Development with greenness evaluation and validation of stability indicating ion-pair reverse phase HPLC method for determination of related substances and assay of Nirmatrelvir drug substance

This research article focuses on development and greenness evaluation of newer drug Nirmatrelvir and related substances. Nirmatrelvir, an antiviral agent, inhibits protease of SARS-CoV-2 and thus suppresses its replication. Considering clinical importance of the Nirmatrelvir, we aim to develop its analysis using novel greener ion-pair reverse phase stability-indicating high performance liquid chromatographic technique. The mobile phase ‘A’ contains an ion-pair reagent, 5 mM 1-octane sulfonic acid sodium salt in water containing Acetonitrile and the mobile phase ‘B’ is Acetonitrile. Zorbax SB-CN 5 μm was selected as the stationary phase and evaluation was performed at wavelength 202 nm with flow rate of 0.8ml/min. The retention time of Nirmatrelvir was about 15.1 min and the relative retention times for Impurity-A and Impurity-B were around 0.30 and 0.71 respectively. Correlation coefficient of 0.999 was observed. For analytes of interest LOD and LOQ were found in range 0.10-0.16 μg/mL and 0.26-0.41 μg/mL respectively. For Impurity-A and Impurity-B, mean recovery was observed in the range of 98.21%-100.53% and 100.30%-100.81% respectively. Results obtained showed that the validated method is within permissible range in all respective parameters. Stress degradation studies indicated the proposed method was stability oriented and is evaluated using greenness tools.