Derivatized samples were analysed using an Agilent 7890B/5977A GC–MS (Agilent Technologies, Cheshire). First, 1 μl of sample was injected in splitless mode with helium carrier gas at a rate of 1.0 ml min−1. Initial oven temperature was held at 100 °C for 1 min before ramping to 170 °C at a rate of 10 °C min−1, followed by a ramp to 220 °C at a rate of 3 °C min−1 and a final ramp to 300 °C at a rate of 10 °C min−1 with a 5 min hold. Compound detection was carried out in single ion monitoring (SIM) mode. Total ion counts of each metabolite were normalised to the internal standard D6-Glutaric acid.
Agilent 7890b 5977a gc ms
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 7890B/5977A GC–MS is an integrated gas chromatography-mass spectrometry system. It combines the powerful separation capabilities of gas chromatography with the sensitive identification and quantification capabilities of mass spectrometry.
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4 protocols using agilent 7890b 5977a gc ms
1
GC-MS Analysis of Derivatized Metabolites
2
GC-MS Analysis of Fatty Acid Methyl Esters
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An Agilent 7890B+5977A GC/MS (Agilent Technologies Inc., USA) was used to determine the composition of FAMEs. The capillary column was HP-88 (60 m × 0.25 mm × 0.2 µm). Initial temperature of the oven was 50 °C and maintained for 2 min, heated at a rate of 25 °C min−1 to 175 °C, and maintained for 5 min, then heated again at a rate of 7 °C min−1 to 210 °C and maintained for 2 min, and finally heated at a rate of 2 °C min−1 to 230 °C and maintained for 1 min. The injector temperature was kept at 250 °C in split (20:1) mode for an injection volume of 1 µL. The auxiliary heater, electron ionization (EI) source, and MS Quadrupole temperature were 250 °C, 230 °C, and 150 °C, respectively. Helium was used as the carrying gas at 1 mL min−1.
3
GC-MS Metabolite Profiling of IPEC-J2 Cells
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IPEC-J2 cells (50 × 104) were seeded in 10 cm dishes for GC-MS analysis as described by Morita et al. [16 (link)]. Briefly, cells were washed with PBS and treated by 0.25% trypsin. And then cells were collected and pelleted at 1000 ×g for 5 min. After being quenched using 500 μL of prechilled 50% (v/v) methanol, cells were centrifuged at 1000 ×g for 5 min and then removed and added 500 μL of prechilled 100% (v/v) methanol. Cells were measured by an Agilent 7890B-5977A GC-MS equipped with HP-5ms (30 m × 250 μm × 0.25 μm) capillary column (Agilent J&W, Santa Clara, CA, USA). All metabolites were previously validated using authentic standards (Sigma).
4
GC-MS Analysis of Metabolites in IPEC-J2 Cells
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IPEC-J2 cells (5 × 105 cells per dish) were seeded in 10 cm dishes for GC–MS analysis as described by our previous study (Xiao et al., 2016 ). Briefly, cells were washed with phosphate buffer saline (PBS) and treated by 0.25% trypsin. And then, cells were collected and pelleted at 1,000 × g for 5 min. After being quenched using 500 L of prechilled 50% (vol/vol) methanol, cells were centrifuged at 1,000 × g for 5 min and then removed and added 500 L of prechilled 100% (vol/vol) methanol. Cells were measured by an Agilent 7890B-5977A GC–MS equipped with HP-5 ms (30 m × 250 m × 0.25 m) capillary column (Agilent J&W, Santa Clara, CA, USA). All metabolites were previously validated using authentic standards (Sigma, St. Louis, MO, USA). The data are expressed relative to the control cells.
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