2.4. Liquid Chromatography/Mass Spectrometry (LC/MS) Analyses of Phospholipids

Lipids were extracted according to Bligh and Dyer [39], and the following internal standards were added: 20 pmol each of 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-di- heptadecanoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoserine, 1,2-dimyristoyl-sn-glycero-3-phosphate, 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine, 1,2-di- heptadecanoyl-sn-glycero-3-phosphocholine, and 1,2-dinonadecanoyl-sn-glycero-phosphocholine.

The samples were re-dissolved in hexanes/2-propanol/water (42:56:2, v/v/v) and injected into a Thermo Scientific Dionex Ultimate 3000 high-performance liquid chromatograph equipped with an Ultimate HPG-3400SD standard binary pump and an Ultimate ACC-3000 autosampler column compartment (Waltham, MA, USA). Separation was carried using a FORTIS HILIC column (150 × 3 mm, 3 µm particle size) (Fortis Technologies, Neston, UK). The mobile phase consisted of a gradient of solvent A (hexanes/isopropanol 30:40, v/v) and solvent B (hexanes/isopropanol/20 mM ammonium acetate in water, 30:40:7, v/v/v). The gradient started at 75% A from 0 to 5 min, then decreased from 75% A to 40% A at 15 min, from 40% A to 5% A at 20 min, was held at 5% until 40 min, and then increased to 75% at 41 min. Then, the column was re-equilibrated holding 75% A for an additional 14-min period before the next sample injection [46]. The flow rate through the column was fixed at 0.4 mL/min. The liquid chromatography system was coupled online to an Sciex QTRAP 4500 Mass Spectrometer equipped with a Turbo V ion source and a TurbolonSpray probe for electrospray ionization (AB Sciex, Framingham, MA, USA). Source parameters were set as follows: ion spray voltage, -4500 V; curtain gas, 30 psi; nebulizer gas, 50 psi; desolvation gas, 60 psi; temperature, 425 °C. Phospholipid species were analyzed in scheduled multiple reaction monitoring mode with negative ionization, detecting in Q3 the m/z of either 303.2 or 331.2, corresponding to AA and AdA, respectively, as [M-H]⁻. Compound parameters were fixed as follows: declustering potential; −45 V (choline glycerophospholipids), −60 V (ethanolamine glycerophospholipids) −30 V (phosphatidylinositol), −50 V (phosphatidylserine), −60 V (phosphatidic acid), −50 V (phosphatidylglycerol); collision energy: −50 V (choline glycerophospholipids), −40 V (ethanolamine glycerophospholipids), −60 V (phosphatidylinositol), −50 V (phosphatidylserine), −45 V (phosphatidic acid), −45 V (phosphatidylglycerol); entrance potential, −10 V; and collision cell exit potential, −8 V. All glycerophospholipids were detected as [M-H]⁻, ions except choline glycerophospholipids, which were detected as [M + CH₃COO]⁻ ions. Quantification was carried out by integrating the chromatographic peaks of each species and comparing these with the peak area of the internal standard that corresponded to each class.