LC-MS/MS–based lipidomics

Plasma was obtained by centrifugation (4,500g, 5 min, 4°C) of the heparinized blood samples. Organ extracts were prepared by (i) homogenization in ice-cold methanol containing indomethacin and BHT, (ii) removal of insoluble remains by centrifugation (4500g, 4°C, 10 min), (iii) evaporation of methanol under a stream of nitrogen at room temperature, and (iv) dissolution of the residue in water.

After the internal standard (PGE₁) was added to plasma, organ extracts, or culture medium, the samples were acidified with 0.1% formic acid. The samples were then subjected to solid-phase extraction in an Oasis mixed-mode anion exchange μElution plate (Waters) in three sequential steps: (i) The plate cartridge was preconditioned with methanol and then with water; (ii) samples were loaded into the cartridge, and unwanted compounds were eluted in water containing 5% of methanol; and (iii) the compounds of interest were eluted in methanol containing 0.1% of formic acid. The prepared samples were then passed through an Acquity UPLC system equipped with a BEH C18 column (1.7-μm particles, 2.1 mm by 50 mm), which was coupled to an Acquity TQD MS/MS detector. The mobile phase consisted of water (solvent A) and acetonitrile (solvent B), both with 0.1% formic acid, and it was pumped at a flow rate of 0.4 ml/min. The gradient elution program was performed in four stages: (i) solvent B at 35% for 6 min, (ii) solvent B at 90% for 1.5 min, (iii) solvent B at 99% for 2.5 min, and (iv) solvent B at 30% for 3 min. The injection mode was full loop, with injection volume of 10 μl. The MS tune parameters were as follows: 0.16 liter/hour for the flow of the collision gas (argon), 3.02 × 10⁻³ mbar for the pressure in the collision cell, and 1000 liters/hour for the flow of the desolvation gas (N₂). The source and desolvation temperatures were 150° and 400°C, respectively. A multiple reaction monitoring mode was used for the quantification of the analytes in negative ionization mode, according to the parameters shown in table S2. The standards were purchased from Cayman and analyzed in concentrations ranging from 0.1 to 50 ng/ml.