Mass spectrometric description of novel oxymetholone and desoxymethyltestosterone metabolites identified in human urine and their importance for doping control

Abstract

The metabolism of two anabolic steroids - oxymetholone and desoxymethyltestosterone - was reinvestigated to identify new targets potentially valuable for the antidoping analysis. Excretion urine samples from the laboratory reference collection were used in this study. Following fractionation of the urinary extract by means of high performance liquid chromatography (HPLC), each fraction was subjected to gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis after trimethylsilylation. About 20 metabolites were found for desoxymethyltestosterone and more than 40 for oxymetholone, with many of them being isomeric compounds. In addition to the well-known reduced and hydroxylated metabolites, 18-nor-17,17-dimethyl and 18-nor-17-hydroxymethyl-17-methyl steroids were also identified. Having evaluated all the metabolites in terms of how long they could be detected, we suggest that 18-nor-2ξ,17β-hydroxymethyl-17α-methyl-5α-androst-13-en-3α-ol is an important marker of oxymetholone abuse. In case of desoxymethyltestosterone, better detectability could be achieved if 18-nor-17,17-dimethyl-5α-androst-13-en-2ξ,3α-diol is monitored. These novel metabolites could be detected using GC-MS/MS at least for 14 days after administration of these anabolic steroids compared to 5-7 days for previously reported metabolites.

Anabolic agents: recent strategies for their detection and protection from inadvertent doping

“…The identification of LTMs of different anabolic steroids was accomplished by means of several different methodologies including fractionation and in-depth elucidation of urine samples collected from human administration studies6 7 and animal model elimination studies 8 9. A novel approach was recently presented, which is based on the administration of deuterated steroids followed by the identification of the respective deuterated urinary metabolites using hydrogen isotope ratio mass spectrometry 10.…”

Determination of mepitiostane metabolites in human urine by liquid chromatography/tandem mass spectrometry for sports drug testing

“…Crystallised epitiostanol sulfoxide (1.7 mg, 24% yield) was obtained and identified by NMR spectroscopy with 1 H, 13 Differences in 13 C chemical shifts between epitiostanol and the corresponding sulfoxide ( ıC in ppm) were −4.80 (C-1), 16.06 (C-2), 15.45 (C-3), −5.04 (C-4) and 5.37 (C-5). These chemical shifts were consistent with the occurrence of a sulphur atom attached to the more electronegative oxygen atom.…”

“…To the best of our knowledge, the endogenous existence of two steroids in human urine is unclear; however, 5␣-androst-2-en-17␤-ol is an isomer of the endogenous steroid 5␣-androst-16-en-3␤-ol in humans. The metabolism of desoxymethyltestosterone (17␣-methyl-5␣-androst-2-en-17␤-ol) in humans consists of the reduction of the double bond and hydroxylation, and 20 metabolites have been reported [13]. It is therefore suggested that several hydroxyl-metabolites from 5␣-androst-2-en-17␤-ol may exist in mepitiostane-administered urine [11].…”

The Potential Role of Oral Fluid in Antidoping Testing

“…Oxymetholone (50 mg), a synthetic testosterone derivative, was orally administered to 3 women [28 (3.5) years old], with plasma and OF collection for 24 h, and oxymetholone quantification by GC-MS (43). At the first OF collection at 30 min, oxymetholone concentrations were 11.3 μ g/L, but were negative by 1 h. Plasma concentrations were positive for 24 h. Recently, new urinary oxymetholone metabolites were identified in 3 males receiving 50 mg/day oral oxymetholone for 5 consecutive days, enabling detection windows in urine for up to 20 days (45). Short detection windows for OF oxymetholone, and perhaps other synthetic steroids, suggest that OF may not be a useful matrix for these doping agents.…”