Chromatographic analysis was carried out on a Prominence HPLC system (Shimadzu, Kyoto, Japan), equipped with an on-line degassing unit (DGU-20A), a quaternary pump (LC--20A), an auto sampler (SIL-20A), a column oven (CTO-20A) and a photodiode array detector (SPD-M20A). Separation was performed on a Waters Nova-Pack C18 column (150 mm×3.9 mm, 4 μm particle size; Milford, CT, USA) at ambient temperature (25 °C) with gradient elution (solution A: 0.1% by volume formic acid in 5% methanol (Merck) and solution B: 0.1% by volume formic acid (Merck) in 100% methanol). The gradient programme was the following: 100% A at 0 min, 75% A for 15 min, 65% A for 40 min, 55% A for 60 min, 50% A for 65 min, 0% A for 90–95 min and 100% A for 110–120 min. The flow rate of the mobile phase was 1 mL/min and the injection volume was 20 μL (23 (link)).
Identification of the individual phenolic compounds was based on the comparison of the retention times and the UV spectra of unknown peaks to those of standard compounds. Quantitative analysis was based on calibration curves constructed at specific wavelengths of reference compounds using the external standard method.
Identification of the individual phenolic compounds was based on the comparison of the retention times and the UV spectra of unknown peaks to those of standard compounds. Quantitative analysis was based on calibration curves constructed at specific wavelengths of reference compounds using the external standard method.
