Box–Behnken Optimized Reversed‐Phase Ultra‐High‐Performance Liquid Chromatography Method for Simultaneous Trace‐Level Determination of Process‐Related Impurities in Sunitinib Drug Substance

ABSTRACT

An RP‐UHPLC method was developed for the simultaneous trace‐level quantification of three process‐related impurities of sunitinib malate: sunitinib amide impurity (IMP‐1), sunitinib carboxylic acid impurity (IMP‐2), and 5‐fluoroindolin‐2‐one (IMP‐3). Systematic optimization proceeded through a three‐factor Box–Behnken design evaluating acetonitrile content (40%–60% v/v), mobile phase pH (3.5–4.5), and column temperature (30°C–40°C) across 17 randomized experimental runs; quadratic polynomial models for all three responses—sunitinib retention time, Rs(IMP‐1/IMP‐2), and Rs(IMP‐2/sunitinib)—yielded R ² ≥ 0.9997, adjusted R ² ≥ 0.9993, and nonsignificant lack‐of‐fit in every case ( p  ≥ 0.05). Numerical optimization via composite desirability maximization ( D  =  0.750) identified 50% acetonitrile in 10 mM ammonium formate (pH 3.8) at 35°C as the operating optimum, affording baseline resolution of all four analytes in 6 min on an Agilent Poroshell 120 EC‐C18 column (2.1 × 100 mm, 1.9 µm). Validation followed the ICH Q2(R2) framework for a Category II quantitative impurity test, and the numbers held up well: linearity across 5–150 ng mL ⁻¹ with r ≥ 0.999; LOQ values between 8.48 and 16.97 ng mL ⁻¹ (a six‐ to twelvefold margin under the 0.1% specification threshold); accuracy of 98.9%–100.4%; and combined precision with %RSD no higher than 1.03%.