Abstract A high performance liquid chromatography coupled with photodiode array detection method
was developed for the identification and quantification of p-hydroxy benzoic acid and agnuside in the
extracts of Vitex negundo and Vitex trifolia. The separation was achieved using acetonitrile and
O-phosphoric acid–water (0.5%, v/v) as the mobile phase in an isocratic elution mode. Mean retention
times of standard p-hydroxy benzoic acid and agnuside were 6.14 and 11.90 min respectively. The
developed method was validated as per the ICH guidelines for limit of detection, limit of quantification,
linearity, accuracy and precision. Good linearity (r
2
Z0.999) was observed for both the compounds in
wide concentration range. Relative standard deviation values for intra-day and inter-day precision studies
were less than 2%. The analytical recoveries of p-hydroxy benzoic acid and agnuside by the developed
HPLC method were 93.07% and 106.11% respectively. Two compounds were identified and quantified in
leaves and bar extracts of V. negundo and V. trifolia using the developed HPLC method