Development and Validation of a Stability‐Indicating Liquid Chromatographic Method for Darolutamide and Its Degradation Impurities and Analytical Quality by Design‐Based Robustness Assessment

ABSTRACT This study is pivotal in identifying and characterizing three previously unreported degradation impurities of darolutamide, a nonsteroidal antiandrogen, through comprehensive forced degradation studies. Utilizing ultrahigh performance liquid chromatography (UHPLC) and liquid chromatography–mass spectrometry, the degradation products were isolated, and their molecular structures elucidated. Separation was achieved on an Acquity Ethylene Bridged Hybrid C18 column (150 × 2.1 mm 2 , 1.7 µm) using a gradient mobile phase system comprising 0.2% orthophosphoric acid buffer (mobile phase A) and a mixture of acetonitrile, methanol, and orthophosphoric acid buffer in a 60:20:20 (v/v/v) ratio (mobile phase B). Chromatographic conditions were optimized at a flow rate of 0.3 mL/min, with ultraviolet (UV) detection at 210 nm, an injection volume of 3 µL, and a column temperature maintained at 35°C. The analytical method was validated in accordance with the International Council for Harmonization guidelines. Linearity was demonstrated over the range from the limit of quantification to 200% of the target concentration with correlation coefficients ( r 2 ) between 0.997 and 0.999. Recovery of the impurities ranged from 93.4% to 103.3%, and precision was confirmed with relative standard deviation values below 5%. Forced degradation studies under various chemical and physical stress conditions revealed that darolutamide is particularly susceptible to chemical degradation. The degradation led to the formation of three distinct impurities, whose molecular masses were identified by liquid chromatography‐mass spectroscopy, and plausible degradation pathways were proposed based on reaction mechanisms. A quality by design approach, incorporating design of experiments, was employed to evaluate robustness under varied analytical conditions. This work presents a novel and validated approach for impurity profiling of darolutamide, contributing valuable insights for regulatory and quality control purposes.