Abstract

A comparative extraction efficiency study was performed for microwave assisted and other conventional extraction techniques. An accurate, precise and validated reverse phase high-performance liquid chromatography method was developed for estimation of bergapten from Pithecellobium dulce. Several parameters which can potentially affect the extraction yield, namely extraction technique, microwave power, extraction time and solid-liquid ratio were studied. The extraction conditions of microwave assisted extraction were optimized at extraction time of 10 min, microwave power of 700 W and solid-liquid ratio of 1:20 g/ml. The amount of bergapten in samples was analysed by reverse phase high-performance liquid chromatography using isocratic solvent system with chromatographic conditions of acetonitrile:water (65:35, v/v), RP column (4.6×250 mm, 5 μm), flow rate of 1.0 ml/min and UV wavelength of 266 nm. Under optimum conditions, linearity was obtained in the range of 0.5-10 µg/ml, with the correlation coefficient (R²) above 0.996. Intraday (n=3) and interday (n=3) precisions for retention time and peak area were found to be less than 0.5 and 1.5%, respectively. The limit of detection and limit of quantification were 1.0 and 3.77 μg/ml, respectively. The recovery expressed in relative standard deviation for the spiked compound at different concentrations (10, 20 and 30 µg/ml) was in the range of 0.95-1.09%. The developed microwave assisted extraction can be used as an alternative procedure to the conventional extraction techniques. The developed analytical method for estimation of bergapten has been demonstrated to be accurate, precise and reliable as per International Conference on Harmonisation guidelines.