Simultaneous estimation of Agnuside and Negundoside in Vitex negundo extract by HPLC

Although a few HPLC methods have been reported for the estimation of agnuside and negundoside individually, no reports are available for simultaneous estimation of these compounds in single run despite of main iridoid glycosides of Vitex negundo. The present study discusses development and validation of an HPLC-DAD method for simultaneous estimation of agnuside and negundoside in methanol extract of V. negundo. An HPLC analysis was carried out using Eclipse XBD C18 column (150 mm x 4.6 mm, 5 µm) and gradient elution using 0.1% trifluoroacetic acid and acetonitrile at flow rate of 1.0 mL/min. Detection was carried out at 256nm. The developed method was found to be linear over the range 0.12 – 75 µg/mL and 1.25 – 75 µg/mL for agnuside and negundoside, respectively. The mean recoveries were found be 104.43%, 103.83% and 97.23% for agnuside; and 95.86%, 108.45% and 112.82% for negundoside. The method was found to be sensitive, precise and robust. The developed and validated HPLC method was utilized for standardization of extracts and herbal formulations containing V. negundo.