Waters 2795 hplc

Analysis of several lipid classes, e.g., triacylglycerols (TG), phospholipids, sphingolipids, and lysophospholipids was performed using liquid chromatography tandem mass spectrometry (LC-MS/MS). In brief, the second portion of the dissolved lipids was analyzed by Waters 2795 HPLC (Waters Corporation, MA, USA) coupled to a triple quadrupole mass spectrometer (Acquity TQ Detector, Water Corporation). Samples were injected into a Hypersil GOLD Phenyl column (150 mm × 2.1 mm i.d, 3 μm particle size, ThermoFisher Scientific, USA) with a 45-min stepwise linear gradient: 5 mM ammonium acetate in MilliQ water was used as mobile phase A and 5 mM ammonium acetate in methanol was used as mobile phase B. The gradient started at 20% B for 2 min followed by an increase from 80% to 99% B in 18 min and then stayed at 99% for 15 min. The re-equilibration step was performed going back to 80% in 1 min and equilibrate for 9 min. One 20-μl aliquot was injected for detection of phosphatidylcholine (PC) and sphingomyelin (SPH) species by multiple reaction monitoring (MRM) mode, and one 20 μl aliquot was injected detecting phosphatidylethanolamine (PE) and lysophosphatidylcholine (LPC) species by MRM. Integration of peaks was performed by MassLynx software, version 4.1. The relative abundance of lipid species was calculated using area percentage.

Prostaglandins in cell supernatants were extracted and analyzed according to Idborg et al. [19 (link)]. Working in duplicates, 450 μl of samples were spiked with 50 μl deuterated internal standards of 6-keto-PGF_1α, PGF_2α, PGE₂, PGD₂, TxB₂, and 15-deoxy-Δ12,14PGJ₂ (Cayman Chemical Company) and made acidic with 500 μl 0.2 % formic acid (FA) in Milli-Q. The samples were loaded on Oasis HLB 1 cc 30 mg cartridge (Waters Corporation, MA, USA) that had been preconditioned with methanol and 0.05 % FA in Milli-Q. Prostaglandins were eluted in 1 ml methanol and evaporated to dryness under vacuum. Samples were stored at −20 °C until resuspended in 50 μl 7 % acetonitrile prior to analysis with LC-MS/MS. The extracted prostaglandins were quantified using a triple quadrupole mass spectrometer (Acquity TQ Detector, Waters Corporation) equipped with a Waters 2795 HPLC (Waters Corporation). Separation was performed on a 100 × 2.0 mm Synergi 2.5 μm Hydro-RP 100 Å column (Phenomenex, CA, USA) with a 45-min stepwise linear gradient (10–90 %) of 0.05 % FA in acetonitrile as mobile phase B and Milli-Q as mobile phase A. Individual prostaglandins were detected in multiple reaction monitoring mode. Data were analyzed using MassLynx Software, version 4.1, with internal standard calibration and quantification to external standard curves.