New long-standing metabolites of 17-methyltestosterone are detected in HepG2 cell in vitro metabolic model and in human urine

Novel metabolites of the anabolic androgenic steroid 17 alpha-methyltestosterone have been detected in HepG2 cell in vitro metabolic model and in human urine. Their detection was accomplished through targeted gas chromatography-(tandem) mass spectrometry analysis that has been based on microscale synthesized standards. The related synthesis and the gas chromatography-(tandem) mass spectrometry characterization of the analytical standards are described. All newly presented metabolites have a fully reduced steroid A-ring with either an 17,17-dimethyl-18-nor-Delta 13 structure or they have been further oxidized at position 16 of the steroid backbone. Metabolites with 17,17-dimethyl-18-nor-Delta 13 structure may be considered as side products of phase II metabolic sulfation of the 17 beta-hydroxy group of methyltestosterone or its reduced tetrahydro-methyltestosterone metabolites 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol and 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol that produce the known epimeric 17 beta-methyl-5 beta-androstane-3 alpha,17 alpha-diol and 17 beta-methyl-5 alpha-androstane-3 alpha,17 alpha-diol metabolites. The prospective of these new metabolites to increase detection time windows and improve identification was investigated by applying the World Anti-doping Agency TD2021IDCR criteria. The new metabolites, presented herein, complement the current knowledge on the 17 alpha-methyltestosterone metabolism and in some cases can be used as additional long-term markers in the frame of sport doping drug testing.