SCFAs were analyzed by GC-MS, essentially as described before [28 (link)]. Briefly, fecal samples were mixed 1:1 (w:w) with PBS, vortexed for 2 minutes and centrifuged at 14,000×g for 10 min. One hundred and fifty μl of the supernatant were mixed with 550 μl internal standard solution, containing methanol, internal standard (2 mg/ml 2-ethyl butyric acid), and formic acid (20%). The analysis was carried out on a GC-MS (8890 GC System; Agilent Technolgies, Amstelveen, the Netherlands) equipped with a PAL3 RSI 85 autosampler (Agilent) by injecting 1 μl sample on a DB-FATWAX Ultra Inert column (30 m, 0.25 mm, 0.25 μm, Agilent). The temperature settings of the injector port, oven, flame-ionization detector and mass spectrometer detector were 250, 200, 275 and 225°C, respectively. The flow rate over the column was 1.2 ml|/min. The MS Quantitative Analysis (Quant-My-Way) software from Agilent was used to determine concentrations of SCFA. LODs were: 0.95 mmol/L for acetate and 0.91 mmol/L for both propionate and butyrate. The lowest concentrations found in the samples were at least 2-fold higher than the LOD.
Db fatwax ultra inert column
Manufactured by Agilent Technologies
Sourced in United States, Germany
The DB-FATWAX Ultra Inert column is a gas chromatography (GC) column designed for the separation and analysis of fatty acid methyl esters (FAMEs) and other lipid-related compounds. The column features an inert stationary phase that is optimized for the sensitive and reproducible separation of these analytes.
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Lab products found in correlation
Pal3 rsi 85 autosampler, by Agilent Technologies (2 mentions)
8890 gc system, by Agilent Technologies (1 mentions)
5975c inert xl msd, by Agilent Technologies (1 mentions)
7693 series autosampler, by Agilent Technologies (1 mentions)
Orion meter model 290, by Thermo Fisher Scientific (1 mentions)
Orion 9512 ammonia sensing electrode, by Thermo Fisher Scientific (1 mentions)
Gc 2010 plus, by Shimadzu (1 mentions)
Pal3 rsi 85, by Agilent Technologies (1 mentions)
5 protocols using db fatwax ultra inert column
1
GC-MS Analysis of Short-Chain Fatty Acids
2
Quantifying Short-Chain Fatty Acids via GC-MS
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SCFAs (acetate, propionate, and butyrate) in the lumen and dialysate samples were analyzed with gas chromatography–mass spectrometry (GC-MS). Samples were prepared for GC-MS as described before [28 (link)]. In short, the samples were centrifuged and formic acid, 2-ethyl butyric acid (internal standard) and methanol were added to the supernatant. The analysis was carried out on a GC-MS (8890 GC System; Agilent Technologies, Amstelveen, The Netherlands) equipped with a PAL3 RSI 85 autosampler (Agilent) by injecting 1 μL sample on a DB-FATWAX Ultra Inert column (30 m, 0.25 mm, 0.25 μm, Agilent). The temperature settings of the injector port, oven, flame-ionization detector, and mass spectrometer detector were 250, 200, 275 and 225 °C, respectively. The flow rate over the column was 1.2 mL/min. With the use of calibration curves of known quantities of standards, quantities of SCFAs in the samples were determined.
3
Rumen Fluid Analysis: NH3 and VFA
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Prior to analysis, samples for NH3 and VFA were thawed. For NH3 concentration, a portable meter (Orion meter model 290; Thermo Scientific Inc., Waltham, MA, USA) coupled to an NH3 ion selective electrode (Orion 9512 ammonia sensing electrode; Thermo Scientific Inc., Waltham, MA, USA) was used. For VFA profile, 1 mL of filtered rumen fluid was mixed with 0.2 mL of an internal standard (2-mg/mL 2-ethyl butyric acid in 25% meta-phosphoric acid (w/v)). Subsequently, samples were centrifuged at 18,000×g for 20 min, and the resulting supernatant was analyzed using a 7890A gas chromatography system equipped with a DB-FATWAX Ultra Inert column (30 m × 0.25 mm, 0.25 µm), a 5975C inert XL MSD with triple-axis mass detector, and a 7693 series autosampler (Agilent Technologies, Santa Clara, CA, USA). Ionization was performed in an electron impact mode at 70 eV and a selected ion monitoring mode was used to acquire ion abundance. Volatile fatty acids were quantified by an internal standard calibration with authentic VFA standards.
4
GC Analysis of Fatty Acid Compounds
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GC was carried out on a Shimadzu GC 2010-Plus (Shimadzu, Duisburg, Germany) with a flame ionization detector and a DB-FATWAX Ultra Inert column (30 m × 0.25 mm, film thickness 0.25 μm, Agilent Technologies, Waldbronn, Germany) with pre-column (deactivated fused silica, 5 m × 0.25 mm). The injection volume was 1 μL, the inlet temperature 220 • C and a split ratio of 10:1 up to 100:1 was applied, depending on the concentration of the sample. The carrier gas was helium at a constant linear velocity of 40.0 cm/s. The detector temperature was 240 • C. The oven temperature program started at 40 • C and increased at a rate of 5 • C/min to 60 • C. This temperature was held for 2 min and then further increased at a rate of 10 • C/min to 220 • C. The final temperature was held for another 10 min. The total runtime was 32 min.
5
GC-MS Analysis of Gut SCFA and BCFA
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SCFA and branched-chain fatty acid (BCFA) production was analyzed via GC-MS, as described by Nuenen et al. [22 (link)]. In brief, we centrifuged TIM-2 lumen samples for 10 min at 14,000 rpm. Then, 150 µL (lumen) of the clear supernatant was transferred to a glass-GC-vial containing 550 µL of the internal standard (a mixture of demineralized water, methanol, 2 mg/mL 2-ethyl butyric acid, and formic acid (20%)). The internal standard guarantees an accurate quantification and minimizes variability of the results. For TIM-2 dialysate samples, 300 µL dialysate was transferred to a glass-GC-vial containing 400 µL of a mixture of the internal standard. Of this mixture 0.5μL was loaded onto a DB-FATWAX Ultra Inert column (30 m, 0.25 mm, 0.25 μm; Agilent, Amstelveen, the Netherlands) in an Agilent 8890 GC System Custom, coupled to an Inert Plus MSD Turbo EI/CI, using an automatic sampler (PAL3 RSI 85, Agilent). We set the temperature settings of the injector port, oven, flame-ionization detector, and mass spectrometer detector to 250 °C, 200 °C, 275 °C, and 225 °C, respectively. Additionally, we maintained a carrier gas flow rate of 1.2 mL/min over the column. Concentrations were determined based on a calibration curve. Total SCFAs refers to the cumulative amount of acetate, propionate, butyrate, valerate, and caproate.
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