Development and Validation of an UHPLC–MS/MS Method for Extended Serum Steroid Profiling in Female Populations

Abstract

<b>Aim:</b> Quantitative endogenous steroid profiling in blood appears as a complementary approach to the urinary module of the World Anti-Doping Agency's Athlete Biological Passport Steroidal Module for the detection of testosterone doping. To refine this approach further, a UHPLC-MS/MS method was developed for the simultaneous determination of 14 free and 14 conjugated steroids in serum. <b>Results:</b> The method was validated for quantitative purposes with satisfactory results in terms of selectivity, linearity range, trueness, precision and combined uncertainty (<20%). The validated method was then applied to serum samples from both healthy women and women diagnosed with mild hyperandrogenism. <b>Conclusion:</b> The UHPLC-MS/MS method showed promising capability in quantifying free and conjugated steroids in serum and determining variations of their concentration/distribution within serum samples from different populations.