High-resolution MS/MS characterization of steroid fragmentation profiles for structural elucidation

The analysis of steroids by liquid chromatography - tandem mass spectrometry (LC-MS/MS) is associated with several analytical challenges, including difficulty in confirming their structures in complex samples, due to structural similarities. Collision-induced dissociation (CID) is commonly employed but many aspects of the fragmentation behavior of steroids remain unexplored. In this study, we report the systematic high-resolution MS/MS characterization of 33 endogenous steroid standards analyzed using a microLC-HRMS/MS workflow on a quadrupole-time-of-flight platform in positive mode, while varying collision energies, for their native forms and Girard P (GP) derivatives. For underivatized steroids, intermediate collision energies produced the most interpretable spectra, enabling the identification of many class-specific diagnostic ions. The multiple collision energies tested revealed strong dependence of both precursor persistence and diagnostic fragment presence related to structural features present in the steroids. Girard P derivatization increased signal intensities and altered their chromatographic retention, with the signal enhancement depending on the presence of specific structural features. Higher collision energies applied to GP-derivatized steroids revealed diagnostic fragments, useful for structural characterization. Fragmentation patterns and collision energy-dependent profiles provide increased confidence for steroid structural classification and isomer discrimination.