Impurity profiling and stability analysis of enzalutamide: Identification , genotoxicity assessment, and development of UHPLC methods for critical impurities

Abstract

This study aims to comprehensively evaluate and control the impurity profile of enzalutamide, an androgen receptor signaling inhibitor used to treat metastatic castration-resistant prostate cancer. Using liquid chromatography-mass spectrometry (LC-MS), twenty impurities were identified based on their mass-to-charge ratios (m/z) during synthetic method development and stress testing studies. Classification of these compounds according to ICH M7 guidelines revealed two potentially genotoxic impurities: Enzal-2 (4-isothiocyanato-2-(trifluoromethyl)benzonitrile), classified as a Class 3 impurity due to its isothiocyanate group, and Enzal-2A (4-amino-2-(trifluoromethyl)benzonitrile), classified as a Class 2 impurity due to a positive Ames test result. Based on the threshold of toxicological concern (TTC) of 1.5 mu g/day and the maximum daily dose of enzalutamide of 160 mg, a control limit of 9.4 ppm was established for Enzal-2 and Enzal-2A to mitigate safety risks. Both Enzal-2 and Enzal-2A were identified as process-related impurities, with Enzal-2A also recognized as a hydrolysis degradation product. Specific ultra-high-performance liquid chromatography (UHPLC) methods were developed and validated for the precise, accurate, and robust quantification of enzalutamide, Enzal-2, Enzal-2A, and related impurities. These methods were applied to enzalutamide samples subjected to various stress conditions, including elevated temperature, daylight, UV radiation, and exposure to oxidative, neutral, alkaline, and acidic environments, as well as under accelerated and long-term stability testing. The findings underscore the importance of comprehensive impurity profiling and validated analytical methods to ensure the safe and effective manufacture, quality control, and use of enzalutamide.