Development and validation of an UHPLC-MS/MS method for extended serum steroid profiling in female populations.

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. 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. 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.