HPLC Analysis of Phenolic Acids and Flavonoids

20 μL of ethanolic extract were injected into the HPLC system (Jasco, Tokyo, Japan; PU-4180 pump, MD-4015 PDA detector, AS-4050 autosampler). The stationary phase was an Agilent (Santa Clara, CA, USA) Zorbax Eclipse Plus C18 reversed-phase column (100 × 3 mm I.D., 3.5 μm). The chromatographic method for the analysis of phenolic acids was adapted from Mattila and Kumpulainen [42 (link)] as detailed in Tubon et al. [34 (link)]. Gradient elution was carried out with a mixture of acidic phosphate buffer (50 mM, pH 2.5) and acetonitrile flowing at 0.7 ml/min. The signals at 254, 280, and 329 nm were used for analyte quantitation. The recovery values of phenolic acids in spiked samples ranged from 78.8 to 92.2% (RSD < 9.8%, n = 6). The chromatographic method for the analysis of flavonoids was adapted from Wojdyło et al. [43 (link)], as previously reported [34 (link)]. Gradient elution was carried out with a mixture of 4.5% formic acid and acetonitrile. Runs were monitored at the following wavelengths: flavan-3-ols and flavanones at 280 nm and flavonol glycosides at 360 nm. PDA spectra were measured over the wavelength range of 200−600 nm in steps of 2 nm. Retention times and spectra were compared with those of pure standards. Calibration curves were constructed for all standards at concentrations ranging from 1.0 to 100.0 μg/ml (r² ≥ 0.9998). Results are expressed as mg/g extract.