Methoxypyridine

Samples for metabolite assays were pretreated as described previously (Jiang et al., 2021 (link); Lv et al., 2021b (link)). Briefly, 20 mg of faeces was added to 800 μL of precooled chromatography grade methanol and then homogenized three times using a Precellys Evolution instrument (Bertin Technologies, USA) at 5,000 rpm for 30 s with 15 s intervals between the rounds for extraction. After centrifugation at 14,000 rpm for 15 min, the supernatant was filtered through a 0.22 µm membrane, and 20 µL of heptadecanoic acid (1 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added to the filtrate as an internal reference and then dried under nitrogen at room temperature. After drying, the samples were methoxymated with methoxypyridine (Sigma-Aldrich, St. Louis, MO, USA) and trimethylsilylated with N,O-bis(trimethylsilyl)acetamide containing 1% trimethylsilyl chloride. The pretreated samples were analysed with an Agilent 7890A-5975C GC-MS system (Agilent, USA). The downstream data were compared with the NIST 17 database programmatically to identify the corresponding metabolites (matching score ≥ 80%).

Sample preparation for metabolic assays was performed as described previously (Lv et al., 2021a (link)). Briefly, a mixture of 20 mg feces and 800 μL methanol was homogenized three times using a Precellys Evolution instrument (Bertin Technologies, United States). After centrifugation at 14,000 rpm for 15 min, the supernatant was filtered through a 0.22 μm membrane, and 20 μL of heptadecanoic acid (1 mg/mL, Sigma–Aldrich, St. Louis, MO, United States) was added as an internal reference and then dried under nitrogen at room temperature. After drying, the samples were methoxymated with methoxypyridine (20 mg/mL, Sigma–Aldrich, St. Louis, MO, United States) and trimethylsilylated with N,O-bistrifluoroacetamide containing 1% trimethylsilyl chloride. The pretreated samples were analyzed with an Agilent 7890A-5975C GC–MS system (Agilent, United States). The downstream data were compared with NIST 17 databases to identify the corresponding metabolites (matching score ≥ 80%).