COMPUTATIONAL MULTI-FACTOR MODELING TO THE SIMULTANEOUS DETERMINATION OF ASSAY AND RELATED SUBSTANCES METHOD DEVELOPMENT, VALIDATION, AND IDENTIFICATION OF MAJOR DEGRADATION PRODUCT OF COVID-19 DRUG MOLNUPIRAVIR BY HPLC: A STRATEGIC RESEARCH USING AQBD

Abstract

Objective: In the current study, a new approach was developed for the simultaneous determination of molnupiravir with its degradation products using RP-HPLC by following the Analytical Quality by Design (AQbD) approach, a proactive strategy for advancing pharmaceutical research.

Methods: Development was carried out by applying the two levels of fractional factorial design (2[5-1] with Resolution: V, four center points, and no replicates) to the five independent factors. The separation was achieved using liquid chromatography, equipped with XBridge BEH Shield (250 mm x 4.6 mm, 3.5 µm) RP18 column at 260 nm with gradient elution. A force degradation study was performed in different stressed conditions and a major degradant was identified using modern analytical techniques.

Results: The dependent factors were assessed against independent factors using the H-N plot, Pareto chart, and 3D surface plot applied through ANOVA. Major degradant obtained from stress study was identified and characterized using LC-MS, NMR, and FTIR techniques. The analytical method was found to be specific, precise (% RSD ≤ 2.0), linear (r = 0.9999) and accurate (99.2-101.4 %) with a 0.2-0.6 mg/ml range. The LOD and LOQ were 0.02 % (0.08 µg/ml, S/N ratio ≥ 3) and 0.05 % (0.2 µg/ml, S/N ratio ≥ 10, % RSD: 2.2) respectively. The analytical solutions were stable for 48 hour in the clear glassware at room temperature. The study depicts the successful development and validation of a simultaneous determination of assay and related substances by RP-HPLC using Design of Experiments (DoE) approach.

Conclusion: A Fractional Factorial Design (FFD) experiment explored the interactions of dependent with independent factors using Design Expert software, version 13.0. This AQbD-driven approach provided an in-depth understanding of chromatographic separation factors, ensuring method reliability and effectiveness for simultaneously determining the purity and impurity of molnupiravir, assisting in regulatory compliance.