Vol. 13, Issue 2 (2025)

The quality control plays an important role in maintaining the quality and efficacy of a medicinally active compound and the formulation which it contains. Method development and its validation gains significance because standard analytical procedures for newer medicinal molecules, especially those which are of natural origin or ayurvedic may not be found in the pharmacopoeias. HPLC and HPTLC method are the most preferred method for the analysis of ayurvedic formulation because of its greater sensitivity for the analyte of interest. These methods developed have been validated according to ICH Harmonized Tripartite Guideline Q2 (R1), Validation of Analytical Procedures: Text and Methodology. The estimation of Vitex negundo Linn based on its marker compound is very important for measuring its most for the therapeutic activity in formulation. A method which is accurate, precise and specific is very important to estimate the accurate content of marker compounds in vitex negundo.Nirgundi consist of dried leaves and roots of Vitex negundo Linn (verbenacaeae) As agnuside, negundoside, p-hydroxy benzoic acid arepolar molecule so its analysis is carried out using reverse phase HPLC method. The column used for separation of principle was Lichrospher ® c18 with a particle size 5µ. The mobile phase used was acetonitrile and gradient buffer. The p-hydroxy benzoic acid, negundoside, agnuside have absorption maxima at 254. Therefore, the wavelengthwas chosen as 254 nm for estimation

It was found that the retention time for p-hydroxy benzoic acid, negundoside, agnuside was 8.6,11.80,15.35 respectively. The method was validated in term of precision and accuracy and specificity. A good linear relationship was observed for p- hydroxy benzoic acid, negundoside, agnuside with correlation of more than 0.987,0.998, 0. 987. The peak in the chromatograms indicate that the method was free from interference. The repeatability and reproducibility indicate that proposed method is precise andreproducible. The percentage recovery of different concentration of extract was found to be in between.