Quantification of Lipid Mediators by LCMS

Lipid mediators were examined by LCMS essentially as described previously (Quehenberger et al., 2011 (link); Tam et al., 2013 (link); Tam et al., 2020 (link)). Before lipid metabolite isolation by solid phase extraction (SPE), deuterated standards (Cayman Chemical) were added to 0.9 mL of BAL Methanol was evaporates and the samples reconstituted in a minimal volume of water/acetonitrile (60/40) containing 0.02% v/v acetic acid. Eicosanoids were separated using a Waters Acquity UPLC BEH 1.7 μm 2.1 × 50 mm column using a 4 minute gradient of 99.9% A/B to 75/25 A/B followed by washing and reconditioning. Solvent A is 50/50 water/acetonitrile containing 0.02% acetic acid and solvent B is 50/50 acetonitrile/isopropanol. Eicosanoids were analyzed by a Waters Synapt G2Si QTOF operated in negative-ionization mode via MS^e. Data analysis was performed using UNIFI 1.6 (Waters), MS-DIAL4 (Tsugawa et al., 2020 (link)), and Mzmine 2.53 (Pluskal et al., 2010 (link)).