Optimisation of chromatographic separation of oxidised phospholipids and detection with targeted approaches provides greater coverage of the oxidised lipidome (Abstract)

Phospholipid oxidation by adventitious damage generates a wide varietyof products with potentially novel biological activities that can modulateinflammatory processes associated with various diseases. To understandthe biological importance of oxidised phospholipids (OxPL) and theirpotential role as disease biomarkers requires precise information aboutthe abundance of these compounds in cells and tissues. There are manychemiluminescence and spectrophotometric assays available for detectingoxidised phospholipids, but they all have some limitations. Mass spectro-metry coupled with liquid chromatography is a powerful and sensitiveapproach that can provide detailed information about the oxidativelipidome, but challenges still remain.The aim of this work is to develop improved methods for detection ofOxPLs by optimisation of chromatographic separation through testingseveral reverse phase columns and solvent systems, and using targetedmass spectrometry approaches. Initial experiments were carried out usingoxidation products generated in vitro to optimise the chromatographyseparation parameters and mass spectrometry parameters. We haveevaluated the chromatographic separation of oxidised phosphatidylcho-lines (OxPCs) and oxidised phosphatidylethanolamines (OXPEs) using C8,C18 and C30 reverse phase, polystyrene–divinylbenzene based mono-lithic and mixed–mode hydrophilic interaction (HILIC) columns, inter-faced with mass spectrometry. Our results suggest that the monolithiccolumn was best able to separate short chain OxPCs and OxPEs from longchain oxidised and native PCs and PEs. However, variation in charge ofpolar head groups and extreme diversity of oxidised species make analysisof several classes of OxPLs within one analytical run impractical. Weevaluated and optimised the chromatographic separation of OxPLs byserially coupling two columns: HILIC and monolith column that providedus the larger coverage of OxPL species in a single analytical run.