SCFA concentrations of fecal contents were determined by mass spectrometry. Briefly, fecal samples were acidified with weight equivalent amount of 3.7% hydrochloric acid. The tubes were sonicated in methanol for 20 minutes before use. Internal standards (14.72 mmol/L butyric acid-d7) were added to the acidified samples, followed by diethyl ether to obtain diethyl ether-fecal extract. The ether extracts were then mixed with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), followed by derivatization by incubating the organic extract-MTBSTFA mixture at room temperature for 1 hour. The derivatized samples were run through 6890N Network GC system (Agilent Technologies, Mississauga, Canada) equipped with DB-17HT (30 m × 0.25 mm ID, 0.15 mm film) and 5973N Mass Selective Detector (Agilent Technologies). Acetic acid, propionic acid, and butyric acid were quantified and reported as nmol/mg of fecal sample.
Db 17ht
Manufactured by Agilent Technologies
Sourced in United States, Canada
The DB-17HT is a high-temperature capillary gas chromatography (GC) column designed for a wide range of applications. It features a 100% dimethylpolysiloxane stationary phase, which provides excellent thermal stability and peak shape for a variety of analytes.
Automatically generated - may contain errors
Lab products found in correlation
6890n network gc system, by Agilent Technologies (2 mentions)
5973n mass selective detector, by Agilent Technologies (2 mentions)
Db 5ms, by Agilent Technologies (2 mentions)
Pegasus 4d, by Leco (1 mentions)
5977b mass selective detector, by Agilent Technologies (1 mentions)
Clarus 400, by PerkinElmer (1 mentions)
6 protocols using db 17ht
1
Quantification of Fecal Short-Chain Fatty Acids
2
SCFA Quantification in Mouse Feces
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The concentrations of SCFAs in feces of mice were determined by gas chromatography–mass spectrometry, as described previously [26 (link)]. Briefly, e-tubes in which fecal samples were acidified with a weight equivalent amount of 3.7% hydrochloric acid were sonicated in methanol for 20 min before use. To the acidified samples, internal standards (14.72 mmol/L butyric acid-d7) were added, followed by the addition of diethyl ether to obtain a diethyl ether–fecal extract. The acidified samples were extracted three times with propyl formate containing butyric acid-d7 as the internal standard, and a 60 µL extract aliquot was derivatized with 25 µL MTBSTFA at 40 °C for 1 h and then analyzed by GCMS Then the derivatized samples were run through the 6890N Network GC system (Agilent Technologies, Mississauga, ON, Canada) equipped with DB-17HT (30 m × 0.25 mm ID, 0.15 mm film) and 5973N Mass Selective Detector (Agilent Technologies). Acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, pentanoic acid, and lactic acid were quantified and reported as nmol/mg of fecal sample. The calibration curves were obtained for all seven targets by injecting all the standards as a mixture.
3
Two-Dimensional GC-MS Analysis
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GC × GC separation was performed using a DB-5 ms (30 m × 0.25 mm internal diameter [ID] × 0.25-µm film thickness, Agilent Technologies) and DB-17ht (2.3 m × 0.10 mm ID × 0.10-µm film thickness, Agilent Technologies) chromatographic column combination for the first and second dimensions, respectively. Helium was used as carrier gas and kept at a constant flow of 1.0 mL/min. A linear temperature program was used, starting at 30 °C (held for 2 min) and increasing at 5 °C/min to 325 °C (15 min) for the first dimension, and starting at 35 °C (held for 2 min) and increasing at 5.2 °C/min to 340 °C (15.3 min) for the second dimension. Modulation time was set to 6 s. Ion source temperature was set at 230 °C, and the transfer line temperature was set at 300 °C. High-resolution accurate mass spectra were acquired with an electron energy of − 70 eV, with a data acquisition rate of 200 spectra per second using a scan range of 35–545 Da. The samples were injected using a cool on-column mode with an injection volume of 0.1 µL. The cool on-column injector temperature was ramped to track the oven temperature with a positive offset of 3 °C.
4
Comprehensive GC×GC-TOF-MS Analysis
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The comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC/TOF-MS) analyses were performed on an Agilent 7890 gas chromatograph equipped with a TOF-MS (Pegasus 4D, LECO Corporation, Joseph, MI, USA) detector and a LECO ChromaTOF program. The chromatographic separation was achieved using two different polar capillary columns, a conventional nonpolar column DB-5MS (29.950 m × 0.25 mm × 0.25 mm) from J&W Scientific (Folsom, CA, USA) and a medium column DB-17HT (1.640 m × 0.1 mm × 0.1 mm) from the same company. The connection between the two GC columns is a typical combination in GC × GC, which could produce ordered and orthometric chromatograms. According to the previous studies, the initial oven temperature was set to 60 °C for 1 min, then the temperature increased by 5 °C/min up to 165 °C and then increased again by 25 °C/min until it reached the final temperature of 280 °C and then held for 14 min. The mass spectrometer was operated at an acquisition rate of 100 spectra per second, ranging from 20 to 400 u. The electron impact ionization energy was 70 eV and the acquisition voltage was 1700 V. The temperature for the ion source was set to 220 °C.
5
GC-MS Analysis of Organic Compounds
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The GC/MS analysis was performed on an Agilent 7820A GC System Plus gas chromatograph coupled with 5977B Mass Selective detector and flame-ionization detector (Agilent Technologies, Palo Alto, CA, USA). A fused silica capillary column, a mid-polar DB-17HT (J&W Scientific, Folsom, CA, USA) with 60 m column length, 0.25 mm i.d., 0.25 μm film thickness, was used. The oven temperature was programmed from 60 °C (2.5 min held) to 100 °C at a rate of 5 °C/min, from 100 to 225 °C at a rate of 2.5 °C/min and from 225 to 275 °C at a rate of 5 °C, 10 min held at the final temperature was applied. Helium (99.999%) was used as a carrier gas at a constant flow rate of 0.8 mL/min. The split ratio was 1:125, the inlet temperature was set to 260 °C and the transfer line temperature was 280 °C. Mass selective detector operated in electron impact ionization (EI) mode at 70 eV electron energy, the ion source temperature was set to 230 °C and the quadrupole temperature was 150 °C. The mass scan range was 45–1050 m/z.
6
Gas Chromatographic Analysis of Essential Oils
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Gas chromatographic analyses were performed on a Perkin-Elmer Clarus 400 gas chromatograph (Shelton, CT, USA) equipped with two flame ionization detectors (FID), a data-handling system, and a vaporizing injector port into which two fused-silica capillary columns with different polarities were installed: a DB-1 (polydimethylsiloxane, 30 m × 0.25 mm i.d., 0.25 µm film thickness) and a DB-17HT (50% phenyl-methyl-polysiloxane, 30 m × 0.25 mm i.d., 0.15 µm film thickness), both from J & W Scientific Inc. (Rancho Cordova, CA, USA). The analytical conditions were an oven temperature from 45 to 175 • C at a rate of 3 • C/min followed by a temperature increase at 15 • C/min until 300 • C and hold during 10 min; injector and detector temperatures, 280 • C and 300 • C, respectively; carrier gas (hydrogen) at a linear velocity of 30 cm/s; and a split ratio of 1:50. The volume injected was 0.1 µL of a n-pentane-EO solution (1:1). The percentage composition of the EOs was computed by the normalization method from the GC peak areas and calculated as mean values of two injections from each EO, without using correction factors, in accordance with ISO 7609 [42] .
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