Comprehensive Profiling of 6α‐Chloro‐Testosterone Metabolism in Human Urine Using Gas Chromatography–Mass Spectrometry

According to its structure, 6α-chloro-17β-hydroxyandrost-4-en-3-one (6-CT) can exhibit both anabolic and antiestrogenic effects, and as such, it is prohibited in sports according to the World Anti-Doping Agency rules. The aim of this paper was to study the metabolism of 6-CT in humans by gas chromatography couple to mass spectrometry in tandem (GC-MS/MS), time-of-flight (GC-qTOF) and isotopic ratio (GC-C-IRMS), to describe changes in the endogenous steroid profile and the influence of 6-CT consumption on the carbon isotope ratio (CIR). One single oral dose of 6-CT (25 mg) was administered to two volunteers, and preadministration and postadministration samples of urine were collected for, at least, 4 days. Halogenated and dehalogenated metabolites were detected considering the accurate mass of the structure, the endogenous steroid profile was quantified and CIR values were measured. All urinary concentrations or areas were adjusted to a specific gravity of 1.020. The results showed halogenated and dehalogenated metabolites of 6-CT, considering the Phase I main reactions of steroids. The potential favoured 5β- reduction pathway allowed the formation of 5β- metabolites that are known to have weak or null androgenic activity (e.g., 6β-hydroxy-etiocholanolone, 5β-dihydrotestosterone and 5β-androstanediol), but the impact on the steroid profile evaluated in the Athlete Biological Passport (ABP) should be carefully assessed.