Development and validation of quality by design stability-indicating method for quantitative determination of...

Analytical Quality by Design (AQbD) enhances analytical methods from the start by integrating scientific principles and risk management. This paper details the development and validation of a high-performance liquid chromatography method for lamivudine and its impurities, using AQbD. The Analytical Target Profile (ATP) was established, followed by a scouting phase and risk assessment. Method parameters were investigated through design of experiments (DoE). The Method Operable Design Region (MODR) was defined using DoE models and Monte Carlo simulations, validation was performed, and a top-down approach estimated measurement uncertainty. During the scouting phase, coelutions were observed. High-risk parameters were explored in the first DoE. Subsequent designs optimized the methanol proportion, buffer concentration and pH. The MODR confirmed the method's robustness. Forced degradation studies showed that lamivudine degrades in acidic, basic and oxidative conditions. The final method utilizes a C18 column at 25 °C, 25 µL injection volume, a flow rate of 1.0 mL/min, a mobile phase A composed by ammonium formate buffer 18,2 mM pH 5.7:methanol (97:3 v/v), and a mobile phase B with a pH of 3.8. An acceptance zone was established using guard bands. AQbD was effectively applied to develop a new analytical method, minimizing the risk of false decisions.