Alternative long-term markers for the detection of methyltestosterone misuse

Methyltestosterone (MT) is one of the most frequently detected anabolic androgenic steroids in doping control analysis. MT misuse is commonly detected by the identification of its two main metabolites excreted as glucuronide conjugates, 17α-methyl-5α-androstan-3α,17β-diol and 17α-methyl-5β-androstan-3α,17β-diol. The detection of these metabolites is normally performed by gas chromatography–mass spectrometry, after previous hydrolysis with β-glucuronidase enzymes, extraction and derivatization steps. The aim of the present work was to study the sulphate fraction of MT and to evaluate their potential to improve the detection of the misuse of the drug in sports. MT was administered to healthy volunteers and urine samples were collected up to 30 days after administration. After an extraction with ethyl acetate, urine extracts were analysed by liquid chromatography tandem mass spectrometry using electrospray ionisation in negative mode by monitoring the transition m/z 385 to m/z 97. Three diol sulphate metabolites (S1, S2 and S3) were detected. Potential structures for these metabolites were proposed after solvolysis and mass spectrometric experiments: S1, 17α-methyl-5β-androstan-3α,17β-diol 3α-sulphate; S2, 17β-methyl-5α-androstan-3α,17α-diol 3α-sulphate; and S3, 17β-methyl-5β-androstan-3α,17α-diol 3α-sulphate. Synthesis of reference compounds will be required in order to confirm the structures. The retrospectivity of these sulphate metabolites in the detection of MT misuse was compared with the obtained with previously described metabolites. Metabolite S2 was detected up to 21 days after MT administration, improving between 2 and 3 times the retrospectivity of the detection compared to the last long-term metabolite of MT previously described, 17α-hydroxy-17β-methylandrostan-4,6-dien-3-one.

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► Three previously unreported methyltestosterone metabolites are detected in urine.
► These metabolites are excreted as conjugates with sulphate.
► The structure of these metabolites has been postulated based on GC–MS and LC–MS/MS.
► These metabolites are useful for the detection of methyltestosterone misuse.
► One of the metabolites improves the retrospectivity up to 2–3 times.