Agilent 7890b gas chromatograph

SCFA contents in BALF supernatant were detected by Metware Biotechnology Co., Ltd. (Wuhan, China) with gas chromatography-tandem mass spectrometry analysis. Briefly, BALF samples were thawed and vortexed for 1 min prior to analysis. A total of 50μL of samples were mixed with 100μL of phosphoric acid (0.5% v/v) solution, vertexing for 3 min and ultrasonicating for 5 min. After that, the mixture was centrifuged at 12000 rpm for 10 min at a temperature of 4°C. The supernatant was collected and used for GC-MS/MS analysis. Agilent 7890B gas chromatograph coupled to a 7000D mass spectrometer with a DB-5MS column (30m length × 0.25mm inner diameter × 0.25μm film thickness; J&W Scientific, Folsom, CA) was used. Helium was used as the carrier gas, at a flow rate of 1.2mL/min. Injections were made in the spitless mode, and the injection volume was 2μL. The oven temperature was held at 90°C for 1 min, raised to 100°C at a rate of 25°C/min, raised to 150°C at a rate of 20°C/min, and held at 150°C for 0.6 min. Then, the temperature was further raised to 200°C at a rate of 25°C/min and held at 200°C for 0.5 min. After running for 3 min, all samples were analyzed in multiple reaction monitoring mode. The temperature of the injector inlet and transfer line were held at 200°C and 230°C, respectively.

The derivatives (0.5 μL) were injected using an Agilent 7693 ALS (Agilent Technologies, Wilmington, DE, USA) in splitless mode. Chromatographic separation was carried out using an Agilent 7890B gas chromatograph (Agilent Technologies) equipped with an RTX-5Sil MS column (Restek, Gellefonte, PA, USA). The oven temperatures were programmed at 50°C for 1 min, ramped at 20°C/min to 330°C, and held constant for 5 min. Mass spectrometry analysis was performed on a Leco Pegasus HT time of flight mass spectrometer controlled by ChromaTOF software 4.50 version (LECO, St. Joseph, MI, USA). Mass spectra were acquired in the mass range of 85–500 m/z at an acquisition rate of 17 spectra/s.
Raw result files were collected and pre-processed using ChromaTOF software, and further process using Binbase, an in-house database. ChromaTOF-specific peg files were converted to generic *.txt result files and additionally as generic netCDF files for further data evaluation. More details can be found in previous reports [13 (link), 14 (link)]. A total of 85 out of 962 metabolic signals were finally reported with occurrence in 50% of the samples per study design group. The quality control was carried out with mixture of 31 pure reference compounds between every 10 samples [13 (link), 15 (link), 16 (link)].

Heating of the samples was carried out in a Tembloc thermostat dry-block (JP Selecta S.A., Barcelona, Spain).
GC analyses of the volatiles were done with an Agilent 7890B Gas Chromatograph (Agilent Technologies, Santa Clara, California) equipped with a Tracer MHS123 2t^® Head Space Sampler and a flame ionization detector (FID). Acquisition of data was done with the Agilent ChemStation for GC System program. The conditions for the GC assays were: SP2380 column (poly 90% biscyanopropyl–10% cyanopropylphenyl siloxane), 60 m length × 0.25 mm internal diameter × 0.20 μm film (Sigma–Aldrich Co. LLC, St. Louis, MO, USA), 500 μL injection volume, hydrogen carrier gas at 1.5 mL min⁻¹ and split injection (50:1 split ratio). The oven temperature programme was: 50 °C (7 min initial time), then rise at 10 °C min⁻¹ to 150 °C and hold 3 min. The injector and detector temperatures were 150 °C and 170 °C, respectively.

Gas chromatography-electron impact mass spectrometry (GC-EIMS) analyses were performed with an Agilent 7890B gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an Agilent HP-5MS (Agilent Technologies Inc., Santa Clara, CA, USA) capillary column (30 m × 0.25 mm; coating thickness 0.25 µm) and an Agilent 5977B single quadrupole mass detector (Agilent Technologies Inc., Santa Clara, CA, USA). The oven temperature program was set to rise from 60 °C to 240 °C at 3 °C/min. Temperatures were set as follows: injector temperature, 220 °C; transfer-line temperature, 240 °C. The carrier gas was He, at 1 mL/min flow. The acquisition was performed with the following parameters: full scan, with a scan range of 35–300 m/z; scan time: 1.0 s; threshold: 1 count. The identification of the constituents was based on the comparison of their retention times (t_R) with those of pure reference samples and of their linear retention indices (LRIs), which were determined relatively to the t_R of a series of n-alkanes (C9–C25). The detected mass spectra were compared with those listed in the commercial libraries NIST 14 and ADAMS, as well as in a homemade mass-spectral library, built up from pure substances and components of EOs of known composition and MS literature data [44 ,50 ].

The SCFA content of fecal samples was determined by MetWare (http://www.metware.cn/) using the Agilent 7890B-7000D GC-MS/MS platform. Stock solutions of standards were prepared at 1 mg/mL in MTBE and stored at −20°C. Immediately prior to analysis, the stock solutions were diluted with MTBE to working concentrations. Fecal samples (20 mg) were weighed and placed in a 2 mL EP tube with 1 mL of aqueous phosphoric acid (0.5% v/v) and a small steel ball. The mixture was ground three times for 10 s each, vortexed for 10 min, ultrasonicated for 5 min, and centrifuged at 12,000 r/min for 10 min at 4°C. A 0.1-mL sample of the supernatant was mixed with 0.5 mL MTBE containing internal standard in a 1.5 mL centrifugal tube and the mixture vortexed for 3 min, ultrasonicated for 5 min, and recentrifuged at 12,000 r/min for 10 min at 4°C. The new supernatant was collected and analyzed by GC-MS/MS using an Agilent 7890 B gas chromatograph coupled to a 7000D mass spectrometer with a DB-FFAP column (30 m length × 0.25 mm i.d. ×0.25 μm film thickness, J&W Scientific, USA).