Overcoming challenges in LC separation of oxysterols through guided design space modelling: A comparison of three stationary phases.

Liquid chromatography-mass spectrometry (LC-MS) is an attractive alternative to GC-MS for oxysterol (OS) analysis, as it eliminates the need for chemical derivatization. However, in-source fragmentation during electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), usually adopted for LC-MS analysis of OS, may lead to the generation of isomeric ions from non-isomeric precursors. This prevents a complete unambiguous identification along the MS dimension and makes chromatographic separation a fundamental step. In this work, analytical design space modelling empowered by a software-based modelling tool (DryLab®) was employed to optimize the reversed-phase LC separations of ten clinically relevant OS, including isomeric ring-oxidized and side chain-oxidized sterols. Three stationary phases, octadecyl (C18), cyanopropyl (ES-CN), and pentafluorophenyl (F5), were compared in terms of separation efficacy, using HPLC columns of identical geometry (150 × 2.1 mm, 2.7 μm) packed with core-shell particles. Gradient steepness, column temperature, and composition of the organic modifier (mixture of methanol and acetonitrile) in the mobile phase were selected as critical method parameters for successive construction of the design spaces on the selected stationary phases. All the resulting optimized methods resolved positional isomers and diastereomers, excepting 25(R/S)-26-hydroxycholesterol epimers. The C18 stationary phase (at 52 °C) offered high selectivity but demanded the longest run times (ca. 80 min). ES-CN (at 50 °C) and F5 (at 25 °C) stationary phases enabled faster effective runs (< 45 min), with ES-CN also reducing retention/chemical artifacts that are presumably triggered by π-π binding interactions with the stationary phase.