Fecal Butyrate Quantification

Butyrate was measured at the CRCHUM Metabolomics core facility by liquid chromatography-mass spectrometry using a protocol adapted from Han et al. (2015). Briefly, samples were homogenized in 50% aqueous acetonitrile containing 2,2-dimethylbutyric acid as internal standard. After centrifugation, water-soluble carbonyl groups found in supernatants were derivatized using 3-nitrophenylhydrazine to produce the corresponding phenylhydrazone derivatives. Derivatized short-chain fatty acids (SCFAs) were separated by high-performance liquid chromatography (Shimadzu Nexera X2 UHPLC System, Columbia, MA, United States) using a Poroshell 120 EC-C18, 2.1 × 75 mm, 2.7 μm particles column (Agilent Technologies, Santa Clara, CA, United States) coupled to a guard column, and a gradient mobile phase composed of formic acid in water (solvent A) and formic acid in acetonitrile (solvent B). Detection was performed after electrospray ionization on a Sciex 6500 mass spectrometer operated in negative-ion mode.