title: Analysis of anabolic androgenic steroids as sulfate conjugates using high performance liquid chromatography coupled to tandem mass spectrometry.
abstract: Improvements in doping analysis can be effected by speeding up analysis time and extending the detection time. Therefore, direct detection of phase II conjugates of doping agents, especially anabolic androgenic steroids (AAS), is proposed. Besides direct detection of conjugates with glucuronic acid, the analysis of sulfate conjugates, which are usually not part of the routine doping control analysis, can be of high interest. Sulfate conjugates of methandienone and methyltestosterone metabolites have already been identified as long-term metabolites. This study presents the synthesis of sulfate conjugates of six commonly used AAS and their metabolites: trenbolone, nandrolone, boldenone, methenolone, mesterolone, and drostanolone. In the following these sulfate conjugates were used for development of a fast and easy analysis method based on sample preparation using solid phase extraction with a mixed-mode sorbent and detection by high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Validation demonstrated the suitability of the method with regard to the criteria given by the technical documents of the World Anti-Doping Agency (WADA). In addition, suitability has been proven by successful detection of the synthesized sulfate conjugates in excretion urines and routine doping control samples.
authors.affiliation: Oslo University Hospital, Norwegian Doping Control Laboratory, Oslo, Norway.
authors.forename: S
authors.initials: S
authors.lastname: Rzeppa
authors.affiliation: Oslo University Hospital, Norwegian Doping Control Laboratory, Oslo, Norway.|University of Münster, Institute of Food Chemistry, Münster, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Heinrich
authors.affiliation: Oslo University Hospital, Norwegian Doping Control Laboratory, Oslo, Norway.|School of Pharmacy, University of Oslo, Norway.
authors.forename: P
authors.initials: P
authors.lastname: Hemmersbach
mesh_terms: D000328:Adult; D045930:Anabolic Agents / Q000652:urine*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D004300:Doping in Sports; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008297:Male; D008655:Mesterolone / Q000652:urine*; D050217:Metabolic Detoxication, Phase II; D058573:Performance-Enhancing Substances / Q000652:urine*; D011237:Predictive Value of Tests; D012015:Reference Standards; D065667:Renal Elimination; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000592:standards; D013256:Steroids / Q000652:urine*; D015813:Substance Abuse Detection / Q000379:methods* / Q000592:standards; D013431:Sulfates / Q000652:urine*; D053719:Tandem Mass Spectrometry / Q000592:standards; D016482:Urinalysis
chemical_list: D045930:Anabolic Agents; D058573:Performance-Enhancing Substances; D013256:Steroids; D013431:Sulfates; D008655:Mesterolone
keywords: anabolic androgenic steroids; doping analysis; liquid chromatography-mass spectrometry; metabolism; sulfate conjugates