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 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 (HR-MS/MS) characterization of 33 endogenous steroid standards analyzed using a micro-LC-HR-MS/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. GP 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.