Analytical Method Validation of Nirmatrelvir (INN) Assay in the Quality Control Laboratory: A USP-Based Study

Background: Paxlovid(R) is a novel COVID-19 treatment comprising nirmatrelvir, a protease inhibitor found to be active in almost all COVID-19 samples that have been tested. It is used in combination with ritonavir for the treatment of mild-to-moderate coronavirus disease 2019 (COVID-19) in patients at high risk of hospitalization. In this study, we have developed and validated a High-Performance Liquid Chromatography (HPLC) method to determine Nirmatrelvir in pharmaceutical formulation, and to determine its quality according to USP. Methods: A reverse-phase HPLC method was established for the quantitative determination of Nirmatrelvir by Zorbax SB-C18 column, mobile phase system is buffer : acetonitrile, diluent is 70% acetonitrile and UV detection at 220 nm. The linearity, precision, accuracy, specificity and sensitivity of the method were validated and the stability-indicating capability was established by performing the stress degradation study. Results: HPLC was an excellent assay (r2=0.999) with the concentration range of 1- 100ug/ml. The Limit of detection (LOD) and Limit of Quantification (LOQ) was determined as 0.5 ug/mL and 1.5 ug/mL respectively. Recovery studies revealed between 98-102% of accuracy. The results from stress degradation studies were in agreement with the method being able to separate Nirmatrelvir from its degradation products. Conclusion: The proposed HPLC method is reliable to determine Nirmatrelvir in bulk, finished products and pharmaceutical formulations for routine quality control analysis after validation. It is also appropriate for stability testing purposes, to ensure that Nirmatrelvir formulations retain their potency during storage.