Ultra-high-performance liquid chromatography method development for the quantification of Molnupiravir and its process-related impurities using Box-Behnken experimental design

Molnupiravir, a promising antiviral agent, has gained significant attention for its potential in treating viral infections, particularly in the context of the coronavirus disease 2019 pandemic. This study aimed to develop and validate an ultra-high-performance liquid chromatography (UHPLC) method for the quantification of Molnupiravir and its associated impurities (Imp-I, Imp-II, Imp-III, and Imp-IV). The method involved a systematic investigation of critical method parameters and their optimization using Quality by Design principles. The percentage of organic modifiers, column temperature, and flow rate were systematically optimized using the Box-Behnken design. The developed method was assessed for specificity, system suitability, accuracy, precision, linearity, and detection limits. The results demonstrate strong specificity, with the analysis remaining accurate even in the presence of impurities. The developed method exhibits excellent precision, with repeatability and intermediate precision showing relative standard deviation values ranging from 0.78% to 1.14% for impurities and 0.82% to 1.91% for Molnupiravir. Furthermore, the method displays exceptional linearity, covering a wide range of concentrations. The linear regression analysis yields high coefficients of determination (r2, 9993-0.9997), confirming the linearity. The developed UHPLC method is well-suited for the accurate and reliable analysis of Molnupiravir and its impurities, making it a valuable tool for quality control and pharmaceutical research applications.