Exionlc ac

The lipids were determined according to our method described earlier [25 (link)] using an ExionLC AC (Sciex, Framingham, MA, USA) UHPLC system and a 4500 Q-TRAP mass spectrometer equipped with an ESI source. A total of 10 µL of lipid extract was injected onto an Eclipse XDB-C18, 50 mm × 4.6 mm, 1.8 µm column (Agilent, Santa Clara, CA, USA), heated at 40 °C, with the flow rate of 500 μL min⁻¹. Ultrapure water (A) and methanol (B) were applied as a mobile phase, with both containing 5 mM ammonium formate. The solvent gradient was initiated at 70% B and, after 0.25 min, increased to 100% B for 1 min; then, it was maintained at 100% B for 6 min before being returning to the initial solvent composition over 2 min. The data analysis was conducted with Analyst v1.6.3 software (Sciex, Framingham, MA, USA). The phospholipids were determined qualitatively using an MRM method with the following standards for each class: phosphatidylethanolamine (PE 14:0/14:0; Merck, Darmstadt, Germany) and phosphatidylglycerol (PG 14:0/14:0; Merck, Darmstadt, Germany).

A rapid resolution liquid chromatograph (ExionLC™ AC; SCIEX, Framingham, MA, USA) was coupled to a 5500 Q-Trap system (SCIEX) with an ESI source. Chromatographic separation was undertaken on a HSS C18 column (2.1 × 100 mm, 1.8 μm; Waters, Milford, MA, USA) at 35 °C. The flow rate was 0.25 mL/min. The injection volume was 2 μL. Mobile phase A was 0.01% formic acid with ammonium acetate (0.1 mM) in Milli-Q water. Mobile phase B was acetonitrile. The linear gradient was the same as that described for untargeted MA (equilibrium duration = 16.1–20 min, 98% A). The Q-Trap system was operated in positive and negative ion modes using MRM. The source-dependent parameters were ion spray voltage = 5 kV (−4.5 kV for negative); vaporizer temperature = 500 °C, nebulizing gas (GS1) = 40 psi; drying gas (GS2) = 45 psi; curtain gas = 30 psi. Acquisition and processing of data were done using Analyst 1.6 (SCIEX).