Development of a Validated RP-HPLC Method for Quantification of Four Bioactive Phenolic Compounds from Mulberry Leaf Infusion

Abstract

The aim of this work was to develop and validate RP-HPLC method for quantification of 4 major polyphenolic compounds of mulberry leaf infusion. The mulberry leaf samples were extracted by simulation of tea infusion beverage preparation. HPLC-DAD analysis combined with column C-18 150 mm x 4.6 mm, 2.7 μm was used to determine bioactive polyphenols such as chlorogenic acid, caffeic acid, rutin, and quercetin. The optimal conditions involved the flow rate of mobile phase at 0.3 ml/min with gradient elution of 0.1% formic acid in water and methanol, column temperature at 35 °C, 2 μl injection volume, and the detection wavelength at 320 nm (chlorogenic acid and caffeic acid) and 360 nm (rutin and quercetin). The retention times of chlorogenic acid, caffeic acid, rutin, and quercetin were 25.68, 28.03, 33.97 and 39.11 minutes, respectively. Analysis of four bioactive compounds was found to be linear with a correlation coefficient > 0.99 each at the tested concentration. All other validation parameters that represented accuracy and precision met the AOAC requirements. The developed analytical method was specific, robust, and accurate for simultaneous determining the stated compounds in mulberry leaf extracted with hot water. Moreover, this method could provide the chromatographic profiles of specific cultivar from specific source that could be used to control the quality of mulberry leaf tea products. Different cultivars and different origins of mulberry leaf in this study were also found to present different content of chlorogenic acid, caffeic acid, and rutin. No quercetin was found in the studied samples.