Agilent 7890b gas chromatography system

A liquid–liquid extraction and LC-MS method was applied to determine the metabolites in isolated neutrophils. Primarily isolated neutrophils were collected by high-speed centrifugation (13,000 g, 20 min) and lyophilized for the following steps by Shanghai Luming Biological Technology Company, China. Each group contained 8 individual samples. In brief, isolated neutrophils were analyzed using an Agilent 7890B gas chromatography system (Agilent Technologies Inc., CA, USA) coupled to an Agilent 5977A Mass Selection Detector system (Agilent Technologies Inc., CA, USA). The data preprocessing and statistical analysis were performed using the Analysis Base File Converter software. Metabolites exhibiting a ford change greater than 1.0 and a P-value less than 0.05 were considered as differential metabolites.

Gas chromatography-mass spectrometry (GC-MS) analysis of the essential oil was performed on an Agilent 7890B Gas Chromatography system (Agilent Technologies, USA) coupled to an Agilent 5977A Mass Spectrometer Detector system (Agilent Technologies). The HP5-MS type capillary column (a non-polar column; 30 m x 0.25 mm id and 0.25 µm film thickness, Agilent Technologies) was employed to separate and analyze individual constituents. Then, 1 µl of diluted sample (1/100; v/v, essential oil in acetone) was injected into the split mode with a 1:20 split ratio. The gas chromatographic conditions were carrier gas helium (1.0 mL/min), initial oven temperature 40 °C for 3 min isothermal, 40 to 150 °C at a rate of 6 °C/min, and 150 to 320 °C at a rate of 10 °C/min, and then held for 3 min. The injector temperature was set to 270 °C. Mass spectra were scanned in the range 40 to 500 amu with EI mode (70 eV) in full scan mode. The percentage composition of essential oil was calculated using the peak normalization method. The essential oil constituents were identified by comparing their retention indices (RI), mass spectra with NIST (National Institute of Standards and Technology), Adams library spectra, Wiley 7 n.1 mass computer library, and in published literature [30 ,31 (link)].

A gas chromatographic-mass spectrometric (GC-MS) analysis of the EO was performed on an Agilent 7890B Gas Chromatography system (Agilent Technologies, Wilmington, NC, USA), coupled to an Agilent 5977A Mass Spectrometer Detector system (Agilent Technologies, Wilmington, USA).

GC–MS analysis was performed using Oebiotech (Shanghai, China). Prior to GC–MS analysis, the fecal samples stored at −80°C were transferred to Eppendorf tubes containing 500 μl of water and centrifuged at 10,000 × g for 15 min. We used the Agilent 7890B gas chromatography system coupled with the Agilent 5977A MSD system (Agilent, USA) to determine metabolites in gut microbiota. The DB‐5MS‐fused silica capillary column (30 m × 0.25 mm × 0.25 μm, Agilent, USA) was used to separate the samples. Helium (>99.999%) was used as the carrier gas at a constant flow rate of 1 ml/min through the column. The oven temperature for GC–MS was initially set to 60°C for 30 s, which was ramped to 125°C at a rate of 8°C per min, 210 °C at a rate of 5°C per min, 270°C at a rate of 10°C per min, 305°C at a rate of 20°C per min, and finally held at 305°C for 5 min. The temperature of MS quadrupole and ion source (electron impact) was set to 150°C and 230°C, respectively. The collision energy was 70 eV. Mass spectrometric data were acquired in a full‐scan mode from 50 to 500 m/z.

We collected microglia from each group, treated the microglia with a mixed reagent of methanol/water = 4:1, and then lysed the cells with an ultrasonic cell disrupter. The samples were then analyzed by GC‒MS+LC‒MS according to the corresponding procedures.
Samples were analyzed in an Agilent 7890B gas chromatography system and an Agilent 5977B MSD system (Agilent Technologies Inc., CA, USA). HP-5MS quartz capillary columns were used to separate derivatives. Nitrogen was passed through the column at a flow rate of 1 mL/min. The temperature of the injector was controlled at 260°C. The initial oven temperature was 60°C, maintained for 30 s, gradually heated at a rate of 8°C/min, and finally maintained at 305°C for 5 min. The MS quadrupole and ion source were set to 150 and 230°C, respectively, with an impact energy of 70 eV. Finally, the generated raw data were analyzed by Progenesis QI v3.0 (Nonlinear Dynamics, Newcastle, UK) software and imported into the KEGG database, the Human Metabolome Database (HMDB), Lipidmaps (v2.3), and the EMDB2.0 database for characterization to obtain the results of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, heatmap, and volcano map analyses regarding each group of differential metabolites.

LC‒MS was performed on the ACQUITY UPLC I-Class system (Waters Corporation, Miford, USA) coupled with VION IMS QT of the high-resolution mass spectrometer (Waters Corporation, Milford, USA). An ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) was employed in both the positive and negative models. GC‒MS was performed on an Agilent 7890B gas chromatography system coupled to an Agilent 5977A MSD system (Agilent Technologies Inc., CA, USA). A DB-5MSf used-silica capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J& W Scientific, Folsom, CA, USA) was utilized to separate the derivatives. To monitor the stability and repeatability of LC‒MS and GC‒MS, QC samples were inserted regularly and analysed in every ten samples.

Untargeted metabolomics profiling of fecal and serum samples was performed. Briefly, 40 mg of each fecal sample from the surgical mice or 80 μl of each serum sample from the FMT mice was extracted with methanol before lyophilization. Then, 15 mg/ml of methoxylamine hydrochloride in pyridine was subsequently added and incubated at 37°C for 90 min. Bis (trimethylsilyl)trifluoroacetamide (BSTFA; with 1% trimethylchlorosilane) and n-hexane were added to the mixture, which was derivatized at 70°C for 60 min. The samples were placed at ambient temperature for 30 min. The derivatized samples were analyzed on an Agilent 7890B Gas Chromatography System coupled to an Agilent 5977AMSD System (Agilent Technologies Inc, CA, USA).