HPLC Analysis of Polyphenol Content

Individual polyphenols were determined by using a reversed-phase HPLC (Waters 2695, Milford, MA, USA), which was equipped with a C18 column (Nova-Pak, 150 × 3.9 mm, 4 μm, Waters), a 600 E multi-solvent delivery system, a Waters U6K sampler and a Waters 2996 photodiode array detector (DAD) [22 (link)]. Briefly, a gradient elution of polyphenols was performed using a binary solvent system (acetonitrile and acidified distilled water (pH 2 with orthophosphoric acid)) under isothermal conditions (25 °C). A detection wavelength range of 210–550 nm was used. The identification of the single compounds was confirmed by comparing their retention times and UV-DAD spectra with those of the corresponding standards, as well as with those reported in the literature [25 (link)]. The amount of each compound (expressed as area unit (AU) × 10⁶), was calculated before and after digestion.