Acquity uplc beh 1.7 μm 2 1 50 mm column

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)).

The metabolites in the crude extracts as well as the fractionated extracts were analyzed on a Waters Acquity I-Class UPLC system coupled to an Acquity TUV detector and Synapt G2Si HDMS mass spectrometer in positive ion mode with a heated electrospray ionization (ESI) source in a Z-spray configuration. LC separation was performed on a Waters Acquity UPLC BEH 1.7 μm 2.1 × 50 mm column using an 0.6 mL/min gradient of 95/5–15/85 A/B in 4 min followed by washing and reconditioning the column. Eluent A is 0.1 % formic acid in water and B is 0.1 % formic acid in ACN. Conditions on the mass spectrometer were as follows: capillary voltage 0.5 kV, sampling cone 40 V, source offset 80 V, source 120 °C, desolvation 250 °C, cone gas 0 L/h, desolvation gas 1000 L/h and nebulizer 6.5 bar. The analyzer was operated in resolution mode and low energy data was collected between 100 and 2000 Da at 0.2 s scan time. MS^e data was collected using a ramp trap collision energy 20−40 V, and masses were extracted from the TOF MS TICs using an abs width of 0.005 Da.