Oasis max μelution plate

AA- and LA-derived lipid mediators detectable by our ultraperformance LC-MS approach were assessed in the mucus samples collected at the time of surgery (see Fig E1 in this article’s Online Repository at www.jacionline.org). Twenty-five to 40 μL of each mucus specimen was placed into a microcentrifuge tube containing 5000 μL 25% methanol in water and internal standard mix (1 ng each deuterated eicosanoid). The sample was vortexed and spun to pellet protein. The supernatant was then extracted on an Oasis MAX μElution plate (Waters Corp, Milford, Mass) as follows: Sample wells were first washed with methanol (200 μL) followed by 25% methanol in water (200 μL). The sample was then loaded into the well and washed with 600 μL 25% methanol. Eicosanoids were eluted from the plate with 30 μL 2-propanol/acetonitrile (50/50, vol/vol) containing 5% formic acid into a 96-well elution plate containing 30 μL water in each well. Samples were analyzed on a Waters Xevo TQ-XS triple quadrupole mass spectrometer connected to a Waters Acquity I-Class ultraperformance LC (Waters Corp). Separation of analytes was obtained using an Acquity PFP column (2.1 × 100 mm), with mobile phase A being 0.01% formic acid in water and mobile phase B acetonitrile. Eicosanoids were separated using a gradient elution beginning with 30% B going to 95% B over 8 minutes at a flow rate of 0.250 mL/min.