Quality by Design–Based RP‐HPLC Method for Simultaneous Estimation of 4‐Hydroxytamoxifen and Thymoquinone in Liposomal Formulation

Developing a method for the simultaneous quantification of drugs in combination formulations is crucial for optimizing combination therapy. 4-Hydroxytamoxifen (4-OHT) and thymoquinone exhibit synergistic activity in breast cancer treatment, yet no robust analytical method exists for their simultaneous estimation in liposomal systems. This study aimed to design, optimize and validate an RP-HPLC method for quantifying both drugs using an analytical quality by design (AQbD) approach. An analytical target profile (ATP) was defined, and risk assessment using an Ishikawa diagram identified mobile phase composition, flow rate and column temperature as critical parameters. Method optimization was performed using a Box-Behnken Design, evaluating effects on retention times, peak areas, tailing factors and resolution. Optimal conditions included a 90:10 methanol:aqueous mobile phase, 0.5 mL/min flow rate and 35°C column temperature. The method was validated per ICH Q2(R1) guidelines, demonstrating excellent sensitivity (LOD 0.69-0.29 μg/mL), accuracy (95%-105% recovery) and precision (RSD < 2%). The method was successfully applied for drug estimation in liposomal formulations, ex vivo permeation, cytotoxicity and cellular uptake studies. Liposomal gels showed high entrapment efficiency, enhanced drug release, permeation and cellular uptake. Analytical Eco-Scale assessment confirmed excellent green performance.