Abstract

ADVANCED LIQUID CHROMATOGRAPHIC STRATEGIES FOR THE SIMULTANEOUS ESTIMATION OF MULTICOMPONENT DIRECTACTING ANTIVIRAL COMBINATIONS IN PHARMACEUTICAL DOSAGE FORMS AND BIOLOGICAL MATRICES: A COMPREHENSIVE REVIEW

Vaidehee Maheshwari*, Anju Goyal

ABSTRACT

The therapeutic management of chronic viral hepatopathies hasadvanced considerably with the introduction ofmulticomponent direct-acting antivirals (DAAs). Fixed-dosecombinations, particularly those combining non-structuralprotein 5A (NS5A) inhibitors with NS3/4A protease inhibitors,demonstrate high sustained virologic response rates acrossdiverse viral genotypes. The physicochemical diversity of theseco-formulated active pharmaceutical ingredients (APIs)presents complex analytical challenges regarding simultaneousquantification, degradation profiling, and bioanalysis.Objective: This review critically evaluates the contemporaryanalytical methodologies developed for the simultaneousestimation of these synergistic antiviral combinations in bothbulk formulations and complex biological matrices between2014 and 2024. Methodology: An extensive literature reviewwas conducted focusing on spectroscopic, high-performanceliquid chromatographic (HPLC), ultra-performance liquidchromatographic (UPLC), and hyphenated mass spectrometric (LC-MS/MS) techniques. Thereview synthesizes data on stationary phase chemistry, mobile phase optimization, andsample extraction protocols. Results/Discussion: While synchronous fluorescence and derivative spectroscopy offer rapid, solvent-minimized screening, reversed-phase HPLC remains the predominant platform for quality control and stability-indicating assays. The transition to UPLC systems featuring sub-2 μm particle sizes has substantially reduced analysis times while increasing peak capacity. For pharmacokinetic profiling, LC-MS/MS operating in multiple reaction monitoring (MRM) mode dominates, achieving lower limits of quantification (LLOQ) in the picogram per milliliter range. Adherence to International Council for Harmonisation (ICH) guidelines (Q2 and Q14) and the implementation of Analytical Quality by Design (AQbD) principles are critically evaluated. Conclusion: The continuous refinement of hyphenated techniques and the integration of AQbD frameworks ensure high method robustness and sensitivity. Future trajectories highlight the necessity of adopting green analytical chemistry (GAC) principles and multi-dimensional chromatography for complex matrix analysis.

Keywords: Multicomponent Analysis, Direct-Acting Antivirals, RP-HPLC, LC-MS/MS, Analytical Quality by Design, Pharmacokinetics, Stability-Indicating Methods.