Bpx5 column

GC-MS analysis of the ethanolic extract of A. sessilis was performed using a QP-2010 Ultra GC-MS spectrometer (Shimadzu, Kyoto, Japan) fused with a BPx5 column (30 × 0.25 μm ID × 0.25 μm df). The oven temperature was programmed from 50°C at 0 min and increased to 300°C and remained constant for 10 min. The stem extract was diluted in methanol. Helium gas (99.999%) was used as a carrier gas with the following conditions: total flow: 11.8 mL/min, column flow: 0.8 mL/min, linear velocity: 32.4 cm/s, purge flow: 3.0 mL/min, and split ratio: 10. Mass spectra were taken with ion source temperature and interface temperature of 200°C and 250°C, respectively. The mass scan parameters had a start time of 2.5 min, with end time 93.0 min. The acquisition (ACQ) parameters involved the following conditions: scan event time: 0.10 s, scan speed: 10000, and mass range: 40 m/z to 700 m/z. The relative percentage of each compound was calculated by comparing its average peak area to the total amount of areas.

For GCMS analysis, 100 μL of oil was dissolved in 2 mL of dichloromethane (DCM). The sample was analysed on a Shimadzu GC-MS system model QP500 with a medium polarity capillary column (BPX-5 column (29.4 m × 0.25 mm), with film thickness of 0.25 μm) with helium as the carrier gas. One microlitre of the sample was injected using splitless injection with injector temperature 300°C according to the following scheme: 50°C for 2 min with 10°C/min up to 300°C. The final temperature was held for 10 min. The total runtime for each sample was 37 min. For MS detection, electron ionization with 70 eV was applied and mass fragments were detected between 40 and 500 m/z. The ion source temperature and transfer line temperature were 200°C and 300°C, respectively. Note that the detector was activated after 5 min.

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).