The HPLC analysis for apigenin (A), luteolin (L), hydroxytyrosol (HT), oleuropein (OP), A7G, and L7G was carried out using a Shimadzu Prominence LC-20 system as previously reported [22 (link)]. The HPLC column was ZORBAX SB-C18 (5 µm, φ 4.6 × 150 mm). Separation was carried out at 40 °C with a gradient elution program at a flow rate of 1.0 mL/min. The mobile phases were 10% formic acid in water (A) and a 1:1 mixture of acetonitrile and methanol (B). The gradient program was 0–100% (B) for 40 min, followed by a re-equilibration duration of 10 min. The monitoring wavelength was configured at 280 nm for A, L, HT, and OP, while for A7G and L7G, it was set at 331 nm. The injection volume of the WOL (50 mg/mL MeOH, 0.22 µm filtration) in the HPLC system was 10 µL.
For apigenin-7-O-rutinoside (A7R) and oleuroside (OS), the analysis condition was modified somewhat. The HPLC column was a TSKgel ODS-100 V (3 µm, φ2 × 150 mm). Separation was carried out at 40 °C with a gradient elution program at a flow rate of 0.2 mL/min. The mobile phases were 0.5% acetic acid (A) and acetonitrile (B). The following multistep linear gradient was applied: 0 min, 5% B; 5 min, 15% B; 25 min, 30% B; 35 min, 95% B. The monitoring wavelength was 311 nm for OS and 254 nm for A7R.
For apigenin-7-O-rutinoside (A7R) and oleuroside (OS), the analysis condition was modified somewhat. The HPLC column was a TSKgel ODS-100 V (3 µm, φ2 × 150 mm). Separation was carried out at 40 °C with a gradient elution program at a flow rate of 0.2 mL/min. The mobile phases were 0.5% acetic acid (A) and acetonitrile (B). The following multistep linear gradient was applied: 0 min, 5% B; 5 min, 15% B; 25 min, 30% B; 35 min, 95% B. The monitoring wavelength was 311 nm for OS and 254 nm for A7R.
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