Development and validation of a novel RP-HPLC method for the simultaneous quantification of ascorbic acid, gallic acid, ferulic acid, piperine, and thymol in a polyherbal formulation

Abstract

A novel, accurate, precise, and linear RP-HPLC method for the simultaneous estimation of ascorbic acid, gallic acid, ferulic acid, piperine, and thymol in a polyherbal formulation was developed and validated as per ICH guidelines. A good chromatographic separation was achieved using gradient profile with the help of Shim-pack RP-HPLC column (250 mm*4.6 mm, 5 µm) and a mobile phase consisting of acetonitrile and 1% acetic acid was used. The column temperature was maintained at 28 °C throughout the run with a wavelength of 272 nm using UV-Visible detector. The retention time of ascorbic acid, gallic acid, ferulic acid, piperine, and thymol was found to be 3.9, 6.7, 21.5, 44.6, and 45.9 min respectively. The linearity of ascorbic acid, gallic acid, ferulic acid, piperine, and thymol was found to be in a range of 10-100, 5-50, 1-10, 5-50, and 2-20 µg/mL, respectively with correlation coefficient >0.99. The high recovery values (98-102%) indicate satisfactory accuracy. The % RSD values were found to be less than 2% in the precision study which reveals that the method is precise. Hence the developed method can be used for quality control and quantitative analysis of extracts and commercial containing these selected phytoconstituents.