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

• Published in: Drug testing and analysis 2024-06, Vol.16 (6), p.604-615
• Main Authors: Angelis, Yiannis S., Sakellariou, Panagiotis, Fragkaki, Argyro G., Karnava, Sophia, Goula, Olga, Kiousi, Polyxeni, Kioukia‐Fougia, Nassia, Georgakopoulos, Costas, Loui, Stella, Chlapana, Fotini, Kletsas, Dimitris
• Format: Article
• Language: English
• Subjects: 17α‐methyltestosterone; Anabolic Agents - metabolism; Anabolic Agents - urine; anabolic androgenic steroids; Androgens - metabolism; Androgens - urine; Chromatography; doping control; Doping in Sports; Gas Chromatography-Mass Spectrometry - methods; Hep G2 Cells; HepG2; Humans; in vitro metabolism; Mass spectrometry; Metabolism; Metabolites; Methyltestosterone - metabolism; Methyltestosterone - urine; Scientific imaging; Steroids; Substance Abuse Detection - methods; Tandem Mass Spectrometry - methods
• ISSN: 1942-7603; 1942-7611
• DOI: 10.1002/dta.3589

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. Novel fully A‐ring‐reduced metabolites of methyltestosterone were predicted and synthesized on a microscale. Their GC‐MS/(MS) behavior was studied to develop a targeted detection method. Targeted analysis of methyltestosterone HepG2 cell incubation supernatants and post‐administration urine samples confirmed the presence of these novel methyltestosterone metabolites in both matrices. Evaluation of their human urine post‐administration excretion profiles revealed that some of them could be used as additional long‐term metabolites for the monitoring of methyltestosterone in the frame of doping control.