Strategic Implementation of Analytical Quality by Design in RP‐HPLC Method Development for Andrographis paniculata and Chrysopogon zizanioides Extract‐Loaded Phytosomes

Andrographis paniculata and Chrysopogon zizanioides have shown promising antiviral potential, drawing attention to traditional medical systems as alternative therapeutic approaches. This study focuses on the development and validation of a quality by design enabled chromatographic method for the simultaneous quantification of ferulic acid (FA) and chlorogenic acid (CGA) as key biomarkers in a phytosomal formulation containing extract. Hydroalcoholic extraction was conducted using a 70:30% v/v. Phytosomes were formulated through thin‐film hydration, followed by sonication to produce small unilamellar vesicles. Chromatographic conditions were optimized using a Box–Behnken Design, evaluating the organic phase ratio, flow rate, and column temperature as independent variables and retention time (RT) and tailing factor (TF) of both biomarkers as dependent variables. A Phenomenex Luna column (5 µm × 250 mm, 4.6 mm) was employed under optimized conditions, which included a mobile phase comprising 0.1% formic acid in water and acetonitrile (70:30 v/v), a flow rate of 1 mL/min, and a column temperature of 30°C, ensuring high‐resolution peak separation. Phytosomes exhibited a particle size of 380 nm with a zeta potential of −34.86 mV. RTs were 8.689 min for FA and 6.435 min for CGA with TFs of 1.212 and 1.213, respectively. Validation per International Council for Harmonization (ICH) Q2 (R1) guidelines confirmed excellent linearity ( R 2 > 0.998), accuracy (96.8%–98.3% recovery), precision (percentage relative standard deviation [%RSD] < 2), limit of detection (LOD), limit of quantification (LOQ), and robustness. Quantification showed CGA 20.43 and FA 63.20 µg. The validated chromatographic method successfully quantifies both biomarkers in phytosomal formulations, ensuring quality control and supporting phytopharmaceutical research for dengue and inflammatory conditions.