Electronic crimper

Furthermore, 5–10 mg of feces was mixed with 90 μL of Milli-Q and 10 μL of 2 mM internal standard containing acetic acid, butyric acid, and crotonic acid for 5 min. The mixture was homogenized with 50 μL of 36% HCl and 200 μL of 97% diethyl ether and was centrifuged at 3000 rpm for 10 min at room temperature. Subsequently, 80 μL of the supernatant organic layer was transferred to a new glass vial and combined with 16 μL of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide as a derivatization reagent. The vials were immediately capped tightly with electronic crimper (Agilent), incubated for 20 min in an 80 °C water bath, and then left at room temperature in the dark for 48 h for derivatization. The derivatized samples were analyzed using a GC-MS-TQ8040 gas chromatograph mass spectrometer (Shimadzu Corporation, Kyoto, Japan), and the injection was performed using an AOC-20i autoinjector (Shimadzu Corporation, Kyoto, Japan). The capillary column was a BPX5 column (0.25 mm × 30 m × 0.25 μm; Shimadzu GLC). Pure helium gas was used as a carrier gas and delivered at a flow rate of 1.2 mL min-1. The head pressure was 72.8 kPa with split (split ratio of 30:1). The injection port and interface temperatures were 230 °C and 260 °C, respectively. This analysis measured the 10 types of fecal SCFAs (C1:0–C6:0).