Quality by Design‐guided Development and Validation of a Sensitive Ultra‐High‐Performance Liquid Chromatography Method for Trace‐Level Determination of Erlotinib Impurities

ABSTRACT

Erlotinib is an epidermal growth factor receptor tyrosine kinase inhibitor widely used in the treatment of non‐small cell lung cancer. During the multistep synthesis of the Erlotinib active pharmaceutical ingredient, several process‐related impurities may be generated, which must be carefully monitored to ensure drug safety and regulatory compliance. In the present study, a rapid and sensitive ultra‐high‐performance liquid chromatography (UHPLC) method was developed and validated for the simultaneous determination of Erlotinib and three process‐related impurities, namely Erlotinib carboxylic acid impurity (ERPI‐1), Erlotinib amine impurity (ERPI‐2), and Erlotinib methoxy impurity (ERPI‐3). Method development employed a design of experiments approach using a three‐factor, three‐level Box–Behnken design to evaluate the influence of acetonitrile (ACN) percentage, mobile phase pH, and column temperature on chromatographic performance, enabling the establishment of a statistically validated Method Operable Design Region. Separation was achieved on an Agilent Poroshell 120 EC‐C18 column (2.1 × 100 mm, 1.9 µm) with a mobile phase consisting of 10 mM ammonium formate and ACN, and ultraviolet detection at 258 nm. Numerical optimization identified the optimal conditions as 50% ACN, mobile phase pH 3.8, and column temperature of 35°C, yielding a total analysis time of 6 min, with retention times of 3.37 min (ERPI‐2), 3.77 min (ERPI‐1), 4.17 min (Erlotinib), and 1.89 min (ERPI‐3), with adequate resolution between all analytes. The method was validated in accordance with International Council for Harmonization Q2(R1) guidelines and demonstrated excellent system suitability, specificity, linearity ( R ² ≥ 0.9989), precision (percentage relative standard deviation ≤2.98%), and accuracy (recoveries within 97%–100%). The limits of detection ranged from 0.01 to 0.01 µg/mL, confirming high sensitivity for trace‐level impurity determination. The developed UHPLC method is rapid, reliable, and quality‐by‐design‐aligned, making it highly suitable for routine quality control and impurity profiling of Erlotinib in pharmaceutical formulations.