LC-HRMS analysis of phospholipids bearing oxylipins

Several oxylipins including hydroxy- and epoxy-polyunsaturated fatty acids (PUFA) act as lipid mediators. In biological samples they dominantly occur esterified in phospholipids (PL). Esterified oxylipins are usually quantified indirectly after alkaline hydrolysis as non-esterified oxylipins. Here, a liquid chromatography high-resolution mass spectrometry (LC-HRMS) method for the direct analysis of oxylipins bound to PL was developed. Optimized reversed phase LC separation enabled the separation of isobaric and isomeric PL from different lipid classes bearing oxylipin positional isomers, by both the polar head groups as well as the fatty acyl chains. Each individual PL bearing oxylipins was tentatively identified based on the combination of retention time, precursor ion and specific product ions. The oxylipin was characterized based on product ions resulting from the characteristic α cleavage occurring at the hydroxy/epoxy group. Phospholipid sn 1/sn 2 isomers were identified based on the neutral loss of the fatty acyl in the sn 2 position as a ketene. A total of 422 individual oxPL species from 7 different lipid classes i.e., PI, PS, PC, PE, PC P, PC O, and PE P, bearing hydroxy- and epoxy-PUFA were detected in human serum and cells. This method was applied to investigate in which PL class supplemented oxylipins are incorporated in HEK293 cells. 153 individual oxPL species were characterized and semi-quantified using one internal standard per lipid class: 20:4;15OH, 20:4;14Ep, and 20:5;14Ep were mostly bound to PI. 20:4;8Ep and 20:5;8Ep were esterified to PC and PE while other hydroxy- and epoxy-PUFA were mainly found in PC. The developed LC-HRMS method enables the direct and comprehensive analysis of PL bearing oxylipins and to investigate their biological role.