Lipid mediator standards

Tributylamine was purchased from Millipore Sigma. LCMS grade water, methanol, isopropanol, chloroform and acetic acid were purchased through Fisher Scientific. All lipid mediator standards were purchased from Cayman Chemical.

All lipid mediator standards were purchased from the Cayman Chemical Company (Ann Arbor, MI, USA). HLB SPE cartridges were purchased from Anbocis Technology (Beijing, China). All HPLC grade solvents were acquired from Sigma–Aldrich (St. Louis, MO, USA) or Merck & Co., Inc. (Kenilworth, NJ, USA). Aliquots of 100 μL of thawed human serum were used. A mixture of deuterium-labelled internal standards was added to each sample, followed by 1.5 mL of cold methanol (MeOH). The samples were vortexed for 5 min and stored at -20°C overnight. Cold samples were centrifuged at 14,000 rpm for 5 min, and the supernatant was then transferred to a new tube. Samples were evaporated under nitrogen to volumes less than 50 μL, after which 1 mL of acidified H₂O (pH 3.5) was added to each sample before HLB SPE extraction as previously described [14 (link)]. In brief, the acidified samples were loaded onto an HLB SPE column, washed with water and hexane, and eluted with methyl formate. The methyl formate fractions were collected, dried under nitrogen, and reconstituted in 100 μL of MeOH:H₂O (2 : 3, v/v). Samples were centrifuged at 20,000 rpm, and 80 μL of the supernatants was subjected to LC–MS/MS lipid mediator metabolomics analysis.

Phospholipid mixtures were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Lipid mediator standards and deuterium-labeled compounds used as internal standards were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Trace element standard solutions, special grade reagents of ammonium bicarbonate, sodium chloride, diethyl ether, and chloroform, nitric acid and perchloric acid of toxic metal determination grade, hydrogen peroxide for atomic absorption spectrometry, and high-performance liquid chromatography-grade solvents (methanol, isopropanol, and acetonitrile) were purchased from Wako Pure Chemical Co. (Osaka, Japan) and from Kanto Chemical Co., Inc. (Tokyo, Japan, for untargeted lipidomics). Deionized water was produced using a Millipore-Q water system (Millipore, Bedford, MA, USA).