2.11. Lipidomic analysis using liquid chromatography‐mass spectrometry (LC‐MS)

Lipid extraction and untargeted LC‐MS lipidomic analysis was performed as described previously, with slight modifications. 32 , 33 In brief, lipids were extracted from biventricular samples (between 10 and 30 mg) after spiking with six internal standards (lysophosphatidylcholine 13:0, phosphatidylcholine 19:0/19:0 and 14:0/14:0, phosphatidylethanolamine 17:0/17:0, phosphatidylglycerol 15:0/15:0, and phosphatidylserine 12:0/12:0; Avanti Polar Lipids Inc, Alabaster, USA). Samples (0.49‐1.52 μL) were injected into a 1290 Infinity HPLC coupled with a 6530 Accurate Mass Q‐TOF (Agilent Technologies Inc, Santa Clara, USA) via a dual electrospray ionization (ESI) source operated in the positive mode. Lipids were eluted on Zorbax Eclipse plus column (C18, 2.1 × 100 mm, 1.8 μm, Agilent Technologies Inc) over 83 minutes at 40°C using a gradient of solvent A (0.2% formic acid and 10 mM ammonium formate in water) and B (0.2% formic acid and 5 mM ammonium formate in methanol/acetonitrile/methyl tert‐butyl ether [MTBE], 55:35:10 [v/v/v]). MS data processing was achieved using Mass Hunter B.06.00 (Agilent Technologies Inc) and an in‐house bioinformatics script that we have developed and encoded in both Pearl and R languages to ensure optimal MS data alignment between chromatographic runs. In addition, frequency and adducts filters were applied, as well as signal intensity normalization (using cyclic loess algorithm) and imputation of missing values with k‐nearest neighbor on scaled data. This yields a data set listing MS features with their mass, retention time, and corrected signal intensity. For subsequent data mining, we used a targeted approach in which lipid features from this data set, including their fatty acids side chains, were annotated by alignment with our in‐house database which contains 498 lipids that were previously identified by MS/MS for the following selected lipid (sub)classes: (a) sphingolipids: ceramides (Cer) and spingomyelins (SM); (b) glycerolipids: diglycerides (DG) and triglycerides (TG); (c) glycerophospholipids: phosphatidylcholines (PC), phosphatidylethanolamines (PE), ether‐linked phosphatidylethanolamines (PE(O)), phosphatidylinositols PI); and (d) sterols: cholesteryl esters (CE).