The digesta sample (2 g) was mixed thoroughly with 4 ml of water (18.2 MΩ) by vigorous vortexing and initially centrifuged at 1500xg for 15 min at 4°C. The obtained supernatant was collected and stored at -80°C until further analysis. After thawing it was cleared by centrifugation at 12,000xg for 10 min at room temperature and analysed for amine concentration using high-performance liquid chromatography and heptylamine as an internal standard, according to the previously described method [24 ]. Amines were derivatised with dansyl chloride and extracted using Waters SEP-PAK serif™ C18 cartridges for solid phase extraction (6 ml, 500 mg; Waters, Watford, Hertfordshire, UK). The separation was carried out using a Finnigan Surveyor Plus HPLC (Thermo Scientific, San Jose, USA) with a photodiode array detector set at 254 nm and a Waters Symmetry Shield RP18 column (150 × 3.9 mm i.d., 5 μm) preceded by a guard column (Waters Symmetry Shield RP18, 20 × 3.9 mm, 5 μm). Amine concentrations were calculated using standard curves prepared for individual compounds.
Waters symmetry shield rp18
Manufactured by Waters Corporation
Sourced in United States
The Waters Symmetry Shield RP18 is a reversed-phase liquid chromatography column designed for the analysis of a wide range of compounds. It features a silica-based stationary phase with a C18 alkyl chain bonded to the surface, providing a highly retentive surface for the separation of both polar and non-polar analytes.
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Lab products found in correlation
Sep pak serif c18 cartridges, by Waters Corporation (2 mentions)
Surveyor plus hplc, by Thermo Fisher Scientific (1 mentions)
Ecd 2465, by Waters Corporation (1 mentions)
Finnigan surveyor plus hplc, by Thermo Fisher Scientific (1 mentions)
1100 series hplc system, by Agilent Technologies (1 mentions)
Lc ms grade, by Merck Group (1 mentions)
4 protocols using waters symmetry shield rp18
1
Amine Profiling of Digesta Samples
2
Quantification of Caecal Amine Concentrations via HPLC
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The concentration of amines in caecal digesta was determined using the HPLC method described previously [17 (link)]. The digesta samples were homogenised in ultra-pure water by intensive vortexing and supernatants were obtained by centrifugation at 10,000 rpm for 15 min. The supernatant was subsequently diluted five-fold with an acetone and water mixture (2:1) and alkalised with borax buffer (0.1 M, pH 10.5). Heptylamine was then added as an internal standard to a final concentration of 5 μg/mL. Subsequently, amines were derivatised by incubation with 1% dansyl chloride in acetone for 25 min at 65 °C in a water bath in the dark and then extracted using Waters SEP-PAK serif™ C18 cartridges for solid phase extraction (6 mL, 500 mg; Waters, Watford, Hertfordshire, UK). Separation was carried out using a Finnigan Surveyor Plus HPLC (Thermo Scientific, San Jose, CA, USA) with a photodiode array detector and a Waters Symmetry Shield RP18 column (150 × 3.9 mm i.d., 5 μm) preceded by a guard column (Waters Symmetry Shield RP18, 20 × 3.9 mm, 5 μm). The mobile phase was composed of 5% acetonitrile in water and 100% acetonitrile, flowing under a gradient elution. Dansyl derivatives of amines were detected by measuring absorbance at 254 nm, and their concentrations were determined from standard curves.
3
Quantification of Monoamine Neurotransmitters in Rat Hippocampus
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In order to detect monamine neurotransmitters and their metabolites, the hippocampus from each rat was weighed and quickly homogenized in 120 μL of pretreatment solution A (0.4 mol/L of perchloric acid) on ice prior to being centrifuged at 12,000 rpm/min for 20 min at 4 °C after standing at room temperature for 30 min. Next, 90 μL of supernatant was collected, and 45 μL of pretreatment solution B (20 mmol/L of potassium citrate, 0.3 mol/L of dipotassium hydrogen phosphate, and 2 mmol/L of EDTA·2Na) was added. The sample was next vortexed and then centrifuged at 12,000 rpm/min for 20 min at 4 °C after also standing at room temperature for 30 min. The supernatants were collected and analyzed by high-performance liquid chromatography (HPLC) coupled with an electrochemical detector (Waters ECD2465, Milford, MA, USA) [64 ]. Here, the chromatographic column Waters symmetry shield RP 18 (150 × 3.9 mm, 5 μm, Waters Atlantis) was maintained at 30 °C, and the flow rate was 0.8 mL/min. The mobile phase (methanol–water, 8:92, v/v) was mixed with sodium acetate, 1-octanesulfonate, citric acid, and EDTA•2Na, and the detector potential was +0.6 V, with an injection volume of 40 μL.
4
LC-MS/MS Analysis of Quorum Sensing Molecules
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For LC-MS/MS analyses an 1100 Series HPLC system (Agilent Technologies, Waldbronn, Germany) was used. The Agilent HPLC components (G1322A degasser, G1312A binary pump, G1329A autosampler, G1315A diode array detector) were connected with an ABSCIEX 3200 Q TRAP LC/MS/MS mass spectrometer (AB Sciex, Germany GmbH, Darmstadt, Germany). For measurements, the following LC gradient was used: Starting with two minutes H2O acidified with 0.025% formic acid (solvent A):acetonitrile (solvent B, LC-MS grade, Sigma Aldrich) (95:5) followed by a gradient to A:B (5:95) in 30 min, flow rate 0.6 mL/min; injection volume 20 μL; column: Waters Symmetry Shield RP18 (5 μm, 250×4.6 mm, Waters GmbH, Eschborn, Germany). MS measurements were performed in positive ionization mode. For precursor ion scan measurements, parameters were optimized for C6-HSL using the “automatic compound optimization” option of the Analyst LC/MS software (AB Sciex, Germany GmbH). As precursor m/z 102.1 was used. The following parameters were optimized: curtain gas 10 psi, temperature 450°C, gas 1 and 2 20 psi, ion spray voltage 5,500 V, declustering potential 46 V, collision energy 13 V, entrance potential 12 V, scan area 50–400 Da. To obtain MS/MS product ion spectra of the AHLs, MS parameters were optimized separately, for each standard.
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