Development and validation of a reverse phase HPLC-DAD method for separation, detection & quantification of rutin and...

Buckwheat has tremendous nutraceutical potential owing to its rutin and quercetin content. The aim of this study was to optimise and validate an analytical method for separating and quantifying these two flavonoids from it. Factors, such as range, linearity, precision, accuracy, limit of detection and limit of quantification, were evaluated for the two compounds using high performance liquid chromatography. On the basis of resolution and symmetry, mobile phase consisting of methanol and methanol:water:acetic acid in the ratio of (100:150:5), flow rate 1.3 ml/min and column temperature 30 °C were found to be optimal analytical conditions. Calibration curves exhibited good linearity with correlation coefficient of 0.995 & 0.9907 over the range 60-180 μg/ml & 2-10 μg/ml for rutin and quercetin respectively. LOD and LOQ values for rutin and quercetin were 6.36, 0.58 and 19.28, 1.77 μg/ml respectively. Recovery values of 96-100.8% confirmed that the method was accurate for rutin and quercetin analysis. This validated method was successfully used to analyse rutin and quercetin in leaves and seeds of buckwheat plant.