Agilent 1260 infinity binary lc

Phytochemicals of AM extract were analyzed by HPLC-diode array. The HPLC was performed using an Agilent 1260 Infinity Binary LC (Santa Clara, CA, USA). The chromatographic conditions were based on a previous study [28 (link)]. The AM extract was separated on a 150 mm × 4.60 mm Purospher^® Star PR-18 end capped column with a particle size of 5 µm. The mobile phase consisted of water containing 0.1% formic acid and acetonitrile (92:8 % v/v) at a flow rate of 0.8 mL/min set for 10 min. The injection volume of sample was 10 µL, and acetonitrile was increased as described here: time (min)/% acetonitrile 24/14, 35/23, and 60/24, respectively. The detection wavelengths were set at 250 nm and 330 nm. Spectra between 200 nm and 400 nm were collected for analysis. Peak identification in the chromatogram was performed by comparing retention times and spectral characteristics with the standard.

Ex-DHF-P utilized standard operating procedures for blood sampling. Fasted blood samples from each patient were drawn on the same time of the day to avoid circadian variation and after a 20 min resting period in supine position. All samples were immediately centrifuged and stored at −80 °C. Targeted metabolomics profiling of the plasma samples was performed using the AbsoluteIDQ p180 Kit (BIOCRATES LifeSciences AG, Innsbruck, Austria). A 10 µL aliquot of each plasma sample was processed as recommended by the manufacturer. The fully automated assay combined flow injection (FIA) and LC-MS/MS selective detection using MRM pairs and quantifies up to 188 metabolites from 5 different compound classes. Via FIA acyl carnitines, phospho- and sphingolipids were measured in positive ionization mode and the sum of hexoses in negative ionization mode. With a LC-MS/MS analytical method, under the use of an Agilent C18 column, amino acids and biogenic amines were detected. MS analyses were performed on an AB SCIEX 5500 QTrap™ mass spectrometer (AB SCIEX, Darmstadt, Germany) with electrospray ionization combined with an HPLC system (Agilent 1260 Infinity Binary LC, Santa Clara, CA, USA). Internal standards (isotope labelled) were partially integrated in the kit plate for metabolite quantification.

Size exclusion (SE)-HPLC was performed on the Agilent 1260 Infinity Binary LC (Agilent, Santa Clara, CA, USA) at the Veterinary Drugs and Biologics Division of the APQA. The LC was equipped with a G1311C quaternary pump with degasser and a G4212B variable wavelength detector with UV monitoring at 254 nm. The chromatograph was fitted with the TSKgel G4000 PWXL analysis column (7.8 mm inner diameter × 30 cm; Tosoh Corporation, Bioscience Division, Tokyo, Japan) and the TSKgel PWX guard column (6.0 mm inner diameter × 4.0 cm, Tosoh Corporation, Bioscience Division, Tokyo, Japan) to extend column life. OpenLab Chromatography data system (CDS) ChemStation Edition Rev. C01.02 was used for analysis. The mobile phase consisted of a mixture of 30 mM Tris-HCl and 400 mM NaCl adjusted to pH 8.0 with hydrochloric acid. Hundred microliters of each sample was injected and eluted via isocratic flow at 0.5 mL/min in the mobile phase. The operating pressure of the HPLC was approximately 40 bars at a column oven temperature of 20 ℃.