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A further insight into methyltestosterone metabolism: New evidences from in vitro and in vivo experiments
Author(s) -
MartinezBrito Dayamin,
Ian Michele,
Tatangelo Maria Antonietta,
Molaioni Francesco,
Torre Xavier,
Botrè Francesco
Publication year - 2020
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.8870
Subject(s) - chemistry , methyltestosterone , metabolite , in vivo , chromatography , hydroxylation , mass spectrometry , urine , excretion , microsome , metabolism , gas chromatography–mass spectrometry , tandem mass spectrometry , cytochrome p450 , in vitro , enzyme , biochemistry , medicine , microbiology and biotechnology , biology
Rationale Although the metabolism of methyltestosterone (MT) has been extensively studied since the 1950s using different techniques, the aim of this study was to investigate the hydroxylation in positions C2, C4 and C6 after in vitro experiments and in vivo excretion studies using gas chromatography time‐of‐flight (GC/TOF) and gas chromatography/tandem mass spectrometry (GC/MS/MS). The results could be influenced by the mass spectrometric analyser used. Methods Incubations were carried out with human liver microsomes and six enzymes belonging to the cytochrome P450 family using MT as a substrate. The trimethylsilyl derivatives of the samples were analysed using GC/TOF and GC/MS/MS once the correct MS/MS transitions had been selected, mainly for 6‐hydroxymethyltestosterone (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 using 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. Conclusions In doping analysis, the formation of 4‐hydroxymethyltestosterone (oxymesterone) from MT 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.