Optimization and Validation of an HPLC–Method for Quality Control of Pueraria Lobata Root

Abstract

Pueraria lobata Willd. (Fabaceae) is a widely used medicinal plant, known as “Kudzu” and “Ge” in Japanese and Chinese traditional medicine, respectively. P. lobata is a rich source of isoflavones with phytoestrogenic properties, and its commercial use is widespread. In this study the optimization and validation of an HPLC-method for quality control of Pueraria root material is presented. By means of this analytical method the major individual constituents, i.e. the isoflavone 8- C-glycosides hydroxypuerarin, puerarin, methoxypuerarin and xylosylpuerarin, the 7- O-glycosides daidzin and genistin, and the aglycones daidzein and genistein could be quantified. The extraction procedure and the extraction solvent composition were optimized in order to ensure the exhaustive extraction of the plant material. The HPLC - conditions were evaluated and optimized for the exact quantification of all individual compounds. The HPLC analysis was carried out on a Agilent XDB RP C18 (150 × 4.6) column eluted with a binary system consisting of water (+ 0.01% formic acid) and methanol (+ 0.01% formic acid) using a linear gradient; detection was at 262 nm. Daidzin, daidzein, genistin and genistein were used as external standards. Due to the great difference in content between the C-glycosides on the one hand, and daidzin, genistin and the aglycones on the other, two separate HPLC runs were necessary for a complete analysis. The final method was fully validated according to the ICH guidelines in terms of linearity, precision and accuracy. Linear relationships for the responses of all four standards were proven. The method was shown to be repeatable for the individual compounds, i.e. RSD% betweendays values were within 2 to 7.5%. The accuracy of the method was demonstrated to be equal to 100% by recovery experiments. These validation results demonstrated the suitability of the method for the precise and accurate determination of Pueraria root isoflavones.