1260 uhplc

All the equipment for the analyses was from Agilent technologies (Santa Clara, CA, United States) and consisted of a 6545 QTOF mass spectrometer equipped with an electrospray ionization interface (ESI) coupled to a 1260 UHPLC, a G4204A quaternary pump, G4226A ALS auto-sampler, and G1316C thermostated column compartment. UHPLC was carried out on a ZORBAX RRHD Eclipse Plus C18, 95Å, 2.1 × 50 mm, 1.8 μm column, with water (0.1% formic acid)-MeCN gradient elution, from 5 to 95% MeCN for 10 min at a flow rate of 0.5 mL/min. Twenty μL of each sample were injected into the LC-MS/MS instrument in triplicates and an average peak area of three analyses was calculated. The water-MeCN solution was injected as a blank within a sequence of samples to confirm that there was no cumulative carryover. Mass spectral parameters were optimized for 3-oxo-C6-HSL by varying the fragmentor voltage of the ion source for scan mode and collision energy for product ion mode (MS/MS), 3-oxo-C8-HSL was quantified under the same parameters. Specific parameters of the ion source were readjusted. The ESI was operated in positive mode. The source temperature was set to 300°C, Nozzle voltage 500 V and ion spray voltage was 5.5 kV.

Nonfasting blood samples were taken by venipuncture in all patients during the early morning hours. Each blood sample was centrifuged immediately to obtain the serum, which was stored in Eppendorf tubes at − 80 °C until the A77 1226 levels were quantified. Serum samples were analysed to quantify A77 1726 levels by a validated high-pressure liquid chromatographic method, using an Agilent 1260 UHPLC (Santa Clara, California, United States of America). Separation of the analytes was accomplished using a reversed-phase column (Phenomenex, C18, 4µ, 150 × 4.6 mm; Torrens, California, United States of America). The column temperature was maintained at 21 °C. The mobile phase consisted of 40 mM (pH 5.0) KH₂PO₄:methanol (1:1). Elution of A77 1726 was achieved at a flow rate of 1.2 mL/min. UV detection was set at 290 nm.