N-aminophthalimide-derived unsaturated PC reaction-assisted analysis of carbon-carbon double bond position and cis/trans isomerism in fatty acid chains based on UHPLC-ESI-MS/MS

Phosphatidylcholines (PCs) are key components of biological membranes and play crucial roles in key physiological processes, where the positions and cis/trans isomerism of carbon-carbon double bonds (C=C) in fatty acyl chains critically influence physiological functions and disease mechanisms. The conventional LC-MS technique cannot simultaneously resolve the positional and cis/trans isomers of phosphatidylcholine. To address the limitations of conventional LC-MS/MS in distinguishing PC isomers, we developed a novel method combining N-aminophthalimide (PhthNH 2 ) derivatization with UHPLC-ESI-MS/MS. This approach leverages the specific reaction between PhthNH 2 and C=C bonds to form N-aziridine adducts, enabling precise localization of double bond positions through characteristic fragment ions while achieving baseline chromatographic separation of cis/trans isomers in monounsaturated PC. A derivatization workflow and data analysis procedure for simultaneously detecting PC C=C bonds positional and cis/tans isomers was successfully established and optimized. The method successfully identified 31 C=C positional isomers in plasma samples and revealed significant differences in PC isomer abundance between colorectal cancer patients and healthy controls, establishing a powerful new tool for investigating PC biology and disease associations. • ●A chemical derivatization method was developed for locating C=C bonds in phosphatidylcholines. • ●A novel fragmentation pathway for determining C=C bonds position was observed for PC-sodium adducts after aziridination. • ●C=C bonds positional and cis/trans isomers of PC can be simultaneously discriminated using UHPLC-ESI-MS/MS.