Tracefinder software version 3.3 sp1

Detection of C3 content in feces were measured by gas chromatography/mass spectrometry (GC/MS) as described 21 , 40 (link). Briefly, 0.1 g feces were homogenized with 0.4 ml ddH2O and centrifuged at 4 °C and 5000 rpm for 5 minutes. Collect an aliquot (0.2 ml) of the supernatant, then add 0.05 ml 50% H2SO4 and 0.25 ml ether containing 50 μg/ml 2-methylvaleric acid. After vortexing for 2 minutes, the mixture was centrifuged at 4 °C and 12000 rpm for another 10 minutes. After incubating for 30 minutes at -20 °C, the supernatant was collected in a vial containing anhydrous sodium sulfate, and then applied to the Thermo TSQ Vantage triple quadrupole mass spectrometer. Data collection and processing were performed with TraceFinderTM software version 3.3 sp1 (Thermo Fisher Scientific Corp., USA). The C3 were quantified using pure standards diluted in ether.

Targeted metabolomics (PA measurement) was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously (33 (link)). Briefly, cecum samples (100 mg) were dissolved in 900 μL of ice-cold water and extracted via sonication in water for 10 min. After ethyl acetate was added and the samples were shaken for 3 min, they were centrifuged at 13000 rpm at 4°C for 10 min. The supernatant was collected and dried with nitrogen and then reconstituted with methanol/ammonium acetate pH4.5 (60:40 v/v) for further computer analysis. The chromatographic separation was performed on the Thermo Scientific Prelude SPLC system, and detection was performed using a Thermo TSQ Vantage triple quadrupole mass spectrometer. Data collection and processing were performed with TraceFinderTM software version 3.3 sp1 (Thermo Fisher Scientific Corp., USA).

Cecal content and liver were collected and a liquid chromatography-mass spectrometry (LC-MS) system was used to analyze SSII levels. Water was added to the sample at 9-fold volume/weight and extracted ultrasonically for 10 min, then methanol was added and the samples were centrifuged at 13000 rpm for 10 min at 4 °C. The supernatant was concentrated by drying under nitrogen, and then dissolved into mobile phase for analysis by HPLC. Chromatographic separation was conducted on a Thermo Scientific Prelude SPLC system, and Thermo TSQ Vantage triple quadrupole mass spectrometer was used for detection. The chromatographic column was Hypersil gold 50*2.1 mm, 1.9 μm. Mobile phase A was comprised of a 0.1% formic acid water solution and mobile phase B was methanol. The gradient elution was carried out according to the following procedure: 0-1.25 min, 90% A and 10% B; 1.25-2.25 min, 20% A and 80% B; 2.25-5.25 min, 5% A and 95% B; 5.25-6.25 min, 90% A and 10% B. Data acquisition and analysis were performed with TraceFinder^TM software version 3.3 sp1 (Thermo Fisher Scientific Corp., USA).