A simple, rapid and specific reversed-phase HPLC method has been developed for analysis of withaferin-A in a polyherbal formulation containing Withania somnifera extracts. HPLC analysis was performed on a C18 column using a 60:40 (v/v) mixture of acetonitrile and water as isocratic mobile phase at a flow rate of 1 ml/min. UV detection was at 230 nm. The method was validated for accuracy, precision, linearity, specificity and sensitivity in accordance with international conference on harmonization guidelines. Validation revealed that method was specific, accurate, precise, reliable and reproducible. Good linear correlation coefficients (r2> 0.9993) were obtained for calibration plots in the ranges tested. Limit of detection was 0.05 and limit of quantification was 0.16 μg. Intra and inter-day RSD of retention times and peak areas were less than 2%. Recovery was between 98.15 and 100.69% obtained for withaferin-A. The established HPLC method was appropriate and withaferin-A was well resolved. The method was successfully used for quantitative analysis of withaferin-A in a polyherbal formulation.
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