The potential of urinary androstdiene markers to identify 4-androstenediol (4-ADIOL) administration in athletes

Doping control laboratories accredited by the World Anti-Doping Agency (WADA) require criteria that allow endogenous steroids to be distinguished from their synthetic analogues in urine. Methodology based on “looking outside the metabolic box” was used in this study to identify diagnostic urinary markers of 4-androstenediol (4-ADIOL) administration. Androst-2,4-diene-17-one and androst-3,5-diene-17-one are proposed to be formed in urine from acid-catalyzed hydrolysis of 4-ADIOL sulfoconjugate, a major phase II metabolic product of 4-ADIOL. The presence of these markers in the routine gas chromatography–mass spectrometry (GC–MS) steroid screen was suitable to identify samples requiring confirmation by gas chromatography–combustion-isotope ratio mass spectrometry (GC–C-IRMS) – to measure the carbon isotope ratio (δ13C) of the androstdiene markers and confirm their likely synthetic origin based on depleted 13C content.