Hp 9000

The HPLC system consisted of a HP 1100 L instrument with a Diode Array Detector and managed by a HP 9000 workstation (Agilent Technologies, Palo Alto, CA, USA). The column was a Synergy max RP (150 mm × 3.0 mm) with a particle size of 4 μm (Phenomenex) maintained at 27°C. The eluents were H₂O at pH 2.0 by formic acid 5% (A) and acetonitrile (B) with a flow rate of 0.4 ml/min. The elution method involved a multistep linear solvent gradient changing from an initial concentration of 95% (A) to 78% (A) in 8 min; 5 min to 74% (A); 12 min to 65% (A) then 5 min to initial conditions. The total time of the analysis was 30 min, equilibration time 5 min. Injected volume of the samples was 5 μl solution. The UV–vis spectra were recorded between 220 and 650 nm in both cases. Chromatographic profiles were registered at 254, 280, 330, 350 and 520 nm.

The quantification of the khellin uploaded in the nanovesicles was performed with a HP 1100 liquid chromatograph using a DAD detector and an HP 9000 workstation (Agilent Technologies Italia Spa, Italy). The column was 150 mm, 4.6 mm i.d. and 3.5 μm Eclipse XDB-C18 and the mobile phase was a gradient with a flow rate of 0.4 mL/min. The gradient was the following: (A) formic acid/water pH 3.2 and (B) CH₃CN using the following gradient: 0–13 min: 50–80% B; 13–15: min 80–50% B; 15–18: min 50–50% B; with a post-time of 5 min. The analyses were performed at 27 ± 2 °C, recording the UV-Vis spectra in the 200–600 nm range and acquiring the spectra at 246 nm. Khellin gave a linear correlation (y = 0.00004144x − 0.00313111; R² ≥ 0.9999) in the range from 0.005 to 1.00 µg.

POE analyses were performed with an HP 1100 HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with DAD detector and a Luna Omega Polar (150 × 3 mm, 5 µm) (all from Agilent Technologies) RP-C18 analytical column. The software was HP 9000 (Agilent Technologies). The mobile phase consisted of (A) formic acid/water pH 3.2 and (B) acetonitrile. The following gradient profile was applied: 0–2 min 95% A and 5% B; 2–8 min 75% A and 25% B; 8–10 min 70% A and 30% B; 10–12 min 70% A and 30% B; 12–16 min 60% A and 40% B; 16–18 min 40% A and 60% B; 18–22 min 5% A and 95% B; 22–23 min 5% A and 95% B; 23–25 min 95% A and 5% B. The flow rate was 0.5 mL/min, injection volume 10 μL, and the temperature 25 °C. The calibration curve was prepared using a standard solution of catechin (0.435 mg/mL) diluted 10, 20, 50, 100, 200, and 500 times. The concentration absorption relationship was above than 0.9996. The chromatographic profile of POE polyphenols was acquired at 260 nm and the total polyphenol content was expressed as catechin, using the external standard method.