Untargeted microLC-HRMS/MS for Measuring Endogenous Steroids in Mouse Plasma and Human Serum Samples Applied to...

Steroids play essential roles in regulating metabolism, response to stress, electrolyte balance, and reproductive function; however, their analysis in complex biological matrices remains challenging. Low endogenous concentrations, structural similarities, and poor ionization efficiencies can limit their detection, and conventional workflows frequently require large sample volumes and/or chemical derivatization, often restricting quantitative applications to targeted analyses. A workflow was developed for the untargeted analysis of endogenous steroids in serum or plasma samples using microflow liquid chromatography coupled to high-resolution tandem mass spectrometry. The Evosep One LC platform, applied widely for bottom-up proteomics, was coupled to a Sciex ZenoTOF 7600 quadrupole-time-of-flight system to enable the separation and detection of unconjugated and sulfated steroids in low-volume serum and plasma samples. The method was tested with a range of analytical standards, demonstrating efficient chromatographic separation and detection with high-accuracy MS/MS for structural confirmation. A total of 13 unconjugated steroids were confirmed and detected from female mouse plasma and/or human serum in positive mode, whereas three sulfated steroids were detected exclusively in human serum using negative mode. The sulfated steroids were confirmed using in vitro incubations of the parent steroids. Considerable interspecies differences were observed, consistent with the known literature on steroid metabolism. In a transgenic mouse model developed to mimic a metabolic subtype of polycystic ovary syndrome (PCOS), significant alterations in corticosteroids were detected. In human serum samples, dehydroepiandrosterone and androstenedione were significantly elevated in PCOS patients compared with healthy volunteers. The observed relative changes in both species showed similarities to the steroid perturbation patterns previously reported in PCOS.