Advanced Liquid Chromatographic Strategies for the Simultaneous Estimation of Multicomponent Direct-Acting Antiviral Combinations in Pharmaceutical Dosage Forms and Biological Matrices: A Comprehensive Review

The therapeutic management of chronic viral hepatopathies has advanced considerably with the introduction of multicomponent direct-acting antivirals (DAAs). Fixed-dose combinations, particularly those combining non-structural protein 5A (NS5A) inhibitors with NS3/4A protease inhibitors, demonstrate high sustained virologic response rates across diverse viral genotypes. The physicochemical diversity of these co-formulated active pharmaceutical ingredients (APIs) presents complex analytical challenges regarding simultaneous quantification, degradation profiling, and bioanalysis. Objective: This review critically evaluates the contemporary analytical methodologies developed for the simultaneous estimation of these synergistic antiviral combinations in both bulk formulations and complex biological matrices between 2014 and 2024. Methodology: An extensive literature review was conducted focusing on spectroscopic, high-performance liquid chromatographic (HPLC), ultra-performance liquid chromatographic (UPLC), and hyphenated mass spectrometric (LC-MS/MS) techniques. The review synthesizes data on stationary phase chemistry, mobile phase optimization, and sample 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.