Synchronised determination of chlorogenic acid and five flavonoids in mulberry leaves using HPLC with photodiode array detection

An analytical method based on high-performance liquid chromatography separation with diode array detection was developed and validated for the synchronised determination of chlorogenic acid and five flavonoids (rutin, isoquercitrin, quercitrin, quercetin, and luteolin) in mulberry leaves (Morus spp.). Chromatographic separation was carried out under gradient elution conditions on a Shim Pack VP-ODS C18 (250 × 4.6 mm, particle size 5 μm) column at a temperature of 30 °C using a mobile phase consisting of 0.5% (v/v) phosphoric acid in water and acetonitrile at a flow rate of 0.9 ml/min. The column eluent process was best at 330 nm (for chlorogenic acid, quercitrin, and luteolin), 350 nm (for rutin and isoquercitrin), and 365 nm (for quercetin). Application of optimum extraction conditions led to extraction of chlorogenic acid and five flavonoids from mulberry leaves with mean recoveries of 97.78-103.24%. The developed method was validated in terms of linearity, recovery, precision, and stability. The relative standard deviation for intra-day precision (n=6) and inter-day precision (n=6) was