Design-of-experiment approach for the development and validation of a high-performance thin-layer chromatography method for the simultaneous estimation of berberine chloride and galangin inTinospora cordifoliaM. andAlpinia galangaL. and their herbal formulations

Abstract

In the present study, a design-of-experiment (DoE) approach Was used to determine optimized mobile-phase compositions for the development of a high-performance thin-layer chromatography (HPTLC) method for the simultaneous estimation of berberine chloride (BER-H) and galangin (GAL) in Tinospora cordifolia M. and Alpinia galanga L., respectively, and their formulations. A Box—Behnken design (BBD) was used to optimize the compositional parameters and evaluate the main effect, interaction effects and quadratic effects of the mobile-phase compositions on the retardation factor (RF) of both drugs. HPTLC separation was performed on aluminum plates pre-coated with silica gel 60 F254 as the stationary phase, using toluene—ethyl acetate—formic acid (3:6:1, v/v) as the mobile phase at a wavelength of 267 nm. A sharp and well-resolved peak was obtained for BER-H and GAL at RF values of 0.17 ± 0.01 and 0.82 ± 0.01, respectively. The calibration curve was in the range of 200–1200 ng per band for both BER-H and GAL, with r2 = 0.984 and r2 = 0.980, respectively. Statistical insight was achieved with analysis of variance (ANOVA). The method was validated for linearity, accuracy, precision, limit of detection, limit of quantification, robustness, and specificity. To provide a better visualization of the statistically significant factors derived from the statistical analysis, the perturbation plot and response surface plot for the effect of independent variables on the RF of BER-H and GAL were evaluated. The developed HPTLC method was found to be simple, accurate, precise, sensitive, and specific for the simultaneous quantification of berberine chloride and galangin in Tinospora cordifolia M. and Alpinia galanga L., respectively, and their formulations.