2.3. Flavonoids Analysis by Reversed-Phase HPLC

The 3 sets of isomer pairs were analyzed by modifying an existing method [8]. CSE and its flavonoids were analyzed using HPLC (Alliance e2695; Waters, Milford, MA, USA) with the Empower 3 software (Waters), a PDA detector (2998, Waters), and a ProntoSIL 120-5-C18-ace-EPS column (4.6 × 250 mm, 5.0 μm; Bischoff, Leonberg, Germany), with the flavonoids and extract monitored at 360 nm. The column temperature was set to 40 °C and an injector volume of 5 μL was used. Gradient elution was carried out with 0.1% (v/v) formic acid in water (solvent A) and acetonitrile (solvent B). All solvents were filtered and degassed. A flow rate of 1.0 mL min⁻¹ was used. The following binary mobile-phase linear gradients were used: 100% A at 0 min, 90% A at 4 min, 86% A at 20 min, 84% A at 30 min, 84% A at 36 min, 80% A at 44 min, 80% A at 50 min, 75% A at 54 min, 30% A at 58 min, 30% A at 62 min, 15% A at 66 min, 15% A at 70 min, 100% A at 72 min, and 100% A at 75 min. Flavonoids were identified by comparing their retention time and ultraviolet (UV) spectra with those of their respective standards. The Q3R was identified as in previous studies and was quantified using the calibration curve for rutin because the standard for Q3R is not commercially available [13,22].