7890b 5977a system, by Agilent Technologies - Product details

1

GC-MS Analysis of Metabolite Derivatization

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For GC-MS analysis, 150 µl of the aqueous phase were dried down in a vial insert, washed twice with 40 µl MeOH and dried again. Samples were then derivatised by methoximation (20 µl of 20 mg/mL methoxyamine in pyridine, RT overnight) before addition of 20 µl of N,O-bis(trimetylsilyl)trifluoroacetamide (BSTFA) + 1% trimethylchlorosilane (TMCS) (Sigma, 33148) for ≥1 h. Metabolite analysis was performed by GC-MS using an Agilent 7890B-5977A system. Splitless injection (injection temperature 270°C) onto a 30 m + 10 m × 0.25 mm DB-5MS+DG column (Agilent J&W) was used, with a helium carrier gas, using electron impact ionization (EI) mode. Oven temperature was initially 70 °C (2 min), followed by a temperature increase to 295 °C at 12.5 °C/min and subsequently to 320 °C at 25 °C/min (held for 3 min). MassHunter Workstation software (B.06.00 SP01, Agilent Technologies) was used for metabolite identification and quantification by comparison to the retention times, mass spectra, and responses of known amounts of authentic standards. Fractional labelling of individual metabolites was calculated as the fraction of carbons in the metabolite pool that were ¹³C atoms after correction for natural abundance.

Fets L., Driscoll P.C., Grimm F., Jain A., Nunes P.M., Gounis M., Doglioni G., Papageorgiou G., Ragan T.J., Campos S., Silva dos Santos M., MacRae J.I., O’Reilly N., Wright A.J., Benes C.H., Courtney K.D., House D, & Anastasiou D. (2018). MCT2 mediates concentration-dependent inhibition of glutamine metabolism by MOG. Nature chemical biology, 14(11), 1032-1042.

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2

GC-MS Analysis of Fecal and Diet Samples

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Fecal or diet samples extracts (1 μL) with split ratio = 10:1 were analyzed in an Agilent 7890B-5977A system fitted with a DB-5MS column (0.25 μm × 0.25 mm × 30 m; Agilent Technologies Inc., Santa Clara, CA, USA). The injection temperature was 300 °C, the transfer interface was set to 280 °C and the ion source was adjusted to 230 °C. The oven temperature program consisted of an initial 70 °C for 3 min followed by an increase to 170 °C at 5 °C min⁻¹, 234 °C at 4 °C min⁻¹, 270 at 5 °C min⁻¹, ramping up to 300 °C at 10 °C min⁻¹ and holding for 5 min. The detector voltage was set to 0.93 kV. The metabolite EI ionization voltage was set to 70 eV. Full scan mode (m/z: 33−600) was used to acquire the mass signals.

Qin D., Wang G., Dong Z., Xia Q, & Zhao P. (2020). Comparative Fecal Metabolomes of Silkworms Being Fed Mulberry Leaf and Artificial Diet. Insects, 11(12), 851.

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3

GC-MS Analysis of Metabolite Derivatization

Check if the same lab product or an alternative is used in the 5 most similar protocols

For GC-MS analysis, 150 µl of the aqueous phase were dried down in a vial insert, washed twice with 40 µl MeOH and dried again. Samples were then derivatised by methoximation (20 µl of 20 mg/mL methoxyamine in pyridine, RT overnight) before addition of 20 µl of N,O-bis(trimetylsilyl)trifluoroacetamide (BSTFA) + 1% trimethylchlorosilane (TMCS) (Sigma, 33148) for ≥1 h. Metabolite analysis was performed by GC-MS using an Agilent 7890B-5977A system. Splitless injection (injection temperature 270°C) onto a 30 m + 10 m × 0.25 mm DB-5MS+DG column (Agilent J&W) was used, with a helium carrier gas, using electron impact ionization (EI) mode. Oven temperature was initially 70 °C (2 min), followed by a temperature increase to 295 °C at 12.5 °C/min and subsequently to 320 °C at 25 °C/min (held for 3 min). MassHunter Workstation software (B.06.00 SP01, Agilent Technologies) was used for metabolite identification and quantification by comparison to the retention times, mass spectra, and responses of known amounts of authentic standards. Fractional labelling of individual metabolites was calculated as the fraction of carbons in the metabolite pool that were ¹³C atoms after correction for natural abundance.

Fets L., Driscoll P.C., Grimm F., Jain A., Nunes P.M., Gounis M., Doglioni G., Papageorgiou G., Ragan T.J., Campos S., Silva dos Santos M., MacRae J.I., O’Reilly N., Wright A.J., Benes C.H., Courtney K.D., House D, & Anastasiou D. (2018). MCT2 mediates concentration-dependent inhibition of glutamine metabolism by MOG. Nature chemical biology, 14(11), 1032-1042.

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4

GC-MS Metabolite Profiling Protocol

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Metabolite analysis was performed by GC-MS using an Agilent 7890B-5977A system. Splitless injection (injection temperature 270°C) onto a 30 m + 10 m × 0.25 mm DB-5MS + DG column (Agilent J&W) was used, using helium as the carrier gas, in electron ionization mode. The initial oven temperature was 80°C (2 min), followed by temperature gradients to 140°C at 30°C/min, to 250°C at 5°C/min and then to 320°C at 15°C/min (held for 6 min). Metabolites were identified by comparison with the retention times and mass spectrum of authentic standards using the MassHunter Workstation software (B.06.00 SP01, Agilent Technologies). Abundance was calculated by comparison to responses of known amounts of standards.

Blassberg R., Macrae J.I., Briscoe J, & Jacob J. (2015). Reduced cholesterol levels impair Smoothened activation in Smith–Lemli–Opitz syndrome. Human Molecular Genetics, 25(4), 693-705.

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7890b 5977a system

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4 protocols using 7890b 5977a system

GC-MS Analysis of Metabolite Derivatization

GC-MS Analysis of Fecal and Diet Samples

GC-MS Analysis of Metabolite Derivatization

GC-MS Metabolite Profiling Protocol

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