New oxymesterone metabolites in human by gas chromatography-tandem mass spectrometry and their application for doping control.

Oxymesterone (17 alpha-methyl-4, 17 beta-dihydroxy-androst-4-ene-3-one) is one of the anabolic androgenic steroids (AAS) banned by the World Anti-Doping Agency (WADA). The biotransformation of oxymesterone is performed in vitro by human heptocytes and human urinarymetabolic profiles are investigated after single dose of 20mgto two adult males aswell. Cell cultures and urine samples were hydrolyzed by beta-glucuronidase, extracted, and reacted with N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide (NH4I), and dithioerythritol. After derivatization, a gas chromatography triple quadruple tandem mass spectrometry (GC-MS/MS) using full scan and MS/MS modes was applied. The total ion chromatographs of the blank and the positive samples are compared, and 7 new metabolites were found. In addition to the well-known 17-epioxymesterone, oxymesterone is metabolized by 4-ene-reduction, 3-keto-reduction, 11 beta-hydroxylation, and 16 xi-hydroxylation. Based on the behavior of the MS/MS results of product ion and precursor ion modes, a GC-MS/MS method has been developed monitoring these metabolites. The structures of metabolite 2 and 4 are tentatively identified as 17 alpha-methyl-3 beta, 17 alpha-dihydroxy-5 alpha-androstane-4-one and 17 alpha-methyl-3 alpha a, 4 xi, 17 beta-trihydroxy-5 alpha-androstane, respectively. Detection of oxymesterone using new metabolitesM2 and M4 can extend the detection window up to 4 days since the parent steroid was not detectable. Copyright (C) 2015 John Wiley & Sons, Ltd.