7890 gas chromatography

A previously described method by Hou et al. [13 (link)] was used to perform the GC-MS analysis for metabolites in A. inebrians roots. In brief, the 7890 gas chromatography (Agilent, CA, USA) coupled with a Pegasus HT^® time-of-flight mass spectrometer (LECO, MI, USA) was used for GC-MS analysis. The raw mass spectra data were processed via ChromaTOF software (versions 4.3×). The metabolites were identified based on the LECO-Fiehn Rtx5 database by matching the mass spectrum and retention index [29 (link)].

The levels of NAD and NAD‐related metabolites including ATP were determined by multiple reaction monitoring (MRM) using Agilent 6460 Triple Quad mass spectrometer coupled to Agilent 1290 HPLC system. MS settings and chromatographic conditions were used as described previously (Yaku et al., 2018). TCA cycle intermediates were analyzed by selected ion monitoring (SIM) using Agilent 5977 MSD Single Quad mass spectrometer coupled to Agilent 7890 Gas Chromatography. A 30‐cm‐long DB5‐MS column with 10‐m DuraGuard precolumn was used for separation by GC/MS. Helium was used as a gas carrier with a constant flow rate of 1.1 mL/min. The temperature setting was started at 60°C for 1 min, then increased at 10°C/min to 325°C, and held at 325°C for 10 min. Amounts of metabolite were calculated by integrated sum of area using MassHunter Quantitative Software (Agilent).

Extraction and measurement of SCFAs in cecal samples was described elsewhere^{43 (link)}. Briefly, the extraction was performed using water, concentrated hydrochloric acid, and diethyl ethel, and the derivatization of samples was carried out using N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) followed by the heating at 80 °C for 20 min. Levels of SCFA were determined by using Agilent 5977 MSD single Quad mass spectrometer coupled to Agilent 7890 Gas Chromatography (GC/MS) with selected ion monitoring (SIM) mode. GC/MS conditions were described previously^{44 (link)}. Quantification of SCFA levels was calculated by integrated sum of area using Mass Hunter Quantitative software (Agilent).