TG-5MS capillary column-equipped TRACE1310-ISQLT equipment (Thermo Fisher Scientific, Wyman Street, Waltham, MA, USA) was used for the analysis. Helium (purity 99.999%) was the carrier gas at a flow rate of 1 mL/min with an ionization voltage of 70 eV, covering a mass range 40–460 m/z. The temperature of the GC oven was kept at 35 °C for 3 min, increased to 150 °C at 3 °C/min, then ramped up to 260 °C at 10 °C/min, before being held at 290 °C at 5 °C/min for 8 min. The constituents were identified by comparing constituents’ mass spectra with the NIST 08, C8-C40 n-alkane standard solution and published mass spectra [35 ]. The peak area normalization was used to determine the relative concentrations of the components.
Trace 1310 isq lt
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TRACE 1310-ISQ LT is a gas chromatography-mass spectrometry (GC-MS) system designed for analytical laboratories. It combines a TRACE 1310 gas chromatograph with an ISQ LT single quadrupole mass spectrometer. The system is capable of performing qualitative and quantitative analysis of complex samples.
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6 protocols using trace 1310 isq lt
1
GC-MS Analysis of Organic Compounds
2
Fecal SCFA Analysis by GC-MS
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SCFAs were detected and analyzed in the fecal samples using gas chromatography–mass spectrometry (GC–MS) technology (Thermo TRACE 1310-ISQ LT, America). Briefly, fecal pellets were homogenized in sterile deionized water (150 µL/sample) and centrifuged at 13,000 rpm, at 4 °C for 5 min, to pellet the particulate matter. A small sample of the supernatant (1 μL) was injected into the column and used for detection by GC–MS.
3
Metabolic Profiling and Histological Analysis
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Plasma insulin and hepatic triglyceride and cholesterol were measured using commercial kits. Glucose tolerance test (GTT), insulin tolerance test (ITT) and histological analyses, including hematoxylin & eosin (H&E) and Alcian blue-periodic acid Schiff (AB-PAS) staining, immunohischemistry of TLR4 (ab13867, Abcam), F4/80 (GB11027, Servicebio), and UCP1 (ab155117, Abcam), immunofluorescence staining of LC3 (14600-1-AP, Proteintech), were performed as described previously.52 (link) Adipocyte size was determined from H&E stained sections and the number of CLSs was manually counted from F4/80-stained sections in five random fields from 5 mice per group by Image J software. Intestinal permeability was assessed in vivo following oral administration of fluorescein-isothiocyanate (FITC)-dextran (46944–500 MG-F, Sigma). Fecal SCFAs levels were detected by GC/MS (Thermo, TRACE 1310-ISQ LT), and the final data were normalized according to the fecal weight.
4
SCFA Extraction and GC-MS Analysis
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The LGG-s was transferred to a 2 mL centrifuge tube to which 50 μL of 15% phosphoric acid, 10 μL of 75 μg/mL internal standard (isocaproic acid) solution, and 140 μL of ether were added, vortexed for 1 min, and centrifuged at 12,000 rpm at 4 °C for 10 min. The supernatant was collected and used for chromatography-mass spectrometry (GC-MS) analysis (Trace 1310/ISQ LT, Thermo Fisher Scientific, Waltham, MA, USA). The SCFA standards were purchased from Sigma-Aldrich.
5
GC-MS Analysis of Colonic SCFAs in Piglets
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The SCFAs (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid) in colonic contents of piglets from three groups were determined by the gas chromatography-mass spectrometry (GC-MS) analysis method. The 50 mg sample was homogenized in the mixed solution (50 μl of 15% phosphoric acid, 100 μl of 125 μg/ml internal standard (isocaproic acid) solution and 400 μl of ether) and centrifuged at 4°C for 10 min at 12,000 rpm. The supernatant was then analyzed by GS-MS (Thermo TRACE 1310-ISQ LT, USA). The chromatographic column Agilent HP-InnoWax column was used in this study. In the operation process of GS-MS, the initial temperature was 90°C. The temperature was heated to 120°C at 10°C/min, then to 150°C at 5°C/min, and finally to 250°C at 25°C/min for 2 min. The temperature of the inlet was 250°C. SCFAs were identified by the retention time of standard compounds.
6
Quantifying Fecal Short-Chain Fatty Acids
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SCFAs were evaluated by the gas chromatography mass spectrometer (GC-MS, TRACE 1310-ISQ LT, Thermo, USA). Briefly, 50 mg of feces samples were suspended in 50 μL of 15% phosphoric acid and 4 μL ether with 125 μg/mL of isocaproic acid as internal standard. Then, the solution was strongly vortexed for 1 min. After centrifuged at 12000 rpm at 4 °C, the supernatant was collected and examined by GC-MS equipped with Agilent HP-INNOWAX column (30 m * 0.25 mm * 0.25 μm). The program was performed as follows: started at 90 °C, heated to 120 °C by 10 °C/min, heated to 150 °C by 5 °C/min, heated to 250 °C by 25 °C/min and held for 2 min. Then, the SCFAs contents were analyzed.
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