Detection of epitestosterone doping by isotope ratio mass spectrometry.

BACKGROUND 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 delta(13)C values for urinary epitestosterone. Sample preparation included deconjugation with beta-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 delta(13)C values were determined for 43 control urines with epitestosterone concentrations > or =40 microg/L (139 nmol/L) and 10 athletes' urines with epitestosterone concentrations > or =180 microg/L (624 nmol/L), respectively. RESULTS The log epitestosterone concentration distribution was gaussian [mean, 3.30; SD, 0.706; geometric mean, 27.0 microg/L (93.6 nmol/L)]. The delta(13)C values for four synthetic epitestosterones were low (less than or equal to -30.3 per thousand) and differed significantly (P <0.0001). The SDs of between-assay precision studies were low (< or =0.73 per thousand). The mean delta(13)C values for urine samples obtained from 43 healthy males was -23.8 per thousand (SD, 0.93 per thousand). Nine of 10 athletes' urine samples with epitestosterone concentrations >180 microg/L (624 nmol/L) had delta(13)C values within +/- 3 SD of the control group. The delta(13)C value of epitestosterone in one sample was -32.6 per thousand (z-score, 9.4), suggesting that epitestosterone was administered. In addition, the likelihood of simultaneous testosterone administration was supported by low delta(13)C values for androsterone and etiocholanolone. CONCLUSIONS Determining delta(13)C values for urinary epitestosterone is useful for detecting cases of epitestosterone administration because the mean delta(13)C values for a control group is high (-23.8 per thousand) compared with the delta(13)C values for synthetic epitestosterones.