The SCFA content of fecal samples was determined by MetWare (http://www.metware.cn/ ) using the Agilent 7890B-7000D GC-MS/MS platform. Stock solutions of standards were prepared at 1 mg/mL in MTBE and stored at −20°C. Immediately prior to analysis, the stock solutions were diluted with MTBE to working concentrations. Fecal samples (20 mg) were weighed and placed in a 2 mL EP tube with 1 mL of aqueous phosphoric acid (0.5% v/v) and a small steel ball. The mixture was ground three times for 10 s each, vortexed for 10 min, ultrasonicated for 5 min, and centrifuged at 12,000 r/min for 10 min at 4°C. A 0.1-mL sample of the supernatant was mixed with 0.5 mL MTBE containing internal standard in a 1.5 mL centrifugal tube and the mixture vortexed for 3 min, ultrasonicated for 5 min, and recentrifuged at 12,000 r/min for 10 min at 4°C. The new supernatant was collected and analyzed by GC-MS/MS using an Agilent 7890 B gas chromatograph coupled to a 7000D mass spectrometer with a DB-FFAP column (30 m length × 0.25 mm i.d. ×0.25 μm film thickness, J&W Scientific, USA).
Agilent 7890b 7000d gc ms ms platform
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 7890B-7000D GC-MS/MS platform is an analytical instrument that combines gas chromatography (GC) with triple quadrupole mass spectrometry (MS/MS). This system is designed to provide sensitive and selective detection of compounds in complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 7890b gas chromatograph, by Agilent Technologies (1 mentions)
7000d mass spectrometer, by Agilent Technologies (1 mentions)
Db ffap column, by Agilent Technologies (1 mentions)
Saturated sodium chloride, by Merck Group (1 mentions)
N hexane, by Merck Group (1 mentions)
Methyl tert butyl ether (mtbe), by Merck Group (1 mentions)
4 protocols using agilent 7890b 7000d gc ms ms platform
1
Quantitative Analysis of Fecal SCFAs
2
Free Fatty Acid Composition Analysis
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Muscle and liver samples were harvested from cyp17a1-/- and cyp17a1+/+ fish for free fatty acid composition analysis, as described in previous research (Tan et al., 2020 (link); Ubhayasekera et al., 2013 (link)). The samples (50 mg) were mixed with 150 µL of methanol (Darmstadt, Germany), 200 μL of methyl tert-butyl ether (Darmstadt, Germany), and 50 μL of 36% phosphoric acid (Sigma-Aldrich, USA)/water (Millipore, USA), then vortexed for 3 min at 580 ×g, followed by centrifugation at 13 500 ×g for 5 min at 4°C. The collected supernatant (200 μL) was transferred to a new centrifuge tube, evaporated to dryness, reconstituted with 300 μL of a 15% boron trifluoride (RHAWN, China) methanol solution, vortexed for 3 min at 580 ×g, and incubated in an oven at 60°C for 30 min. After cooling to room temperature, 500 μL of n-hexane (Darmstadt, Germany) and 200 μL of saturated sodium chloride (Sigma-Aldrich, USA) solution were added, after which the solution was vortexed for 3 min and centrifuged at 13 500 ×g for 5 min at 4°C, with 100 μL of n-hexane layer solution then taken for subsequent GC-MS analysis using the Agilent 7890B-7000D GC-MS/MS platform (Agilent, USA). Fatty acids were identified by retention times of standard mixtures (Personal Bio, China). Details on GC-EI-MS/MS procedures and assays are provided in the Supplementary Materials.
3
Metabolomic Profiling of Flower Buds
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Three ower buds of every repeat at ve chilling points were crushed by a crusher (MM 400 Retsch)
containing zirconia beads at 30 Hz. A total of 20 mg powder was added into 500 μL of methanol: isopropanol: water (3: 3: 2, V/V/V) solution. The supernatant (50 μL) were taken and evaporated in nitrogen after being vortexed (3 min) and sonicated (30 min), with adding internal standard. After evaporated in a nitrogen stream and freeze-drying, the residue was further derivated as follows: Firstly, the small molecule carbohydrate was mixed with methoxine hydrochloride solution (100 μL) in the 1.5 mL tubes. Secondly, bistri uoroacetamide (100 μL) was added to the solution at 37ºC for 2 h. After vortexed, the mixture was incubated at 37ºC for 30 min. N-Hexane was used as dilution fusion. Then, mixture was detected by MetWare (http://www.metware.cn/) based on the Agilent7890B-7000D GC-MS/MS platform, and the parameters were set as reported by Gómez-González et al. [66] .
containing zirconia beads at 30 Hz. A total of 20 mg powder was added into 500 μL of methanol: isopropanol: water (3: 3: 2, V/V/V) solution. The supernatant (50 μL) were taken and evaporated in nitrogen after being vortexed (3 min) and sonicated (30 min), with adding internal standard. After evaporated in a nitrogen stream and freeze-drying, the residue was further derivated as follows: Firstly, the small molecule carbohydrate was mixed with methoxine hydrochloride solution (100 μL) in the 1.5 mL tubes. Secondly, bistri uoroacetamide (100 μL) was added to the solution at 37ºC for 2 h. After vortexed, the mixture was incubated at 37ºC for 30 min. N-Hexane was used as dilution fusion. Then, mixture was detected by MetWare (http://www.metware.cn/) based on the Agilent7890B-7000D GC-MS/MS platform, and the parameters were set as reported by Gómez-González et al. [66] .
4
Targeted Metabolite Extraction and Derivatization
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Three ower buds of every repeat at ve chilled phase were crushed by a crusher (MM 400,Retsch)
containing zirconia beads at 30 Hz. A total of 20 mg powder was added into 500 µL of methanol: isopropanol: water (3: 3: 2 V/V/V) solution. After vortexed for 3 min and sonicated for 30 min, 50 µL of the supernatant were taken and evaporated in nitrogen, with adding internal standard. After evaporated in a nitrogen stream and freeze-drying, the residue was further derivated as follows: Firstly, the small molecule carbohydrate was mixed with 100 µL methoxine hydrochloride solution in the 1.5 mL Eppendorf tubes. Secondly, 100 µL of bistri uoroacetamide was added to the solution after 37 °C for 2 h. After vortexed, the mixture was incubated at 37 °C for another 30 min. N-Hexane was used as dilution fusion.
The mixture was detected by MetWare (http://www.metware.cn/) based on the Agilent7890B-7000D GC-MS/MS platform [63].
containing zirconia beads at 30 Hz. A total of 20 mg powder was added into 500 µL of methanol: isopropanol: water (3: 3: 2 V/V/V) solution. After vortexed for 3 min and sonicated for 30 min, 50 µL of the supernatant were taken and evaporated in nitrogen, with adding internal standard. After evaporated in a nitrogen stream and freeze-drying, the residue was further derivated as follows: Firstly, the small molecule carbohydrate was mixed with 100 µL methoxine hydrochloride solution in the 1.5 mL Eppendorf tubes. Secondly, 100 µL of bistri uoroacetamide was added to the solution after 37 °C for 2 h. After vortexed, the mixture was incubated at 37 °C for another 30 min. N-Hexane was used as dilution fusion.
The mixture was detected by MetWare (http://www.metware.cn/) based on the Agilent7890B-7000D GC-MS/MS platform [63].
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