Gas chromatography tandem mass spectrometry (GC-MS/MS) analysis of SCFAs

The caecal contents (50 mg) were homogenized for 1 min with 50 μL of phosphoric acid (15%, v,v) and ether (400 μL) containing isocaproic acid (125 μg/mL, 100 μL) as an internal standard. After centrifugation for 10 min (12,000 rpm/min, 4℃), the supernatant was collected, and the concentrations of SCFAs in the samples were determined by GC/MS, equipped with an Agilent HP-Innowax capillary column (30 m × 0.25 mm; ID, 0.25 μm) in a trace 1310 GC system. Helium was used as the carrier gas, with a flow rate of 1 mL/min. Injection was made in split mode at 10:1 with an injection volume of 1 μL and injector temperature of 250℃. The temperature of the ion source and GC/MS interface were 230°C and 250°C, respectively. The oven temperature was programmed to proceed from an initial temperature of 90°C to 120℃ at 10℃/min, and then to 150℃ at 5℃/min, and ultimately to 250℃ at 25℃/min, where it was held for 2 min. The mass spectrometer was operated in the electron impact ionization mode and selective ion monitoring mode with a collision energy of 70 eV.
A mixture of SCFA standards (MilliporeSigma) that included acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid was used to prepare calibration curves at a concentration range of 0.02 to 500 μg/mL. The concentration of total SCFAs was calculated as the sum of the measured concentrations of the 7 SCFA standards.