High performance liquid chromatography (hplc)

Lysine, cadaverine, and AMV were analyzed by HPLC (HITACHI) equipped with a reverse-phase Diamonsil C18 column (Diamonsil 5 μm, 250 × 4.6 mm) and UV–VIS detector. Samples were centrifuged at 7700 × g for 10 min. The supernatant was reacted with phenyl isothiocyanate, filtered through 0.22 μm film, and used for HPLC analysis. Solvent A was methanol and solvent B was water with 0.1% formic acid. The column temperature was set at 40 °C. The following gradient was used at a flow rate of 1 mL min⁻¹: 32% to 80% solvent A for 20 min, 80% to 32% solvent A for 2 min, and 32% solvent A for an additional 5 min. Quantification was based on the peak areas at specific wavelengths (254 nm). The analysis of glutarate and glucose was performed by HPLC (HITACHI) equipped with an Organic Acid Analysis Column (Amine HPX-87H Ion Exclusion Column, 300 mm × 7.8 mm) and refractive index detector. The mobile phase was 5 mM H₂SO₄ at a flow rate of 0.5 mL min⁻¹ and the oven temperature was set at 60 °C. The product glutarate was analyzed by ESI-MS and the molecular weight was in accordance with that of the glutarate standard (Supplementary Fig. 7).

Freshly collected fecal samples were mixed with 70% ethanol solution at a ratio of 1 mg of fecal sample: 10 μL 70% ethanol, and then homogenized with appropriate amounts of glass beads (1.0 mm in diameter; Biospec Products) by vortexing at 3000 rpm for 10 min. Homogenized samples were centrifuged at 14,000× g for 10 min, and the supernatants were collected for fatty acid derivatization, according to a previously described method [53 (link)]. Derivatized supernatants were filtered using a 0.22-μm polycarbonate syringe filter (Millipore, St. Charles, MO, USA). SCFAs were separated and quantified using high-performance liquid chromatography (HITACHI, Tokyo, Japan) on a C18 HTec column (NUCLEODUR, Macherey-Nagel, Düren, Germany), with column temperature 40 °C, flow rate 1 mL/min, and detection wavelength 400 nm.