Isq lt

GC–MS was carried out on Direct Probe Controller Inlet part to Single Quadrupole mass analyzer (Thermo Scientific; GC–MS model ISQ LT) using Thermo X-Caliber software. The Mass spectroscopy system was used to predict the subcomponents of plant SFE extracts using RTX-2330 (fused silica) 30 m capillary column of 0.25 µm internal diameter and df (µm) 0.20 µm. The column was operated at an initial temperature of 160–250 °C at the rate of 5 °C/min and was held for 30 min. The injector and detector temperatures were 240 °C and 250 °C, respectively. The carrier gas (nitrogen) was supplied at a total flow rate of 50 ml/min with a split ratio of 20:0 and subcomponents were identified by comparison with a linked library.

Acetic acid, propionate acid and butyric acid (the charged modes were all positively charged) concentration of the plasma collected from different participants was performed using trace 1310 gas chromatograph (Thermo Fisher Scientific, USA) and ISQ LT (Thermo Fisher Scientific, USA). GC-MS instrument conditions: sample inlet temperature 250°C; Ion source temperature 230°C; Transmission line temperature 250°C, quadrupole rods temperature 150°C. Electron bombardment ionization (EI) source, full scan and SIM scan mode, electron energy 70eV. The peak area and retention time were extracted using MSD ChemStation software, and a standard curve was drawn to calculate the short-chain fatty acid concentration.

Sample preparation was adapted from protocol of Zheng et al. (15 (link)). Extraction steps were carried out at 4°C. Briefly, 1 ml of human milk were used and suspended with 500 µl solution of NaOH at 0.005 M including internal standard mix of acetate-D3, butyrate-13C2, propionate-D2 and valerate-D9 at 235 µM, 88 µM, 82 µM and 41 µM, respectively. 500 µl of propanol/pyridine mix (3:2 v/v) were added to the samples and then vortexed. 50 µl of propylchloroformate (PCF) was successively added twice to the solution and vortexed. The biphasic solution was formed after addition of 300 µl of hexane and sonicated and centrifuged at 2000xg and 4°C during 5 min. 300 µl of organic phase were transferred to GC/MS vials before their injections. SCFAs were quantified by GC/MSusing an ISQ LT™ equipped with a Triplus RSH (Thermo Fisher Scientific, Illkirch, France) and a fused-silica capillary column with a (5%-phenyl)-methylpolysiloxane phase (DB-5ms, J&W Scientific, Agilent Technologies Inc., USA) of 50 m×0.25 mm i.d coated with 0.25 µm film thickness. Peaks of SCFAs were quantified using XCalibur QuanBrowser software (Thermo Fisher Scientific, Illkirch, France). Details on chromatography, calibration range, limit of detection and retention time of SCFAs are reported in supplementary materials (Supplementatary Table S1).

Headspace solid phase microextraction/gas chromatography-mass spectrometry (HS-SPME/GC–MS) (GC:TRACE 1310, MS:ISQ-LT, Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA) was utilized to determine the volatile profiles with and without fermentation. One gram of sodium chloride and 50 μL of 3-octanol were added to 5 mL samples, mixed into 15 mL headspace bottles, and incubated overnight at 4 °C. Then, the sample was placed into the TriPlus RSH Autosampler-SPME system for extraction, adsorbed at 60 °C for 30 min, and held for 5 min.
One milliliter of the above mixture was added to 20 mL headspace injection vials. Volatile substances were isolated using a DB-WAX chromatographic column (30 m × 0.25 mm × 0.25 μm). Gas chromatography (GC) spectrometry parameters were set as follows: the injection temperature was 25 °C, and He served as the carrier gas at a flow rate of 1.2 mL/min. The injection volume was 1 μL, with split injection (40:1). The temperature was kept at 40 °C for 3 min, then increased to 180 °C at a rate of 6 °C/min, held for 2 min, and increased to 230 °C at a rate of 10 °C/min, held for 6 min.
The mass spectrometry parameters were set as follows: electron bombardment ion source, ionization energy was set as 70 eV, ion source temperature was 200 °C, interface temperature was 250 °C, and scanning range was 33.00~450.00 amu.

50 μl of 15% phosphoric acid, 100 μl internal standard (isohexanoic acid, 125 μg/ml) solution, 400 μl of ether were added in an appropriate amount of fecal sample from rats; The Sample was homogenized for 60 s at 60 Hz (Repeated 2 times), and centrifuged at 12,000 rpm (4°C) for 10 min. The supernatant was collected and stored at −20°C until further processing. Concentrations of SCFAs were quantified by Gas Chromatography-Mass Spectrometry (GC-MS) (GC: Thermo, TRACE 1310; MS: Thermo, ISQ LT) detection method according to established protocols (Hsu et al., 2019 (link); Zhang S. et al., 2019 (link)).

The GC–MS analysis was performed on a gas chromatograph (Trace 1310, Thermo Scientific, United States) equipped with a single quadrupole mass spectrometer (ISQ LT, Thermo Scientific, United States). The separation was conducted using a DB-5 capillary column (30 m × 0.25 mm i. d., film thickness 0.25 μm, Agilent). The oven temperature program was initially set at 65°C for 2 min, warmed to 140°C at 5°C/min for 10 min, then heated to 180°C at 3°C/min for 10 min, and finally, increased to 250°C at 20°C/min for 5 min. The electron impact (EI)-MS was operated at 70 eV, and the scan range (rate) was set as 45–500 amu (0.20 s per scan). The injector, MS transfer line, and ion source were kept at 220, 280, and 230°C, respectively. The splitless injection mode was used for analysis. Identification of volatiles was achieved by comparing the mass spectra and RI values with those of the known compounds in the standard NIST 11 library and WILEY275 library.