Green and Robust RP-HPLC for Simultaneous Quantification of Gallic, Ferulic, and Caffeic Acids in Human Plasma via...

This study aimed to develop and validate a sustainable reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous quantification of Gallic Acid (GA), Ferulic Acid (FA), and Caffeic Acid (CA) in human plasma, aligning with green analytical chemistry principles and regulatory guidelines.

Chromatographic conditions were optimized using a Box-Behnken design under the framework of response surface methodology, evaluating the influence of acetonitrile concentration, mobile-phase pH, and flow rate on retention time, peak symmetry, and efficiency. Method validation was performed in accordance with ICH M10 and EC 2002/657/EC guidelines, including assessment of linearity, accuracy, precision, recovery, stability, and matrix effects. The environmental impact of the analytical workflow was assessed using ComplexGAPI, AGREE, AGREEprep, Eco-Scale, and BAGI metrics.

The optimized chromatographic parameters, 35% acetonitrile, pH 2.8, and a flow rate of 0.6 mL/min, produced well-resolved peaks with retention times of 2.039 min for GA, 3.077 min for FA, and 3.990 min for CA, while yielding minimal deviations (<5%) between predicted and experimental responses. The method demonstrated excellent linearity (R² > 0.999), with limits of detection and quantification ranging from 0.151-1.236 μg/mL and 0.453-3.70 μg/mL, respectively. Accuracy, precision, and recovery were within acceptable regulatory limits. The analytes exhibited robust stability under stress and storage conditions with negligible matrix interference.

The integration of chemometric optimization and multi-tool environmental evaluation confirmed the method's analytical efficiency and ecological compatibility. Compared with conventional RP-HPLC protocols, the proposed workflow significantly reduces solvent usage and hazardous waste generation while maintaining analytical performance.

The developed RP-HPLC method provides a validated, reliable, and environmentally sustainable analytical platform for quantifying phenolic acids in human plasma. Its regulatory compliance, robustness, and reduced ecological footprint make it suitable for routine clinical and pharmacokinetic studies.