For 13C-glutamine tracing, 2 × 105 cells were seeded in 6 cm plates containing DMEM supplemented with 10% FBS and cultured overnight. Cells were washed with PBS twice and cultured in glutamine-free DMEM (15-017-CV, Corning) supplemented with 10% dialyzed FBS containing 13C-glutamine (4 mM; Cambridge Isotope Laboratory). After culturing for 24 h, the whole cell extract was prepared and dried using the same protocol as glucose tracing. 50 μl MOX (10 mg/ml in pyridine, 226904 Sigma) was added and the mixture was incubated at 42 °C for 1 h. After the samples were allowed to cool down, 100 μl TBDMS (394882, Sigma) was added and samples were incubated at 70 ˚C for 1 h. Then samples were transferred to GC vials and analyzed by Agilent 5977B GC-MSD- using HP-5MS UI47 (link).
Agilent 5977b gc msd
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 5977B GC/MSD is a gas chromatograph-mass spectrometer system. It is designed for the separation, identification, and quantification of chemical compounds in complex samples. The system combines gas chromatography (GC) for the separation of compounds and mass spectrometry (MS) for their identification and analysis.
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Lab products found in correlation
Tbdms, by Merck Group (1 mentions)
Sp 2560 capillary column, by Merck Group (1 mentions)
Turbo mass gold, by PerkinElmer (1 mentions)
Turbomass 5.1 spectrometer, by PerkinElmer (1 mentions)
Elite 1, by PerkinElmer (1 mentions)
Db 5ms, by PerkinElmer (1 mentions)
Agilent 7890b gc, by Agilent Technologies (1 mentions)
Agilent 7890b, by Agilent Technologies (1 mentions)
6 protocols using agilent 5977b gc msd
1
Tracing Cellular Metabolism with 13C-Glutamine
2
Fatty acid identification by GC-MS
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The extracted fatty acids were prepared with fatty acid methyl esters (Berton et al., 2016 (link)). The fatty acids were separated by an Agilent 5977B GC/MSD (Agilent, CA, United States) using a 100 m × 0.25 mm diameter SP-2560 capillary column of 0.02 mm thickness (Supelco, Bellefonte, PA) (Ebrahimi et al., 2014 (link)). Helium was used as a carrier gas with a spit ratio of 9:1. The experimental conditions for mass spectrometry were as follows: full scan mode, a solvent delay of 11 min, a gain factor of 10, an ion source temperature of 230°C, and a quadrupole temperature of 150°C. Qualitative Analysis B.07.00 software (Agilent) was used with the National Institute of Standards and Technology (NIST) database to identify the compound information for the reference standard (Sigma-Aldrich, United States) and samples. The results are expressed as a percentage of total fatty acids.
3
GC-MS Analysis of Extracted Samples
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Gas chromatography–mass spectrometry (GC-MS) analysis of the extracted sample was performed using an Agilent 5977B GC/MSD (Agilent, Santa Clara, CA, USA). Gas Chromatograph was equipped and coupled to a mass detector Turbo Mass Gold, PerkinElmer Turbo Mass 5.1 spectrometer with an Elite-1 (100% dimethylpolysiloxane), DB-5MS, 30 m × 0.25 mm i.d., 0.25 μm film thickness of capillary column. The instrument was set to an initial temperature of 80 °C and maintained at this temperature for 1 min. At the end of this duration, the oven temperature was raised to 300 °C, at the rate of an increase of 15 °C/min, and maintained for 9 min. The injection port temperature was ensured at 290 °C, and the helium flow rate was one mL/min. The ionization voltage was 70 eV. Samples were injected in split mode as 10:1. Mass spectral scan range was set at 30–600 (m/z) [30 (link)].
4
Extraction and GC-MS Analysis of Algae
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Clean Bulung Boni and Bulung Anggur were air-dried in the shade for 2 days and then dried in the oven for 7 days at 45 °C. Dried samples were then powdered using an electric blender. The samples of each alga (15 g) were macerated in ethanol solvent (150 mL). The mixtures were kept for 96 h, then filtered and concentrated using a rotary vacuum evaporator to produce the crude extract.
The crude extract was then subjected to phytochemical analysis using GC-MS. Gas chromatography analysis was carried out on the Agilent 7890B GC (Santa Clara, CA, USA) coupled with a mass detector Agilent 5977B GC/MSD (Santa Clara, CA, USA). A measure of 1.0 µL of the extract was injected into the chromatograph at an injector temperature of 250 °C. The column (Wakosil-II5C18 4.6*200 mm, Richmond, VA, USA) oven temperature was programmed to increase from 70 °C to 290 °C at a rate of 10 °C/min. The run time for GC was 17 min. The identification of chemical compounds and the interpretation of mass spectra of GC-MS were carried out using the Wiley Spectral library.
The crude extract was then subjected to phytochemical analysis using GC-MS. Gas chromatography analysis was carried out on the Agilent 7890B GC (Santa Clara, CA, USA) coupled with a mass detector Agilent 5977B GC/MSD (Santa Clara, CA, USA). A measure of 1.0 µL of the extract was injected into the chromatograph at an injector temperature of 250 °C. The column (Wakosil-II5C18 4.6*200 mm, Richmond, VA, USA) oven temperature was programmed to increase from 70 °C to 290 °C at a rate of 10 °C/min. The run time for GC was 17 min. The identification of chemical compounds and the interpretation of mass spectra of GC-MS were carried out using the Wiley Spectral library.
5
GC-MS Analysis of Metabolite Levels
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GC-MS measurement of relative metabolite levels and isotope enrichment was performed as previously described [32] . Dried samples were derivatized with equal amounts of methoxylamine (20 mg/mL in pyridine) and N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA) or N-Methyl-N-(trimethylsilyl)-trifluoracetamide (MSTFA) using a derivatization robot (Gerstel MPS). A sample volume of 1µL was injected in a SSL injector in splitless mode at 270 °C. GC-MS analysis was performed by an Agilent 7890B gas chromatogram system coupled to an Agilent 5977B GC/MSD (MSD, Agilent Technologies), equipped with a 30 m DB-35MS + 5m Duraguard capillary column (0.25 mm inner diameter, 0.25 µm film thickness). Helium was used as the carrier gas with a constant flow rate of 1 mL/min. Metabolites were detected in either full scan or selected ion mode. Processing of chromatograms and calculation of mass isotopomer distributions and relative quantification of metabolites were performed using the Metabolite Detector software [36] .
6
GC-MS Analysis of Methanolic Bupleurum nivosa Extract
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GC-MS analysis was conducted to
identify the compounds
present in the methanolic extract of B. nivosa. Sample derivatization was carried out using the trimethylsilyl
derivatization method. Initially, 20 μL of the sample was allowed
to evaporate in an oven at 80 °C for 25 min. Subsequently, the
dried sample was dissolved in an 80 μL solution of methoxyamine
hydrochloride in pyridine (2 mg/100 mL). The mixture was thoroughly
mixed using a vortex and incubated at 30 °C for 30 min before
analysis.26 (link) GC-MS analysis of the methanolic
extract of B. nivosa was performed
using Agilent Technologies equipment, specifically the Model No: 7890B
GC system and Agilent 5977B GC/MSD. Helium gas was employed as the
carrier gas at a flow rate of 1 mL/min. The injector temperature was
maintained at 270 °C. The oven temperature profile was designed
to gradually increase from 70 to 200 °C (with a 10 °C rise
in temperature every minute), followed by a hold at 310 °C for
5 min, with an additional 10 °C increase every minute. MS was
operated in electron ionization mode at 70 eV, using an electron multiplier
voltage of 1859 V. The retention times of the detected compounds were
determined by using Chemstation software. The confirmation of various
phytochemicals was established by comparing their spectral data with
those of authenticated compounds in the National Institute of Standards
and Technology (NIST) library.
identify the compounds
present in the methanolic extract of B. nivosa. Sample derivatization was carried out using the trimethylsilyl
derivatization method. Initially, 20 μL of the sample was allowed
to evaporate in an oven at 80 °C for 25 min. Subsequently, the
dried sample was dissolved in an 80 μL solution of methoxyamine
hydrochloride in pyridine (2 mg/100 mL). The mixture was thoroughly
mixed using a vortex and incubated at 30 °C for 30 min before
analysis.26 (link) GC-MS analysis of the methanolic
extract of B. nivosa was performed
using Agilent Technologies equipment, specifically the Model No: 7890B
GC system and Agilent 5977B GC/MSD. Helium gas was employed as the
carrier gas at a flow rate of 1 mL/min. The injector temperature was
maintained at 270 °C. The oven temperature profile was designed
to gradually increase from 70 to 200 °C (with a 10 °C rise
in temperature every minute), followed by a hold at 310 °C for
5 min, with an additional 10 °C increase every minute. MS was
operated in electron ionization mode at 70 eV, using an electron multiplier
voltage of 1859 V. The retention times of the detected compounds were
determined by using Chemstation software. The confirmation of various
phytochemicals was established by comparing their spectral data with
those of authenticated compounds in the National Institute of Standards
and Technology (NIST) library.
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