Abstract

*Drx. Krishnakant Ajit Choure, Dr. Sushama Vaishnav, Dr. Sachin S Bhusari, Dr. Pravin S Wakte

ABSTRACT

This study focuses on the development and Analytical Quality by Design (AQbD)–driven optimization of a stability-indicating reverse-phase high-performance liquid chromatographic (RP-HPLC) method for the simultaneous quantitative estimation of 5-Fluorouracil (5-FU) and Betanin in both bulk drug substances and sustained-release pharmaceutical formulations. A comprehensive risk assessment using Ishikawa fishbone analysis identified critical method parameters, which were systematically optimized using a 3² factorial Design of Experiments (DoE) approach. Critical method variables including mobile phase composition (methanol:acetonitrile ratio), buffer pH, and flow rate were investigated to establish their influence on chromatographic performance parameters such as resolution, tailing factor, and retention time. Response Surface Methodology (RSM) facilitated the development of predictive mathematical models and establishment of a Method Operable Design Region (MODR) ensuring robust analytical performance. Comprehensive forced degradation studies under acidic, alkaline, oxidative, thermal, and photolytic stress conditions confirmed the method's stability-indicating capability with adequate resolution between parent compounds and their degradation products. The developed method was rigorously validated according to ICH Q2(R2) guidelines, demonstrating excellent linearity (R² > 0.999), accuracy (recovery 98.5-100.5%), precision (RSD < 1.0%), specificity, robustness, and appropriate sensitivity (LOQ < 1 µg/mL). The optimized method achieved baseline resolution (Rs > 5.0) with analysis time under 10 minutes using a C18 column with methanol:acetonitrile:phosphate buffer (70:25:5 v/v, pH 4.0) at 1.0 mL/min flow rate. This rapid, economical, and scientifically robust analytical method is suitable for routine quality control, stability testing, and pharmacokinetic studies of 5-FU and Betanin in combined pharmaceutical formulations.