Identifying 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-OHC) remained challenging to separate due to similar retention behavior, they were distinguishable by their unique MRM transitions. We applied this method to investigate oxysterol changes in a longitudinal mouse study comparing a normal diet to a high-fat diet. Liver and brain samples were analyzed, revealing distinct distribution patterns between the two organs. Notably, 24(S)-hydroxycholesterol levels, a signature cholesterol metabolite exclusively produced in the brain, increased with age independent of diet. In contrast, 5,6α-epoxycholesterol production in the liver was influenced by both age and dietary factors. Our method provides a robust tool for studying oxysterol variation and its implications in aging and diet, offering new insights into cholesterol-derived lipid regulation across different physiological conditions.