The quantification was based on a modified method of the fast GC method reported by Tsochatzis et al. [1 (link)] using styrene and PS oligomer standards, which were prepared in CHCl3, and external calibration was applied, according to a previously reported method [1 (link)]. The organic CHCl3 extracts were filtered with PTFE 0.22 μm filters, and a volume of 1 μL was injected by the GC-TOF-MS system in split-less mode into an Agilent Technologies 7890B gas chromatography system coupled to an Agilent Technologies 7200 Accurate-Mass Q-ToF mass Spectrometer (Agilent Technologies, Waldbronn, Germany). Separation of the metabolites was performed on an HP-5MS capillary column (30 m, 0.250 mm i.d., 0.25 μm film thickness, Agilent Technologies). The ion source temperature was set to 230 °C, and the MS analysis was performed in scan mode with a quadruple temperature of 150 °C and a fragmentation voltage of 70 eV (pressure of 21.4 psi, a gas saver of 20 mL/min and 3 mL/min purge flow). The GC oven temperature program followed the temperature program previously reported in [1 (link)]. Briefly, an isotherm of 60 °C was held for 2 min, followed by a temperature ramp of 10 °C/min up to a temperature of 320 °C that was held for 5 min. The total analysis time was 34 min.
7890b gas chromatography system
Manufactured by Agilent Technologies
Sourced in United States
The 7890B gas chromatography system is a versatile analytical instrument designed for the separation and identification of complex chemical mixtures. It features a precision-engineered oven, sensitive detectors, and advanced software for data acquisition and analysis. The 7890B is capable of separating and quantifying a wide range of organic compounds with high accuracy and reproducibility.
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Lab products found in correlation
5977a msd system, by Agilent Technologies (2 mentions)
7200 accurate mass q tof mass spectrometer, by Agilent Technologies (1 mentions)
Hp 5ms capillary column, by Agilent Technologies (1 mentions)
Adb 5ms fused silica capillary column, by Agilent Technologies (1 mentions)
Db waxetr column, by Agilent Technologies (1 mentions)
Combi pal, by CTC Analytics (1 mentions)
Hp 5 m gc column, by Agilent Technologies (1 mentions)
J w dbffap fused silica capillary column, by Agilent Technologies (1 mentions)
7 protocols using 7890b gas chromatography system
1
Quantification of Styrene and PS Oligomers
2
GC-MS Analysis of Complex Samples
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An Agilent 7890B gas chromatography system combined with a 5977 AMSD system (Agilent Technologies Inc., Santa Clara, CA, USA) was used for GC-MS. The samples were separated using an ADB-5MS fused-silica capillary column (Agilent J&W Scientific, Folsom, CA, USA). Helium (>99.999%) was passed through the column at a rate of 1 mL/min. The injection volume was 1 μL, and the injector was operated in a splitless mode at 260 °C. The initial temperature of the oven was 60 °C, which was maintained for 0.5 min, and then it was gradually increased to 125 °C at a rate of 8 °C/min; to 210 °C/min at 5 °C/min; to 270 °C at 10 °C/min; and to 305 °C at 20/min, which was maintained for 5 min. The MS quadrupole and ion source (electron impact) temperatures were set to 150 °C and 230 °C, respectively. The collision energy was 70 eV. Mass spectral data were collected in full-scan mode (m/z 50–500) with the solvent delay time set to 5 min. The QC samples were injected at regular intervals (every 10 samples) during the run to assess reproducibility.
3
Comprehensive VOCs Analysis by GC-MS
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VOCs analysis was performed with a 7890B gas chromatography system (Agilent, Palo Alto, CA, USA) equipped with a CombiPal auto-sampler (CTC Analytics, Zwingen, Switzerland) coupled to an Agilent 5975C triple quadrapole mass detector. The SPME fibre was desorbed into an ultra-inert straight SPME liner (0.75 mm, Agilent Technologies Inc., USA) at 250 °C in splitless mode for 2 min, and separation of compounds achieved through a DB-Waxetr column (60 m × 250 µm inner diameter × 0.25 µm film thickness; Agilent Technologies Ltd., USA) with helium at a flow rate of 1 mL min−1. The oven temperature was set to 40 °C for 3 min and then ramped from 40 to 90 °C at 10 °C min−1, 90 to 180 °C at 5 °C min−1, 180 to 250 °C at 20 °C min−1 and held for 2 min, resulting in a total run time of 31.5 min. The mass spectrometer was operated in an electron impact (EI) ionization at 70 eV with an ion source temperature of 250 °C, to scan a mass range from 35 m/z to 350 m/z.
4
Analysis of Cecal SCFA Composition
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The fecal SCFA levels were measured by a modified method originally reported by García-Villalba et al. (2012) (link). The sample (0.1 g) was placed in a bead tube and 0.9 ml of 0.5% phosphoric acid solution was added. The tube was mixed and then heat-treated at 85 °C for 15 min. The sample was cooled after grinding, centrifuged (14,000 × g for 10 min), and the supernatant was transferred to a new tube, mixed with an equal volume of ethyl acetate, and centrifuged again (14,000 × g, 10 min). The ethyl acetate layer was transferred to a vial and the internal standard (4-methylvaleric acid) was added to generate the sample solution. After extraction the concentration of SCFA in each sample was measured by gas chromatography using the 7890B Gas Chromatography System (Agilent Technologies Inc., CA, USA). Helium was used as the carrier gas at 1.2 mL/min.
The detector temperature was kept at 250°C. The oven temperature program was as follows: 50°C; then 10°C/min to 90°C; 15°C /min to 150°C; 5°C /min to 170°C; 20°C/min to a final temperature of 250°C, held for 4 min. One microliter of extract was injected in the splitless mode. In this study, butyric acid, acetic acid, and propionic acid were detected as the major cecum SFCAs.
The detector temperature was kept at 250°C. The oven temperature program was as follows: 50°C; then 10°C/min to 90°C; 15°C /min to 150°C; 5°C /min to 170°C; 20°C/min to a final temperature of 250°C, held for 4 min. One microliter of extract was injected in the splitless mode. In this study, butyric acid, acetic acid, and propionic acid were detected as the major cecum SFCAs.
5
Metabolomic Analysis of Cecal Content by GC-MS
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All samples were measured using the Agilent Technologies 7890B Gas chromatography system coupled with the Agilent Technologies 5977A MSD system (Agilent Technologies, Santa Clara, CA, USA). 60 mg cecal content sample was added to Eppendorf tube. Samples were homogenized with cold methanol (360 μL) and internal standard (40 μL, 0.3 mg/mL 2-chlorophenylalanine) and were sonicated for 30 min. After the samples were rotated for 2 min and sonicated with 300 μL chloroform and 600 μL water for 30 min, and the mixture was centrifuged at 13,000 rpm for 10 min at 4 °C. Finally, the supernatant was dried under vacuum, mixed with 80 μL of methoxyamine, and oximated for 90 min in the dry supernatant; then 80 μL BSTFA and 20 μL n-hexane solution were added for 60 min at 70 °C. Metabonomics analysis was performed by gas chromatography-mass spectrometry (GC-MS).
The peak recognition, peak alignment, waveform filtering and missing interpolation of the original data were analyzed by using MS-DIAL software and metabolite modeling was based on the Lug database. The data matrix sample information, the peak name of each substance, the retention time, the mass-to-charge ratio and the signal strength were acquired. Results were analyzed using the OE biotechnology cloud platform (https://cloud.oebiotech.com/task/ ) (accessed on 22 January 2023).
The peak recognition, peak alignment, waveform filtering and missing interpolation of the original data were analyzed by using MS-DIAL software and metabolite modeling was based on the Lug database. The data matrix sample information, the peak name of each substance, the retention time, the mass-to-charge ratio and the signal strength were acquired. Results were analyzed using the OE biotechnology cloud platform (
6
Glycan Compositional and Linkage Analysis
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Aliquots (10%) of the Pronase glycopeptide fractions were dried and mixed with 1 nmol scyllo-inositol internal standard and subjected to methanolysis, re-N-acetylation and trimethylsilylation and GC-MS monosaccharide composition analysis of the resulting 1-O-methyl-glycoside TMS derivatives, as described in (50 ). Remaining 90% of the samples were used for methylation linkage analysis (50 ). Briefly, the samples were dried and subjected to permethylation using the sodium hydroxide method. The permethylated glycans were then subjected to acid hydrolysis, NaB(2H)4 reduction, and acetylation to generate partially methylated alditol acetates (PMAAs). The 1-O-methyl-glycoside TMS derivatives and the PMAAs were analyzed by GC-MS (Agilent Technologies, 7890B Gas Chromatography system with 5977A MSD, equipped with Agilent HP-5 m GC Column, 30 m × 0.25 mm, 0.25 μm) as described in (50 ).
7
Lipid Extraction and GC-FID Analysis
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Cells transfected with siRNA (as well as corresponding controls) were trypsinized for 5min, collected and spun at 1200rpm for 5min to pellet hepatocytes. HepG2 cell pellets were washed in 1x PBS. Lipid extractions were conducted using previously described protocols [44, 45] . Samples were analyzed using a 7890B gas chromatography system (Agilent Technologies) with a flame ionization detector, and samples were separated on a J&W DBFFAP fused-silica capillary column (15m, 0.1µm film thickness, 0.1mm i.d.;
Agilent Technologies). Fatty acid peaks were identified by comparison with retention times of fatty acid methyl ester standards. To estimate SCD1 activity, we calculated the product-to-precursor fatty acid ratio (i.e., 18:1n9/18:0 and 16:1n7/16:0), as previously reported [46, 47] . Fatty acid data were normalized to protein concentrations for each treatment condition and reported as µg fatty acid per µg/µl protein.
Agilent Technologies). Fatty acid peaks were identified by comparison with retention times of fatty acid methyl ester standards. To estimate SCD1 activity, we calculated the product-to-precursor fatty acid ratio (i.e., 18:1n9/18:0 and 16:1n7/16:0), as previously reported [46, 47] . Fatty acid data were normalized to protein concentrations for each treatment condition and reported as µg fatty acid per µg/µl protein.
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