The qualitative and quantitative evaluation of fecal SCFAs was performed using the Agilent gas chromatography-mass spectrometry (GC-MS) system composed of a 5971 single quadrupole mass spectrometer, a 5890 gas-chromatograph, and a 7673 autosampler, through our previously described GC-MS method[10 (link)].
Just before the analysis, fecal samples were thawed and combined with 10 mM sodium bicarbonate solution (1:1 w/v) in a 1.5 mL centrifuge tube. Then, the obtained suspension was sonicated for 5 min and centrifuged at 5000 rpm for 10 min, and then the supernatant was collected. Finally, SCFAs were extracted as follows: an aliquot of 100 µL of sample solution (corresponding to 0.1 mg of stool sample) was added to 50 μL of internal standards mixture, 1 mL of tert-butyl methyl ether, and 50 µL of 1.0 M HCl solution in a 1.5 mL centrifuge tube. Subsequently, each tube was shaken in a vortex apparatus for 2 min and centrifuged at 10000 rpm for 5 min, and finally the solvent layer was transferred to an autosampler vial and analyzed three times.
Just before the analysis, fecal samples were thawed and combined with 10 mM sodium bicarbonate solution (1:1 w/v) in a 1.5 mL centrifuge tube. Then, the obtained suspension was sonicated for 5 min and centrifuged at 5000 rpm for 10 min, and then the supernatant was collected. Finally, SCFAs were extracted as follows: an aliquot of 100 µL of sample solution (corresponding to 0.1 mg of stool sample) was added to 50 μL of internal standards mixture, 1 mL of tert-butyl methyl ether, and 50 µL of 1.0 M HCl solution in a 1.5 mL centrifuge tube. Subsequently, each tube was shaken in a vortex apparatus for 2 min and centrifuged at 10000 rpm for 5 min, and finally the solvent layer was transferred to an autosampler vial and analyzed three times.
