany the process via the App for remote testing. This eliminates the need for direct contact between athletes and third parties. Within the framework of the present research project, the DBS samples are stored at the Institute for Biochemistry, German Sport University Cologne. In case of suspicion of tampering, sabotage or contamination, an analysis of the stored DBS samples will be initiated. The introduction of remote testing as a new testing method alongside the classic urine, blood and plasma samples will make the testing system more variable and increase the unpredictability of test dates to a considerable extent. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3194 Loria F , Stutz A , Rocca A , Grabherr S , Kuuranne T , Pruijm M , Leuenberger N Monitoring of hemoglobin and erythropoiesis-related mRNA in athletes and patients dried blood spots Swiss Laboratory for Doping Analysis, Lausanne University Hospital, Lausanne, Suisse ; Service of Nephrology, Lausanne University Hospital, Lausanne, Switzerland ; University Center of Legal Medicine, Lausanne University Hospital, Lausanne, Switzerland Abstract We assessed the feasibility of monitoring hematological parameters, such as hemoglobin (HGB) and reticulocyte (RET) mRNA in dried blood spots (DBS), to improve the Athlete Biological Passport (ABP) and patient care. Here, we measured HGB and erythropoiesis-related mRNA from ALAS2 and CA1 in venous blood (VB) and DBS from healthy athletes and hemodialysis patients on MIRCERA treatment. Finally, EPO was directly measured in DBS using ELISA. Changes in HGB over time were well captured with both VB and DBS. When combining HGB and mRNA analysis in DBS, the DBS off-score was more sensitive than the classical ABP off-score to detect EPO use. Moreover, DBSs are more efficient also for direct EPO detection. To conclude, DBS represents a practical new tool for the analysis of HGB and off-score calculation, and could help to enhance blood doping detection and predict the response to EPO in hemodialysis patients. Published as: Loria F, Stutz AP, Rocca A, Grabherr S, Kuuranne T, Pruijm M, Leuenberger N. Monitoring of hemoglobin and erythropoiesis-related mRNA with dried blood spots in athletes and patients. Bioanalysis. 2022 Mar;14(5):241-251. doi: 10.4155/bio-2021-0252. 1 2 1 3 1 2 1 1 2 3 ® MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3195 Salamin O , Nicoli R , Saugy M , Pitteloud N , Kuuranne T Dried blood spot as alternative matrix for the blood steroid profile and the detection of testosterone doping in women Laboratoire Suisse d'Analyse du Dopage Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Epalinges, Suisse ; Research and Expertise in antiDoping sciences (REDs), University of Lausanne, Lausanne, Switzerland ; Service of Endocrinology, Diabetology and Metabolism, Lausanne University Hospital, Lausanne, Switzerland Abstract While the implementation of the ‘urinary steroidal module’ of the Athlete Biological Passport (ABP) improved the testosterone detection capability, various confounding factors may influence the urinary steroid profile complicating its interpretation and decreasing its sensitivity, notably for targeting the confirmatory isotope ratio mass spectrometry (IRMS) analysis. In addition, it is believed that athlete rather resort to low doses of topical testosterone, which significantly reduces peaks of urinary concentrations that are difficult to discriminate from natural variability. To overcome those limitations, recent studies highlighted the great potential of the blood steroid profile as a sensitive complementary approach to the urinary steroid profiling for the detection of testosterone doping, especially in women. However, the application of serum steroid profile is associated with some pre-analytical constraints, such as the need of a trained phlebotomist or specific shipping conditions among others. The use of dried blood spot (DBS) as surrogate matrix for the blood steroid profile offers a convenient and valuable strategy with simplified collection and shipping conditions and allows for more frequent anti-doping sampling. In the present work, a sensitive UHPLC-MS/MS method was developed and validated for the simultaneous determination of 11 free and 8 conjugated steroids in DBS [1]. It was applied for the analysis of samples collected weekly in 14 healthy women during a normal menstrual cycle (control phase) followed by a 28- days testosterone gel treatment (treatment phase) and another menstrual cycle. DBS samples were either collected at the fingertip with a volumetric HemaXis DB-10 device or generated with a calibrated micropipette using whole blood samples collected at the same time. Hematocrit-corrected concentrations were then were compared with those obtained from concurrent serum samples collected simultaneously. For most of the quantified compounds, the results demonstrated a high correlation between DBS and serum concentrations with the exception of testosterone during and just after T gel administration. For these samples, in comparison to serum, surprisingly high testosterone concentrations were observed in capillary DBS. It also seemed that the testosterone values increased as the treatment progressed suggesting a potential accumulation. On the contrary, DBS generated using whole blood collected in EDTA, representing the systemic concentration, demonstrated excellent agreement with testosterone concentration in serum. When the mean testosterone concentrations measured throughout the study were compared between both matrices, they demonstrated similar kinetics with no significant difference. In addition, the other analytes demonstrated satisfactory correlation with serum matrix during and after treatment. Therefore, we made the assumption that residual T gel T is trapped locally in the stratum 1 1 2 3 1 1 2 3 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3196 corneum of the finger acting as a reservoir. This residual testosterone could then cause such extreme testosterone values in capillary blood when collected by finger prick. This hypothesis was further confirmed in a preliminary study by collecting DBS from various sites of collection before and after testosterone gel contact. In the control condition, DBS were collected by finger prick, or on the upper arm with TAP and Tasso devices and generated with whole blood collected by venipuncture. Then a small amount of testosterone gel was applied on an inert surface with the right hand with no glove. After 1 and 6 hours, DBS were collected by finger prick on the right hand corresponding to the applicator, on the left hand covered with a glove in-between, on the upper arm and generated with whole blood collected with venipuncture. Following the analysis of the samples, we observed that extremely high testosterone values were measured in the DBS collected from the right hand used for application and that this concentration remained elevated until 6h after the contact with testosterone gel (Figure 1). On the contrary, testosterone concentration measured in the DBS from the left hand, covered with a glove, exhibited a slight increase 1h after testosterone gel contact which corresponds to the small amount of testosterone absorbed at the systemic level. Similar results were obtained with DBS generated from whole blood and collected with TAP and Tasso on the upper arm. These observations confirm the hypothesis that testosterone is persisting in the finger skin and that local testosterone could interfere and generate high testosterone concentration in capillary blood collected by finger-prick. Nevertheless, further studies should be carried out to confirm this hypothesis by combining testosterone gel application and multiple capillary blood collection sites on several subjects. Figure 1. Testosterone concentration measured in different DBS collected at various sites before and after (1 and 6 hours) testosterone gel contact References 1. O. Salamin, R. Nicoli, C. Xu, J. Boccard, S. Rudaz, N. Pitteloud, M. Saugy, T. Kuuranne. Steroid profiling by UHPLC-MS/MS in dried blood spots collected from healthy women with and without testosterone gel administration. J. Pharm. Biomed. Anal., 2021, 114280 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3197 Garzinsky A, Thomas A, Guddat S, Görgens C, Dib J, Thevis M Dried blood spots for doping controls – development of a comprehensive initial testing procedure with fully automated sample preparation Institute of Biochemistry, German Sport University, Cologne, Germany Abstract Over the past two decades, extensive research has been conducted on the applicability of dried blood spots (DBS) as a complementary matrix for the detection of doping substances, owing to several benefits compared to blood and urine. Given the small sample volume required, DBS provide a minimally invasive and fast sampling procedure and enable space-saving transport and storage. Among additional advantages, DBS as a sample matrix could allow for remote testing in the future, which has gained interest during the ongoing pandemic. Following the publication of a technical document by the World Anti-Doping Agency (WADA) and the associated harmonization of sampling and analysis procedures, DBS became particularly relevant for routine application. The objective of this study is the extension of an already established protocol to a comprehensive Initial Testing Procedure (ITP) that includes various substances from all groups of the Prohibited List. The sample preparation is accomplished in a fully automated procedure by using a multi-purpose sampler connected to a DBS autosampler. Subsequent analysis of extracted substances is conducted using high-resolution tandem mass spectrometry (MS) in Full MS and Data-Independent Acquisition mode. According to the criteria established by WADA, the protocol is validated in terms of selectivity, detection limit, carryover, reproducibility and stability of the sample extract within an ongoing process. For proof of context, DBS collected after the administration of representatives of various substance classes such as stimulants, glucocorticoids and beta-blockers were tested for signals triggering a Confirmation Procedure. The automated sample preparation as well as the chromatographic separation has been successfully optimized for a wide range of analytes and the selected MS modes allow flexible adaptation and extension of the substance list. In total, the validation process included over 200 substances that are tested for reasonable LODs, which was confirmed by the analysis of post-administration samples for several compounds. Prospectively, the sample preparation can be adjusted for certain substance classes to achieve improved sensitivity. Overall, the incorporation of the ITP developed within this study into routine practice accomplishes the requirements arising from an anticipated future expanded use of DBS in doping controls. The details of this study will be published elsewhere. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3198 Okano M, Ikekita A, Sato M, Kageyama S, Inoue T, Akiyama K, Aoi A, Miyamoto A, Momobayashi A, Ota M, Saito M, Sakurai H, Shiomura S, Takemine M, Watanabe Y, Hikota T Doping control analyses during the Tokyo 2020 Olympic and Paralympic Games Anti-Doping Laboratory, LSI Medience Corporation, Tokyo, Japan Abstract We summarise the doping control analyses performed at the XXXII Olympic Games (Jul. 23- Aug. 8, 2021) and the XVI Paralympic Games (Aug. 24- Sep. 5, 2021) held in Tokyo, Japan after a year of delay due to the COVID-19 pandemic. A new satellite facility of the existing WADA accredited Tokyo laboratory was established and fully operated by 278 staff, including 90 Tokyo laboratory analysts/administrative staff, 49 international experts and 139 Japanese temporally staff from five universities. All urine samples for all substances on the standard analysis menu but also for small peptides and Myo-inositol trispyrophosphate were analyzed. Upon requested by the ITA and the IPC, the selected urine samples were analyzed for large peptides, EPOs and endogenous AAS when administered exogenously. The laboratory also had the capacity to analyze for rhGH in serum (i.e. Both isoform and biomarkers tests) and for EPOs in serum and plasma, as well as for ABP markers, haemoglobin-based oxygen carriers and homologous blood transfusion (HBT). In addition, for the first time ever, we applied a new PCR method for detection of cDNA-EPO doping during the Olympics. The laboratory also analyzed blood samples for the presence of steroid esters following the spotting of collected intravenous EDTA blood onto dried blood spot (DBS) cards at the laboratory. Moreover, full scan/data acquisition analysis by GC-HRMS and LC-HRMS were conducted for all urine samples collected during the Olympics, which might be possible to find traces of doping substances that are not currently being analyzed in future data processing prior to sample reanalysis. For the Olympics, 5,079 urine samples and 1,104 blood samples were analyzed. In 19 samples, the presence of a prohibited substance was confirmed, resulting in 8 atypical findings (ATFs), and 11 adverse analytical findings (AAFs) were reported including HBT (2 cases) and rEPO in blood (1 case). During the Olympics, B-sample confirmation analyses were performed 3 times. By using the paperless chain-of- custody system, laboratory could ready for analysis in advance, which allowed improving fast turnaround time reporting. For the Paralympics, 1,695 urine samples and 479 blood samples were analyzed. In 12 urine samples, the presence of a prohibited substance was confirmed, resulting in 2 ATFs and 10 AAFs. Further analysis requested by APMUs were ~250 samples for the Olympics and ~60 samples for the Paralympics. WADA double blind EQAS samples were received/analyzed, and correctly reported during the Olympics (6 urine samples) and the Paralympics (4 urine samples). We would like to express our sincere gratitude to everyone involved in this big project. Published as: Okano M et al. Doping control analyses during the Tokyo 2020 Olympic and Paralympic Games. Drug Test Anal. 2022 Nov;14(11-12):1836-1852. doi: 10.1002/dta.3381 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3199 Zhang L, Wang Y, Xing Y, Zhang Y, Wang S, Shen L, Wang Z Operation of the doping analysis laboratory for Beijing 2022 Winter Olympic and Paralympic Games under COVID-19 pandemic National Anti-Doping Laboratory, Beijing Sport University, Beijing, People´s Republic of China Abstract Doping analysis with fast turnaround time of 24/48 hours reporting is a “traditional” requirement for major competitions such as Olympic Games, which requires tremendously increased allocation of resources, especially under the worldwide pandemic of COVID-19. The “closed-loop” concept and operation mode established by the Beijing Organizing Committee for the 2022 Olympic and Paralympic Winter Games (BOCOG) provided a relatively isolated environment to the non-Games related civilians. To maintain this system, more than 200 persons were included as supporting crew of the laboratory with massive logistic resources allocated. The National Anti-Doping Laboratory (NADL) in Beijing carried out the analysis mission of the Beijing 2022 Olympic and Paralympic Winter Games. 3165 samples were analyzed during the Winter Olympics while 679 samples were analyzed for the Paralympics. The workforce accomplishing this work was composed of 36 domestic analysts, 20 international experts from other World Anti-Doping Agency (WADA) accredited laboratories and 61 university students of suitable majors, and 12 on-site instrumental engineers. This article is summarizing the achievements from the laboratory’s preparation phase, in-Games operational details such as instruments, methods, workforces and facility and the Quality Assurance measures to maintain the integrity and correctness of results reported to the Result Management Authority, with the effect of the pandemic and "closed-loop" situation during the whole process highlighted. Acknowledgements The success of the doping analysis work of Beijing 2022 Winter Olympics and Paralympics is attributed to the collaboration of the International Olympic Committee (IOC), the World Anti-Doping Agency (WADA), the International Testing Agency (ITA) and the Beijing Organizing Committee for the 2022 Olympic and Paralympic Winter Games (Beijing 2022). We would like to express our genuine gratitude to all the WADA experts for the assessments and suggestions which improved the operation of our laboratory, all the experts including lab directors from other WADA accredited laboratories who came in person or supported us from remote with extensive expertise, in particular under the challenge of the COVID-19 pandemic, all the logistics personnel from the Beijing Sport University, the volunteers from Peking University, and all the on-site engineers from Agilent and Thermofisher. We’d also like to give our special thanks to the Tokyo Anti-Doping Laboratory who supported us with the hGH Isoform instrument, the Rome Anti-Doping Laboratory for a batch of dozens of reference material solution of MRL substances, and the Cologne Anti-Doping Laboratory for the Dynabeads required in large peptides analysis. MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3200 Mareck U, Fußhöller G, Schertel T, Petring S, Thevis M Risk of unintentional antidoping rule violations by consumption of hemp products Center for Preventive Doping Research / Institute of Biochemistry, German Sport University Cologne, Cologne, Germany Abstract Hemp products receive continuously growing consumer and market attention, and an expanding scope of applications is recognized, supported by suppliers operating through different distribution channels with the Internet being a major retail platform. Hemp products are prepared from cannabis plants and, therefore, might contain a variety of different natural cannabinoids. According to the regulations of the World Anti-Doping Agency (WADA), all natural and synthetic cannabinoids are prohibited in-competition, with the explicit exemption of cannabidiol (CBD). Due to the fact that hemp products prepared from cannabis plants may contain natural cannabinoids, possibly leading to unintentional violations of anti- doping regulations, an investigation of 23 hemp products for presence of cannabinoids was performed. An assay for the detection of 16 cannabinoids in nutritional supplements was established. The sample preparation consisted of QuEChERS extraction, trimethyl-silylation and analysis by gas chromatography / tandem mass spectrometry (GC-MS/MS). A total of 23 commercially available hemp products was analyzed, and assay characteristics such as selectivity, limit of detection (LOD), limit of identification (LOI), limit of quantification (LOQ), linearity, precision, recovery and accuracy were determined. Twenty of 23 hemp products showed a variety of cannabinoids in occasionally high concentrations with four products covering the complete analysed cannabinoid spectrum. An ethical committee-approved controlled single dose administration study was conducted with commercially available hemp products, and 16 cannabinoids were targeted in urine samples collected after consumption of the hemp products. Variable patterns of cannabinoids or their metabolites were observed in those urine samples, where 30% of the specimens collected 8 hours after consumption exhibited the presence of a prohibited cannabinoid. Those findings would have resulted in an unintentional violation of anti-doping regulations if observed in an athlete’s doping control sample. Published as: Mareck U, Fusshöller G, Schertel T, Petring S, Huestis MA, Thevis M. Risk of unintentional antidoping rule violations by consumption of hemp products. Drug Test Anal. 2023 Jan;15(1):27-41. doi: 10.1002/ dta.3327 MANFRED DONIKE WORKSHOP 2022 Lecture RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3201 Keiler A , König S , Rzeppa S , Thieme D Agreement of steroid profiles in Athlete Biological Passport residues and corresponding serum samples Institute of Doping Analysis & Sports Biochemistry, Kreischa, Germany ; Environmental Monitoring and Endocrinology, Technische Universität Dresden, Dresden, Germany Abstract Standard steroid module of the Athlete Biological Passport (ABP) consists of a multicomponent analysis of urinary samples. However, the urinary steroid concentrations may be affected by confounders like microbial degradation, UGT2B17 gene polymorphisms affecting glucuronidation, insufficient conjugate hydrolysis or possible co-administration of diuretics as masking agents resulting in significant biological variations [1]. Therefore, it can be helpful to use other matrices to quantify steroids. Aim of the study was to investigate the feasibility to re-use plasma obtained from athlete ABP blood samples for measuring a steroid profile, based on testosterone and androstenedione initially. Therefore, intra- individual matching ABP blood and serum samples (originally collected for hGH testing; n = 36) were precipitated with methanolic zinc trifluoroacetate, centrifuged and the supernatant was directly injected into a HPLC-MS-MS system. In spite of the different storage duration of ABP blood samples (at least one month at 4°C) and serum samples (three months at -18°C), testosterone and androstenedione concentrations showed an unexpectedly high correlation and revealed an adequate agreement according to Bland-Altman analysis. Furthermore, significant haemolysis didn’t invalidate the quantified parameters. In conclusion, athlete ABP blood from the haematological module might be additionally used for steroid profiling. Published as: König S, Rzeppa S, Thieme D, Keiler AM. Agreement of steroid profiles in Athlete Biological Passport residues and corresponding serum samples. Drug Test Anal. 2022 Sep 6. doi: 10.1002/dta.3365 References 1. Mazzarino M, Abate MG, Alocci R, Rossi F, Stinchelli R, Molaioni F, de la Torre X, Botrè F. Urine stability and steroid profile: towards a screening index of urine sample degradation for anti-doping purpose. Anal Chim Acta. 2011; 683(2):221-6. 1,2 1 1 1 1 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3202 Stojanovic B , Rasic J , Andjelkovic M , Dikic N , Forsdahl G , Gmeiner G Characterization of the urinary excretion profile of higenamine after multiple dose oral administration utilizing on-line SPE LC with HRMS detection Doping Control Laboratory, Seibersdorf Labor GmbH, Seibersdorf, Austria ; Beo-Lab Laboratories, Belgrade, Serbia ; University of Singidunum, Belgrade, Serbia ; University of Tromsø, Tromsø, Norway Abstract Since 1 January 2017, higenamine is added on the World Antidoping Agency’s (WADA) Prohibited List under group S3: beta-2 agonists and is banned at all times for athletes. Higenamine is present in various natural plants such as Nandina domestica, Aconitum carmichaelii, Annona squamosa, Nelumbo nucifera, etc. Also, higenamine can be found in dietary supplements used as fat burners. Consequently, there are many different sources of higenamine and a high probability of its intake by athletes. To characterize the excretion profile of higenamine after oral intake and to define the window of opportunity for the detection of higenamine and its metabolite coclaurine is an important task to evaluate application schemes as communicated by athletes. The aim of this study was to characterize the urinary excretion profile of higenamine after multiple dose oral application of higenamine capsules. For this purpose, a double blind study including 12 female basketball players aged 28-41 was performed. Participants of this study were not included in national testing pools. The study design shows 6 players in the higenamine group and 6 players in the placebo group. Applications lasted 21 days with a dose of 3 x 25 mg daily before meals. During treatment period one, one urine sample of every player was collected every morning, while after 21 day all urine samples during the following 48 h were collected. For the detection of higenamine and its main metabolite in urine samples, a new, fast and highly sensitive quantitative on-line SPE LC HRMS method was developed and validated. Finally, the method was applied for the quantification of higenamine in urine and the excretion pattern of higenamine after multiple dose application was defined. Results obtained show substantial inter-individual differences in the excretion profile of higenamine. The concentrations for all six volunteers exceeded 10 ng/mL for at least 20 hours after the last administration of higenamine. For 3 volunteers, the urinary concentrations exceeded 10 ng/mL for more than 40 hours. In this case, higenamine could be detected for the entire post-administration collection time of 48 hours and the elimination half-life was estimated to be 17 hours. The details of this study will be published elsewhere. 1 2 3 3 1,4 1 1 2 3 4 st st MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3203 Göschl L , Gmeiner G , Gärtner P , Steinacher M , Forsdahl G Detection of DHCMT long-term metabolite glucuronides with LC-MSMS as an alternative approach to conventional GC-MSMS analysis Doping Control Laboratory, Seibersdorf Labor GmbH, Seibersdorf, Austria ; Department of Pharmacy, University of Tromsø - The Arctic University of Norway, Tromsø, Norway ; Institute of Applied Synthetic Chemistry, Technical University of Vienna, Vienna, Austria Abstract Introduction Fast and accurate analysis of dehydrochloromethyltestosterone (DHCMT) as one of the most detected illicit used anabolic-androgenic steroids in professional sports is of great importance for a constructive fight against doping abuse. The conventional method, GC-MSMS, is sensitive and selective but also very time- and resource-consuming. A new approach for simple detection with LC-HRMSMS is introduced in the presented work. Two newly described phase-II metabolites of the important DHCMT long-term metabolite 4-chloro-18-nor-17β-hydroxymethyl-17α-methyl-5β-androst-13-en-3α-ol (M3) were identified as suitable targets for analysis with an online-SPE-LC-MS approach. Experimental Identification of new phase-II metabolites: An online-SPE-LC-HRMSMS method described earlier was used for the identification of potential M3- glucuronide signals in DHCMT positive urine samples. Parallel reaction monitoring (PRM) runs using negative ionization mode with precursor mass m/z = 513.2255, which correspond to theoretical species [DHCMT-M3-mono-glucuronide – H]-, were carried out. Three potential signals, I-III, were identified. The corresponding high-resolution MSMS spectra provided strong evidence for the existence of three distinct M3 and/or M3-epimer glucuronide conjugates. Subsequently, peaks I-III were isolated by MS-online- fractionation and confirmed with the conventional GC-MSMS method using reference standards for DHCMT metabolite M3. WADA identification criteria were fulfilled by comparing retention times and two MS/MS transitions. Structure elucidation: After clearly identifying two potential metabolite M3 glucuronide conjugates, the following derivatization experiment was performed to distinguish between the two conjugation sites. Trityl chloride, known to selectively protect primary alcohols in the presence of secondary alcohols, was used. Only the metabolite with the glucuronide conjugation on position 3 is expected to be etherified with trityl chloride. The reaction was performed by mixing 500µl concentrated and evaporated sample with 5ml tritylation agent (trityl chloride in dimethylformamide and triethyl amine) and stirring for 48h at room temperature. The reaction was quenched with sat. aq. NaHCO3. After evaporating the solvents, the brown-yellowish residue was dissolved in MQ, centrifuged, and the supernatant was analyzed with the online-SPE-LC-MS mentioned above. 1,2 1 3 3 1,2 1 2 3 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3204 Results M3 glucuronide identification: The parent molecule with a mass of m/z = 513.2255 was detectable in all three signals (I-III). The two most specific product ions, m/z = 477.2488, which is formed by the loss of hydrogen chloride (HCl) and m/z = 301.2168, created by the cleavage of the glucuronic acid and the loss of HCl, were generated in all three peaks. Isolation, enzymatic hydrolysis and analysis with GC-MSMS of the 3 signals confirmed that peak I and peak II correspond to glucuronic acid conjugates of the DHCMT long-term metabolite M3. Peak III appears to be a glucuronic acid conjugate of an M3-epimer of unknown exact structure. Elucidation of conjugation sites: A positive urine sample was measured before and after the derivatization reaction with trityl chloride with the LC-HRMSMS method described above to visualize the successful derivatization, as shown in Figure 1. Peak II disappeared entirely after the derivatization. Peak I and III, on the other hand, remained utterly unharmed. If our assumption is correct, this is a clear sign that selective derivatization of signal II has occurred, and conversely, I and III remained unchanged in this reaction. Considering these findings and the theoretical structures of these metabolites allows the conclusion to be drawn that peak I represents DHCMT-M3-17-hydroxymethyl-glucuronide and peak II represents DHCMT-M3-3-glucuronide. Conclusion A new approach for simple detection of the important DHCMT metabolite M3 with LC-HRMSMS is introduced with the presented work. Therefore, LC-HRMSMS, GC-MSMS, fractionation, and derivatization experiments were combined to identify and characterize for the first time two new different glucuronide- acid conjugates of the DHCMT long-term metabolite M3. Figure 1. Resultsof derivatization experiment with tritylchloride; XIC, m/z 513.2255 -> 301.2168 (35eV), ESI-, 5 ppm mass tolerance Published as: L. Göschl, G. Gmeiner, P. Gärtner, M. Steinacher, G. Forsdahl. (2022) Detection of DHCMT long-term metabolite glucuronides with LC-MSMS as an alternative approach to conventional GC-MSMS analysis. Steroids, 2022 Apr;180:108979. doi: 10.1016/j.steroids.2022.108979 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3205 Thieme D , Anielski P , Keiler AM Unusual steroid findings: steroid design or synthetic accidents? Institute of Doping Analysis & Sports Biochemistry, Kreischa, Germany ; Environmental Monitoring and Endocrinology, Technische Universität Dresden, Dresden, Germany Abstract Forensic analyses of confiscated doping agents and ‘doping analyses’ - conducted in urine, blood or hair of accused individuals to verify self consumption or abstinence - are recurrently performed at the IDAS. Corresponding results are often insightful, as to the consumption habits of bodybuilders and the availability of steroids, but are hardly statistically representative. The regional black market is clearly dominated by the traditional anabolic steroids. The percentage of ‘atypical findings’, e.g. SARMs, growth hormone stimulating agents or unusal steroids as ‘dienedione’ or ‘methylstenbolone’ is well below 1%. Recently, putative metabolites of 18-methyl-(13β-ethyl)-nortestosterone were identified in a steroid user’s urine sample. In the following investigations, 4 confiscated ‘nutrition supplements’ were identified which should - according to their declaration - contain numerous designer steroids, including 13-ethyl structures or diol ‘prohormones’ of dehydrochlormethyltestosterone (‘halodrol’). Only four out of the 11 steroids declared could be analytically substantiated. 1 1 1,2 1 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3206 Toscano Bayona L , Chaves DC , Martinez Ramirez JA , Cárdenas Cuadros PA Biotransformation of metandienone with an in-vitro model with the fungus Cunninghamella elegans Laboratorio de Control al Dopaje de Colombia, Bogota, Kolumbien ; Departamento de Medicina, Universidad Nacional de Colombia, Bogotá, Kolumbien Abstract According to WADA, metandienone accounts for 10% of adverse analytical findings in anabolic steroids. Its metabolism has been studied through different in-vivo and in-vitro models to establish the metabolites that are markers of substance use. The Cunnhingamella elegans fungus has been used as a biotransformation model for different types of molecules. The aim of this study was to investigate the ability of Cunnhingamella elegans to produce metabolites of metandienone. Its characterization was carried out using gas chromatography coupled to mass spectrometry (GC-MS/EI) in the scan mode. We found that this fungal model can reproduce phase I reactions and four monohydroxylated metabolites are proposed with modifications at positions 6,7 and 14. The modification at position 6 was identified as 6β-OH metandienone by comparison with reference material; the hydroxyl at position 14 is considered typical of fungi and has not been previously reported for metandienone. We are expecting to use this model to study the metabolism of similar molecules. 1,2 2 2 1 1 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3207 Sahu PL , Rani S , Sahu K , Soni A , Mahajan S , Teja Illa G , Nandi U , Prakash Gupta A , Ahmed QN , Reddy DS Synthesis and characterization of etamivan sulfate: PK study of etamivan to decipher its metabolites National Dope Testing Laboratory, New Delhi, India ; CSIR-Indian Institute of Integrative Medicine, Jammu, India Abstract In today’s professional sports, regular testing of athletes for doping abuse has become an indispensable part. As these drugs are tremendously improving the potency of athletes, WADA and its related organizations are continuously innovating new ways to detect these drugs. In the field of anti-doping analysis, the discovery, characterization, and detection of new metabolites of different drugs is the prime focus of WADA and the group. Etamivan (Emivan, Analepticon and Vandid) is one of the few non-steroidal drugs that belong to S6.B specified stimulants listed in the WADA prohibited list [1]. Primarily, it has been used as a respiratory stimulant drug and is analogous to nikethamide. Etamivan shows similar actions as doxapram hydrochloride [2]. It has been mainly used to treat barbiturate overdose, perinatal asphyxia, postsurgical respiratory depression, and hypoventilation during thoracic surgery and in patients with respiratory failure. Despite having such a good profile, this drug is enlisted in S6.B specified stimulants, as WADA suspects the misuse of Etamivan by athletes to enhance their performance. In the fight against doping the laboratories are confronted to perform urine and blood analysis. In this particular case, employing LC-MS analysis studies, Etamivan sulphate was identified as an important long-term metabolite [3]. Thereby, it is always advisable to establish a synthetic route for these metabolites as reference standards.In this context, we optimized a method for the synthesis of Etamivan sulfate. Etamivan was synthesized in lab from commercially available vanillic acid using conventional coupling method with diethylamine in presence of N-(3-Dimethylaminopropyl)N′-ethylcarrbodiimide hydrochloride, anhyd. hydroxybenzotriazole and diisopropylethylamine in dry DMF at room temperature. Further, to avoid the format-ion of side product [4-(diethylcarbamoyl)-2-methoxyphenyl 4-hydroxy-3-methoxy- benzoate], the reaction mixture on hydrolysis followed by acidification resulted in the formation of desired Etamivan in good yield. Etamivan on further treatment with pyridine sulfur trioxide complex in dry pyridine at 94 °C for 7 h resulted in complete conversion to desired sulfated product. However, due to its labile nature during work up, we were successful to isolate pure Etamivan sulfate in 10% yield (Figure 1). The desired product was characterized by H NMR, C NMR, DEPT C-NMR, HRMS and TGA analysis. In addition to this, the mass fragmentation analysis of Etamivan and Etamivan sulfate was also performed and their fragments were interpreted. Further Snap PK study of Etamivan was performed after oral administration to Wistar rat. Results showed that peak of Etamivan disappeared from plasma after 30 min. Further, fine tuning of LC conditions using same PK study samples are ongoing for separation of generated metabolites, which are likely to be Etamivan sulfate and Etaviman glucuronide based on MS data. 1 1 1 1 2 2 2 2 2 2 1 2 1 13 135 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3208 Figure 1. Synthesis of Etamivan sulfate from Vanillic acid References 1. World Anti-Doping Code International Standard Prohibited List. (2022) https://www.wada- ama.org/sites/default/files/resources/ files/ 2022list_final_en.pdf 2. Aronson J K. (2016) Meyler’s Side Effects of Drugs (Sixteenth Edition), The International Encyclopedia of Adverse Drug Reactions and Interactions, 155. 3. Parr MK, Orlovius AK, Guddat, Gütschow, Thevis, Schänzer. (2007) Sulfoconjugates of heavy volatile nitrogen containing doping substances for improved LC-MS/MS screening, In Schänzer W, Geyer H, Gotzmann A, Mareck U. (eds.) Recent Advances in doping analysis (15), Sportverlag Strauß, Köln, 97-102. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3209 Kraiem S , Bouabdallah S , Touil S , Beotra A , Mohamed Ali V , Al-Maadheed M Elimination profile of 20-hydroxyecdysone (20-OHE) in urine: Liquid- liquid extraction and dilute and inject methodology using UHPLC/ HRMS. A comparative study Antidoping Lab Qatar, Doping Analysis Lab, Doha, Qatar ; Département de chimie, Faculté des sciences de Bizerte, Université de Carthage, 7021 Zarzouna, Tunisie, Laboratoire des composés hétéro-organiques et des matériaux nanostructurés (LR18ES11), Bizerte, Tunisia ; Centre for Metabolism and Inflammation, Division of Medicine, University College London, London, United Kingdom Abstract Ecdysteroids are of interest as potenial sport performance enhancers due to their anabolic effects. Several studies have been reported on the elimination profile of ecdysterone in urine and blood by applying different sample extraction and analysis techniques [1-4]. After the inclusion of ecdysterone in the monitoring list of WADA in 2020, many of the antidoping laboratories introduced ecdysterone in their screening procedures, which can be liquid-liquid extraction, solid phase extraction or dilute and inject as per the extraction protocol followed in routine testing. The aim of the present study was to compare liquid-liquid extraction and dilute and inject procedure using UHPLC high resolution mass spectrometry for the detection of supplement derived 20-OHE and its metabolite after administration to healthy volunteers.Two different supplement preparations were administered to two volunteers and their excretion profile was evaluated, An UHPLC-MS/MS method was developed for the detection of the parent compound and its metabolite 14-deoxy 20-OHE. The chromatographic separation was performed on an Acquity UPLC BEH C18 column (2.1 mm x 100, particle size 1.7 µm), the mass spectrometer was operated in positive mode ionisation (ESI+) with acquisition in full scan and MSMS mode simultaneously. References 1. Kraiem S, Al-Jaber MY, Al-Mohammed H, et al. Analytical strategy for the detection of ecdysterone and its metabolites in vivo in uPA(+/+)-SCID mice with humanized liver, human urine samples, and estimation of prevalence of its use in anti-doping samples. Drug Test Anal. 2021; 13: 1341–1353. doi.org/10.1002/dta.3032 2. Ambrosio G, Joseph JF, Wuest B, Mazzarino M, de la Torre X, Diel P, Botrè F, Parr MK. Detection and quantitation of ecdysterone in human serum by liquid chromatographycoupled to tandem mass spectrometry. Steroids (2020), 157:108603, doi.org/10.1016/j.steroids.2020.108603 3. Parr MK, Ambrosio G, Wuest B. et al. Targeting the administration of ecdysterone in doping control samples. Forensic Toxicol 38, 172–184 (2020). doi.org/10.1007/s11419-019-00504-y 4. Ambrosio G, Yuliandra T, Wuest B, Mazzarino M, de la Torre X, Botrè F, Diel P, Isenmann E, Parr MK. Urinary Elimination of Ecdysterone and Its Metabolites Following a Single-Dose Administration in Humans. Metabolites. 2021; 11(6):366. doi.org/10.3390/metabo11060366 1 2 2 1 1,3 1,3 1 2 3 ® MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3210 Min H, Son J, Seo Y, Park J Analysis of intact glycopeptide in erythropoietin for doping control using liquid chromatography-mass spectrometry Korea Institute of Science and Technology, Doping Control Centre, Seoul, Korea Abstract Recombinant erythropoietin (EPO), a representative glycoprotein hormone, has been misused and abused to improve athletic performance in sports, and the World Anti-Doping Agency has designated recombinant EPO as a prohibited drug. Recombinant EPO has a glycan structure of tetra-sialic acid, unlike endogenous EPO, and we tried to confirm this specific structure in recombinant EPO designated as a prohibited drug, especially Biological Reference Preparation for erythropoietin (BRP). In this experiment, to analysis recombinant EPO, using glycopeptide enrichment technique and intact glycopeptide of recombinant EPO was analyzed by LC-MS. As a result of the analysis, a target for glycan structure of tetra-sialic acid found only in recombinant EPO was obtained using the PMi software, and a high score and high repeatability peak from this candidate group was selected as a target for detecting recombinant EPO. In addition, a detection experiment was performed by applying the recombinant EPO in urine sample through the optimization process of the recombinant EPO detection assay. The ELISA method was used for detection EPO in urine samples, and by optimizing the analysis method, the experimental time was reduced to less than one day, so that the peak distinguishing recombinant EPO from endogenous EPO could be analyzed more efficiently and quickly. In this study, it was possible to analyze an intact glycopeptide with a tetra-sialic acid glycan structure present only in recombinant EPO through LC-MS analysis. It was predicted that the assay could be used to quickly detect recombinant EPO, which has been designated as a prohibited substance in sports competitions. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3211 Joon-Yeop Y , Minyoung K , Byung-Gee K , Junghyun S , Changmin S CRISPR/dCas9 based erythropoietin variant detection assay: Simple visualization method for single nucleotide polymorphism of erythropoietin Korea Institute of Science and Technology, Doping Control Centre, Seoul, Korea ; Interdisciplinary Program of Bioengineering, Seoul National University, Seoul, Korea ; Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea ; Bio-Max/N-Bio Institute, Seoul, Korea ; School of Chemical and Biological Engineering, Seoul, Korea ; Institute for Sustainable Development, Seoul, Korea Abstract Single nucleotide polymorphisms (SNPs), which provide different phenotypes for a single gene, appear to be a potential analysis target for diseases, but in the field of doping, SNP of the erythropoietin gene has inherent risk in doping testing procedures. Although the mutant EPO encoded by this variant is 27 amino acids longer than the wild-type EPO, the mutant EPO band on the electrophoretic gel is similar to that of recombinant EPO. SAR-PAGE method of EPO doping analysis based on molecular weight differences can lead to false-positive results. In order to avoid the risk of misinterpretation, we developed a simple EPO SNP detection assay using sequence specific binding ability of CRISPR/dCas9 system. DNA fragment containing EPO SNP suitable for detection is amplified using a fast-PCR protocol using 1 µL whole blood. Subsequently, a complex of sgRNA having the complemental sequence of the EPO SNP region and nuclease-deficient dCas9 protein binds to the PCR product. All reaction samples, including positive and negative controls, were analyzed by electrophoretic mobility shift analysis (EMSA), which allows easy visualization of DNA-protein complexes. As a result, the presence or absence of the SNP of the EPO gene can be detected within 3 hours without a multi-step sample preparation procedure, and the reproducibility was shown in several experimental conditions. Keywords: Single nucleotide polymorphism, Visualization, Human erythropoietin, Doping control, Sports * This year's Manfred Donike Award for the best poster presentation went to Korean researcher Joon- Yeop Xi. The identification of a natural erythropoietin polymorphism that can interfere with the urine doping control analytical assay for recombinant erythropoietin has required additional investigations into the genetic disposition of selected athletes, and the approach presented here is an elegant alternative to standard sequencing approaches. A simple, comparatively rapid and specific method has been developed that could simplify anti-doping testing protocols, addressing a new and particular specific need. 1,2,3 1 2,3,4,5,6 1 1 1 2 3 4 5 6 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3212 Joon-Yeop Y , Minyoung K , Byung-Gee K , Junghyun S , Changmin S CRISPR/dCas9-based high-throughput gene doping analysis (HiGDA) for exogenous human erythropoietin Korea Institute of Science and Technology, Doping Control Centre, Seoul, Korea ; Interdisciplinary Program of Bioengineering, Seoul National University, Seoul, Korea ; Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea ; Bio-Max/N-Bio Institute, Seoul, Korea ; School of Chemical and Biological Engineering, Seoul, Korea ; Institute for Sustainable Development, Seoul, Korea Abstract A genetic approach to improving athletic performance is called “gene doping” and is prohibited by the World Anti-Doping Agency. The development of a doping gene detection method is essential as there is currently no standard assay for gene doping validation. Recently, clustered regularly interspaced short palindromic repeats associated protein (Cas)-related assays have been used for nucleic acid detection in several fields. Furthermore, dCas9, a nuclease-deficient mutant of Cas9, can act as a sequence-specific DNA binding protein with a target-specific single guide RNA. Based on this principle, we developed a dCas9-based high-throughput gene doping analysis for exogenous gene validation. The assay comprises two distinctive dCas9s, a magnetic bead immobilized capture dCas9 for exogenous gene isolation and a biotinylated dCas9 with streptavidin–polyHRP that enables rapid signal amplification. Compared to the existing RT-PCR-based gene detection system, we succeeded in detecting the target gene in a concentration as low as 12.3 fM (1.23 amol) and up to 10 nM (1 nmol) in a whole blood sample within 1 h with HiGDA. The HiGDA is not only enables the direct detection of gene doping but also can successfully quantify the extent of gene doping, is a viable method for gene doping validation. 1,2,3 1 2,3,4,5,6 1 1 1 2 3 4 5 6 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3213 Zhou X , He S , Liu X , Wu D Detection of de-N-glycosylated EPO with SDS-PAGE: A complementary confirmation procedure for recombinant EPO in blood samples National Anti-Doping Laboratory, Beijing Sport University, Beijing, People´s Republic of China ; Shanghai Anti-doping laboratory, Shanghai University of Sport, Shanghai, People´s Republic of China Abstract Variant c.577del in the EPO gene is a frameshift variant that can result in the extension of the amino acid sequence of EPO by invalidating the termination codon. As the molecular weight of its encoded protein EPO (VAR-EPO) is similar to that of recombinant EPO (rEPO), the World Anti- Doping Agency has published annex B to the TD2022EPO which can protect “clean” athletes with variant c.577del. However, it is still necessary to develop a confirmation method for rEPO that can discriminate rEPO in all individuals directly. Based on the glycosylated characteristic of EPO, we selected the detection of de-N-glycosylated EPO as a complementary confirmation method for rEPO in blood samples. All samples were analyzed for both intact EPO and de-N-glycosylated EPO with SDS-PAGE, including rEPO spiked blood samples and blank samples. The results showed that, after de-N-glycosylation, a single-band was detected in samples collected from non-variant carriers, no matter whether the sample was spiked with rEPO or not. In samples collected from variant carriers, a double-band was detected. The ratio of lower band to upper band increased significantly corresponding to the concentration of rEPO. We calculated a series of cut-off values by normality distribution function to discriminate rEPO. Neither false positive in blank samples nor false negative result in spiked samples at Minimum Required Performance Levels were found. This indicates that this method could be adopted as a complementary confirmation method for rEPO in blood samples. A revised testing strategy was also proposed, which would discriminate rEPO directly without further investigation. Published as: He S, Liu X, Wu D, Zhou X. Detection of de-N-glycosylated EPO with SDS-PAGE: A complementary confirmation procedure for recombinant EPO in blood samples. Drug Test Anal . 2022 Nov;14(11-12): 1974-1983. doi: 10.1002/dta.3324 1 1 1 2 1 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3214 Thomas A, Thilmany S, Hofmann A, Thevis M Probing for peptidic drugs (2-10 kDa) in doping control blood samples Institute of Biochemistry, German Sport University, Cologne, Germany Abstract Bioactive peptides with a molecular mass between 2 and 10 kDa represent an important class of substances banned in elite sports, which has recognized with an increasing number and variety of substances by anti-doping organizations. Also, the annually renewed list of prohibited substances of the World Anti-Doping Agency (WADA) explicitly mentions more and more of these peptides, and efficient testing procedures are required. Even under simplified sample preparation conditions, liquid chromatography coupled to high resolution mass spectrometry (with resolution properties > 100 000 FWHM) offers the suitable conditions for this task and can therefore be used as an initial testing procedure. In contrast to urine, blood analysis essentially relies on the detection of intact peptide hormones, and the expected concentrations are commonly higher in blood samples than in urine. This facilitates the analysis, and a generic sample preparation by means of mixed-mode solid-phase extraction could be realized in this study. Co-extraction and analysis of several different peptides such as insulins (human, lispro, aspart, glulisine, tresiba, detemir, glargine, bovine insulin, porcine insulin), growth hormone releasing hormones (sermorelin, CJC-1295, tesamorelin), insulin-like-growth factors (long-R -IGF-I, R -IGF-I, Des -IGF-I) and mechano growth factors (human MGF, MGF-Goldspink) with criteria that fulfil the requirements of the WADA documents (TD2022 MRPL) for doping controls. The proof of principle was shown by the analysis of post administration samples after treatment with synthetic insulin analogs. Published as: Thomas A, Thilmany S, Hofmann A, Thevis M. Probing for peptidic drugs (2–10 kDa) in doping control blood samples. Anal Sci Adv. 2022; 3: 235– 243. doi.org/10.1002/ansa.202200027 3 3 1-3 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3215 Rahaman KA , Muresan AR , Kim KH , Lee KM , Min H , Kim HJ , Sung C , Kang M , Lee J , Son J , Kwon O Increased 5-oxoproline and 5-oxoprolinase level in stored RBCs: Biomarkers for homologous blood doping Korea Institute of Science and Technology, Doping Control Centre, Seoul, Korea ; Korea University of Science and Technology, Division of Bio-Medical Science & Technology, KIST School, Seoul, Korea Abstract Blood transfusion increases red blood cells (RBCs) and significantly improves physical performance. Heterogenous blood transfusion can be tested and detected in the doping labs. However, homologous blood doping is still critical for blood doping laboratories. Many doping labs have been investigating for markers related to homologous blood doping tests. In the first study, we have stored human blood at 4 °C in 2 different groups. One group was kept for 3 days, and the other group was kept for 20 days. Later, we have collected the storage buffer from both groups and measured the 5-oxoprolinase levels by ELISA. In the second experiment, we have stored RBCs at 4 °C in a cell stabilizing buffer for 14 days. RBCs and buffer samples were collected daily and were stored at -20 °C until further analysis. Later, we measured 5-oxoproline, glutamate, GSH concentrations in RBCs by LC-MS/MS, and 5-oxoprolinase levels in the buffer in which the erythrocytes were stored by ELISA. We found that the 5-oxoprolinase released in the cell stabilizing buffer was significantly higher on day 20 than on day 3. In RBC cells, we also found a time kinetic increase of 5-oxo- proline and glutamate from day 1 to day 14. On the other hand, the 5-oxoprolinase levels increased significantly in the storage buffer until day 13. The level of increased 5-oxoproline in cells and 5-oxo- prolinase in the buffer showed a significant correlation. We conclude that storage aging has a significant relationship with 5-oxoproline and 5-oxoprolinase levels in RBC. The average human body does not have 5-oxoproline and 5-oxoprolinase at the detectable level. In homologous blood doping, athletes take their preserved RBCs for doping. The intake of storage RBCs in the blood increases 5-oxoproline and 5-oxoprolinase to the detectable level, thus establishing a biomarker for homologous blood doping. Further experiments on humans are needed to confirm these doping markers to include them in daily laboratory detection routines. 1,2 1,2 1 1 1 1 1 2 1 1 1 1 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3216 Ota M, Miyamoto A, Sato M, Kageyama S, Okano M Doping control analysis of trimetazidine in DBS Anti-Doping Laboratory, LSI Medience Corporation, Tokyo, Japan Abstract Trimetazidine is classified as a banned substance in sports, and being misused by athletes. Trimetazidine can be detected in urine after administration of the permitted drug lomerizine. Therefore, laboratories shall analyse a lomerizine-specific metabolite (M6) to confirm the origin of trimetazidine in urine whenever trimetazidine is identified in urine. Dried blood spot (DBS) analysis has been a part of sports drug testing since 2021. In this study, application studies were conducted herein to develop dried blood spot (DBS) analysis method for trimetazidine using LC–MS/MS for doping control purposes. After oral administration of trimetazidine, venous and capillary blood (fingertip and upper arm) were spotted on cellulose paper (DMPK-C card). Trimetazidine could be identified in DBS, and there were no concerns regarding the qualitative analysis of trimetazidine in DBS using either fingertip or upper arm blood sampling, confirming the applicability of both sampling methods to sports doping testing. After administering lomerizine, the intact lomerizine has a strong peak intensity in blood compared to trimetazidine. Notably, the M6 metabolite was less detectable in blood compared to trimetazidine. Based on the results, laboratories should confirm intact lomerizine when trimetazidine is identified in DBS. Published as: Okano M, Miyamoto A, Ota M, Kageyama S, Sato M. Doping control analysis of trimetazidine in dried blood spot. Drug Test Anal. 2022; 1-11. doi:10.1002/dta.3414 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3217 Dos Santos L, Anselmo C, Pereira H, Carneiro M, Carneiro AC Development and validation of a dried blood spot assay for the analysis of stimulants and glucocorticoids Brazilian Doping Control Laboratory - LBCD, Rio de Janeiro, Brazil Abstract Dried Blood Spot (DBS) is a type of Dried Matrix Spot (DMS) where a biological sample (blood) is spotted on an appropriate paper for further analysis. A DBS sampling tends to be less invasive, besides reducing storage and shipping costs. Regarding doping control, DBS could be useful for substances prohibited in competition as an additional matrix complementary to urine, enabling the possibility of correlating the pharmacological effects of doping agents within its urinary concentration. Therefore, the objective of this work is to develop and validate a DBS assay with the purpose of analyzing stimulants (ritalinic acid, aranthol, isometheptene, sibutramine, didesmethylsibutramine, desmethylsibutramine, OH-didesmethyl- sibutramine, methylphenidate) and glucocorticoids (prednisone, prednisolone, 20b-dihydroprednisolone, 6b-OH-prednisolone) by LC-HRMS. In developing the DBS method, the following parameters were evaluated: extraction solutions, recovery of the paper Whatman 903 Protein Saver Card, the hematocrit influence, and the limit of detection (LOD) of stimulants and glucocorticoids by LC-HRMS. The methanol:acetonitrile:aqueous acetic acid 2% (v/v/v) extraction solution showed higher (60-80%) recoveries than the other solution without aqueous acid. Performed tests of the influence of hematocrit (n=7) with values of 30, 40, 50, and 60% showed a relative standard deviation (RSD) lower than 15% and accuracy between 85 and 115%, which is considered acceptable by the literature. Concentrations from 0.15 to 2 ng/mL were obtained as a preliminary evaluation of LOD. The present method is in the validation stage to be used in analysis by DBS of capillary blood samples from volunteers who will ingest drugs containing the stimulants and glucocorticoids targets of this study. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3218 González-Rubio S , Ballesteros-Gómez A , Muñoz G , Rubio S Cubosomic supramolecular solvents: synthesis, characterization and potential for high throughput multiclass testing of banned substances in urine Department of Analytical Chemistry, Institute of Fine Chemistry and Nanochemistry, University of Córdoba, Córdoba, Spain ; Madrid Anti Doping Laboratory, Madrid, Spain Abstract The search for sample treatments in human sport drug testing, able to efficiently extract multiclass prohibited substances while keeping utmost selectivity and sample throughput, is a major challenge yet unsolved. In this paper, this challenge was addressed by using supramolecular solvents (SUPRASs) made up of cubosomes. These SUPRASs, here firstly reported, were synthesized by the salt-induced coacer- vation of 1,2-hexanediol in urine. The formation of square and rounded cubosomes with a size range of 140-240 nm was confirmed by electron microscopy. These nanostructures consisted of 1,2-hexanediol, salt and a high water content (36-61%, w/w). Their applicability in multiclass determinations was investigated by the extraction of 92 prohibited substances (log P from - 2.4 to 9.2) belonging to ten categories of the World Anti-doping Agency (WADA) list. Variables influencing both recoveries and matrix effects were optimized. Cubosomic SUPRASs showed a high extraction efficiency and interference removal capability which was attributed to their large hydrophilicity and surface area. Both features were superior to that of other eleven SUPRAS that were based on sponge droplets and inverted hexagonal aggregates and to that of conventional organic solvents. A sport drug testing method based on cubosomic SUPRASs-LC-ESI-MS/MS was proposed and validated. For the ten urine samples analyzed, around 82-95% were efficiently extracted (recoveries 70-120%) and 81-92% did not present matrix effects. Method detection limits (0.001-4.2 ng/mL) were all far below WADA's limits. The proposed SUPRAS-based sample treatment is as simple as QuEChERS but the distinctive features of cubosomes confer them high capability in multiclass determinations. 1 1 2 1 1 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3219 Rubio A , Görgens C , Guddat S , Piper T , Garzinsky AM , Krug O , Thevis M Chiral analysis of selected enantiomeric drugs relevant in doping controls Institute of Biochemistry, German Sport University, Cologne, Germany ; EuMoCEDA, European Monitoring Center for Emerging Doping Agents, Cologne / Bonn, Germany Abstract Various substances classified by the World Anti-Doping Agency (WADA) as prohibited in sports feature one or more chiral centers. The enantiomers of these chiral drugs frequently exhibit stereoselectivity in their pharmacology and pharmacodynamic properties and, in most cases, the desired pharmacological activity is attributable to only one enantiomer, while the other remains less active or, in rare instances, even exerts toxic effects. Amongst those, few analytes exist that are so-called threshold substances, for which also enantiomerically pure drugs are available. The commonly employed non-chiral analysis of these compounds does not allow for differentiating between the use of a racemic mixture from their enantiomerically pure analogs. In order to support identifying the exclusive use of the pharmacologically active compound, a multi- analyte chiral chromatography-based quantitative approach considering the β -agonists salbutamol, formoterol, and fenoterol, the stimulant methamphetamine, the β-blockers propanolol, pindolol, and metoprolol, and the anabolic agent clenbuterol was developed. The test method employed liquid chromatography with a chiral column comprising a stationary phase based on the macrocyclic glycopeptide antibiotic teicoplanin as chiral selector. The liquid chromatograph was interfaced via electrospray ionization to a high resolution / high accuracy mass spectrometer, and urine samples were prepared for analysis following a protocol including enzymatic hydrolysis and subsequent liquid-liquid extraction. The method was characterized in accordance with the WADA International Standard for Laboratories guidelines concerning initial testing procedures for threshold substances, with specific focus on relevant concentration ranges. For proof-of-concept, authentic urine samples and WADA´s samples for External Quality Assessment Scheme (EQAS) containing the target compounds were analyzed, showing satisfactory results for chiral separation, and their enantiomeric composition was assessed (Figure 1). The herein presented approach, which can be expanded to include further target analytes if required, proved to be suitable for the chiral separation of a total of eight selected enantiomeric doping agents, allowing to determine their ratio at urinary concentrations relevant for sports drug testing purposes, i.e. between 0.01 and 2 ng/mL, and providing the tool to overcome the limitations of non-chiral approaches in routine doping analysis. Additionally, after enantiomeric ratio evaluation, differences in pharmacokinetics amongst both enantiomers could be observed in some cases (e.g. salbutamol), showing the capability of this approach to offer support in investigations where questions of pharmacokinetics and stereo- selectivity are to be addressed for result management and decision-making processes, thus complementing existing derivatization strategies with chiral derivatizing reagents (e.g. Marfey´s Reagent). 1 1 1 1 1 1,2 1,2 1 2 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3220 Figure 1. Extracted ion chromatograms with diagnostic precursor-product ion pairs for the target analytes (MS/MS experiments) and precursor ion (full MS) for related ISTDs showing the separation of target analytes in urine sample extracts containing: formoterol (A), salbutamol (B), clenbuterol (C), methamphetamine (D), and propranolol (E). Blank urine specimens are shown in black, authentic urine samples containing the target analytes in red, related ISTDs in green, and the enantiopure reference standards (when available) in blue. Except for the methamphetamine enantiomers, baseline separation was accomplished. Published as: Rubio A, Görgens C, Guddat S, Piper T, Garzinsky AM, Krug O, Thevis M. (2021) Chiral analysis of selected enantiomeric drugs relevant in doping controls. J. Chromatogr. Open. 1, 100017. doi.org/10.1016/j.jcoa. 2021.100017 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3221 Lee J, Jeong TY, Kang M, Jang H, Kim M Mobile-Phase Composition Map (MPC Map) for ionization efficiency and chromatographic behavior of 311 prohibited substances in LC- ESI/MS analysis Korea Institute of Science and Technology, Doping Control Centre, Seoul, Korea Abstract The sensitivity is of great importance in analysis of prohibited substances using liquid chromatography- electrospray ionization/mass spectrometry (LC-ESI/MS). The ionization efficiency in ESI and chroma- tographic behavior in LC are core factors that affect LC-ESI/MS sensitivity and mobile-phase composition (MPC) is a key parameter for achieving the best ionization efficiency and chromatographic behavior of analytes. This study aims to investigate the effect of the mobile-phase composition on the ionization efficiency and chromatographic behavior of 311 prohibited substances to achieve the best sensitivity. For this purpose, formic acid (0.01~0.5%), acetic acid (0.01~0.5%), ammonium formate (0.1~5 mM), ammonium acetate (0.1~5 mM), ammonium fluoride (0.1~5 mM) and no additive were evaluated as mobile-phase additives under methanol as an organic modifier. Based on results, we present MPC Map for 311 prohibited substances and this MPC Map would provide comprehensive information on optimal mobile-phase composition for LC-ESI/MS analysis of prohibited substances. This work was supported by Korea Institute of Science and Technology (2V09270). MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3222 Kim KH, Kim SH, Jeong W Qualitative analysis applied with derivatization for formoterol and salbutamol in human urine by liquid chromatography-mass spectrometry Korea Institute of Science and Technology, Doping Control Centre, Seoul, Korea Abstract Formoterol and salbutamol are considered to have a stimulating effect on respiration and growth- promoting action in athletes, so they were specified as a prohibited drug by the World Anti-Doping Agency. Formoterol and Salbutamol were quantitatively analyzed to prevent doping. but they exist as a pair of enantiomers and only the active isomer is extracted and sold. Therefore, it is necessary to check which drug was administered in accordance with the World Anti-Doping Agency's regulations. However, in a reversed-phase column, it is difficult to analyze each drug due to the characteristics of enantiomers, so the development of a qualitative analysis method for chiral separation is required. Most doping laboratories have limited equipment and time, so the indirect method that can be applied to the existing method among chiral separation methods is more advantageous than the direct method that requires additional equipment and setup. In this study, a qualitative analytical method using an indirect method was developed. and we optimized derivatization conditions for the pH and concentration of the triethylamine buffer, the concentration of the derivatization reagent, and the reaction temperature. Optimized analytical methods were validated for limits of detection, limits of identification, linearity, matrix effects, and precision. The developed method can be used for the analysis of other chiral isomers in urine and can also be helpful in the study of qualitative analysis methods for various chiral drugs. MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3223 Ayotte C, Charlebois A, Couture M, Desjardins M, Lalonde K Presence of β -agonists growth promoters in human urine samples. GC-MS/MS evaluation of the excretion profiles of ractopamine administered in microdoses Laboratoire de contrôle du dopage INRS - Centre AFSB, Laval, Canada Abstract β -adrenergic agonists having the potential to be misused to enhance performance for their thermogenic and anabolic properties are prohibited in sports. Clenbuterol, ractopamine and zilpaterol are principally utilised legally or illegally as growth promoters added to feed the animals raised for their meat. There are no withdrawal times imposed after the last administration of ractopamine prior to slaughter, and residues are detected in the meat, livers, kidneys of treated animals, which constitutes a risk of inadvertent consumption. Despite its widespread utilisation in Canada and the U.S.A. for instance, only one study in humans seems to have been done and its report is inaccessible. There is therefore insufficient information available on the fate of ractopamine in humans, and to implement efficient methods for their detection and identification in urine. Ractopamine is detected by the intense and specific ion-transition 322.2 -> 130.1 of the tetra-TMS derivative formed by the initial GC-MS/MS procedure generally applied for anabolic agents (LOD at 0.05 ng/mL), following the enzymatic hydrolysis of its glucuronide. To fulfill the identification criteria in the confirmation procedure (LOI at 0.15 ng/mL), ractopamine after the hydrolysis of glucuronides and sulfates, is converted to its tri-TMS derivative with BSTFA with 1% TMCS. With this method, ractopamine sulfate was found to form between 85% to 97% of total ractopamine excreted from the analysis of athletes’ urine samples collected for routine doping controls or following the administration of a micro-dose of 2.5 µg to volunteers. Although there is important inter-individual variation in the excretion profiles, peak levels were reached at 2 to 6 h, and decreased rapidly below 1 ng/mL 10 h after dosing. For two subjects, 50% to 60% of the dose administered was excreted within one day, 28% in the third one, with 80% in the first 6 h to 9,5 h. Ractopamine when detected in athletes’ samples as the hydrolysed glucuronide was estimated in levels lower than 100 pg/mL in 95% of the cases, with a mean concentration of 48 pg/mL ± 34 pg/mL. With one exception, the highest level estimated of total ractopamine (hydrolysed glucuronides and sulfates) in athletes' samples was 1.2 ng/mL. Considering the very low proportion excreted in the free and glucuroconjugated forms, the LOD of the ITP must be inferior to 0.5 ng/mL to confirm total ractopamine at the minimum required performance limit (MRPL) set by WADA at 1 ng/mL. Published as: Ayotte C, Couture M, Lalonde K, Charlebois A. Presence of β -agonist growth promoters in human urine samples: GC-MS/MS evaluation of the excretion profiles of ractopamine administered in microdoses. Drug Test Anal. 2022; 14(11-12):1825-1835. doi: 10.1002/dta.3395. 2 2 2 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3224 Breuer J, Thomas A, Geyer H, Thevis M Probing for the presence of semenogelin in human urine by immunological and chromatographic-mass spectrometric methods in the context of sports drug testing Institute of Biochemistry, German Sport University, Cologne, Germany Abstract It is suspected and debated that an increasing number of adverse analytical findings (AAFs) in routine doping controls are likely due to intimate contact with ejaculate that may facilitate the transfer of banned substances. Therefore two test methods were established for determining trace levels of semenogelin I, an important and specific component of semen, in female urine samples. For the first assay, a kit for the rapid identification of Semenogelin (RSID™-Semen), was used comprising of an immunochromatographic strip test with lateral flow. Secondly, a liquid chromatography/tandem mass spectrometry (LC-MS/MS)- based method was adapted using solid-phase extraction of urine, trypsinization of the retained protein content, and subsequent detection of semenogelin I-specific peptides. The two analytical approaches were characterized with regard to sensitivity, specificity, and reproducibility, as well as recovery, linearity, precision, and identifiability. Both assays were used to determine the stability of the analyte in urine (at 3 μL/mL) at different storage conditions (room temperature, +4°C, and -20°C). For the confirmatory procedure, a series of urine samples were collected before and after sexual intercourse and analyzed according to the LC-MS/MS method. Both analytical test methods were specific for this application since no signals for semenogelin were observed in blank urine. The analytical assays reached a limit of detection of 1 µL (immunochromatographic test) and 10 nL (LC-MS/MS) of ejaculate per mL of urine and was characterised concerning stability, intraday and interday imprecision (4.5-10.7% and 3.8- 21.6%, respectively), recovery (44%), and linearity within the working range of 0-100 nL/mL. Samples collected after sexual intercourse were tested positive for semenogelin I up to 55-72 h. Overall, both analytical test methods can detect semenogelin in urine samples and thus demonstrate whether ejaculate is present in the urine sample or not. Published as: Breuer J, Thomas A, Geyer H, Thevis M. Probing for the presence of semenogelin in human urine by immunological and chromatographic mass spectrometric methods in the context of sports drug testing. Anal Sci Adv. 2022; 3: 21-28. doi.org/10.1002/ansa.202100058 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3225 Sahu PL , Kalita S , Kumar J , Pawar S , Sethi K , Radhakrishnanand P , Murty USN , Dubey S , Sahu K , Upadhyay A , Kori RK , Kumar P Synthesis and characterization of reference materials of certain drugs and their metabolites National Dope Testing Laboratory, New Delhi, India ; National Institute of Pharmaceutical Education and Research, Guwahati, Assam, India, Guwahati, India Abstract Substances prohibited for use in sports such as drugs and hormones can be taken intentionally by athletes to increase their performance, which is against the ethics and spirit of sports. Reference materials play a vital role in doping control analysis. Certain metabolite reference materials are not available commercially and their detection limit is also very low. Therefore, it is difficult to conclude that either sport person is doped or not. Therefore, it was planned to develop metabolite reference materials via synthetic routes to meet the requirements of quality control during Olympics. Therefore, our team has developed six reference materials for para-hydroxy prenylamine [1], nor-ethylmorphine HCl [2], carboxy toremifene [3], octopamine sulfate, norfenefrine sulfate and etilfrine sulfate which may be used for routine dope testing. Octopamine sulfate, norfenefrine sulfate and etilfrine sulfate have structural similarity and few steps are also common in synthetic reactions [4]. The audience may see subsequent publications in literature for more details. An Indian patent also has been filed for octopamine sulfate and carboxy toremifene synthetic scheme because of the novelty to protect invention rights [5,6]. All six reference materials have been synthesized and characterized by established analytical techniques such a s H-NMR, C-NMR, HRMS, UV, IR and TGA. All developed reference materials have been tested for purity assessments using RP-HPLC-DAD. Hence, it is concluded that all developed six reference materials are feasible for synthesis and the same can be used in sports dope testing analysis. Developed reference materials will strengthen the anti-doping society to maintain the clean sport program. References 1. Joseph A, et al. Analytical developments of p-hydroxy prenylamine reference material for dope control research: Characterization and purity assessment. Drug Testing and Analysis. 2022 Feb;14(2):224-32. 2. Pawar SD, et al. Synthesis, characterization, method development, and validation of nor-ethylmorphine hydrochloride reference material using established analytical techniques for dope control analysis. Drug Testing and Analysis. 2022 Feb;14(2):388-92. 3. Kumar GJ, et al. Process development for the total synthesis of the novel drug metabolite Carboxy toremifene as a standard reference material along with characterization and purity assessment for the Antidoping quality Control Purposes. Drug Test Anal. 2022 Oct 13. doi: 10.1002/dta.3387 4. Kalita SJ, et al. Synthetic and analytical developments of Octopamine sulfate, Norfenefrine sulfate, and Etilefrine sulfate reference standards. Archiv Der Pharmazie. Manuscript ID ardp.202200496. Status: Under review 5. Process to prepare octopamine sulfate. 2022. Subarna Jyoti Kalita, Sachin Dattram Pawar, Prachi Vernekar, Mayur Arun Pawar, Veena K. S., Km Abha Misra, Kalyan Kumar Sethi, Pullapanthula Radhakrishnanand, Upadhyayula Suryanarayana Murty, Puran Lal Sahu, Sachin Dubey, Kapendra Sahu, Awanish Upadhyay, Pramod Kumar, Application No.: 202231000058 and filing date: JANUARY 01, 2022. 6. Process to prepare carboxy-toremifene. 2021. Gangasani Jagadeesh Kumar, Sachin Dattram Pawar, Pullapanthula Radhakrishnanand, Upadhyayula Suryanarayana Murty, Puran Lal Sahu, Sachin Dubey, Kapendra Sahu, Awanish Upadhyay, Pramod Kumar. Application No. : 202131058419 Filing Date: December 15, 2021 1 2 2 2 2 2 2 1 1 1 1 2 1 2 1 13 MANFRED DONIKE WORKSHOP 2022 Poster RECENT ADVANCES IN DOPING ANALYSIS (30) ISBN 978-3-86884-048-3226 Orie NN , Raees A , Alijaber MY , Mohamed-Ali N , Bensmail H , Hamza MM , Al-Ansari N , Beotra A , Mohamed-Ali V , Al-Maadheed M 20-Hydoxyecdysone dilates muscle arterioles in a nitric oxide- dependent, estrogen ER-β receptor-independent manner Antidoping Lab Qatar, Doping Analysis Lab, Doha, Qatar ; Qatar Computing Research Institute, Hamad bin Khalifa University, Doha, Qatar ; Centre for Metabolism and Inflammation, Division of Medicine, University College London, London, United Kingdom Abstract 20-hydroxyecdysone is an ecdysteroid with anabolic and favorable metabolic potentials [1-5], which have made it attractive to athletes and raised concern about their potential use for doping purposes. Although the efficacy and mechanisms of its anabolic effects in humans are unclear, 20-hydroxyecdysone binds to estrogen receptor beta (ER-β) at low concentrations [6], which makes it a potential vasodilator. Here we tested the hypothesis that 20-hydroxyecdysone dilates muscle arterioles by activating estrogen ER-β receptors. This would effectively enhance muscle blood blow and performance with the potential to enhance athletic performance. Direct effects of the compound on arteriolar tone were assessed by wire myography in ovine abdominal muscle and mesenteric arterioles. The roles of endothelial nitric oxide synthase (NOS3), cyclooxygenase (COX) and estrogen ER-β receptor (ER-β) in its effects were determined with specific blockers and by expression analyses in human coronary artery endothelial cells (HCAECs) and humanized liver tissues from uPA+/+-SCID mice (transplanted with human hepatocytes) for effects on NOS3 mRNA and protein. Comparable dose-dependent relaxations were recorded for 20-hydroxyecdysone in both muscle and mesenteric arterioles with maximum relaxations of 46.94 ± 5.84% and 56.88 ± 7.04% respectively, which were not statistically different. Similar relaxation was recorded for β-estradiol in both arterioles. In addition, NOS inhibition with 100 µM L-NAME attenuated the relaxation to both 20-hydroxyecdysone (p

M. THEVIS H. GEYER U. MARECK (EDITORS) RECENT ADVANCES IN DOPING ANALYSIS (29) Proceedings of the Manfred Donike Workshop 39th Cologne Workshop on Dope Analysis 22nd to 26th March 2021 SPORTVERLAG Strauß - Hellenthal 2021 Bibliografische Information Der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über abrufbar. Thevis, Mario; Geyer, Hans; Mareck, Ute (Eds.) Recent Advances in Doping Analysis (29). Proceedings of the Manfred Donike Workshop, 39th Cologne Workshop on Dope Analysis, 22nd to 26th March 2021 / [MDI e.V.] – 2021 Sportverlag Strauß. ISBN 978-3-86884-047-6 ©SPORTVERLAG Strauß Neuhaus 12 – 53940 Hellenthal Tel. +49 (0)2448/2470040 – Fax +49 (0)2448/9195610 E-mail: info@sportverlag-strauss.de www.sportverlag-strauss.de Satz: Autorensatz 2 MANFRED DONIKE WORKSHOP 2021 TABLE OF CONTENTS PAGE LECTURES Hullstein I, Yu Q, Dehnes Y: Carbon isotope ratio determination of seized nandrolone preparations in comparison to results from analyses of 19-norandrosterone in urine samples ............................................ 13-18 Thieme D: 'Operation Bloodletting' - practical insights from a major doping trial? ................................... 19-23 Mazzoni I, Ventura R, Daley-Yates P, Collomp K, Saugy M, Buttgereit F, Rabin O, Stuart M: Defining permitted and prohibited use of glucocorticoids in the anti-doping context Part I. Glucocorticoids: new approach and new regulations ..................................................... 24-29 Daley-Yates P, Ventura R, Mazzoni I, Collomp K, Saugy M, Buttgereit F, Rabin O, Stuart M: Defining permitted and prohibited use of glucocorticoids in the anti-doping context Part II. Approach for defining acceptable and unacceptable use of glucocorticoids in sport .............................................................................................................................................. 30-33 POSTER PRESENTATIONS Haenelt N, Lourens L, Fußhöller G, Geyer H, Goldmann L, Schult C, Schwenke A, Hülsemann F, Gougoulidis V, Blatt C, Thevis M: Follow-up investigations of atypical passport findings for the ratio 5a-Androstane- 3a,17b-diol/5b-Androstane-3a,17b-diol ..................................................................................... 34-37 Fußhöller G, Geyer H, Haenelt N, Hülsemann F, Gougoulidis V, Blatt C, Thevis M: Additional investigations in connection with atypical findings for 19-norandrosterone - a case study ................................................................................................................................... 38-40 Mareck U, Geyer H, Fußhöller G, Haenelt N, Thevis M: Results of confirmation procedures in the scope of the steroidal athlete biological passport in the Cologne laboratory from 2017-2019................................................................... 41-44 3RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6 PAGE Piper T, Geyer H, Toboc A, Ahrens B, Pohanka A, Thevis M: Recent findings on 7-oxo-DHEA and its improved detection based on reference population-derived thresholds for 7β-OH-DHEA and 5αandrostane-3β,7β-diol-17-one ........... 45-49 Piper T, Panto S, Thevis M: Highly sensitive low resolution GCxGC-TOF as a complement in doping control analysis of anabolic androgenic steroids.................................................................................................... 50-54 Krug O, Thomas A, Thevis M: Identification and characterization of urinary isopropylnorsynephrine metabolites ................ 55-59 Geisendorfer T, Athanasiadou I, Tsivou M, Gmeiner G: Long-term urinary excretion profile after a single oral administration of acetazolamide ........ 60-63 Fernández-Alvarez M, Serrano E, Muñoz G: Specific urinary metabolites of non-prohibited mebeverine: LC-MS/MS monitoring of MAC and DMAC on reporting of p-hydroxyamphetamine ............................................................... 64-68 Albertsdóttir AD, van Gansbeke W, van Eenoo P, Polet M: Intact phase II AAS metabolites on GC-MS .................................................................................. 69-72 Rubio A, Geyer H, Costa Padilha M, Pereira H, Cameron L, Thevis M: Higenamine quantification and investigation into structural characteristics of its metabolites in urine samples........................................................................................................ 73-77 Martinez Brito D, Leogrande P, de La Torre X, Botrè F: Analysis of 7-oxo-DHEA metabolites by liquid chromatography mass spectrometry................ 78-82 Camuto C, Guglielmelli A, De-Giorgio F, de La Torre X, Mazzarino M, Marti M, Botrè F: An insight into the metabolism of New Psychoactive Substances: targeted and untargeted metabolic profile of a new mephedrone analogue .................................................. 83-87 Camuto C, De-Giorgio F, Fiacco I, Marti M, Mazzarino M, Botrè F: In vivo metabolism of JWH-175: blood and urine detection of JWH-018 in mice ...................... 88-91 Pühringer M, Gmeiner G: Detection of S-23 metabolites in urine after a single oral administration using liquid chromatography high resolution mass spectrometry ................................................................. 92-95 Thomas A, Fox J, Slade S, Kislyuk S, Gastall H, Thevis M: Preliminary data for ion mobility separation of recombinant and synthetic insulin variants on a cyclic IMS mass spectrometer .............................................................................. 96-100 4RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6 PAGE Stacchini C, Botrè F, de la Torre X, Mazzarino M: The individual longitudinal profile of IGF-1 in capillary blood: a new ABP parameter? ........ 101-106 Martín-Navas P, Saavedra MJ, Cortés N, McPherson Medina A, Polo M, Fiallo Fernández T, Rodríguez G, Muñoz G, Montes de Oca Porto R: A Fit-for-Purpose approach with the Cuban monoclonal antibody CBSSEPO for the ERAs analysis in urine samples .......................................................................................................... 107-111 Biasini GM, de La Torre X, Botrè F, Donati F: Detection of human Peroxiredoxin-2 in stored erythrocytes: potential biomarker of Autologous Blood Transfusions in doping control ................................................................... 112-116 Akiyama K, Kageyama S, Okano M: DNA analysis of dried blood spots and urine for doping control purposes ............................ 117-121 Krug O, Geyer H, Thomas A, Walpurgis K, Piper T, Thevis M: Black market products suspected to contain doping relevant ingredients - report for 2019-2020 .............................................................................................................................................. 122-127 Mareck U, Fußhöller G, Haenelt N, Thevis M: Risk of unintentional antidoping rule violations by consumption of hemp products ............. 128-132 Cantón MI, Garcia PP, Serrano E, Muñoz G: Comparison of the separation of 2-fluoroamphetamine, 3-fluoroamphetamine and 4-fluoroamphetamine by gas chromatography-mass spectrometry using different columns and derivatization agents ................................................................................................... 133-138 Berghes B, Radu M, Cristea CD, Toboc A, Stan C: Optimization of a cocaine and benzoylecgonine identification method using the linear ion trap .................................................................................................................................................. 139-143 Moleme BJ, Grobbelaar E, Du Preez H: The influence of gas filter saturation on the chromatographic sensitivity of GC-MS/MS analysis .................................................................................................................................................. 144-147 Inthong T, Nimsoongnern S, Wilairat P, Kongpatanakul S: Workflow Management and Sample Tracking for Doping Analysis ........................................... 148-151 Leogrande P, Jardines Garcia D, Domenici E, de La Torre X, Parr MK, Botrè F: Low-energy electron ionization for steroidomics analysis using highresolution mass spectrometry ........................................................................................................................................ 152-156 Wicka M, Grucza K, Stanczyk D, Drapala A, Kowalczyk K, Konarski P, Burstein K, Kwiatkowska D: Development and validation of a method for the detection of benzodiazepines, barbiturates, imidazopyridine and derivates of cyclopyrrolone in blood by means of LC-MS/MS ............................................................................................................................................. 157-162 5RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6 PAGE Mareck U, Fußhöller G, Geyer H, Thevis M: Simplified confirmation analysis for Carboxy-THC ........................................................................ 163-166 Rubio A, Sigmund G, Piper T, Geyer H, Thevis M: Evaluation of doping control samples to determine the prevalence of nicotine use by German elite athletes ......................................................................................................................... 167-170 Montes de Oca Porto R, Martinez Brito D, Correa Vidal T, Fiallo Fernández T, Hernández Domínguez D: hGH levels and gender, sport, and endogenous corticosteroids in a Cuban population. Preliminary results ............................................................................................................................... 171-176 Wicka M, Kaliszewski P, Grucza K, Stanczyk D, Drapala A, Konarski P, Kowalczyk K, Kwiatkowska D: Determination method of 27 prohibited glucocorticosteroids in human urine ....................... 177-181 PRESENTATIONS ABSTRACTS - LECTURES Salamin O, Nicoli R, Langer T, Schweizer Grundisch C, Boccard J, Rudaz S, Xu C, Pitteloud N, Saugy M, Kuuranne T: Longitudinal evaluation of multiple biomarkers for the detection of testosterone gel administration in women with normal menstrual cycle ......................................................... 182-183 de Wilde L, van Renterghem P, van Eenoo P: Long-term stability study and evaluation of intact steroid conjugate ratios after the administration of endogenous steroids .......................................................................................... 184 Piper T, Geyer H, Nieschlag E, Thevis M: Carbon isotope ratios of endogenous steroids found in human serum – method development, validation, and reference population-derived thresholds .............................. 185-186 Piper T, Haenelt N, Fusshöller G, Geyer H, Thevis M: Sensitive detection of testosterone and testosterone prohormone administrations based on urinary concentrations and carbon isotope ratios of androsterone and etiocholanolone ........................................................................................................................ 187-188 Matos R, Anselmo C, Lopez N, Magalhães A, Sardela V, Pereira H: Zebrafish water tank (ZWT) model as a tool for metabolic studies. New results for anabolic agents ................................................................................................................................. 189 Göschl L: Detection of phase-II glucuronides of exogenous anabolic androgenic steroids exemplified by stanozolol ................................................................................................................ 190 6RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6 PAGE Görgens C, Ramme A, Guddat S, Schrader Y, Winter A, Dehne E, Horland R, Thevis M: Organ-on-a-chip: Determine feasibility of a liver microphysiological model to assess long-term steroid metabolites in sports drug testing ..................................................................... 191 Wagener F, Möller T, Guddat S, Görgens C, Angelis YS, Petrou M, Lagojda A, Kühne D, Thevis M: Elimination profiles of microdosed SARM LGD-4033 mimicking contaminated product ingestion ........................................................................................................................................... 192 Krombholz S, Thomas A, Piper T, Thevis M: Elimination profile of orally administered phenylethylamine ....................................................... 193 Albertsdóttir AD, van Gansbeke W, van Eenoo P, Polet M: Non-hydrolysed sulfated metabolites in routine doping control screening, enabled by GC-LE-EI-QTOF-MS ............................................................................................................................ 194 Lange T, Thomas A, Görgens C, Bidlingmaier M, Schillbach K, Fichant E, Delahaut P, Thevis M: Comprehensive insights into the formation of metabolites of the ghrelin mimetics capromorelin, macimorelin and tabimorelin as potential markers for doping control purposes ........................................................................................................................................... 195 Euler L, Gillard N, Delahaut P, Mürdter T, Schwab M, Thomas A, Thevis M: Are contaminated eggs a potential source of minute amounts of clomiphene in doping control samples? .............................................................................................................................. 196 Keiler A, Zschiesche A, Savill R, Chundela Z, Thieme D: Biosynthesis of long-term metabolites using HepG2 cells ............................................................. 197 Loria F, Cox H, Voss SC, Rocca A, Miller G, Townsend N, Georgakopoulos C, Eichner D, Kuuranne T, Leuenberger N: The Use of RNA-Based 5’-Aminolevulinate Synthase 2 Biomarkers in Dried Blood Spots to Detect Recombinant Human Erythropoietin Micro-Doses ........................................................ 198 Martin L, Ericsson M, Marchand A: Multiplexed detection of Agents Affecting Erythropoiesis (AAEs) and overall strategy for optimized analysis ............................................................................................................... 199-200 Leuenberger N, Rocca A, Martin L, Marchand A, Ericsson M, Kuuranne T A fast screening method for the detection of CERA in dried blood spots ..................................... 201 Thomas A, Krombholz S, Wolf C, Thevis M: Determination of ghrelin and desacylghrelin in plasma and urine by means of LC-MS for doping controls ........................................................................................................................... 202 Gavrilović I, Memdouh S, Cowan D, Abbate V: Improving the detection of peptide hormones for anti-doping purposes..................................... 203 7RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6 PAGE Voss SC, Yassin M, Grivel J, Al Hmissi S, Allahverdi N, Nashwan A, Merenkov Z, Al Malki A, Raynaud C, Elsaftawy W, Al Kaabi A, Donati F, Botre F, Mohamed Ali V, Georgakopoulos C, Al Maadheed M: RBC derived extracellular vesicles as markers for autologous blood doping – a clinical trial .................................................................................................................................................... 204 Goucher E: Alternative Approaches Towards Sports Anti-Doping – a Focus on Dried Spot Analysis .......................................................................................................................... 205 Levernæs M, Broderstad L, Zandy E, Dehnes Y: Comparison of dried blood spots and urine as sample matrices in doping control ...................... 206 Garzinsky AM, Thomas A, Thevis M: Insights into the pulmonary elimination of beta-blockers, glucocorticoids and stimulants obtained from post-administration exhaled breath samples ...................................... 207 Marchand A, Roy D, Lewis J, Mcguire R, Ericsson M: Development of a miniaturized multiplex immunoassay for Growth Hormone (GH) detection .................................................................................................................................... 208-209 Mareck U, Fußhöller G, Geyer H, Huestis MA, Scheiff AB, Thevis M: Preliminary data on the potential for unintentional anti-doping rule violations by permitted CBD use ........................................................................................................................... 210 Danaceau J, Gavilovri? I, Christensen P, Wood M: Analysis of doping agents by UPC -MS/MS ..................................................................................... 211 Paßreiter A, Thomas A, Grogna N, Delahaut P, Thevis M: First Steps toward Uncovering Gene Doping with CRISPR/Cas by Identifying SpCas9 in Plasma via HPLC−HRMS/MS ............................................................................................................ 212 Honesova L, Polet M, van Eenoo P: A uniform sample preparation procedure for gas chromatography combustion isotope ratio mass spectrometry for all human doping control relevant anabolic steroids using online 2/3-dimensional liquid chromatography fraction collection ....................................... 213-214 Gavrilovic I, Cowan D, Parr M, Botre F, de La Torre X, Wüst B: SFC-MS – A New Tool for Anti-Doping Analysis .............................................................................. 215 Knoop A, Geyer H, Lerch O, Rubio A, Schrader Y, Thevis M: Detection of anti-SARS-CoV-2 antibodies in dried blood spots in support of the management of the COVID-19 pandemic in the context of sport .................................................. 216 8RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6 PAGE Cavalcanti G, Carneiro G, Borges R, Padilha M, Pereira H: Variable Data Independent Acquisition (vDIA) and Feature-Based Molecular Networking Analysis for Untargeted Screening of Synthetic Cannabinoids ...................................................... 217 Deventer K, van Gansbeke W, Hooghe F, Polet M, van Eenoo P: Investigation of the urinary excretion of prednisolone and metabolites after nasal administration: Relevance to doping control ................................................................................. 218 Ventura R, Daley-Yates P, Mazzoni I, Collomp K, Saugy M, Buttgereit F, Rabin O, Stuart M: Defining permitted and prohibited use of glucocorticoids in the antidoping context Part III. Establishing reporting levels and washout periods for glucocorticoids ........................... 219 Buisson C, Robin B, Frelat C, Amiot V, Narduzzi L, Ericsson M, Collomp K: Study on hydrocortisone misuse: Topical versus oral administration .................................... 220-221 PRESENTATIONS ABSTRACTS - POSTERS Bressan C, Celma A, Alechaga &, Monfort N, Sancho JV, Ventura R: Collision cross-section measurements for the structural characterization of sulfate and glucuronide metabolites of anabolic steroids ................................................................................. 222 Andersson A, Pohanka A: The shared fate of norethisterone and levonorgestrel .................................................................. 223 Ponzetto F, Nonnato A, Settanni F, Nicoli R, Kuuranne T, Ghigo E, Mengozzi G: Extended steroid profiling by single-run UHPLC-MS/MS analysis: first insights into conjugated androgens plasma levels ....................................................................................... 224-225 Jardines Garcia D, Botrè F, de La Torre X: Coupling longitudinal steroid profile (ABP – steroid module) with isotopic ratio mass spectrometry values. Athletes cases studies .................................................................................. 226 Buisson C, Elloumi A, Figadère B, Ericsson M, Beniddir MA: The sniffing dog/anti-doping dog Molly - A new dimension of doping controls .................... 227-228 Martinez Brito D, Leogrande P, de La Torre X, Colamonici C, Curcio D, Botrè F: Should Arimistane be considered a direct metabolite of 7-oxo-DHEA? ........................................ 229 Pfeffer S, Gmeiner G, Gärtner P: Synthesis, characterization and application of a marker substance for monitoring 17-keto-modifications in endogenous steroids caused by microbiological activity ..................... 230 9RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6 PAGE Kwon OS, Muresan AR, Rahaman KA, Rafique FB, Kim KH, Lee KM, Min H, Kim HJ, Sung C, Lee J, Son J: Metabolism of bolasterone by LC-MS/MS and GC-MS/MS .................................................... 231-232 Kwon OS, Rahaman KA, Muresan AR, Rafique FB, Kim KH, Lee KM, Min H, Kim HJ, Sung C, Lee J, Son J: Discovery of in vitro generated metabolites of Thymosin β4 by UHPLCQ-Exactive Orbitrap MS ............................................................................................................................... 233-234 Dubey S, Sah S, Singh AK, Jamal H, Singh S, Sahu PL: Investigation of meclofenoxate stability and profiling of its degradation products in urine for human sports doping control purposes .................................................................... 235-236 Mazzarino M, Camuto C, Comunità F, Stacchini C, Botrè F: Evaluation of the metabolic behaviour of novel bath salt type drugs by data-independent acquisition mass spectrometry: The case of N-ethyl heptedrone ................................................. 237 Uçaktürk E, Selbes Y, Demirel HA: Investigation of ibutamoren and its metabolites in urine samples ............................................... 238 Marchand A, Martin L, Kafi R, Zhou X, Zhang L, Ericsson M: Detection of rEPO biosimilar Jimaixin after administration in healthy subjects ........................... 239 Reihlen P, Blobel M, Weiß P, Wittmann J, Leenders F, Walpurgis K, Thevis M: Introduction of a PEGylated EPO-conjugate as internal standard for EPO analysis in doping controls ................................................................................................................................. 240 Kempkes R, Schoeps S, Reihlen P, Geyer H, Gotzmann A, Thevis M: The Cologne hematological APMU: A collaboration between the Cologne laboratory and the National Anti Doping Agency Germany ............................................................................. 241 Marchand A, Roulland I, Semence F, Ericsson M: A simple method for EPO transgene detection in whole blood and dried blood spots ......... 242-243 Mareck U, Geyer H, Schertel T, Petring S, Krug O, Thevis M: Findings of non-declared doping substances in nutritional supplements in follow up investigations of positive doping cases ........................................................................................... 244 Dobrescu M, Danila G, Stan C, Toboc A: A gas chromatography – tandem mass spectrometry (GC-MS) method for the identification of 5-methylhexan-2-amine in food supplements .................................................... 245 Rzeppa S, Große J, Thieme D: Δ8-Tetrahydrocannabinol: Emergence of a less common cannabinoid with a challenging detection .................................................................................................................................... 246-247 10RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6 PAGE Kwiatkowska D, Grucza K, Kowalczyk K, Konarski P, Drapala A, Chajewska K, Wicka M: Ecdysterone - excretion study after ingestion of spinach ....................................................... 248-249 Guddat S, Goergens C, Sobolevsky T, Thevis M: Meldonium residues in milk- a possible scenario for inadvertent doping in sports? ................... 250 Judák P, Coppieters G, Deventer K, van Eenoo P: Application of online automatic filtration and filter back-flush solid phase extraction in routine doping control analysis ....................................................................................................... 251 González-Rubio S, Ballesteros-Gómez AM, Carreras D, Muñoz G, Rubio S: A comprehensive study on the performance of different retention mechanisms in sport drug testing by liquid chromatography tandem mass spectrometry ............................................ 252 de Wilde L, Roels K, Deventer K, van Eenoo P: Automated identification of cocaine and benzoylecgonine at 1 ng/mL using turbulent flow online SPE LC-MS/MS .............................................................................................................. 253 Khelifi S, Saad K, Vonaparti A, Mahieddine S, Saleh A, Salama S, Al-Mohannadi M, Al-Thaiban H, Lommen A, Horvatovich P, Beotra A, Abushareeda W, Al Maadheed M, Georgakopoulos C: Ultra-fast retroactive processing by MetAlign of liquid chromatography/high- resolution full-scan Orbitrap mass spectrometry data in WADA Human Urine Sample Monitoring Program ......................................................................................................................... 254 Vonaparti A, Salama S, Mahieddine S, Saleh A, Saad K, Al-Thaiban H, Khelifi S, Maryam A, Saghbazarian S, Al Maadheed M, Georgakopoulos C: Dilute and shoot screening of doping agents by UHPLC/HR-Orbitrap-MS .................................... 255 Knoop A, Fusshöller G, Thevis M: Identification of hydrafinil metabolites in doping controls ........................................................... 256 Sobolevsky T, Ahrens B: Biotin as a masking agent in chorionic gonadotropin assays utilizing biotinylated antibodies ......................................................................................................................................... 257 Anselmo C, Matos R, Pereira H, Martucci ME: Metabolomic retrospective analysis of HRMS data from cathinones metabolism in zebrafish (Danio rerio) ..................................................................................................................... 258 11RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6 SCIENTIFIC BOARD E. Benetka, Seibersdorf (Austria) C. Buisson, Paris (France) D. Cowan, London (United Kingdom) H. Cox, Los Angeles (USA) X. de la Torre, Rome (Italy) H. Geyer, Cologne (Germany) G. Gmeiner, Seibersdorf (Austria) U. Mareck, Cologne (Germany) M. Mazzarino, Rome (Italy) G. Muñoz, Madrid (Spain) R. Montes de Oca Porto, Havana (Cuba) M. Okano, Tokyo (Japan) H. Pereira, Rio de Janeiro (Brazil) T. Piper, Cologne (Germany) C. Reichel, Seibersdorf (Austria) W. Schänzer, Cologne (Germany) M. Thevis, Cologne (Germany) D. Thieme, Kreischa (Germany) P. van Eenoo, Ghent (Belgium) R. Ventura, Barcelona (Spain) 12RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6 Hullstein I, Yu Q, Dehnes Y Carbon isotope ratio determination of seized nandrolone preparations in comparison to results from analyses of 19-norandrosterone in urine samples Norwegian Doping Control Laboratory, Oslo University Hospital, Oslo, Norway Abstract Determining the origin of anabolic androgenic steroids (AAS), that also are produced endogenously in the human body, is a major issue in doping control. In some cases, the presence of nandrolone metabolites may be a result of bacterial degradation of endogenous androsterone [1]. The GC-C-IRMS technique provides the capability to measure the carbon isotope ratio (CIR) in order to determine the origin of these metabolites. Published studies indicate, however, that there are nandrolone preparations available in the black market showing δ C values in, or close to, the range of values seen in endogenous steroids [3,4]. In this study, the aim was to measure δ C values of nandrolone in preparations seized in Norway between 2013 and 2020, to investigate if there were changes compared to preparations that were part of a previous study [4]. In addition, 39 urine samples containing 19-norandrosterone (19-NA) and 19-nor‐ etiocholanolone (19-NE), were analysed. The urine samples were collected in Norway and Denmark and originated mainly from fitness centres. A total of 28 seized preparations were analysed. The nandrolone preparations showed δ C values in the range of -19.9 ‰ to -30.4 ‰. The urine samples showed values for 19-NA between -22.2 ‰ and -31.1 ‰. These results, as well as the degree of conformity between the results for urinary concentrations of 19-NA and 19-NA/19-NE and the IRMS δ C values, will be presented. Introduction Determining the origin of anabolic androgenic steroids (AAS) that are also produced endogenously in the human body, is a major issue in doping control. Nandrolone esters are widely used for their anabolic effects. The main metabolites of nandrolone are 19-norandrosterone and 19-noretiocholanolone. In some cases, the presence of low levels of these nandrolone metabolites may be a result of bacterial demethylation of endogenous androsterone and etiocholanolone [1] or pregnancy [2]. Analysis by GC-C- IRMS technique provides the capability to measure the carbon isotope ratio (CIR) to determine the origin of these metabolites. Published studies indicate, however, that there are nandrolone preparations available on the black market showing δ C values in, or close to, the range of values seen in endogenous steroids [3,4]. In this study, the aim was to measure δ C values of nandrolone in preparations seized in Norway by Norwegian police authorities between 2013 and 2020, to investigate if there were changes compared to preparations that were part of a previous study [4]. In addition, urine samples collected in Norway and Denmark containing 19-NA and 19-NE, were analyzed. The samples originated mainly from fitness centers. 13 13 13 13 13 13 MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-613 Experimental All solvents were analytical grade or HPLC grade and were purchased from Sigma-Aldrich (Oslo, Norway). Water was purified in-house (Milli-RO, Millipore DirectQ). Nandrolone and steroidal reference materials were obtained from Cerilliant Corporation (Round Rock, TX, USA) and Sigma-Aldrich (Oslo, Norway). A mix of steroids CU34-3, MX018-1 and MX018-2 with certified carbon isotope delta values were provided by National Measurement Institute, department of Industrial, Science, Energy and Resource, Australian Government. Sample preparation: Seized material, either powder or oil preparations, was dissolved in methanol and hydrolysis of the esters was performed for 1 hour at 60°C in 1M methanolic KOH. The solution was neutralized with 1 M HCl, followed by solid phase extraction using 500 mg Bond Elute cartridges [4]. Urine samples were prepared using our in-house routine method which is based on the method for endogenous steroid published by the Anti-Doping Laboratory in Rome [5]. In brief, 1-21 mL urine was hydrolyzed using β-glucuronidase before liquid-liquid extraction by tert-butyl-methyl ether. The HPLC clean-up was performed using an ACE 5 C18 (250 mm × 4.6 mm, 5 µm) column. The separation was performed at 38°C. The mobile phase consisted of acetonitrile and purified water. Five fractions were collected, see Table 1. Pregnanediol (PD), androsterone (A), 11-Ketoetiocholanolone (11-Keto) and pregnanetriol (PT) were collected for use as endogenous reference compounds (ERC) in the analysis. The GC-C-IRMS analyses of both preparations and urine samples, were performed on a Thermo Delta V Plus coupled to ISQ single quadrupole mass spectrometer, using an Agilent J&W HP5 MS UI (30 m, i.d. 0.25 mm, film thickness 0.25 μ m) column as previously described [5]. 1-3 µL were injected using splitless mode. Table 1. Fractions collected during the HPLC clean-up Results and Discussion A total of 31 seized preparations were analyzed. Figure 1 shows the δ C of these preparations. The left panel (yellow) shows the δ C values measured in a previous study conducted in our laboratory in 2014 [5]. The average δ C in ERCs observed in our laboratory in a Norwegian/Danish reference population is 23.3 ‰, as indicated in Figure 1 with a solid black line. 13 13 13 13 MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-614 The nandrolone preparations showed δ C values in the range of -19.9 to -30.4 ‰. The urine samples showed values for 19-NA between -22.2 and -31.1 ‰. Preparations seized before 2015 showed δ C values in a range different from the average observed for ERCs in a Norwegian/Danish population. After administration of these preparations the detected 19-NA in urine would be expected to fulfill the criteria for an Adverse Analytical Finding (AAF) for 19-NA [6]. In 2015, this picture changed and for nandrolone esters received for analysis in 2016 and 2017 we observed delta values close to the expected endogenous range in approximately 50% of the preparations. After 2018, the majority contains nandrolone with δ C close to this range. This constitutes a clear challenge for doping analysis as the possibility for false negative results for 19-NA in samples analyzed by GC-C-IRMS is greatly increased. Figure 1. Distribution of δ C in the seized preparations A total of 39 urine samples containing 19-NA were analyzed by GC-C-IRMS. Three of these had been collected in 2011-2014 and 36 in the period of 2017-2020. All except one were collected in fitness centers, or similar. Several of these samples also contained other anabolic androgenic steroids. 13 13 13 13 MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-615 Table 2. Results including δ C values measured in urine samples containing 19-NA estimated to 15 ng/mL or lower Table 2 shows the results for the samples with estimated 19-NA between 5 and 15 ng/mL (corrected for SG according to [6]). According to TD2021NA, an IRMS analysis would be mandatory for these samples. The results for 11 of the 15 samples showed δ C that would not fulfill the criteria for an AAF according to the technical document [6] when using PD as ERC. However, when compared to the measured δ C in androsterone (A), the differences in eight of the samples indicate that 19-NA has a different origin, and the presence of 19-NA cannot be a result of bacterial demethylation of A. These subjects had been using testosterone preparations in addition to nandrolone which will influence the δ C values of androsterone. Accordingly, A is not a valid ERC if its δ C value indicates administration of testosterone or testosterone precursor(s) (TD2021NA [6]). Table 2 also shows the 19-NA/19-NE concentration-ratio measured in the samples. If the IRMS results do not fulfill the criteria for exogenous origin of the 19-NA, the ratio 19-NA/19-NE must be taken into consideration. According to TD2021NA, the result constitutes an Atypical Finding (ATF) if this ratio is higher than 3. However, if the ratio is ≤ 3, the result will be reported as negative. When this is applied to the samples in Table 2, eight of the samples would have been reported as ATF. In addition, two samples show 19-NE lower than LOQ, which also would result in a high ratio. The evaluation of the 19-NA/19-NE ratio can indicate the exogenous origin of 19-NA, but this would have to be verified by GC-C-IRMS [6]. 13 13 13 13 13 MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-616 Table 3. Results including δ C values measured in urine samples containing 19-NA estimated to higher than 15 ng/mL. For samples 1, 13 and 20, 11-Keto was used as ERC. Table 3 shows the results for 24 samples with 19-NA estimated to higher than 15 ng/mL (corrected for SG according to (6)). Of these 24, 18 samples showed 19-NA with δ C within the range we see for ERCs in urine samples from Norway and Denmark. Only six samples would have fulfilled the criteria given in TD2021NA (6) if the result from the GC-C-IRMS was to be used as decision for an AAF. However, when comparing the measured δ C values for A and 19-NA, the difference is too large to be compatible with in situ demethylation of A as origin for the presence of 19-NA in 11 of the samples. The table is also showing the 19-NA/19-NE ratio for the samples with 19-NA concentration > 15 ng/mL. For samples with 19-NA > 15 ng/mL, the evaluation of the ratio is of less relevance as it will reflect the metabolism of nandrolone in the body, and not potential bacterial degradation of A and Etio. The first choice of ERC in this study was pregnanediol (PD). However, in three samples (1, 13 and 20 in Table 3) the concentration of PD was too low for reliable results. The second choice would normally be androsterone (A), but for all these samples the δ C of A was influenced by concomitant use of testosterone. For this reason, A was excluded as ERC and 11-Keto was used. 13 13 13 13 MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-617 Conclusions According to this study, 70% of nandrolone preparations seized in Norway after 2017 show δ C within the range observed for ERCs in Norway and Denmark, i.e., the normal endogenous range of δ C isotopic signatures of urinary steroid metabolites. This is reflected in the analysis of urine samples with confirmed the presence of 19-NA collected in Norway and Denmark. Analysis of 19-NA in these samples reveals that 74% would not have fulfilled the positivity criteria used by the WADA-accredited doping control laboratories (TD2021NA). References 1. Grosse J, Anielski P, Hemmersbach P, Lund H, Mueller RK, Rautenberg C, Thieme D. Formation of 19- norsteroids by in situ demethylation of endogenous steroids in stored urine samples. Steroids. 2005 Jul;70(8):499-506. 2. Mareck-Engelke U, Schultze G, Geyer H, Schänzer W. The appearance of urinary 19-norandrosterone during pregnancy. Eur J Sport Sci. 2002;2:1-7. 3. Brailsford AD, Majidin WNM, Wojek N, Cowan DA, Walker C. IRMS delta values ( C) of nandrolone and testosterone products available in the UK: Implications for anti-doping. Drug Test Anal. 2018Nov;10(11- 12):1722-1727. 4. Carbon isotope ratios of nandrolone, boldenone, and testosterone preparations seized in Norway compared to those of endogenously produced steroids in a Nordic reference population. Hullstein I, Sagredo C, Hemmersbach P. Drug Test Anal. 2014 Nov-Dec;6(11-12):1163-9. 5. de la Torre X, Colamonici C, Curcio D, Molaioni F, Botrè F. A comprehensive procedure based on gas chromatography-isotope ratio mass spectrometry following high performance liquid chromatography purification for the analysis of underivatized testosterone and its analogues in human urine. Anal Chim Acta. 2012 Dec 5;756:23-9. 6. World Anti-Doping Agency. Technical Document, TD2021NA, Harmonization of Analysis and Reporting of 19-Norsteroids Related to Nandrolone, Montreal 2021. www.wada-ama.org/sites/default/files/ resources/files/td2021na_final_eng_v2.0_m.pdf (access date 08.10.2021) Acknowledgements The steroid preparations were kindly provided by the Norwegian National Criminal Investigation Service (Kripos). Part of the study (seizures analyzed before 2014) was financially supported by WADA, grant number 11A26CS. 13 13 13 MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-618 https://www.wada-ama.org/sites/default/files/resources/files/td2021na_final_eng_v2.0_m.pdf Thieme D 'Operation Bloodletting' - practical insights from a major doping trial? Institute of Doping Analysis and Sports Biochemistry (IDAS) - Dresden, Kreischa, Germany Abstract Organized blood doping in endurance sports was subject of an expensive trial which was held on more than 20 days from September 2020 to January 2021. The exhaustive investigations revealed useful insights into courses of action, skills, logistic and pharmacological precautions, networking, secondary substance abuse, and other aspects. Approximately 20 athletes were involved into accused doping activities from 2014-2018. Extra to the ‘conventional’ autologous blood doping, its deception by using erythropoietin or HIF-1a stabilizers was described in detail. Moreover, the use of numerous other doping agents and/or methods, e.g. hGH, IGF-1, Synacten, AICAR, SR9009, TB500 and TB1000 have either been attempted or accomplished. Strategies to avoid or ‘beat’ doping tests were recurrently discussed and their efficacy could be evaluated by assessing positive doping cases amongst corresponding athletes or comparisons between respective blood passports with confirmed blood manipulations. Introduction The 'Operation Bloodletting' represented a multi-national police investigation which had a focus on endurance sports and culminated during the Nordic Ski Worldcup in Seefeld, 20 February to 3 March 2019. Triggered by a TV interview given by a whistleblower, there were criminal investigations by Austrian and German police officers, who could rapidly identify structures of organized doping. The major protagonists, a German physiologist and few assistants, had completely underestimated the effects of the investigative TV report and continued their activities after a very short break. The core activity consisted in the supply of interested athletes with autologous blood transfusions. The default strategy consisted in blood infusion immediately prior to start and withdrawal of after competition, i.e. in the resting period before blood tests maybe conducted. There has never been any conflict with DCOs or chaperons who intended to monitor activities between contest and doping control. This may well have been favored by the fact, that none of the corresponding athletes reached outstanding (podium) placements. Speculations in the public that only secondary athletes were punished while (in particular German) top athletes remained untouched seemed non conclusive, as complete records (surveillance of telephone and text communications, numerous witness statements) were evaluated within an international criminal investigation. Experimental Autologous Blood Transfusion Whole blood was only collected and reinfused in few exceptional cases. For instance during Olympic Winter Games when blood was reinfused to the athletes prior to departure (flight to Seoul) next to the MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-619 injection of anticoagulant medications (Enoxaparin) and multiple withdrawal – transfusion cycles of whole blood were organized between the competitions. The default ‘service’ utilized erythrocyte concentrates and included: withdrawal of blood (1-2 bags) online- or offline formation of erythrocyte concentrates, 1 bag = 180 g erythrocytes, by apheresis machines (Alyx, MCS + 9000), storage at freezers (-80 °C) reconstitution of erythrocyte concentrates by an Automated Blood Cell Processor (ACP 215) on site reinfusion. The instrumental processes were well controlled, instruments were properly maintained. Expiration dates of consumables were observed. In contrast, on site operations (blood withdrawal and infusions at competition locations) were often performed under highly critical conditions, i.e. hotel rooms or backseats of cars. Moreover, the conventional labelling of blood bank (using nicknames) and the lack of initial tests for blood group compatibility were points of major concern. Alternative attempts to adjust blood profiles (instead of withdrawal post competition) were made in few cases for logistic reasons, typically if athletes or nurse assistants were not available in time. In those cases, the protocol included drinking of water and saline (500 mL each) or administration of albumin, at least if upcoming doping controls are assumed. Individual blood profiles – i.e. data retrieved from WADA database (ADAMS), complemented by own hematological tests in case of lacking doping controls – were carefully monitored in all cases. Critical deviations of relevant parameters were discussed between athletes and physician, e.g.: Concerned athlete: "Hi, off score 130!!! 16,1 hb u 0,27 reti in%. " Physician (original in German): "... need to raise Retis by E (po) …" According to the physician’s statements in court, erythropoietin was only applied in low doses (e.g. 2x 300 IU) to increase relative amount of reticolycytes, i.e. to adjust blood profiles and not aiming to increase of oxygen transport capacity. MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-620 Figure 1. Blood Passport of an athlete during long-term blood doping. (Poor quality according to the court records). In spite of numerous blood tests, there was only one suspicious record which could be dismissed by pretending the use of an oxygen tent. Based on the criminal investigations, at least 13 proven blood infusions could be substantiated (red arrows) one of it took place the day before an ABP doping control (longer red arrow) but none of them raised any suspicion. HIF-1a-stabilizers HIF-1a-stabilizers gained a certain popularity as a save (undetectable) alternative to the administration of EPO, i.e. to adjust blood profiles. In theory, the organizers of the doping activities - i.e. the physician and a small group of trainers or consultants of athletes – were informed about the biological effects, potential availability and detectability of roxadustat (FG-4592), molidustat (BAY 85-3934) and daprodustat (GSK1278863). The focus switched immediately to the next compound after the first report of doping cases related to roxadustat (2015) and molidustat (2017). In practice, it remained unclear if the athletes could really purchase the intended (pure?) HIF-1a- stabilizers, which were delivered as unlabeled powders from unclear sources. The doubts are supported by the inconsistent appearance (color) as well as missing biological effects (unchanged reticulocyte levels after a three weeks roxadustat administration cycle). MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-621 Figure 2. According to the clinical data, i.e. high dosages and considerable half-live [1] and the significant prices (e.g. Cayman), HIF-1a stabilizers seemed to become less interesting for cheating athletes than erythropoietin. Additional Doping Activities All other doping activities (i.e. outside blood transfusion and epo supplementation) were based on hearsay, poor level of reliable information or lack of any conclusive concept. Clear misunderstandings lead to the assumption, that: the HIF-1 a protein (rather than its HIF stabilizers) was purchased and could be used as endogenous (hence undetectable) alternative for blood doping or extracellular hemoglobin, which was purchased at Sigma Aldrich, would be a suitable alternative to enhance oxygen transportation. In the latter situation, the unmodified human hemoglobin was thought to be a kind of HBOCs and – beside the qualitative fact, that extracellular hemoglobin cannot release the bound oxygen at the target tissue and does not markedly contribute to oxygen transport – there was an inexplicable quantitative confusion. It was thought that 10 g (accidentally confused with milligrams) hemoglobin is equivalent to one bag (‘Human Hämoglobin from Sigma …. Ampulle 10 mg compares zu 1 bag’). This misunderstanding is hard to explain, as ABP data – known to respective athletes and consultants – indicate that normal Hb concentrations (~15 g/dL in healthy athletes resulting) corresponds to approximately 70 g Hb per bag. Luckily, the irresponsible attempt to infuse hemoglobin (presumably 10 g, dissolved in saline) to a volunteer athlete, ended with moderate side effects (i.e. haematuria, temperature malregulation, allergic reactions) – presumably due to the miscalculation of dosage. Numerous other doping substances gained interest of the group. The administration of Somatropin (Gentropin, Norditropin or Go-Quick purchased from pharmacies) was confirmed. Dose recommendations were provided (e.g. every other day 0.027 mg/kg) in accordance by published detection capabilities (Bidlingmaier M and Strasburger CJ [2]) but there were no systematic concepts or strategies discussed. Further doping agents such as IGF-1 (from Sigma), AICAR, TB-500 and TB-1000 became presumably available. There was no apparent concept with respect to useful dosage, time of application, desirable effects or risk of detection. MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-622 Numerous other compounds were under consideration, but it remains highly questionable if any of those has been purchased or applied, e.g. ITPP, IGF-1 (mecasermin), IGF-1-LR3, GRF 1-29, GHRP6, Gonadorelin, Synacten, AICAR, SR9009. At least a systematic application within the doping-network is not assumed. Conclusions Application of the straight forward infusion-withdrawal (so called ‘IN-OUT’) approach proved to be an easy and widely unnoticed strategy of blood doping. Access of athletes and their doping consultants to ABP data was found to be an effective way to avoid detection of blood doping. Erythropoietin was applied to adjust reticulocytes in cases of critical blood profiles (off-scores). HIF-1a-stabilizers gained temporal popularity as an alternative but was soon devalued after upcoming doping cases. Any complementary doping attempts – i.e. outside autologous blood transfusion – were carried out in a surprisingly unprofessional manner. The logistic effort (travelling assistants, instrumentation, freezer capacity) to maintain this version of blood doping was considerable. Balancing the available resources, it deems likely that the 23 athletes convicted of blood doping represent the vast majority of the network. All of the 45 confiscated blood bags could be attributed to these athletes. References 1. Neil S. Sanghani and Volker H. Haase, Hypoxia-Inducible Factor Activators in Renal Anemia: Current Clinical Experience Adv Chronic Kidney Dis. 2019;26(4):253-266 2. Bidlingmaier M, Strasburger CJ. Nat, Technology insight: detecting growth hormone abuse in athletes. Clin Pract Endocrinol Metab. 2007 MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-623 Mazzoni I , Ventura R , Daley-Yates P , Collomp K , Saugy M , Buttgereit F , Rabin O , Stuart M Defining permitted and prohibited use of glucocorticoids in the anti- doping context Part I. Glucocorticoids: new approach and new regulations Science & Medicine, WADA, Montreal, Canada ; Catalonian Antidoping Laboratory Fundació Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain ; GSK, London, UK ; Université D'Orléans, Orléans, France ; Université Paris-Saclay, Orsay, France ; AFLD, Chatenay-Malabry, France ; REDs, Lausanne, Switzerland ; Charité University Medicine, Berlin, Germany ; University College London, London, UK Abstract The use of glucocorticoids (GC) is prohibited in sports competitions when administered by oral, intravenous, intramuscular or rectal routes, and they are allowed by other routes for therapeutic purposes. There are no restrictions of use in out-of-competition periods. The ability to differentiate between permitted and prohibited administrations was needed, and a temporary reporting level of 30 ng/mL was initially established by WADA. However, different studies have shown the need of establishing compound-specific reporting levels. Additionally, local injections of GC result in urinary and plasmatic concentrations similar to those obtained after prohibited routes, indicating systemic distribution of the drug. As a consequence, the status of local injections of GC in the WADA Prohibited List needed to be re-evaluated. A novel approach for defining permitted and prohibited use of GC in sport based on the potential for performance enhancement and risk to health has been developed. Known performance enhancing doses of GC are expressed in terms of daily cortisol-equivalent doses and, thereby, the dose which may be potentially performance enhancing for any GC and route of administration can be derived. The model supports that local injections produce similar systemic effects than intramuscular administration. In consequence, local injections will be included in the list of prohibited routes of administration from 1 January 2022. Based on administration studies available in the literature, revised and substance-specific urinary reporting levels are proposed to better distinguish between prohibited and permitted GC use in sport. In addition, washout periods are presented to enable clinicians to use GC safely and to avoid the risk of athletes testing positive for a doping test. Introduction Glucocorticoids (GC) have been prohibited in sports since 1996 by the International Olympic Committee (IOC) [1], banned except for topical use (aural, dermatological and ophthalmological except rectal), by 1 2 3 4,5,6 7 8 1 9 1 2 3 4 5 6 7 8 9 st MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-624 inhalation, or by intra-articular or local injection. A written notification was needed prior to competition for some non-prohibited routes (inhaled, local or intra-articular injections). In 2000 it was clarified that “The systemic use of glucocorticosteroids is prohibited when administered orally, rectally, or by intravenous or intramuscular injection” [2] implying that these 4 routes of administration were the ones recognized as systemic. In 2004, WADA published the first International Standard for the List of Prohibited Substances and Methods (the List) [3] and GC remained prohibited in competition because they fulfil at least 2 of 3 criteria established by the World Anti-Doping Code [4]: a- Proven or potential to enhance sport performance: GC use results in central nervous system effects (euphoria), hyperglycemia, increase in energy mobilization and stimulation of erythropoiesis. In this regard, some studies demonstrated performance enhancement (PE) of short-term use of oral GC in different exercise models [5-7]. b- Use represents an actual or potential health risk: even if GC are widely used in medical practise, they also produce serious adverse effect like cortisol suppression, increases in blood pressure, cholesterol or glucose, water retention, headaches, dizziness, mood swings, osteoporosis, immunosuppression and lengthening in wound healing [8-11]. c- Use is detrimental to the spirit of sport. The List maintained prohibited the same routes of administration as the IOC, as they were assumed to produce the systemic effects linked to performance enhancement (PE) and risk for health. Results and Discussion With the publication of the 1 List by WADA, a temporary urinary reporting concentration (Minimum Required Performance Level = MRPL) of 30 ng/mL was established to distinguish permitted and prohibited routes of administration and differentiate in- and out-of-competition use. This concentration was empirically based on doping control practice as there was limited information on urinary excretion of GC. However, it was evident that a unique MRPL would unlikely be adequate for all GC, as the numerous GC approved worldwide for medical use have different potencies, pharmacokinetic properties, doses and routes of administration. For example, the plasma half life of methylprednisolone is 2.5 h, and of dexamethasone is 4.0 h; the relative affinity to the GC receptor, taking cortisol as 100, is 5 for prednisone, 220 for prednisolone and 1200 for methylprednisone. Consequently, different metabolism, excretions rates, and urinary concentrations should be expected [12]. In order to improve the GC MRPL of 30 ng/mL, WADA awarded in 2004 three research projects to Dr Martial Saugy, Dr Ray Kaslauskas and Dr Jacques de Ceaurriz from the anti-doping laboratories at Lausanne, Sydney and Paris, respectively, to perform excretion studies of the most used GC and routes of administration [13-15]. In addition, 3 research projects were subsequently funded to study the PE aspects of GC [16-18]. The initial results confirmed the variability in excretion patterns and concentrations for common pharmacological doses and routes of administration (Fig. 1)[13]. st MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-625 Figure 1. Examples of the variability in urinary excretion of commonly used pharmacological doses for the same route of administration: 2 mg oral betamethasone (A) and 16 mg oral triamcinolone (B), or different routes of administration: 100 µg inhaled prednisolone acetonide (C). In the examples shown, the maximum urinary concentration of a therapeutic dose of oral betamethasone is ca. 100 ng/mL, while that of oral triamcinolone is in the order of 8 µg/mL range, and inhaled triamcinolone acetonide would not surpass the single digit ng/mL values (from Saugy M, Avois L. Criteria setting for the misuse of glucocorticosteroids; Study LSDD-Lausanne 2008). However, the initial results from the local injections (e.g. intra-articular, peri-articular, peritendinous) were unexpected, as it revealed that the urinary levels attained were similar to the systemic routes (see example Fig. 2). From this, it could be predicted that local GC injections did not only remain confined to the site of injection but also distributed systemically. This generated a contradiction in the List, as most likely systemic effects (i.e. PE and risk for health) similar to the prohibited routes would occur following local injections but they were permitted. In addition, since the urinary concentrations were indistinguishable, this created problems for the results management. For example, in Figure 2, an estimated urine concentration of 100 ng/mL betamethasone could be either due to an intra-articular injection administered the day of the competition, which was allowed, or an oral administration of betamethasone within 24 h of the doping control, which was prohibited and would lead to a sanction. MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-626 Figure 2. Examples of the similarity in urinary excretion of commonly used pharmacological doses of beta- methasone for a prohibited route of administration: 2 mg oral betamethasone (A), and a permitted route of administration: 7 mg intra-articular betamethasone (B) (from Saugy M, Avois L. Criteria setting for the misuse of glucocorticosteroids; Study LSDD-Lausanne 2008) By 2008-2009 all three initial WADA-funded studies were completed and based on the new data, the List Expert Group (EG) discussed whether to prohibit local injections. However, there were concerns as these injections were broadly used in sport and could be perceived as negatively interfering with medical practice. In addition, the requests for TUE could increase. Therefore, more studies were needed to confirm and consolidate the data before taking any further actions. From 2010 onwards, WADA awarded three more studies to Dr Rosa Ventura from the Barcelona doping control laboratory [19-21]. In addition, other studies independent from WADA were conducted in other anti-doping laboratories, such as Rome, Athens and New Delhi to name a few [22-24]. The complete list of studies used to propose the prohibition of local injections in the 2022 List can be found in part III of this trilogy on “Defining permitted and prohibited use of glucocorticoids in the anti-doping context” by Rosa Ventura et al. By 2016, there were enough results confirming the overlap in excretion of local injections and the prohibited routes of administration. From this, a systemic distribution was not only inferred but also demonstrated by published results showing cortisol suppression following local injections [10,25]. Consequently, the List EG formed a first GC Working Group (GCWG) which reviewed the data and recommended to prohibit local injections. Therefore, the draft 2017 List proposed prohibiting all GC injections, keeping the MRPL of 30 ng/mL and including a 72 h washout period to allow elimination of the GC administered out-of-competition. However, upon circulation of the draft List to the stakeholders, more than 50% of responders did not support the change, some because they believed that these injections were not used for doping, others because it would interfere with medical practice and/or increase the number of TUEs while others argued that the reasons for the change were not extensively explained. A second GCWG was formed to review the overall status of GC and its place in the List. This GCWG recommended keeping GC prohibited in competition but proposed prohibiting them and establishing reporting levels based on PE. Due to the requirement to link pharmacokinetics of the different GC with PE, and the likeliness of reviewing the MRPL, a third (current) GCWG was established with a more technical composition at the analytical, pharmacological, pharmacokinetic, GC therapy and PE levels. The third GCWG met initially in 2018 and evaluated the previous recommendations. The diversity of GC with different potencies, effective doses, pharmacokinetics, elimination time, affinity and length of occupancy for GC receptor was considered. In addition, the revised 2021 Code defines in-competition as MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-627 the period commencing at 11:59 p.m. on the day before a competition until the end of competition and the sample collection. Therefore, the in-competition period would be variable as well, and there could be single or multiple competitions in a day, with different durations. In view of this as well the variability in GC excretion patterns, it was concluded that it was not realistic or achievable to establish MRPL based purely on PE. In addition, if the prohibition was based only on PE, it would be in conflict with the World Anti-Doping Code, as it is necessary to fulfil any 2 of 3 aforementioned criteria to consider prohibiting a substance. Instead, during 2018 to 2020 the third GCWG developed a novel approach to define acceptable and not acceptable use of GC in sport based on cortisol equivalents (see part II-presentation by Peter Daley-Yates for details) linked to PE and risk to health. From this analysis it was concluded that local GC injections were equivalent to the prohibited routes of administration and consequently, should be prohibited in- competition. Once the permitted and prohibited routes were identified, the new MRPL for different GC were established as well. Finally, washout periods were determined for safe medical use of GC in sport to avoid an Adverse Analytical Finding when a GC is administered out-of-competition (see part III- presentation by Rosa Ventura). The proposal “All glucocorticoids are prohibited (in-competition) when administered by any injectable, oral, or rectal route” was included in the draft 2021 List. Examples of injectable routes include intravenous, intramuscular, periarticular, intra-articular, peritendinous, intratendinous, epidural, intrabursal, intradermal, subcutaneous. The proposal, circulated in April 2020, was supported by the majority of stakeholders. However, due to the widespread use of GC in medical practice, the WADA Executive Committee decided, in September 2020, to implement the prohibition for the 2022 List to be able to thoroughly communicate the change, allow medical personnel to get acquainted with the implementation of the washout periods, enable laboratories to update their analytical procedures and give time to sports authorities to develop educational tools for Athletes. Conclusions After many years of gathering data on GC excretion, a novel strategy based on cortisol equivalents was developed to assess the prohibited or permitted status of GC routes of administration and to establish new and more suitable MRPLs that reflect the pharmacokinetics of different GC, plus washout periods that take into consideration the permitted use of GC out-of-competition. This comprehensive work allowed to solve the incongruency between the status of different GC injections, produced more fit-for- purpose MRPLs and will guide physicians in their choice and timing of GC administration. Overall, the new rules to be implemented in 2022 will impact and benefit all aspects of the use of GC in sport. References 1. Prohibited Classes of Substances and Prohibited Methods, 1996, International Olympic Committee Medical Commission, Lausanne, Switzerland, 1996. 2. Prohibited Classes of Substances and Prohibited Methods, 2000, Olympic Movement Anti-doping Code, Appendix A, International Olympic Committee Medical Commission, Lausanne, Switzerland, 2000. 3. The Prohibited List International Standard, 2004, World Anti-Doping Agency, Montreal, Canada, https://www.wada-ama.org/sites/default/files/resources/files/WADA_Prohibited_List_2004_EN.pdf (access date 07.2021) 4. World Anti-Doping Code (2021). Eds: World Anti-Doping Agency, Montreal, Canada, 2021. MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-628 https://www.wada-ama.org/sites/default/files/resources/files/2021_wada_code.pdf (access date 07.2021) 5. Arlettaz A, Portier H, Lecoq A-M, Rieth N, De Ceaurriz J, Collomp K. (2007) Effects of short-term prednisolone intake during submaximal exercise. Med Sci Sports Exerc; 39 (9), 1672-1678. 6. Panse BL, Le Panse B, Thomasson R, Lecoq AM, Amiot V, Rieth N, De Ceaurriz J, Collomp K. (2009) Short- term glucocorticoid intake improves exercise endurance in healthy recreationally trained women. Eur J. Applied Physiol . 107(4), 437-443. 7. Collomp K, Arlettaz A, Portier H, Lecoq AM, Le Panse B, Rieth N, De Ceaurriz J. (2008) Short-term glucocorticoid intake combined with intense training on performance and hormonal responses. Br J Sports Med;42(12), 983-988. 8. Buttgereit F. (2020) Views on glucocorticoid therapy in rheumatology: the age of convergence. Nat Rev Rheumatol. 16(4), 239-246. 9. 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Collomp K, Arlettaz A, Pelle A, Portier H, Rieth N, Fontayne P, Laure P, Le Scanff C, Lecoq AM (2005) Potential direct and indirect ergogenic effects of glucocorticoids https://www.wada- ama.org/sites/default/files/resources/files/collomp-potential_direct_and_indirect_.pdf (access date 07.2021) 17. Do MC, Collomp K, Prieur F, Gagey O (2011) Effects of glucocorticoid during repeated bouts of high- intensity exercise https://www.wada-ama.org/sites/default/files/resources/files/review_dr_do_11d7md_0.pdf (access date 07.2021) 18. Collomp K, Do MC, Lasne F (2015) Exogenous DHEA administration and performance: Possible mechanisms of action and metabolic signature https://www.wada-ama.org/sites/default/files/resources/files/final_ report_14d05kp_pr._collomp.pdf (access date 07.2021) 19. Ventura R, Segura J, Matabosch X, Pozo O, Berges Casas R, Monfort N (2013) Evaluation of alternative glucocorticosteroid metabolites for the discrimination between legal and forbidden administration routes. https://www.wada-ama.org/sites/default/files/resources/files/research_13d22rv_final.pdf (access date 07.2021) 20. Ventura R, Matabosch X, Coll S (2016) Studies on intra-articular and peri-articular administrations of glucocorticoids https://www.wada-ama.org/sites/default/files/resources/files/16c11rv_dr_ ventura_summary.pdf (access date 07.2021) 21. Ventura R (2020) Studies of glucocorticoids after oral administration: evaluation of reporting levels and washout periods https://www.wada-ama.org/en/resources/research/studies-of-glucocorticoids-after-oral- administration-evaluation-of-reporting (access date 08.2021) 22. Mazzarino M, Piantadosi C, Comunità F, de la Torre X, Botrè F. (2019) Urinary excretion profile of prednisone and prednisolone after different administration routes. Drug Test Anal. 11(11-12), 1601-1614. 23. Ahi S, Beotra A, Dubey S, Upadhyay A, Jain S. (2012) Simultaneous identification of prednisolone and its ten metabolites in human urine by high performance liquid chromatography-tandem mass spectrometry. Drug Test Anal. 4(6) ,460-467. 24. Athanasiadou I, Vonaparti A, Dokoumetzidis A, Saleh A, Mbeloug M, Al-Maadheed M, Valsami G, Georgakopoulos C. (2019) Effect of hyperhydration on the pharmacokinetics and detection of orally administered budesonide in doping control analysis. Scand J Med Sci Sports 29(10), 1489-1500. 25. Matabosch X, Llorente-Onaindia J, Carbó ML, Pérez-Mañá C, Monfort N, Monfort J, Ventura R. (2019) Elimination profile of triamcinolone hexacetonide and its metabolites in human urine and plasma after a single intra-articular administration. Drug Test Anal. 11(11-12), 1589-1600. Acknowledgements All the scientist who contributed to research on glucocorticoids and the review of data in particular: anti- doping laboratories and members of the Working Groups and List Expert Groups for the time dedicated to review data and make recommendations on this complex issue. MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-629 Daley-Yates P , Ventura R , Mazzoni I , Collomp K , Saugy M , Buttgereit F , Rabin O , Stuart M Defining permitted and prohibited use of glucocorticoids in the anti- doping context Part II. Approach for defining acceptable and unacceptable use of glucocorticoids in sport GSK, London, UK ; Catalonian Antidoping Laboratory Fundació Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain ; WADA, Montreal, Canada ; Université D'Orléans, Orléans, France ; Université Paris-Saclay, Orsay, France ; AFLD, Chatenay-Malabry, France ; REDs, Lausanne, Switzerland ; Charité University Medicine, Berlin, Germany ; University College London, London, UK Abstract The use of glucocorticoids (GC) is prohibited in sports competitions when administered by oral, intravenous, intramuscular or rectal routes, and they are allowed by other routes for therapeutic purposes. There are no restrictions of use in out-of-competition periods. The ability to differentiate between permitted and prohibited administrations was needed, and a temporary reporting level of 30 ng/mL was initially established by WADA. However, different studies have shown the need of establishing compound-specific reporting levels. Additionally, local injections including peri-articular and intra-articular of GC result in urine and plasma concentrations similar to those obtained after prohibited routes, indicating systemic distribution of the drug. As a consequence, the status of local injections of GC in the WADA Prohibited List needed to be re-evaluated. A novel approach for defining permitted and prohibited use of GC in sport based on the potential for performance enhancement and risk to health has been developed. Known performance enhancing doses of GC are expressed in terms of daily cortisol-equivalent doses and, thereby, the dose which may be potentially performance enhancing for any GC and route of administration can be derived. The model supports that local injections produce similar systemic effects than intramuscular administration. In consequence, local injections will be included in the list of prohibited routes of administration from 1 January 2022. Oral and injectable GC, when used at their approved therapeutic doses, are likely to produce total GC exposures above the performance enhancing threshold. However, inhaled, intranasal, dermal or other topical GC, when used at their approved therapeutic doses, are unlikely to exceed the performance enhancing threshold. Introduction Glucocorticoids (GC) and their synthetic analogues have a wide range of potencies and pharmacokinetic properties [1]. In man, the normal daily output of the naturally occurring GC (cortisol) is ≈ 18-22 mg/day 1 2 3 4,5,6 7 8 3 9 1 2 3 4 5 6 7 8 9 st MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-630 with an estimated upper normal physiological range of ≈ 26.4 mg [2,3]. Administering GC drugs can result in a total GC exposure (exogenous + endogenous) that exceeds the upper physiological range and hence is potentially performance enhancing. Whereas GC drug use that does not exceed this upper physiological threshold can reasonably be regarded as not performance enhancing [3]. The administration of GC drugs by topical routes such as inhaled, intranasal, ophthalmological, perianal and dermal, are unlikely to reach performance enhancing levels even at maximum licensed therapeutic doses. However, other routes of administration (e.g. oral and parenteral) have been shown to be potentially performance enhancing within the normal therapeutic dose ranges. These performance- enhancing doses can be expressed in terms of cortisol-equivalent doses and thereby the dose which may be potentially performance enhancing for any GC and route of administration can be determined [3]. Experimental This approach was applied to define the GC doses and routes of administration that should be acceptable or unacceptable for use in sport. To make this assessment, we converted the administered exogenous GC dose into a cortisol equivalent dose using: the bioavailability of the systemically absorbed fraction of the dose for the route of delivery and formulation; the relative GC potency in terms of the GC-receptor binding affinity or GC activity; the rate at which active GC is cleared from the body via metabolism and/or excretion; the plasma clearance of cortisol; and the plasma clearance of the exogenous GC [3]. These parameters are mostly available for commonly used GC formulations and routes of administration. This may not be known for some topical routes (e.g. skin, eye, ear) and older molecules, however, even when assuming 100% bioavailability the exposure is estimated to be below the upper physiological exposure threshold over the entire therapeutic dose range. Also, for intradermal, peri-articular and intralesional, routes bioavailability was assumed to be 100%. However, for intra-articular injections absorption from the injection site can be prolonged. Although 100% bioavailability may eventually be attained, we estimated the fraction absorbed in each 24h period post-dose from the absorption half-life. We also considered the impact of cortisol suppression that can occur following exogenous GC administration. However, following single doses this is not relevant because cortisol already present in the body is not immediately removed but is part of the total GC pool. Following chronic administration cortisol suppression is relevant, but not in the performance enhancing range because endogenous cortisol makes only a small contribution to the total GC pool [3]. Results and Discussion For the approved dose ranges for all the commonly used GC drugs, formulations, and routes of administration the conversion was made into cortisol equivalents. These were compared with the upper end of normal and supraphysiological cortisol thresholds (Figure 1). Comparisons of the cortisol equivalents dose estimates were also made with GC dose regimens known to be performance enhancing based on published data [3]. MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-631 Figure 1. Physiological, supra-physiological and performance enhancing GC exposure Using this approach, two categories were defined: (i) Acceptable GC use, defined as a dose of exogenous GC (cortisol equivalent dose ≤ 5.28 mg/day) that when added to upper normal physiological daily GC exposure (26.4 mg/day) results in a total exposure (endogenous + exogenous) that does not exceed supraphysiological levels (cortisol equivalent dose > 32 mg/day) (Figure 1). (ii) Unacceptable GC use, defined as a dose of exogenous GC that is equivalent to a dose demonstrated to be performance enhancing based on published data, e.g. 4 mg oral dexamethasone (32.6 mg in cortisol equivalents) [3]. This is ≈ 6 times the 5.28 mg/day acceptable dose defined above and results in a total GC exposure (26.4 mg endogenous + 32.6 mg exogenous) of ≈ 60 mg/day. Between these two categories, there are insufficient data to assess potential for performance enhancement. However, despite this limitation this approach allows clear guidance on acceptable and unacceptable use since many of the widely used GC doses and formulations fall into either the acceptable or unacceptable category [3]. Conclusions Although clinical data demonstrating performance enhancement are only available for 4 mg oral dexamethasone, 50 mg and 60 mg oral prednisolone, these correspond to 32.6 mg, 80 mg, and 96 mg in cortisol equivalents, respectively. Based on extrapolation, 20 mg oral prednisolone and 8.5 mg intra- articular triamcinolone acetonide are estimated to all have cortisol equivalent systemic exposure equivalent to 4 mg oral dexamethasone and hence are considered potentially enhancing. Oral and injectable routes (e.g. intravenous, intramuscular, subcutaneous, intra-articular) when used at their approved doses are likely to produce total GC exposures of > 32mg/day except at the lowest doses that are rarely used clinically. For example, oral prednisolone (> 3.3mg) and oral dexamethasone (> 0.65mg) and intra-articular triamcinolone acetonide (> 1.4mg) are estimated to produce total GC exposures of MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-632 > 32 mg/day. However, none of the inhaled, intranasal, dermal or other topical GCs when used at their approved doses would exceed the 32 mg/day potentially performance enhancing threshold. References 1. Daley-Yates PT. (2015) Inhaled corticosteroids: potency, dose equivalence and therapeutic index Br J Clin Pharmacol 80(3):372-380. 2. Kraan GP, Dullaart RP, Pratt JJ, et al. (1988) The daily cortisol production reinvestigated in healthy men. The serum and urinary cortisol production rates are not significantly different. J Clin Endocrinol Metab 83(4):1247-1252. 3. Ventura R, Daley-Yates PT, Mazzoni I, et al. (2020) A novel approach to improve detection of glucocorticoid doping in sport with new guidance for physicians prescribing for athletes. British J Sports Med 55:631-642. MANFRED DONIKE WORKSHOP 2021 Lecture RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-633 Haenelt N, Lourens L, Fußhöller G, Geyer H, Goldmann L, Schult C, Schwenke A, Hülsemann F, Gougoulidis V, Blatt C, Thevis M Follow-up investigations of atypical passport findings for the ratio 5a-Androstane-3a,17b-diol/5b-Androstane-3a,17b-diol Institute of Biochemistry, German Sport University Cologne, Cologne, Germany Abstract According to the WADA technical document TD2016EAAS, the ratio 5α-Androstane-3α,17ß-diol/5ß- Androstane-3α,17ß-diol (5αAdiol/5βAdiol) > 2.4 is an atypical passport finding (ATPF) and triggers a Suspicious Steroid profile-Confirmation Procedure Request (SSP-CPR). The confirmation procedure (CP) includes a confirmation of the steroid profile and an additional analysis with GC/C/IRMS. In the years 2016-2018, a total of 82 samples with 5αAdiol/5βAdiol > 2.4 underwent CPs in the Cologne laboratory, of which only one yielded a positive GC/C/IRMS result. This sample showed additionally a 5α- dihydrotestosterone concentration above a laboratory-internal population-based threshold. The characteristics of the GC/C/IRMS negative samples (IC/OOC samples, bacterial activities, other steroid profile ratios, sex of athletes, pH etc.) were evaluated. Introduction Since the implementation of the WADA technical document TD2016EAAS [1], the ratio 5αAdiol/5βAdiol > 2.4 triggers a Suspicious Steroid Profile-Confirmation Procedure Request (SSP-CPR) in case no further steroid profiles of the athlete are availabe in the steroidal athlete biological passport module (steroidal ABP). The confirmation procedure (CP) includes a confirmation of the steroid profile and an additional analysis with GC/C/IRMS. In the following results of such CPs from the years 2016–2019 are presented. Experimental The initial testing procedure (ITP) and confirmation procedure (CP) of the steroid profiles were conducted according to the method described by Thevis [2] with consideration of the rules described in the TD EAAS [1]. The IRMS analyses were conducted according to the method described by Piper et al. [3]. Results and Discussion In the years 2016-2019, a total of 82 samples with 5αAdiol/5βAdiol > 2.4 in the initial testing procedure (ITP) underwent CPs in the Cologne laboratory. Only one sample yielded a positive GC/C/IRMS result. This sample showed, additionally to the increased ratio 5αAdiol/5βAdiol (ITP: 2.73; CP 2.65), an increased ratio androsterone/etiocholanolone (A/Etio; ITP: 5.25; CP: 4.72) and a 5α-dihydrotestosterone (DHT) concentration of 35.3 ng/mL, which is above the laboratory-internal population-based thresholds for female and male athletes of 18 ng/mL and 21 ng/mL, respectively [4]. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-634 The characteristics of the 81 GC/C/IRMS negative samples are presented in the following: 67 samples (83%) showed bacterial activities based on the ratio of free testosterone over total testosterone ≥ 0.05, obtained in the CP. These samples were invalidated for the use in the steroidal ABP according to the TD EAAS [1]. None of the samples exceeded the thresholds 5α-androstanedione/ androsterone and/or 5β-androstanedione/etiocholanolone ≥ 0.1, which are indicators of strong bacterial activities [1]. Nine samples showed pH values above 7.0. The majority of the samples (91%) originated from male athletes. 87% of the samples were collected in-competition, and 52% of the samples could be attributed to combat or strength sports. All samples showed DHT concentrations below the laboratory- internal population-based thresholds for female and male athletes [4]. The DHT concentrations showed a median value of 2.20 ng/mL with an interquartile range from 1.3 to 3.2 ng/mL. It was not yet evaluated, if further samples of the athletes with increased ratios 5αAdiol/5βAdiol show also increased ratios, i.e. have naturally increased 5αAdiol/5βAdiol ratios. No correlation between the concentrations of 5αAdiol and 5βAdiol and increased ratios of 5αAdiol/5βAdiol could be found. In Figure 1a) and b), the distribution of the ratios 5αAdiol/5βAdiol and A/Etio are presented. 98% of the samples showed A/Etio ratios > 2, and 81% showed A/Etio ratios > 3. The ratios 5αAdiol/5βAdiol obtained in the ITP and CP showed no significant difference (Wilcoxon test, p=0.2533; see Figure 2). Figure 1. Distribution of the ratios 5αAdiol/5βAdiol (a) and A/Etio (b) in the 81 samples with 5αAdiol/5βAdiol > 2.4 and negative GC/C/IRMS results MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-635 Figure 2. Boxplots of the ratios 5αAdiol/5βAdiol of the 81 samples of the ITP and CP (no significant difference; p=0.2533; Wilcoxon test) Conclusions It cannot be excluded that many of the increased ratios 5αAdiol/5βAdiol (and A/Etio) are naturally increased ratios. But as all the analyses were triggered by SSP-CPRs, no further steroid profiles of the athletes were available to verify this assumption. Based on the fact that 83% of the samples with the ratio of 5αAdiol/5βAdiol > 2.4 showed a ratio of free testosterone to total testosterone ≥ 0.05 and 87% were collected in-competition, it may be concluded that bacterial activities and/or mental stress contributed to the increased ratios and not a doping scenario. As the only sample, which led to a positive GC/C/IRMS out of the 82 samples contained additionally a high concentration of DHT, we propose that only a combination of an increased ratio 5αAdiol/5βAdiol > 2.4 with a high concentration of DHT may trigger an SSP-CPR. This proceeding may prevent unnecessary, time consuming and expensive CPs with GC/C/IRMS analyses. References 1. WADA Technical Document - TD 2016 EAAS. Endogenous Anabolic Androgenic Steroids Measurement and Reporting, www.wada-ama.org/sites/default/files/resources/files/wada-td2016eaas-eaas-measurement-and- reporting-en.pdf (access: 16.03.2021) 2. Thevis M. Mass Spectrometry in Sports Drug Testing - Characterization of Prohibited Substances and Doping Control Analytical Assays. Wiley, New Jersey, 2010. 376 pages. ISBN: 978-0-470-41327-2 3. Piper T, Mareck U, Geyer H, Flenker U, Thevis M, Platen P, Schänzer W. Determination of C/ C ratios of endogenous urinary steroids: method validation, reference population and application to doping control purposes. Rapid Commun. Mass Spectrom. 2008, 22, 2161-2175 4. Mareck U, Geyer H, Opfermann G, Thevis M, Schänzer W. Factors influencing the steroid profile in doping control analysis. J Mass Spectrom, 2008, 43, 877-891. Acknowledgements The authors acknowledge support from the Federal Ministry of the Interior, Community and Building of MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-636 the Federal Republic of Germany (Berlin, Germany) and the Manfred Donike Institute for Doping Analysis (Cologne, Germany) MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-637 Fußhöller G, Geyer H, Haenelt N, Hülsemann F, Gougoulidis V, Blatt C, Thevis M Additional investigations in connection with atypical findings for 19-norandrosterone - a case study Institute of Biochemistry, German Sport University Cologne, Cologne, Germany Abstract In this case study, two atypical findings for 19-norandrosterone (NA) from an athlete are reported, i.e. NA concentrations between 2.5 and 15 ng/mL and IRMS values for NA indistinguishable from those of endogenous steroids. The result management authority (RMA) decided to conduct further studies and performed monthly doping controls for eight months. In these samples a continuous decrease of NA excretion could be shown. Based on these data and on the knowledge that nandrolone preparations with a pseudo-endogenous IRMS signature are available and that the injection of nandrolone esters can be detected for several months via urinary NA, the RMA of this case decided to declare an anti-doping rule violation for the use of a prohibited substance. Introduction Case report: In a doping control sample 19-norandrosterone (NA) was identified with an estimated concentration of 11.7 ng/mL (10.2 ng/mL adjusted to a specific gravity of 1.020). According to TD2019NA [1] GC/C/IRMS was conducted, which led to results, that did not confirm an exogenous origin of 19-NA. In accordance with TD2019NA, the sample was reported as atypical finding (ATF) and further doping controls were recommended to the testing authority. In total eight doping control samples were collected within a time period of eight months. Experimental The analyses of NA was conducted according to the method described by Hülsemann et al. [2]. The IRMS analyses were conducted according to the method described by Piper et al. [3]. Both methods are accredited for the use in doping control in the scope of the ISO17025 and WADA accreditation. Results and Discussion After a report of the atypical finding for NA, the RMA conducted further target controls. To evaluate the variation of the urinary NA excretion, the NA concentration, the NA concentration adjusted to the specific gravity of 1.020, the ratio 19-norandrosterone/androsterone (NA/A) and the ratio 19-norandrosterone/19- noretiocholanolone (NA/NE) were evaluated. The obtained results are presented in Table 1. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-638 Table 1. Results of follow-up investigations of an ATF for 19-norandrosterone (NA) of sample 1. S.G.: specific gravity; adj. conc. NA: 19-norandrosterone concentration adjusted to the specific gravity of 1.020; NA/A: ratio 19-norandrosterone/androsterone x 1000; NA/NE: ratio 19-norandrosterone/19-noretiocholanolone; δ C NA, A, PD: GC/C/IRMS values of 19-norandrosterone, androsterone and pregnanediol In total, the RMA collected eight doping control samples within eight month. Based on the NA concentration > 5 ng/mL in sample 3 , this sample was also analysed with GC/C/IRMS and led to values for NA indistinguishable from that of endogenous steroids. In the samples, a decrease in NA excretion within eight months could be shown. The ratio 19-nor- androsterone/androsterone x 1000 (NA/A) showed a continuous decrease, whereas the other parameters, concentration of NA (conc. NA) and concentration of NA adjusted to the specific gravity of 1.020 (adj. conc. NA), showed a discontinuation in sample 2. Most probably the best parameter to compare the excretion of NA in different samples of an individual is the ratio NA/A. Based on the knowledge that nandrolone preparations with a pseudo-endogenous IRMS signatures are available [4] and that the injection of nandrolone esters can be detected for several months via urinary NA [5,6], the only explanation for these results was an injection of a nandrolone ester with a pseudo- endogenous IRMS signature with the last injection before the collection of sample 1. The result managing authority of this case decided to declare an anti-doping rule violation for the use of the prohibited anabolic androgenic steroid nandrolone. In this case the GC/C/IRMS analysis of NE in sample 1, according to a method of Iannella et al. 2021 (7) would not have clarified the situation, as there was no problem with the GC/C/IRMS measurement of NA in sample 1 and 3. Conclusions In case of atypical findings for NA, the collection and analyses of further samples may provide information about the source of NA and may support the RMA in the decision making process. According to this study, a good parameter for monitoring and comparison of the NA excretion is the ratio NA/A. Similar to the GC/C/IRMS measurements, where the the delta value of the target compound NA is compared to the delta value of the endogenous reference compound A, in the ratio NA/A, the concentration of the target compound NA is compared with the concentration of the endogenous reference compound A. 13 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-639 References 1. WADA Technical Document TD 2019 NA: Harmonization of analysis and reporting of 19-norsteroids related to nandrolone. www.wada-ama.org/sites/default/files/td2019na_final_eng_clean.pdf (access: 26.01.2021) 2. Hülsemann F, Gougoulidis V, Schertel T, Fusshöller G, Flenker U, Piper T, Thevis M. Case Study: Atypical δ C values of urinary norandrosterone. Drug Test Anal. 2018 Nov;10(11-12):1728-1733. doi: 10.1002/dta.2498 3. Piper T, Emery C, Saugy M. Norandrosterone analysis by GC/C/IRMS. In: Schänzer W, Thevis M, Geyer H, Mareck U (eds.) Recent advances in doping analysis (20). Sportverlag Strauß, Köln (2012) 201-204 4. Brailsford AD, Majidin WNM, Wojek N, Cowan DA, Walker C. IRMS delta values ( C) of nandrolone and testosterone products available in the UK: Implications for anti-doping. Drug Test Anal. 2018 Nov;10(11- 12):1722-1727 5. Palonek E, Ericsson M, Gårevik N, Rane A, Lehtihet M, Ekström L. Atypical excretion profile and GC/C/IRMS findings may last for nine months after a single dose of nandrolone decanoate. Steroids. 2016 Apr;108:105-11 6. Mareck-Engelke, U., Geyer, H., Schänzer, W: 19-Norandrosterone - Criteria for the Decision Making Process. W. Schänzer, H. Geyer, A. Gotzmann, U. Mareck-Engelke (eds.) Recent advances in doping analysis (6). Sport und Buch Strauß, Köln (1999) 119-130 7. Iannella L, Colamonici C, Curcio D, Botrè F, de la Torre X. Detecting the abuse of 19-norsteroids in doping controls: A new gas chromatography coupled to isotope ratio mass spectrometry method for the analysis of 19-norandrosterone and 19-noretiocholanolone. Drug Test Anal. 2021 Apr;13(4):770-784 Acknowledgements The authors acknowledge the support from Sport Ireland (Dublin, Ireland), the Federal Ministry of the Interior, Community and Building of the Federal Republic of Germany (Berlin, Germany) and the Manfred Donike Institute for Doping Analysis (Cologne, Germany) 13 13 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-640 Mareck U, Geyer H, Fußhöller G, Haenelt N, Thevis M Results of confirmation procedures in the scope of the steroidal athlete biological passport in the Cologne laboratory from 2017-2019 Center for Preventive Doping Research / Institute of Biochemistry, German Sport University, Cologne, Germany Abstract The steroid module of the Athlete Biological Passport (ABP) uses the Adaptive Model to identify Atypical Passport Findings (ATPF) and Suspicious Steroid Profiles (SSP), which can trigger Confirmation Procedure (CP) requests. CPs include the identification and quantification of all markers of the steroid profile as well as Gas Chromatography – Combustion – Isotope Ratio Mass Spectrometry (GC-C-IRMS) and confirmation of confounding factors. A total of 1378 (2%) doping control urine samples – analyzed in the Cologne anti-doping laboratory – for which CPs were requested in 2017, 2018 and 2019, was evaluated. Overall, 41 (3%) of these urine specimens were reported as Adverse Analytical Findings (AAFs) exclusively confirmed by IRMS, most of them originating from strength sports and male athletes. Fourteen specimens were identified solely due to ATPFs. The high number of negative IRMS results (97%) may be attributed to various reasons; e.g. the influence of ethanol consumption on the ratio testosterone/epitestosterone (T/E) and other steroid profile parameters, a possible influence of mental stress on 5α-androstane-3α,17ß-diol/5β-androstane-3α,17ß- diol (Adiol/Bdiol) and androsterone/etiocholanolone (A/Etio), the influence of bacterial activities on Adiol and/or Bdiol, and the possible use of substances with pseudo-endogenous IRMS signature. Based on the results it can be concluded that revisiting the criteria triggering CPs is warranted. Introduction Endogenous anabolic androgenic steroid (EAAS) concentrations and their ratios constitute the urinary “steroid profile”, which may be altered following the administration of synthetic forms of EAAS, corresponding precursors or active metabolites, as well as epitestosterone [1]. The steroid module of the Athlete Biological Passport (ABP) utilizes the Adaptive Model to identify Atypical Passport Findings (ATPF) and Suspicious Steroid Profiles (SSP), which can trigger Confirmation Procedure (CP) requests. This elaborate and expensive CP includes the identification (in compliance with TD IDCR2015 [2]) and quantification of all markers of the “steroid profile” as well as Gas Chromatography – Combustion – Isotope Ratio Mass Spectrometry (GC-C-IRMS). In addition, the presence or absence of confounding factors (ethyl glucuronide (ETG), signs of microbial degradation including the presence of the free forms of testosterone etc.) has to be confirmed [1]. The identification and quantification of the steroid profile components is important for Adverse Passport Findings (APF) [3,4], where negative GC-C-IRMS results of ATPFs may result from the administration of testosterone preparations with carbon isotope ratios within the range reported for endogenous steroids [5-7]. However, no APF has been reported since the introduction of the ABP in 2014. On the other hand, GC-MS analysis is required to ensure the identity of the peaks of the relevant Target Compounds and MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-641 Endogenous Reference Compounds and the absence of significant interference prior to reporting an Adverse Analytical Finding or an Atypical Finding based on GC/C/IRMS results [8]. An evaluation of the doping control urine samples – analyzed in the Cologne anti-doping laboratory – for which the Confirmation Procedure was requested in 2017, 2018 and 2019 was performed. Experimental In total, the steroid profiles of 73589 doping control urine samples from national and international federations were analyzed in 2017, 2018 and 2019 in the Cologne anti-doping laboratory. Out of these samples, 1378 samples returned a CP request. The initial testing procedure (ITP) and CP of the steroid profiles were conducted according to the method described by Thevis [9] with consideration of the rules described in the TD EAAS [1]. The IRMS analyses were conducted according to the method described by Piper et al. [10]. Results and Discussion A total of 1378 (2%) doping control urine samples triggered CP requests for the steroid profile based on ATPFs, SSPs (Suspicious Steroid Profiles) findings, or TA-CPRs (Testing Authority Confirmation Procedure Requests). The mandatory identification of the six steroid profile markers Androsterone (A), Etiocho- lanolone (Etio), 5α-Androstane-3α,17ß-diol (Adiol), 5β-Androstane-3α,17ß-diol (Bdiol), Testosterone (T) and Epitestosterone (E) was performed in each of the 1378 urine samples, accounting for a total of 8268 time-consuming identifications. Forty-one (3%) of these urine specimens were reported as Adverse Analytical Findings (AAFs) exclusively confirmed by IRMS, most of them originating from strength sports (Table 1), 16 samples resulting from SSP-CPRs, 11 from ATPF-CPRs and 14 from TA-CPRs. Fourteen specimens (4 ATPF-CPRs and 10 TA-CPRs) showed no suspicious steroid profile parameter as depicted in TD2018EAAS. In summary: 14 of 41 reported AAFs were detected based exclusively on CPRs. In general, the majority of AAFs originated from male athletes. In 2018, an almost even distribution between male and female athletes was observed, due to the fact that testosterone doping was detected in a large number of female Asian weightlifters (Figure 1). The reasons for the high number of negative IRMS results (97%) of the CPs may be various confounding factors, which lead to alterations of the steroid profile [11]. Ethanol consumption is a frequently detected source of steroid profile alterations [12-15]. The oral intake of ethanol can increase the T/E ratio and decrease the A/T ratio by an elevated excretion of T-glucuronide and decreased elimination of A- glucuronide. This effect was found more pronounced in female than in male volunteers, whereas the changes in the steroid profile ratios were always connected with the presence of ethanol in urine. In the current study, 20% of the 1378 urine samples contained ethyl glucuronide (ETG) greater than 5 µg/mL, none of them tested positive by GC-C-IRMS. The application of oral contraceptives leads to an increase of the T/E ratio resulting from a suppression of the E excretion, whereas the ratios A/Etio, Adiol/Bdiol as well as the excretion rates of A and T are not influenced. Following cessation of administration, a decrease of the T/E ratio is observed resulting from an increase of the E excretion [16]. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-642 Participating in competition (IC) is, for the majority of athletes, associated with mental stress. This may result in a general significant difference between samples collected IC and out of competition (OOC). As proof of concept, Piper et al. [17] showed elevated ratios of A/Etio and Adiol/Bdiol in samples collected IC. A possible influence of bacterial activities on the ratio Adiol/Bdiol was examined by Haenelt et al. [18] as part of follow-up investigations of atypical passport findings. Resulting from the fact that 83% of the samples with the ratio Adiol/Bdiol ≥ 2.4 showed a ratio of free testosterone to total testosterone ≥ 0.05 and 87% were collected in-competition, it was postulated that bacterial activities and/or mental stress contributed to the increased ratios. Also, Geisendorfer et al. [19] observed possible alterations of the steroid profile based on bacterial activity. This is partly taken into account by the monitoring of markers of bacterial activity, like 5α- and 5ß-androstanedione. Ratios to their precursors A and Etio correlate with oxidative activity at the 3-position of the A-ring. The reductive activity at the 17-position of the D-ring may cause a tremendous effect on the 5α- and 5ß-androstanedione concentrations. Further, Pfeffer et al. [20] demonstrated that alterations in position 17 of the endogenous steroids were caused by microbiological activity. The 17-keto group was reduced to a 17ß-hydroxy group, leading to changes in concentrations of the urinary steroid profile. Last not least negative GC-C-IRMS results of ATPFs may also result from the possible use of substances with pseudo-endogenous IRMS signature [5-7]. Table 1. Sport disciplines with number of positive IRMS results Figure 1. Distribution by sex in AAF specimens MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-643 Conclusions 14 samples would not have been detected without the Adaptive Model to identify Atypical Passport Findings. Only 3% of CPs lead to AAFs. The ratio between success (positive IRMS results) and analytical effort necessitates improvement. References 1. World Anti-Doping Agency. Technical Document TD 2018EAAS, v.1.0. www.wada-ama.org/sites/default/files/resources/files/td2018eaas_final_eng.pd (access 26.05.2020) 2. World Anti-Doping Agency. Technical Document TD2015IDCR, v. 1.0. www.wada-ama.org/sites/default/files/resources/files/td2015idcr_-_eng.pdf (access 26.05.2020) 3. World Anti-Doping Agency. International Standard Testing and Investigations (ISTI) 2019. www.wada-ama.org/sites/default/files/resources/files/isti_2019_en_new.pdf (access 26.05.2020) 4. World Anti-Doping Agency. ISTI, ISL Athlete Biological Passport Operating Guidelines, Version 7.1, June 2019. www.wada-ama.org/sites/default/files/resources/files/guidelines_abp_v71.pdf (access 26.05.2020) 5. Cawley A, Collins M, Kazlauskas R, Handelsman DJ, Heywood R, Longworth M, Arenas-Queralt A (2010) Stable isotope ratio profiling of testosterone preparations. Drug Test Anal. 2, 557-567. 6. Forsdahl G, Östreicher C, Koller M, Gmeiner G (2011) Carbon isotope ratio determination and investigation of seized testosterone preparations. Drug Test Anal. 3, 814-819 7. Brooker L, Cawley A, Drury J, Edey C, Hasick N, Goebel C (2014) Stable carbon isotope ratio profiling of illicit testosterone preparations – domestic and international seizures. Drug Test Anal. 6, 996-1001 8. World Anti-Doping Agency. Technical Document TD 2019IRMS, v 1.0. www.wada-ama.org/sites/default/files/td2019irms_final_eng_clean.pdf (access 26.05.2020) 9. Thevis M. Mass Spectrometry in Sports Drug Testing – Characterization of Prohibited Substances and Doping Control Analytical Assays. Wiley, New Jersey, 201. 376 pages. ISBN: 978-0-470-41327-2 10. Piper T, Mareck U, Geyer G, Flenker U, Thevis M, Platen P, Schänzer W (2008) Determination of C/C ratios of endogenous urinary steroids: method validation, reference population and application to doping control purposes. Rapid Commun. Mass Spectrom. 22, 2161-2175 11. Mareck U, Geyer H, Opfermann G, Thevis M, Schänzer W (2008) Factors influencing the steroid profile in doping control analysis. Journal of Mass Spectrometry, 43, 877-891 12. Falk O, Palonek E, Björkhem I (1988) Effect of ethanol of the ratio between testosterone and epitestosterone in urine. Clinical Chemistry, 34, 1462-1464 13. Karila T, Konsunen V, Leinonen A, Tähtelä R, Seppälä T (1996) High doses of alcohol increase urinary testosterone-to-epitestosterone ratio in females. J Chromatogr B Biomed Appl. 687:109-16 14. Mareck-Engelke U, Geyer H, Schindler U, Flenker U, Iffland R, Donike M: Influence of ethanol on steroid profile parameters. M Donike, H Geyer, A Gotzmann, U Mareck-Engelke (eds.) Recent advances in doping analysis (3). Sport und Buch Strauß, Köln (1995) 143-155 15. Geyer H, Mareck U, Haenelt N, Schänzer W: Atypical steroid profiles in connection with ethanol findings in urine. W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent advances in doping analysis (17). Sport und Buch Strauß, Köln (2009) 261 - 264 16. Mareck-Engelke U, Flenker U, Schänzer W: Stability of steroid profiles (6): the influence of oral contraceptives on steroid profiles. W Schänzer, H Geyer, A Gotzmann, U Mareck-Engelke (eds.) Recent advances in doping analysis (4). Sport und Buch Strauß, Köln (1996) 139-157 17. Piper T, Geyer H, Haenelt N, Hülsemann F, Schänzer W, Thevis M (2021) Current Insights into the Steroidal Module of the Athlete Biological Passport. Int J Sports Med, 442(10): 863-878 18. Haenelt N, Lourens L, Fußhöller G, Geyer H, Goldmann L, Schult C, Schwenke A, Hülsemann F, Gougoulidis V, Blatt C, Thevis M: Follow-up investigations of atypical passport findings for the ratio 5α-androstane- 3α,17ß-diol/5ß-androstane-3α17ß-diol. M Thevis, H Geyer, U Mareck (eds.) Recent advances in doping analysis (29) Sport und Buch Strauß, Köln (2021) 19. Geisendorfer T, Göschl L, Benetka E, Gmeiner G: Monitoring of bacterial activity: increase of Diol concentrations. Lecture presented at the 35 Cologne Workshop 2017 20. Pfeffer S, Gmeiner G, Gärtner P: Synthesis, characterization and application of a marker substance for monitoring 17-keto-modifications in endogenous steroids caused by microbiological activity. Poster presented at the 39 Cologne Workshop 2021 Acknowledgements The authors thank the Manfred Donike Institute for Doping Analysis e.V. (Cologne, Germany) and the Federal Ministry of the Interior, Building and Community (Berlin, Germany) for supporting the study. th th MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-644 Piper T , Geyer H , Toboc A , Ahrens B , Pohanka A , Thevis M Recent findings on 7-oxo-DHEA and its improved detection based on reference population-derived thresholds for 7β-OH-DHEA and 5α- androstane-3β,7β-diol-17-one Institute of Biochemistry, German Sport University, Cologne, Germany ; Romanian Doping Control Laboratory, Bucharest, Romania ; UCLA Olympic Analytical Laboratory, Los Angeles, United States of America ; Doping Control Laboratory, Karolinska University Hospital, Stockholm, Sweden Abstract The administration of 7-oxo-DHEA is forbidden according to WADA´s Prohibited List. Even if no official urinary concentration threshold for 7-oxo-DHEA or its main metabolite 7β-OH-DHEA has been established so far, laboratories detect significantly elevated concentrations for these steroids from time to time. Therefore, an isotope ratio mass spectrometry (IRMS)-based method was developed and validated to enable the differentiation between endogenous and exogenous 7-oxo-DHEA and metabolites. During studies on the metabolism of 7-oxo-DHEA, a novel metabolite was detected (5α-androstane-3β,7β-diol- 17-one, 5aM) and preliminarily included in the IRMS method. The developed method was further improved to enable the detection of both metabolites, 5aM and 7β-OH-DHEA, validated and finalized by investigations on a reference population encompassing n=88 males and females. The derived population-based thresholds will enable to deal with results of samples suspicious for the administration of 7-oxo-DHEA in accordance with the relevant Technical Document issued by WADA. In 2019, three cases with elevated concentrations of 7β-OH-DHEA were detected in Bucharest and Cologne and demonstrated to show exogenous carbon isotope ratios (CIR) for both 5aM and 7β-OH- DHEA. In 2020, four additional cases apeared in the United States of America, Sweden and Germany. Again, all samples exhibited exogenous CIR and were reported as atypical analytical findings as no positivity criteria have been established so far. Samples were forwarded into long-term storage where applicable to enable re-analysis once positivity criteria have been adopted. Introduction The administration of 7-oxo-DHEA (OXO) is forbidden according to WADA´s Prohibited List [1]. Even if no official urinary concentration threshold for OXO or its main metabolite 7β -OH-DHEA (OH) has been established so far, laboratories detect significantly elevated concentrations for these steroids from time to time. Therefore, an isotope ratio mass spectrometry (IRMS)-based method was developed and validated to enable the differentiation between endogenous and exogenous OXO and metabolites [2]. Experimental Improved HPLC-clean up The already developed method [2] lacks the ability to separate OH from the novel metabolite of OXO, 5α- 1 1 2 3 4 1 1 2 3 4 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-645 androstane-3β,7β-diol-17-one (5aM), which complicated the determination of both analytes. Switching to an isocratic approach for the second HPLC on acetylated compounds solved this issue as depicted in Figure 1. The method parameters have been added to the given chromatogram. Figure 1. HPLC chromatogram of a standard containing OH and 5aM together with method parameters and fraction collection times Linear mixing models As the HPLC parameters were substantially changed, the novel method was partially revalidated in order to test the suitability of the new clean up. Two experiments were conducted: In the first trial samples were fortified with OH and both OH and 5aM were measured to accomplish the mixing model for OH and to verify that the values of 5aM are stable with different amounts of OH. In the second trial 5aM was added vice versa. Reference population based investigations A reference population encompassing n=88 males and females was investigated in order to estimate potential reference limits to differentiate between endogenous and exogenous OH and 5aM. Samples were derived from athletes with already confirmed negative IRMS results and fresh aliquots of 20 mL each were processed for the population based investigations. Validation With each batch processed for the population determinations a positive quality control urine (QCP) MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-646 was prepared resulting in 10 individual preparations. A combined measurement uncertainty (m ) was calculated on the basis of this long-term reproducibility and the results obtained in the linear mixing models. Results and Discussion Linear mixing models The obtained results are shown in Figure 2 (upper part), demonstrating that the method is fit-for- purpose. Reference population based investigations Out of the 88 samples, 4 showed a too low concentration of 5aM and 14 a too low concentration or co- elution for OH. The obtained results are depicted in Figure 2 (lower part). Both distributions were found Gaussian shaped and the reference limits were calculated by adding the threefold standard deviation to the mean value in accordance with the recommendations of the International Federation of Clinical Chemistry [3]. The limits are also shown in Figure 2. Figure 2. Upper part - linear mixing models performed on OH (left) and 5aM (right), lower part - densityplots of the Δ-values obtained within the reference population for PD-OH (left) and PD-5aM (right) u MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-647 Validation The results obtained on the QCP are listed in Table 1. These results combined with those of the linear mixing models enabled calculations of the m . For OH a m of 0.61 ‰ and for 5aM a m of 0.48 ‰ was determined. The limit of detection (LOD) of the IRMS method could only be estimated as no validated method for the quantification of both metabolites was available. For OH, the LOD was at 25 ng/mL and for 5aM at 8 ng/mL. These values were derived from the blank urine employed in the linear mixing models. Taking into account the results obtained on suspicious samples so far, these limits seem to be adequate. Table 1. Results obtained for the QCP over a time period of 3 months. All values in δ C [‰] Samples investigated so far In the last 2 years several samples were found to be suspicious for the administration of OXO in Europe and America (Table 2). Five out of these six samples showed significantly elevated concentrations for OH. In sample number 2 derived from Los Angeles only 50 ng/mL of OH were estimated but the sample additionally showed the presence of traces of androst-3,5-diene-7β-ol-17-one and a specific gravity of only 1.002. Small amounts of androst-3,5-diene-7β-ol-17-one can be expected after the administration of OXO as has been described earlier [4]. The carbon isotope ratios found in all six samples clearly showed the exogenous origin of OXO and its metabolites. Table 2. Results obtained on samples showing elevated urinary concentrations of OH and OXO. All values in δ C [‰] u u u 13 VPDB 13 VPDB MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-648 Conclusions All samples under investigation so far were reported as Atypical Findings and forwarded to long-term storage were applicable. Despite carbon isotope ratios clearly demonstrate the exogenous origin of OXO and its metabolites, these samples were not reported as Adverse Analytical Finding as no decision criteria supported by WADA are available. This also holds true for the urinary concentrations which may trigger an IRMS-based confirmation. As both, OXO and OH, are explicitly listed on the Prohibited List [1] the current state is unsatisfactory. These new results may contribute to a timely solution of this problem, especially if concentration thresholds for the ITP would additionally be set in place. First investigations here demonstrated that OH will be the most promising analyte and a potential threshold at 700 ng/mL may be used as a basis to improve the detection of OXO-misuse [2,5]. References 1. WADA Prohibited List 2021. https://www.wada-ama.org/sites/default/files/resources/files/2021list_en.pdf, accessed 26.07.21 2. Piper T, Fusshöller G, Geyer H, Toboc A, Danila MG, Thevis M. Detecting the misuse of 7-oxo-DHEA by means of carbon isotope ratio mass spectrometry in doping control analysis. Rapid Commun Mass Spectrom 2020; 34: e8776. 3. Solberg HE. Approved recommendation (1987) on the theory of reference values. Part 5. Statistical treatment of collected reference values. Determination of reference limits. J Clin Chem Clin Biochem. 1987;25:645–56. 4. Martinez-Brito D, de la Torre X, Colamonici C, Curcio D, Botre F. 7-Keto-DHEA metabolism in humans. Pitfalls in interpreting the analytical results in the antidoping field. Drug Test Anal 2019;11:1629–1643. 5. Danila G, Pop A, Toboc A, Stan C. 7-Keto-DHEA – a case study. In: Thevis M, Geyer H, Mareck U (Eds). RECENT ADVANCES IN DOPING ANALYSIS (28). SPORTVERLAG Strauß - Hellenthal 2020. Acknowledgements This research project was supported by the Manfred-Donike Institute for Doping Analysis (Cologne, Germany) and the Federal Ministry of the Interior, Building and Community (Berlin, Germany). MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-649 Piper T , Panto S , Thevis M Highly sensitive low resolution GCxGC-TOF as a complement in doping control analysis of anabolic androgenic steroids Institute of Biochemistry, German Sport University, Cologne, Germany ; LECO European Application and Technology Center, Berlin, Germany Abstract The current approach in sports drug testing to detect the misuse of anabolic androgenic steroids (AAS) mainly relies on extensive sample preparation followed by gas chromatography/triple quadrupole mass spectrometry (GC/MS/MS)-based measurements. These determinations encompass the sensitive detection of exogenous AAS, i.e. their urinary metabolites, and the accurate quantification of endogenous steroids covering a wide range of urinary concentrations. Unfortunately, numerous substances show comparable physico-chemical properties and, therefore, similar GC retention times. Using MS/MS-based techniques allowed for improving both sensitivity and peak purity in most instances. However, the number of ion transitions that can be monitored simultaneously is limited and, consequently, the number of different analytes to be included in routine testing procedures is restricted, too. Employing two-dimensional GCxGC hyphenated via a thermal modulator adds the necessary additional chromatographic dimension to achieve sufficient resolution and peak purity that enables to relinquish the MS/MS-based purification. This enhanced chromatographic resolution is a prerequisite to use a time-of- flight mass spectrometer (TOFMS). The TOFMS enables very high acquisition rates up to 500 spectra/s and always acquires full scan spectra. This does not only enable to select those ions offering optimal purity (or signal/noise ratios) for peak identification but also to use the routine data for a downstream retrospective data mining whenever new doping agents (presumably) enter the market or if novel long- term metabolites of a known substance have been detected. Furthermore, monitoring of prohibited substances can easily be employed as the number of ion transitions per time frame in the chromatographic run is not limited. In order to test the suitability of a GCxGC-TOF system to fulfil the requirements of routine sports drug testing, the chromatographic conditions of a Pegasus BT4D TOF coupled to an Agilent 7890 GC were optimized and tested by parallel measurements of doping control samples including the detection of exogenous steroids and the quantification of endogenous steroids in the same run. Introduction The current approach in sports drug testing to detect the misuse of anabolic androgenic steroids (AAS) mainly relies on extensive sample preparation followed by gas chromatography/triple quadrupole mass spectrometry (GC-MS/MS)-based measurements. These determinations encompass the sensitive detection of exogenous AAS, i.e. their urinary metabolites, and the accurate quantification of endogenous steroids covering a wide range of urinary concentrations. Unfortunately, numerous 1 2 1 1 2 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-650 substances show comparable physico-chemical properties and, therefore, similar GC retention times. Using MS/MS-based techniques allowed for improving both sensitivity and peak purity in most instances. However, the number of ion transitions that can be monitored simultaneously is limited and, consequently, the number of different analytes to be included in routine testing procedures is restricted, too. One possible solution may offer employing two-dimensional GC in combination with time-of-flight mass spectrometry (GCxGC-TOFMS) as has already been demonstrated in the past [1]. Experimental Urine samples were prepared twice in accordance with our accredited method and subjected either to GC-MS/MS determinations or GCxGC-TOFMS measurements [2,3]. The GCxGC principle is shown in Figure 1. Within the first dimension compounds of interest are separated with a conventional temperature program employing a Rxi-1ms GC column (20 m x 0.18 mm i.d. x 0.18 μm coating (Restek, Bad Homburg, Germany). In the second dimension after the QuadJet thermal modulator a more polar column (Rxi-17Silms, 0.45 m x 0.15 mm ID x 0.15 μm coating (Restek)) has been installed to enable separation of peaks that co-elute on the first column. The Table embedded in Figure 1 summarizes the GCxGC-TOFMS method parameters. TM MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-651 Figure 1. Principle of GCxGC target analyte separation (upper part) and applied method parameters (lower part) Results and Discussion Exogenous steroids The GCxGC-TOFMS was tested for its suitability to detect metabolites of exogenous doping agents like for example stanozolol. As desired, the second dimension allowed for separation of co-eluting peaks as demonstrated exemplarily in Figure 2. In order to compare the performance of the GCxGC-TOFMS to the routine method 5 excretion study samples (stanozolol, metenolol, boldenone, mesterolone and oxandrolone) were prepared according to the established protocol and submitted to analysis. Only the oxandrolone metabolite was not detected which was presumably due to well known derivatization artefacts within the samples under investigation. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-652 Figure 2. Example for the improved separation achieved by adding a second dimension. A) Two-dimensional total ion chromatogram of a standard mixture (Std. Metaboliten) containing the majority of relevant target analytes. B) Magnification of the region where 4-chloro-3α-ol-androst-4-en-17-one (Clostebol metabolite) elutes together with co-eluting target analytes. C) Obtained mass spectra of Clostebol metabolite compared to a library entry. Endogenous steroids - steroid profile One of the main challenges in routine doping controls on steroids is the necessity for simultaneous detection of exogenous compounds and accurate quantification of endogenous steroids. The GCxGC- TOFMS demonstrated a high sensitivity and good linearity over a broad working range. At the lowest calibration level applied (2.5 ng/mL) the signal-to-noise was still > 500 for testosterone and epitestosterone. Regarding the upper limit of the working range, the limitation was based rather on the chromatographic conditions than on the dynamic range of the TOFMS. As demonstrated in Figure 3, a significant peak distortion was noted for androsterone and etiocholanolone at 5000 ng/mL resulting in a linear range for quantification up to 2000 ng/mL. Figure 3. Overload of the chromatographic system found for androsterone and etiocholanolone MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-653 Ten urine sample were quantified on both the GCxGC-TOFMS and the GC-MS/MS system and results are listed in Table 1. In general, good agreement was found albeit some differences were significant. Here a detailed and more sophisticated method optimization may further improve the results and lead to comparable values in the future. Table 1. Urinary concentrations measured in different urine samples employing the routine set-up (black) and the novel GCxGC-TOFMS-based approach (blue). All values given in ng/mL. Conclusions On the basis of these preliminary results, GCxGC-TOFMS seems to be a promising complement for sports drug testing. The sensitive detection of exogenous steroid is comparable to GC-MS/MS and beneficially full scan data can be acquired. The quantification of endogenous steroid was also promising albeit further steps in method optimization may be necessary here. A weak point is the current limited linear range for concentrated steroids like androsterone and etiocholanolone. Employing different GC columns with a thicker film may enable to overcome these limitations. References 1. Silva Jr. AI, Pereira HMG, Casili A, Conceicao FC, Aquino Neto FR. Analytical challenges in doping control: Comprehensive two-dimensional gas chromatography with time of flight mass spectrometry, a promising option. J Chrom A 2009;1216:2913–2922. 2. Mareck U, Geyer H, Opfermann G, Thevis M, Schänzer W. Factors influencing the steroid profile in doping control analysis. J Mass Spectrom 2008;43:877-891. 3. Thevis M, Fusshöller G, Schänzer W. Zeranol: doping offence or mycotoxin? A case related study. Drug Test. Analysis 2011;3:777–783. Acknowledgements This research project was supported by the Manfred-Donike Institute for Doping Analysis (Cologne, Germany) and the Federal Ministry of the Interior, Building and Community (Berlin, Germany). MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-654 Krug O, Thomas A, Thevis M Identification and characterization of urinary isopropylnorsynephrine metabolites Institute of Biochemistry, German Sport University, Cologne, Germany Abstract Isopropylnorsynephrine (isopropyloctopamine, deterenol, 4-(1-hydroxy-2-(isopropylamino)ethyl)phenol), a beta-selective and direct-acting adrenergic agonist, has been reported in the past as declared as well as non-declared ingredient of dietary supplements. The proven biological activity and the structural similarity of isopropylnorsynephrine to substances classified as prohibited compounds according to the World Anti-Doping gency’s (WADA’s) regulations could necessitate the inclusion of this sympathomimetic amine into routine doping control analytical assays. Therefore, information on urinary metabolites is desirable in order to allow for an efficient implementation of target compounds into existing multi- analyte testing procedures, enabling the unequivocal identification of the administration of isopropylnorsynephrine by an athlete. In a pilot study setting, urine samples were collected prior to and after the oral application of ca. 8.7 mg of isopropylnorsynephrine, which were subjected to liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry. The intact drug, hydroxylated and/or glucurono- or sulfo-conjugated isopropylnorsynephrine were detected up to 48h post-administration, with isopropylnorsynephrine sulfate representing the most abundant urinary target analyte. No relevant amounts of the dealkylation product (octopamine) were observed, indicating that merely moderate adaptations of existing test methods (or data evaluation strategies) are required to include isporpoylnorsynephrine in anti-doping analytics, if required. Introduction Isopropylnorsynephrine (IPNS, isopropyloctopamine, deterenol, 4-(1-hydroxyl-2-(isopropylamino)ethyl)- phenol, WIN 833, Fig. 1) is described as fatburner [1], and it is available as ingredient of nutritional supplements [2]. The mechanisms of action as stimulant are related to agonism of beta- and antagonism of alpha-adrenergic receptors [3]. The structural similarity to octopamine and synephrine, which are prohibited and monitored respectively by WADA [4] led to the assumption that metabolites of IPNS may overlap with these compounds. The pilot study´s aim is the examination of first results concerning IPNS- metabolism, supporting decisions for implementation of target compounds into testing procedures [5]. Experimental The Cologne Anti-doping Laboratory ordered a nutritional supplement labeled to contain IPNS via internet. The analysis of the product via LC-MS showed a drug-content of 1.58 mg/g. Furthermore the product was tested negative concerning octopamine, synephrine and potential metabolites of IPNS. Two MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-655 healthy male volunteers (46-47 years) ingested 5.5 g (half of one serving, which corresponded to 8.7 mg IPNS) of the nutritional supplement. Urine samples were collected before ingestion, and up to 48h after ingestion. The urine samples were aliquoted (0.5 mL), fortified with 100 ng of internal standard (IPNS- d7), diluted 1:1 (aq.), centrifuged and analyzed by means of high performance liquid chromatography / high resolution (high accuracy) mass spectrometry (HPLC-HRMS) in full scan- and hcd-mode. The identification was conducted by an Vanqish HPLC-system linked via heated electrospray ionization (HESI) with a Thermo Orbitrap Exploris 480 mass spectrometer. The LC was equipped with a Thermo Accucore C-8 (100 x 2.1 mm, 2.7 µm article size) column. The LC was conducted using 5mM aqueous ammonium acetate, containing 0.1% acetic acid (solvent A) and acetonitrile (solvent B). The elution started with 100 % A, decreasing from 1 to 9 min to 50 % A, followed by decreasing to 0 % during 0.5 min, after 1.5 min 0 % A, the re-equilibration was running for 4 min. The total runtime was 15 min. For quantification of IPNS in urine, 6 blank samples were fortified with IPNS reference material in a range from 0.01 to 1 µg/mL. The IPNS concentrations in authentic samples were calculated baseing on this external calibration. To yield more detailed information for metabolite structure eludication samples were methylated selectively with Iodomethane [6] and additionally a sulfo-conjugate of IPNS was synthezised in microscale in accordance to established protocols [7]. Results and Discussion After ingestion of IPNS, the unmodified compound and five metabolites (see Fig. 1) could be detected and identified: One hydroxy-metabolite of IPNS at m/z 212.12 [M+H]+, two glucuronides of the hydroxylated IPNS at m/z 388.16 [M+H]+, one sulfo-conjugate of IPNS with m/z at 276.09 [M+H]+, and one sulfo-conjugate of O-IPNS with m/z at 290.07 [M+H]+. The extracted ion chromatograms and related product ion mass spectra of the metabolites are shown in Figure 2. Figure 1. Isopropylnorsynephrine and identified metabolites M1 to M4 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-656 Figure 2. MS²-product ion spectra of Isopropylnorsynephrine metabolites @ ce 30 The metabolite with the highest abundance was sulfoconjugated IPNS, the product ion mass spectrum showed the loss of H O that suggested the sulfonation predominantly at the phenolic OH-group. The product ion mass spectrum of synthezised IPNS-sulfate matches plausibly the spectrum of M3. (Fig. 2, Fig. 3). The selective methylation showed that two methyl groups could be inserted under formation of a quarternary amine (Fig. 3). Also, a signal attributable to the sulfo-conjugate of IPNS N-oxide (M4) was observed with a product ion mass spectrum shown in (Fig. 2), supported by characteristic product ions observed at m/z 248 (-propene), m/z 210 (-SO3), and m/z 203 (assigned to sulfoconjugated hydroxyl-(4- hydroxy-phenyl)methylium). 2 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-657 In case of the glucurono-conjugates, the considerably higher abundance of the extracted ion chromatogram of glucuronide M2b in comparison to M2a and the related product ion spectra indicated that the glucuronidation of the hydroxylated sympathomimetic amine occurs mainly at the aliphatic hydroxyl group. In M2a, the loss of water due to the free aliphatic hydroxy group can be observed, which doesn´t occur in M2b. Figure 3. MS²-product ion spectra of synthesized (A) Isopropylnorsynephrine sulfate (M3) and (B) methylated Isopropylnorsynephrine sulfate The values depicted in diagrams of excretion profiles (Fig. 4) were specific gravity-adjusted. Isopropyl- norsynephrine could be detected up to 36h in post-administration samples, the highest urinary concentration was observed 1h after ingestion (1.1 to 2.3 µg/mL). Due to the absence of reference material for metabolites, area ratios of metabolite- and ISTD-abundance were calculated. Sulfoconjugate M3 was the most intensive metabolite and was detectable for 36h, the glucurono-conjugate M2b reached 1/5 of M3-abundance and was also detectable up to 36h. Figure 4. Urinary excretion of Isopropylnorsynephrine and metabolites MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-658 Conclusions This recent investigation of isopropylnorsynephrine-metabolism shows five metabolites, but neither octopamine nor synephrine could be observed in collected urine samples. Hence, it seems to be unlikeliy that the ingestion of IPNS results in adverse analytical findings for octopamine and synephrine. References 1. J. Mercader, E. Wanecq, J. Chen, C. Carpéné: Isopropylnorsynephrine is a stronger lipolytic agent in human adipocytes than synephrine and other amines present in Citrus aurantium (2011) J Physiol Biochem Vol. 67: 443–452 2. B. Venhuis, P. Keizers, A. Van Riel, D. de Kaste: A cocktail of synthetic stimulants found in a dietary supplement associated with serious adverse events (2014) Drug Test Anal 6; 578-581 3. W. G. Anderson: The sympathomimetic activity of N-isopropyloctopamine in vitro (1983) J Pharmacol Exp Ther 225; 553-558 4. https://www.wada-ama.org/sites/default/files/resources/files/2021list_en.pdf 5. O.Krug, A. Thomas, M. Thevis: Mass spectrometric identification and characterization of urinary metabolites of isopropylnorsynephrine for doping control purposes (2021) Anal Sci Adv 1-8 6. M. Thevis, H. Schmickler, W. Schänzer: Effect of the location of hydrogen abstraction on the fragmentation of diuretics in negative electrospray ionization mass spectrometry (2003) J Am Soc Mass Spectrom 14; 658-670 7. A.K. Orlovius, S. Guddat, M.K. Parr et al.: Terbutaline sulfoconjugate: characterization and urinary excretion monitored by LC/ESI-MS/MS (2009) Drug Test Anal 1;568-575 Acknowledgements The authors want to thank the Manfred Donike Institute, and the Federal Ministry of the Interior for financial support, and the colleagues at the Institute of Biochemistry for support in theory and practice. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-659 Geisendorfer T, Athanasiadou I, Tsivou M, Gmeiner G Long-term urinary excretion profile after a single oral administration of acetazolamide Doping Control Laboratory, Seibersdorf Laboratories, Seibersdorf, Austria Abstract Acetazolamide is the key substance in pharmaceutical formulations recommended for the prophylaxis of acute mountain sickness symptoms in altitudes above 2500 m. An acetazolamide excretion study was conducted, triggered by two positive cases claiming the use of acetazolamide to combat altitude sickness. A single dose of 250 mg acetazolamide was orally administered to a healthy male volunteer. Urine samples were collected up to three months post-dose. Urine sample preparation was conducted according to the validated initial testing procedure (ITP) using LC-MSMS. Based on data evaluation, acetazolamide is mainly excreted unchanged in human urine and could be detected up to 30 days post- dose at concentrations ranging from ca. 0.2 ng/mL to ca. 500 µg/mL. Introduction Acetazolamide is the key substance in pharmaceutical formulations recommended for the prophylaxis of acute mountain sickness symptoms in altitudes above 2500 m [1]. Acetazolamide represents 2% of positive findings of class S5 of the WADA Prohibited List [2] according to the World Anti-Doping 2019 Lab Statistics [3]. The current excretion study of acetazolamide was triggered by two positive cases (1.6 µg/mL and 672 ng/mL) claiming the use of acetazolamide to combat altitude sickness with an aim to provide more information to the Testing Authority on the eventual administration time by the athletes. Experimental Subject One healthy, recreationally active Caucasian male subject participated (47 years old, non-smoker). Written consent was provided prior to participation. Study design A three-month clinical study was conducted. A single oral dose of 250 mg of acetazolamide (1 tablet; Diamox; Amdipharm Limited) was administered. Urine samples were collected at every micturition during the first three days and once per day up to three months post-dose. In total 46 urine samples were collected. Sample preparation Preliminary laboratory measurements including SG were also performed for all urine samples using an Atago 3464 Refractometer (Atago, Tokyo, Japan). Urine sample preparation was conducted according to the validated initial screening (ITP) and confirmation (CP) procedures with LODs 0.5 ng/mL and MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-660 0.05 ng/mL, respectively. Briefly, 5 mL urine spiked with 100 μL of the ISTDs mixture solution were enzymatically hydrolyzed by β‑glucuronidase (E. coli, 25 μL) after addition of 1.0 mL of phosphate buffer (pH 6.8) at 50°C for 1 h. A liquid-liquid extraction was performed with 7 mL of ethyl acetate followed by shaking for 10 mins. After centrifugation at 3000 rpm for 5 min, samples were frozen at -80°C. The organic layer was then separated, transferred into a clean glass tube and evaporated to dryness. The dry residue was reconstituted with 200 μL of MeOH/MQ H O (30/70, v/v), heated at 60°C for 10 mins and transferred into an autosampler vial. The injection volume was 10 μL. For the CP procedure, a dilute-shoot method has followed using 5 mL urine spiked with 100 μL of the ISTDs mixture solution after addition of 1.0 mL of acetate buffer (pH 4.8). A liquid-liquid extraction was performed with 6 mL of ethyl acetate followed by shaking for 20 mins. After centrifugation at 3000 rpm for 5 min, samples were frozen at -80°C. The organic layer was then separated, transferred into a clean glass tube and evaporated to dryness. The dry residue was reconstituted with 150 μL of MeOH/MQ H O (10/90, v/v) with 0.1% formic acid, heated at 60°C for 10 min. Then, transferred into an autosampler vial and 10 μL was injected for analysis. Instrumentation Chromatographic separation was conducted using a UHPLC system (Thermo Scientific) coupled with a vacuum degasser, a high-pressure binary pump, an autosampler with a temperature controlled sample tray set at 7°C, and a column oven set at 25°C. The LC analysis time for both ITP and CP was 10 minutes. The injection volume was 10 μL. The instrument and mass spectrometric characteristics are summarized in Table 1. Table 1. Instrument and mass spectrometric characteristics 2 2 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-661 Results and Discussion Acetazolamide was mainly excreted unchanged in human urine and with the currently applied ITP (samples #00-33) could be detected up to 30 days post-dose at concentrations ranging from ca. 0.5 ng/mL to ca. 500 µg/mL (Fig. 1). The C (530 µg/mL) was measured 10 hours after administration (TAD). When the CP was applied to urine samples #33-46, then acetazolamide could be detected up to 3‑months post-dose with a concentration of ca. 0.05 ng/mL. According to the WADA TL24 - Minimum reporting level for certain diuretics that are known contaminants of pharmaceutical products (released after the present results were presented) acetazolamide together with other five diuretics (namely, bumetanide, furosemide, hydrochlorothiazide, torasemide, and triamterene) should not be reported as Adverse Analytical Findings at concentrations below 20 ng/mL. Figure 1. Chroamtograms of Azetazolamide in urine samples analysed with ITP at pre-dose (blank sample), PQC Spiked at 5 ng/mL, samples (#21-33) from day 8 up to one-month after oral administration Figure 2. Chromatograms of Acetazolamide in urine samples analysed with CP: blank sample, PQC spiked at 0.5 ng/mL, samples 1-month (#33), 2-months (#40), 3-months (#46) after oral administration (TAD) max MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-662 Figure 3. Concentration-time profile of orally administered Acetazolamide up to 30 days (blue line) and up to 3-months (red line) Conclusions The present long-term excretion study addresses the question how long acetazolamide could be present in human urine at detectable levels. The results showed that acetazolamide was identified up to 3‑months post-dose at concentration levels of ca. 0.05 ng/mL. References 1. Urinary excretion of acetazolamide in healthy volunteers after short- and long-term exposure to high altitude. Ritschel, W.A., Paulos, C., Arancibia, A., Agrawal, M.A., Wetzelsberger, K.M., Luecker, P.W. Methods Find Exp Clin Pharmacol 1998, 20(2): 133 2. World Anti-Doping Agency. The 2021 Prohibited List. International Standard, Montreal (2021) 3. https://www.wada-ama.org/sites/default/files/resources/files/2021list_en.pdf (access date 29.09.2021) 4. World Anti-Doping Agency. WADA Lab Statistics 2019. https://www.wada- ama.org/sites/default/files/resources/files/2019_anti-doping_testing_figures_en.pdf (access date 29.09.2021) 5. World Anti-Doping Agency. TL24 Minimum reporting level for certain diuretics that are known contaminants of pharmaceutical products. https://www.wada-ama.org/sites/default/files/resources/files/tl24_diuretics _eng_2021_0.pdf (access date 29.09.2021) MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-663 Fernández-Alvarez M, Serrano E, Muñoz G Specific urinary metabolites of non-prohibited mebeverine: LC-MS/MS monitoring of MAC and DMAC on reporting of p-hydroxyamphetamine Doping Control Laboratory, Spanish Agency for Health Protection in Sport, Madrid, Spain Abstract Amphetamine derivatives have been detected in urine of irritable bowel disease patients treated with mebeverine, a non-prohibited antispasmodic drug. Accordingly, the World Anti-Doping Agency (WADA) demands accredited laboratories to exclude mebeverine administration before reporting an Adverse Analytical Finding (AAF) based on the detection of p-hydroxyamphetamine (p-OH-A). In order to confirm or dismiss mebeverine consumption, it is essential to monitor specific urinary metabolites and reject those ones (e.g. vanillic acid, isovanillic acid and protocatechuic acid) which might also be excreted due to the ingestion of certain fruits and vegetables. In the present work, mebeverine metabolism was studied via Liquid Chromatography-Quadrupole Time- of-Flight (LC-QToF) by analysing one urine sample collected after administration of this drug and provided by the World Association of Anti-Doping Scientists (WAADS). Eight specific markers were tentatively identified on the basis of their exact masses and MS/MS spectra. Among them, mebeverine acid (MAC) and desmethylmebeverine acid (DMAC) were chosen as the most useful indicators of mebeverine intake. A simple and rapid procedure based on dilute-and-shoot followed by Liquid Chromatography-Triple Quadrupole (LC-QQQ) detection was validated to confirm MAC and DMAC occurrence in urine. Validation results in this matrix have not been previously reported. The suitability of the proposed methodology was proved in terms of selectivity, specificity, carryover, robustness and reliability of detection at the limit of identification (LOI : 2 ng/mL, LOI : 1 ng/mL). Introduction Figure 1 summarizes mebeverine metabolic pathways. After oral ingestion, mebeverine ester bond is quickly and easily cleaved to mebeverine alcohol (MAL) and veratric acid (VA) which, in turn, undergo further metabolism [1-3]. N-de(hydroxybutylation) of MAL and its subsequent O-demethylation and N-de- ethylation justify the occurrence of amphetamine derivatives in urine of patients receiving a mebeverine treatment, namely p-methoxyethylamphetamine (PMEA), p-hydroxyethylamphetamine (p-OH-EA), p‑methoxyamphetamine (PMA) and p-OH-A [3-5]. There are disagreements over which compound is the main metabolite of mebeverine [2,6,7]; in any case, anti-doping laboratories must focus on the monitoring of the most specific markers of this drug, regardless of their relative abundance. VA and its O-demethylated biotransformation products (vanillic acid, isovanillic acid and protocatechuic acid) are not-specific metabolites because they can be originated from the ingestion of certain food [3]. MAC DMAC MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-664 Figure 1. Mebeverine metabolic pathways according to literature [1-9] Experimental MAC and DMAC were purchased from TLC Pharmaceutical Standards and diphenylamine (ISTD) from Sigma-Aldrich. Methanol and acetonitrile were acquired from Fisher Chemical, formic acid from Scharlau, potassium dihydrogen phosphate and di-sodium hydrogen phosphate from Merck, and β-glucuronidase (E. Coli) from Roche Diagnostics. Stock and working solutions were prepared in methanol. For the tentative identification of specific mebeverine metabolites, QA_2017B WAADS sample (0-24h pooled urine collected after administration of a 135 mg mebeverine pill) and blank urines were analysed by centrifugation and direct injection into LC-QToF. Experiments including a previous hydrolysis step (pH 7 phosphate buffer, β-glucuronidase, 52.5°C, 1h) were also carried out to reveal the conjugated state of the suggested metabolites. Samples were analysed in positive mode in a 1290 Infinity HPLC coupled to a 6550 iFunnel Q-TOF analyser with an electrospray ionization (ESI) source with Agilent Jet Stream technology (Agilent Technologies). Data were acquired in full scan or target MS/MS mode. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-665 Sample preparation for MAC and DMAC confirmation was performed as follows: 20 µL of ISTD solution (1 µg/mL) were added to 1 mL of sample on a glass tube. Once vortex-mixed, 200 µL of the sample were transferred to a vial and diluted with 800 µL of ultrapure water. Finally, the vial was capped and shaken. 31 different urine samples (from individuals of both sexes, pH 5.1-8.7, specific gravity 1.002-1.030 g/mL) were used during validation. Analysis were carried out in an Agilent 1260 HPLC with an AB SCIEX QTRAP 6500 detector. Compounds were separated employing an Agilent Poroshell 120 EC-C18 column (2.1 x 50 mm, 2.7 μm). The mobile phase (0.4 mL/min) consisted of water (A) and acetonitrile (B), both containing 0.2% formic acid. The gradient was: 0-1 min, 1% B; 1-7 min, to 60% B; 7-7.1 min, to 100% B; 7.1-9.1 min, 100% B; 9.1- 9.2 min, to 1% B; 9.2-11.0 min, 1% B. Detection was done using positive ESI source and MRM mode. Results and Discussion Eight specific Mebeverine metabolites were tentatively identified on the basis of their exact masses and MS/MS spectra obtained from LC-QToF analysis (Figure 2). Regarding the free and glucuronide conjugated metabolites, hydrolysed vs. non-hydrolysed results showed that: OH-MAL, OH-DMAL and N-desethyl-MAL were only excreted as conjugates. MAL, DMAL and N-desethyl-DMAL were mostly excreted as conjugates. MAC and DMAC were mostly excreted as free compounds. In fact, overloaded peaks were obtained in both experiments; consequently, sample dilution (1:50) and injection of lower volumes were required for their appropriate identification. Among those compounds, MAC and DMAC were chosen as the most useful mebeverine markers due to their specificity, their high-intensity signals, the no need of a hydrolysis step and the availability of reference materials. In particular, in the QA_2017B WAADS sample, the concentration of both metabolites was around 10 µg/mL, while p-OH-A was detected at a considerably lower level (roughly 10 ng/mL). MAC was first proposed by Stockis et al. as a valuable indicator of oral exposure to mebeverine both in urine and plasma [6]. The quantification of MAC and DMAC in plasma has been previously validated due to their usefulness to study clinical pharmacokinetics of mebeverine [6,8,9]. The proposed procedure for the confirmation of MAC and DMAC in urine by LC-QQQ was validated (Table 1). Validation results in this matrix have not been reported before. Selectivity was evaluated by individual analysis of 10 different blank samples obtained from healthy human volunteers. Regarding specificity, it was checked by processing blank specimens spiked with commonly encountered stimulants in doping control samples. No interfering peaks at the retention times of the target analytes were observed in neither of the two sets of samples. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-666 Figure 2. Extracted ion chromatograms (EIC), Product Ion (target MS/MS) spectra, structures and fragment interpretation of the suggested mebeverine metabolites Table 1. Summary of LC-QQQ parameters and validation results for DMAC and MAC The limit of identification (LOI) was determined by spiking 9 different blank urines at concentration levels between 1 and 100 ng/mL and subsequent analysis with the confirmation assay. Reliability of detection was verified at LOIs (Figure 3), carryover from 500 ng/mL was not observed and robustness of the method was confirmed. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-667 Figure 3. LC-QQQ (MRM) DMAC and MAC chromatograms of a blank urine and a urine spiked at LOI Conclusions Despite the initial lack of the corresponding reference materials and thanks to the exact mass capabilities of the LC-QToF system, eight specific mebeverine metabolites were tentatively identified by analysing a urine sample collected after administration of this drug. MAC and DMAC standards were subsequently purchased and a simple LC-QQQ based procedure was validated to exclude (or confirm) mebeverine intake before reporting an AAF based on the detection of p-OH-A, as required by WADA [10]. References 1. Dickinson RG, Baker PV, Franklin ME, Hooper WD. (1991) Facile hydrolysis of mebeverine in vitro and in vivo: negligible circulating concentrations of the drug after oral administration. J Pharm Sci. 80 (10), 952–7. 2. Kristinsson J, Snorradóttir I, Jóhannsson M. (1994) The metabolism of mebeverine in man: identification of urinary metabolites by gas chromatography/mass spectrometry. Pharmacol Toxicol. 74, 174–180. 3. Kraemer T, Bickeboeller-Friedrich J, Maurer HH. (2000) On the metabolism of the amphetamine-derived antispasmodic drug mebeverine: gas chromatography-mass spectrometry studies on rat liver microsomes and on human urine. Drug Metab Dispos. 28(3), 339-347. 4. Kraemer T, Wenning R, Maurer HH. (2001) The antispasmodic drug mebeverine leads to positive amphetamine results by fluorescence polarization immunoassay (FPIA)-Studies on the toxicological analysis of urine by FPIA and GC-MS. J Anal Toxicol. 25, 333-338. 5. Zaitsu K, Katagi M, Kamata T, Kamata H, Shima N, Tsuchihashi H, Hayashi T, Kuroki H, Matoba R. (2008) Determination of a newly encountered designer drug ‘‘p-methoxyethylamphetamine’’ and its metabolites in human urine and blood. Forensic Sci Int. 177, 77-84. 6. Stockis A, Guelen PJM, de Vos D. (2002) Identification of mebeverine acid as the main circulating metabolite of mebeverine in man. J Pharm Biomed Anal. 29, 335-340. 7. Bergeron M, Bergeron A, van Amsterdam P. (2013) Use of polarity switching for the simultaneous bioanalysis of analytes with three orders of magnitude difference in concentration by LC–MS/MS Bioanalysis. 5 (15), 1911–1918. 8. Khatri CA, Phanikumar CV, Jayaveera KN, Reddy KY. (2012) Development and validation of bioanalytical method for simultaneous quantification of veratric acid, mebeverine acid and desmethyl mebeverine acid in human EDTA plasma by using LC-MS/MS. J Pharm Chem. 6(4), 11-18. 9. Moskaleva NE, Baranov PA, Mesonzhnik NV, Appolonova SA. HPLC–MS/MS method for the simultaneous quantification of desmethylmebeverine acid, mebeverine acid and mebeverine alcohol in human plasma along with its application to a pharmacokineticsstudy. J Pharm Biomed Anal. 138, 118-125. 10. World Anti-Doping Agency. WADA Technical Letter TL-02 (version 3.0). Mebeverine metabolism (2020) www.wada-ama.org/sites/default/files/resources/files/tl02_mebeverine_metabolism_eng_2021_1_0.pdf (access date 09.08.2021) MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-668 Albertsdóttir AD, van Gansbeke W, van Eenoo P, Polet M Intact phase II AAS metabolites on GC-MS DoCoLab Universiteit Gent-UGent, Ghent, Belgium Abstract Research conducted on anabolic androgenic steroids (AAS) has repeatedly demonstrated the importance that phase II metabolites play on their detection for anti-doping purposes. The detection of these metabolites in their intact (non-hydrolysed) form has largely been conducted using LC-MS as the general consensus is that, in their intact form, these compounds are incompatible with GC-MS. Recently, our group presented work where the general GC-MS behaviour of non-hydrolysed sulfated AAS was mapped and the detection of these metabolites of metenolone and mesterolone exceeded the detection time provided by the conventional approach i.e., hydrolysed glucuronides. In order to maximise the chances of detecting AAS compounds, one must be aware of what is possible. It is known how non-hydrolysed sulfates behave when analysed on GC-MS, but the question remains how compatible non-hydrolysed glucuronides are with GC-MS? To provide an answer to that question drostanolone (2α-methyl-5α-androstan-17β-ol-3-one) was used as a trial model. Our data show that, contrary to general understanding, phase II metabolites are indeed compatible with GC-MS. Introduction The general consensus is that hydrolysis is required prior to analysing non-hydrolysed (intact) phase II metabolites of AAS on GC-MS.[1] However, recently published work maps the GC-MS behaviour of non- hydrolysed sulfated AAS. Here, contrary to their LC-MS behaviour, the intact sulfate is not detected as the sulfate group is cleaved off in the injector and a detectable artefact is formed [2,3]. Furthermore, their value has been demonstrated where analysing non-hydrolysed sulfated metabolites of metenolone [4] and mesterolone [2] provided increased detection time over the conventional metabolites. The question remained whether non-hydrolysed glucuronated metabolites could be analysed in the same manner so reference material of non-hydrolysed phase II drostanolone metabolite as well as extracted administration samples were analysed with GC-LE-EI-QTOF-MS and the results are presented below. Experimental Glucuronated drostanolone metabolite (Gluc-DrostM) and drostanolone metabolite were purchased from the National Measurement Institute (Australia) while sulfated drostanolone metabolite (Sulf-DrostM) reference material was synthesised according to previously published work [5]. The source of other reagents and materials are listed in our previous publications [4,6]. The excretion samples were from a previous publication [4] where, briefly, 25 mg of drostanolone propionate was administered to a volunteer (healthy male, 31 years, 80 kg) and a pre-administration sample (blank) and post-administration samples were collected. In this work, the blank and post- MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-669 administration sample at day 3 were extracted. The non-hydrolysed sulfated and glucuronated metabolites were extracted from the administration samples using a previously published single liquid-liquid extraction (LLE) using ethyl acetate at pH 9.5 [2] and 2 [6], respectively. After rolling for 20 min and centrifugation the organic phase was transferred to a separate tube and evaporated to dryness. All samples were derivatise prior to analysis using the routine derivatisation procedure where 60 μL of MSTFA/ethanethiol/NH I (500:4:2, v/v/v) was added to the dried residue. After brief vortexing (2 s), the liquid was transferred to a vial and incubated at 80°C for 30 min. 1.4 μL were injected onto an Agilent 7250 GC-QTOF-MS (Agilent Technologies, Palo Alto, CA, USA), equipped with back flush system and an Agilent 7693A ALS autosampler, was utilized. The MS operated in low energy electron ionization (LE-EI) mode at 17 eV. The same conditions were used as in our previously published work [7] except the method was prolonged by 2.8 min at 325°C. Results and Discussion The derivatised non-hydrolysed reference material was injected on the GC-LE-EI-QTOF and the data is presented in Figure 1. Figure 1. In both cases, the same peak is observed in the reference material and in the excretion urine sample while it is absent in the pre-administration sample (blank). The chromatograms were obtained by using the theoretical mass and a 20 ppm window. 4 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-670 Furthermore, to verify that the result could be replicated in authentic samples, a pre-administration (blank) and post-administration sample (3 days) were extracted using slightly different LLE to maximise the extraction efficiency of the respective metabolites and the results are presented in Figure 1. For both phase II metabolites the same peak is observed in the post-administration sample and the reference material whereas it is absent in the pre-administration sample. This clearly demonstrates that what is observed in the reference material is present in the excretion samples and therefore one can conclude that non-hydrolysed phase II AAS metabolites do not necessarily require hydrolysis for GC-MS analysis. Despite both phase II metabolites show compatibility with the GC-MS, they behave differently as the sulfate group is cleaved off in the injector, leading to shorter retention times, while the glucuronide maintains its bond with the drostanolone metabolite structure and eludes much later. In theory, it is possible that a portion of the non-hydrolysed glucuronide gets cleaved off in the injector leading to the same artefact as for the sulfated steroid. Using excretion samples is not feasible to determine if this hypothesis is correct as, independent of the applied sample procedure, one will always extract a small amount of non-hydrolysed glucuronate along with the non-hydrolysed sulfate. Therefore, both reference materials were spiked separately at 50 ng/mL in negative urine and extracted using LLE with ethyl acetate at pH 9.5 and the data are presented in Figure 2. Figure 2. Glucuronated drostanolone metabolite does not lead to a false positive response for sulfated drostanolone metabolite and the same is true vice versa MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-671 As is evident from Figure 2, the non-hydrolysed phase II metabolites only produce a positive response at their expected retention time. Conclusions When non-hydrolysed sulfated and glucuronated drostanolone metabolites were analysed using GC-MS, the same behaviour was observed in the reference material and the excretion samples. Furthermore, when the reference material is spiked separately in negative urine and extracted using the sample preparation used to extract sulfates, as expected, the glucuronates get extracted as well. However, they do not produce a false positive signal for the sulfated metabolite and vice versa. Therefore, contrary to general understanding, non-hydrolysed phase II metabolites are compatible with GC-MS. Finally, in order to facilitate the implementation of non-hydrolysed phase II metabolites into the anti- doping routine work it is vital that a greater variety of standard reference material is made available as the dependence on excretion urine or material made in-house inhibits the establishment of validation parameters for these compounds. References 1. Shackleton, C., Pozo, O. J., & Marcos, J. (2018). GC/MS in Recent Years Has Defined the Normal and Clinically Disordered Steroidome: Will It Soon Be Surpassed by LC/Tandem MS in This Role? Journal of the Endocrine Society, 2(8), 974–996. https://doi.org/10.1210/js.2018-00135 2. Polet, M., Van Gansbeke, W., Albertsdóttir, A. D., Coppieters, G., Deventer, K., & Van Eenoo, P. (2019). Gas chromatography−mass spectrometry analysis of non-hydrolyzed sulfated steroids by degradation product formation. Drug Testing and Analysis, 11(11–12), 1656–1665. https://doi.org/10.1002/dta.2606 3. Sakellariou, P., Kiousi, P., Fragkaki, A. G., Lyris, E., Petrou, M., Georgakopoulos, C., & Angelis, Y. S. (2020). Alternative markers for Methylnortestosterone misuse in human urine. Drug Testing and Analysis, 12(11– 12), 1544–1553. https://doi.org/10.1002/dta.2887 4. Albertsdóttir, A. D., Gansbeke, W. V., Coppieters, G., Balgimbekova, K., Eenoo, P. V., & Polet, M. (2020). Searching for new long-term urinary metabolites of metenolone and drostanolone using gas chromatography–mass spectrometry with a focus on non-hydrolysed sulfates. Drug Testing and Analysis, 12(8), 1041–1053. https://doi.org/10.1002/dta.2818 5. Waller, C. C., & McLeod, M. D. (2014). A simple method for the small scale synthesis and solid-phase extraction purification of steroid sulfates. Steroids, 92, 74–80. https://doi.org/10.1016/j.steroids.2014.09.006 6. Pozo, O. J., Van Eenoo, P., Van Thuyne, W., Deventer, K., & Delbeke, F. T. (2008). Direct quantification of steroid glucuronides in human urine by liquid chromatography–electrospray tandem mass spectrometry. Journal of Chromatography A, 1183(1), 108–118. https://doi.org/10.1016/j.chroma.2008.01.045 7. Polet, M., Van Gansbeke, W., & Van Eenoo, P. (2018). Development and validation of an open screening method for doping substances in urine by gas chromatography quadrupole time-of-flight mass spectrometry. Analytica Chimica Acta, 1042, 52–59. https://doi.org/10.1016/j.aca.2018.08.050 Acknowledgements The Partnership for Clean Competition (grant no. 2019R2000409G) is gratefully acknowledged for the funding for this work. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-672 Rubio A , Geyer H , Costa Padilha M , Pereira H , Cameron L , Thevis M Higenamine quantification and investigation into structural characteristics of its metabolites in urine samples Institute of Biochemistry, German Sport University, Cologne, Germany ; Brazilian Doping Control Laboratory (LBCD - LADETEC / IQ - UFRJ), Rio de Janeiro, Brazil ; Universidade Federal do Estado, Rio de Janeiro, Brazil ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract Higenamine, also known as norcoclaurine, is a non-selective β2-agonist naturally occurring in different plants (e.g. Nandina domestica, Tinospora crispa) and may be found in dietary supplements. In the herein project, a quantitative method for the free compound in urine is characterized following direct injection approach. The samples were analyzed by means of liquid chromatography – tandem mass spectrometry (LC-MS/MS). Besides, an estimation of the contribution of the conjugated metabolites was assessed. For that purpose, enzymatic hydrolysis (i.e. β-glucuronidase and arylsulfatase) was performed and the samples were analyzed according to the above mentioned approach, leading to a clear major contribution from the sulfo-conjugates compared to the glucuronide conjugates. Further evaluation of the conjugated metabolites was conducted; solid-phase extraction was performed and the herein obtained samples were fractionated by means of HPLC collection, and the subsequent LC-MS/MS analysis of these fractions showed the presence of at least three different potential glucuronide conjugates and two sulfo- conjugates for higenamine. The results of this research project can be of great value for anti-doping routine work, as they may contribute to ensure fair result management and decision-making processes in case of higenamine findings in sports drug testing programs. Introduction The use of higenamine in sports is prohibited at all times [1]. A reporting level of 10 ng/mL (50% of the Minimum Required Performance Level (MRPL)) applies for the detection of β2-agonists in urine and, in case of higenamine, it refers to the free compound only [2]. In the herein project a quantitative method for the free compound in urine is fully characterized [3]. The contribution of higenamine metabolites has been previously tested [4]; in this case, enzymatic hydrolysis was performed in order to estimate the specific contribution of both conjugate groups. Finally, the investigation of conjugated metabolites structural characteristics was conducted. Experimental The quantitative method for urinary determination of higenamine was fully characterized following a direct injection approach using 90 μL of urine and isoxsuprine-d5 was used as ISTD. Measurements were performed by means of LC-MS/MS on a Vanquish UHPLC system coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher). The LC was equipped with an EC 4/3 Nucleodur C-18 Pyramid 5 µm pre- 1 1 2 2 3 1,4 1 2 3 4 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-673 column (Macherey-Nagel) and EC 50/3 Phoroshell 120 C-18 Pyramid 2.7 µm analytical column (Agilent); mobile phases were 0.1 % formic acid (A) and acetonitrile containing 0.1 % formic acid (B). After one minute at 99%, elution was performed with a gradient from 99% to 60% A within 8 min at 400 µL/min followed by a gradient from 60% to 20% A within 2 min at the same flow rate. Re-equilibration was conducted at 99% A for 2 min (overall run time of 12 min. The mass spectrometer was operated in ESI positive mode. Data were acquired in full MS (m/z 100-1000; 45,000 FWHM at m/z 100) and tMS . tHCD experiments were performed at a resolution of 30,000 FWHM (at m/z 200), dynamic scan range starting at m/z = 80, and isolation window of m/z = 1. The contribution of urinary conjugated metabolites was assessed using samples from previous single dose elimination study (50 mg higenamine/dose dietary supplement; 136 h collection time), and enzymatic hydrolysis (i.e. 20 µL β-glucuronidase E. coli and 10 µL arylsulfatase P. aeruginosa) was performed. For structure elucidation, solid-phase extraction was performed using Oasis HLB 3 cc cartridges (Waters) and the herein obtained samples were fractionated on an Agilent 1100 HPLC system (150 x 4.6 mm phenyl-hexyl 3 µm column – Thermo Fisher) coupled to a Teledyne Isco Foxy R1 fraction collector. Subsequent LC-MS/MS analysis was conducted according to the method described. Results and Discussion The method employed for the quantitative determination of higenamine in urine was comprehensively characterized and the results are summarized in Table 1. The approach was found to be highly specific and linear from 0 to 100 ng/mL (R > 0.99) with an estimated LOD of 0.6 ng/mL (S/N > 3). Ion supression effects ranged from 42 to 68%, and the method´s intra- and interday imprecision were determined at three different concentration levels and varied from 5 to 11% and 8 to 11%, respectively. 2 2 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-674 Table 1. Assay characterization results concerning higenamine Urine samples from a previous elimination study (oral administration of 50 mg of higenamine) were re- evaluated following the method described. Higenamine was observed to be mainly excreted in its conjugated forms as presented in Figure 1. The herein presented urine sample corresponds to the first sampling time point (i.e. 4 h after ingestion) with a concentration of 16.7 ng/mL of free higenamine. After enzymatic hydrolysis with β-glucuronidase and arylsulfatase, a major contribution from the sulfo- conjugates compared to the glucuronide conjugates was observed. In this case, enzymatic hydrolysis led to a concentration of 101.7 ng/mL (free + glucuronide conjugates) and 6,034.2 ng/mL (free + sulfo- conjugates), respectively; free higenamine + glucuronide conjugates represents 1.68 % of the sulfo- conjugates contribution. The subsequent LC-MS/MS analysis of semi-preparative HPLC-fractionated signals suggests the presence of three (or more) different potential glucuronide conjugates and at least two sulfo-conjugates for higenamine as supported by characteristic dissociation patterns and diagnostic product ions such as 340.1021 for glucuronide conjugates, and 187.0056 and/or 244.0271 for sulfo-conjugates (Figure 2), which would correspond to the potential presence of disulfo-conjugate(s) (RT: 4.53) and monosulfo- conjugate (RT: 4.75), respectively. CID experiments were performed indicating again the potential presence of disulfo-conjugate(s) and one monosulfo-conjugate. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-675 Figure 1. Extracted ion chromatograms obtained after LC-MS/MS analyses before and after enzymatic hydrolysis. Urine sample collected 4 h after ingestion of 50 mg higenamine is shown as an example. Figure 2. Extracted ion chromatograms obtained after LC-MS/MS analysis of the HPLC collected fractions MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-676 Conclusions The herein obtained data show a major urinary excretion of higenamine in its conjugated form, and higenamine sulfo-conjugate(s) are presented as potential suitable marker(s) of the consumption of higenamine. However, further studies with significant number of volunteers should follow. For future testing purposes, further investigation into structural characteristics of higenamine metabolites and their synthesis as reference material appears advisable. References 1. World Anti-Doping Agency, The World Anti-Doping Code - International Standard: Prohibited List 2021 2. World Anti-Doping Agency, WADA Technical Document – TD2019MRPL: Minimum required performance levels for detection and identification of non-threshold substances. 2019. 3. H. Wagner, M. Reiter, W. Ferstl. New drugs with cardiotonic activity I Chemistry and pharmacology of the cardiotonic active principle of Annona squasrnosa L. Planta medica - Journal of Medical Plant Research. 1980, 40, 77-85 4. K. Grucza, D. Kwiatkowska, K. Kowalczyk, M. Wicka, M. Szutowski, P. Cholbinski. Analysis for higenamine in urine by means of ultra-high-performance liquid chromatography-tandem mass spectrometry: Interpretation of results. Drug Test Anal. 2018; 10: 1017-1024 Acknowledgements The authors thank the Manfred Donike Institute for Doping Analysis, the Federal Ministry of the Interior, Community, and Building of the Federal Republic of Germany (Berlin, Germany), and the World Anti- Doping Agency (Montreal, Canada, grant #T20M01MT) for supporting the presented study. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-677 Martinez Brito D, Leogrande P, de La Torre X, Botrè F Analysis of 7-oxo-DHEA metabolites by liquid chromatography mass spectrometry Laboratorio Antidoping FMSI, Federazione Medico Sportiva Italiana, Rome, Italy Abstract 7-oxo-DHEA undergoes an extensive phase I metabolism including reductions, oxidations, and hydroxy‐ lations. In the context of doping analysis 7α- and 7β-hydroxylated metabolites of DHEA and 7ξ-hydroxy- androstenedione, androsterone or epiandrosterone have been described as the main in vivo metabolites. Besides, we recently described additional urinary metabolites after the analysis of separated glucuronated and sulfated fractions with specific derivatization. The goal of this work was to evaluate the 7-oxo-DHEA metabolism by accurate mass spectrometry and triple quadrupole both coupled to liquid chromatography (LC-qTOF and LC-QqQ) avoiding derivatives formation. One single oral dose of 7-oxo-DHEA was administered to four volunteers and samples collected before and up to 10 hours after the administration were analyzed. The urinary combined fraction (free + glucuronated) was analyzed after hydrolysis with β-glucuronidase from E. coli and extraction with TBME. The extracts were reconstituted in water/acetonitrile before analysis. At least ten metabolites were determined by LC-qTOF analysis (accurate mass error from -5.4 to 2.4 ppm). After product ion scan experiments, several transitions could be selected for the identification of these metabolites by LC-QqQ. The assesment of 7-oxo-DHEA metabolism by liquid chromatography- mass spectrometry showed that some metabolites that are not physiologically present in urine could be good markers for the administration of 7-oxo-DHEA and could avoid the need for IRMS confirmation, as currently required by WADA for endogenous steroids. Introduction The metabolism of 7-oxo-DHEA includes reductions, oxidations and hydroxylation reactions. In the context of doping analysis, 7α-and 7β-hydroxylated metabolites of DHEA and 7ξ-hydroxy- androstenedione, androsterone or epiandrosterone have been described as the main metabolites. Recently, we described new findings after the analysis by GC-qTOF. Urinary purified fractions were obtained by preparative-HPLC and specific derivatization was applied to discriminate between hydroxyl or keto groups. Several potential metabolites and potential degradation products were described as well as the influence of the derivatization reagent and injector port temperature [1-4]. This investigation aimed to evaluate 7-oxo-DHEA metabolites already described by GC-qTOF [2] using accurate mass spectrometry and triple quadrupole, both coupled to liquid chromatography, avoiding the derivatization step and high temperatures. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-678 Experimental LC-QqQ Agilent 1290 Infinity II LC System (Agilent Technologies Italia Spa, Cernusco sul Naviglio, Milan, Italy). Chromatographic column RP Agilent Eclipse plus C18 (10 cm; 2.1 mm; 1.8 µm) was kept at 30°C. Mobile phase: solvent A (water: 0.1% formic acid) and solvent B (acetonitrile: 0.1% formic acid) was set at 0.3 mL/min. The mass spectrometer was an Agilent Ultivo LC/TQ equipped with a Jet Stream Electrospray Ionization (AJS ESI) Ion Source, operated in the following conditions: fragmentor energy 135 V, nebulizer gas pressure N 40 psi, gas temperature 150°C, capillary voltage 3500 V and nozzle voltage 500 V. LC-qTOF The same chromatographic conditions were used. The mass spectrometer was a qTOF (Agilent 6545) operating with an electrospray ionization source. The acquisition was in positive mode and full scan data from 50 to 930 Da were acquired. Constant mass calibration was obtained by monitoring m/z 121.0509 (C H N ) and m/z 922.0098 (C H O N P F ). Urinary excretion study Five volunteers (two female, three male, average age 38 ± 10 years, 67 ± 8 kg body weight, and normal BMI) were administered with a single oral dose of 100 mg of 3-acetyl-7-keto-DHEA (Now Foods, Blooming​‐ dale, IL, USA). Capsules composition was controlled and no additional signals different to 3-acetyl-7-keto- DHEA were detected. Only traces of 7-keto-DHEA was observed. Sample preparation To 2 mL of urine, 20 μL methyltestosterone (10 μg/mL, internal standard), 750 μL phosphate buffer (0.8 M, pH7) and 50 μL β-glucuronidase (E. coli) were added and hydrolysis occurred during 1h at 55°C. Then pH was adjusted to 9-10 (500 μL carbonate-bicarbonate buffer, 20%). Double liquid-liquid extraction was done with tert-butylmethylether; the dry extract was reconstituted in 50 μL of water: acetonitrile (1:1, V:V). Samples were analyzed by LC-qTOF for metabolite detection and errors ± 10 ppm were accepted. LC- QqQ was used to evaluate the product ion scan (PIS) and MRM experiments. Results and Discussion The evaluation data was based on a 7-oxo-DHEA metabolism study earlier described using GC-qTOF [2]. Results obtained after the evaluation of twelve potential metabolites of 7-oxo-DHEA, their molecular formula, error from the accurate mass, retention time, and the selected transitions obtained after PIS and MRM experiments using pre- and post-administration urines are shown in Table 1. 2 5 4 4 18 18 6 3 3 24 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-679 Table 1 . Data of the proposed metabolites observed in urine after the administration of 7-oxo-DHEA to five volunteers. Data include formulae, theoretical mass and error from the theoretical mass, retention time (RT) and proposed transitions after the analysis by LC-qTOF and LC-QqQ The analyses of all samples showed the presence of 7-oxo-DHEA ( III) and the proposed isomer C3α- (IV). Two potential isomers with a combination of two keto and one hydroxy groups on C3, C7 and C17 were observed at 7.9 and 8.1min (I and II). Two compounds were found at 6.1 min (V) and 6.45 min (VI) showing m/z 305. They fit with androst-5- ene-3α,17[α/β]-diol-7-one structure as referred previously by GC-qTOF. It is important to mention that 6α-OH-T (with similar MS) elutes at 6.4 min. Therefore, special attention has to be paid when chromatographic conditions are not optimal. An additional compound at 6.55 min (VII) with an evident loss of water (m/z 287) was observed. This metabolite could correspond with the GC-qTOF proposed structure androst-5-ene-3α,7ξ-diol-17-one but no suitable transitions could be obtained. 7-oxo-DHEA mono-hydroxylated metabolites previously proposed as androst-3,5-diene-3,7ξ,17ξ,16ξ- tetraol and androst-5-ene,7-oxo,3α,17ξ,16ξ-triol were also observed. Three signals at 4.5 (X), 5.2 (XI) and 5.35 min (XII) corresponding to that mass were observed by LC/MS. One potential additional metabolite, not described by GC-qTOF [2], eluted at 5 min. Considering its early RT, it could be a reduced-hydroxylated 7-oxo-DHEA metabolite (VIII, m/z 323). In the MS, no molecular mass was observed but a prominent loss of water could not be discarded due to the polarity of the structure [5]. The molecule fragmentation is quite similar to that obtained for the known metabolite 7β- hydroxy-epiandrosterone (IX). Figures 1 to 3 show the results for samples collected before and after 7-oxo-DHEA administration to one volunteer. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-680 Figure 1. Chromatogram of the selected transitions for compounds I to V in urines collected before, 3 and 9 hours after administration of a single dose of 7-oxo-DHEA Figure 2. Chromatogram of the selected transitions for compounds VI to IX in urines collected before, 3 and 9 hours after administration of a single dose of 7-oxo-DHEA MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-681 Figure 3. Chromatogram of the selected transitions for compounds X to XII in urines collected before, 3 and 9 hours after administration of a single dose of 7-oxo-DHEA Conclusions The analysis of 7-oxo-DHEA is still a challenge for antidoping laboratories as it is an endogenous compound and therefore a confirmatory IRMS analysis is required by WADA. It is widely metabolized after redox and hydroxylation reactions (mainly) and a considerable number of metabolites can be found in the urine after the administration. This metabolic approach for 7-oxo-DHEA using LC-MS showed that some metabolites not physiologically present in urine could be good markers of the 7-oxo-DHEA administration. The excretion of the hydroxylated metabolites in urine has been described previously by Martinez-Brito et al. by using GC instrumentation. References 1. Martinez-Brito D, de la Torre X, Parr MK, Botrè F. Mass spectrometric analysis of 7-oxygenated androst-5- ene structures. Influence in trimethylsilyl derivative formation. Rapid Commun Mass Spectrom. 2020;34(17):1-8. doi:10.1002/rcm.8834 2. Martinez-Brito D, de la Torre X, Colamonici C, Curcio D, Botrè F. 7-keto-DHEA metabolism in humans. Pitfalls in interpreting the analytical results in the antidoping field. Drug Test Anal. 2019;(August):1629- 1643. doi:10.1002/dta.2734 3. Delbeke FT, Van Eenoo P, Van Thuyne W, Desmet N. Prohormones and sport. J Steroid Biochem Mol Biol. 2002;83(1-5):245-251. doi:10.1016/S0960-0760(02)00274-1 4. Cawley AT, George A V. Complementary stable carbon isotope ratio and amount of substance measurements in sports anti-doping. Drug Test Anal. 2012;4(12):897-911. doi:10.1002/dta.1378 5. Marcos J, Pozo OJ. Current LC–MS methods and procedures applied to the identification of new steroid metabolites. J Steroid Biochem Mol Biol. 2016;162:41-56. doi:10.1016/j.jsbmb.2015.12.012 Acknowledgements This investigation was performed thanks to the World Anti-Doping Agency Grant Number 19A09XD. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-682 Camuto C , Guglielmelli A , De-Giorgio F , de La Torre X , Mazzarino M , Marti M , Botrè F An insight into the metabolism of New Psychoactive Substances: targeted and untargeted metabolic profile of a new mephedrone analogue Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Rome, Italy ; Department of Health Care Surveillance and Bioetics, Section of Legal Medicine, Catholic University of Rome, Rome, Italy ; Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy ; Department of Translational Medicine, Section of Legal Medicine and LTTA Center, University of Ferrara, Ferrara, Italy Abstract Mexedrone is a synthetic cathinone structurally related to the well-known mephedrone, belonging to the class of the N-alkyl derivatives of cathinone. Mexedrone appeared after extensive advertising on the web, two years before it was launched on the market as a legal substitute for mephedrone. Mexedrone was presented in 2015 with a specific website and soon its effects were described in online forums. To date, the metabolic pathway of mexedrone is unknown, making its detection a challenge in routine drug tests or anti-doping analysis, as it belongs to the class of cathinones that has been on the WADA list since 2014. The present study aimed to evaluate the phase I metabolic pathway of mexedrone and to identify its suitable marker(s) of intake. Mexedrone was incubated in the presence of human liver microsomes (HLM) and different CYP450 isoforms. First, the phase I profile of mexedrone was defined through an untargeted high-resolution technique, based on UHPLC-QTOF. The metabolic profile and chemical structures of the metabolites were subsequently confirmed by a targeted triple quadrupole mass spectrometric technique (LC-QqQ). The main phase I metabolic reactions were hydroxylation and N-/O-dealkylation. The CYP450 isoforms most involved in mexedrone metabolism were the CYP2C19, followed by CYP2D6 and CYP1A2. The hydroxylated metabolite and the parent compound appear to be the most suitable markers of intake. Introduction Mexedrone was introduced in 2015 through a specific website, and soon its effects were described in online forums [1]. The structure of mexedrone was first identified and characterized in 2016 in a powder purchased online [2], and in the same year its presence was confirmed in post-mortem biological fluids of a 27 year old man [3]. The effects of mexedrone were first reported in 11 confirmed cases of mexedrone intake in the City Hospital of Birmingham (UK). The most common effects were agitation, sinus tachycardia and psychosis [4]. To the best of our knowledge, no data are presently available on the metabolism of mexedrone. 1 1 2,3 1 1 4 1 1 2 3 4 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-683 Experimental Mass spectrometric strategy The elemental composition of mexedrone and of the compounds formed after incubation of the parent compound in the presence of HLM was defined by using the Time-of-Flight analyzer (QTOF) and full scan as acquisition mode. MS/MS experiments were then performed at different collision energies by low- resolution triple quadrupole analyzer (QqQ) operating in product ion scan mode to characterize the fragmentation behavior of mexedrone. The characteristic fragments of the molecule (structural markers) were then selected to set up the Multiple Reaction Monitoring (MRM) method and to propose the chemical structures of the metabolites identified. Protocols for the in vitro metabolism studies and sample pre-treatment were developed from a protocol already in use in our laboratory to perform metabolism studies of similar substances [5,6], testing different enzymatic protein concentrations, time of incubation and substrate concentration as well as solvents, volume of solvents, and pH of extraction. Instrumental conditions Untargeted HRMS Samples from in vitro metabolism studies were analyzed by using an Agilent 1290 infinity II series UHPLC instrument equipped with a: Zorbax C18 (100 mm x 2.1 mm, 1.8 μm) coupled with an orthogonal acceleration time-of-flight mass spectrometer 6545 (Agilent Technologies) equipped with an ESI source. The solvents used were: ultrapurified water (eluent A) and acetonitrile (eluent B), both containing 0.1% formic acid. The flow rate was set to a constant flow rate of 400 µL/min starting at 2% of B. Targeted MS/MS: The same samples were analyzed using an Agilent 1200 series HPLC instrument equipped with a SUPELCO Discovery C18 column (150 mm x 2.1 mm x 5 µm) coupled with an API4000 QqQ mass spectrometer (Sciex) with an ESI source. The solvents were the same described in the above section. The flow rate was set to a constant flow rate of 250 µL/min starting at 5% of B. Results and Discussion In vitro investigation Identification of metabolites After 4 hours of incubation at 37°C, the parent compound and three metabolic products were identified. The extracted ion chromatogram and the relative spectra of metabolites of mexedrone are reported in Figure 1. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-684 Figure 1.a) Extracted ion chromatogram of a representative sample after 4h of incubation with HLM Hydroxylated (M1) and two dealkylated M2 and M3. The extracted mass spectra of M1 b), M2 c) and M3 d) were reported. The metabolite M1 presents a molecular ion at m/z 224.1281 with elemental composition C H NO attributed to the hydroxylation of mexedrone. The metabolite M2 and M3 with the same m/z 194.1175 and elemental composition C H NO were attributed to the N-/O- dealkylation of mexedrone. The results obtained after the MS/MS experiment allow us to propose three structures of mexedrone metabolites, shown in Figure 2 with their fragmentation pathways. The same metabolic reactions were reported for the in vitro metabolism of mephedrone [7]. See Table 1 for the elemental composition, product ions, and relative collision energies of mexedrone and its metabolites. 12 17 3 11 15 2 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-685 Figure 2. Proposed structures and mass spectra pattern for Hydroxylated (M1), the O-dealkylated (M2) and the N-dealkylated (M3) metabolites of mexedrone with characteristic diagnostic ion transitions, the dashed linefragmentations Table 1. Mass spectra parameters for mexedrone and M1-M3 metabolites, precursor and monitored product ions of the MRM method, and relative collision energies Individual contribution of CYP isoforms The relative contribution of individual CYP450 isoforms to the phase I metabolic reactions of mexedrone was evaluated with five different CYP isoforms (i.e.,CYP1A2, CYP2C9, CYP3A4, CYP3A5, CYP2D6). The results showed that CYP3A4 and CYP3A5 were not involved in mexedrone metabolism while CYP1A2 and CYP2D6 formed only the hydroxylated metabolite M1. The isoform CYP2C9 was involved in the formation of all the metabolites (M1-M3) and is the isoform most involved in mexedrone metabolism. These results differ from mephedrone for which the role of CYP2D6 in the formation of demethylated products has been reported [8]. The CYP2C9 is a highly polymorphic gene, with isoforms characterized by a marked decrease in enzyme activity. In metabolism studies, the presence of subjects with allelic variations that reduce metabolic activity means that metabolites formed by CYP2C9 may not be detected. These suggest that M1 is the most informative metabolites and potential in vivo marker for mexedrone intake. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-686 Conclusions The phase I metabolic pathway of mexedrone was defined through in vitro studies carried out employing HLM and five different CYP450 isoforms. The metabolic reactions involved in mexedrone metabolism were dealkylation and hydroxylation. Three different in vitro metabolites were identified the detected metabolites are hydroxy-mexedrone (M1), O-dealkyl-mexedrone (M2), N-dealkyl-mexedrone (M3). The formation of M1 involved three CYP450 isoforms (i.e., CYP2C9, CYP2D6, CYP1A2) while M2 and M3 were formed only after incubation with CYP2C9. These results suggest that M2 and M3 may have a greater influence on allelic variants that may compromise their detection. Further studies are needed using in vivo models to confirm the in vitro observation and to define the windows of detection of mexedrone and its metabolites. References 1. G. McLaughlin, N. Morris, P. V. Kavanagh, J. D. Power, G. Dowling, B. Twamley, J. O’Brien, B. Talbot, D. Walther, J. S. Partilla, M. H. Baumann, S. D. Brandt. Synthesis, characterization and monoamine transporter activity of the new psychoactive substance mexedrone and its N-methoxy positional isomer, N- methoxymephedrone. Drug Test. Anal., 2017, 9, 358–368. 2. Z. Qian, W. Jia, T. Li, C. Liu, Z. Hua. Identification and analytical characterization of four synthetic cathinone derivatives iso-4-BMC, β-TH-naphyrone, mexedrone, and 4-MDMC. Drug Test. Anal., 2017, 9, 274–281. 3. S. P. Elliott, S. D. Brandt, C. Smith. The first reported fatality associated with the synthetic opioid 3,4- dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide (U-47700) and implications for forensic analysis. Drug Test. Anal., 2016, DOI 10.1002/dta.1984. 4. L. Roberts, L. Ford, N. Patel, J. A. Vale, S. M. Bradberry. 11 Analytically Confirmed Cases of Mexedrone Use Among Polydrug Users. Clin. Toxicol., 2017, 55, 181–186. 5. C. Chieffi, C. Camuto, F. De-Giorgio, X. de la Torre, F. Diamanti, M. Mazzarino, C. Trapella, M. Marti, F. Botrè. Metabolic profile of the synthetic drug 4,4′-dimethylaminorex in urine by LC–MS-based techniques: selection of the most suitable markers of its intake. Forensic Toxicol. , 2020, DOI 10.1007/s11419-020- 00544-9. 6. C. Camuto, S. Pellegrini, F. De-Giorgio, X. de la Torre, M. Marti, M. Mazzarino, F. Botrè. Urinary excretion profile of methiopropamine in mice following intraperitoneal administration: A liquid chromatography– tandem mass spectrometry investigation. Drug Test. Anal., 2020, DOI 10.1002/dta.2900. 7. E. Olesti, M. Farré, E. Papaseit, A. Krotonoulas, M. Pujadas, R. De Torre, Ó. J. Pozo. Pharmacokinetics of Mephedrone and Its Metabolites in Human by LC-MS / MS. 2017, 19, DOI 10.1208/s12248-017-0132-2. 8. E. Olesti, M. Farré, M. Carbó, E. Papaseit, C. Perez-Mañá, M. Torrens, S. Yubero-lahoz, M. Pujadas, Ó. J. Pozo, R. de la Torre. Response Pharmacological Study of Mephedrone and Its Metabolites: Pharmacokinetics , Serotoninergic Effects, and Impact of CYP2D6 Genetic Variation. 2019, 106, DOI 10.1002/cpt.1417. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-687 Camuto C , De-Giorgio F , Fiacco I , Marti M , Mazzarino M , Botrè F In vivo metabolism of JWH-175: blood and urine detection of JWH-018 in mice Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Rome, Italy ; Department of Health Care Surveillance and Bioetics, Section of Legal Medicine, Catholic University of Rome, Rome, Italy ; Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy ; Department of Chemistry and Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy Abstract The in vitro metabolism of JWH-175 is known in the literature and shows the formation of the active metabolite JWH-018. However, it is still unclear whether the in vivo formation of JWH-018 and its metabolites is possible and could be detected after the intake of JWH-175. Our aim was to evaluate the metabolic profiles of JWH-175 in urine and blood to verify the in vivo formation of JWH-018 after administration of a dose of 10 mg/kg of JWH-175 to ICR (CD-1 ) mice. For this purpose, two groups of mice were selected and JWH-175 was administered. The first group was used for the collection of urine samples, the second group for collection of blood samples at different time points. Samples were analyzed by LC-QqQ. The formation of JWH-018 and its metabolites was confirmed by comparison with the results obtained after incubation of JWH-018 in the presence of human liver microsomes (HLM). Introduction JWH-175 is a synthetic cannabinoid (SC) of the naphthylmethylindole family. It was first synthesized by John W. Huffman in 2005 and it is structurally related to JWH018, with a ketone linker replaced by a methylene one [1]. Experiments in mice indicate that JWH-175 may have abuse potential while similar SCs as JWH-176 and JWH-030 have a low potential for abuse [2]. The in vitro metabolism of JWH-175 is known in the literature, showing the formation of the active metabolite [3], which could explain these differences of activity in mice. However, it is still unclear whether the in vivo formation of JWH-018 and its metabolites is possible and could be detected after the intake of JWH-175. Experimental Protocol for the in vivo studies Sample collection For the in vivo studies, two different groups of mice were selected. To both groups a dose of 10 mg/kg of JWH-175 was administered. For the first group pooled urine samples were collected in the range of 0‑6 hours after the intake. For the second group blood sample were collected in the time interval of 0‑300 min after the intake. 1 2,3 1 4 1 1 1 2 3 4 ® MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-688 Sample pre-treatment For blood metabolic profile, samples were centrifugated at 9000 rpm for 8 min to obtain plasma. The supernatant was added with 500 μL of acetonitrile and centrifugated at 13000 rpm for 3 min. The aqueous layer was next collected and added with 50 μL of internal standard solution (10 μg/mL) and 500 μL of phosphate buffer (0.8 M, pH 7.4). Samples were then extracted with 5 mL of ethyl-acetate. For the urinary excretion studies a sample pre-treatment protocol was developed starting from protocols already used by our laboratory for similar studies [4-5]. Instrumental conditions Samples were analyzed using an Agilent 1200 series HPLC instrument equipped with a SUPELCO C18 column (15 cm x 2.1 mm x 5 µm) coupled with a Sciex 5500QTRAPtriple quadrupole mass spectrometer (Sciex, Milan, Italy) with an ESI source operating in positive ionization mode. Analyses were carried out at a constant flow rate of 250 µL/min using as mobile-phase ultra-purified water, 0.1% formic acid (A), and acetonitrile 0.1% formic acid (B). The mass spectrometric parameters were optimized by infusing the standard solution of JWH-175, JWH- 018 and JWH-210 at a concentration of 10 μg/mL. Multiple reaction monitoring (MRM) was used as acquisition mode. The MRM method was optimized starting from the protocol routinely employed in our laboratory [6]. Results and Discussion Blood kinetic Analysis of the blood samples revealed the presence of JWH-018 as the main metabolite of JWH-175. These unequivocally confirm the in vivo formation of JHW-018. The blood concentration of JWH-018 showed a maximum of excretion at 180 min after administration of the parent compound while JWH-175 was detected only in traces, see Figure 1. Results are calculated from the concentration extrapolated from the calibration curve (R> 0.990) for each mouse collecting point (i.e., 30, 180 and 300 min) and range between 150 and 350 ng/mL of JWH-018. Figure 1. Extracted chromatogram of a representative blood sample 30 min after the intake of JWH-175 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-689 Urinary excretion Samples from the mice excretion study were analyzed and the results obtained show the formation of mono-, di-hydroxylated and carboxylated or dehydrogenated-mono-OH metabolites of JWH-018. To confirm the formation of JWH-018 metabolites, a standard solution of JWH-018 was incubated with HLM. The in vitro metabolic profile obtained was compared with those obtained from in vivo excretion studies. The results show the formation of ten metabolites of JWH-018 in common between the in vitro and in vivo samples. In detail, three di-hydroxylated (M1-M3), three mono-hydroxylated (M8-M10) and four carboxylated or dehydrogenated-mono-OH (M4-M7) metabolites were identified in both studies (Figure 2). These suggest an extensive metabolism of JWH-018 after its formation from JWH-175. Furthermore, the parent compound JWH-018 and JWH-175 were not detected in urine. Figure 2. Comparison of in vitro (a-c) and in vivo (d-f) metabolism of JWH-018. The metabolites reported are three di-hydroxylated M1-M3 a and d, four carboxylate or dehydrogenate-mono-OH (M4-M7) b and e and three mono-hydroxylated (M8-M10) c and f. Conclusions Our results demonstrate the formation of JWH-018 in vivo. JWH-018 was detected in blood samples and its formation was supported by the detection of JWH-018 metabolites in urine. Specifically, the 10 metabolites detected in urine are: six hydroxylated and four carboxylated/ monohydroxy-dehydrogenated. JWH-018 is detected in blood in significantly higher amounts than the parent compound JWH-175 in all blood samples. The formation of the active metabolite JWH-018 could explain the differences in activity tested in mice compared to JWH-176 [2]. Furthermore, these results highlight that the detection of JWH-018 and its metabolites in biological fluids of athletes cannot be attributed only to the intake of JWH-018. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-690 References 1. Huffman JW., Padgett LW. Recent developments in the medicinal chemistry of cannabimimetic indoles, pyrroles and indenes. Curr Med Chem. 2005;12(12):1395-411. DOI 10.2174/0929867054020864. 2. Tampus R et al. Assessment of the Abuse Liability of Synthetic Cannabinoid Agonists JWH-030, JWH175, and JWH-176. Biomol Ther (Seoul). 2015; Nov;23(6):590-6. DOI 10.4062/biomolther.2015.120. 3. Fietzke, M., Thomas, A., Beike, J. et al. In vitro elucidation of the metabolic profile of the synthetic cannabinoid receptor agonists JWH-175 and JWH-176. Forensic Toxicol 34, 353–362, 2016. DOI 10.1007/s11419-016-0322-0 4. Chieffi C, Camuto C, De-Giorgio F, et al. Metabolic profile of the synthetic drug 4,4′-dimethylaminorex in urine by LC–MS-based techniques: selection of the most suitable markers of its intake. Forensic Toxicol. 2020 DOI 10.1007/s11419-020-00544-9 5. Camuto C, Pellegrini S, De-Giorgio F, et al. Urinary excretion profile of methiopropamine in mice following intraperitoneal administration: A liquid chromatography–tandem mass spectrometry investigation. Drug Test Anal. 2020. DOI 10.1002/dta.2900 6. Mazzarino M, Torre X de la, Botrè F. A liquid chromatography-mass spectrometry method based on class characteristic fragmentation pathways to detect the class of indole-derivative synthetic cannabinoids in biological samples. Anal Chim Acta. 2014 DOI 10.1016/j.aca.2014.06.003 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-691 Pühringer M, Gmeiner G Detection of S-23 metabolites in urine after a single oral administration using liquid chromatography high resolution mass spectrometry Doping Control Laboratory, Seibersdorf Laboratories, Seibersdorf, Austria Abstract Selective androgen receptor modulators (SARMs) are non-steroidal anabolic therapeutics acting on the androgen receptor which are currently developed for the treatment of numerous hormonal diseases. Due to their anabolic properties and the minimizing of androgenic adverse side effects in contrast to conventional steroidal anabolic agents as well as their oral applicability, SARMs drew attention to professional athletes but also to hobby athletes with regard to their performance enhancing properties. S-23 is an arylpropionamide-based SARM whose metabolism has already been extensively studied in vitro [1]. Here we describe the detection of S-23 metabolites in human urine after administration of a single oral dose of a nutritional supplement containing 10 mg S-23. Urine samples were analyzed by means of liquid-chromatography coupled to high resolution mass spectrometry. Glucuronide conjugated S-23 metabolites proved to be a useful and unambiguous target for the detection of S-23 abuse. Using a solid-phase extraction (SPE) sample preparation method, phase II metabolites could be detected over a period of up to 55 days. Introduction SARMs are a chemical diverse group of compounds all acting on the androgen receptor (AR) which is essential for the function of secondary sexual organs as well as muscle and bone growth [2]. By selectively stimulating the AR in muscle and bones while leaving AR in other tissues unstimulated, adverse side effects of traditional steroid replacement therapies such as prostate and cardiovascular disease as well as acne and increased growth of body hair, should be avoided [3]. S-23 is an arylpropionamide-based SARM developed by the pharmaceutical company GTx, Inc. The metabolism of S-23 has already extensively been studied in vitro [1]. In this study, the detection of S-23 and its metabolites in human urine after a single oral dose administration is investigated. Experimental Sample preparation: Dilute-and-shoot: 100 µL of urine samples were diluted with 900 µL 0.1% (v/v) formic acid. Ostarine was added as internal standard. Solid-phase extraction: The solid-phase extraction was performed using an Oasis HLB 3 cc 60 mg column (Waters, Milford, Massachusetts, USA). The 2 mL urine aliquots were spiked with the internal standard MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-692 ostarine. Cartridges were conditioned with methanol followed by MQ-water. 2 mL of urine samples were applied on the columns, washed with MQ-water and eluted using methanol. The solvent was evaporated and samples re-suspended in 150 µL of methanol-water (30:70). Samples were heated for 20 min at 60°C, transferred in a vial and injected into the LC-MS. Chromatographic separation: The Vanquish™ UHPLC system (Thermo Scientific) was equipped with a Kinetex Biphenyl column (100 Å; 30 x 2.1 mm; 2.6 µm) protected by an Accucore Ph/Hex (10 x 3.0 mm; 2.6 µm) pre-column. As mobile phase A 0.2% formic acid and as mobile phase B 0.1% formic acid in methanol were used at a flow rate of 280 µL/min. The chromatographic gradient was as follows: 0-2 min isocratic at 90% A; 2-5 min gradient from 90% A to 0% A; isocratic at 0% from 5-8 min; equilibration at 90% A from 8-11 min. Mass Spectrometry: QExactive focus hybrid quadrupole-orbitrap mass spectrometer (Thermo Scien- tific) operated in full-scan mode (100-800 m/z) at a resolution of 70000 and parallel reaction monitoring (CE = 30 eV) at a resolution of 17500 targeting S-23 metabolites. Ionization was achieved using electro‐ spray ionization (ESI) in negative mode. The mass accuracy was < 5 ppm throughout the measurements. Results and Discussion A total of 3 metabolites of S-23 were identified in urine samples at different concentrations and time periods after administration. S-23 was found to undergo phase II metabolism by being conjugated with glucuronic acid. Two such phase II metabolites, the glucuronide of S-23 (M1) and the glucuronide after hydroxylation of S-23 (M2), were detected in urine samples as the most prominent and long-term metabolites. Arylpropionamide SARMs such as S-23 have in common an aromatic moiety connected to the nitrogen of an amide bond. These kinds of substructures are substrates to human hydrolytic metabolism [4]. For S-23, one such a metabolite (M3) was detected in the excretion study. Figure 1. Structure of S-23 phase II metabolites identified in human urine samples ® MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-693 Table 1. Masses of parent and diagnostic product ions of S-23 M1-3 and respective retention times The two long-term phase II metabolites of S23, M1 and M2, were detectable using two different approaches: 1.) dilution of the urine samples, direct injection into the chromatographic system and analysis using automated pre-cleaning of the sample using on-line SPE; 2.) manual SPE sample preparation followed by the addition of the solvent and injection into the LC-MS system. In Figure 2, the excretion profile of S-23 metabolites M1, M2 and M3 using the manual SPE method is depicted. S-23 glucuronide (M1) was detected over a time period of 55 days after administration. M2 was detectable in urine for 42 days and M3 for 27 days after oral administration using the SPE sample preparation method with an aliquot of 2 mL urine. In dilute-and-shoot samples using online-SPE prior to the chromatography M1 was identified for 26 and M2 for 21 days. Figure 2. Relative abundances of S-23 M1-3 peak areas compared to the internal standard (ISTD) S-22 and specific gravity (SG) correction using the manual sample preparation method. The excretion profile of S-23 M1 is depicted 0-20 days (left) and 20-56 days (right) after a single oral dose administration of S-23. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-694 Conclusions The detection of illicit therapeutics abuse in sports drug testing requires the knowledge of the excretion of the drug and/or its metabolites in human urine samples. In the present study, 3 metabolites of the SARM S-23 were detected in urine samples using two different sample preparation methods namely dilute-and-shoot and SPE. S-23 glucuronide proved to be a useful long term metabolite for the detection of S-23 abuse and was detected over a period of 55 days after a single oral administration. References 1. Thevis M et al. (2010) Characterization of in vitro generated metabolites of the selective androgen receptor modulators S-22 and S-23 and in vivo comparison to post-administration canine urine specimens. Drug Test Anal. 2, 589–598. 2. Narayanan R, Coss C C, Dalton J T. (2018) Development of Selective Androgen Receptor Modulators (SARMs). Mol Cell Endocrinol. 465, 134–142. 3. Bhasin S et al. (2006) Drug Insight: Testosterone and selective androgen receptor modulators as anabolic therapies for chronic illness and aging. Nat Clin Pract Endocrinol Metab. 2, 146–159. 4. Bradshaw P R et al. (2018) Metabolic Hydrolysis of Aromatic Amides in Selected Rat, Minipig, and Human in Vitro Systems. Sci Rep. 8, 1–8. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-695 Thomas A , Fox J , Slade S , Kislyuk S , Gastall H , Thevis M Preliminary data for ion mobility separation of recombinant and synthetic insulin variants on a cyclic IMS mass spectrometer Institute of Biochemistry, German Sport University, Cologne, Germany ; Waters Coporation, Waters, Wilmslow, UK Abstract Synthetic insulins represent prohibited substances in professional sport according to the list of the World Anti-Doping Agency. Due to their peptidic character and sharing a largely common amino acid sequence, their physico-chemical properties are very similar. Thus, their mass spectrometric and liquid chromatographic properties are very similar too. For example, recombinant human insulin and insulin lispro differ in the amino acid sequence at position B28/29 only and share the identical molecular weight. Obvioulsy, separation of these analogs represents a considerable challenge. Nevertheless, definite identification of these target peptides is crucial in doping controls as well as in forensics or related disciplines. Considering these facts, the present approach shows the separation performance of a new cyclic ion mobility mass spectrometer for several rapid acting insulin analogues. The cyclic Ion mobility separation (IMS) mass spectrometer was able to separate two synthetic forms (aspart and lispro) of insulin from recombinant human insulin by ion mobility. For insulin aspart, the drift time separation complements the separation in m/z, and will give greater confidence in the identification of these variants in unknown samples. For insulin lispro, its shorter drift time profile distinguished it from the isobaric human sample. These peptides currently require retention time separation and MS/MS experiments to be identified. Both lispro and human insulin could be identified in a mixture of the two peptides, without the need for retention time separation in LCMS. Introduction Synthetic insulins represent prohibited substances in professional sport according to the list of the World Anti-Doping Agency. Due to their peptidic character and sharing a largely common amino acid sequence, their physico-chemical properties are very similar. Figure 1 shows the amino acid sequence of human insulin and the two rapid acting synthtic insulin analogs insulin lispro resp. insulin aspart in comparison. Noteworthy, recombinant human insulin and insulin lispro differ only in the amino acid sequence at position B28/29 and share the identical molecular weight. Obvioulsy, separation of these analogs represents a considerable challenge. Nevertheless, definite identification of these target peptides is crucial in doping controls as well as in forensics or related disciplines. 1 2 2 2 2 1 1 2 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-696 Figure 1. Structures of a) human insulin, b) insulin lispro and c) insulin aspart Experimental For infusion experiments, 2.1 µM insulin (human, aspart or lispro) in 33% MeOH were directly infused by means of a syringe. Waters SELECT SERIES Cyclic IMS (Manchester, UK) was used for all IMS experiments. Here the fivefold protonated precursor ions were monitored at a mass to charge ratio of m/z 1162 for human insulin and insulin lispro resp. m/z 1166 for insulin aspart. Six to nine passes on the cyclic ion mobility cell (Fig. 2) were performed in order to reach the ion mobility separation for the multiple charged precursors. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-697 Results and Discussion By means of cyclic ion mobility coupled to high resolution mass spectrometry all three insulin variants were separated even without any chomatography or tandem mass spectrometry. Figure 2 shows the separation for human insulin and insulin aspart, which are anyhow different due to their different molecular weights and their different precursor ions (m/z 1162 vs 1166). In case of human insulin and insulin lispro even the 1:1 mixture of these isobaric peptides are separated sufficiently (nearly baseline) within 9 passes (corr. to 28.55 ms) in the cyclic ion mobility cell. The synthetic insulin lispro shows a considerable shorter mobility profile compared to human insulin. The separation of the two variants is shown in Figure 3 and supports the possibility to distinguish the two isobaric peptides within this millisecond experiment. Figure 2. Ion mobility separation of human insulin and insulin aspart based on their fivefold protonated precursor ions. Separation time = 15.31 ms (6 passes) MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-698 Figure 3. Ion mobility separation of human insulin and insulin lispro as mixture (1:1) based on their fivefold protonated precursor ions. Separation time = 28.55 ms (9 passes) Noteworthy, in case of confirmatory analysis also chromatographic and tandem mass spectrometric data will provide additional information. Earlier studies using a non-cyclic (conventional TriWave ion mobility cell) showed only a slight separation of human insulin and insulin lispro (see Fig. 4 and Ref. [1]). Figure 4. Ion mobility separation (m/z vs drift time) of human insulin (2H10-labelled, used as ISTD) and insulin lispro in a plasma sample using a conventional (TriWave) ion mobility cell [1] MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-699 Conclusions Separation capability of cyclic ion mobility cell is significantly enhanced The cyclic IMS mass spectrometer was able to separate two synthetic forms (aspart and lispro) of insulin from recombinant human insulin by ion mobility. For insulin aspart, the drift time separation complements the separation in m/z, and will give greater confidence in the identification of these variants in unknown samples. For insulin lispro, its shorter drift time profile distinguished it from the isobaric human sample. These peptides currently require retention time separation and MS/MS experiments to be identified. Both lispro and human insulin could be identified in a mixture of the two peptides, without the need for retention time separation in LCMS. References 1. Thomas A, Schänzer W, Thevis M., Determination of human insulin and its analogues in human blood using liquid chromatography coupled to ion mobility mass spectrometry (LC-IM-MS). Drug Test Anal. 2014 Nov- Dec;6(11-12):1125-32. doi: 10.1002/dta.1710. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6100 Stacchini C , Botrè F , de la Torre X , Mazzarino M The individual longitudinal profile of IGF-1 in capillary blood: A new ABP parameter? Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Rome, Italia ; Chimica e Tecnologia del farmaco, Sapienza Università di Roma, Rome, Italia Abstract The insulin-like growth factor 1, IGF-1, is a peptide hormone with anabolic action, which is always prohibited, both in-competition and out-of-competition. The structure of exogenous and endogenous IGF‑1 is identical, and, to date, the WADA-accredited antidoping laboratories cannot report an adverse analytical finding for the illicit administration of IGF-1, since it is not possible to establish a reliable reporting threshold of its concentration, due to the significant differences between individuals. From literature data, many factors are known to alter the individual concentration of circulating IGF-1, including insulin levels, age, gender, stress status, body mass index, disease state, ethnicity, and finally the administration of xenobiotics. Here we are reporting a preliminary evaluation of the circadian fluctuations of IGF-1 to consider its inclusion in the endocrinological module of the athlete biological passport as a tool to detect its abuse. To this end, capillary blood samples were collected from healthy volunteers three times a day for 5 days. Samples were then analyzed by LC-MS using a bottom-up approach. Stability studies were performed to verify the stability of IGF-1 in whole blood samples at different temperatures: 4°C for up to 72 hours, to ensure sample integrity in the transfer and storage phases from sample collection to analysis, and the stability of IGF-1 in plasma at 4°C, -20°C and -80°C for up to 3 months to select the best storage conditions. Introduction Many factors alter the concentration of IGF-1, including insulin levels, age, gender, disease; individually, IGF-1 appears to be a stable hormone, whose levels seem constant in blood [1]. Furthermore, considering that IGF-1 is an indirect marker for the detection of GH doping, the analysis of the longitudinal profile of IGF-1 could be an instrument not only for the illicit detection of IGF-1 as such, but also of GH [2]. Our study aims to evaluate the circadian fluctuation of IGF-1, to consider the inclusion of its monitoring in the athlete’s biological passport (ABP) as a tool to detect its abuse. To this purpose, we have preliminarily evaluated individual profiles of subjects of different ages and gender, also with an endocrine disorder. Experimental Sample collection and sample pre-treatment Two hundred microliters of capillary blood samples were collected by finger pricking from male and 1,2 1 1 1 1 2 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6101 female volunteers, who gave written consent. The sampling involves the use of a personal lancing device (OneTouch UltraSoft ) with microneedles (Microlet ), and the blood drops were collected with Microvette CB300 (Lithium Heparin). Samples were stored at 4°C for no longer than 72 hours, then the plasma was separated by centrifugation and stored at -80°C until analysis. Four volunteers, 2 males (volunteers 1 and 2; 31 and 40 years old) and 2 females (volunteers 3 and 4: 28 and 31 years old; volunteer 4 with insulin-dependent type 1 diabetes), were recruited. For the definition of the circadian profile, samples were collected three times a day for five days. Following the method accredited in the WADA guidelines, samples are pre-treated and subjected to tryptic digestion overnight, then injected into the LC-MS system [3]. LC-MS conditions LC-MS system: Agilent Technologies 1200 Series; column: C18 (ID 2.1 mm, L. 5 cm, 5 µm); mobile phases: ultra-purified water (A) and acetonitrile (B), both containing 0.1% formic acid; gradient; flow rate: 300 µL/min; injection volume: 50 µL. API 4000 (AB Sciex) triple quadrupole; source: ESI positive; acquisition mode: selective reaction monitoring (SRM). Stability studies Stability studies were conducted to assess the optimal storage conditions by comparing different storage times (up to 90 days) and temperatures (4°C, -20°C, -80°C) (see Figure 1). Figure 1. Outline of stability studies: a) long-term stability, up to 90 days at -20°C and -80°C; b) "transfer" stability, up to 72 hours, at 4°C (WB = whole blood; P = plasma) Validation parameters The method, once optimized, was validated according to WADA's guidelines for Human Growth Hormone Biomarkers Test [4], which involves the detection of IGF-1 by LC-MS/MS using a bottom-up approach with the identification of two peptides (T1 AA 1-21; T2 AA 22-36). The parameters considered were specificity, linearity, sensitivity in terms of limit of detection (LOD), limit of identification (LOI) and limit of quantification (LOQ), repeatability (S ), intermediate precision (S ), accuracy, and carry-over were evaluated. ® TM ® ® r w MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6102 Results and Discussion Validation To assess specificity, as IGF-1 is an endogenous hormone, blank rat plasma samples were analyzed: no interferences were detected. Linearity was defined in the range between 50 to 1000 ng/mL, with a correlation coefficient R > 0.995 for the linear relationship of the calibration data. The values related to other validation parameters are reported in Table 1. As shown, the CV% is never higher than 15 for both the within-assay repeatability and the intermediate precision. The analysis of rat blank plasma samples after the analysis of rat plasma samples spiked at the highest concentration (1000 ng/mL), showed the absence of carry-over. The difference between IGF-1 T1 and IGF-1 T2 concentrations is acceptable according to WADA assay requirements [4]. Table 1. Validation parameters Stability studies results The stability of IGF-1 in the transportation phase was assessed by comparing the IGF-1 concentrations of the same pool of capillary blood stored under different conditions: (i) at the temperature of 4°C up to 72 h in the Microvette CB300 that are used for the sample collection; (ii) at a temperature of -80°C as plasma, centrifuged and separated immediately after collection, and stored in Eppendorf LoBind tube. The results are reported in Table 2: the CV% of the concentration values never exceeds 10 which indicates that the analyte of interest does not undergo degradation under the conditions studied. 2 ® MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6103 Table 2. Results of the storage and long-term stability studies IGF-1 circadian profiles The results obtained for the circadian profile of the four volunteers studied are reported in Figure 2. Being a preliminary study, the individual ranges were calculated as the interval between the mean of the concentrations plus and minus three times the standard deviation (mean ± 3SD). The variability of IGF-1 concentrations for each volunteer never exceeded 20%, confirming the possibility of its inclusion in the ABP monitoring as an effective tool for doping control. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6104 Figure 2. Circadian profile of volunteer 1 (male 31 y.o) (a); volunteer 2 (male 40 y.o) (b); volunteer 3 (female 28 y.o); (c), volunteer 4 (female 31 y.o, insulin-dependent type 1 diabetes) (d): mean and range of normality are reported for both peptides (n=number of analytical replicates) MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6105 Conclusions Preliminary results of the IGF-1 profiles show that it is possible to determine normal ranges of IGF-1, that remain constant, without significant fluctuations. Capillary blood collection is a minimally invasive, inexpensive technique that does not require specialized personnel. Samples can be collected at any time without affecting athletic performance. Stability studies have shown that both transport and storage phases are easy to manage: no significant differences in the IGF-1 concentration in the samples stored at 4°C up to 72 hours; plasma samples at -20°C and at -80°C are stable for up to 3 months. The validated method is specific, sensitive (LOD: 20 ng/mL; LOI: 25 ng/mL; LOQ: 50 ng/mL), linear (range 50-1000 ng/mL, R > 0.9950), and repeatable, with no carryover effects. References 1. Laron Z. (2001) Insulin-like growth factor 1 (IGF-1): a growth hormone, Molecular Pathology, 54(5): 311–316 2. World Anti-Doping Agency. The 2018 Prohibited List. 2021 www.wada-ama.org/en/resources/science- medicine/prohibited-list-documents (access date 21.08.21) 3. Cox HD, Lopes F, Woldemariam GA, Becker JO, Parkin JO, Thomas A, Butch AW, Cowan DA, Thevis M, Bowers LD, Hoofnagle AN. (2014) Interlaboratory Agreement of Insulin-like Growth Factor 1 Concentrations Measured Mass Spectrometry, Clinical Chemistry, 60:3: 41–548 4. World Anti-Doping Agency. Laboratory Guidelines - Human Growth Hormone (hGH) Biomarkers Test 2021 https://www.wada-ama.org/en/resources/laboratories/guidelines-human-growth-hormone-hgh-biomarkers- test (access date 21.08.21) 2 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6106 Martín-Navas P , Saavedra MJ , Cortés N , McPherson Medina A , Polo M , Fiallo Fernández T , Rodríguez G , Muñoz G , Montes de Oca Porto R A Fit-for-Purpose approach with the Cuban monoclonal antibody CBSSEPO for the ERAs analysis in urine samples Madrid Anti-Doping Laboratory, Madrid, Spain ; Havana Antidoping Laboratory, La Habana, Cuba Abstract A comparison between the monoclonal mouse anti-human EPO clone AE7A5 (R&D Systems MAB2871) and the new Cuban monoclonal antibody CBSSEPO (Center of Genetics Engineering and Biotechnology Sancti Spiritus - CIGB SS) is performed in urine samples as a Fit for Purpose approach for the analysis of ERAs. Several SAR-PAGE ERAs analyses in urine (single-blotting and double-blotting) are shown, using different internal quality controls as samples spiked with different types of ERAs at around 20 pg/mL: BRP EPO, Aranesp™, Mircera™, EPO-Fc (ProSpec) and Retacrit™. The use of the Cuban CBSSEPO monoclonal antibody for the analysis of ERAs in urine needs to be deeply studied in order to clarify a series of variables: spots, intense unspecific bands (globulins, EPO degradation fragments), high background and elevated time of exposure indicate that the sensitivity of the antibody needs to be improved to fulfill with the TD2021EPO and its MRPL for the different types of ERAs. Apparently the Cuban CBSSEPO antibody could be use with single-blotting and double-blotting, as in both cases there are some results that could be characterized. However, the practice in this approach indicates that their use with single-blotting could be highly convenient, as the results are clearly more satisfactory. The Cuban CBSSEPO antibody binds with more affinity to the Goat Anti-Mouse IgG-HRP 2 antibody than to the Goat Anti-Mouse IgG-Biotin 2 antibody. For this reason, a good point could be the use of the Cuban CBSSEPO as an alternative to the primary monoclonal antibody clone AE7A5 for the initial testing procedure of ERAS (after further evaluation), although the confirmation procedure would require the use of clone AE7A5. Introduction In order to improve the sensitivity and specificity of the EPO-receptor agonists (ERAs) analysis, the development of new primary monoclonal antibodies is a continuous target in the antidoping field. The search of other options to the clone AE7A5 is an interesting aim for the upcoming future. A comparison between the monoclonal mouse anti-human EPO clone AE7A5 (R&D Systems MAB2871) and the new Cuban monoclonal antibody CBSSEPO (Center of Genetics Engineering and Biotechnology Sancti Spiritus - CIGB SS) is performed in urine samples as a Fit for Purpose approach for the analysis of ERAs. Several SAR-PAGE ERAs analyses in urine (single-blotting and double-blotting) are shown, using different internal quality controls as samples spiked with different types of ERAs at around 20 pg/mL: BRP EPO, Aranesp™, Mircera™, EPO-Fc (ProSpec) and Retacrit™. The ERA-MIX standards are DYN-NESP- CERA 0.1% insulin/PBS. Some EPO-Fc, EPO-BRP and DYN+NESP standards are used as well. 1 1 1 2 1 2 1 1 2 1 2 nd nd MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6107 The analysis were carried out in the DCL of Madrid (Spain) and in the Antidoping Laboratory of La Habana (Cuba). The CBSSEPO Cuban monoclonal antibody was supplied by the Antidoping Laboratory of La Habana (Cuba). Figure 1: Cuban monoclonal antibody CBSSEPO (AcM CBSSEPO.1) from the Center of Genetics Engineering and Biotechnology Sancti Spiritus - CICGB SS (La Habana, Cuba) Experimental The steps of the procedure is shown in the following flow diagram (Fig. 2): Figure 2: Steps of the procedure To sum up, the different steps are: - Concentration / Immunopurification (ELISA Stem Cell) - SAR PAGE (125V / 25W / Consntant voltage / 3h / Cold bath - Immunoblotting (Single blotting + Blot Cycler or Double Blotting) - Chemiluminiscence detection (FEMTO reagent) MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6108 Results and Discussion Figure 3: Single blottingg and BlotCycler Both gels (Figures 3A and 3B) have the same configuration and were handled at the same time. (Clone AE7A5 versus CBSSEPO). ERA-MIX (DYN+NESP+CERA) standards are shown in lanes 1, 2, 9, 10 and 11. A standard of EPO-Fc is shown in lane 12. The configuration of the internal quality controls is as follow: QCN (lane 3), QCP BRP (lane 4), QCP NESP (Aranesp™, lane 5), QCP CERA (Mircera™, lane 6), QCP EPO-Fc (ProSpec, lane 7) and QCP ZETA (Retacrit™, lane 8). Lane 8 (Retacrit™) is shown too as individual and circled in the GASepo global image (Figure 3C). Antibody Goat Anti-Mouse IgG-HRP was used as 2 antibody. It provides high sensitivity through signal amplification as can bind to both 1st monoclonal antibodies (Clone AE7A5 and CBSSEPO). Both gels show a matrix effect in the QCPs corresponding to CERA and EPO-Fc. These bands are slightly more scrolled up in the QCPs than in the ERA-MIX. Background is more intense in the gel treated with CBSSEPO than in the gel treated with clone AE7A5. Some spots due to non-specific bindings are shown too in this gel. A band due to an urinary excretion protein appears in the gel treated with CBSSEPO. This band is shown with an arrow below the endogenous band in all the samples. Different urinary globulins such as α -1-microglobulin or β-2- microglobulin have a molecular weight under 30kDa. The degradation products of the endogenous EPO also have a molecular weight under 30 kDa. The time of exposure for the acquisition of the image was significantly different for each antibody: 0,5 seg / Auto Mode for Clone AE7A5 and 8 seg / Auto Mode for CBSSEPO. Background and spots in the CBSSEPO gel, as well as the high intensity of the urinary globulins, make faint bands for the samples in the image acquisition. Nonetheless, all the QCN and QCPs are observed. QCP Zeta (Retacrit™, lane 8, circled) is shown too as an individual picture (Figure 3C) as it is slightly fainter than the others bands. All the standards are observed too. nd MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6109 Figure 4. Double blotting Gels from Figures 4A and 4B have the same configuration and were handled at the same time. (Clone AE7A5 versus CBSSEPO). ERA-MIX (DYN+NESP+CERA) standards are shown in lanes 1, 2, 9, 10 and 11. A standard of EPO-Fc is shown in lane 12. The configuration of the internal quality controls in Figures 4A and 4B is as follow : QCN (lane 3), QCP BRP (lane 4), QCP NESP (Aranesp™, lane 5), QCP CERA (Mircera™, lane 6), QCP EPO-Fc (ProSpec, lane 7) and QCP ZETA (Retacrit™, lane 8). Antibody Goat Anti- Mouse IgG-Biotin was used always (Madrid and La Habana) as 2 antibody. An incubation with HRP conjugated-Streptavidin is required in order to provide a signal (enzimatic activity) for the chemiluminsicent reagent. The time of exposure for the acquisition of the image was significantly different for each antibody: 2,1 seg / Auto Mode for Clone AE7A5 and 11,2 seg / Auto Mode for CBSSEPO. There is no background but none of the samples are observed in the CBSSEPO gel (Figure 4B). Some unespecific bands are shown in the ERA-MIX area (circled) and in the samples area (arrows), but the characterization and identification is no possible. Figures 4C and 4D show the same gel. It was incubated first with the Cuban monoclonal antibody CBSSEPO (Figure 4C, left) and, after a washing treatment with PBS, the PVDF membrane was incubated with the monoclonal mouse anti-human EPO clone AE7A5 (Figure 4D, right). The configuration of the nd MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6110 internal quality controls in Figures 4C and 4D is as follow: QCN (lanes 2, 4, 6, 9 and 11), QCP BRP (lanes 3 and 5). EPO-BRP (standard) is shown in lanes 1 and 12. DYN+NESP standard is shown in lane 10. A big background and some unspecific bands (circled) are shown in Figure 4C. No characterization of samples is possible after treatment with the Cuban monoclonal antibody CBSSEPO. However, some defined bands (arrows) appear in the lanes of the standards. BRP, DYN and NESP could be somehow identified. Conclusions The use of the Cuban CBSSEPO monoclonal antibody for the analysis of ERAs in urine needs to be deeply studied in order to clarify a series of variables: Spots, intense unspecific bands (globulins, EPO degradation fragments), high background and elevated time of exposure indicate that the sensitivity of the antibody needs to be improved to fulfill with the TD2021EPO and its MRPL for the different types of ERAs. Apparently the Cuban CBSSEPO antibody could be use with single-blotting and double-blotting, as in both cases there are some results that could be characterized. However, the practice in this approach indicates that their use with single-blotting could be highly convenient, as the results are clearly more satisfactory. The Cuban CBSSEPO antibody binds with more affinity to the Goat Anti-Mouse IgG-HRP 2nd antibody than to the Goat Anti-Mouse IgG-Biotin 2nd antibody, though the robustness in double-blotting should be further evaluated. For this reason, a good point could be the use of the Cuban CBSSEPO as an alternative to the primary monoclonal antibody clone AE7A5 for the initial testing procedure of ERAs (more studies will be necessary), although the confirmation procedure would require the use of clone AE7A5. References Reichel C, Gmeiner G, Reihlen P, Thevis M, Schänzer W. SARCOSYL-PAGE: Optimized Protocols for the Separation and Immunological Detection of PEGylated Proteins. Methods Mol Biol. 2019; 1855:131-149. doi: 10.1007/978-1-4939-8793-1_14. PMID: 30426415. Reichel C. Detection of peptidic erythropoiesis-stimulating agents in sport. Br J Sports Med. 2014 May; 48(10):842-7. doi: 10.1136/bjsports-2014-093555. Epub 2014 Mar 27. PMID: 24677025. Acknowledgements We would like to thank to the Center of Genetics Engineering and Biotechnology Sancti Spiritus from La Habana, Cuba, for the supply of the monoclonal antibody and to the people of the Antidoping Laboratory of La Habana (Cuba) for their kindly willingness to collaborate in this work. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6111 Biasini GM, de La Torre X, Botrè F, Donati F Detection of human Peroxiredoxin-2 in stored erythrocytes: potential biomarker of Autologous Blood Transfusions in doping control Laboratorio Antidoping, Federazione Medico Sportiva Italiana (FMSI) - Antidoping Laboratory, Roma, Italia Abstract Despite being banned by the World Anti-Doping Agency (WADA), autologous blood transfusions (ABT) may be attempted by athletes to enhance their physical performance. Currently, a direct detection method for ABT has not yet been developed. A possible detection strategy might be based on the morphological and biochemical changes of the red blood cells (RBCs) during the storage period in blood bags. The increased oxidative stress, due to storage, causes severe damages to erythrocytes and compromises their survival. The thiol protein Peroxiredoxin-2 (PRDX2) is the major RBCs antioxidant, responsible for the degradation of reactive oxygen species (ROS). Previous studies from our research group demonstrated that PRDX2 levels in RBCs membrane increase during storage, due to migration, accumulation phenomena and increased oxidative stress. The aim of the present study was to evaluate whether PRDX2 levels change in different RBCs subpopulations. PRDX2 levels were analyzed in the three fractions and aged erythrocytes showed higher levels of PRDX2 compared to younger ones, since its migration to the membrane is an age and stress-related phenomenon. Higher PRDX2 levels in stored blood samples may depend on the augmented population of aged-RBCs over storage time. Therefore, PRDX2 could be a promising biomarker for the direct detection of ABT misuse for doping purpose. Moreover, we analyzed PRDX2 levels in dried blood spots (DBS) compared to whole blood samples, demonstrating that DBS is a suitable matrix for PRDX2 analysis, although with lower recovery than whole blood samples. Introduction One of the greatest challenges for anti-doping authorities is to develop a direct method to detect the illicit recourse to ABT [1] to improve physical performances. During storage, many biochemical and physiological alterations occur to RBCs: loss of their characteristic shape, membrane damages, premature ageing and increased oxidative stress [2,3]. These alterations affect also protein structures and functions. PRDX2, the main erythrocytes antioxidant, migrates to the membrane and accumulates in its oxidized form, losing its ability to reduce ROS [4]. Our aim is to analyze PRDX2 levels in blood fractions, to evaluate its suitability as a biomarker of storage. Moreover, since PRDX2 seems a promising biomarker for the direct detection of ABT, we explored the feasibility of its analysis in DBS matrix. Experimental Isolation of RBCs fractions and sample preparation Whole blood samples (1 mL) from 6 individuals were purified from plasma and leukocytes. RBCs were MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6112 washed twice with a solution 8 mM HEPES (ThermoFisher Scientific, USA) of RPMI 1640 Medium (EuroClone SPA, Italy) and resuspended at 25% hematocrit. RBCs fractions were isolated through centrifugation 1075 g for 20 minutes over a discontinuous Percoll (Sigma-Aldrich, USA) gradient (density varying from 1.117/1.052 g/mL) [5,6]. This provides three fractions: the lightest-top-young-RBCs, the densest-old-RBCs and a middle fractions populated by middle-aged-RBCs [7]. To obtain ghost- membranes, isolated RBCs fractions (n = 18) were washed, resuspended in N-ethylmaleimide (NEM) 0.1M in PB solution and incubated for 15 minutes at RT. After incubation, a solution of 0.1M of NEM in PB/2% cOmplete Protease Inhibitor Cocktail (Roche, Switzerland) was added to samples, and cells were lysed mechanically through a homogenizer. Protein concentration from ghost-membrane was estimated through Bradford assay. DBS protein extraction Blood samples (20 μL) were spotted on Whatman FTA DMPK-C cards (Sigma-Aldrich, USA) and let dry for 24 hours. The full spot was excised using an 8 mm punch and quartered with a razor blade. Spots were transferred into 11 mm diameter well and washed for 1 hour with 200 μL of phosphate-buffered saline (PBS). Each eluted blood sample underwent sample preparation as described above and were analyzed through SDS-PAGE and immunoblotting assay. SDS-PAGE and Western Blotting Denatured protein samples (n = 22) were analyzed by SDS-PAGE. 5 µg of each sample were loaded in the gel (NuPAGE 4-12% Bis-Tris Gel). PRDX2 standard was loaded as control. The western blot analyses were performed on PVDF membranes with primary antibody against PRDX2 (Anti-h/m/r Peroxiredoxin2 at 1/1600). Membranes were incubated with secondary antibody (Gt-anti-MS IgG (H+L) cross-adsorbed HRP- conjugate) for 90 minutes. Images were acquired through the ImageQuant LAS4000. Bands of interest were quantified by densitometry through GASepo software. Results and Discussion Figure 1 shows the separation of the three fractions after centrifugation over Percoll gradient. At the top there is a small fraction of low density and younger eryhrocytes, at the bottom there is a more consistent layer of high density and aged eryhrocytes, while in the middle there is a layer made by middle-aged RBCs. ® ® ® ® MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6113 Figure 1. Representative image of the layers obtained through centrifugation of blood, purified from plasma and leukocytes, over a discontinuous Percoll gradient. The thin layer on the top consists of low density and youn RBCs, the layer on the bottom contains high density and aged cells, while the thick layer in the middle is the most aboundant fractions, middle density and middle aged RBCs. Our results demonstrated that levels of PRDX2 in ghost-membranes are significatively higher in the aged RBCs compared to the youger ones (p = 0.05) (Figure 2a). This result can be explained with the effects of the increased oxidative stress, that occurs with ageing, on the protein PRDX2. Due to the increased oxidative stress PRDX2 undergo to phenomena of accumulation and migrates from the cytosol to the membrane, losing its ability to reduce ROS species. On the contrary no changes have been reported on Band 3 levels, the major intermembrane protein of erythrocytes, used as a control protein in the three RBCs fractions (Figure 2b). The difference between protein abundance among the three RBCs fractions is even more clear in the ratio between the analyzed proteins (Figure 2c). Figure 2. (a) Levels of Band 3, the main intermembrane protein of RBCs, used as a control, do not change between erythrocytes fractions. (b) Levels of PRDX2 are significantly higher in aged RBCs fraction membranes compared to the younger ones (p=0.05). (c) Ratio between Band3/PRDX2 in the three RBCs fraction. ® MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6114 Moreover, it has been evaluated the suitability of DBS matrix for the analysis of PRDX2. This matrix has many advantages such as the easier collection, storage shipping, the small volume of blood (20 µL) used and there is no need for a phlebotomist [8]. Our data demonstrated that DBS are a suitable matrix for PRDX2 analysis, despite the protein recovery is lower compared to whole blood samples (Figure 3). Figure 3. (a) Representative PRDX2 bands obtained from western blot assay from DBS or whole blood matrix, compared with the standard (25 ng). (b) Istograms representing levels of PRDX2 obtained from DBS or whole blood of the same individual. In both cases with DBS the recovery is less compared to whole blood samples. Conclusions From the data we obtained the following conclusions can be drawn: 1. PRDX2 levels are significantly higher in aged erythrocytes membranes and it can be considered a suitable biomarker of ageing. 2. The analysis of the alteration of Band3/PRDX2 ratio rather than changes of only one protein, can strongly improve the detection of stored and aged RBC. The aged RBCs fraction may play a key role for the detection of transfused blood samples to develop a direct method to detect ABT. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6115 3. PRDX2 levels in RBCs fractions will be evaluated in each fraction after different periods of storage. 4. Despite the impossibility to fractionate RBCs subpopulation, DBS it is a suitable matrix for PRDX2 analysis. References 1. The World Anti-Doping Agency. The Prohibited List 2020 International Standard. WADA, Montreal, 2019. 2. Mustafa I, Al Marwani A, Mamdouh Nasr K, Abdulla Kano N, Hadwan T. (2016) Time Dependent Assessment of Morphological Changes: Leukodepleted Packed Red Blood Cells Stored in SAGM. Biomed Res Int. 2016:4529434. 3. Putter JS, Seghatchian J. (2017) Cumulative erythrocyte damage in blood storage and relevance to massive transfusions: selective insights into serial morphological and biochemical findings. Blood Transfus. 15(4):348-356. 4. Marrocco C, Pallotta V, D'alessandro A, Alves G, Zolla L. (2012) Red blood cell populations and membrane levels of peroxiredoxin 2 as candidate biomarkers to reveal blood doping. Blood Transfus. 10 Suppl 2:s71-7. 5. Risso A, Mansutti E, Mehrishi JN. (2015) Decreased expression of CD47 and CD55 surface molecules on density-based subsets of red cells of β-thalassaemia intermedia patients compared to red cells of healthy blood donors. J Biol Phys Chem. 15(2): 45-53. 6. Rennie CM, Thompson S, Parker AC, Maddy A. (1979) Human erythrocyte fraction in "Percoll" density gradients. Clin Chim Acta. 98(1-2):119-25. 7. Huang YX, Wu ZJ, Mehrishi J, Huang BT, Chen XY, Zheng XJ, Liu WJ, Luo M. (2011) Human red blood cell aging: correlative changes in surface charge and cell properties. J Cell Mol Med. 15(12):2634-42. 8. Björkesten J, Enroth S, Shen Q, Wik L, Hougaard DM, Cohen AS, Sörensen L, Giedraitis V, Ingelsson M, Larsson A, Kamali-Moghaddam M, Landegren U. (2017) Stability of Proteins in Dried Blood Spot Biobanks. Mol Cell Proteomics. 16(7):1286-1296. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6116 Akiyama K, Kageyama S, Okano M DNA analysis of dried blood spots and urine for doping control purposes Anti-Doping Laboratory, LSI Medience Corporation, Tokyo, Japan Abstract Sample substitution, which is the process of substituting an athlete's urine with that of someone else, is prohibited in doping control. Some athletes have even resorted to injecting the urine from someone else directly into their own bladder. Therefore, identification by DNA analysis (STR analysis: short tandem repeat) is recommended to prove that a collected urine sample belongs to the same athlete. In sports drug testing, urine is primarily collected; however, the use of dried blood spots (DBS) is beginning to be implemented in terms of low invasiveness, strong preservation stability, and less storage space. In this study, we developed a DNA analysis method using DBS for the purpose of establishing a DNA analysis system to detect unfair manipulation, including sample substitution. Moreover, we confirmed the authenticity of a urine sample using DBS as reference. In DBS samples collected from fingertip blood collection on the DMPK-C card, we used a direct amplification method without DNA extraction in GlobalFiler PCR Amplification Kit. A 1.2 mm small punch disc of the DBS card with the Prep-N-Go™ lysis buffer can be placed directly into an amplification tube, purified, and amplified. The PCR reagents were prepared as per the GlobalFiler User Guide and added to the 96 well plate wells containing samples giving a final reaction volume of 25 μL. In urine sample (2 mL), DNA was extracted and purified using the PrepFiler Forensic DNA Extraction Kit. DNA amplifications were carried out with the GlobalFiler PCR Amplification Kit similarly to DBS. In DBS samples (n = 20), all alleles were detected in all the samples. The heterozygote peak height ratio, which is an index of DNA sample suitability for the PCR conditions, were 68 - 92%. The long-term stability of DNA was also investigated using the DBS samples stored at 4°C for three years after sample collection. No deletion of the alleles was observed in samples stored for three years after collection, and identification was possible in all samples. In the identification of urine samples (n = 20), allele deficiency was observed in 50% of the urine samples stored for three years after collection, and allele deficiency was found in > 250 bps. The heterozygote peak height ratios in the urine samples were 66 - 87%. Although allele deficiency was observed, identification of urine sample using DBS as reference were possible in all samples. This study shows that the developed DNA analysis system could be utilized for individual identification using DBS and urine samples. It is considered that individual identification across sample types was useful to detect unfair manipulation, including sample substitution. Introduction Identification by DNA analysis is recommended to prove that a sample belongs to the same athlete [1]. In doping testing, DNA analysis using urine is beginning to be implemented [2]. However, it is very difficult to store all collected urine specimens for long periods. Also, urine samples have a small number MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6117 of cells and low DNA yields. Alternatively, DBS sample is of low invasiveness, strong preservation stability, requires less storage space, and has high DNA yields. Therefore, DBS is considered an appropriate sample to solve the problem. We studied the development of a DNA analysis method using DBS for the purpose of establishing a DNA analysis system to detect unfair manipulation, including sample substitution. Moreover, we conducted identification of urine sample using DBS as reference. Experimental Human study A total of 20 volunteers (9 females and 11 males) participated, and the study was performed in accordance with the ethical guidelines approved by LSI Medience. Capillary blood sampling using a lancet was performed, and samples collected from the fingertip were spotted on FTA DMPK-C cards (GE Healthcare) immediately. The card was stored at 4°C until analysis. Spot urine was collected and stored at -20°C until analysis. Analytical method In the DBS sample, a direct amplification method was utilized without DNA extraction using the GlobalFiler PCR Amplification Kit. A 1.2 mm small punch disc of the DBS card with the Prep-N-Go™ lysis buffer can be placed directly into an amplification tube, purified, and amplified. The PCR reagents were prepared as per the GlobalFiler User Guide and added to the 96 well plate wells containing samples giving a final reaction volume of 25 μL. Urine samples (2 mL) were centrifuged at 2,240 g for 30 min and the supernatant was removed. DNA was extracted and purified using the PrepFiler Forensic DNA Extraction Kit. Each DNA extract was subjected to real-time DNA quantification for amplification of the human specific DNA sequence D17Z1 (GenBank accession M13882), based on previously published protocols [3]. PCR amplification was carried out as per the GlobalFiler User Guide but the cycle numbers were adjusted based on the sample type. Instead of 29 cycles, 27 cycles were used in the DBS sample, and 30 cycles were used in the urine sample. PCR products were prepared by adding 1 μL amplified product to 9.6 μL Hi-Di™ Formamide (Life Technologies) and 0.4 μL GeneScan 600 LIZ Size Standard v2.0 (Life Technologies). Capillary electro- phoresis and detection of amplicons was performed in a 3500 Genetic Analyzer using POP-4 (Life Technologies), 36-cm capillary, 1.2 kV injection voltage, and 24 s injection time. Results and Discussion DBS identification In DBS samples (n = 20), all alleles were detected in all the samples (Figure 1A and 1B). The peak height ratios are to be used as a measure of how well both alleles at a locus amplify during the PCR. The heterozygotes peak height ratio, which is an index of DNA samples suitable for PCR conditions, were 68 - 92% (Table 1). The long-term stability of DNA analysis was also investigated using the DBS samples stored at 4°C for three years after collection. No deletion of the allele was observed in samples stored for three years after collection, and identification was possible in all samples. Thus, DBS samples are considered to be effective for DNA analysis. DNA analysis was possible with a small amount of blood. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6118 Figure1. Identification profiles and noise detection rate. Percentage of full profiles (A) and noise detection rate (B) obtained from DBS and urine samples. Table 1. Heterozygote peak height ratio obtained from DBS and urine samples. The PCR amplification was performed from DBS and urine. The analysis of the 3500 run data used a GeneMapper ID-X 1.6 software. The peak height of the lower peak was divided by that of the higher peak. N: The number of heterozyous samples at a given locus. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6119 Individual identification across sample types (urine sample vs DBS as reference) In the identification of urine samples (n = 20), the amount of DNA ranged from 2.5 pg to 1,980 pg. Some factors (such as sex, the time of day, number of urinations, etc.) could all contribute to the large variation in quantity of DNA observed [3]. In DNA analysis, full profiles were obtained for 50% of the urine samples (Figure 1A and 1B). Allele deficiency was observed in 50% of the urine samples stored for three years after collection, and allele deficiency was found in > 250 bps (Figure 2). Use of the GlobalFiler PCR Amplification Kit ensures that even partial profiles may provide a high power of discrimination. The heterozygote peak height ratios in the urine samples were 66–87%. Although allele deficiency was observed, identification of urine sample using DBS as reference were possible in all samples. These results suggested that the method retains a high accuracy and reliability when applied to actual athlete samples tested in an anti-doping laboratory. Figure 2. Illustration of the allele deficiency was found in > 250 bps in urine samples stored for three years after collection. Electropherograms obtained from urine samples stored at -20°C for three years after collection and reference sample (DBS). Arrowheads indicate allele deficiency. Conclusions We established a DNA analysis system using DBS to detect unfair manipulation, including sample substitution. DBS samples are considered to be effective for the long-term stability of DNA analysis, and suitable as reference samples. The results of individual identification across sample types (Urine sample vs DBS as reference) showed that the developed DNA analysis system could be utilized for individual identification of DBS and urine samples. It is considered that individual identification across sample types was useful to detect unfair manipulation, including sample substitution for doping control purposes. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6120 References 1. Nicolas J, Francois M, Natalie S, Matt S, Alain L, Vincent C, Patrice M. (2011) Use of forensic investigations in anti-doping. Forensic Sci Int., 213(1-3), 109-113 2. Devesse, L., Court, D. S., Cowan, D. (2015) Determining the authenticity of athlete urine in doping control by DNA analysis. Drug Testing Anal. 7(10), 912-918 3. Tetsushi K, Koji F, Hiroaki N, Natsuko M, Kentaro K, Naoto Y, Kazumasa S. (2013) Estimation of the detection rate in STR analysis by determining the DNA degradation ratio using quantitative PCR. Legal Medicine, 15(1), 1-6 Acknowledgements We would like to thank the Corporation Shinanokai Shinanozaka Clinic for providing blood and urine specimens. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6121 Krug O, Geyer H, Thomas A, Walpurgis K, Piper T, Thevis M Black market products suspected to contain doping relevant ingredients - report for 2019-2020 Institute of Biochemistry, German Sport University, Cologne, Germany Abstract The black market for performance enhancing drugs, including original pharmaceuticals as well as faked products, is a common source for recreational/mass sport athletes. The analysis of confiscated products and products from test purchases is an essential part of monitoring the black market regarding developments to novel performance enhancing drugs. The European Monitoring Center for Emerging Doping Agents (EuMoCEDA) analyzed a total of 73 products qualitatively and quantitatively throughout 2019 and 2020. Another strategy of EuMoCEDA is the collection of information via internet research to monitor developments of the black market of suspected performance enhancing products. Anabolic agents, stimulants, growth factors, natural and synthetic insulins, IGF-1 and synthetic analogs as well as growth hormone releasing factors were determined by high performance liquid chromatography/high resolution mass spectrometry (HPLC-HRMS)-experiments in full-scan mode. For gas chromatography/(high resolution) mass spectrometry (GC-(HR)MS) – experiments, analytes were derivatized and measured in full-scan mode. Included substances were anabolic agents, stimulants, beta- 2-agonists and narcotics. For the analysis of peptides and proteins, aliquots were separated by polyacrylamide gel electrophoresis and subsequently stained with coomassie blue. By bottom-up proteomic approaches including tryptic digestion and nano liquid chromatography/tandem high resolution mass spectrometry, proteinogenic ingredients were identified. Analytes included, but were not limited to human growth hormone (hGH), growth factors (e.g.: FGF, MGF, etc.), various erythropoietins (EPO), and growth hormone releasing factors. During 2020, an internet research was conducted by monitoring trading platforms for putative doping- relevant products. The systematic procedure was designed as google search in English and German as well. The keywords “Anabolic agents, Anabolika, SARMs, HIF-stabilizers, HIF-Stabilisatoren, stimulants, Stimulanzien, peptide hormones, Peptid Hormone” in combination with „supplier, buy, Anbieter, kaufen“ led to numerous online trading platforms. The product lines of 19 platforms were monitored and analyzed concerning substance classes of WADA´s Prohibited List. Additionally, test purchases were conducted. In 2019 and 2020, a total of 73 suspicious (illicit) black market products were analyzed at the Center for Preventive Doping Research. Doping-relevant findings accounted in 78 cases for 32 different drugs (multi-findings included), from which 50% of the ingredients were not or falsely declared. 77% of the identified doping relevant compounds accounted for anabolic agents (predominantly testosterone esters); 17% accounted for hormone and metabolic modulators, and 1% related to beta-2-agonists, and 3% related to peptide hormones, growth factors, related substances, and stimulants, respectively. The internet research showed that 19 online trader offered products with 97 doping-relevant drugs. Test purchases confirmed the desolate quality of black market products and hence the health risk for recreational/mass sports athletes. The apportionment concerning substance classes partially reflects MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6122 analytical results of confiscated black market products. The overrepresentation of stimulants may be derived from a wide product range of new psychoactive substances (NPS) as designer drugs. Introduction The source of performance enhancing drugs for recreational/mass sport athletes is still the black market [1-3]. The analysis of confiscated products, as well as products from test purchases is an important and integral part of monitoring the black market regarding developments to novel performance enhancing drugs. The European Monitoring Center for Emerging Doping Agents (EuMoCEDA) analyzed a total of 73 products qualitatively and quantitatively throughout 2019 and 2020. Another aproach of EuMoCEDA includes the collection of information via internet research to monitor developments of the black market of suspected performance enhancing products. Experimental Depending on the formulation (oily solution, lyophilized, etc.), samples were solved or extracted with water, acetic acid (2% aq.), and/or acetonitrile (50:50 v/v) and subsequently diluted to yield an adequate concentration of labeled drug content. For gas chromatography, extracted and afterwards dried samples were reconstituted in ethyl acetate, derivatized with N-methyl-N-(trimethylsilyl)-trifluoro-acetamide (MSTFA), or a mixture of MSTFA/ethanethiol and ammonium iodide, respectively [1]. The samples were screened by HPLC-ESI-MS using an Accela 1250 series HPLC interfaced via electrospray to a Thermo Scientific TSQ Vantage system. For HRMS experiments a Thermo Q-Exactive plus, a Thermo Exploris, and an Agilent 6550 iFunnel Q-TOF mass spectrometer were used. GC-MS experiments were performed on a Trace 1310 Gas Chromatograph in combination with a TSQ 8000 Evo Triple Quadrupole Mass Spectrometer from Thermo. To screen the most common target analytes in black market products, high performance liquid chromatography/mass spectrometry (HPLC-MS) experiments were conducted in single-reaction- monitoring (SRM) mode. Anabolic agents, stimulants, growth factors, natural and synthetic insulins, IGF-1 and synthetic analogs as well as growth hormone releasing factors could be determined by high performance liquid chromatography/high resolution mass spectrometry (HPLC-HRMS)- experiments in full-scan mode. Qualification and quantification of analytes were obtained by conducting product-ion scans with substance specific fragmentation pathways. For gas chromatography/mass spectrometry (GC-MS) experiments, analytes were derivatized and measured in full-scan mode. Qualitative and quantitative analysis were accomplished by using reference substances and/or reference databases. Included substances were anabolic agents, stimulants, beta-2-agonists and narcotics [1]. For the analysis of peptides and proteins, aliquots were separated by polyacrylamide gel electrophoresis and subsequently stained with coomassie blue. By bottom-up proteomic approaches including tryptic digestion and nano liquid chromatography/ tandem high resolution mass spectrometry, proteinogenic ingredients were identified. Analytes included, but were not limited to human growth hormone (hGH), growth factors (e.g.: FGF, MGF, etc.), various erythropoietins (EPO), and growth hormone releasing factors [4]. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6123 Internet research During 2020 an internet research was conducted by monitoring trading platforms for putative doping- relevant products. The systematic procedure was designed as google search in Englich and German as well. The keywords “Anabolic agents, Anabolika SARMs, HIF-stabilizers, HIF-Stabilisatoren, stimulants, Stimulanzien, peptide hormones, Peptid Hormone” in combination with „supplier, buy, Anbieter, kaufen“ led to numerous online trading platforms. The product line of 19 platforms were monitored and analyzed concerning substance classes of WADA´s Prohibited List. Additionally test purchases were conducted. Results and Discussion In 2019 and 2020, a total of 73 suspicious (illicit) black market products were analyzed at the Center for Preventive Doping Research. Doping-relevant findings accounted in 78 cases for 32 different drugs (multi-findings included), from which 50% of the ingredients were not or falsely declared (Tab. 1). As shown in Fig. 2, 77% of the identified doping relevant compounds accounted for anabolic agents (predominantly testosterone esters); 17% accounted for hormone and metabolic modulators, and 1% related to beta-2-agonists, and 3% related to peptide hormones, growth factors, related substances, and stimulants, respectively. The analytes, which were currently not doping relevant, were stimulating, dermatologic, and virilizing agents as well as amino acids, fatty acids, and vitamins. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6124 Table 1. Identified drugs in black market products MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6125 Figure 1. Apportionment of identified doping relevant drugs in analyzed black market products 2019-2020 The internet research showed that 19 online trader offered products with 97 doping-relevant drugs. Test purchases confirmed the desolate quality of black market products and hence the health risk for recreational/mass sports athletes. The Apportionment concernig substance classes reflects analytical results of confiscated black market products partially. The overrepresentation of stimulants may be derived from a wide product range of NPS as designer drugs. Figure 2. Apportionment of doping-relevant agents in product lines of 19 online trading platforms Conclusions The athletes of recreational/mass sport risk their health by misusing black market products. Faked and falsely labeled preparations represent a particular problem. Anabolic agents and hormones are still the most popular products to improve body shape. The Cologne Anti-Doping Laboratory´s commitment under the umbrella of EuMoCEDA, yielded the analysis of traded drugs, as well as the compilation of informations concerning availability, handling and forms of misuse of black market products. This shows again the requirement and relevance of monitoring the black market and the investigation of distributed products under the umbrella of EuMoCEDA. References 1. O. Krug, A. Thomas, K. Walpurgis, T. Piper, G. Sigmund, W. Schänzer, T. Laußmann, M. Thevis: Identification of black market products and potential doping agents in Germany 2010-2013 (2014) Eur J Clin Pharmacol, Vol. 70, 1303-1311 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6126 2. C. Weber, O. Krug, M. Kamber, M. Thevis: Qualitative and Semiquantitative Analysis of Doping Products Seized at the Swiss Border. (2017) Substance Use & Misuse , Vol 52, (6) 742-753 3. C. Weber, M. Kamber, V. Lentillon-Kaestner: Are doping substances imported into Switzerland mainly to increase athletic performance? (2016) Performance Enhancement & Health. Vol 5(2), 66–76. 4. M. Thevis, Y. Schrader, A. Thomas, G. Sigmund, H. Geyer, W. Schänzer: Analysis of Confiscated Black Market Drugs Using Chromatographic and Mass Spectrometric Approaches (2008) J Anal Toxicol, Vol. 32, 232 Acknowledgements The authors want to thank the Manfred Donike Institute, and the federal ministery of the Interior for financal support, and the collegues at the Institut of Biochemestry for collaboration. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6127 Mareck U, Fußhöller G, Haenelt N, Thevis M Risk of unintentional antidoping rule violations by consumption of hemp products Center for Preventive Doping Research / Institute of Biochemistry, German Sport University, Cologne, Germany Abstract Hemp products receive a continuously growing consumer and market attention, and an expanding scope of applications is recognized, supported by suppliers operating through different distribution channels with the Internet being a major retail platform. Hemp products are prepared from cannabis plants and, therefore, might contain a variety of different natural cannabinoids. According to the regulations of the World Anti-Doping Agency (WADA), all natural and synthetic cannabinoids are prohibited in-competition, with the explicit exemption of cannabidiol (CBD). Based on these facts the objective of the study was to investigate if the consumption of hemp products can lead to unintentional violations of anti-doping regulations by the ingestion and subsequent excretion of natural cannabinoids. Eight different commercially available hemp products such as beer, tea, oil, cookies and marzipan were included in the investigation. Following approval by the local ethics committee, controlled single dose administration studies were conducted to probe for the presence of cannabinoids in urine samples collected after consumption of the hemp products. Variable patterns of cannabinoids or their metabolites were observed in the urine samples. In 5 of 8 individuals (63%), urine samples collected 8 hours after consumption yielded findings of a prohibited cannabinoid that would have resulted in an unintentional violation of anti-doping regulations. Introduction Since the prohibition of the cultivation of plants of the species Cannabis sativa L (so-called fibre hemp) with minor content of the psychoactive Δ -tetrahydrocannabinol (THC) was lifted in 1996, a large number of hemp-containing foods have been manufactured. An increasing popularity is recognized, potentially based on the assumption that the consumption of such food products causes psychoactive effects. Hemp products are trend foods with supposedly positive nutritional properties and health-promoting effects [1]. The variety of consumables is ranging from hemp-based snacks, cereals, energy bars, oils, beverages, and sweets to food supplements and diverse food stuff. The products are available in different kinds of walk-in shops and via the Internet. In Figure 1, typical examples for hemp products are shown. According to the World Anti-Doping Agency (WADA) regulations, cannabinoids use is prohibited in competition except for cannabidiol (CBD) [2]. Recently performed administration studies with CBD products showed a substantial risk of an antidoping rule violation for athletes [3]. Similar to CBD products, an increase in advertising and consumption is likewise noticeable with hemp products. Resulting from the fact that hemp products are also obtained from processed cannabis plants, these products may also contain prohibited cannabinoids. 9 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6128 In this study it should be investigated, if the consumption of hemp products leads to adverse analytical findings (AAF) in doping controls. Figure 1. Examples of hemp products Experimental Eight different commercially available hemp products (Table 1) were purchased, and single application elimination studies were performed with healthy volunteers who consumed the hemp products following the manufacturer´s dosage recommendations and provided urine samples, collected before and 8, 16 and 32 hours after product administration. The excretion study was performed with approval (number 122/2020) of the ethics committee of the German Sport University Cologne (Germany) and written informed consent was obtained from all participants. Table 1. Hemp products for administration studies MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6129 Urine samples were analyzed for the presence of 16 doping-relevant cannabinoids (Table 2) according to an established method by means of GC-MS [3]. The cannabinoid concentrations were adjusted to a urine specific gravity (SG) of 1.020 [4]. Table 2. Analyzed cannabinoids and internal standards (marked in grey) Results and Discussion In Table 3, the results for urine samples collected 8 hours after administration are presented. Neither THC nor its metabolite carboxy-THC were detectable. This may be due to the fact that in Europe only the cultivation of hemp plants with minor content of the psychoactive Δ -tetrahydrocannabinol is permitted. Conversely, CBD was detected in all but one urine specimen. The application of two out of three hemp seed oils (product numbers 5 and 6) resulted in urinary findings concerning CBD and cannabigerol (CBG). Hemp oil is commonly generated from cannabis seeds, which do not contain any cannabinoid [4]. Hence, if cannabinoids are found in oil and seeds, its origin is most likely attributable to external contact of the seed hulls with cannabinoid-containing resins in bracts and leaves during maturation, harvesting, and processing [5]. Urine samples collected after the consumption of cookies and marzipan showed various prohibited cannabinoids. For products number 1 (beer), 2 and 3 (tea), 7 (cookies), and 8 (marzipan), the ingredient “hemp bud” is declared on the label. Cannabinoid findings in post-administration samples with these 9 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6130 products may result from hemp bud resins, which (as well as hemp leaves) are usually removed prior to food manufacturing processes. In urine samples collected later than 8 hours (16 respectively 32 hours), merely CBD was detectable (data not shown). The most abundant cannabinoid was CBG followed by cannabidivarin (CBDV). Similar results were obtained after consumption of CBD products [3]. Alarming however is that in 5 of 8 individuals (63%) the 8 hour urine sample contained cannabinoids, which would constitute an AAF if the sample was collected from an athlete in-competition. Table 3. Cannabinoids (ng/mL) in excretion study urine samples, collected 8 hours after application of hemp products according to the recommended manufactureres dosage Conclusions The consumption of hemp products can lead to findings of prohibited cannabinoids in urine e.g. cannabidivarin (CBDV), cannabichromene (CBC), cannabidivarinic acid (CBDVA), cannabinol (CBN), cannabigerol (CBG), cannabinolic acid (CBDA) and cannabigerolic acid (CBGA). Comprehensive information and thorough education of athletes concerning the risk associated with the consumption of hemp products is necessary and important. Revisiting reporting levels for cannabinoids might be warranted. References 1. Lachenmeyer D, Bock V, Deych A, Sproll C, de Rezende T, Walch S (2019) Hanfhaltige Lebensmittel – ein Update. Deutsche Lebensmittelrundschau: Zeitschrift für Lebensmittelkunde und Lebensmittelrecht. 351- 372 2. World Anti-Doping Agency. The 2021 Prohibited List. International Standard, Montreal (2021). https://www.wada-ama.org/sites/default/files/prohibited_list_2021_en.pdf (access date 12.03.2021) 3. Mareck U, Fußhöller G, Geyer H, Huestis MA, Scheiff AB, Thevis M (2021) Preliminary data on the potential for unintentional antidoping rule violations by permitted cannabidiol (CBD) use. Drug Test Anal. 13,539-549 4. World Anti-Doping Agency. Technical Document TD2019DL v.2.0. Decision Limits for the Confirmatory Quantification of Threshold Substances. https://www.wada-ama.org/sites/default/files/resources/files/td2019dl_v2_finalb.pdf (access date 04.05.2020) 5. Matthäus B, Brühl L (2008) Virgin hemp seed oil: An interesting niche product. Eur. J. Lipid Sci. Technol., 110, 655-661 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6131 Acknowledgements The authors thank the Manfred Donike Institute for Doping Analysis e.V. (Cologne, Germany), the Federal Ministry of the Interior, Building and Community (Berlin, Germany) and the Doping Autoriteit Netherlands (Capelle aan den Ijssel, Netherlands) for supporting the study. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6132 Cantón MI, Garcia PP, Serrano E, Muñoz G Comparison of the separation of 2-fluoroamphetamine, 3-fluoro- amphetamine and 4-fluoroamphetamine by gas chromatography-mass spectrometry using different columns and derivatization agents Madrid Anti-Doping Laboratory, Madrid, Spain Abstract In sport, the word stimulant usually refers to agents stimulating the central nervous system (CNS), affecting mood, alertness, locomotion and appetite, or targeting the sympathetic nervous system causing particularly cardiovascular actions [1]. This way, these substances can provide an unfair advantage in many events: increased alertness, diminished fatigue and cardiovascular activation. Stimulants are included in class S6 of the World Anti-Doping Agency (WADA) Prohibited List. Many of the stimulants are amphetamine derivatives, including agents with abuse potential as recreational drugs, such as fluoroamphetamines (FA), which are not expressly listed in S6, but they are compounds with similar structure to those in that class, so an anti-doping laboratory may include them in its testing procedures. The current laboratory method for the detection of stimulants by gas chromatography-mass spectrometry (GC-MS) is not able to discriminate between the isomers 2-fluoroamphetamine (2FA), 3‑fluoroamphetamine (3FA) and 4-fluoroamphetamine (4FA). In this study, GC-MS with different columns and different forms of derivatizing [4,5] has been used for the separation of 2FA, 3FA and 4FA. Free bases, their Schiff base derivatives, acylated and silylated derivatives were subjected to analysis using dimethylpolysiloxane (Ultra 1) and 5%-phenyl-methyl- polysiloxane (Ultra 2) capillary columns. Ultra 2 column and trifluoroacetyl derivatives (TFA) together with a modification of the ramp showed the best results. Introduction Being able to identify the largest number of doping substances and methods is the goal of an anti-doping laboratory to protect clean athletes from cheaters. Section S6 of the WADA Prohibited List indicates that other substances with a similar chemical structure or biological effect are also prohibited. For this reason, it is important that substances such as FA are introduced into laboratory procedures. The different isomers 2FA, 3FA and 4FA are difficult to separate in our laboratory with the detection method by GC-MS currently in use (extraction pH13 tert-butyl methyl ether, no derivatization, Ultra1 conditions injection) due to the small structural difference between them (Figure 1). This fact leads to the search for improvements in sample preparation and chromatographic conditions. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6133 Figure 1. 2FA, 3FA and 4FA structures Experimental 2FA and 4FA reference compounds were purchased from Cerilliant and 3FA from NMI. Diphenylamine (ISTD), tert-butyl methyl ether (TBME), sodium hydroxide, sodium sulfate and acetone were obtained from Merck, MBTFA (N-methyl-bis(trifluoroacetamide)) and MSTFA (N-methyl-N-trimethylsilyl- trifluoroacetamide) from Macherey Nagel. Columns Ultra 1 and Ultra 2 were obtained from Agilent. To 5 mL of sample in a glass tube, 15 µL ISTD (100 µg/mL), 2 drops NaOH (pH13), 2 mL TBME and 1g of Na SO were added. Once vortex-mixed (20 min), centrifuged (5 min) and frozen, samples were transferred to a vial and brought to dryness under N . Different derivatization procedures were carried out: 1. 50 µL acetone (Schiff base derivatives) 2. 100 µL MSTFA (80°C 10 min) + 10 µL MBTFA (80°C, 10 min]) [3] (acylated derivatives) 3. 50 µL derivatizating agent (120 mg DTT (1,4-dithioerythritol) + 60 mg NH I + 30 mL MSTFA) (65°C, 30 min) (silylated derivatives) 4. no derivatization (free bases) To discriminate between 2FA, 3FA and 4FA, spiked urines at 100 ng/mL and blank urines were analyzed by Agilent 6890GC-5975MSD instruments. Agilent columns, Ultra 1 (25 m, 0.2 mm, 0.11 µm) and Ultra 2 (25 m, 0.2 mm, 0.33 µm) were employed. Injection volume 2 µL; split, SIM acquisition modes. Ultra 1 Conditions: Initial temperature 90°C, rate: 10°C/min, final temp 300°C and final time 3.00 min, run time 24.00 min 2 4 2 4 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6134 Ultra 2 Conditions: Ramp 1: Initial temperature 70°C; rate 1: 2.0°C/min, final temp 110°C and final time 3.00 min; rate 2: 60.0°C/min, final temp 250°C and final time 0.00 min; rate 3: 100°C/min, final temp 300°C and final time 2.00 min; run time 25.83 min Ramp 2: Initial temperature 70°C; rate 1: 2.0°C/min, final temp 110°C and final time 3.00 min; rate 2: 30.0°C/min, final temp 250°C and final time 0.00 min; rate 3: 100°C/min, final temp 300°C and final time 2.00 min; run time 28.17 min Results and Discussion Negative and spiked urines with 2FA, 3FA and 4FA at 100 ng/mL (Minimun Required Performance Level for Detection, MRPL, established by WADA for stimulants) were analyzed by GC-MS with different derivatizing protocols (Schiff base derivatives, acylated derivatives, silylated derivatives and free bases) and different gas chromatographic conditions (Ultra 1, dimethylpolysiloxane and Ultra 2, 5%-phenyl- methyl-polysiloxane, this one using two ramps). The Schiff base derivatives procedure was chosen due to its specificity and simplicity. Acylated derivatives procedure seemed to be an option for getting better stability and chromatographic peaks. The usual silylated derivatives procedure used in the laboratory was also tested. Free bases were analyzed too. Results showed silylated derivatives (TMS) did not allow the detection of suitable concentration levels in any column (they were not detectable at MRPL, maybe due to matrix effects). For Ultra 1 column free bases and Schiff base derivatives were barely separated. The acylated derivative of 2FA eluted at a lower retention time than the pair 3FA/4FA, but this pair could not be enough resolved. For Ultra 2 column, the pair of free bases 3FA/4FA could not be differentiated, while Schiff base and acylated derivatives allowed discrimination between all the fluoroamphetamines, including the pairs 2FA/3FA and 3FA/4FA (Figure 2), respectively. After that, a slowed down ramp was optimized achieving a better resolution between peaks, specially for the acylated derivatives of pair 3FA/4FA. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6135 Figure 2. 2FA, 3FA and 4FA comparative in Ultra 1 and Ultra 2 column with different derivatization agents MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6136 Conclusions The best results were obtained for the Ultra 2 column. Schiff base derivatization allowed an optimal separation between the pair 2FA/3FA (resolution 2FA/3FA around 0.85) [2] and 4FA, but there were not enough diagnostic ions for a reliable identification (only ions m/z 84, m/z 109). Trifluoroacetyl derivatives (TFA) showed an excellent separation between 2FA and the pair 3FA/4FA and an acceptable resolution of the pair 3FA/4FA (resolution around 0.85), allowing in addition the identification with at least three diagnostic ions (Figure 3). After modifying the temperature ramp in column, a resolution of 1 was reached (Figure 4). This highlights the importance of optimizing the gas chromatographic conditions (column selection and temperature ramp) and the use of a proper derivatizing agent. Figure 3. a: Mass spectrum of Schiff base; b: Mass spectrum of TFA derivative MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6137 Figure 4. 2FA, 3FA and 4FA results in Ultra 2 column with ramp 1 and ramp 2 References 1. Docherty J.R. (2008) Pharmacology of stimulants prohibited by the World Anti-doping Agency (WADA) British J. Pharmacology 154, 606-622 2. Foley Joe P. (1991) Resolution equations for column chromatography. Analyst 116, 1275-1279 3. Kaewklum M, Kaewklum S, Dithayam S, Wilairat P, Kongpatanakul S. (2020) Separation and identification of regioisomers of fluoroamphetamine (FA) and fluoromethamphetamine (FMA) in doping control by gas chromatography-mass spectrometry. In: Thevis M, Geyer H, Mareck U (eds.) Recent advances in doping analysis (28). Sportverlag Strauß, Köln, 103-107 4. Parkinson D.R. (2014) Analytical Derivatization Techniques. Reference module in chemistry, molecular sciences and chemical engineering, 1-38 5. Kataoka H. (1996) Derivatization reactions for the determination of amines by gas chromatography and their applications in environmental analysis. J. of Chromatography A 733, 19-34 6. Donike M. (1973) Acylierung mit Bis(Acylamiden); N-Methyl-Bis-(Trifluoroacetamid) und Bis(Trifluoroacetamid), zwei neue Reagenzien zur Trifluoracetylierung[N-methyl-bis-(trifluoroacetamide) and bis(trifluoroacetamide), two new reagents for triflouroacetylation]. J of Chromatography 78, 273-9 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6138 Berghes B, Radu M, Cristea CD, Toboc A, Stan C Optimization of a cocaine and benzoylecgonine identification method using the linear ion trap Doping control laboratory, Bucharest, Romania Abstract Cocaine is an alkaloid that is obtained from coca leaves (Eritroxilon Coca). Cocaine use leads to increased energy, capacity to achieve great physical effort and a decreased need for sleep. According to the WADA technical guidance from 25 January 2021, the detection of benzoylecgonine (BZE) as a target analyte is mandatory while cocaine (COC) is an optional target analyte for the Initial Testing Procedure (ITP). In the Confirmation Procedure (CP), both KOK and BZE are mandatory target analytes. This paper presents the identification of COC and BZE (BZE was previously validated in CP by solid phase extraction (SPE) on XAD2 resin). A detection method of COC and BZE was developed using liquid chromatography coupled with a mass spectrometer (LC/MS/MS). Liquid-liquid extraction (LLE) with ethyl acetate is performed at pH 9.6. Acquisition mode for BZE was MRM and for COC MRM and MS . Introduction Cocaine is prohibited in sports and it is included in the World Anti-Doping Agency (WADA) Prohibited List at section S6. Stimulants (non-specific stimulants) [1]. Methods were developed using LLE, SPE or direct injection, followed by LC/MS/MS techniques or gas chromatograph-mass spectrometry techniques (GC/MS)[2-7]. For CP, every presumptive adverse analytical finding (PAAF) for COC shall be confirmed at every level (at least 1 ng/mL). Also, every PAAF for BZE ≥ 50 ng/mL shall be confirmed. The identification method was optimized using LLE with ethyl acetate at pH 9.6, followed by injection in ABSciex Qtrap 5500/Agilent 1290 Infinity system, using mixed MRM and MS acquisition mode. Experimental Materials and methods Cocaine, benzoilecgonine and mefruside were purchased from LGC Standards (Wesel, Germany). Ethyl acetate and acetonitrile were purchased from VWR Chemicals (Bioaqua Group LTD, Romania), potassium bicarbonate and potassium carbonate were purchased from Merck (Redox, Romania). Ultrapure water was obtained using a Milli-Q Q-POD equipment from Merck. Sample preparation methods are presented in Figure 1. Equipment used was ABSciex Qtrap 5500/Agilent 1290 Infinity. The chromatographic column was Zorbax SB-C18 2.1 x 50 mm, 5 µm with SecurityGuard ULTRA Cartridges UHPLC C18 guard column. The A solvent was 5 mM amonium formate, 1‰ formic acid in water; B solvent was 5 mM amonium formate, 1‰ formic acid in 90% acetonitrile + 10% water. Chormatographic and MS conditions are presented in Table 1. 3 3 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6139 Figure 1. Sample preparation Table 1. Chormatographic and MS conditions Validation The methods were investigated for matrix effects, limit of identification, carry-over, and robustness. Matrix effects: 10 blank urine samples were analyzed and monitored for interferences. Limit of identification: 10 blank urines spiked with 0.5 ng/mL and 1 ng/mL for COC and with 25 ng/mL and 50 ng/mL for BZE were analyzed and compliance with TD2021IDCR criteria was verified. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6140 Carry-over was evaluated with the consecutive injection of a sample fortified with 500 ng/mL and two blank samples. Robustness was evaluated by modifying column temperature, source temperature, flow, injection volume, ethyl acetate volume, reconstitution volume, and buffer solution volume. Results and Discussion Initially, the identification of COC using MRM acquisition mode was attempted (results not shown). Since the concentration level is low, IDCR criteria could not be met due to matrix interferences and high background noise. For BZE, MRM acquisition mode was sufficient due to its much higher concetration. The low levels of the COC could be identified in urine matrix using MRM + MS due to the high specificity of this acquisition mode, which greately reduces matrix interferences and background noise. Mass spectrum in MS acquisition mode of COC is shown in Figure 2. Figure 2. MS spectrum of cocaine The method is specific for COC and BZE, with no interfering signals on the target compounds signals. The LOI is compliant with WADA TD2019MRPL, ISL 2021 documents and WADA Letter for cocaine findings, 25 January 2021. A spiked urine at LOI and a blank urine are shown in Figure 3. Carry-over was observed at a concentration of 500 ng/mL only for COC, but it’s lower than the limit of identification. The method is robust for the investigated parameters for both analytes. 3 3 3 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6141 Figure 3. Chromatograms of a spiked urine at LOI (top) and a blank urine (bottom) Conclusions In this work we present a method for the identification of COC and BZE in urine using MRM + MS acquisition mode for COC and MRM for BZE. The specificity and the LOI show that the method can be used to identify COC at 1 ng/mL and BZE at 50 ng/mL in doping control samples. The method is robust against small variations of column temperature, source temperature, flow, injection volume, ethyl acetate volume, reconstitution volume, and buffer solution volume. References 1. World Anti-Doping Agency. The 2021 Prohibited List. International Standard, Montreal (2021) https://www.wada-ama.org/sites/default/files/resources/files/2021list_en.pdf (access date 19.02.2021) 3 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6142 2. Görgens C, Guddat S, Thomas A, Wachsmuth A, Orlovius AK, Sigmund G, Thevis M, Schänzer W. (2016) Simplifying and expanding analytical capabilities for various classes of doping agents by means of direct urine injection high performance liquid chromatography high resolution / high accuracy mass spectrometry, J. Pharm. Biomed. Anal, 131:482-496 3. Thomas A, Sigmund G, Guddat S, Schänzer W, Thevis M. (2008) Determination of selected stimulants in urine for sports drug analysis by solid phase extraction via cation exchange and means of liquid chromatography-tandem mass spectrometry, Eur. J. Mass Spectrom, 14:135-143 4. Deventer K, Pozo OJ, Verstraete AG, Van Eenoo P. (2014) Dilute-and-shoot-liquid chromatography-mass spectrometry for urine analysis in doping control and analytical toxicology, Trends in Analytical Chemistry, 55:1-13; 5. Jeanville PM, Estape ES, Needham RS, Cole MJ. (2000) Rapid Confirmation/Quantitation of Cocaine and Benzoylecgonine in Urine Utilizing High Performance Liquid Chromatography and Tandem Mass Spectrometry, J Am Soc Mass Spectrom, 11:257–263 6. Snozek CLH, Bjergum MW, Langman LJ. (2012) Cocaine and Metabolites by LC-MS/MS. In: Langman LJ, Snozek CLH (eds.) LC-MS in Drug Analysis: Methods and Protocols, Methods in Molecular Biology (902). Springer Science+Business Media; 7. Maquille A, Guillarme D, Rudaz S, Veuthey JL. (2009) High-Throughput Screening of Drugs of Abuse in Urine by Supported Liquid-Liquid Extraction and UHPLC Coupled to Tandem MS, Chromatografia 70:1373- 1380. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6143 Moleme BJ, Grobbelaar E, Du Preez H The influence of gas filter saturation on the chromatographic sensitivity of GC-MS/MS analysis South African Doping Control Laboratory, University of the Free State, Bloemfontein, South Africa Abstract Anti-Doping analysis utilizes a large variety of analytical instruments. Historically and still today, GC-MS and GC-MS/MS techniques are applied daily in Anti-Doping analysis. Anabolic steroids, selective androgenic receptor modulators, and other compounds are analyzed using these techniques. Quality control samples are analyzed with every analytical batch to ensure system sensitivity, repeatability, and identification capability. Recently, the laboratory observed an issue with the quality control samples on a specific GC-MS/MS system. The quality control samples did not show the presence of zilpaterol, stanozolol, and prostanozol metabolites as expected. Furthermore, the GC-MS/MS system's sensitivity was low compared to the other GC-MS/MS systems. Resolution of the issue through standard troubleshooting procedures was not possible. An in-depth investigation covering the preparation of reference standards, quality controls, extraction, derivatization, and the instrument was unable to identify the problem. The laboratory consulted service engineers to resolve the issue. The replacement of one of the two gas filters on the carrier gas line resolved the issue. The instrument's sensitivity increased, and zilpaterol, stanozolol, and prostanozol metabolites were detected in the quality control samples. The system checks, as prescribed by the manufacturer, did not indicate saturation or a fault even from the gas filter indicator. Introduction Anti-Doping analysis utilizes a large variety of analytical instruments. Historically and still today, GC-MS and GC-MS/MS techniques are applied daily in Anti-Doping analysis. Anabolic steroids, selective androgenic receptor modulators and other compounds are analyzed using these techniques. Quality control samples are analyzed with every analytical batch to ensure system sensitivity, repeatability and identification capability. Routine maintenance procedures are performed before running every analytical batch to ensure that the system meets the laboratory's criteria and those from the manufacturer. Recently, the laboratory observed an issue with the quality control samples on a specific GC-MS/MS system. The quality control samples did not show the presence of zilpaterol, stanozolol and prostanozol metabolites as expected. Furthermore, the GC-MS/MS system's sensitivity was low compared to the other GC-MS/MS systems. Standard troubleshooting procedures and an in-depth investigation covering the preparation of reference standards, quality controls, extraction, derivatization and instrument were unable to identify the problem. The replacement of one of the two gas filters, specifically connected to the carrier gas line, resolved the issue even though it showed no signs of saturation from the gas filter indicator. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6144 Experimental Routine maintenance was performed before running the samples. The liner and septa were replaced and a Checktune was performed (Fig. 1). Two quality control samples (QCs), namely QC-T and QC-Super, were injected on the GC-MS/MS system in question before and after the gas filter change. The sample names included either before or after to indicate the time of injection using one sequence. The QCs were injected three (3) more times after the gas filter change. A second Checktune was performed after changing the gas filter before running the samples (Fig. 2). The samples were injected on an Ultra-1 Crosslinked methyl silicone (16.5 m x 0.20 mm i.d. x 0.11 μm film thickness) column using the routine ITP GC-MS/MS screening method. Figure 1. Air and Water Check from Checktune before replacing the gas filter Figure 2. Air and Water Check from Checktune after replacing the gas filter Results and Discussion The instrument sensitivity increased with the first (1 ) injection of the QCs following the gas filter change, in both the abundance and resolution of chromatographic peaks increased. The chromatographic peaks for zilpaterol, stanozolol and prostanozol metabolites were observed with the first injection of the QCs after the gas filter change (Figures 3 and 4). st MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6145 Figure 3: Chromatographic comparison of ability to detect Zilpaterol after the Gas Clean Filter replacement in QC-T sample Figure 4: Chromatographic comparison of ability to detect 3’OH-Stanozolol after the Gas Clean Filter replacement in QC-T sample The sensitivity and resolution of chromatographic peaks kept improving with more injections as the water was flushed from the system. If the loss of sensitivity and chromatographic resolution is not resolved by liner replacement, source cleaning, column replacement, or cutting, a saturated gas filter may be the problem. The water content in the GC-MS/MS system showed an approximate decrease of 50% after the gas filter change (refer to Figures 1 and 2). Conclusions The study shows that a saturated gas filter decreases the sensitivity and the detection capability of a GC- MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6146 MS\MS system, specifically with regards to the substances in the scope Anti-Doping analysis. An acceptable Air and Water check report with a helium gas filter that shows no saturation on the indicator cannot be solely relied upon as the indicator could be faulty. Lastly, the study recommends that a combination of the Air and Water Check together with the sensitivity and chromatography of polar compounds (zilpaterol, stanozolol and prostanozol) be used to assess the GC-MS/MS system's suitability for quality analysis. References 1. World Anti-Doping Agency. The 2021 Prohibited List. International Standard, Montreal (2021), www.wada-ama.org/sites/default/files/wada_2021_english_prohibited_list_0.pdf (access date 17.03.2021) Acknowledgements We would like to thank Agilent Technologies and the Service Engineers at Chemetrix for support and assitance. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6147 Inthong T, Nimsoongnern S, Wilairat P, Kongpatanakul S Workflow Management and Sample Tracking for Doping Analysis National Doping Control Centre, Analytical Science and National Doping Test Institute, Mahidol University, Bangkok, Thailand Abstract The data management system has workflow and data tracking support features as mandated by WADA since ISL 2019. The low-cost Microsoft Access was employed for sample tracking, with data exchange capabilities between the Laboratory and ADAMS. A 50-inch LCD screen displays the sample status starting from sample receipt to reporting in ADAMS. The status is changed with each step of users’ activities by embed computer commands. The status is color coded, with orange for closing to report within due date, and red indicating due now or overdue. If some methods of analysis are not completed in closing to report due date, the Laboratory Manager will step in to correct the problems. When the results are reported in to ADAMS, the tracking stops and disappears from the screen. The Laboratory Manager can check how many remaining samples in the lab to estimate the capacity in taking sample analysis the next day. The system has also been developed for sample tracking of special requests, e.g. ATPF-CPR from ADAMS, for samples that already have results in ADAMS. The tracking system needs to be re-activated by the Laboratory Manager who will assign the new report date which is shown on the monitor screen with color status. The input of the confirmation results of steroid profile and/or IRMS results in to ADAMS triggers the cessation of the tracking and the status disappears from the screen. This system has been shown to be effective in preventing delays in reporting the results. Microsoft Access is easy to use, and scientists who have little knowledge in database and computer programming can easily be trained to maintain and update the system. Introduction The data management system has workflow and data tracking support features as mandated by WADA since ISL 2019[1]. The low-cost, flexible and easy to use Microsoft Access has also been used for the Laboratory Information Management System (LIMS) in doping control laboratories [2,3]. The system can be employed for sample tracking, with data exchange capabilities between the Laboratory and ADAMS. A 50-inch LCD screen displays the sample status starting from sample receipt to reporting. The status is changed with each step of users’ activities by embed computer commands. The status is tracked as color code. If some methods of analysis are not completed by report due date, the Laboratory Operation Manager (LOM) will step in to correct the problems. The system has also been developed for sample tracking of special requests, e.g. ATPF-CPR notification from ADAMS. LOM also can check how many remaining samples in the lab to estimate the capacity for accepting new samples. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6148 Experimental Event Display Control The tracking starts at sample receipt and continues until the results are reported into ADAMS, when the tracking stops and disappears from the screen. The system has also been developed for sample tracking of special requests, e.g. ATPF-CPR from ADAMS, for samples that already have results sent to ADAMS. Figure 1. Flow diagram for sample tracking and sample status MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6149 The tracking system then needs to be re-activated by the Laboratory Manager who will assign a new report date which is shown on the monitor screen with color status. The entering of confirmation results of steroid profile and/or IRMS results into ADAMS triggers the cessation of the tracking and the sample status disappears from the screen. Results and Discussion Visual Control A 50-inch LCD screen hung on the wall next to the entrance corridor displays sample status, status tracking during the analysis, and sample flow management. The main record composes of Receipt No., Lab code set with the same activities, sample status, report due date which is shown as a color code: orange indicates reporting within due date, and red indicates due or overdue. Sub-record indicates the remaining analysis procedure(s). The status is changed automatically with each step of users’ activities by embed computer commands. The screen will be refreshed and updated accordingly. Figure 2. Display of LCD screen monitor for sample tracking MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6150 Conclusions The screen shows only reception number to link to the sample information. After each analytical procedure is completed, the results are sent on line using secure password access and summarized to prepare the analytical report by using hierarchical action and result. The status of analysis of a sample between Lab and ADAMS is displayed on a screen which is effective in preventing miss-reporting of pending results. The due dates of reporting are indicated by color codes with respect to each sample which is effective to prevent delays in reporting the results. It also accommodates special analysis request such as ATPF-CPR. Moreover, this system is useful for estimating the current capacity of workload in the Laboratory. The future plan is the development of the system for automatic assignment of initial testing procedures according to sex, sport, competition type (IC, OOC) and based on the ITP results assignment of confirmation procedure. References 1. The World Anti-Doping Code, International Standard for Laboratories. November 2019, 5.2.3.4 Control of Data and Computer Security 2. Inthong T, Pinitprapha W, Wilairat P, Kusamran T, Anukarahanonta T and Chaikum N. Database System for Monitoring Chain of Custody of Samples in Doping Analysis, In: W. Schänzer, H. Geyer, A. Gotzmann, U. Mareck-Engelke. 29 Cologne Workshop on Doping Analysis. Sport und Buch Strauß, Köln (2011) 270- 273. 3. Nazir J, Cowan D A, Caldwell R and Walker C. A Menu Driven Laboratory Information Management System (DCLIMS) for Anti-Doping Laboratories. 38 Cologne Workshop on Doping Analysis. Sport und Buch Strauß, Hellenthal (2020) Page 200. Acknowledgements This project has been carried out with the support of the National Doping Control Centre (NDCC), Analytical Science and National Doping Test Institute, Mahidol University. th th MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6151 Leogrande P , Jardines Garcia D , Domenici E , de La Torre X , Parr MK , Botrè F Low-energy electron ionization for steroidomics analysis using high- resolution mass spectrometry Laboratorio Antidoping FMSI, Federazione Medico Sportiva Italiana, Rome, Italy ; Institute of Pharmacy, Freie Universität Berlin, Berlin, Germany Abstract The coupling of gas chromatography to mass spectrometry has proven to be the analytical technique of choice for steroidomics analysis, allowing excellent separation efficiency and a wide range of coverage and providing valuable structural information. Systematic electron ionization fragmentation studies of steroids have been performed to elucidate their characteristic fragmentation pattern. However, the energy provided to the electron is much higher than the ionization potential of most organic compounds, leading to extensive fragmentation in the source. This work presents a multifactorial study on optimizing a low-energy electron ionization source to maximize molecular ion formation while minimizing the extension of fragmentation to increase the versatility of the analytical technique used. The effects of electron energy, emission current, and source temperature on steroid fragmentation pathways were studied in full factorial experimental designs, using 27 steroid reference materials chosen to cover the urinary steroid profile. Two response parameters were evaluated using screening designs to identify the most relevant factors and/or interactions: degree of fragmentation and relative abundances of the molecular ion. Finally, the peak width was evaluated to minimize the experimentally observed chromatographic peak tail of the extracted ion. By following this experimental approach, it is possible to select optimal conditions more quickly; to examine multiple factors simultaneously, to highlight possible interactions; to estimate the effects of each factor at different levels of the other factors; to increase the molecular ion intensity, from which the elemental composition can be calculated with high-resolution data. Introduction Coupling gas chromatography to mass spectrometry is the technique of choice for steroid detection in the antidoping field. Gas chromatographic separation allows excellent separation efficiency and a wide coverage range, providing complementary information to that obtainable with LC-based methods. Although the use of the electron ionization (EI) source with an electron energy of 70 eV allows selective and highly reproducible mass spectra, the applied ionization energy is much higher than the ionization potential (7-15 eV) of most organic compounds, leading to extensive fragmentation in the source [1]. Applying a low-energy EI (LE-EI) source preserves the carbon skeleton of the molecules, reducing their fragmentation extension and increasing the intensity of high m/z fragments, simplifying their structure elucidation and molecular weight assignment [2]. 1,2 1 1 1 2 1 1 2 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6152 Experimental Aliquots of 3 μL of each standard (1 mg·mL ) were used to prepare the reference solutions of the target steroids (Table 1), which were dried by evaporation under a nitrogen stream and then derivatized by using 50 μL of a mixture of MSTFA, NH I, and 2-mercaptoethanol in a ratio of 1000:4:6 (V/w/V). The experimental strategy followed the consecutive steps: (i) screening design that narrows the range of variables to be evaluated; (ii) optimization design that studies the response of each combination of factors and their levels to identify the conditions under which the process is close to optimization. Three full factorial designs (FFDs) were performed to evaluate a total of 648 experiments. Table 1. Classification and coding of reference materials chosen to develop DoEs The designs of the experiments (DoEs) were run using in-house scripts on the open-access software “The R Project for Statistical Computing R” (version 4.0.3). -1 4 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6153 The factors considered, both individually and in combination, were: (i) the emission current (EC), measured between the filament and the input split; (ii) the collision energy (CE), required to activate the fragmentation process; and (iii) the source temperature (T), to avoid sample condensation. A range of variability was estimated for each factor and all possible experimental combinations were run (Table 2). Table 2. Experimental combinations performed for full factorial designs (FFDs, °C_eV_A). Response parameters evaluated using the screening designs were the degree of fragmentation (consisting of the number of fragments with relative abundances > 3%) and the relative abundance of the molecular ion. The parameters found to be most relevant were used to minimize the chromatographic peak tail of the extracted ion by monitoring the peak width and find the best compromise for simultaneous analysis of all steroids included in the design table. Results and Discussion The responses represent the parameters considered to improve the information that can be extracted from the data and reduce the time required for the mass spectra deconvolution process. The degree of fragmentation and the molecular ion were evaluated by screening designs which highlighted collision energy and source temperature as the most influential factors, while emission current always showed a non-significant influence. In the case of the fragmentation degree, when the two factors are analyzed individually, much less variation in response is observed between T levels than between CE levels, with the latter being the most significant factor (Fig. 1). MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6154 Figure 1. Plot of the screening DoE corresponding to the effects of T (a) and CE (b) on the degree of fragmentation of the steroids considered in the design table (indicated by the numbers 1 to 27 on the x-axis). This result is not certainly new, but rather confirmed the soundness of the applied approach. The monitoring of the molecular ion response was one of the key points of this work as it represented a way to increase the versatility and the applicability of this analytical technique for steroidomics analysis. An optimization design was performed to select the final source setting by searching for the combination of factors that minimize peak tailing by combining molecular ion, degree of fragmentation, and peak width into a single response. The 10 eV collision energy resulted in the most impactful level on the studied process. However, its application reduces the fragmentation pattern of some classes (especially androgens) to the molecular ion only, losing any structural information. To overcome this limitation, the peak width response was studied separately because high chromatographic peak resolution is necessary for data processing. Finally, the optimal source set up to exploit the enhanced response of some steroid classes, such as glucocorticoids for which the use of the LE-EI source allowed the preservation of the specific fragmentation pathway without sacrificing that of the others, led to the combination of the collision energy at 15 eV and the temperature at 230°C (Fig. 2). MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6155 Figure 2. Plot of the effect of T and CE on the chromatographic peak width, evaluating all target steroids using an optimization design. Conclusions Optimization of the LE-EI source configuration by monitoring multiple inputs at the same time identified temperature and collision energy as the most relevant factors for the steroids electron ionization process. These involved both mass-spectrometric and gas chromatographic conditions of the analytical technique used, allowing to maximize the information that may be extracted from the data for all classes of steroids considered and reduce the time required for their statistical processing. The best compromise to preserve the specific fragmentation pattern of the most thermally labile steroids, increasing the versatility and range of coverage of LE-EI-GC-HRMS for steroidomics analysis was found to be 230°C and 15 eV, while the emission current always showed a non-significant influence. References 1. Mairinger T., Sanderson J., Hann S. (2019) GC–QTOFMS with a low-energy electron ionization source for advancing isotopologue analysis in 13 C-based metabolic flux analysis, Analytical and Bioanalytical Chemistry. 411, 1495–1502. 2. Polet M., van Gansbeke W., Albertsdóttir A.D., Coppieters G., K. Deventer, van Eenoo P. (2019) Gas chromatography-mass spectrometry analysis of non-hydrolyzed sulfated steroids by degradation product formation, Drug Testing and Analysis. (11), 1656–1665. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6156 Wicka M , Grucza K , Stanczyk D , Drapala A , Kowalczyk K , Konarski P , Burstein K , Kwiatkowska D Development and validation of a method for the detection of benzodiazepines, barbiturates, imidazopyridine and derivates of cyclopyrrolone in blood by means of LC-MS/MS Polish Anti-Doping Laboratory, Warsaw, Poland ; Medical, Medical University of Warsaw, Warsaw, Poland Abstract The growing availability of various classes of psychoactive drugs, such as benzodiazepines, barbiturates, imidazopyridine and cyclopyrrolone derivatives present a challenge for clinical assays, forensics, and toxicological studies. The presented study aimed to propose an analytical method allowing the detection of the aforementioned compounds in human blood using LC-MS/MS. The proposed method was developed and validated to comply with the requirements typical for the fields mentioned. Gathered characteristics of the method included parameters as matrix effects, extraction recovery, process efficiency, limit of detection (LOD), limit of quantification (LOQ), limit of identification (LOI), relative retention times (RRT), linearity, intraday precision, and accuracy. Based on the analysis of the collected data, the described method is suitable for both the initial testing procedure and confirmatory procedure. Introduction Benzodiazepines, barbiturates, imidazopyridine, and cyclopyrrolone are drugs that are usually prescribed to treat various kinds of medical conditions including but not limited to anxiety, insomnia, seizures, and depression [1]. Lately, in addition to standard benzodiazepines, the so-called ‘designer drugs’ became more common. They are cheap and easy to access, e.g. online. Very often, they exhibit a stronger effect than normal benzodiazepines and thus are more dangerous [2]. The determination of these psychoactive drugs in biological fluids is essential in many fields such as clinical assays, forensics, and toxicological studies [3]. The presented work aimed to address this problem by developing and validating an LC-MS/MS method for the detection of 24 psychoactive drugs in human blood, for some of which no other methods have been published so far. The described method is suitable for both the initial testing procedure and the confirmatory procedure. Experimental Sample pre-treatment The method for the analysis of whole blood was based on a double extraction with 1-chlorobutane, and then with a mixture of dichloromethane and ethyl acetate (70:30, v/v). The residue was reconstituted in 80 µL of mobile phase (acetonitrile/water, 4:6 v/v), transferred in a vial and 15 µL were injected into the LC-MS/MS system. 1 1 1 1 1 1 2 1 1 2 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6157 Instrumental analysis All analyses were conducted using a LC Waters Alliance 2695XC/MS liquid chromatography system. The LC system was equipped with Atlantis dC18 (3 µm, 100 mm x 2.1 mm) from Waters. The mobile phase consisted of 0.1% acetic acid in acetonitrile (A) and 0.1% acetic acid in water (B), and an LC gradient was employed at the constant flow rate of 300 µLmin at 45°C. MRMs of the studied substances were traced with the Micromass Quattro Micro API mass spectrometer equipped with an ESI source. All analytes were investigated in the ESI mode. Desolvation gas flow was set at 600 L/h at 350°C with ion source temperature set at 120°C. Collision energies were optimized for each ion transition as summarized in Table 1. Method validation The method was investigated for matrix effects, extraction recovery, process efficiency, limit of detection (LOD), limit of quantification (LOQ), limit of identification (LOI), relative retention times (RRT), linearity, intraday precision, and accuracy. Within this study, the matrix effect was evaluated by analyzing the analytes added into seven blood samples at concentrations of 5 and 20 ng/mL versus the analyte added directly to a neat solvent. For identification capability, 7 different blood samples were spiked at various concentrations ranging from 0.2 to 100 ng/mL. Recovery [%] was estimated at 5 ng/mL and 20 ng/mL (n=7). The analytical curve was constructed using blank blood spiked with standard solutions in the range between 0.2 and 100 ng/mL. Results and Discussion MRMs of the studied substances are shown in Table 1. Table 1. List of precursor ions [M+H] and ion transitions for screening and confirmation analysis for 24 substances and 5 internal standards + + MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6158 The results gathered during method validation are as follows: Correlation coefficients were > 0.991 for all analytes, covering concentration ranges from 0.2 to 100 ng/mL (Table 2A). The LODs ranged between 0.15 ng/mL and 2.44 ng/mL (Table 2B). Table 2. A – Linear regression parameters and linearity range for analyzed compounds, B – Characteristics of the validation method MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6159 The extraction recovery ranged between 14.3% and 89.8% for concentration of 5 ng/mL, and 15.1% to 76.3% for concentration of 20 ng/mL (Table 3A). The repeatability standard deviations S (P) have reached values below 0.90. Pyrazolam showed the highest intra-serial variability of 7,94% CV (P). For the other compounds, a coefficient of variation of 5% was obtained. The standard reproducibility variation coefficients S (O) values varied between 0.07 and 1.02 (Table 3B). Table 3. A – Extraction recoveries, matrix effect, and proces efficiency, B – intra-day precision and accuracy (n=7) r r r MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6160 MRM chromatograms of selected analytes tested in blood are shown in Figure 1. Figure 1. MRM chromatogram of analytes tested in blood for QC concentration 10 g/mL MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6161 Conclusions An analytical method allowing the detection of 24 psychoactive drugs in blood was developed and validated. The high precision and accuracy of the method, combined with the excellent linear behavior of the analytical curve over the examined concentration range and relatively low LOD and LOQ values make the described method suitable for both initial testing procedure and confirmatory procedure. Among the analytes, there are substances for which no methods of determination in blood have been published so far, and given their mechanism of action and the likelihood of abuse, they are particularly important for medical and toxicological analysis. References 1. Mei V, Concheiro M, Pardi J, Cooper G. Validation of an LC-MS/MS Method for the Quantification of 13 Designer Benzodiazepines in Blood. J Anal Toxicol., 2019. 17;43(9):688-695. DOI: 10.1093/jat/bkz063. 2. Moosmann B, Auwärter V. (2018). Designer benzodiazepines: Another class of new psychoactive substances. In Handbook of Experimental Pharmacology. 2019. 252:383–410. Springer New York LLC. DOI: 10.1007/164_2018_154. 3. Qriouet Z, Qmichou Z, Bouchoutrouch N, Mahi H, Cherrah Y, Sefrioui H. Analytical Methods Used for the Detection and Quantification of Benzodiazepines. J Anal Methods Chem. 2019. 2019:2035492. DOI: 10.1155/2019/2035492. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6162 Mareck U, Fußhöller G, Geyer H, Thevis M Simplified confirmation analysis for Carboxy-THC Center for Preventive Doping Research / Institute of Biochemistry, German Sport University, Cologne, Germany Abstract In order to optimize and streamline confirmation procedures concerning carboxy-THC (THC-COOH), the utility and sufficiency of a single point calibrator enabling the estimation of the THC-COOH concentration for routine sports drug testing purposes was assessed. An evaluation of 14 in competition doping control urine samples, analyzed in the Cologne anti-doping laboratory in 2019 and 2020 and reported as adverse analytical finding (AAF) based on THC-COOH values greater than 180 ng/mL, was performed. THC-COOH values were calculated based on existing confirmation data utilizing both the entire calibration curve as well as single point calibration (150 ng/mL) employing D -THC-COOH as internal standard. THC-COOH concentrations computed from single point calibration and calibration curve exhibit only minor differences, indicating that a single point calibration would be sufficient for the confirmation of THC- COOH concentrations. Introduction According to regulations of the World Anti-Doping Agency (WADA), the use of cannabinoids is forbidden in competition (IC) [1]. In doping controls, the detection of cannabinoid misuse is based on the analysis of the pharmacologically inactive metabolite 11-nor-delta-9-carboxy-tetrahydrocannabinol-9-carboxylic acid (carboxy-THC = THC-COOH), and urinary concentrations greater 180 ng/mL constitute an adverse analytical finding (AAF) [2]. The confirmation procedure includes the time-consuming analysis of urine specimen in triplicate and calculation of the target analyte concentration by means of an appropriate calibration curve [2]. Additionally, the installation of methods for the confirmation of threshold substances requires elaborative efforts for validation and maintenance [3]. During the period 2015-2018, cannabis was reported by the WADA-accredited anti-doping laboratories worldwide with a percentage of adverse analytical findings ranging between 3-4% [4-7]. In order to optimize and streamline confirmation procedures concerning THC-COOH, the utility and sufficiency of a single point calibrator enabling the estimation of the THC-COOH concentration for routine sports drug testing purposes was assessed. An evaluation of IC doping control urine samples – analyzed in the Cologne anti-doping laboratory in 2019 and 2020 – and reported as AAF based on THC-COOH values greater than 180 ng/mL, was performed. Experimental Fourteen IC doping control urine specimens from national and international federations, analyzed in 2019 and 2020 in the Cologne anti-doping laboratory, reported as AAF for THC-COOH (> 180 ng/mL), were part of the investigation. THC-COOH values were calculated based on existing confirmation data utilizing both the entire calibration curve as well as single point calibration (150 ng/mL) employing D -THC-COOH as 9 9 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6163 internal standard (ISTD). The initial testing procedure (ITP) consisted of enzymatic hydrolysis, liquid-liquid extraction, trimethylsilylation, and analysis by gas chromatography / tandem mass spectrometry utilizing 17α- methyltestosterone as ISTD [8]. For confirmation purposes, an optimized method was used. The extraction was performed at pH 7 and an adequate deuterated standard (D -THC-COOH) was utilized. The THC-COOH concentration (mean value from triplicate determination) was calculated using a calibration function with at least 5 points, encompassing the estimated concentration level of the suspicious sample. The confirmation procedure was applied to those urine specimens showing an estimated urinary target analyte level greater than 130 ng/mL (cut-off). Results and Discussion Urinary THC-COOH values obtained from either ITP, CP with single point calibration (SPC), or CP with calibration curve are compared in Table 1. Table 1. Comparison of THC-COOH values (ng/mL) ITP:initial testing procedure CP: confirmation procedure The THC-COOH concentrations computed from SPC and calibration curve exhibit only minor differences (Figure 1), indicating that a single point calibration would be sufficient for the confirmation of THC-COOH concentrations. As illustrated in Figure 1, SPC-derived urinary concentrations of THC-COOH deviate from corresponding calibration curve-derived values with less than 10%. All samples determined with CPs employing SCP or conventional calibration curves resulted in AAFs. This also applied to two samples showing ITP THC-COOH concentrations less than the DL. Generally, samples with ITP-derived THC-COOH concentrations below the threshold value of 150 ng/mL or the DL of 180 ng/mL may result in AAFs, and the comparison of the ITP and CP results showed considerably larger differences (Figure 2) attributed to various reasons. Lower ITP-determined concentrations may result from limitations of the analytical method, where the ISTD is 17α-methyltestosterone. Since the liquid- liquid extraction is performed at pH 9.6, incomplete recoveries of THC-COOH are not appropriately 9 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6164 compensated by the ISTD, which exhibits substantially deviating physico-chemical properties. Further, high target analyte concentrations can result in saturation of the analytical setup, contributing to inaccurate estimations in ITPs. Such urine samples are hence diluted before performing the CP to match the working range of the calibration curve (50 – 500 ng/mL). Figure 1. Comparison of CP results obtained with calibration curve (red line) and single point calibration (blue dots) Figure 2. Comparison of CP results obtained with calibration curve (red line) and ITP (black dots) MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6165 Conclusions Proposed proceeding: CPs for samples exceeding cut-off 130 ng/mL THC-COOH in ITP after specific gravity (SG) correction Identification of THC-COOH according to WADA TD2021IDCR [9] SPC at 150 ng/mL with certified reference material Appropriate deuterated internal standard (e.g. D -THC-COOH) Use of a negative QC sample and a positive QC sample (150 ng/mL) References 1. World Anti-Doping Agency. The 2021 Prohibited List. International Standard, Montreal (2021). www.wada-ama.org/sites/default/files/resources/files/2021list_en.pdf (access date 12.03.2021) 2. World Anti-Doping Agency. Technical Document TD2019DL, v.2.0. Decision Limits for the Confirmatory Quantification of Threshold Substances. www.wada-ama.org/sites/default/files/resources/files/td2019dl_v2_finalb.pdf (access date 04.05.2020) 3. World Anti-Doping Code. International Standard for Laboratories (ISL), January 2021, Montreal (2021). www.wada-ama.org/sites/default/files/resources/files/isl_2021.pdf (access date 12.03.2021) 4. World Anti-Doping Agency, 2015 Anti-Doping Testing Figures. www.wada-ama.org/sites/default/files/resources/files/2015_wada_anti-doping_testing_figures_report_0.pdf (access date 26.05.2020) 5. World Anti-Doping Agency, 2016 Anti-Doping Testing Figures. www.wada-ama.org/sites/default/files/resources/files/2016_anti-doping_testing_figures.pdf (access date 26.05.2020) 6. World Anti-Doping Agency, 2017 Anti-Doping Testing Figures. www.wada-ama.org/sites/default/files/resources/files/2017_anti-doping_testing_figures_en_0.pdf (access date 26.05.2020) 7. World Anti-Doping Agency, 2018 Anti-Doping Testing Figures. www.wada-ama.org/sites/default/files/resources/files/2018_testing_figures_report.pdf (access date 26.05.2020) 8. Thevis M. Mass Spectrometry in Sports Drug Testing – Charcterization of Prohibited Substances and Doping Control Analytical Assays. Wiley, New Jersey, 2010. 376 pages. ISBN: 978-0-470-41327-2 9. World Anti-Doping Agency. Technical Document TD2021IDCR, v. 1.0 https://www.wada-ama.org/sites/default/files/resources/files/td2021idcr_final_eng_0.pdf (access date 04.06.2021) Acknowledgements The authors thank the Manfred Donike Institute for Doping Analysis e.V. (Cologne, Germany) and the Federal Ministry of the Interior, Building and Community (Berlin, Germany) for supporting the study. 9 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6166 Rubio A , Sigmund G , Piper T , Geyer H , Thevis M Evaluation of doping control samples to determine the prevalence of nicotine use by German elite athletes Institute of Biochemistry, German Sport University, Cologne, Germany ; European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany Abstract Up to now, nicotine is not included in the World Anti-Doping Agency´s (WADA´s) Prohibited List. However, it was added to WADA´s Monitoring Program in 2012, categorized as a stimulant “in-competition only”. To assess the prevalence of nicotine among German elite athletes from different sports, a total of 2,406 in-competition doping control samples collected under the authority of the National Anti-Doping Agency (NADA) Germany in 2017 were analyzed via full scan GC-MS/NPD experiments and evaluated for the presence of urinary nicotine and cotinine. Out of the 2,406 evaluated samples, 301 were found to contain nicotine/cotinine (12.5 %). The highest numbers of occurrences were observed in team sports such as baseball (50.0 %), ice hockey (37.0 %), handball (25.8 %), and football (18.4 %). All estimated concentrations were above the conservative concentration limit for active exposure (> 50 ng/mL). While most of the nicotine/cotinine containing samples showed approximate concentrations between 1 and 5 µg/mL, abnormally high values (> 6 µg/mL) were observed in some samples from different sports. The herein obtained data add to the trends observed for nicotine use among athletes that has been reported especially over the last two decades. Introduction The ergogenic effect of nicotine may be beneficial in sports. Smokeless tobacco ( e.g. snus) would be the source most likely considered by athletes, assuming that smoking and sport practice at top level are not compatible [1]. The use of nicotine among athletes has been reported as an increasing trend since the late 1970s [2,3]. As a consequence of the suspected abuse and the arguably performance-enhancing effects, nicotine was included in WADA’s Monitoring Program since 2012 [4]. The German press recently claimed that nicotine, via the application of snus, may be a potential doping substance especially in football [5,6]. In the herein pilot project the prevalence of nicotine and the main nicotine metabolite cotinine among German elite athletes has been assessed. Experimental Data of 2,406 NADA in-competition doping control samples of German athletes from different sports collected in 2017 and analyzed via full scan GC-MS experiments according to the method described by Thevis et al. 2007 [7], were evaluated for the presence of urinary nicotine and the main nicotine metabolite cotinine. The WADA rules for research with anonymized doping control samples were followed 1 1 1 1 1,2 1 2 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6167 [8]. As nicotine is not included in WADA´s Prohibited list, a full method validation was not applicable to this study. However, the LOD was assessed ( i.e. 20 ng/mL) in order to guarantee a proper identification of target analytes, and robustness of the results was ensured by the use of stable analytical conditions over the entire time of data acquisition. SAMPLE PREPARATION Figure 1. Samples were analyzed following the illustrated liquid-liquid-extraction (LLE) approach. SAMPLE ANALYSIS GC-MS analysis was performed on an Agilent 6890/5973 system (Waldbronn, Germany) equipped with an additional Nitrogen-Phosphorous Detector (NPD) and two separate Agilent HP-5 MS columns (inner diameter: 0.25 mm, film thickness: 0.25 µm). In order to obtain matching retention times, the column directed to the Mass Selective Detector (MSD) was 28 m long while the other column connected to the NPD was 24 m long. The injection volume was 5 µL (split ratio 1:5), the GC carrier gas was helium (constant pressure at 18 psi) and a temperature gradient was employed starting at 82°C for 0.45 min, increasing to 330°C with 30°C min . Final temperature was kept for 4.5 min. The mass spectrometer was operated with EI and full scan analysis (m/z 40-400). In order to detect nicotine and cotinine, characteristic ions were extracted at m/z 84 for nicotine, m/z 98 for cotinine and m/z 114 for ISTD. Semi- quantitative analysis was performed for both analytes using a single-point calibration. For this purpose, the ISTD-normalized peak areas of the most abundant product ion were used and quality standards with known concentrations of both analytes were used for comparison. Results and Discussion Out of the 2,406 evaluated samples, 301 were found to contain nicotine/cotinine (12.5 %). The highest numbers of occurrences were observed in team sports such as baseball (50.0 %), ice hockey (37.0 %), handball (25.8 %), and football (18.4 %). All estimated concentrations were above the conservative concentration limit for active exposure (> 50 ng/mL). According to the definition of the conservative concentration limit, the nicotine consumption is considered to have happened immediately before or during the competition. While most of the nicotine/cotinine containing samples showed approximate concentrations between 1 and 5 µg/mL, abnormally high values (> 6 µg/mL) were observed in some -1 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6168 samples from different sports. As such acute exposure to nicotine is hardly achievable for even a regular consumer, concentrations above 6 µg/mL increase the likelihood of a scenario intended to pharmacologically manipulate athletic performance. Figure 2. Nicotine/cotinine findings in sports with at least 20 samples. Figure 3. Concentrations [μg/mL] of nicotine + cotinine in the 301 samples MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6169 Conclusions The herein obtained data add to the trends observed for nicotine use among athletes that has been reported especially over the last two decades by several studies. The alleged high prevalence of nicotine use in football, e.g. via snus, could not be confirmed. However it is noticeable that among the 15 samples with the highest concentrations of nicotine + cotinine, 8 samples originated from football players. It may be considered as the basis for future investigations aiming at the determination if the detected nicotine levels are due to tobacco smoking or due to the consumption of smokeless tobacco via dried blood spots (DBS). References 1. Benowitz et al. Nicotine absorption and cardiovascular effects with smokeless tobacco use: comparison with cigarettes and nicotine gum. Clinical Pharmacology & Therapeutics 1988, 44, 23 2. Marclay et al. Determination of nicotine and nicotine metabolites in urine by hydrophilic interaction chromatography-tandem mass spectrometry: Potential use of smokeless tobacco products by ice hockey players. Journal of Chromatography A 2010, 1217, 7528 3. Marclay et al. A one-year monitoring of nicotine use in sport: Frontier between potential performance enhancement and addiction issues. Forensic Science International 2011, 213, 73 4. World Anti-Doping Agency. The 2012 monitoring program, Montreal. 2012. Available at: http://www.wada- ama.org 5. A. Langrock. Snus: Die Liga im Rausch. https://www.zeit.de/sport/2018-03/snus-fussball-eishockey- aufputschmittel-trenddroge, acc. 26.02.2021 6. V. Kunzmann. Snus ist die Droge der Fußballstars: Athleten im Rausch. https://web.de/ magazine/sport/fussball/snus-droge-fussballstars-athleten-rausch-33197004; access 26.02.2021 7. Thevis et al. Determination of tuaminoheptane in doping control urine samples. European Journal of Mass Spectrometry 2007, 13(213), 2013 8. WADA Code of Ethics: https://www.wada-ama.org/sites/default/files/resources/files/isl_2021.pdf; access: 03.01.2021 Acknowledgements The authors thank the Manfred-Donike Institute for Doping Analysis (Cologne, Germany) for supporting the study. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6170 Montes de Oca Porto R, Martinez Brito D, Correa Vidal T, Fiallo Fernández T, Hernández Domínguez D hGH levels and gender, sport, and endogenous corticosteroids in a Cuban population. Preliminary results Antidoping Laboratory Sports Medicine Institute, Havana, Cuba Abstract Considering the factors that influence its levels, WADA has set decision limits for the ratio of growth hormone (hGH) isoforms of pituitary to that recombinant for a positive result. On the other hand, it has been described the difference in several endogenous steroid concentrations according to the sport, and the relationship between GH and stress process. The main goal of this work was to observe preliminarily the behavior of the concentration of the isoforms and their ratios in Cuban athletes subdivided by sports and gender, as well as the correlation between hGH concentrations and endogenous corticoids as tetrahydrocortisol and cortisol. Concentrations of hGH isoforms in serum and concentrations of endogenous corticoids in urine were measured following laboratory SOPs. Descriptive statistics were obtained by sport and sex. A non- parametric test Mann-Whitney U test (α = 0.05) and a non-parametric correlation test Spearman rho and Kendall´s Tau tests (α=0.05) were applied. The main conclusions were: (i) significant differences between sexes for both isoform concentrations (lower in female) and no significant differences in recGH/pitGH ratio for both sexes were observed; (ii) in spite of the short number of samples, the lowest concentrations of both isoforms were observed in rowing, softball and handball, and the highest values for swimming (female and male). Ratio recGH/pitGH showed higher values for softball (female and male), (iii) no correlation was observed between isoform concentrations and endogenous corticoids probably influenced by the sample collection scheme. Introduction It is known that variations in hGH occurs with age, gender, physical exercise, among others. Also, the difference in concentrations of several steroidal hormones according to the sport, and the relationship between hGH and stress process has been described [1-5]. In order to study the behavior of hGH isoforms in Cuban athletes, a preliminary evaluation of hGH levels was done. The aims of the study were (i) to observe the concentration levels of pitGH and recGH and ratio between them in the Cuban population of athletes, (ii) to study the potential differences of the hGH levels among the sports and sexes and (iii) to study the correlation between hGH concentrations and two endogenous corticoids in urine. Experimental AutoLumat LB 953 Multi-Tube Luminometer from Berthold. hGH LIA Kit 2 were obtained from CMZ-Assay. Serum samples were prepared as described by the manufacturer. Recombinant and pituitary MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6171 isoform concentrations (ng/mL) were obtained by the instrument software and then, the ratio was calculated as recGH to pitGH (1:1). Gas chromatograph HP 6890 coupled to mass spectrometer single quadrupole HP 5973. Capillary column HP1 (17 m, 200 μm internal diameter, 0,11 μm thick stationary phase). Acquisition mode in SIM mode (m/z 636 for tetrahydrocortisol-TMS and m/z 632 for cortisol-TMS)[6]. Urine sample preparation was done according to Laboratory SOP to detect steroids in free + glucuroconjugate fractions in urine. Hydrolysis with β-glucuronidase (E. coli) and liquid-liquid extraction with tert-butyl methyl ether was done. Trimethylsilyl derivatives were obtained after the reaction of dry extract with MSTFA/NH I/2-mercaptoethanol [6]. Descriptive statistics were obtained by sport and sex. A non-parametric comparison was performed by applying the Mann-Whitney U test (α = 0.05) and a non-parametric correlation was performed by application of Spearman rho and Kendall´s Tau tests (α = 0.05) between cortisol and tetrahydrocortisol with hGH isoform concentrations and the ratio recGH to pitGH. Results and Discussion Application of Mann-Whitney U test (α=0.05) to compare GH isoforms concentrations showed that there are significant differences between female and male. Female showed lower concentrations of recGH and pitGH than male (Table 1 and Figure 1). This agrees with previous resports [3,4]. The ratio recGH/pitGH isoforms showed no significant differences between sexes in the studied group of Cuban athletes. Additionally, both male and female showed mean values considerably lower than the decision limit setting for this kit (male: 1.91 and female: 1.59) [7]. Today, the decision limits described in TD2021GH take into account the athlete´s sex and the used kit [7]. Following the results of the present study (in spite of the size of sample), the absence of significant differences between males and females should be considered in the future setting of the decision limit values. Statistical differences among sports were not addressed because of the small number of samples. Nevertheless, it can be observed that sports as rowing, softball and handball showed the lowest concentrations of recGH and pitGH, and swimming showed the highest values for both females and males. The ratio recGH/pitGH showed higher values for softball (females and males) (Table 2 and Fig. 2). It is noted that age was not taken into consideration introducing an important bias [3]. Even when the literature has been described the relationship between stress process and GH levels [5], no correlation was observed after correlating GH isoform concentrations and the ratio recGH/pitGH with endogenous levels of tetrahydrocortisol and cortisol (Spearman rho and Kendall´s Tau tests, α = 0.05). It is known the circadian cycle and excretion pulse mode of the studied hormones and therefore, these results are linked directly to the fact that the collection of samples (serum and urine) does not occur at the same time. 4 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6172 Table 1 . Statistics results obtained for recGH, pitGH, and recGH to pitGH ratio for the studied Cuban athletes. Percentile are presented both sexes Figure 1. Quantile-Quantile Plot and statistical results after comparison of male and female group by application of Mann-Whitney U test MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6173 Table 2. Descriptive statistics by sexes and sports, of the ratio recGH to pitGH. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6174 Figure 2. Box-Plot for concentrations of recGH and pitGH as well as ratio recGH to pitGH by sexes and sports. DL represents the decision limit for ratio recGH to pitGH (kit 2: female: 1.59 and male: 1.91) Conclusions A preliminary study was carried to evaluate the behavior of hGH isoform concentrations, the recGH/pitGH ratio, and the relationship to two endogenous corticosteroids in both sexes and eight sports of Cuban athletes. The main results were: (i) Significant differences between sexes for both isoform concentrations (lower in female) and no significant differences in the recGH/pitGH ratio for both sexes were observed. (ii) In spite of the small number of samples, the lowest concentrations of both isoforms were observed in rowing, softball and handball, and the highest values for swimming (female and male). The ratio recGH/pitGH showed higher values for softball (female and male). (iii) No correlation was observed between isoform concentrations and endogenous corticoids, probably influenced by the sample collection scheme. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6175 References 1. Erotokritou-Mulligan I, Bassett EE, Kniess A, Sönksen PH, Holt RIG. Validation of the growth hormone (GH)- dependent marker method of detecting GH abuse in sport through the use of independent data sets. Growth Horm IGF Res. 2007;17(5):416-423. doi:10.1016/j.ghir.2007.04.013 2. Thieme D, Hemmersbach P, eds. Handbook of Experimental Pharmacology: Doping in Sports. Vol 196. Springer-Verlag Berlin Heidelberg; 2010. doi:10.1007/978-3-642-00663-0 3. McHugh CM, Park RT, Sönksen PH, Holt RIG. Challenges in detecting the abuse of growth hormone in sport. Clin Chem. 2005;51(9):1587-1593. doi:10.1373/clinchem.2005.047845 4. Nelson AE, Meinhardt U, Hansen JL, et al. Pharmacodynamics of growth hormone abuse biomarkers and the influence of gender and testosterone: A randomized double-blind placebo-controlled study in young recreational athletes. J Clin Endocrinol Metab. 2008;93(6):2213-2222. doi:10.1210/jc.2008-0402 5. Saugy M, Robinson N, Saudan C, Baume N, Avois L, Mangin P. Human growth hormone doping in sport. Br J Sports Med. 2006;40(SUPPL. 1):35-40. doi:10.1136/bjsm.2006.027573 6. Martínez Brito D, Correa Vidal MT, Oropesa Rodríguez R, González Pérez O, Ledea Lozano OE. Cuantificación simultánea de andrógenos, estrógenos, corticoides y pregnanos mediante cromatografía de gases acoplada a espectrometría de masas. Rev Cuba Farm. 2014;48(4):550-561. 7. World Anti-Doping Agency (WADA). WADA Technical Document – TD2021GH Human Growth Hormone (hGH) Isoform Differential Immunoassays. 2021:1-9. MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6176 Wicka M, Kaliszewski P, Grucza K, Stanczyk D, Drapala A, Konarski P, Kowalczyk K, Kwiatkowska D Determination method of 27 prohibited glucocorticosteroids in human urine Polish Anti-Doping Laboratory, Warsaw, Poland Abstract Glucocorticosteroids (GC) are naturally produced steroid hormones or synthetic compounds that inhibit inflammatory processes. They are produced from cholesterol by the adrenal band layer. Clinically, GC are mainly used in the treatment of inflammatory conditions such as arthritis and dermatitis and as an adjuvant therapy for autoimmune diseases. GC are often abused in sport because of their anti- inflammatory performance. This work describes an analytical method to determine synthetic glucocorticosteroids in human urine. Urine samples were analyzed by means of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) using a UniSpary source. A method for the detection of 27 glucocorticosteroids in human urine was developed and validated. The method is suitable for routine analysis in doping control. Introduction Glucocorticosteroids (GC) are naturally produced steroid hormones or synthetic compounds that inhibit the inflammatory process [1]. They are produced from cholesterol by the adrenal band layer [2]. In humans, GC regulate a wide variety of biological processes, including energy metabolism, reaction to inflammation, and cardiac output [3]. Clinically, GC are mainly used for the treatment of inflammatory conditions such as arthritis and dermatitis and as an adjuvant therapy for autoimmune diseases [3]. The pharmacological activity of the various synthetic glucocorticosteroids is calculated on the basis of a pharmacological scale designed in comparison to hydrocortisone [2]. All synthetic GC are strong anti- inflammatory agents compared to hydrocortisone and regulate many processes. GC drugs should always be used under the supervision of a doctor and following the recommendations. Their improper long-term use or overdose increases the risk of adverse side effects [4]. GC are often abused in sport because of their anti-inflammatory performance. The aim of the presented work was to develop and validate an LC- MS/MS method for the detection of 27 synthetic glucocorticosteroids in human urine. The list of presented substances does not include compounds indicated in the WADA Prohibited List [5]. The novelty of the approach was the use of the UniSpray source. A compareison of the UniSpray source to electrospray will be presented in a separate publication. Experimental Chemicals and reagents Standards were purchased from SIGMA-Aldrich (Saint Louis, Missouri, USA), Toronto Research Chemicals (Toronto, Canada), LGC Standards (Lomianki, Poland), Dr. Ehrenstrofer GmbH (Augsburg, Germany), USP MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6177 Reference Standards (Basel, Switzerland), Steraloids (USA), NMIA (Canberra, Australia), and Cayman Chemical (Ann Arbor, USA). Solvents were from Fisher Chemical (Hampton, USA). Sample pre-treatment The sample preparation is a two-step procedure involving enzymatic deconjugation of glucuronides and then liquid-liquid extraction with 6 mL of methyl tert-butyl ether. The residue was reconstituted in 100 µL of mobile phase (acetonitrile/water 1/1 V/V), transferred in a vial and 5 µL was injected into the LC-MS/MS system. Instrumental analysis Chromatographic separation was conducted using a Waters Acquity I-Class UPLC System liquid chromatography with BEH C18 (1.7 µm, 100 mm x 2.1 mm) from Waters. The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B), and LC gradient was employed at the constant flow rate of 300 µL/min at 45°C. MRMs of the studied substances were traced with a Xevo TQ-XS mass spectrometer equipped with an UniSpray source. All analytes were investigated in the US mode. Desolvation gas flow was set at 1000 L/h at 600°C with ion source temperature at 150°C. The capillary voltage was 3.0 kV (Table 1). Table 1. Selected tandem mass spectrometry transition for qualitative and quantitative analysis + MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6178 Results and Discussion The characteristics of the test method, including linearity, sensitivity, limit of detection (LOD), limit of quantification (LOQ), analyte recovery, specificity, were determined by different experiments. All compounds were identified and their retention times ranged from 3.02 to 9.73 min (Figure 1). Figure 1. A plot of the chromatographic distribution of all 27 glucocorticosteroids in the analytical method The assay was linear in the range of concentration 0.75 ng/mL - 30 ng/mL (criterion: r ≥ 0.950). The sensitivity of the method was determined by quantifying the LOD and LOQ. The extraction efficiency of the compounds analyzed varied between 65.4% and 103.9% (Table 2). MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6179 Table 2. A- Calibration data for glucocorticosteroids ; B - Recovery percentage of glucocortycosteroids in urine ; C - LOD and LOQ values Specificity was assessed by analyzing 12 blank samples from different individuals (6 males and 6 females) and other different 73 blank samples. Figure 2 shows an example chromatogram for the strongest MRM for each glucocorticosteroid (for the final concentration of 30 ng/mL). 1 2 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6180 Figure 2. Typical MRM chromatograms of urine samples (A) and analyzed glucocorticosteroids (B) Conclusions All compounds (exception: prednisolamate and tixocortol) were fragmented into at least 4 ions. All the selected ionic transitions have proved to be specific. No interfering signals from the matrix were observed for each of the MRM - high selectivity of the method. The method is suitable for the use in complex biological matrices (% RSD 0.8, α‑PVP / Q2 > 0.7, methylone). In addition to the metabolites already described for alpha-PVP, new metabolite candidates were proposed using this straightforward metabolomics workflow, including a putative metabolite that possibly comes from the hydroxylated metabolites previously described. Regarding methylone, which in the original work was not possible to observe metabolites produced by zebrafish, putative metabolites from reduction, loss of ketone, and dehydrogenation were suggested by the metabolomic approach. The proposed workflow in association with ZWT may be interesting for future analyzes that seek to assess the in vivo metabolism of doping agents. 1 1 1 2 1 2 MANFRED DONIKE WORKSHOP 2021 Poster RECENT ADVANCES IN DOPING ANALYSIS (29) ISBN 978-3-86884-047-6258 RECENT ADVANCES IN DOPING ANALYSIS 2021 (29) Table of contents Lectures Poster Presentations Presentations Abstracts - Lectures Presentations Abstracts - Posters Scientific Board Contributions 1_Lectures 2_Posters 3_Presentations Abstracts - Lectures 4_Presentations Abstracts - Posters

Einen intensiven Austausch der Wissenschaft mit Sport und Politik zur Situation von Trainerinnen im deutschen Leistungssport gab es am Donnerstag den 2. Juli 2026. Im Rahmen des Trainerinnen-Symposiums im Rhein-Energie-Stadion in Köln kamen Akteurinnen und Akteure zusammen um auf Basis der Forschungsergebnisse des QualiFT-Projektes zu diskutieren, wie die Situation von Trainerinnen verbessert werden kann. Am 2. Juli 2026 fand im Rhein-Energie-Stadion das Trainerinnen-Symposium statt, das einen intensiven Austausch von Sport, Politik und Wissenschaft zur Situation von Trainerinnen im deutschen Leistungssport ermöglichte. Das Symposium fand im Rahmen des vom Bundesinstitut für Sportwissenschaft geförderten Forschungsprojekts „Trainerinnen – Qualifizierung von Frauen für den Trainerberuf“ (QualiFT) unter der Leitung von Prof. Dr. Astrid Schubring und Prof. Dr. Karen Petry statt. Ziel der Veranstaltung war es, nachhaltige Veränderungen im deutschen Sport anzustoßen, indem relevante Stakeholder zusammenkamen, um die Situation von Trainerinnen strategisch zu betrachten. Dabei wurden zentrale Projektergebnisse präsentiert, Handlungsempfehlungen für Politik, Sport und Wissenschaft entwickelt und internationale Ansätze zur Förderung von Frauen im Trainerberuf vorgestellt. Am Vorabend der Veranstaltung gab es ein Get-Together, und am Haupttag standen zwei hochkarätig besetzte Podiumsdiskussionen in Anlehnung an die Forschungsergebnisse des QualiFT-Projekts im Mittelpunkt. Anschließend folgten Workshops zu Themen wie faire Vergütung für Trainerinnen, Vereinbarkeit von Karriere und Familie sowie Kommunikationsstrategien für Frauen. Die Grußworte wurden von Dr. Babette Kibele, Leiterin der Abteilung Sport und Ehrenamt des Bundeskanzleramts, und Prof. Dr. Ansgar Thiel, Rektor der Deutschen Sporthochschule Köln, gehalten. Dr. Kibele betonte die Unterstützung der Politik bei der Finanzierung und Trainerausbildung und verwies auf eine weitere Befassung mit dem Thema am 7. Juli im Rahmen der Trainer-Offensive. Prof. Thiel mahnte die geringen Veränderungen in der Förderung von Trainerinnen in den letzten zwanzig Jahren an und betonte die Notwendigkeit, das Potenzial gut ausgebildeter Trainerinnen zu nutzen. Er betonte die Verpflichtung des organisierten Sports, seine Strukturen so anzupassen zu müssen, dass sich alle Menschen mit ihrem Potential voll entfalten können. In der ersten Podiumsdiskussion wurde von Dr. Babette Kibele die Bedeutung von Vorbildern hervorgehoben. Katja Kosak vom österreichischen Bundesministerium stellte das Gender Trainee-Programm vor und betonte die Notwendigkeit von Systemänderungen in den Fachverbänden in Personalentwicklung. Wiebke Fabinski vom DOSB unterstrich die Relevanz einer bildungspolitischen Anerkennung des Berufsbildes, und Karl Schwarzenbrunner vom Deutschen Eishockey-Bund verwies auf die Notwendigkeit, dass Fachverbände einen Nutzen in der Förderung von Trainerinnen erkennen. Prof. Schubring präsentierte Best-Practice-Beispiele aus dem internationalen Raum, darunter Quoten in der Schweiz, incentive-basierte Fördergelder in Neuseeland und Sichtbarkeit von Trainerinnen bzw. Storytelling in Australien. In der zweiten Podiumsdiskussion wurden Strategien zur Stärkung des Berufsbildes Trainerin sowie das Thema Familienvereinbarkeit diskutiert. Holger Hasse (Präsident, Trainer*innen Deutschland) und Lothar Linz (Direktor, Trainer Akademie Köln) betonten die Notwendigkeit struktureller Veränderungen und verbesserter Arbeitsbedingungen. Isabelle Sawade (DTB Teamchefin Rhythmische Sportgymnastik) und Lisa Brennauer (Bund Deutscher Radfahrer) teilten ihre Erfahrungen als Trainerinnen mit Vereinbarkeit von Leistungssport und Familie, während Karin Orgeldinger (Stiftung Deutsche Sporthilfe) und Lea Krüger (Athleten Deutschland) die Unterstützung von Athletinnen auf dem Weg zur Trainerin und die Erhöhung der Attraktivität des Trainerinnenberufs hervorhoben. In den anschließenden Workshops konnten die Teilnehmenden zwischen drei themenspezifischen Inhalten wählen. Die Ergebnisse wurden im Plenum berichtet, und die Teilnehmenden teilten ihre geplanten Schritte zur Förderung von Frauen in der Trainerinnentätigkeit. Der Tag endete mit einem Ausklang bei kühlen Getränken und Snacks. Zur QualiFT Projektseite Fotos: Jörn Wolter

Im Rahmen der PE1.14 „Internationale Sportpolitik und Entwicklungszusammenarbeit“ findet am kommenden Samstag, den 04.07.2026 eine „sportpolitische Messe“ zu verschiedenen spannenden Themen statt. Die Themen werden von den Studierenden in interaktiven Messeständen aufbereitet, präsentiert und diskutiert. Alle Interessierten sind herzlichen zwischen 10:00 und 13:00 Uhr in die Seminarräume 96 und 97 eingeladen. Eine Anmeldung ist nicht erforderlich. Wir freuen uns auf einen spannenden Tag mit vielen Einblicken in die nationale und internationale Sportpolitik. Mehr erfahren…

Am vergangenen Mittwoch verwandelte sich der Keller des Hockey- und Judozentrums in eine Nachrichtenzentrale. Im Rahmen eines Pop-Up-Newsroom-Projekts berichteten Studierende der University of Brighton und des SPJ der Deutschen Sporthochschule Köln gemeinsam über die WM-Spiele Schweiz gegen Kanada und Bosnien-Herzegowina gegen Katar. In den Seminarräumen wurden die beiden Begegnungen übertragen und von den Studierenden per Live-Blog, Statistik-Feed, Social-Media-Update und Audio-Kommentar begleitet. Zudem waren Reporterteams in der Kölner Innenstadt unterwegs, um Fanreaktionen einzufangen und direkt in den Newsroom zu übertragen. Die Inhalte wurden bilingual auf Deutsch und Englisch produziert und über die Kanäle der Universität Brighton ausgespielt. Dr. Inga Oelrichs, SPJ-Koordinatorin und Mitorganisatorin des Newsrooms, fasst zusammen: „Es war schön zu sehen, wie die einzelnen Inhalte ineinandergegriffen haben und sich die Studierenden damit gegenseitig unterstützt haben.“ Das Projekt mit Owen Evans fand im Rahmen des Besuchs von BA- und MA-Studierenden der University of Brighton an der Deutschen Sporthochschule statt. Die Gäste waren vom 22. bis zum 26. Juni in Köln. Arbeitsproben zum Medienprojekt gibt es hier: University of Brighton video coverage: https://x.com/OverTime_Online/status/2069879026949234927 German Sport University video coverage: https://x.com/OverTime_Online/status/2069870669878157735 Switzerland 2-1 Canada: https://overtimeonline.co.uk/switzerland-vs-canada-as-it-happened/ Schweiz 2-1 Kanada: https://overtimeonline.co.uk/schweiz-2-1-kanada-der-live-blog-zum-nachlesen/ Bosnia 3-1 Qatar: https://overtimeonline.co.uk/bosnia-herzegovina-vs-qatar-as-it-happened/ Bosnien-Herzegowina 3-1 Katar: https://overtimeonline.co.uk/bosnien-mit-historischem-21-sieg-gegen-katar/ Social media coverage: https://x.com/OverTime_Online

Institut für Kreislaufforschung und Sportmedizin, Abt. II Wie beeinflussen deine Zyklussymptome deine sportliche Leistungsfähigkeit und Trainingsbeteiligung im normalen Trainingsalltag? ZIEL DER STUDIE Wir möchten besser verstehen, wie sich Zyklussymptome im Trainingsalltag bemerkbar machen. Dafür untersuchen wir, ob und wie Zyklussymptome deine wahrgenommene sportliche Leistungsfähigkeit, dein Belastungsempfinden und deine Trainingsbeteiligung beeinflussen. Ergänzend werden Hormonanalysen durchgeführt, um die erhobenen Daten umfassender analysieren zu können. WAS DICH ERWARTET Wir begleiten dich über 4 aufeinanderfolgende Zyklen und erfassen täglich über kurze Online-Abfragen, welche Zyklussymptome aufgetreten sind, wie du deine sportliche Leistungsfähigkeit einschätzt, wie anstrengend dein Training war und ob du wie geplant trainieren konntest. Zusätzlich wird dir für die Hormonanalysen 1x pro Zyklus Blut abgenommen. → Dein normaler Trainingsalltag bleibt unverändert: Du trainierst wie gewohnt, ohne zusätzliche sportliche Tests oder Vorgaben durch die Studie. WER KANN TEILNEHMEN? → Sportlich aktive Frauen im Alter von 18-40 Jahren, die nicht Rauchen und keine langwierigen Verletzungen aufweisen. DEINE VORTEILE KONTAKT Wenn wir dein Interesse geweckt haben oder du Fragen an uns hast, melde dich gerne bei uns! → franziska.moser@stud.dshs-koeln.de Abschlussfragebogen Vorgespräch Tägl. Online Abfrage (max. 5 min) über 4 Zyklen 1 Termin zur Blutentnahme pro Zyklus Natürlicher Zyklus → regelmäßiger Zyklus → inkl. Kupferspirale/-kette/-ball Hormonelle Verhütung → orale Verhütungsmittel (Kombi-/Mini-Pille) → Hormonspirale Zyklusstörung → diagnostizierte Zyklusstörung (z.B. Ausbleiben der Periode, zu kurzer/ zu langer Zyklus, PMS, PMDD) Kostenloses, umfangreiches HORMONPROFIL → z.B. Sexualhormone, Knochenmarker Individuelle zyklus-basierte TRAININGSEMPFEHLUNGEN → spezifisch für deinen Zyklus & Trainingsalltag Erkenne & Verstehe individuelle ZYKLUSMUSTER und Zusammenhänge mit deinem Training besser → Wir führen mit dir ein umfangreiches Zyklustracking durch