Carbon stable isotope analysis of 7-Keto-DHEA doping targeting both glucuronated and sulfoconjugated metabolites

Code: 242A12JFN

7-keto-DHEA is a performance enhancing anabolic androgenic steroid on the WADA prohibited list and abuse by athletes can be detected unambiguously using carbon stable isotope analysis after urine sample purification and isotope ratio mass spectrometric analysis. Stable isotope confirmation is required as 7-keto-DHEA and its primary metabolites can be formed through microbial transformation of naturally produced endogenous steroids. As the 13C content of steroids is conserved during their metabolism, the 13C content of the metabolites and parent compound excreted in urine will reflect that of the source of the compounds (either endogenous through in situ microbial reworking or exogenous through doping). Existing methods for detecting 7-keto-DHEA doping target multiple metabolites found principally as free form or glucuronidated metabolites. Due to method artifacts, it is possible that the proportion of sulfoconjugated metabolites of 7-keto-DHEA is currently being under reported. During initial scouting investigations, a significant increase in recovery of the analytes of interest has been observed using an acidic hydrolysis involving tetrahydrofuran and methanol as solvents. We aim to develop and validate a GC/C/IRMS confirmation method targeting the principal metabolites of 7-keto-DHEA in both the sulfoconjugated and glucruonidated steroid fractions. We will analyze a series of anonymized athlete urine samples which contain high concentrations of either 7α-OH-DHEA and 7β-OH-DHEA during our routine initial testing procedure over the last 5 years. This will serve as a reference population for North American athletes and will be of use in the context of determining positivity thresholds for inclusion in future WADA technical documents.