Comprehensive metabolic profiling of fluoxymesterone in vivo by GC-Orbitrap HRMS: a integrated strategy for novel biomarkers discovery in doping analysis.

Fluoxymesterone (9α-fluoro-11β,17β-dihydroxy-17α-methyl-androst-4-en-3-one) is a prohibited anabolic androgenic steroid (AAS) under the regulations of the World Anti-Doping Agency (WADA). Comprehensive elucidation of its metabolic pathways and the identification of novel metabolites are essential for improving doping control. In this study, two healthy male volunteers were orally administered a single 10 mg dose of fluoxymesterone, and urine samples were systematically collected over a 30-day period. An optimized three-step pretreatment procedure combined with gas chromatography-Orbitrap high-resolution mass spectrometry (GC-Orbitrap-HRMS) enabled the comprehensive detection of free, glucuronide-conjugated, and sulfate-conjugated metabolites. In total, the parent compound and ten metabolites were identified, including five novel structures reported for the first time. The primary metabolic transformations involved 4-ene reduction, 3-keto reduction, 6β-hydroxylation, 11β-hydroxy oxidation, and skeletal rearrangement. Excretion profiling demonstrated substantial interindividual variability in metabolite concentrations and clearance kinetics. Notably, the newly identified metabolite F-M7 (9α-fluoro-17α-methyl-3ξ,11β,17β-triol-5ξ-androstane) was detectable as a glucuronide conjugate for up to 5 days post-administration, highlighting its potential as a reliable biomarker. These findings expand the understanding of fluoxymesterone metabolism in vivo and propose promising analytical targets for enhancing the sensitivity and reliability of anti-doping strategies.