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

Abstract

Oxymesterone (17α-methyl-4, 17β-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 urinary metabolic profiles are investigated after single dose of 20 mg to two adult males as well. Cell cultures and urine samples were hydrolyzed by β-glucuronidase, extracted, and reacted with N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide (NH4 I), 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, 11β-hydroxylation, and 16ξ-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 17α-methyl-3β, 17β-dihydroxy-5α-androstane-4-one and 17α-methyl-3α, 4ξ, 17β-trihydroxy-5α-androstane, respectively. Detection of oxymesterone using new metabolites M2 and M4 can extend the detection window up to 4 days since the parent steroid was not detectable. Copyright © 2015 John Wiley & Sons, Ltd.

Identification and characterization of novel long‐term metabolites of oxymesterone and mesterolone in human urine by application of selected reaction monitoring GC‐CI‐MS/MS

“…In 1992, 17‐epioxymesterone (EpiOxy) was identified as a metabolite . Recently, a second publication reported two new metabolites (17α‐methyl‐3,17β‐dihydroxy‐5α‐androstane‐4‐one and 17α‐methyl‐3α,4β,17β‐trihydroxy‐5α‐androstane) which were detectable up to 4 days, whereas the parent compound (PC) could only be detected up to 2.25 days …”

“…[21] Recently, a second publication reported two new metabolites (17α-methyl-3,17β-dihydroxy-5α-androstane-4-one and 17αmethyl-3α,4β,17β-trihydroxy-5α-androstane) which were detectable up to 4 days, whereas the parent compound (PC) could only be detected up to 2.25 days. [19] Mesterolone (1α-methyl-5α-androstan-17β-ol-3-one) is a synthetic AAS that is still frequently used to treat infertility, hypogonadism and low androgen levels in males, but it is still abused by athletes as well. [9,22] To detect abuse, doping control laboratories usually focus on screening for the PC and/or its metabolite 1α-methyl-5α-androstan-3α-ol-17-one after hydrolysis with β-glucuronidase as both are excreted as glucuronide conjugates.…”

“…Oxymesterone (17α‐methyl‐4,17β‐dihydroxyandrost‐4‐ene‐3‐one) is a synthetic AAS that became available in the 1990s and was commercialized by Farmitalia Carlo Erba Ltd (Italy) as Oranabol . Oxymesterone is 17α‐methylated which increases the oral bioavailability of the compound by reducing its breakdown by the liver .…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…A differentiation whether dimethazine or methasterone was administered might however remain difficult. The AAS oxymesterone was subjected to in vitro and in vivo metabolism studies in order to complement the metabolic profile of the substance in humans . Hepatocytes were used to mimic human metabolic reactions, and urine samples were collected up to 30 days after oral administration of 20 mg of the steroid.…”

“…The AAS oxymesterone was subjected to in vitro and in vivo metabolism studies in order to complement the metabolic profile of the substance in humans. [68] Hepatocytes were used to mimic human metabolic reactions, and urine samples were collected up to 30 days after oral administration of 20 mg of the steroid. A total of seven formerly unreported metabolites resulting predominantly from reduction and hydroxylation reactions was described, with two candidates tentatively identified as 17α-methyl-5αandrostane-3β,17β-diol-4-one and 17α-methyl-5α-androstane-3α,4ξ,17β-triol.…”