The analysis of SCFA was performed by gas chromatography in order to determine the concentrations of acetate, propionate, isobutyrate, butyrate, isovalerate and valerate. Cell free-supernatants (100 μl) from fecal homogenates, prepared as indicated formerly, were mixed with 450 μl methanol, 50 μl internal standard solution (2-ethylbutyric 1.05 mg/ml), and 50 μl 20% v/v formic acid. This mixture was centrifuged and the supernatant obtained was used for quantification of SCFA by GC in a system composed of a 6890NGC injection module (Agilent Technologies Inc., Palo Alto, Ca, USA) connected to a flame injection detector (FID) and a mass spectrometry (MS) 5973N detector (Agilent), as described elsewhere40 (link).
5973 n detector
Manufactured by Agilent Technologies
Sourced in United States
The 5973 N detector is a key component of Agilent's gas chromatography-mass spectrometry (GC-MS) systems. It is a high-performance quadrupole mass spectrometer designed for reliable and sensitive detection of a wide range of analytes. The detector features advanced electronics and a robust design to ensure consistent and accurate performance.
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Lab products found in correlation
6890ngc injection module, by Agilent Technologies (3 mentions)
6890ngc module, by Agilent Technologies (1 mentions)
Cp sil 8cb, by Agilent Technologies (1 mentions)
6890 plus gas chromatograph, by Agilent Technologies (1 mentions)
Masshunter software, by Agilent Technologies (1 mentions)
Chemstation, by Agilent Technologies (1 mentions)
6 protocols using 5973 n detector
1
Quantification of Short-Chain Fatty Acids
2
Quantification of Cecal Short-Chain Fatty Acids
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The cecal concentration of the SCFAs acetate, propionate, iso-butyrate, butyrate, iso-valerate, and valerate was quantified by gas chromatography in a system comprised of a 6890NGC module (Agilent Technologies Inc., Palo Alto, CA, USA) connected to a flame ionization detector and a mass spectrometry 5973 N detector (Agilent), as previously described [23 (link)]. Briefly, samples were obtained from cecal content homogenates, prepared as a 1:5 dilution in PBS (w/v), and 100 μl of cell free-supernatants from the homogenates were mixed with 450 μl methanol, 50 μl internal standard solution (2-ethylbutyric 1.05 mg/ml), and 50 μl 20% v/v formic acid. Supernatants obtained following centrifugation of this mixture were used for SCFA quantification by GC.
3
SCFA Quantification by GC-FID-MS
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Short-chain fatty acids (SCFA) were analyzed by gas chromatography (GC). Cell-free supernatants from fecal homogenates were mixed with methanol, 20% (v/v) formic acid, and an internal standard solution (2-ethylbutyric) in a 38:46:8:8 (v/v) proportion. This mixture was used for SCFA quantification in a system composed of a 6890NGC injection module (Agilent Technologies Inc., Palo Alto, CA, USA) connected to a flame injection detector (FID) and a mass spectrometry (MS) 5973N detector (Agilent), as described elsewhere [24 (link)].
4
Determination of Fatty Acids by GC-MS
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The determination of FAs and α-HFAs was performed on an Agilent 6890 Plus Gas Chromatograph interfaced with a single quadrupole 5973 N detector (Palo Alto, CA, USA). The GC separation was carried out on an Agilent capillary column CP sil 8 CB (15 m × 0.25 mm i.d., 0.25 µm film thickness). The Mass Spectrometer worked with Electron Ionization (EI) by Single Reaction Monitoring (SIM) mode. The acquisition spectra were elaborated using Agilent ChemStation (Palo Alto, CA, USA). The Quantitative Analysis platform of the Masshunter software (Agilent Technologies) was used to process the analytical data.
5
SCFA Analysis by Gas Chromatography
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The analysis of SCFAs was performed by Gas Chromatography (GC) in the fecal culture supernatants to quantify acetic, propionic, butyric acid (major SCFAs), as well as isobutyric and isovaleric acid (branched chain fatty acids: BCFAs). 250 µL of culture supernatants collected at time 0 and 24 h of incubation were mixed with 0.3 mL methanol, 0.05 mL of the internal standard solution (2-ethylbutyric acid 1.05 mg/mL), and 0.05 mL of 20% (v/v) formic acid. The mixture was centrifuged, and the supernatant was collected for SCFA quantification in a system composed of a 6890N GC injection module (Agilent Technologies Inc., Palo Alto, Ca, USA) connected to a flame injection detector (FID) and a mass spectrometry (MS) 5973N detector (Agilent), as described previously [24 (link),25 (link)]. Samples were analyzed in triplicate and results were expressed in µg/mL. Increments (∆) in the levels of these compounds at 24 h of incubation with respect to the basal conditions (time 0) were calculated for each fermentation batch with the different 2′FL preparations tested.
6
GC-MS Quantitation of HHCs in Samples
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Since no HHCs reference standards were available in our laboratory, their obtaining was performed as described at paragraph 2.3. Once HHC reference compounds were obtained, a GC-MS method was developed and validated for their identification and quantitation in the abovementioned samples, according to FDA guidelines for drugs. 13 Our GC-MS routine method for cannabinoids determination was adapted for HHC analysis. 12 (link) HHC analysis was performed on an Agilent 6890 Plus gas chromatograph interfaced with a single quadrupole 5973 N detector (Palo Alto, CA, USA). The GC separation was carried out on an Agilent capillary column CP sil 8 CB (15 m x 0.25 mm i.d., 0.25 µm film thickness) using the following conditions: from 120 °C to 200 °C at 40 °C/min, then to 250 °C at 6 °C/min and finally to 300 °C at 60 °C/min; injector temperature: 280 °C; ion source temperature: 230 °C; carrier gas (helium) flow 1.1 mL/min; injection mode: splitless; injection volume: 0.2 μL; run time: 13 min; mass spectrometer mode: electron ionization by Single Reaction Monitoring (SIM) mode, using the target and qualifier ions shown in Table 1.
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