Lipid extraction and FA lipidomic analysis

For lipid hydrolysis, extracted lipids were resuspended in 200 µl of ethanol and incubated with 0.1 M KOH at RT for 24 h for saponification. The reaction was stopped by addition of 0.2 M HCl. Lipids were extracted as described above with ²H₃₁ palmitic acid as an internal standard.

FA lipidomic analysis was performed on a Dionex Ultimate 3000 LC system (Thermo Fisher Scientific) coupled to a TSQ Quantiva mass spectrometer (Thermo Fisher Scientific). Solvent A consisted of 95:5 water:methanol. Solvent B was 70:25:5 isopropanol:methanol:water. For negative mode, solvents contained 0.028% ammonium hydroxide. An XBridge C8 column (5 µm, 4.6 mm × 50 mm; Waters) was used. The gradient was as described in the section “Lipid extraction and untargeted lipidomics.” MS analyses were performed using electrospray ionization in negative ion mode, with spay voltages of −2.5 kV, ion transfer tube temperature of 325°C, and vaporizer temperature of 200°C. Sheath, auxiliary, and sweep gases were 40, 10, and 1, respectively. Pseudo-multiple reaction monitoring was performed for all FAs.