Comprehensive analysis of oxylipins using reverse phase liquid chromatography and data dependent acquisition workflow...

Oxylipins - involved in inflammatory processes - are reported in several diseases, in biological, pharmacological, and physiological fields. To face the structural complexity of oxylipins, the study of isomers and isobars species relied on Selected Reaction Monitoring (SRM) and Multiple Reaction Monitoring (MRM) in tandem mass spectrometry such as triple quadrupole, quadrupole-Time of Flight (TOF). Unfortunately, false positive signals in cellular matrix could occur using MRM or SRM mode since the MS/MS spectrum of each molecule is not acquired with the previous mode to help molecule confirmation. Using the versatile ability of LTQ-Orbitrap® Velos Pro mass spectrometer, we developed a novel method based on data dependent acquisition (DDA) workflow for oxylipins analysis. To reach sufficient data points per peak and a better sensitivity to quantify oxylipins traces, an optimization of the acquisition frequency was carried out both on linear trap and Orbitrap analyzers. A segmentation of the chromatographic profile and an optimization of the collision energies by HCD (higher energy collision dissociation) for each eicosanoid increased the acquisition frequency significantly and the detection threshold: around 2 pg for some prostanoids and 0.02-2 pg for some leukotrienes and oxidized species. We validated our method in terms of specificity (RSD <10%), sensitivity, accuracy and precision. The intra and inter-day accuracy were between 86.56% and 114.93%. Besides, a relative standard deviation less than 15% as intra- and inter-day precision were obtained for almost all molecules. A linear range between 2.5 and 12,500 pg was reached. DDA approach on LTQ-Orbitrap® constitutes an alternative to MRM mode on triple quadrupole for eicosanoids quantification in complex matrices. Finally, this method helped us to compare for the first time the amount of prostanoids released by J774 and THP-1 macrophages under lipopolysaccharide (LPS) stimulation.