Gas chromatography-mass spectrometry (GC-MS) is an analytical method that combines the features of gas-chromatography and mass spectrometry to identify different substances within a test sample [16] . GC-MS analysis was carried out to identify some of the potent volatile and semi-volatile constitutes present in the ethanol extract of Hellenia speciosa. GC-MS analysis was carried out on the GC-MS-5975C Agilent system comprising an autosampler and gas chromatograph interfaced to a mass spectrometer employing the following condition. The sample was injected into the injected port of the Gas chromatography (GC) device. The GC instrument vaporizes the sample and then separates and analyses the various components. Each component produces a speci c spectral peak that may be recorded on a paper chart electronically. The time elapsed between elution and injection is called the "retention time". Differentiate between some compounds was identi ed using the Retention time. The peak is measured from the base to the tip of the peak [17] .
Agilent 5975c gc ms
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 5975C GC/MS is a gas chromatograph-mass spectrometer system. It is designed to analyze and identify chemical compounds in complex samples. The system combines a gas chromatograph for sample separation and a mass spectrometer for compound identification and quantification.
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Lab products found in correlation
Clm 1396, by Cambridge Isotopes (1 mentions)
Db 5ms dg, by Agilent Technologies (1 mentions)
Carboxen polydimethylsiloxane fiber, by Merck Group (1 mentions)
N tert butyldimethylsilyl n methyltrifluoroacetamide, by Merck Group (1 mentions)
5973 network mass selective detector, by Agilent Technologies (1 mentions)
2 ethylbutyric acid, by Merck Group (1 mentions)
7 protocols using agilent 5975c gc ms
1
GC-MS Analysis of Hellenia speciosa Extracts
2
GC-MS Analysis of Hellenia speciosa Extracts
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Gas chromatography-mass spectrometry (GC-MS) is an analytical method that combines the features of gas-chromatography and mass spectrometry to identify different substances within a test sample [16] . GC-MS analysis was carried out to identify some of the potent volatile and semi-volatile constitutes present in the ethanol extract of Hellenia speciosa. GC-MS analysis was carried out on the GC-MS-5975C Agilent system comprising an autosampler and gas chromatograph interfaced to a mass spectrometer employing the following condition. The sample was injected into the injected port of the Gas chromatography (GC) device. The GC instrument vaporizes the sample and then separates and analyses the various components. Each component produces a speci c spectral peak that may be recorded on a paper chart electronically. The time elapsed between elution and injection is called the "retention time". Differentiate between some compounds was identi ed using the Retention time. The peak is measured from the base to the tip of the peak [17] .
3
Metabolic profiling of LPS-activated BMDMs
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BMDMs were incubated in custom DMEM containing 10 mM U-13C6 heavy labelled glucose (CLM-1396, Cambridge Isotope Laboratories) and 2 mM unlabelled glutamine and activated with 100 ng/ml LPS for 8 hours. Cells were washed three times with ice-cold saline and lysed in 80% methanol. Cell lysates were dried down using a speed-vacuum concentrator and stored at -80°C. Cellular metabolites were extracted and analysed by gas chromatography-mass spectrometry (GC-MS) using protocols described previously (52 (link), 53 (link)). Briefly, metabolite extracts were derived using N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA). D-myristic acid (750 ng/sample) was added as an internal standard to metabolite extracts, and metabolite abundance was expressed relative to the internal standard. GC/MS analysis was performed using an Agilent 5975C GC/MS equipped with a DB-5MS + DG (30 m × 250 µm × 0.25 µm) capillary column (Agilent J&W, Santa Clara, CA, USA). Metabolite measurements were performed at the Rosalind and Morris Goodman Cancer Research Centre Metabolomics Core Facility supported by the Canada Foundation for Innovation, The Dr. John R. and Clara M. Fraser Memorial Trust, the Terry Fox Foundation (TFF Oncometabolism Team Grand 116128) and McGill University. Mass isotopomer distribution was determined using a custom algorithm developed at McGill University (52 (link)).
4
Snapdragon Floral Volatiles Profiling
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Headspace solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) technologies were combined to collect and examine the floral volatile compounds of snapdragon. Briefly, using 10 ng⋅mL–1 3-octanol as the internal standard, 1 g of lobes infected was placed in an airtight bottle equipped with a 100 μM Carboxen/polydimethylsiloxane fiber (SigmaAldrich) at 25°C for 5 min, followed by 40°C for 40 min. The trapped floral scent compounds were subsequently desorbed and transferred to an Agilent 5975C GC-MS (Agilent Technologies) equipped with an HP−1MS fused-silica capillary column (0.25 mm diameter, 30 m length, and 0.25 μm film thickness) with helium as the carrier gas. The desorption program was designed as follows:
Isothermal column temperature at 40°C for 3 min, then the temperature was increased at a rate of 5°C min−1 to 120°C for 1 min, and the temperature was then further increased at a rate of 10°C min–1 to 180°C for 3 min, and then increased to 280°C for 1 min at a rate of 10°C min–1. The volatiles were identified by comparing the mass spectra and retention time with the NIST 2008 mass spectra library and standard samples. Total ion chromatogram (TIC) was analyzed to detect flower volatiles.
Isothermal column temperature at 40°C for 3 min, then the temperature was increased at a rate of 5°C min−1 to 120°C for 1 min, and the temperature was then further increased at a rate of 10°C min–1 to 180°C for 3 min, and then increased to 280°C for 1 min at a rate of 10°C min–1. The volatiles were identified by comparing the mass spectra and retention time with the NIST 2008 mass spectra library and standard samples. Total ion chromatogram (TIC) was analyzed to detect flower volatiles.
5
Qualitative and Quantitative GC-MS Analysis
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On an Agilent 5975C GC/MS (Agilent Technologies, Santa Clara CA, USA) with a HP-5MS column (5% phenyl methyl siloxane, 30 m × 0.25 mm, 0.25 μm), the volatiles were subjected to GC analysis. At a flow rate of 1 mL/min, helium was utilized as the carrier gas. After being set at 45 °C for 1 min, the temperature was elevated to 250 °C at 10 °C/min, maintained at 250 °C for 50 min, and then programmed to hold at 280 °C for 1 min. A split ratio of 40:1 was used with a 1 μL sample injection in split mode. The quadrupole was heated to 220 °C, and the ion source temperature was adjusted to 280 °C. The multi-channel plate voltage of 70 eV was applied, and the detector was set to operate in EI mode with a m/z range of 50–500.
All compounds identification was performed using the mass spectra, linear retention index (LRI), and Kováts retention index (KRI) data. The retention indices (RI) of every compound were calibrated with C5–C30 alkanes, comparing them to the mass spectra in the NIST libraries. By using peak area normalization [37 (link),38 (link),39 (link)], the relative amounts of the identified volatile compounds were quantified.
All compounds identification was performed using the mass spectra, linear retention index (LRI), and Kováts retention index (KRI) data. The retention indices (RI) of every compound were calibrated with C5–C30 alkanes, comparing them to the mass spectra in the NIST libraries. By using peak area normalization [37 (link),38 (link),39 (link)], the relative amounts of the identified volatile compounds were quantified.
6
Quantifying Fecal Short-Chain Fatty Acids
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Fecal SCFAs were evaluated by a gas chromatography mass spectrometer (GC-MS). Briefly, 200 mg of frozen feces was suspended in 1 mL of 1% HCl and strongly vortexed for 1 min. 2-Ethylbutyric acid (Sigma Aldrich, USA) was added as internal standard in a final concentration of 2 mM. The samples were centrifuged at 5000×g for 5 min and the supernatant was acidified to pH 0 with HCl (10 mol/L). Each sample was extracted at 4 °C using an equal volume of diethyl ether. Aliquots (80 μL) of extracts were added with 16 μL N-tert-butyldimethylsilyl-N methyltrifluoroacetamide (Sigma Aldrich) and incubated at 40 °C for 2 h. The SCFAs contents of each samples were analyzed on an Agilent 5975C GC-MS (Agilent Technologies, Palo Alto, CA, USA) equipped with a HP-5MS column (0.25 mm × 30 m × 0.25 μm) and a 5973 Network Mass Selective Detector. The GC program was performed as follows: started at 40 °C, heated to 70 °C by 5 °C /min and held for 3.5 min, then ramped at 20 °C/min to 160 °C followed by 35 °C /min to 280 °C and held for 3 min. The m/z ratios of monitored ions were as follows: 117 (acetate), 131(propionate), 145 (butyrate), and 173 (2-Ethylbutyric acid). SCFAs were quantified with a five-point calibration curve.
7
TCA Cycle Metabolite Profiling in Myotubes
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Myotubes were treated in the same manner as the glucose oxidation experiments, except radiolabeled glucose was not added. Following the 2-h incubation, cell pellets were washed with 1× PBS twice, collected in 1 ml 1× PBS, spun down at 2000 × g speed to discard the supernatant and stored at -80 °C freezer for metabolomics analyses.
TCA cycle-targeted metabolomics was performed at Mayo Clinic Metabolomics Core Facility. Briefly, before analysis, the cell pellets were lysed in 50 µl of 1×PBS after spiking in 20 µl of internal solution containing U- 13 Clabeled analytes. The proteins were removed by adding 250 µl of chilled methanol and acetonitrile solution to the sample mixture. After drying the supernatant, the sample was derivatized as described previously [18] before it was analyzed on an Agilent 5975C GC/MS (gas chromatography/mass spectrometry). Concentrations of lactic acid (m/z 261.2), fumaric acid (m/z 287.1), succinic acid (m/z 289.1), oxaloacetic acid (m/z 346.2), ketoglutaric acid (m/z 360.2), malic acid (m/z 419.3), cis-aconitic acid (m/z 459.3), citric acid (m/z 591.4), isocitric acid (m/z 591.4), and glutamic acid (m/z 432.4) were measured against a seven-point calibration curves that underwent the same derivatization. Data were normalized to total protein content.
TCA cycle-targeted metabolomics was performed at Mayo Clinic Metabolomics Core Facility. Briefly, before analysis, the cell pellets were lysed in 50 µl of 1×PBS after spiking in 20 µl of internal solution containing U- 13 Clabeled analytes. The proteins were removed by adding 250 µl of chilled methanol and acetonitrile solution to the sample mixture. After drying the supernatant, the sample was derivatized as described previously [18] before it was analyzed on an Agilent 5975C GC/MS (gas chromatography/mass spectrometry). Concentrations of lactic acid (m/z 261.2), fumaric acid (m/z 287.1), succinic acid (m/z 289.1), oxaloacetic acid (m/z 346.2), ketoglutaric acid (m/z 360.2), malic acid (m/z 419.3), cis-aconitic acid (m/z 459.3), citric acid (m/z 591.4), isocitric acid (m/z 591.4), and glutamic acid (m/z 432.4) were measured against a seven-point calibration curves that underwent the same derivatization. Data were normalized to total protein content.
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