Quantitative Analysis of Phenolic Compounds

Dried leaves (200 mg) were extracted with 10 mL of methanol containing 0.1 N HCl for 12 h at room temperature. After filtration, the extract was analyzed using an Agilent LC-MS system (Agilent Technologies, Palo Alto, CA, USA), consisting of an analytical 1200 HPLC system with a Shiseido MG II C₁₈ analytical column (250 × 4.6 mm i.d., 5 μm particle size) and a 6120-quadrupole mass spectrometer with electrospray ionization (ESI). The mobile phase was made up of acetonitrile (A) and water (B), with 0.3% formic acid, respectively. The gradient was run as follows: 15% solvent A for 10 min, 15–40% solvent A for 18 min, 40–90% solvent A for 7 min, held at 90% solvent A for 5 min, and then returned to 15% solvent A for column equilibration. The separated components were detected at 330 nm for CQAs and flavonoids, and 520 nm for anthocyanins. Mass data were collected in full scan mode in positive ion mode from m/z 50 to 1000 under a 5 mL min^-1 drying gas flow, 150°C vaporizing temperature, 60 psi nebulizing gas (N₂) pressure, and 30°C drying gas temperature.
To determine and quantify phenolic compounds, we used the same HPLC conditions described above. An Agilent 1200 HPLC system equipped with a binary pump (G1312A), auto sampler (G1347B), PDA detector (G1315D), column oven (G1316A), and ChemStation, using an external standard method with a calibration curve, was used for HPLC analysis. In total, ten phenolic compounds, including four hydroxycinnamic acids, two flavonols, two flavones, and two anthocyanins, were used for calibration curve conduction in the range of 31.25 to 250 ppm. 2’’-Acetylhyperoside was quantified as a relatively equivalent value to hyperoside. All experiments were performed in triplicate.