Detection of analytes was achieved using a 7890B GC and 7010B GC/MS triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), as detailed in [47 (link)]. Samples (1 μL) were injected in splitless mode, and the analytes were separated with a VF-5 ms GC column (60 m × 250 μm × 0.25 μm, Agilent Technologies) and ionized with an electron ionization source. The column head pressure was set at 21.5 psi with a constant flow rate of 1.2 mL/min using helium gas. Initial column temperature was held at 70 °C for 5 min, increased to 150 °C at 50 °C/min, ramped to 280 °C at 4 °C/min, and then held for 15 min. The total run time was 42.1 min. The injector temperature was set at 250 °C. The ion source and auxiliary transfer line temperatures were 300 °C. Electron multiplier voltage was set at 1884 V. Nitrogen gas was used as the collision gas for all MS/MS experiments, and the pressure of collision gas was set at 16.8 psi.
Vf5ms gc column
Manufactured by Agilent Technologies
Sourced in United States
The VF5ms GC column is a gas chromatography column designed for the separation and analysis of a wide range of organic compounds. It features a 5% phenyl-95% dimethylpolysiloxane stationary phase that provides excellent inertness and separation performance across a broad range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 7890 gc system, by Agilent Technologies (2 mentions)
Agilent 5975c inert xl mass selective detector, by Agilent Technologies (2 mentions)
Ctc pal, by Agilent Technologies (2 mentions)
Ez guard, by Agilent Technologies (1 mentions)
Agilent 7890b gc system, by Agilent Technologies (1 mentions)
Agilent 7010 b, by Agilent Technologies (1 mentions)
Acquity uplc pump system, by Waters Corporation (1 mentions)
5 protocols using vf5ms gc column
1
GC-MS Analyte Detection Protocol
2
GC-MS Analysis of Metabolites
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GC-MS samples from the above extraction were re-dissolved and derivatized. Eight microliters of a retention time standard mixture (0.029% v/v n-dodecane, n-pentadecane, n-nonadecane, n-docosane, n-octacosane, n-dotracontane, and n-hexatriacontane dissolved in pyridine) was added. The sample set also included a reference quality control of authentic metabolite standards (1 mg ml−1 each) (Additional file 2 : Table S1A). Volumes of 1 μL were then injected onto 30-m VF-5 ms GC column with 0.25 mm i.d., film thickness of 0.25 μ m, and + 10 m EZ-Guard (Agilent) in splitless and split mode (32:1) allowing a more accurate comparison of highly abundant metabolites (e.g. tartarate, sugars, and inositol). The GC-MS system consisted of an AS 3000 autosampler, a TRACE GC ULTRA gas chromatograph, and a DSQII quadrupole mass spectrometer (Thermo-Fisher ltd). The parameters of the machine were exactly as described in [69 (link)]. Spectral searching was done by consulting the National Institute of Standards and Technology (NIST, Gaithersburg, USA) algorithm incorporated in the Xcalibur® data software (version 2.0.7) against RI libraries from the Max-Planck Institute for Plant Physiology in Golm, Germany (http://www.mpimp-golm.mpg.de/mms-library/ ) and finally normalized by the total metabolites and correcte d for the dilution factor.
3
GC-MS Analysis of Metabolites
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Amino acids, organic acids, and sugars were analyzed by GC-MS by an Agilent 7890 GC system (Agilent, Santa Clara, CA, USA) coupled to an Agilent 5975C inert XL Mass Selective Detector (Agilent, Santa Clara, CA, USA). Samples were first derivatized by methoxyamine hydrochloride dissolved in dry pyridine at room temperature overnight. Amino acids and organic acids were silylated to trimethylsilyl (TBDMS) derivatives by adding N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide with 1% (w/v) tert-butyldimethylchlorosilane and incubated at 60 °C overnight. Sugars were silylated to trimethylsilyl (TMS) derivatives by adding N,O-bis(trimethylsilyl)trifluoroacetamide with 1% (w/v) trimethylchlorosilane and incubated at 60 °C overnight. Metabolites were separated by an Agilent VF5ms GC column (Agilent, Santa Clara, CA, USA). The inlet temperature and MS transfer line temperature were set at 230 °C and 300 °C, respectively. The oven temperature was initially held at 40 °C for 1 min and then raised at 40 °C/min to 80 °C, 10 °C/min to 240 °C, and 20 °C/min until reaching 320 °C, which was maintained for 5 min. Electron ionization (EI) was at 70 eV, and the mass scan range was 50 to 600 amu.
4
Comprehensive GC-MS Analysis of Metabolites
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Amino acids, organic acids, and sucrose were analyzed using a GC-MS system. Amino acids and organic acids were derivatized by methoximation, followed by tert-butyldimethylsilylation. Sucrose was derivatized by methoximation, followed by trimethylsilylation. Samples were analyzed by an Agilent 7890 GC system (Agilent, Santa Clara, CA, USA) coupled to an Agilent 5975C inert XL Mass Selective Detector (Agilent, Santa Clara, CA, USA) with an autosampler (CTC PAL; Agilent, Santa Clara, CA, USA). Metabolites were separated by an Agilent VF5ms GC column, 30 m × 0.25 mm × 0.25 m with 10 m guard column (Part number: CP9013; Agilent, Santa Clara, CA, USA). For amino acids and organic acids, 1 μL of the derivatized sample was injected into 10 split mode with helium carrier gas with a flow rate of 1.2 mL min−1. The oven temperature gradient was: 100°C (4 min hold), increased by 5°C/min to 200°C, then by 10°C/min to 320°C, and held at 320°C for 10 min. Electron ionization (EI) is at 70 eV and the mass scan range was 100–600 amu. The ionization source temperature was set at 150°C and the transfer line temperature at 300°C. The fragment ions used for the isotopologue analysis are described in the Supplemental Table S2 .
5
Mass Spectrometry-Based Multi-Omics Analysis
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Mass spectrometry for anion exchange LC-MS/MS and GC-EI-MS were carried out using the methods described in ref. 12 (link) and detailed in Dataset S5 . Reverse-phase LC-MS/MS and GC-chemical ionization (CI)-MS had the following changes: Samples for reverse-phase liquid chromatography-tandem mass spectrometry were analyzed by an ACQUITY UPLC pump system (Waters) coupled with Waters XEVO TQ-S ultra-performance liquid chromatography tandem mass spectrometry (Waters) by the method described in ref. 12 (link). Samples for gas chromatography-electron ionization-mass spectrometry were analyzed by an Agilent 7890B GC system (Agilent) coupled to an Agilent 7010B triple quadrupole gas chromatography-electron ionization-mass spectrometer with an autosampler (CTC PAL) (Agilent). An Agilent VF5ms GC column, 30 m × 0.25 mm × 0.25 m with 10-m guard column was used. One microliter of the derivatized sample was injected with helium carrier gas at a flow rate of 1.2 mL⋅min−1. The oven temperature gradient was: 40 °C (1-min hold), increased at 40 °C/min to 150 °C, then a 10 °/min to 250 °C, then a 40 °C/min to 320 °C, and finally held at 320 °C for 4.5 min. CI was used, and the mass scan range was 150 amu to 650 amu with step size 0.1 amu. The ionization source temperature was set at 300 °C, and the transfer line temperature was 300 °C.
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