2695 c18 column

The phenolic compounds were analyzed by HPLC (Waters 2695; C18 column (250 × 4.6 mm × 5 μm size particle) and the absorbance was measured at 280 nm by a Waters 2478 Dual λ Absorbance Detector. The mobile phase of acidified water containing 1% formic acid (A) and acetonitrile (B) was used. The setting procedure of the gradient was as follows: 0 min, 20% B; 17 min, 21.5% B; 17.5 min, 68% B; 40 min, 68.3% B; 41 min, 100% B; 51 min, 20% B, and held for 2 min. The volume of injection was 20 μL and the flow rate was 1.0 mL/min. All HPLC-grade standards (ferulic acid, epicatechin, catechins, phloridzin, phloretin, caffeic acid, rutin, and chlorogenic acid) were purchased from Sigma Chemicals. Identification of each peak was performed on the basis of comparing their retention times with their known standards. The concentration of phenolic contents was expressed as milligrams of each compound per liter of juice.

The HPLC–DAD–MS technique was used to analyze the I. obliquus extract. A Waters 2695 C₁₈ column (250 × 4.6 mm, 5 μm) was used with a MeOH (A) and water (B) mobile phase at a 0.5 mL/min flow rate. The elution procedure was as follows: 0–30 min, 24–56% solvent A; 30–35 min, 56–76% solvent A. Online ultraviolet spectra were detected at 300 nm. An LCQ Fleet ion-trap mass spectrometer was connected to the photo-diode array instrument by an ESI interface and used to perform the MS and MSⁿ analyses. The mass spectrometer was operated in the positive ion mode, and the flow rate was 0.15 mL/min. The capillary voltage was set at −20 V in the positive ion mode. A full scan was performed using selected reaction monitoring mode-based identification at 4.5 kV spray voltage, 350 °C capillary temperature, and 10 psi pressure. A 100–1200 m/z scan range at a resolution of 17,500 was employed.