ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF A NOVEL HPTLC (METHOD) FOR THE SIMULTANEOUS ESTIMATION OF BERBERINE, GALLIC ACID, QUERCETIN AND PIPERINE IN A POLYHERBAL FORMULATION

Abstract

A novel, accurate, precise High Performance Thin Layer Chromatography (HPTLC) method for the simultaneous estimation of berberine, gallic acid, quercetin and piperine in a polyherbal formulation has been developed and validated as per ICH guidelines. A good chromatographic separation was achieved using mobile phase consisting of toluene, ethyl acetate, methanol and formic acid in the ratio of 6: 6: 2: 1 (V/V/V/V) with a wavelength of 254 nm using UV-Visible (UV-Vis) detector. The retention factors of berberine, gallic acid, quercetin and piperine were found to be 0.37, 0.51, 0.72 and 0.86, respectively. The calibration graph was found to be linear within the tested range of 1-10 µg band-1 for all the selected phytoconstituents with a 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.