100 mm c18 reverse phase column

Serum metabolite profiling was performed on an Agilent 1290 Infinity Liquid Chromatography System (Agilent Technologies, Santa Clara, CA, USA) equipped with a 2.1 × 100 mm C18 reverse-phase column with 1.8 μm particle size (Waters Corp., Milford, MA, USA). The column was maintained at 40°C; the injected sample volume was 4 μL. Gradient conditions were 0–2 min 5% B, 2–17 min linear gradient from 5 to 95% B, and 17–19 min 95% B. Solvent A was 0.1% formic acid-water; solvent B was 0.1% formic acetonitrile. The flow rate was 400 μL/min. MS experiments were performed on an Agilent 6530 Accurate-Mass Q-TOF/MS (Agilent Technologies) equipped with electrospray ionization source. Data for each ionization technique were acquired in positive ion mode. The measurement conditions were capillary voltage 4.0 kV, cone voltage 35 kV, ion source temperature 100°C, and vaporizer temperature 350°C. Nitrogen was used as the nebulizer gas and delivered at a flow rate of 50 L/h; the desolvation gas (nitrogen) was delivered at a flow rate of 600 L/h. The scan range was m/z 50–1000.

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).