Detection of Epitestosterone Doping by Isotope Ratio Mass Spectrometry

Abstract

AbstractBackground: Epitestosterone is prohibited by sport authorities because its administration will lower the urinary testosterone/epitestosterone ratio, a marker of testosterone administration. A definitive method for detecting epitestosterone administration is needed.Methods: We developed a gas chromatography-combustion-isotope ratio mass spectrometry method for measuring the δ13C values for urinary epitestosterone. Sample preparation included deconjugation with β-glucuronidase, solid-phase extraction, and semipreparative HPLC. Epitestosterone concentrations were determined by gas chromatography-mass spectrometry for urines obtained from a control group of 456 healthy males. Epitestosterone δ13C values were determined for 43 control urines with epitestosterone concentrations ≥40 μg/L (139 nmol/L) and 10 athletes’ urines with epitestosterone concentrations ≥180 μg/L (624 nmol/L), respectively.Results: The log epitestosterone concentration distribution was gaussian [mean, 3.30; SD, 0.706; geometric mean, 27.0 μg/L (93.6 nmol/L)]. The δ13C values for four synthetic epitestosterones were low (less than or equal to −30.3‰) and differed significantly (P <0.0001). The SDs of between-assay precision studies were low (≤0.73‰). The mean δ13C values for urine samples obtained from 43 healthy males was −23.8‰ (SD, 0.93‰). Nine of 10 athletes’ urine samples with epitestosterone concentrations >180 μg/L (624 nmol/L) had δ13C values within ± 3 SD of the control group. The δ13C value of epitestosterone in one sample was −32.6‰ (z-score, 9.4), suggesting that epitestosterone was administered. In addition, the likelihood of simultaneous testosterone administration was supported by low δ13C values for androsterone and etiocholanolone.Conclusions: Determining δ13C values for urinary epitestosterone is useful for detecting cases of epitestosterone administration because the mean δ13C values for a control group is high (−23.8‰) compared with the δ13C values for synthetic epitestosterones.