A validated high performance liquid chromatography method for determination of three bioactive compounds p-hydroxy benzoic acid, negundoside and agnuside in Vitex species

Abstract

A validated rapid and simple isocratic HPLC-PDA method was developed for identification and quantification of p- hydroxy benzoic acid and two iridoids negundoside and agnuside in the extracts of two Vitex species, Vitex negundo and Vitex trifolia. The separation of the three compounds was achieved on a RP-18 (250 mm X 4 mm, 5µm) column at 25o C using acetonitrile (15%) and 0.05% trifluoroacetic acid (TFA) in water (85%). Limit of detection (LOD) were 1, 2.5 and 2.5 μg/ml for p- hydroxy benzoic acid, negundoside and agnuside respectively. Similarly, limit of quantification (LOQ) were 2.5, 5 and 5 μg/ml for p- hydroxy benzoic acid, negundoside and agnuside respectively. Good linearity (r2 > 0.999) was observed for all the three compounds in wide concentration range. Using the developed HPLC method, the three compounds were identified and quantified in leaves and bark extracts of Vitex negundo and Vitex trifolia. The novelty of the developed HPLC method is that it does not require complex sample processing such as use of solid phase extraction as well as use of buffer in mobile phase. This is the first report of a validated HPLC method for the simultaneous determination of p- hydroxy benzoic acid, negundoside and agnuside in Vitex species.