4.3. Lipidomics Analysis

The chloroform phase was used to detect the lipidome using LC–MS on an Orbitrap Exactive (Thermo Scientific, Waltham, MA, USA) in line with an Ultimate 3000 LC (Thermo Scientific, Waltham, MA, USA) [37]. Each sample was analyzed in positive and negative modes, in top 5 automatic data-dependent MS/MS mode. Column hardware consisted of a Biobond C4 column (4.6 × 50 mm, 5 μm, Dikma Technologies, Foothill Ranch, CA, USA). Flow rate was set to 100 μL/min for 5 min with 0% mobile phase B, then, switched to 400 μL/min for 50 min, with a linear gradient of mobile phase B from 20% to 100%. The column was then washed at 500 μL/min for 8 min at 100% mobile phase B before being re-equilibrated for 7 min at 0% mobile phase B and 500 μL/min. For positive mode runs, buffers consisted, for mobile phase A, of 5 mM ammonium formate, 0.1% formic acid, and 5% methanol in water, and, for mobile phase B, of 5 mM ammonium formate, 0.1% formic acid, 5% water, and 35% methanol in Isopropanol. For negative runs, buffers consisted, for mobile phase A, of 0.03% ammonium hydroxide and 5% methanol in water, and, for mobile phase B, of 0.03% ammonium hydroxide, 5% water, and 35% methanol in isopropanol. Each of the spectra for each lipid identified with Lipidsearch^© software (version 4.1.16, Mitsui Knowledge Industry, University of Tokyo) was manually examined for the presence of the head group characteristic fragment, and, if present, for the side chain’s fragment. Each ID was based on the fragments founds in the literature and compiled in the Lipidsearch database. Intensity signals are reported as area of the parent ion. Integrations and peak quality were curated manually before exporting and analyzing the data in Microsoft Excel.

Since the number of samples in the runs were low, the instrument sensitivity did not change significantly, and no repeated quality control sample injections were needed. The instrument was running a constant internal calibration with lock masses for mass accuracy, insuring no drift in the mass accuracy over the span of the sample runs.