Optimisation of electron-induced dissociation parameters for molecular annotation of glycerides and phospholipids in fast LC-MS

Abstract

Electron-induced dissociation methods, particularly electron impact excitation of ions from organics (EIEIO), offer enhanced capabilities for lipid structural elucidation over traditional collision-induced dissociation (CID). Despite their analytical promise, the practicality of EIEIO within routine liquid chromatography-mass spectrometry (LC-MS) workflows remains largely unexplored. In this study, we optimised LC-EIEIO-MS analysis for the rapid and detailed structural annotation of glycerides and phospholipids. We evaluated the effects of reaction times, accumulation times, and electron kinetic energies using lipid standards from multiple classes and at varying concentrations. Our results revealed that short reaction times of 30 ms consistently yielded stronger diagnostic signals crucial for lipid class identification and sn-position discrimination at concentrations as low as 200 pg on column. To systematically infer the position of double bonds from EIEIO spectra, we introduced LipidOracle, a software that tests all possible isomers and correctly accounts for missing data, noise, and crowded spectra. We demonstrated that longer accumulation times of 200 ms were most effective for determining carbon–carbon double bond (CC) positions, particularly in polyunsaturated lipids. Finally, we evaluated the performance of EIEIO with liver and plasma extracts. Overall, we demonstrate that comprehensive lipid structural characterisation, including sn-position and double bond locations in fatty acyl chains, is achievable within typical LC-MS timescales (∼0.2 s). Our findings outline practical guidelines for high-throughput analysis of complex lipid samples by EIEIO.