Agilent 1290 infinity liquid chromatography system

Two groups of mice (Xh-CL and WT animals, 24 mice in each group) were used for pharmacokinetic analysis. The mice received a single dose of 1 mg/kg TP by oral gavage. Blood was collected at 5, 10, 15, 20, 30, 45, 60, and 120 min (n = 3 for each time point). Around 0.5 mL blood was collected from the ocular sinus at each time point. The mice were euthanized with CO₂ immediately after blood collection. Plasma was prepared by centrifugation at 900×g for 10 min and kept at − 80 °C until analysis. TP was then extracted from 200 µL plasma with 2 × 600 µL ethyl acetate and dried under nitrogen. The residues were reconstituted in 100 µL methanol for analysis. TP concentrations were quantified with an Agilent 1290 Infinity Liquid Chromatography system equipped with Agilent ZORBAX Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm) (Agilent Technologies, Santa Clara, CA, USA) for pharmacokinetic analysis. The mobile phase consisted of acetonitrile and water (30:70), with a flow rate of 0.2 mL/min and a sample volume of 2 µL. The compound was measured at 218 nm, and the column was maintained at 25 °C. The pharmacokinetic parameters were analyzed with the software package DAS 3.2 (Mathematical Pharmacology Professional Committee of China, Shanghai, China).

Hepatic metabolite profiles were analysed using an Agilent 1290 Infinity Liquid Chromatography System (Agilent Technologies) equipped with a 2.1 × 100 mm C18 reverse-phase column with 1.8-μm particle size (Waters Corp., Milford, MA, USA) as described previously40 (link). Mass spectrometry was performed on an Agilent 6530 Accurate-Mass QTOF/MS (Agilent Technologies) equipped with an electrospray ionisation source. Data for each ionisation technique were acquired in positive and negative ion modes. LC data were acquired and processed using Mass Hunter Qualitative Analysis Software (version B.03.01; Agilent Technologies). The MS analysis system was used to identify metabolites corresponding to those in the METLIN database (http://metlin.scripps.edu). SIMCA-P+ 11.0 software (Umetrics AB, Umea, Sweden) and online tool MetaboAnalyst 3.0 (http://www.metaboanalyst.ca/MetaboAnalyst) were used for PCA, partial least squares discriminant analysis (PLS-DA) and OPLS-DA analyses. A t-test was used to identify those candidate metabolites obtained from PLS-DA modelling that were statistically different from those in the control group.
Fatty acids in liver tissue were measured by gas chromatography as previously described41 (link)42 (link). TCA cycle metabolites in liver tissue were assayed with a Shimadu QP-2010 ultra GC/MS43 (link).