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

Abstract

Novel metabolites of the anabolic androgenic steroid 17α‐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‐Δ13 structure or they have been further oxidized at position 16 of the steroid backbone. Metabolites with 17,17‐dimethyl‐18‐nor‐Δ13 structure may be considered as side products of phase II metabolic sulfation of the 17β‐hydroxy group of methyltestosterone or its reduced tetrahydro‐methyltestosterone metabolites 17α‐methyl‐5β‐androstane‐3α,17β‐diol and 17α‐methyl‐5α‐androstane‐3α,17β‐diol that produce the known epimeric 17β‐methyl‐5β‐androstane‐3α,17α‐diol and 17β‐methyl‐5α‐androstane‐3α,17α‐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 17α‐methyltestosterone metabolism and in some cases can be used as additional long‐term markers in the frame of sport doping drug testing.