Determination of 13C/12C ratios of urinary epitestosterone and its main metabolites 5α‐ and 5β‐androstane‐3α, 17α‐diol

Abstract

AbstractEpitestosterone (17α‐hydroxy‐androst‐4‐en‐3‐one, EpiT) belongs to the list of prohibited substances of the World Anti‐Doping Agency (WADA). Although it possesses no anabolic effect, it is presumed to be misused by athletes in order to mask administration of testosterone (T) by lowering the urinary T/EpiT ratio.To improve detection, an excretion study with 40 mg of orally administered EpiT was conducted focusing on the metabolites of EpiT: 5α‐ and 5β‐androstane‐3α,17 α‐diol (5aEpiD and 5bEpiD). A reference population of n = 74 volunteers was investigated to elucidate the urinary concentrations of these steroids.In order to prove whether an unusual finding in urinary concentrations or ratios is due to an illicit intake of steroids or due to physiological elevation, determination of carbon isotope ratios is advisable. A method for isotope ratio determination was developed to enable 13C/12C ratios of EpiT, 5bEpiD, 5aEpiD, pregnanediol and androsterone and etiocholanolone to be measured from a single urine specimen. The method's validity was tested by applying linear mixing models and specificity was ensured by means of gas chromatography/mass spectrometry analysis. δ13C values at natural levels were determined with a reference population and both Δ values and corresponding reference limits were calculated.Considering the implemented EpiT‐metabolites, a more than twofold extension of the detection time of EpiT administration was achieved with both the urinary concentration thresholds and the 13C/12C ratios. Copyright © 2009 John Wiley & Sons, Ltd.