Chromatographic method selection strategy for first-line antituberculosis fixed-dose combinations

Background Fixed-dose combinations (FDCs) containing Rifampicin, Isoniazid, Pyrazinamide, and Ethambutol remain the cornerstone of first-line antituberculosis therapy. However, their simultaneous chromatographic determination presents significant analytical challenges due to marked differences in lipophilicity (logP), ionization constants (pKa), chromophoric properties, chemical stability, and dose ratios. These disparities complicate retention control, selectivity optimization, peak symmetry, and detection sensitivity, particularly in stability-indicating method development. Main body This work outlines a mechanism-guided strategy for chromatographic method selection tailored to first-line antituberculosis FDCs. The approach integrates physicochemical profiling with rational selection of separation mode, stationary phase chemistry, mobile phase pH control, and detection strategy. Reversed-phase liquid chromatography (RP-LC) is justified as the primary separation mode due to its compatibility with ultraviolet and mass spectrometric detection, regulatory acceptance, and suitability for stability-indicating analysis. Stationary phase selection (e.g., base-deactivated or polar-embedded C18) is emphasized to minimize silanol interactions and compress the retention window between highly polar and lipophilic analytes. Buffered gradient elution under acidic conditions is recommended to balance retention, improve peak symmetry, and enhance robustness. Detection strategies are aligned with chromophoric disparities and dose imbalance, highlighting the complementary roles of diode array detection and LC–MS techniques. Special consideration is given to degradation pathways and interaction products, particularly under acidic conditions, necessitating forced degradation studies and peak purity assessment to ensure stability-indicating capability. Conclusion A mechanistically informed chromatographic selection strategy enables robust, selective, and stability-indicating simultaneous determination of first-line antituberculosis FDCs. Integrating physicochemical understanding with systematic optimization enhances method reliability, regulatory compliance, and analytical efficiency.