2.5. Lysophosphatidylcholine Quantification
SEPARATION, RESOLUTION
Stock lipid standards were prepared by dissolving them in dichloromethane (DCM)/methanol (MeOH; 2:1 Thirty microliters of internal standard mixture were added to 30 μL of sample followed by 190 μL of MeOH. Samples were then vortexed for 20 s. Next, 380 μL of DCM was added, the sample was vortexed for 20 s, and 120 μL of water was added to induce phase separation. The samples were then vortexed for 10 s and allowed to equilibrate at room temperature for 10 min before centrifugation at 8000× Chromatographic separation was performed on a Vanquish UHPLC system with an Accucore C30, 2.6 μm column (2.1 mm id × 150 mm) coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap High Resolution Mass Spectrometer (Thermo Scientific). The mobile phase consisted of (solution A) 60% ACN, 40% water, 10 mM ammonium formate with 0.1% formic acid and (solution B) 90% IPA, 10% ACN, 10 mM ammonium formate with 0.1% formic acid. The gradient was as follows: 0 to 1.5 min, 32% solvent B; 1.5 to 4 min, 32 to 45% solvent B; 4 to 5 min, 45 to 52% solvent B; 5 to 8 min 52 to 58% solvent B; 8 to 11 min, 58 to 66% solvent B; 11 to 14 min, 66 to 70% solvent B; 14 to 18 min, 70 to 75% solvent B; 18 to 21 min, 75 to 97% solvent B; 21 to 25 min, isocratic 97% solvent B; 25 to 25.1 min, 97 to 32% solvent B; followed by 4 min of re-equilibration of the column before the next run. The flow rate was 260 μL/min. All of the samples were analyzed by electrospray ionization in data-dependent MS/MS mode.Nitrogen was used as the nebulizing gas, with sheath, auxiliary, and sweep gas flows set at 50, 5, and 1 arbitrary units, respectively. Other conditions included the following: resolution, 120,000 full width at half maximum; automatic gain control target, 3 × 10Acquired data were processed using LipidSearch™ software version 4.3 (Thermo Scientific) with the following workflow: first, the individual data files were searched for product ion MS/MS spectra of lipid precursor ions. MS/MS fragment ions were predicted for all precursor adduct ions measured within ±5 ppm. The product ions that matched the predicted fragment ions within a ±5 ppm mass tolerance were used to calculate a match-score, and those candidates providing the highest-quality match were determined. Next, the search results from the individual positive or negative ion files from each sample group were aligned within a retention time window (±0.1 min) and the data were merged for each annotated lipid. The annotated lipids were then filtered to reduce false positives.