Design of experiments-based development and validation of RP-HPLC and UV spectroscopic methods for the simultaneous determination of citicoline and nimodipine in pharmaceutical formulations

A Design of Experiments (DoE) approach was applied for the development and validation of RP-HPLC and UV spectrophotometric methods for the simultaneous determination of citicoline and nimodipine in a synthetic pharmaceutical dosage form. UV spectrophotometric analysis was performed using a simultaneous equation method in methanol, with detection at 239 nm for nimodipine and 271 nm for citicoline. RP-HPLC method development was carried out using a Central Composite Design to investigate the effects of buffer pH, flow rate, and organic solvent composition on critical chromatographic responses. Multi-response optimization was achieved using Derringer's desirability function, considering retention time, capacity factor, and resolution. The optimized chromatographic conditions consisted of methanol (40%) and phosphate buffer (60%) at pH 3.8 (adjusted with orthophosphoric acid), a flow rate of 1.0 mL/min, and UV detection at 225 nm using a C18 column. Citicoline and nimodipine were well resolved with retention times of 3.007 and 5.741 min, respectively. Both methods were validated in accordance with ICH Q2 (R1) guidelines. Statistical evaluation using ANOVA confirmed the significance of model terms and the reliability of the developed RP-HPLC method. The proposed methods are simple and reliable, with the DoE-guided approach providing improved method understanding and robustness, making them suitable for routine quality-control analysis of pharmaceutical formulations.