Method Development and Validation for Ritonavir with Comprehensive Impurity and Toxicity Profiling

Abstract

Introduction / Objective: The selectivity, potency, and stability of antiviral compounds, including Ritonavir (RTV), require high-precision analytical testing and impurity profiling for confirmation. As a widely used pharmacokinetic enhancer in COVID-19 therapy, the regulation and clinic validation of impurities and their toxicity are of the utmost importance. The main goal of the present work was to develop and confirm a simple, accurate, and stability, indicating HPLC method for determining the Ritonavir content and its related impurity (Impurity, A Free Base) includes theoretical and experimental toxicity assessments. Methods: Chromatographic separation was performed on a Phenomenex C18 column (250 4.6 mm, 5 m). The mobile phase was 20:80 of water and acetonitrile, flow rate 1.0 mL/min. The method validation tested different things using ICH Q2(R1) rules, Accuracy, Precision, Specificity, and Linearity. The toxicity assessment for Impurity A was done by the insilico prediction tools (PROTOX 3.0) and checked with an in vitro study of toxicity in PBMC cells. Results: The method demonstrated (R = 0.9999) in a range of 1.64 to 29.94 g/mL, with recovery rates from 92% to 99% and a relative standard deviation below 2%. Ritonavir and its impurity- A with distinct retention times is 4.3 min and 2.3 min respectively. In silico studies stated that the drug has a medium acute oral toxicity as low with a LD50 values of more than 2000 mg/kg with mild hepatotoxic and neurotoxic while MTT results showed an IC50 value >500 µg/mL, suggesting low cytotoxicity. Discussion: The validated HPLC method is a reliable, accurate, and economical way for the routine analysis of Ritonavir and its contaminants. The use of computational and in vitro toxicity assessments used to identify impurity hazards and comply with quality control standards of antiviral drugs. Conclusion: A consistent, precise, and cost-effective HPLC method was successfully developed and validated for Ritonavir and its related impurity. The integration of in silico and in vitro toxicity studies strengthens impurity risk assessment and supports the safe and effective quality control of antiviral pharmaceutical products.