Abstract

Testosterone (T) is an anabolic androgenic steroid (AAS) that is widely used as doping agent. The World Anti-Doping Agency (WADA) has determined criteria to consider for T abuse, based on the steroid profile, including precursor, epimer and metabolites of T. These steroids are extensively metabolized in urine as phase II metabolites (glucuronide and sulfate conjugates), but the direct quantification of these intact metabolites that may help to gather worthy information on endogenous T metabolism is still challenging. In this study, a method coupling ultra-high pressure liquid chromatography (UHPLC) to hybrid quadrupole time-of-flight (QTOF) mass spectrometry was developed and validated, UHPLC offers high chromatographic performance by using columns packed with small particles (i.e. sub-2μm), and QTOF mass analyzer enables exact mass determination on molecular and fragment ions over the entire selected mass range. After a sample preparation by solid phase extraction (SPE), a highly selective chromatographic separation was performed with baseline resolution between pairs of isomers. The analytes were detected in the electrospray negative mode and 2 functions were acquired simultaneously in the MSE mode. This allowed the quantification of the investigated metabolites by assessing the molecular ion obtained in the first function at low collision energy (5 eV), while the second function acquired at ramped collision energy (5 to 70 eV) afforded a rich fragmentation pattern helping the identification of T analogs. This development revealed the promising opportunity to supply a broader steroid profiling including an extensive monitoring of endogenous metabolites (steroidomics). Chemometric tools were used to highlight biomarkers of interest and underlined the ability of this analytical strategy to extend the detection window of T doping. Verf.-Referat