Microwave‐Assisted Meta‐Chloroperoxybenzoic Acid Epoxidation for Qualitative and Quantitative Analysis of Unsaturated Lipid Double‐Bond Positional Isomers by Liquid Chromatography–Tandem Mass Spectrometry

Unsaturated lipid double‐bond (C═C) positional isomers are closely related to lipid structure and biological function, but their reliable qualitative and quantitative analysis remains challenging by conventional liquid chromatography–mass spectrometry (LC‐MS) methods. In this study, we developed a microwave‐assisted meta ‐chloroperoxybenzoic acid (m‐CPBA) epoxidation method coupled with LC–tandem MS for the identification and quantification of unsaturated lipid C═C positional isomers. The reaction conditions were optimized to facilitate the formation of fully epoxidized products, simplify the chromatographic peak profiles, and enhance their suitability for targeted quantitative analysis. The developed method showed good analytical performance, including satisfactory linearity ( r 2 > 0.99), precision (relative standard deviation <15%), recovery (75.5%–106.5%), and acceptable matrix effects (86.0%–117.3%). The method was successfully applied to rat brain tissue and plasma samples from a rat depression model, enabling the quantification of multiple unsaturated lipids and the characterization of their C═C positional isomers in complex biological matrices. These results demonstrate that microwave‐assisted m‐CPBA epoxidation provides a rapid, efficient, and practical strategy for LC‐MS‐based analysis of unsaturated lipid isomers.