Greenness Assessment of High‐Performance Liquid Chromatography Method for Simultaneous Estimation of Apigenin, Apocynin, and Gallic Acid With Quality by Design Approach

ABSTRACT This study presents the development of an eco‐friendly high‐performance liquid chromatography (HPLC) method for the simultaneous determination of apigenin, apocynin, and gallic acid, integrating analytical quality by design (AQbD) and green analytical chemistry (GAC) principles. The objective was to enhance method efficiency while reducing environmental impact, energy consumption, and solvent use. An AQbD approach was employed, starting with quality risk assessment and scouting analysis, followed by optimization using Box–Behnken design (BBD) to screen three key chromatographic parameters. The optimal chromatographic conditions were determined using a C18 column (250 mm × 4.6 mm, 5 µm) with a mobile phase of methanol: water (80%:20% v/v), a flow rate of 1 mL/min, ultraviolet detection at 228 nm, and a temperature of 25°C. The method demonstrated linearity over a concentration range of 1–5 µg/mL for the target compounds, with gallic acid as an internal standard. The method's greenness profile was assessed using various evaluation tools, confirming its compliance with GAC principles. The results indicate that the proposed HPLC method is not only effective and reliable but also environmentally sustainable, making it suitable for routine use in quality control laboratories. The integration of AQbD and GAC principles highlights the method's contribution to more sustainable and efficient analytical practices.