AQbD-Optimised RP-UHPLC Multi-Marker Method for Andrographolide in Andrographis Tablets: A Green and Validation-Oriented Analytical Study

Abstract

A rapid, green, and stability-indicating reversed-phase ultra-high-performance liquid chromatographic method was developed for quantification of andrographolide with simultaneous monitoring of neoandrographolide, 14-deoxyandrographolide, and 14-deoxy-11, 12-didehydroandrographolide in Andrographis tablets. Analytical Quality by Design was applied through definition of an analytical target profile, risk assessment, and Box-Behnken optimisation of aqueous-phase pH, ethanol strength, and flow rate. Separation was achieved on a C18 column (100 × 2.1 mm, 1.7 µm) using water containing 0.1% acetic acid and ethanol under gradient elution at 0.36 mL/min, 35 °C, and 223 nm. The four markers eluted within 6.5 min, and the critical 14-deoxyandrographolide/14-deoxy-11,12-didehydroandrographolide resolution was 2.65. Calibration coefficients exceeded 0.9999. Mean recoveries were 99.22–100.88%, repeatability and intermediate-precision RSD values remained below 0.64%, and robustness testing retained a minimum critical resolution of 2.47. Forced degradation generated resolved degradants under acid, alkali, oxidative, neutral, thermal, and photolytic conditions, with alkaline hydrolysis producing the greatest overall loss. Three commercial batches contained 98.7–101.2% of the declared andrographolide content. Eco-Scale and AGREE scores of 83 and 0.79, respectively, supported a favourable comparative greenness profile.