Strategies to minimize artificial lipid oxidation in mass spectrometry based epilipidomics analysis

(Per)oxidized lipids represent a well-studied component of the epilipidome, a subset of the native lipidome formed through both enzymatic and non-enzymatic lipid oxidation. Given their diverse biological roles, including cellular signalling, regulation of immune responses, and modulation of cell death pathways, accurate detection of lipid peroxidation products is essential. Mass spectrometry-based approaches have become the method of choice for the sensitive, multiplexed detection and structural characterization of oxidized lipids across a variety of biological samples. However, the structural features of lipids, particularly the presence of acyl chains containing multiple double bonds, render them susceptible not only to endogenous oxidation but also to artificial oxidation during sample preparation prior to analysis. Consequently, special care must be taken throughout sample collection, storage, and lipid extraction to minimize artefacts arising from lipid autoxidation. Here, we describe protocols developed in our laboratory over recent years aimed at preventing artificial lipid oxidation during sample preparation, with examples spanning biological materials derived from cell culture, animal and human tissue biopsies, and biofluids. Finally, we propose internal quality control procedures to assess the effectiveness of these measures in preventing lipid autoxidation.