Quantification of Short-Chain Fatty Acids

The analysis of SCFA was performed by gas chromatography in order to determine the concentrations of acetate, propionate, isobutyrate, butyrate, isovalerate and valerate. Cell free-supernatants (100 μl) from fecal homogenates, prepared as indicated formerly, were mixed with 450 μl methanol, 50 μl internal standard solution (2-ethylbutyric 1.05 mg/ml), and 50 μl 20% v/v formic acid. This mixture was centrifuged and the supernatant obtained was used for quantification of SCFA by GC in a system composed of a 6890NGC injection module (Agilent Technologies Inc., Palo Alto, Ca, USA) connected to a flame injection detector (FID) and a mass spectrometry (MS) 5973N detector (Agilent), as described elsewhere^{40 (link)}.