Finnigan surveyor plus hplc

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.

Phytochemicals in RHE were identified using a Thermo Finnigan Surveyor Plus HPLC connected to a LCQ Advantage Max ion trap spectrometer through an ElectroSpray Ionization (ESI) source (Thermo Fischer Scientific, Waltham, MA, USA). The analyses were performed using the same experimental chromatographic conditions reported in 2.5 section. The ion trap operated in data-dependent, full scan (50–1500 m/z), and MSⁿ to obtain a fragment ion width of 35% and an isolation width of 3 m/z. Chlorogenic acid and kaempferol-3-O-glucoside (10 ppm in 0.1% formic acid-methanol solution, 50:50, v/v) were used to set up MS parameters: sheath and auxiliary gas flow: 20 and 10 arbitrary units, respectively; ionization voltage, 5 kv; capillary temperature, 300 °C. The analyses were performed in positive e negative ionization modes. The software used was Thermo Fischer Scientific Excalibur 2.1.