Discrimination of Lipid Hydroperoxide Positional Isomers Using SFC– MS / MS With Sodium Ion: Its Application to Evaluating the Oxidation Mechanisms of Oxidized Oils

Advanced lipid profiling relies on liquid chromatography–tandem mass spectrometry (LC–MS/MS), but long analysis times remain a challenge. Supercritical fluid chromatography (SFC)–MS/MS using supercritical carbon dioxide offers a faster and more environmentally friendly alternative. However, the identification of positional isomers of oxidized lipids (lipid hydroperoxide [LOOH]) has not yet been achieved with SFC–MS/MS. Our research group has previously demonstrated that using sodium ion in LC–MS/MS induces specific fragmentation at the hydroperoxide group of LOOH, enabling the discrimination of LOOH positional isomers. This is essential information for identifying oxidation mechanisms such as autoxidation and photo–oxidation. In this study, we applied this method to SFC–MS/MS and attempted to distinguish six positional isomers of ethyl linoleate hydroperoxide (ELAOOH). Under non–column conditions, flow injection analysis with sodium acetate addition revealed the formation of sodium adducts of ELAOOH and specific fragmentation at the hydroperoxy group. Subsequent column evaluation showed that an octadecylsilane (ODS) column provided almost no retention, whereas Inertsil ODS–EP, which is an ODS column with a polar functional group, allowed good retention and separation. Through optimization, we successfully analyzed all six ELAOOH positional isomers. Finally, we analyzed canola oil that had undergone autoxidation or photo–oxidation under optimized conditions. In autoxidation, ELAOOH isomers with hydroperoxy groups at the 9– and 13–positions increased, while in photo–oxidation, isomers were generated at the 10– and 12–positions. These profiles demonstrated that the optimized SFC–MS/MS method using sodium ion can identify lipid oxidation mechanisms in actual samples. This method has the potential to become a powerful and environmentally friendly tool that can provide valuable information on lipid oxidation while significantly reducing the use of organic solvents compared to conventional methods.