Steroid profiling in urine of intact glucuronidated and sulfated steroids using liquid chromatography-mass spectrometry.

Detection of endogenous anabolic androgenic steroids (EAAS) misuse is a major challenge in doping control analysis. Currently, a number of endogenous steroids, which constitute the steroid profile, are quantified using gas chromatography (GC). With this methodology, only the sum of the free and glucuronidated steroids is measured together. A dilute-and-shoot LC-MS method, which is compliant with the quality requirements for measuring EAAS established by the World Anti-Doping Agency (WADA), was developed and validated containing glucuronidated and sulfated steroids in order to gain some extra information and to expand the existing steroid profile. The developed method is, to the best of our knowledge, the first method to combine both steroid glucuronides and sulfates, which is compliant with the quality standards of the technical document on EAAS, established by WADA. The first advantage of this new steroid profile is the reduced sample preparation time, as it is a direct injection method of diluted urine. A second advantage is the ability of the used gradient to separate 5α-androstane-3α,17β-diol-3-glucuronide (5ααβdiol3G), 5α-androstane-3α,17β-diol-17-glucuronide (5ααβdiol17G), 5β-androstane-3α,17β-diol-3-glucuronide (5βαβdiol3G) and 5β-androstane-3α,17β-diol-17-glucuronide (5βαβdiol17G) allowing to gain specific information on these isomers, which cannot be accomplished in GC-MS screening due to hydrolysis. This steroid profile also contains free testosterone, 5α-androstane-3,17-dione and 5β-androstane-3,17-dione as markers of degradation. In total, 17 compounds and 10 isotopically labelled internal standards are included in this method.