A Box-Behnken design-assisted RP-HPLC method development and optimization for simultaneous determination of four...

The advent of COVID-19 has highlighted the value of repurposing existing antiviral medications and the importance of developing efficient analytical methods to assist their quality control. Simultaneous determination of multiple agents saves time and resources in pharmaceutical analysis. Moreover, the analytical demand during health emergencies underscores the need for validated, rapid, and reliable methods that can support both therapeutic evaluation and regulatory decisions. In this study, we developed and optimized a reversed-phase high-performance liquid chromatography (RP-HPLC) method to measure four repurposed antiviral drugs: favipiravir, sofosbuvir, ledipasvir, and remdesivir. A Box-Behnken design (BBD) approach was employed to study how buffer pH, the amount of organic solvent in the mobile phase, and flow rate affect key outcomes like resolution and run time. The separation was done on INERTSIL ODS-3 C18 column (5 μm, 250 × 4.6 mm) under isocratic elution conditions using acetonitrile (50:50, v/v) and ammonium formate buffer (0.02 M, adjusted to pH 2.8 with formic acid) as the mobile phase at flow rate 1 mL/min. Detection was performed at 240 nm. The method demonstrated good system suitability and met ICH Q2 (R2) validation standards, exhibiting satisfactory linearity (R² > 0.999), accuracy (recoveries within 98-102%), and precision (RSD < 2%). The sustainability of the proposed method was comprehensively evaluated using AGREE, MoGAPI, BAGI, and RAPI tools to assess its greenness, blueness, and whiteness. The proposed method offers a practical and efficient analytical platform for quality control laboratories, enabling reliable routine analysis of multiple antiviral agents in pharmaceutical products, particularly for post-pandemic preparedness and therapeutic surveillance.