2.5. Sample Preparation and LC-MS Analysis of Lipid Mediators

For LC-MS analyses, lung and cortex tissues were homogenized using zirconium beads in a high-frequency oscillator (Precellys, Bertin Technologies, Montigny-le-Bretonneux, France) in phosphate-buffered saline (50 mM phosphate, pH 7.2, and 0.9% sodium chloride). Protein content was quantified using the BCA method. Recovery and quantitation homogenates containing 1–2 mg protein were spiked with 5 ng each of 15(S)-HETE-d8,14(15)-EpETrE-d8, Resolvin D2-d5, Leukotriene B4-d4, and Prostaglandin E1-d4 as internal standards. The samples were then purified using C18 extraction columns as described earlier [36,37,38]. Briefly, the internal standard spiked samples were applied to conditioned C18 cartridges, washed with 15% methanol in water followed by hexane, and dried under vacuum. The cartridges were eluted with 0.5 mL methanol. The eluate was dried under a gentle stream of nitrogen. The residue was redissolved in 50 µL methanol-25 mM aqueous ammonium acetate (1:1) and subjected to LC-MS analysis.

HPLC separation (Prominence XR system, Shimadzu) was achieved using a Luna C18 (3µ, 2.1 × 150 mm) column; the mobile phase consisted of a gradient between A: methanol-water-acetonitrile (10:85:5 v/v) and B: methanol-water-acetonitrile (90:5:5 v/v), both containing 0.1% ammonium acetate, the flow rate was 0.2 mL/min. The gradient program concerning the composition of B was as follows: 0–1 min, 50%; 1–8 min, 50–80%; 8–15 min, 80–95%; and 15–17 min, 95%. The eluate was directly introduced to ESI source of QTRAP5500 mass analyzer (AB Sciex, Framingham, MA, USA) in the negative ion mode with the following conditions: Curtain gas: 35 psi, GS1 & GS2: 35 psi, Temperature: 600 °C, Ion Spray Voltage: −1500 V, Collision gas: low, Declustering Potential: −90 V. The eluate was monitored by the Multiple Reaction Monitoring method to detect unique molecular ion—daughter ion combinations for each of the lipid mediators using a scheduled MRM around the expected retention time for each compound. Optimized Collisional Energies (18–35 eV) and Collision Cell Exit Potentials (7–10 V) were used for each MRM transition. Spectra of each peak detected in the scheduled MRM were recorded using Enhanced Product Ion scan to confirm the structural identity. T Analyst 1.6.3 software and the MRM transition chromatograms were utilized to collect the data that were quantified by MultiQuant software (both from AB Sciex). The internal standard signals in each chromatogram were used for normalization, recovery, as well as relative quantitation of each analyte [36,37,38].