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Martial Saugy, Reds - University of Lausanne Federico Ponzetto, Swiss Laboratory for Doping Analysis - University of Lausanne

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Martial Saugy, Reds - University of Lausanne Federico Ponzetto, Swiss Laboratory for Doping Analysis - University of Lausanne ABP Steroidal Module in blood Prof. Martial Saugy, REDs - University of Lausanne Federico Ponzetto, Swiss Laboratory for Doping Analysis - University of Lausanne 05.11.18 ABP Symposium - Rome A step forward… URINE DRAWBACKS BLOOD MATRIX Confounding factors • Not easy to manipulate • Reduced bacterial contamination • Snapshot of athletes’ physiological condition ENDOGENOUS EXOGENOUS • Accurate pharmacokinetics information UGT2B17 Bacterial contamination • Trend in clinical analyses (evaluation/expertise) polymorphism Ethanol consumption • 1 Sample -> 2 ABP modules GC-MS Sensitivity • Few data in doping context T patch & gel administration • Invasive sampling (ethics) • Sample stability (48h) T. Kuuranne et al. • Transportation strategy Confounding factors and genetic polymorphism in the evaluation of • Small sample volume (4mL) individual steroid profiling. Br. J. Sports Med. (2014) 48(10):848-55 Blood Steroid Profiling – Target Compounds H C O O 3 O CH CH OH 3 3 OH CH3 SampleUHPLC Preparation-MS/MS H HO 14 TARGET ANALYTES CH CH3 CH 3 3 Acquity BEH C18 100 x 2,1 mm , ID 1.7 µm Flow rate 400ISOLUTEµL/min SLE+ Temperature 30°C(Biotage) O O O Inj. volume2 Progestogens10µL Progesterone Deoxycorticosterone Corticosterone H C O O 3 O Phase A H O + 0.1% FA OH 4 Glucocorticoids2 OH CH3 CH CH OH 3 3 Loading 200 µL serum + 200µL H2O OH OH Phase B5 AndrogensACN + 0.1% FA HO Elution 700 µL DCM CH CH CH Gradient 0.5 min 25% B 3 3 3 Evaporation3 Estrogens6 min 58% B 100 µL MeOH/H2O Reconstitution 8 min 98% B 50/50 (v/v) O O O 11 min Total Run Time 17α-Hydroxyprogesterone 11-Deoxycortisol Cortisol O O O CH3 CH3 CH3 CH CH 3 3 OH CH3 HO O HO OH DHEA Androstenedione Estrone OH OH OH CH3 CH3 CH3 HO CH CH 3 3 Estriol O O HO H DHT Testosterone Estradiol Reference Intervals LLOQ REFERENCE INTERVAL COMPOUND 1879 Elite Athletes (nmo/L) (nmol/L) (1056 M, 824 F) 6.67 – 30.53 Testosterone 0.07 ♂ 0.26 – 1.80 ♀ Testosterone Androstenedione 0.17 1.08 – 6.36 50 45 0.05 – 0.32 Progesterone 0.05 ♂ 40 0.06 – 39.59 ♀ 35 17αOH-progesterone 0.30 0.34 – 7.71 30 ♀ 25 ♂ DHEA 1.73 3.04 – 22.41 samples of 20 ° 0.46 – 2.76 N 15 DHT 0.17 ♂ n.d. – 0.92 ♀ 10 5 Corticosterone 0.29 0.85 – 38.47 0 0 3 6 9 12 15 18 21 24 27 30 Cortisol 2.76 64.82 – 696.69 1.5 4.5 7.5 10.5 13.5 16.5 19.5 22.5 25.5 28.5 11-deoxycortisol 0.07 n.d. – 2.20 Concentration [nmol/L] Testosterone Clinical Study Population: 19 healthy men (UGT2B17: 7 ins/ins, 7 ins/del, 5 del/del) Sun Mo Sun Mo Sun Mo Sat Tue Sat Tue 2 weeks Sat Tue Fri Wed Fri Wed Fri Wed Thu Thu Thu CONTROL PATCH WASH OUT ORAL 0h, 2h, 4h, 8h, 12h 0h, 2h, 4h, 8h, 12h = 2 x 2.4mg/24h T 0h, 2h, 4h, 8h, 12h 24h 24h 24h 48h, 60h 48h, 60h 72h = 2 x 40mg TU 72h 96h 96h Multivariate Statistical Analysis H C O O 3 O CH CH OH 3 3 OH CH3 H HO CH CH CH Parallel Factor 3 3 3 Analysis (PARAFAC) O O O Progesterone Deoxycorticosterone Corticosterone H C O O 3 O Circadian Rhythm OH OH CH3 CH CH OH 3 OH 3 HO OH CH CH CH Testosterone Intake 3 3 3 O O O 17α-Hydroxyprogesterone 11-Deoxycortisol Cortisol O O O CH3 CH3 CH3 CH CH 3 3 OH CH3 HO O HO OH DHEA Androstenedione Estrone OH OH OH CH3 CH3 CH3 HO CH CH 3 3 Estriol O O HO H DHT Testosterone Estradiol Longitudinal Monitoring ORAL TRANSDERMAL Testosterone Testosterone 40 60 35 48h 3X 50 30 ins/ins 40 25 20 30 ins/del 15 20 10 NO AUGMENTATION 10 del/del 5 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 F. Ponzetto et al. Longitudinal monitoring of endogenous DHT DHT steroids in human serum by UHPLC- 4.0 3.5 MS/MS as a tool to detect testosterone 2 -12h 4X 3.0 48h 3X 3.0 abuse in sports. 2.5 Anal. Bioanal. Chem.l. (2016), 408 (3):705-719 2.0 2.0 1.5 1.0 1.0 T intake 0.5 0.0 0.0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 2 case reports Female athlete 1 Female athlete 2 Untargeted Approach - Steroidomics Endogenous Sample Preparation SPE glucuronides OASIS HLB (Waters) Endogenous Loading 200 µL serum + 200µL H3PO4 4% sulfates Washing 400 µL H2O/MeOH 95/5 (v/v) + 0.1% NH4OH Elution 50 µL DCM Nucleosides & Reconstitution 50 µL MeOH/H2O Derivatives 90/10 (v/v) UHPLC-HRMS Kinetex C18 150 x 2,1 mm , ID 1.7 µm Flow rate 300 µL/min Temperature 30°C ? Inj. volume 10µL Organic acids Phase A H2O + 0.1% FA Phase B ACN + 0.1% FA Gradient 0.5 min 25% B 16.8 min 95% B Total Run Time 27 min About 40’000 detected peaks Focus on Steroid Compartment Endogenous glucuronides FILTRATION Endogenous In-house Database (130 steroidal compounds) sulfates Online Database NucleosidesEndogenous & DerivativesFree steroids (Ret. Time prediction) ? Literature Organic acids Scientific Knowledge About 250 putative steroids Biomarkers Molecular Annotatio Feature m/z rT Adduct ID Rank formula n Level N125 465.2497 8.42 C25H38O8 1 [M-H]- Androsterone Gluc 1 N96 481.2452 7.43 C25H38O9 3 [M-H]- Hydroxyandrosterone/Hydroxyetiocholanolone Gluc isomer 2 N85Phase481.2453II metabolites7.18 C25H38O9 3 [M-H]- Hydroxyandrosterone/Hydroxyetiocholanolone Gluc isomer 3 N30 increase477.213 more5.82 than C25H34O9 3 [M-H]- Hydroxyandrostenedione Gluc isomer 4 N120 465.2497 8.27 C25H38O8 1 [M-H]- Etiocholanolone Gluc 5 N168 Free463.1958 steroids13.27 C24H32O9 3 [M-H]- Hydroxyestradiol Gluc isomer 6 N112 467.2656 7.86 C25H40O8 1 [M-H]- 5(a/b)-Androstan-3a,17b-diol-17-Gluc * 7 N39 479.2295 6.15 C25H36O9 3 [M-H]- Hydroxytestosterone/HydroxyDHEA Gluc isomer 8 N61 477.2126 6.67 C25H34O9 3 [M-H]- Hydroxyandrostenedione Gluc isomer 9 N77 463.2354 7.07 C25H36O8 1 [M-H]- Testosterone Gluc 10 N28 479.2299 Glucuronides5.79 C25H36O9 3 [M-H]- Hydroxytestosterone/HydroxyDHEA Gluc isomer High presence of 11 N62 481.2452 6.73 C25H38O9 3 [M-H]- Hydroxyandrosterone/Hydroxyetiocholanolone Gluc isomer 12 N66 479.2297 increase6.81 moreC25H36O9 than 3 [M-H]- Hydroxytestosterone/HydroxyDHEA Gluc isomerHydroxylated 13 N58 381.1386 6.54 C19H26O6S 3 [M-H]- Hydroxyandrostenedione Sulf isomer 14 N74 479.2293 7.01SulfatesC25H36O9 2+ [M-H]- 11-Ketoetiocholanolone Gluc compounds 15 N123 369.1743 8.37 C19H30O5S 1 [M-H]- Androsterone/Etiocholanolone Sulf* 16 N111 463.2339 7.86 C25H36O8 3 [M-H]- Androstanedione Gluc isomer 17 N67 451.2342 6.82 C24H36O8 3 [M-H]- 17b-hydroxy-5b-estran-3-one Gluc 18 N14 479.2295 5.4 C25H36O9 3 [M-H]- Hydroxytestosterone/HydroxyDHEA Gluc isomer 19 N32 381.1388Urinary5.89 C19H26O6S 3 [M-H]- Hydroxyandrostenedione Sulf isomer 20 N51 Steroidal453.1576 Markers6.45 C22H30O8S 3 [M-H]- 11b,20-Dihydroxy-3-oxopregn-4-en-21-oic acid Sulf 21 N115 369.1736 8.11 C19H30O5S 1 [M-H]- Epiandrosterone Sulf 22 N16 also increasing453.1566 5.44in bloodC22H30O8S 3 [M-H]- 11b,20-Dihydroxy-3-oxopregn-4-en-21-oic acid Sulf 23 N53 541.2662 6.46 C27H42O11 3 [M-H]- Tetrahydrocortisol Gluc isomer 24 N50 385.1696 6.45 C19H30O6S 3 [M-H]- Hydroxyandrosterone/Hydroxyetiocholanolone Sulf isomer 25 Longitudinal Monitoring NEW BIOMARKERS 5α(β)Adiol Gluc. Etiocholanolone Gluc. 800,000 1,000,000 24 h 9X 800,000 8 h 3X 600,000 600,000 400,000 400,000 200,000 200,000 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 0 1 2 3 4 5 6 7 8 9 10 11 12 13 T intake Androsterone Gluc. Epiandrosterone Sulf. 2,000,000 300.000 12 h 7X 250.000 48 h 20X 1,500,000 200.000 1,000,000 150.000 100.000 500,000 50.000 0 0.000 0 1 2 3 4 5 6 7 8 9 10 11 12 13 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Conclusions & Perspectives Longitudinal monitoring of blood T & DHT showed no differences between UGT2B17 genotypes Compared to urinary T/E, increased sensitivity for transdermal administration Steroidomics highlighted additional biomarkers of T intake (phase II met.) Development and validation of a new quantitative UHPLC-MS/MS method Application of the method on athletes’ samples to obtain reference values (first point ABP) and check stability across time Aknowledgements COLLABORATORS Dr. Tiia Kuuranne Dr. Julien Boccard Dr. Raul Nicoli Prof. Serge Rudaz FUNDING.
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