Luna 3 μm silica 2 100

MS analysis of phospholipids was performed on an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher). Phospholipids were separated on a normal phase column (Luna 3 μm Silica (2) 100 Å, 150 × 2.0 mm, Phenomenex) at a flow rate of 0.2 ml min⁻¹ on a Dionex Ultimate 3000 HPLC system. The column was maintained at 35 °C. Analysis was performed using gradient solvents (A and B) containing 10 mM ammonium acetate. Solvent A contained propanol:hexane:water (285:215:5, vol/vol/vol) and solvent B contained propanol:hexane:water (285:215:40, vol/vol/vol). All solvents were LC–MS grade. The column was eluted for 0–23 min with a linear gradient of 10–32% B; 23–32 min using a linear gradient of 32–65% B; 32–35 min with a linear gradient of 65–100% B; 35–62 min held at 100% B; 62–64 min with a linear gradient from 100% to 10% B followed by and equilibration from 64 to 80 min at 10% B. The instrument was operated with the electrospray ionization probe in negative polarity mode. Analysis of LC–MS data was performed using software package Compound Discoverer (ThermoFisher) with an in-house generated analysis workflow and oxidized phospholipid database. Lipids were further filtered by retention time and confirmed by fragmentation mass spectrum.

Lipids were extracted using the Folch procedure, and phosphorus was determined by a micro-method as described previously [3 (link)]. Phospholipids were analyzed by LC/MS using a Dionex Ultimate 3000 HPLC system coupled on-line to the Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) using a normal phase column (Luna 3 μm Silica (2) 100 Å, 150 × 2.0 mm, (Phenomenex)) as described previously [3 (link)].