Structure Elucidation and In Silico Safety Assessment of a Degradation Impurity in Molnupiravir Capsule Formulation: Development of a Stability-Indicating Method for Related Substances.

BACKGROUND

Molnupiravir, an FDA-approved antiviral for the treatment of COVID-19, requires reliable analytical methods to ensure its quality and safety due to its therapeutic importance.

OBJECTIVES

This study presents the development of a stability-indicating RP-HPLC method for estimating molnupiravir-related impurities in capsule formulations. An unknown impurity is structurally elucidated using LC-TQ/MS and 1H and 1³C NMR spectroscopy. Its potential safety profile is also assessed in silico using the admetSAR tool.

METHODS

Chromatographic separation was carried out using a Zorbax Eclipse Plus C18 column (250 mm × 4.6 mm, 5 µm) with a mobile phase consisting of 0.1% formic acid in water and acetonitrile (85:15, v/v), delivered at a flow rate of 1.0 mL/min at 25 °C. Detection was performed at 236 nm using a photodiode array (PDA) detector.

RESULTS

The method demonstrated linearity (R2 ≥ 0.9990), precision (RSD < 1.34%), and sensitivity, with an LOD of 0.2 µg/mL and an LOQ of 0.4 µg/mL. Forced degradation revealed an unknown impurity at 2.4 min, which was structurally identified as N-hydroxycytidine using LC-MS/MS and NMR. In silico analysis indicates potential hepatotoxicity, mitochondrial toxicity, and reproductive toxicity.

CONCLUSIONS

The validated RP-HPLC method successfully detects and quantifies molnupiravir impurities, supporting routine quality control and regulatory compliance. The unknown impurity's toxicity profile suggests the need for in vivo safety studies before official monograph inclusion.

HIGHLIGHTS: This research introduces a precise RP-HPLC method to evaluate molnupiravir's quality for COVID-19 treatment. It identified an impurity, characterized it, and predicted its safety to enhance our understanding of ensuring quality control for this vital drug.