Validation and method development for Identification of phenolic acids in ethanolic extract of Vitex trifolia.L using HPLC

Vitex trifolia.L is a rich source of phenolic compounds. In the present study, ethanolic extract of Vitex trifolia.L was subjected to high performance liquid chromatography for the identification and validation of known phenolic acids (p-methoxybenzoic acid, p-hydroxybenzoic acid, and p- coumaric acid). The method developed was achieved using acetic acid:acetonitrile (1% v/v) as the mobile phase in an isocratic elution mode. Retention times of standard markers of p-methoxybenzoic acid, p-hydroxybenzoic acid, and p- coumaric acid were 2.144, 2.789 and 10.233 min respectively. By optimizing Inertsil ODS (150 x 4.6mm, 5µm) as ideal column at flow rate of 1.0 ml/min flow and detection wavelength at 254 nm. The developed method identified p-methoxybenzoic acid, p-hydroxybenzoic acid, and p- coumaric acid present in the plant extract by assuming the retention time matching with the plant extract chromatograms and validated as per the ICH guidelines for limit of detection, limit of quantification, linearity, accuracy and precision. All compounds have shown good linearity in wide concentration range (0.999). Intra-day and inter-day precision studies has given less than 2% RSD values, which were in accepted range. The analytical recoveries of p-methoxybenzoic acid, p-hydroxybenzoic acid, and p- coumaric acid by the developed HPLC method were 93.07 %, 95.24% and 99.11 % respectively.