Optimized Quantification of Flavonoid Standards

The quantitative analysis of flavonoid standards—fisetin and quercetin (Sigma-Aldrich, USA) was optimized using a lab solution software and an HPLC system of LC2030 (Shimadzu, Japan) on the C18 column (Luna Omega 3 um Polar C18 100, 250 × 4.6 mm, Phenomenex, USA) with slight modification [25 , 26 ]. The solvents used for mobile phase A and phase B were HPLC grade water (Thermo Fisher Scientific, India) with pH-2.15 and acetonitrile respectively. The solvents were degassed for 30 min using a sonicator (Faithful Instrument, China). The gradient elution condition was set at 5 min, 35% B; at 10 min, 40% B; at 15 min, 50% B; and stopped at 20 min. The flow rate was 0.5 ml/min with an autosampler injection volume of 20 µl. The column temperature was 40 °C, and the UV–visible detector was set at 340 nm.