Lipidomic profiling of targeted oxylipins with ultra-performance liquid chromatography-tandem mass spectrometry

摘要

Oxylipins are bioactive mediators that play diverse roles in (patho) physiology. We developed a sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous profiling of 57 targeted oxylipins derived from five major n-6 and n-3 polyunsaturated fatty acids (PUFAs) that serve as oxylipin precursors, including linoleic (LA), arachidonic (AA), alpha-linolenic (ALA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids. The targeted oxylipin panel provides broad coverage of lipid mediators and pathway markers generated from cyclooxygenases, lipoxygenases, cytochrome P450 epoxygenases/hydroxylases, and non-enzymatic oxidation pathways. The method is based on combination of protein precipitation and solid-phase extraction (SPE) for sample preparation, followed by UPLC-MS/MS. This is the first methodology to incorporate four hydroxy-epoxy-octadecenoic acids and four keto-epoxy-octadecenoic acids into an oxylipin profiling network. The novel method achieves excellent resolution and allows in-depth analysis of isomeric and isobaric species of oxylipin extracts in biological samples. The method was quantitatively characterized in human plasma with good linearity (R = 0.990-0.999), acceptable reproducibility (relative standard deviation (RSD) 20 for the majority of analytes), accuracy (67.8 to 129.3) for all analytes, and recovery (66.8-121.2) for all analytes except 5,6-EET. Ion enhancement effects for 28 of the analytes in tested concentrations were observed in plasma, but were reproducible with RSD 17.2. Basal levels of targeted oxylipins determined in plasma and serum are in agreement with those previously reported in literature. The method has been successfully applied in clinical and preclinical studies.