Development of a stability-indicating HPLC method for vitamin B12 in 3D-printed formulations and in vitro release studies using AQbD approach

An analytical quality-by-design (AQbD) approach was employed to develop a stability-indicating HPLC–UV method for the determination of cyanocobalamin (B12) in 3D-printed formulations and in vitro drug release studies. Forced degradation experiments were performed to generate prior knowledge of the degradation behavior of B12 and to support the development of a stability-indicating analytical method. Risk assessment followed by a screening design identified three critical method parameters (buffer pH, initial methanol content, and gradient slope), which were subsequently optimized using a Box–Behnken design. A multidimensional operating region satisfying the predefined chromatographic acceptance criteria was identified from the response models, and a working point within this region was selected by desirability-based numerical optimization. Robustness around the selected working point was further challenged by Monte Carlo simulation and process capability analysis. The developed models exhibited excellent predictive performance, with coefficients of determination (R 2 ) exceeding 0.9939. Method validation demonstrated satisfactory precision (RSD < 5.7%) and accuracy, with recoveries ranging from 98.8% to 115.2%. The validated method was successfully applied to impurity profiling of B12 API, determination of drug loading in 3D-printed formulations, and quantification of in vitro drug release, demonstrating its suitability for quality control and pharmaceutical development.