Mass spectrometry-based techniques have emerged as the analytical methods of choice for oxysterol (OS) characterization in complex biological matrices due to their exceptional sensitivity and versatility [1]. While gas chromatography-mass spectrometry (GC-MS) remains the gold standard, liquid chromatography-mass spectrometry (LC-MS) offers a compelling alternative by eliminating the need for chemical derivatization and its associated analytical artifacts [2]. However, the coexistence of multiple ionization pathways in electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) necessitates effective chromatographic separation prior to mass spectrometric detection to fully exploit the analytical potential of these techniques. This study presents a systematic comparison of three reversed-phase liquid chromatography (RPLC) stationary phases: octadecyl (C18), cyanopropyl (ES-CN), and pentafluorophenyl (F5) for the separation of ten clinically relevant oxysterols: 25-hydroxycholesterol, 24(S)-hydroxycholesterol, 25(R)-26-hydroxycholesterol, 25(S)-26-hydroxycholesterol, 3β,5α,6β-cholestantriol, 7-ketocholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, 5β,6β-epoxycholesterol, and 5α,6α-epoxycholesterol. Analytical design space modelling empowered by DryLab® software was employed to optimize separation conditions through multivariate experimentation. Critical chromatographic parameters including gradient steepness, column temperature, and mobile phase composition (water with methanol or acetonitrile) were systematically evaluated while maintaining operation within conventional HPLC pressure limits (<400 bar). Comparative analysis revealed that both ES-CN and F5 columns provided superior separation efficiency and reduced analysis time compared to the conventional C18 stationary phase. However, the ES-CN column demonstrated the most favourable performance profile, exhibiting greater resistance to retention artifacts and chemical interactions that can compromise data quality in π-π binding stationary phases such as F5. These findings provide a rational framework for method development in oxysterol analysis, demonstrating that polar-embedded stationary phases offer significant advantages over traditional C18 columns for routine clinical and research applications requiring robust, high-throughput oxysterol profiling.
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Dias I.H.K, Wilson S.R., Biochimie, 153 (2018), pag. 3-12, DOI: 10.1016/j.biochi.2018.05.004.