Xcalibur quantitative software

The profiling experiment was performed with a Dionex Ultimate 3000 UHPLC system (Thermo Scientific) coupled to a Q-Exactive (Thermo Scientific) equipped with an electrospray source operating in both positive and negative mode and full scan mode from 100 to 1200 m/z. The Q-Exactive parameters were: sheath gas flow rate 55 au, auxiliary gas flow rate 15 au, spray voltage 3.3 kV, capillary temperature 300°C, S-Lens RF level 55 V. The mass spectrometer was calibrated with sodium acetate solution dedicated to low mass calibration.
10 μL of sample were injected on a SB-Aq column (100 mm × 2.1 mm particle size 1.8 μm) from Agilent Technologies, protected by a guard column XDB-C18 (5 mm × 2.1 mm particle size 1.8 μm) and heated at 40°C by a Pelletier oven. The gradient mobile phase consists of water with 0.2% of acetic acid (A) and acetonitrile (B). The flow rate was set to 0.3 mL/min. Initial condition is 98% phase A and 2% phase B. Molecules were then eluted using a gradient from 2% to 95% phase B in 22 min. The column was washed using 95% mobile phase B for 2 minutes and equilibrated using 2% mobile phase B for 4 min.
The autosampler was kept at 4°C.
Peak detection and integration were performed using the Thermo Xcalibur quantitative software (3.1.).

About 50 mg of liver samples were supplemented with 400 μL mixture (methanol: water = 4:1). An LC-ESI-MS/MS system (UHPLC-QE, Thermo Q Exactive) with a Waters BEH C18 Liquid chromatography column (100 × 2.1 mm, 1.7 μm) was used to detect the extracted samples. Automatic identification and integration of ion fragments, as well as manual inspection, were performed by the Xcalibur quantitative software (Thermo, Waltham, MA, USA). With the analytes’ mass spectrum peak area as the ordinate and its concentration as the Abscissa, a linear regression standard curve was established. The analytes’ peak area was substituted into a linear equation to calculate the concentration. SCFA (μg/mg) = (C × V)/M. Note: C denotes GC-MS-determined concentration, V is the volume of constant volume, while M denotes sample weight at extraction time, per unit mg.

The profiling experiment was performed with a Dionex Ultimate 3000 UHPLC system (Thermo Scientific) coupled to a Q-Exactive (Thermo Scientific, Waltham, MA, USA) equipped with an electrospray source operating in both positive and negative mode and full scan mode from 100 to 1200 m/z. The Q-Exactive parameters were: sheath gas flow rate 55 au, auxiliary gas flow rate 15 au, spray voltage 3.3 kV, capillary temperature 300° C, S-Lens RF level 55 V. The mass spectrometer was calibrated with sodium acetate solution dedicated to low mass calibration. 10 μL of sample were injected on a SB-Aq column (100 mm × 2.1 mm particle size 1.8 μm, Agilent Technologies), protected by a guard column XDB-C18 (5 mm × 2.1 mm particle size 1.8 μm) and heated at 40° C by a pelletier oven. The gradient mobile phase consists of water with 0.2% of acetic acid (A) and acetonitrile (B). The flow rate was set to 0.3 mL/min. Initial condition is 98% phase A and 2% phase B. Molecules were then eluted using a gradient from 2% to 95% phase B in 22 min. The column was washed using 95% mobile phase B for 2 minutes and equilibrated using 2% mobile phase B for 4 min. The autosampler was kept at 4° C. Peak detection and integration were performed using the Thermo Xcalibur quantitative software (version 2.2).