2.4. Quantitative Analysis of Compound 1–7 Using Reversed-Phase HPLC

Quantitative analysis of the flavonoids was performed using reversed-phase high-performance liquid chromatography (HPLC) (Alliance e2690; Waters Corp., Milford, MA, USA) with a C18 column (Poroshell 120 SB-C₁₈; 120 Å, 2.7 μm, 4.6 × 150 mm; Agilent Technologies, Santa Clara, CA, USA). The column oven temperature was 40 °C, the sample injection volume was 5 μL, and the detection wavelength was set to 360 nm. Solvent A (H₂O: formic acid = 99.9: 0.1, v/v) and solvent B (acetonitrile) were used in the mobile phase, and the flow rate was set to 0.8 mL/min. The solvent elution was graded as follows: 95% A/5% B at 0 min, 95% A/5% B at 1 min, 80% A/20% B at 3 min, 80% A/20% B at 8 min, 77% A/23% B at 10 min, 77% A/23% B at 13 min, 72% A/28% B at 15 min, 72% A/28% B at 20 min, 20% A/80% B at 22 min, 95% A/5% B at 24 min, and 95% A/5% B at 26 min. For the quantitative analysis of compound 1–7 isolated from the CLF extract, 1 mg of each flavanone was accurately weighed and dissolved in MeOH to obtain stock solutions with a concentration of 1.0 mg/mL. Calibration curves were developed for each standard with six different concentrations (100, 50, 25, 12.5, 6.25, and 3.125 µg/mL). A volume of 1 milligram obtained from the CLF extract was also accurately weighed and dissolved in 80% (v/v) aqueous MeOH to create stock solution with a concentration of 5.0 mg/mL. The quantitative analysis was repeated three times.