Integrated Analytical Quality by Design and In Silico Modeling for Stability‐Indicating Ultra‐High‐Performance Liquid Chromatography Method Development and Validation for Impurity Profiling of Gilteritinib Fumarate

Abstract

Impurity profiling of targeted anticancer agents is essential to ensure product quality, patient safety, and regulatory compliance. Despite the clinical importance of gilteritinib fumarate, a potent FMS‐like tyrosine kinase 3 inhibitor used for relapsed or refractory acute myeloid leukemia, existing chromatographic methods are limited to assay determination or degradation product characterization, and none employ an integrated quality‐by‐design framework for simultaneous quantification of specified process‐related impurities. This study describes the development and validation of a stability‐indicating ultra‐high‐performance liquid chromatography method for the simultaneous determination of four specified impurities (N‐oxide, N‐methyl N‐oxide, desmethyl, and nitrile) in gilteritinib fumarate. The development strategy combined in silico retention modeling with experimental optimization using a three‐factor, three‐level Box–Behnken design to evaluate critical method parameters and establish a statistically justified method operable design region. The optimized method achieved baseline separation of all analytes within 20 min, with a critical‐pair resolution exceeding 2.7 and tailing factors below 1.35. Validation in accordance with International Council for Harmonisation guidelines demonstrated excellent linearity, accuracy (recoveries 98.5%–101.3%), and precision, with detection limits of approximately 0.08 µg mL −1 for all impurities. Forced degradation studies confirmed stability‐indicating capability through satisfactory mass balance (97.39%–99.94%) and peak purity. Monte Carlo simulation verified a high probability of meeting predefined performance criteria within the established design region. A comprehensive greenness assessment indicated favorable environmental profiles across multiple metrics. The validated workflow provides a robust, lifecycle‐oriented strategy that advances beyond existing methods by offering simultaneous impurity profiling with demonstrated design space robustness, supporting routine quality control and stability assessment of gilteritinib fumarate drug substance.