LC–High Resolution Mass Spectrometry Analysis of Fatty Acids.

Samples (20 μl) were injected onto a Hypersil Gold Vanquish C18 column (2.1 mm × 150 mm, 1.9 μm; Thermo Fisher) operating at 40°C with a flow rate of 0.3 ml min⁻¹. Chromatographic separation was achieved using a system composed of solvent A: 10 mM ammonium acetate (pH 6.8) and solvent B: CH₃CN. Gradient conditions were set up as follows: 45% B (0–2 minutes), linear gradient of B increasing to 75% (2–15 minutes), linear gradient of B increasing to 95% (15–16 minutes), held at 95% B (16–18 minutes), return to initial condition (5% B) for column equilibration (18–20 minutes). The UPLC was coupled to the same mass spectrometer with a heated electrospray ionization electrospray ionization source (Thermo Fisher). Analysis was performed in the negative ionization mode only using the following: 1) full MS scan range m/z 100–600 Da and 2) parallel reaction monitoring scanning at the exact mass of each of the parent molecules and its corresponding mono-oxygenation product with 30 eV collision energy and a m/z 1.6 isolation window. Both scans used the same 30,000 resolution, 2e⁵ AGC target, and 200-ms IT value. The tune file source parameters were the same as used in tissue extract analyses.