Development of an HPLC Method for Simultaneous Determination of Chlorogenic Acid, Cryptochlorogenic Acid, Rutin, Isoquercitrin in Leaves of Morus alba

Abstract

An HPLC-DAD method was developed for the simultaneous quantification of four main substances in leaves of Morus alba: chlorogenic acid, cryptochlorogenic acid, rutin, and isoquercitrin. The method was executed on a Phenomenex C18 column (250 × 4.6 mm, 5 µm) with a mobile phase solvent system of acetonitrile and 0.1% formic acid in water, eluted according to a gradient program. The flow rate was maintained at 0.85 mL/min, with an injection volume of 10 µL. Detection wavelengths were set at 324 nm for chlorogenic acid, cryptochlorogenic acid, and 353 nm for rutin, isoquercitrin. This analytical method was validated and met the requirements according to the ICH guidelines on specificity, system suitability, linearity range, precision, and accuracy. The established method was applied to determine the content of eight leaf samples of Morus alba collected from Phu Tho, Hanoi. The results indicated that the average content of chlorogenic acid, cryptochlorogenic acid, rutin, and isoquercitrin in these samples was respectively 11.96 - 12.72, 1.81 - 2.63, 1.66 - 1.81, 6.09 - 6.29  mg/g based on dry leaf weight.