biostudies-literatureSubramaniyan INational Cancer Institute (NCI)Cancer Prevention Research Institute of Texas Core Facilities Support Award (CPRIT)e70274https://www.ebi.ac.uk/biostudies/studies/S-EPMC12480644biostudies-literatureUnknown48(10)Structurally similar oxysterols such as 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol; 5,6α- and 5,6β-epoxycholesterol; and 24(R/S)-hydroxy cholesterol, 25-hydroxy cholesterol, and 27-hydroxycholesterol are traditionally difficult to resolve using reversed-phase liquid chromatography (RPLC). We present a simple yet highly optimized method for the simultaneous quantification of eight oxysterols using RPLC coupled with mass spectrometry (MS) without derivatization. Optimal separation of most oxysterols was achieved at a lower column temperature (25°C), with specific combinations of stationary and mobile phases enhancing resolution, particularly for isomeric pairs such as 7α-/7β-OHC, 5,6α-/5,6β-EC, 24 R/S-OHC, and 25-OHC. Although certain analytes (e.g., 24S-OHC and 27-OJournal of separation scienceIdentifying Oxysterols Associated With Age and Diet in Mice Using Optimized Reversed-phase Liquid Chromatography-Mass Spectrometry (RPLC-MS).PMC12480644RP210209R01CA282339Gollahon LLa-Beck NMSubramaniyan IBarr BLi LJanesko BGfalseIdentifying Oxysterols Associated With Age and Diet in Mice Using Optimized Reversed-phase Liquid Chromatography-Mass Spectrometry (RPLC-MS).Structurally similar oxysterols such as 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol; 5,6α- and 5,6β-epoxycholesterol; and 24(R/S)-hydroxy cholesterol, 25-hydroxy cholesterol, and 27-hydroxycholesterol are traditionally difficult to resolve using reversed-phase liquid chromatography (RPLC). We present a simple yet highly optimized method for the simultaneous quantification of eight oxysterols using RPLC coupled with mass spectrometry (MS) without derivatization. Optimal separation of most oxysterols was achieved at a lower column temperature (25°C), with specific combinations of stationary and mobile phases enhancing resolution, particularly for isomeric pairs such as 7α-/7β-OHC, 5,6α-/5,6β-EC, 24 R/S-OHC, and 25-OHC. Although certain analytes (e.g., 24S-OHC and 27-O2025-01-01T00:00:00Z2025 Oct2026-06-04T00:00:04.941Z2026-05-03T03:11:56.802ZS-EPMC124806444102437410.1002/jssc.70274