Benzophenone used as the photochemical reagent for pinpointing C=C locations in unsaturated lipids through shotgun and liquid chromatography-mass spectrometry approaches.

Unsaturated lipids exhibit different physiological significances due to the different locations of the carbon-carbon double bond (C=C). Identifying lipid isomers with mass-based methods remains challenging. Xia's group has been successfully employed Paternò-Büchi (PB) reaction, a photochemical reaction with UV irradiation, coupled with tandem mass spectrometry (MS/MS) to identify and quantify unsaturated lipids in complex mixtures. However, the existing PB reagents possess certain demerits. In this regard, a new PB reagent that is compatible with various lipidomic analysis platforms must be screened. In this study, we comprehensively evaluated the conditions of the PB reaction and screened benzophenone as a new PB reagent. Benzophenone possesses unique advantages, such as a relative high PB yield; the PB products could be readily distinguished from the reacted lipids based on the added high molecular weight (182 Da); and the benzophenone does not affect the lipids appearance interval for reversed-phase column separation. Furthermore, we optimized the reaction conditions by using benzophenone as a PB reagent and summarized the molecular formulas of the diagnostic ions according to the fragment rules. The proposed PB method has been implemented in shotgun and LC-MS lipidomics. To our best knowledge, this work is the first to report the integrated PB reaction with LC-MS lipidomics for identification of lipid isomers.