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

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. Despite their analytical promise, the practicality of EIEIO within routine liquid chromatography-mass spectrometry (LC-MS) workflows remains largely unexplored. In this study, we optimized 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 100 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 showed that longer accumulation times of 200 ms were most effective for the determination of carbon-carbon double bond (C=C) positions, particularly in polyunsaturated lipids. Overall, we demonstrate that comprehensive lipid structural characterization, 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 and establish a two-tiered analytical strategy for high-throughput analysis of complex lipid samples by EIEIO.