Development and Validation of a Stability-Indicating RP-HPLC Method for Gilteritinib Using a Quality by Design

Research Article

Abstract

A robust, stability-indicating reversed-phase high-performance liquid chromatography method was developed and validated for the quantitative determination of the dual FLT3/AXL inhibitor gilteritinib in pharmaceutical dosage forms utilizing an analytical Quality by Design framework. Separation was achieved on a Phenomenex  column (150  4.6 mm, 5 µm) with a mobile phase composed of 0.01 M potassium dihydrogen phosphate buffer (adjusted to pH 3.0 containing 0.1% v/v triethylamine) and acetonitrile in a 59:41 v/v ratio. Chromatographic conditions were systematically optimized utilizing a three-factor, three-level Central Composite Design to evaluate the impact of flow rate, column temperature, and organic modifier concentration on critical quality attributes, including retention time, theoretical plates, and peak asymmetry. Under optimal conditions of 0.93 mL/min flow rate and 27^°C column temperature, gilteritinib eluted as a symmetrical peak at 2.66 min. The method showed a linear response over the concentration range of 10 to 60 µg/mL with a high correlation coefficient of 0.999. The limits of detection and quantitation were determined to be 0.17 µg/mL and 0.53 µg/mL, respectively. Forced degradation studies conducted under hydrolytic, oxidative, photolytic, and thermal stress conditions showed that the method effectively resolved gilteritinib from its primary degradation products eluting at 1.65 and 2.99 min, confirming its stability-indicating capability. Application to commercial formulations resulted in a quantitative drug recovery of 100.25%, showing excellent precision and accuracy for high-throughput pharmaceutical quality control