Series hp 1100

To determine the OLE composition, high-performance liquid chromatography
(HPLC) analysis was performed using a Hewlett-Packard Series HP 1100
equipped with a diode array detector and a C18 LiChrospher 100 analytical
column (250 × 4 mm). The flow rate was adjusted to 1 mL min^–1, and the change of absorbance was displayed at 280
nm wavelength. The mobile phases were used as 2.5/97.5 (v v^–1) of acetic acid/water (A) and acetonitrile (B). A linear gradient
was run for 60 min in total, from 95% A–5% B to 75% A–25%
B for 20 min, and then it was changed into 50% A–50% B for
20 min and 20% A–80% B for 10 min, respectively. At last, re-equilibration
was performed for 10 min to have the initial composition.
The
oleuropein level in OLE was also determined by HPLC analysis using
an oleuropein calibration curve (Figure S3). As an internal standard, coumarin was utilized for the measurement
of antioxidant oleuropein.

The HPLC equipment that was used was a Hewlett-Packard Series HP 1100 that was equipped with a diode array detector. The stationary phase was a C₁₈LiChrospher 100 analytical column (250 × 4 mm i.d.) with a particle size of 5 nm (Merck, Darmstadt, Germany) thermostated at 25 °C.
For the elucidation and quantification of bioactive compounds in the Citrolive™, the extract was dissolved in dimethylsulfoxide (DMSO) at a ratio of 5 mg/mL and this solution was filtered through a 0.45-nm nylon membrane. The flow rate was 1 mL/min and the absorbance changes were monitored simultaneously at 280 and 340 nm. The mobile phases for chromatographic analysis were as follows: (A) acetic acid/water (2.5:97.5) and (B) acetonitrile. A linear gradient was run from 95% (A) and 5% (B) to 75% (A) and 25% (B) during the first 20 min; which changed to 50% each of (A) and (B) after another 20 min (40 min in total); which, after 10 more minutes, changed to 20% (A) and 80% (B) (50 min in total), and finally equilibrated over the last 10 min (60 min in total) to the initial composition.