Comparison of the Hydrolysis and Esterification Methods for the Determination of Genotoxic 5-Chlorothiophene-2-Carbonyl Chloride in Rivaroxaban Using HPLC

This paper describes the development and comparison of the hydrolysis and esterification methods for the determination of 5-chlorothiophene-2-carbonylchloride (CTCC), a potential genotoxic impurity, in the drug rivaroxaban (RIVA) drug substance. Poor recovery of CTCC in the hydrolysis method occurred due to the high percentage of acetonitrile, which suppressed the hydrolysis reaction, but was solved by adding Na2CO3. In the esterification approach, the problem of poor recovery was also encountered because of the competition between the hydrolysis and esterification reactions, and a stable recovery of CTCC under different water content conditions was obtained by adding glacial acetic acid. A high-performance liquid chromatography (HPLC) method was developed to separate the hydrolysis product, 5-chlorothiophene-2-carboxylic acid (CTCA), and the esterification product, methyl-5-chlorothiophene-2-carboxylate (MCTC), from RIVA, process-related impurities and degradation products of RIVA. Standard curves were linear (r = 0.9999) for the hydrolysis and esterification methods in the ranges of 16.7–280.3 ppm and 15.3–255.0 ppm, respectively, and limit of quantitation (LOQ) of CTCA and MCTC was 16.7 and 15.3 ppm, respectively. The accuracy (% recovery) was 98.08–100.5 % for the hydrolysis method and 97.86–99.05 % for the esterification method. Both methods had good repeatability and stability. During the real sample analysis, it was found that the hydrolysis method could not differentiate CTCC and the inherent CTCA impurity, leading to overestimation of residual CTCC in the RIVA. The esterification method was a better solution for the accurate measurement of residual CTCC in RIVA.