Thermo trace 1310 isq gc ms

Fecal samples were collected and added to 50 μL of 15% phosphoric acid (Aladdin Biochemical Technology Co., Ltd., Shanghai, China) on the 14th day. Then, 100 μL of a 125 μg/mL internal standard (isohexanoic acid, Aladdin Biochemical Technology Co., Ltd., Shanghai, China) solution and 400 μL of diethyl ether (Aladdin Biochemical Technology Co., Ltd., Shanghai, China) were added, and the samples were homogenized for 1 min. The supernatant was collected for testing after centrifugation at 12,000 rpm and 4 °C for 10 min. Thermo TRACE 1310-ISQ GC-MS (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with an Agilent HP-INNOAX (Agilent Technologies, Co., Ltd., Beijing, China) capillary column (30 m × 0.25 mm ID × 0.25 μm) was used to determine the SCFA contents.
Chromatographic conditions included the following: injection volume, 1 μL; shunt ratio, 10:1; inlet temperature, 250 °C; ion source temperature, 230 °C; transmission line temperature, 250 °C; quadrupole temperature, 150 °C; program with an initial temperature of 90 °C, 90–120 °C at 10 °C/min, 120–150 °C at 5 °C/min, and then 150–250 °C at 25 °C/min maintained for 2 min; carrier gas, helium; and carrier gas flow rate, 1.0 mL/min.
MS conditions included the following: electron bombardment ionization (EI) source; SIM scanning mode; and electron energy, 70 eV.

For measurement of the SCFAs, each sample contained the whole gut content of one tadpole (Stages 30–36), and eight samples were prepared for each group. For each sample, we ground and homogenized 50 mg of the gut contents in a tube with 50 μl of 15% phosphoric acid, 100 μl of water (with 125 μg/ml 4-methylvaleric acid as an internal standard), and 400 μl of ethyl ether for 1 min. We centrifuged the mixture for 10 min (12 000 rpm, 4°C) and collected the supernatant for gas chromatography–mass spectrometry (GC–MS) analysis. We performed the chromatography using a Thermo TRACE 1310-ISQ GC–MS (Thermo, USA) equipped with an Agilent HP-INNOWAX column (Agilent Technologies, USA). The parameters and abundance table are detailed in Supplementary Data 1 and 3.