Dried metabolites were derivatized for 2 h at 42°C in 50 μl of methoxyamine hydrochloride 10 mg/ml (Sigma) and 100 μl N‐tert‐Butyldimethylsilyl‐N‐methyltrifluoroacetamide (Sigma) for 90 min at 72°C (Dai et al, 2020 ). Metabolites were analyzed using an Agilent 7890B gas chromatograph (Agilent, CA, USA) networked to an Agilent 5977B mass selective detector. Retention times and mass fragmentation signatures of all metabolites were validated using pure standards. To determine the relative metabolite abundance across samples, the area of the total ion current peak for the metabolite of interest was normalized for protein content. The mass isotopomer distribution analysis measured the fraction of each metabolite pool that contained every possible number of 13C atoms: a metabolite could contain 0, 1, 2, …n 13C atoms, where n = the number of carbons in the metabolite. For each metabolite, an informative fragment ion containing all carbons in the parent molecule was analyzed by MATLAB software (MathWorks, CA, USA). The abundance of all mass isotopomers was integrated from m+0 to m+n, where m = the mass of the fragment ion without any 13C. The abundance of each mass isotopomer was then corrected mathematically to account for natural abundance isotopes and finally converted into a percentage of the total pool.
Agilent 5977b mass selective detector
Manufactured by Agilent Technologies
Sourced in United States, Australia
The Agilent 5977B mass selective detector is a laboratory instrument used for the detection and analysis of chemical compounds. It is designed to accurately identify and quantify a wide range of molecules in complex samples. The 5977B utilizes mass spectrometry technology to separate and detect compounds based on their unique mass-to-charge ratios.
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Lab products found in correlation
N tert butyldimethylsilyl n methyltrifluoroacetamide, by Merck Group (1 mentions)
Agilent 7890b gas chromatograph, by Agilent Technologies (1 mentions)
Matlab software, by MathWorks (1 mentions)
Methoxyamine hydrochloride, by Merck Group (1 mentions)
Merlin microseal, by Agilent Technologies (1 mentions)
2 protocols using agilent 5977b mass selective detector
1
Metabolite Derivatization and GC-MS Analysis
2
Gas Chromatography-Mass Spectrometry Analysis
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The analysis was carried out using an Agilent 7890B gas chromatograph equipped with Agilent 5977B Mass Selective Detector (Agilent, Mulgrave, Australia) and Gerstel ODP‐3 Olfactory Detector Port (Gerstel, Lasersan Australasia Pty. Ltd., Australia). The separation was conducted using BP‐5MS capillary column (30 m × 0.250 mm × 0.25 µm), (Trajan Scientific and Medical, Australia). The split/splitless inlet was fitted with a Merlin Microseal (Agilent, Santa Clara, CA, USA), and a splitless SPME liner (i.d. 0.75 mm, Agilent, USA), and operated in splitless mode. High purity helium (99.999 %) was used as a carrier gas under a constant flow of 1.4 ml/min. The column oven temperature started at 40°C with a 4 min hold time, and then ramped at 8.6°C/min to 200°C with an 8 min hold time (total analysis time 30.6 min). MS conditions were scan range 35–450 m/z., solvent delay time 0 min, electron impact mode at 70 eV, MS source 230°C, and MS quad 150°C. For ODP, the column was connected with splitter and the effluent was split 1:1 using two deactivated fused silica capillary tubes (1.43 m × 0.10 mm i.d. to MS and 0.57 × 0.10 mm i.d. to ODP, Trajan Scientific and Medical, Australia). The ODP transfer line was maintained at 250°C and the makeup gas was nitrogen.
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