A Single-Aliquot Strategy for Simultaneous Carbon Isotope Confirmation of 6-Oxo-Androstenedione and Testosterone...

A method compliant with World Anti-Doping Agency (WADA) requirements was validated for carbon isotope ratio (CIR) analysis enabling the simultaneous confirmation of 6-oxo-androstenedione (6-oxo) metabolites and testosterone steroid profile markers from a single urine sample aliquot. The approach allows differentiation between endogenous and exogenous steroid origins when 6α-hydroxy-androst-4-ene-3,17-dione (6α-OH), a marker of misuse of 6-oxo or testosterone prohormones, is detected in athlete samples. CIR analysis is laborious and usually requires large sample volumes. Therefore, this study aimed to validate a method capable of simultaneously performing both confirmations from a single sample aliquot. Based on a previously validated method for testosterone confirmation, 6α-OH, its 6β-epimer, and 6-oxo were included and validated as markers of 6-oxo intake. The proposed method, anchored in two liquid-chromatography clean-up steps, yielded peaks of adequate purity, negligible isotopic fractionation, and amplitudes suitable for CIR steroid analysis within typical linear ranges. Limits of quantification were 5.0 ng/mL for 6α- and 6β-OH and 15 ng/mL for 6-oxo, with combined uncertainties of 0.6‰, 0.5‰ and 0.4‰, respectively. Application to authentic samples demonstrated the method's fitness for purpose in differentiating negative from positive samples. Overall, the outcomes show that all WADA method validation requirements were met and that the proposed strategy for simultaneous CIR analysis of 6-oxo and testosterone markers in a single analytical workflow was successful.