Qualitative and quantitative evaluation of fecal SCFAs (acetic, propionic, butyric, isobutyric, 2-methylbutyric, isovaleric and valeric acids) was performed using an Agilent gas chromatography-mass spectrometry (GC-MS) system consisting of a single quadrupole mass spectrometer (model 5971), gas chromatograph (model 5890) and autosampler (model 7673), using our previously described method [24 (link)].
Briefly, fecal pellets were thawed just before analysis and added to a 1.5 mL centrifuge tube with 0.25 mM sodium bicarbonate solution (1:1 w/v). The obtained suspensions were then sonicated for five minutes, centrifuged at 5000 rpm for 10 min and the supernatants collected. The SCFAs were then extracted as follows: a 100 µL aliquot 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 HCl 6 M + 0.5 M NaCl solution in a 1.5 mL centrifuge tube. Each tube was then shaken in a vortex apparatus for two minutes and centrifuged at 10,000 rpm for five minutes. The solvent layer was then transferred to an autosampler vial and processed three times.
Briefly, fecal pellets were thawed just before analysis and added to a 1.5 mL centrifuge tube with 0.25 mM sodium bicarbonate solution (1:1 w/v). The obtained suspensions were then sonicated for five minutes, centrifuged at 5000 rpm for 10 min and the supernatants collected. The SCFAs were then extracted as follows: a 100 µL aliquot 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 HCl 6 M + 0.5 M NaCl solution in a 1.5 mL centrifuge tube. Each tube was then shaken in a vortex apparatus for two minutes and centrifuged at 10,000 rpm for five minutes. The solvent layer was then transferred to an autosampler vial and processed three times.
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