Design of Experiment Approach for the Development and Validation of a Stability-Indicating High-Performance Thin-Layer Chromatography Method for the Estimation of Metformin Hydrochloride and Ursodeoxycholic Acid in Pharmaceutical Dosage Form

Abstract

In the present study, a design of experiment (DoE) approach was used to optimize chromatographic conditions for the development of a high-performance thin-layer chromatography (HPTLC) method for the simultaneous estimation of metformin hydrochloride (MET) and ursodeoxycholic acid (URSO) in pharmaceutical dosage form. The critical factors were identified using a Taguchi design, and after identification of critical factors, optimization was done using Box–Behnken design (BBD). BBD was used to optimize the compositional parameters and to evaluate the main effect, interaction effects, and quadratic effects of the mobile phase compositions, development distance, and saturation time 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-ethanol-acetone-formic acid (4.5:2:2.5:0.85, V/V) as the mobile phase at a wavelength of 234 nm and 700 nm for MET and URSO, respectively. A sharp and well-resolved peak was obtained for MET and URSO at RF values of 0.19 and 0.80 min, respectively. The calibration curve was in the range of 5000–40000 ng per spot and 1500–12000 ng per spot for MET and URSO, with r2 = 0.984 and r2 = 0.980, respectively. The method was validated for linearity, accuracy, precision, limit of detection, limit of quantification, 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 MET and URSO were evaluated. Stability study was performed under different stress conditions such as acid and alkali hydrolysis, oxidation, and temperature. The developed method was able to resolve drugs and their degradation products formed under the afore-mentioned conditions.