Sep pak serif c18 cartridges

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.

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.