Db 1701 capillary column

For the first experiment in which we quantified butyrate levels butyric acid concentrations were determined from feces. For this purpose, feces were collected and then frozen at -80°C until measurement. Fecal samples were then lyophilized and rehydrated in distilled water. After pH was adjusted (between 2 and 3 with 5M HCl), 1 ml chloroform was added, and samples were centrifuged for 25 min at 4,000 × g. Afterward, the concentration of butyric acid was determined by gas chromatography using a GC2010 apparatus (Shimadzu, Japan). In this procedure, a DB-1701 capillary column with 30 m length, 0.25 mm inner diameter, and 0.25 mm film thickness (Agilent, Santa Clara, CA, USA) was used. The conditions for performing this procedure were as follows—oven: 250°C, split ratio: 20, colum: 95°C (Isothermal), FID: 300°C, make up gas helium: 4 ml/min, H2 flow: 40 ml/min, airflow: 400 ml/min, retention time butyric acid: 3.5–3.6 min. For quantification of SCFA, an external standard (SupelcoTM WSFA-1 Mix, Supelco Sigma Aldrich Co., Bellefonte PA, USA) was used. Results were reported as nanograms/dry weight.

The samples were preheated in a water bath at 45 °C for 20 min. The volatile compounds with different experimental conditions were extracted at 50 °C for 30 min by HS-SPME with a DVD/Car/PDMS fiber (50/30 μm, Supelco, Bornem).
GC-MS analyses were performed using an Agilent 8890 GC coupled with an Agilent 5977B quadrupole mass selective detector (MSD, Agilent Technologies, Diegem, Belgium) with a Varian DB-1701 capillary column (30 m length × 0.25 mm i.d.; 0.25 μm film thickness). The working conditions of GC-MS were as follows: the transfer line to MSD was maintained at 250 °C; the carrier gas (He) flow rate was 1.0 mL min⁻¹; the electron ionization (EI) was 70 eV; the scanned acquisition parameter was ranged from 30 to 550 m/z; the initial oven temperature was 40 °C, held 2 min; the temperature increased from 40 to 100 °C at a rate of 10 °C min⁻¹ and held for 5 min, and then raised to 220 °C at a rate of 10 °C min⁻¹ and held for 15 min; the equilibrium time was 0.5 min; the injection port was in split mode and the split ratio was 30 : 1. The volatile components were identified by comparison of the mass spectrum with mass spectral libraries (NIST 2017). The formation of pyrazines was calculated by the absolute peak area of each individual pyrazine in a semi-quantitative way.^{26 (link)}