7890 gas chromatograph system

The VOCs were characterized with HS-SPME/GC-MS. Briefly, 500 μL of samples was added into 20 mL headspace bottle, and 10 μL 2-octanol was added as an internal standard. They were incubated at 60 °C for 49 min in a PAL rail system SPME cycle. Subsequently, an Agilent 7890 gas chromatograph system coupled with a 5977B mass spectrometer was used for GC-MS analysis, and the mass spectrogram data were obtained in a scanning mode with a range of 20 to 400 m and a solvent delay of 0 min.

The serum sample preparation procedure and GC-TOF-MS analysis were similar to the previously published procedure with slight modification (Gu et al., 2015 (link); Geng et al., 2020 (link); Yu et al., 2020 (link)). In brief, take 50 μl of sample and mix it with 5 μl of internal standard (L-2-chlorophenylalanine) into 1.5-ml Eppendorf (EP) tubes, and 200 μl of methanol was added. Treat with ultrasound for 10 min (incubated in ice water). Centrifuge for 15 min at 12,000 rpm and 4°C. Transfer the supernatant of 180 μl into fresh 1.5-ml EP tubes. Take 20 μl from each sample and pool as QC sample. The dried samples (in a vacuum concentrator) were then incubated with 30 μl of methoxy amination hydrochloride at 80°C for 30 min. Subsequently, 40 μl of the bis (trimethylsilyl)trifluoroacetamide (BSTFA) reagent was added to the sample aliquots and incubated for 1.5 h at 70°C. Then, 5 μl of fatty acid methyl esters (FAMEs) (in chloroform) was added to the QC sample at room temperature. Finally, an Agilent 7890 gas chromatograph system coupled with a Pegasus HT time-of-flight mass spectrometer was used to perform GC-TOF-MS analysis. The energy was −70 eV in electron impact mode. After 4.7 min of solvent delay, mass spectrometry data were obtained at full-scan mode (m/z 50–500) at a rate of 12.5 spectra per second.

Samples were analyzed using an Agilent 7890 gas chromatograph system coupled with a Pegasus HT time-of-flight mass spectrometer and a DB-5MS capillary column coated with 5% diphenyl cross-linked with 95% dimethylpolysiloxane (30-m×250-μm inner diameter, 0.25-μm film thickness, J&W Scientific, Folsom, CA, USA). A 1-μL aliquot of sample was injected in splitless mode. The initial temperature was maintained at 50℃ for 1 min, then increased to 310℃ at a rate of 10℃/min for 8 min. Helium was used as the carrier gas at a rate of 1 mL/min. The injection, transfer line, and ion source temperatures were maintained at 280℃, 280℃, and 250℃, respectively. An electron impact of 70 eV was used for the ionization. After a solvent delay of 6.17 min, mass spectra were obtained in the m/z range of 50 to 500 at 12.5 scans/s.

An Agilent 7890 gas chromatograph system equipped with a Pegasus HT time-of-flight mass spectrometer was used to perform GC-TOF-MS analysis. The 1 μl analyte was analyzed using the splitless mode. The carrier gas was Helium, and 3 ml min⁻¹ was set as the front inlet purge flow, and 1 ml min⁻¹ was set as the gas flow rate through the column. The initial temperature was maintained at 50 °C for 1 min, followed by increase at a rate of 10 °C min⁻¹, until reaching 310 °C, when it was maintained for 8 min. Injection temperature was 280 °C, the transfer line temperature was 270 °C, and the ion sources temperature was 220 °C. The energy of electron impact mode was −70 eV. Mass spectrometry data were generated in full-scan mode using an m/z range of 50–500, with 20 spectra per second following a 366-s solvent delay.

Fecal samples were softened with dH₂O, homogenized, ultrasound-treated, and centrifuged. Supernatants were mixed with 0.1 mL 50% H₂SO₄ and 0.8 mL 2-methylvaleric acid (25 mg/mL stock in ethyl ether) as internal standard. Samples were placed on an oscillating shaker, ultrasonicated, and centrifuged, and the upper ether solution was subjected to GC–MS analysis on An Agilent 7890 gas chromatograph system and an Agilent 5975C mass spectrometer. Data were gathered in multiple reaction monitoring modes with characteristic fragment ions of SCFAs obtained by references. Quantitative measurements of SCFAs in feces were performed according to the data acquired and standard curves from references.