Agilent 7890b 5977

The chromatographic analysis of the component fingerprint was performed on an Agilent 7890B/5977 GC-MS system (Agilent Technologies, Palo Alto, CA, USA) using a capillary column 5MS (30mm × 0.25mm × 0.25 μm). Helium C-60 was used as the carrier gas at a constant flow rate of 1.0 mL/min. Samples (2 μL) were injected in split mode (ratio 1:20), and the injector temperature was 250 °C (held for 20 min). The gas chromatography settings were as blows. Total separation run time was 93.174 min. The oven temperature was fixed at 60 °C for 1 min, then increased to 100 °C (rate 4 °C/min) and held for 0 min, then increased to 120 °C (rate 2 °C/min) and held for 0 min, then increased to 180 °C (rate 1 °C/min) and held for 0 min, then increased to 230 °C (rate 23 °C/min) and held for 10 min. The MS detector was operated in ESI mode (70 eV). The ESI temperature was 150 °C. Data acquisition was performed in full scan mode with a mass range between 35 and 450 m/z. Sample preparation and GC-MS acquisition were randomized to avoid analytical bias.

Fecal butyrate concentrations were measured by gas chromatography-mass spectrometry. Briefly, cecal contents (150 mg) were homogenized in 2.5 mL of dichloromethane using a vortex mixer for 30 min and centrifuged at 8000 rpm for 5 min at 4°C, and the supernatant was collected. The extraction was repeated once. The volume was diluted to 5 ml with dichloromethane, with 0.9 ml taken and added to 0.1 ml BSTFA, derivatized at 60°C for 50 min, and finally passed through a 0.22 µm filter membrane for detection by GC-MS (Agilent 7890B-5977) with the following parameters: injector volume, 2 μL; split ratio, 10:1; injector temperature, 250°C; and carrier gas, helium.