Gamle prøver nye muligheter Kromatografi i hverdagen Spesialkurs NITO Oslo, 4.2.2015 Peter Hemmersbach Oslo universitetssykehus og Farmasøytisk institutt Universitetet i Oslo 1 3 Dilemmaet for antidopingarbeidet Doperne ligger vel alltid et hestehode foran. Brødrene Grimms eventyr: Der Hase und der Igel. International Standard for Laboratories 2015 ISL 5.2.2.6: If the Testing Authority has arranged for storage of the Samples for a period from three months to ten years, the Laboratory shall ensure that the Samples are stored in a secure location under continuous chain of custody;. long-term storage of Samples (ISL 5.2.2.12) shall be conducted and recorded under the Laboratory Internal Chain of Custody. 2 4
Intention of long term storage and re-analysis Extended knowledge about metabolism of doping agents Development of analytical techniques for new substances Improvement of existing analytical techniques Changes in technical documents based on the above mentioned developments Further analysis restricted to substances and methods which were prohibited at the time of sample collection 5 WADAs Dopingliste 2015 I. STOFFER OG METODER SOM ER FORBUDT BÅDE I OG UTENFOR KONKURRANSE S0: Ikke godkjente stoffer S5: Diuretika og andre S1: Anabole stoffer maskeringsmidler S2: Peptidhormoner, vekstfaktorer M1: Forbedring av oksygenopptak/oksygentransport og relaterte substanser S3: Beta-2-agonister M2: Kjemisk of fysisk S4: Hormon- og metabolske manipulasjon modulatorer M3: Gendoping II. STOFFER OG METODER SOM ER FORBUDT I KONKURRANSE I tillegg til kategoriene S1-S5 og M1-M3 definert over, er også følgende grupper forbudt i konkurranse: S6: Stimulerende midler S7: Narkotiske stoffer S8: Cannabis (cannabinoider) S9: Glukokortikoider 7 Intention of long term storage and re-analysis Extended knowledge about metabolism of doping agents Development of analytical techniques for new substances Improvement of existing analytical techniques Changes in technical documents based on the above mentioned developments Further analysis restricted to substances and methods which were prohibited at the time of sample collection 6 Anabol-androgene steroider effektive dopingmidler 8
Screening methods in doping analysis SCR IV Anabolic agents Narcotics Glucucorticoids Beta-2 agonists Cannabinoids Hormone and mebolic modulators SCR V/VII Diuretics Narcotics Stimulants Beta-blockers SCR VI Masking agents (HES, dextrane) SCR IX Masking agents (Glycerol) SCR X/XI Proteins, peptides (Insulins, GHRP s) SCR hcg VIII EPO, GH Hydrolysis LLE dilute and shoot SPE GC- MS/MS LC-MS/MS Biochemical methods 9 11 Fastfase-ekstraksjon (C 18 ) Steroidanalyse i urin Enzymatisk hydrolyse Væske-væske ekstraksjon Inndampning Inndampning Derivatisering MSTFA:NH 4 I:Ethanthiol Oppløsning i vann/acetonitril GC-MS/MS LC-MS/MS ESI, positiv polaritet Metandienone long term metabolites 11 17 -Metyl-17 -hydroksymetyl- 18-norandrosta-1,4,13-trien-3-on 10 12
Increased detection times for metandienone metabolites 10 9 8 11 W. Schänzer et al. Rapid Comm. Mass Spectrom. 2006, 20, 2252. 13 SRM-Identification of a long term CH metandienone metabolite 2 OH O 15 H O LC-MSMS-Identification of a long term metandienone metabolite Athlete sample Negative control sample Positive control sample 14 Methyltestosterone towards new long term metabolites O H O H O O H H O H M2 H 16 M1
Methyltestosterone metabolism Gómez et al. TrAC 53, 106-116 (2014) Norges laboratorium at o Gómez for dopinganalyse et al. Steroids 78 (1), 44-52 (2013). 17 Comprehensive study of anabolic-androgenic steroid (AAS) metabolism by mass spectrometric techniques Gómez et al. TrAC 53, 106-116 (2014) 19 Detection times of the different metabolites of methyltestosterone O H HO 3 SO H 17 -Methyl-5 -androstane-3,17 -diol-3-sulphate Gómez et al. Steroids 78 (1), 44-52 (2013). 18 New long term metabolites (LTM) implemented into GC/LC-MSMS screening procedures Geyer H, et al. Br J Sports Med 2014;48:820 826 20
Adverse analytical findings for metandienone (a), dehydrochloromethyltestosterone (b) and stanozolol (c)* a) b) c) *) in the WADA-lab in Cologne, Geyer H, et al. Br J Sports Med 2014;48:820 826 21 23 Intention of long term storage and re-analysis Extended knowledge about metabolism of doping agents Development of analytical techniques for new substances Improvement of existing analytical techniques Changes in technical documents based on the above mentioned developments Further analysis restricted to substances and methods which were prohibited at the time of sample collection 22 EPO, fra første til tredje generasjon 2007 1987 2001 24
Isoelektrisk fokusering (IEF) 25 Re-analysis of samples http://www.insidethegames.biz/sports/winter/biathlon/1024592-russian-biathletes-facingextended-bans-after-samples-re-analysed 27 Intention of long term storage and re-analysis Extended knowledge about metabolism of doping agents Development of analytical techniques for new substances Improvement of existing analytical techniques Changes in technical documents based on the above mentioned developments Further analysis restricted to substances and methods which were prohibited at the time of sample collection 26 28
IEF: Isoelectric focussing SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis SAR-PAGE: sarcosyl polyacrylamide gel electrophoresis 29 Konklusjon Prøveoppbevaring på opp til 10 år Mer effektiv dopingkontroll Ny kunnskap om metabolisme Utvikling av nye analyseteknikker Nytt regelverk Forbedring av eksisterende analyseteknikker 31 SDS-PAGE Images and SAR-PAGE Images Mads, det er bedre å arbeide innenfra! 30 32