*2.9. Determinations of Individual Phenolic and Anthocyanin Compounds*

Individual phenolic and anthocyanin compounds were determined according to Durst and Wrolstad [34] by an HPLC system equipped with a pump Nexera X2 (LC-30 AD), an autosampler system (SIL-30AC), a diode array detector (SPDM20A) (Shimadzu, Kyoto, Japan) and a Macherey–Nagel HPLC column C18 (250 × 4.6; 5 μm, Nucleodur PolarTec at 30 ◦C. An aliquot of 5 mL of the extract of phytochemicals (point 2.7) was evaporated under N2 stream at 37 ◦C and the residue was dissolved in 1 mL MeOH (HPLC grade). The extract was filtered through a Chromafil AO-45/25 polyamide filter (0.45 μm pore size), 20 μL was injected and the flow rate was set at 1 mL min−1. The elution solvents were (A) 100% acetonitrile and (B) aqueous formic acid 1%. The separation of the compounds was achieved according to the gradient: 0–15 min, 35% A; 15–30 min, 10% A; 30–80 min, 15% A; 80–100 min, 50% A; and finally washing and reconditioning of the column (equilibration time), 100–105 min 5% A. Identification of compounds was carried out by comparing retention times and their UV–Vis spectra from 200 to 700 nm. Each compound was quantified in comparison with a multipoint calibration curve obtained from the corresponding authentic standard (Extrasynthese, Genay, France) and expressed as mg g−<sup>1</sup> DW. Chlorogenic and neochlorogenic acid were monitored at 320 nm, flavan-3-ols at 280 nm and anthocyanins at 510 nm. The data analyses were carried out using LabSolutions LC/GC 5.82 (SkyCom, Tokyo, Japan).
