LC-MS/MS analysis of lipid mediators.

Lipids were extracted using the Folch procedure (64), and phospholipid phosphorus was determined by a micro-method (65). To prevent oxidation of lipids during extraction and sample preparation, a chloroform-methanol mixture supplemented with 0.01% butylated hydroxytoluene was used. LC-MS analysis of phosphatidylethanolamines (PEs) and their respective oxygenated species was performed on a Thermo Fisher Scientific HPLC system coupled to a Thermo Fisher Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer. Lipids were separated on a normal phase column [Luna 3 μm Silica (2) 100 Å, 150 × 1.0 mm, Phenomenex) as described previously (66). Comprehensive analyses of PE and their oxygenated metabolites were performed with high accuracy by exact masses. The Compound Discoverer software package (Thermo Fisher Scientific) with an in-house–generated analysis workflow and oxidized phospholipid database was used to evaluate LC-MS data. Peaks with a signal/noise ratio greater than 3 were identified and searched against the database of oxidized phospholipids. Lipid signals were further filtered by retention time and m/z values and confirmed by tandem mass spectrometry (MS/MS) fragmentation analysis. Principal component analysis and orthogonal projection of latent structures–discriminant analysis (OPLS-DA) of PE profiles were performed using SIMCA 18.0 software (Sartorius).