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Lipids play a major role in platelet signaling and activation. In this study, we analyzed the platelet lipidome in an untargeted manner by reversed-phase UHPLC for lipid species separation coupled to high-resolution QTOF-MS/MS in data-independent acquisition (DIA) mode with sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) for compound detection. Lipid identification and peak picking was supported by the characteristic regular elution pattern of lipids differing in carbon and double bond numbers. It was primarily based on post-acquisition targeted feature extraction from the SWATH data. Multiple extracted ion chromatograms (EICs) from SWATH data of diagnostic ions on MS1 and MS2 level from both positive and negative ion mode allowed to distinguish between poorly resolved isomeric lipids based on their distinct fragment ions, which were used for relative quantification at a molecular lipid species level. It supports assay specificity for relative lipid quantitation via multiple quantifiably ions unlike to data-dependent acquisition methods which rely on precursor ions only. This approach was used to analyze human platelet samples. 457 lipids were annotated. Concentrations of lipids were estimated by stable isotope-labelled lipid class-specific internal standards as surrogate calibrants. Heatmaps of lipid concentrations in dependence on carbon and double bond numbers for the distinct lipid classes revealed a snapshot of the platelet lipidome in the resting state with lipid species distributions within classes supporting some functional interpretations. As expected, activation of the platelets by thrombin has led to significant alterations in the platelet lipidome as proven by univariate (volcano plot) and multivariate (PLS-DA) statistics. Several lipids were significantly up-regulated (lysophosphatidylinositols, oxylipins such as thromboxane B2 (TXB2), hydroxyheptadecatrienoic acid (HHT), hydroxyeicosatetraenoic acid (HETE), hydroxyoctadecadienoic acid (HODE), sphingoid-bases, (very) long chain saturated fatty acids) or down-regulated (lysophosphatidylethanolamines, polyunsaturated fatty acids, phosphatidylinositols). Several of them are well known as biomarkers of platelet activation while others may provide some further insights into pathways of platelet activation and platelet metabolism.

Oxylipins play an important role in cell signaling and they act as auto- and paracrine factors. There are numerous reports on the analysis of oxylipins in biofluids, especially in plasma. Only a limited number of studies addressed the analysis of oxylipins in platelets using modern, sensitive LC MS methods, even though these compounds have a huge impact on platelet functions and thrombo-inflammation. In this work, a new method based on superficially porous particle (2.7 μm) capillary column (0.5 mm ID) and micro-liquid chromatography coupled to tandem mass spectrometry (μUHPLC-ESI-QqQ-MS/MS) method has been developed, optimized and validated. It has finally been successfully applied for human plasma and platelet analysis. The method allows the precise and accurate simultaneous quantification of 42 oxylipins with 13 deuterated internal standards. Solid phase extraction with Bond Elut Certify II provides good extraction recoveries (on average around 75 %). The μUHPLC-MS/MS method is selective, sensitive (LOQs between 30 and 150 pg/mL) and shows good linearity. Limits of detections for most of the compounds are between 2 and 250 fmol on column. Twenty-three oxylipins have been detected in plasma and 19 in non-activated (resting) platelets (all samples were from healthy donors). The μUHPLC-MS/MS method uses very low volume of mobile phase (less than 250 μL of organic solvents in mobile phase per analysis), and therefore is considered environmentally friendly. It also turned out to be robust enough for routine analysis.