Quantification of Lipid Mediators in Plasma

Lipid mediators in human plasma were analysed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) based on protocols published previously [12] (link), [13] (link). Briefly, samples were collected and stored immediately at −80°C. Plasma samples (500 µL) were defrosted on ice and adjusted to 15% (v/v) methanol: water (final volume 4 mL). Internal standards, PGB2-d4 (40 ng) and 12-HETE-d8 (40 ng) (Cayman Chemical Company, Ann Arbor, USA) were added and the pH of resulting solutions adjusted to 3.0 (1 M HCL). Acidified samples were immediately applied to preconditioned solid-phase cartridges (C18-E, Phenomenex, Macclesfield, UK) and lipid mediators eluted with methyl formate. LC/ESI-MS/MS analysis was performed on a HPLC pump (Waters Alliance 2695) coupled to an electrospray ionisation triple quadrupole mass spectrometer (Quattro Ultima, Waters, UK). Chromatographic separation was performed on a C18 Luna column (5 µm, 150×2.0 mm, 21 Phenomenex) for eicosanoids and a C18 Kinetex column (2.6 µm, 100×2.1 mm, Phenomenex) for hydroxy-fatty acids. Analytes were monitored on multiple reaction monitoring mode as reported [12] (link), [13] (link) with the following additions: 15-hydroxyeicosatrienoic acid (HETrE) m/z 321>221, 10-hydroxydocosahexaenoicacid (HDHA) m/z 343>153, 14-HDHA m/z 343>161, 13-HDHA m/z 343>193 and 17- HDHA m/z 343>201.