Introduction: The objective of this study was to develop and optimize a rapid, sensitive, and reproducible Reverse-Phase High-Performance Liquid Chromatography (RPHPLC) method for the simultaneous quantification of two anticancer agents, Exemestane (Exe) and Bosutinib (Bos). As these drugs are increasingly used in combination therapeutic strategies, establishing a reliable analytical method is essential for formulation development, pharmacokinetic investigations, and quality control applications. Methods: A Box–Behnken design was employed to optimize key chromatographic variables, including the mobile phase ratio, flow rate, and column temperature. Chromatographic separation was achieved using a C18 column with acetonitrile and water (60:40, v/v) as the optimized mobile phase, operating at a flow rate of 1.0 mL/min under ambient temperature. Detection was performed at 256 nm using a PDA detector. The method was validated according to ICH Q2 guidelines, assessing linearity, precision, accuracy, robustness, and detection limits. Results: The optimized conditions produced distinct and well-resolved peaks with retention times of 5.345 minutes for Bos and 9.825 minutes for Exe. The method demonstrated excellent linearity within the 5–80 µg/mL concentration range for both drugs (R² > 0.999). Validation results confirmed high precision, acceptable accuracy, and robust performance under deliberate variations of analytical conditions. LOD and LOQ values indicated strong sensitivity suitable for quantitative applications. Discussion: The established method provides an efficient analytical platform capable of accurately resolving and quantifying Exe and Bos simultaneously. Its robustness and reproducibility make it applicable not only to naïve drug solutions but also to complex formulations Conclusion: The validated RP-HPLC method offers a reliable tool for simultaneous estimation of Exe and Bos and is suitable for routine applications, including assay analysis, drug loading, entrapment efficiency determination, and dissolution studies.
Priya Singh (Wed,) studied this question.