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.

中文翻译：

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开发和验证稳定性的高效薄层色谱法估算药物剂型中盐酸二甲双胍和熊去氧胆酸的实验方法设计

在本研究中，实验设计（DoE）方法用于优化色谱条件，以开发用于同时估算盐酸二甲双胍（MET）和熊去氧胆酸（URSO）的高性能薄层色谱（HPTLC）方法）的药物剂型。使用Taguchi设计确定了关键因素，确定了关键因素后，使用Box–Behnken设计（BBD）进行了优化。BBD用于优化组成参数并评估流动相组成的主要作用，相互作用作用和二次作用，显影距离和饱和时间对延迟因子（RF）两种药物。在预涂硅胶60 F254作为固定相的铝板上进行HPTLC分离，使用甲苯-乙醇-丙酮-甲酸（4.5：2：2.5：0.85，V / V）作为流动相，波长为MET和URSO分别为234 nm和700 nm。MET和URSO在R F值分别为0.19和0.80 min时获得一个清晰的峰。MET和URSO的校准曲线在每点5000–40000 ng和每点1500–12000 ng的范围内，r 2 = 0.984，r2 = 0.980。验证了该方法的线性，准确性，精密度，检测限，定量限和特异性。为了更好地可视化从统计分析得出的统计上重要的因素，评估了自变量对MET和URSO的R F的影响的扰动图和响应面图。稳定性研究是在不同的应力条件下进行的，例如酸和碱的水解，氧化和温度。所开发的方法能够解析在上述条件下形成的药物及其降解产物。