Profiling of Cholesteryl Esters by Coupling Charge-Tagging Paternò-Büchi Reaction and Liquid Chromatography-Mass Spectrometry.

The profile of cholesteryl esters (CEs) is increasingly used in metabolic disease monitoring due to the roles of CE in regulating the cholesterol level. While electrospray ionization-tandem mass spectrometry is routinely applied for the identification and quantitation of CE, it has a limitation of not being able to provide the location of carbon-carbon double bond (C═C) within unsaturated fatty acyls. In this study, we paired offline 2-acetylpyridine (2-AP) Paternò-Büchi (PB) reaction and reversed-phase liquid chromatography-tandem mass spectrometry to achieve highly sensitive and structural informative CE analysis from complex mixtures. The 2-AP PB reactions of CE standards provided 20-30% conversion but resulted in enhanced ion signal relative to that of intact CE detected as ammonium adduct ions. MS/MS of 2-AP derivatized CE via collision-induced dissociation produced two abundant diagnostic ions for each C═C in a fatty acyl, leading to both sensitive identification and quantitation of C═C location isomers. Twelve saturated and twenty-seven unsaturated CEs were profiled in pooled human plasma; of the latter group, relative quantitation of 6 groups of C═C location isomers was achieved. A dehydrocholesteryl ester, DHE 18:2 (Δ9,12), was confidently differentiated from coexisting compositional isomers: CE 18:3 (Δ9,12,15) and CE 18:3 (Δ6,9,12). The above results represented improved CE coverage at the C═C location level over those reported by gas chromatography MS or acetone PB-MS/MS methods.