2.5. HPLC Analysis of Antioxidant Compounds

To investigate phenolic compounds in EAA and EAF (5 g), the modified method of Ahn et al. was applied [23]. Each extract was redissolved in water and fractionated with ethyl acetate/ether (1 : 1 = v/v) to obtain the phenol-rich fraction. Each fraction was concentrated under reduced pressure, dissolved in methanol (10 mg/mL each), filtered through a 0.22 μM polyvinylidine difluoride (PVDF) membrane, and analyzed by HPLC.

Antioxidant components and phenolic compounds were analyzed by a Waters HPLC 2790/5 system equipped with PDA by Waters 2996. HPLC separation of compounds for qualitative and quantitative analysis was performed using a reverse phase system with an INNO column at 35°C. Homogentisic acid, gallic acid, protocatechuic acid, chlorogenic acid, (+)-catechin, caffeic acid, phloretic acid, p-coumaric acid, ferulic acid, veratric acid, salicylic acid, naringin, hesperidin, quercetin, cinnamic acid, naringenin, kaempferol, and hesperidin were used as phenolic compound standards. All standards were prepared in methanol and filtered through a 0.22 μM PVDF membrane. The mobile phase consisted of solvent A (0.5% acetic acid in water) and solvent B (0.5% acetic acid in acetonitrile). The gradient program with the mobile phase was modified [23]. The elution program was as follows (B %): 8–10%, 2 min; 10–30%, 27 min; 30–90%, 50 min; 90–100%, 51 min; 100%, 60 min; 100–8%, 62 min; and 8%, 70 min. The injection volume was 10 μL, the flow rate was 1.0 mL/min, and phenols were detected at 280 nm using UV light.