Shim pack giss column

An aliquot of 150 μL of acetonitrile was added to the plasma samples (50 μL), and the mixture was shaken for 60 seconds. After centrifugation for 6 min at 9000 g, the supernatant was collected and a 25 μL aliquot was directly injected into the LC MS/MS system. The analytical system consisted of a Nexera UFLC system coupled to a LCMS-8040 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan). The ESI-MS/MS parameters were set in positive and negative ion mode (polarity switch, 25 msec) as follows: capillary voltage, positive 4500 V and negative 3000 V; desolvation line temperature, 200 °C; heating block temperature, 500 °C; ); and m/z 506.0 → m/z 207.5 for detection of ATP ([M-H] -). The chromatographic separation was conducted with a Shim-pack GISS column (2.1 x 100 mm, 1.9 μm particle size) (Shimadzu, Kyoto, Japan) eluted with flow rate of 0.3 mL/min. The gradient mobile phase system consisted of water (solvent A) and acetonitrile (solvent B) both fortified with 0.2% acetic acid and 0.1% tributylamine as follow: 0 -4.5 min, 10 -90% of B; 4.5 -5.5 min, 90% of B; 5.5 -5.6 min, 90 -10% of B; 5.6 -10 min, 10% B.
The column oven was kept at 30 °C. The data were processed using LabSolutions software (Shimadzu, Kyoto, Japan). In addition, LPS concentration was determined by quantitation of 3-hydroxytetradecanoic acid as described by Teixeira and coworkers (17) .