Fecal SCFA Extraction and GC-MS Analysis

The qualitative and quantitative evaluation of fecal SCFAs and the preparation of standard curves was performed by an Agilent GC-MS system composed of a 5971 single quadrupole mass spectrometer, 5890 gas chromatograph and 7673 autosampler, through our previously described GC-MS method [29 (link)]. Briefly, the SCFAs were extracted as follows: an aliquot of 100 µL of fecal extract solution (corresponding to 0.1 mg of stool sample) was added to 50 μL of an ISTDs 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, centrifuged at 10,000 rpm for 5 min, and finally the solvent layer was transferred to an autosampler vial and processed three times.