A further insight on methyltestosterone metabolism: new evidences from in vitro and in vivo experiments.

Rationale Although the metabolism of methyltestosterone (MT) has been extensively studied since the 1950s by different techniques, the goal of this investigation was to study the hydroxylation in positions C2-, C4- and C6- after in vitro experiments and in vivo excretion studies analysed by GC/TOF and GC/MS/MS. The results could be influenced by the mass spectrometric analyser used. Methods Incubations were carried out with human liver microsomes and 6 enzymes belonging to cytochrome P450 family (CYP) using MT as substrate. The trimethylsilyl (TMS) derivatives of the samples were analysed by GC/TOF, and by GC/MS/MS once the correct MS/MS transitions had been selected, mainly for 6-hydroxy-MT (6-OH-MT) to avoid artefact interferences. A urinary excretion study was then performed after the administration of a 10 mg single oral dose of MT to a volunteer. Results The formation of hydroxylated metabolites of MT in the C6-, C4- and C2- positions after both in vitro and in vivo experiments was observed. Sample evaluation by GC/TOF showed an interference for 6-OH-MT that could only be resolved in GC/MS/MS by monitoring specific transitions. The transitory detection of these hydroxylated metabolites in urine agrees with previous investigations that had described this metabolic route as being of little significance. Conclusion In doping analysis, the formation of 4-hydroxy-methyltestosterone (oxymesterone) from methyltestosterone cannot be underestimated. Although it is only detected as a minor and short-term excretion metabolite, it cannot be overlooked as it was found in both in vitro and in vivo experiments. The use of a combination of different mass spectrometric instruments allowed reliable conclusions to be reached, and it was shown that special attention must be given to artefact formation.