The detection of androstenedione abuse in sport: a mass spectrometry strategy to identify the 4‐hydroxyandrostenedione metabolite

Studies have shown that the administration of androstenedione (ADIONE) significantly increases the urinary ratio of testosterone glucuronide to epitestosterone glucuronide (T/E) – measured by gas chromatography/mass spectrometry (GC/MS) – in subjects with a normal (≈1) or naturally high (>1) initial values. However, the urinary T/E ratio has been shown not to increase in subjects with naturally low (<1) initial values. Such cases then rely on the detection of C₆‐hydroxylated metabolites shown to be indicative of ADIONE administration. While these markers may be measured in the routine GC/MS steroid profile, their relatively low urinary excretion limits the use of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to specifically confirm ADIONE administration based on depleted ¹³C content. A mass spectrometry strategy was used in this study to identify metabolites of ADIONE with the potential to provide compound‐specific detection. C₄‐hydroxylation was subsequently shown to be a major metabolic pathway following ADIONE administration, thereby resulting in urinary excretion of 4‐hydroxyandrostenedione (4OH‐ADIONE). Complementary analysis of 4OH‐ADIONE by GC/MS and GC/C/IRMS was used to confirm ADIONE administration. Copyright © 2008 Commonwealth of Australia. Published by John Wiley & Sons, Ltd.