UHPLC Method Development for the Trace Level Quantification of Anastrozole and Its Process‐Related Impurities Using Design of Experiments

A sensitive, selective, and robust ultra-high-performance liquid chromatography method was developed for the simultaneous quantification of anastrozole and five process-related impurities at trace levels (≤ 0.1%). Traditional univariate optimization was replaced with a systematic chemometric approach using Box-Behnken Design to evaluate critical chromatographic parameters-mobile phase pH (3.0-4.0), column temperature (25°C-35°C), and flow rate (0.20-0.60 mL/min). Response Surface Methodology was employed to model retention behavior, resolution, and peak symmetry, with desirability function optimization identifying the ideal conditions (pH 3.20, 29.3°C, 0.33 mL/min). The method achieved baseline separation on an ACQUITY BEH C18 column (2.1 × 50 mm, 1.7 μm) using 10-mM ammonium formate (pH-adjusted) and acetonitrile as the mobile phase. Validation per International Council for Harmonisation Q2(R1) guidelines confirmed excellent linearity (R² ≥ 0.999), precision (%RSD ≤ 1.5% for ANZ; ≤ 5.3% for impurities), accuracy (recoveries: 94%-101%), and sensitivity (limit of detection: 0.011-0.014 μg/mL; limit of quantitation: 0.020-0.025 μg/mL). This study reports the first validated ultra-high-performance liquid chromatography method enabling trace-level quantification of two newly identified (IMP-1 and IMP-2) and three previously reported process-related impurities of anastrozole in a single run, using a Design of Experiments-based optimization approach.