A validated RP-HPLC-UV method for quantitative determination of puerarin in Pueraria tuberosa DC tuber extract

BACKGROUND: Pueraria tuberosa (Fabaceae) is a well-known medicinal herbs used in Indian traditional medicines. The puerarin is one of the most important bioactive constituent found in the tubers of this plant. Quantitative estimation of bioactive molecules is essential for the purpose of quality control and dose determination of herbal medicines. The study was designed to develop a validated reversed phase high-performance liquid chromatography (RP-HPLC) method for the quantification of puerarin in the tuber extract of P. tuberosa. MATERIALS AND METHODS: The RP-HPLC system with Luna C18 (2) 100 Å, 250 × 4.6 mm column was used in this study. The analysis was performed using the mobile phase: 0.1% acetic acid in acetonitrile and 0.1% acetic acid in water (90:10, v/v) under column temperature 25°C. The detection wavelength was set at 254 nm with a flow rate of 1 ml/min. The method validation was performed according to the guidelines of International Conference on Harmonization. RESULTS: The puerarin content of P. tuberosa extract was found to be 9.28 ±0.09%. The calibration curve showed good linearity relationship in the range of 200-1000μg/ml (r(2)>0.99). The LOD and LOQ were 57.12 and 181.26μg/ml, respectively and the average recovery of puerarin was 99.73% ±1.02%. The evaluation of system suitability, precision, robustness and ruggedness parameters were also found to produce satisfactory results. CONCLUSIONS: The developed method is very simple and rapid with excellent specificity, accuracy and precision which can be useful for the routine analysis and quantitative estimation of puerarin in plant extracts and formulations.