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

Area of Science:

• Analytical Chemistry
• Green Chemistry
• Pharmacokinetics

Background:

• Phenolic acids like Gallic Acid (GA), Ferulic Acid (FA), and Caffeic Acid (CA) are important biomarkers.
• Accurate quantification in human plasma is crucial for pharmacokinetic studies.
• Existing analytical methods may not align with green chemistry principles or regulatory demands.

Purpose of the Study:

• To develop and validate a sustainable reversed-phase high-performance liquid chromatography (RP-HPLC) method.
• To simultaneously quantify GA, FA, and CA in human plasma.
• To ensure the method adheres to green analytical chemistry principles and regulatory guidelines (ICH M10, EC 2002/657/EC).

Main Methods:

• Optimization of chromatographic conditions (acetonitrile concentration, mobile-phase pH, flow rate) using Box-Behnken design and response surface methodology.
• Comprehensive method validation including linearity, accuracy, precision, recovery, stability, and matrix effects.
• Environmental impact assessment using multiple green metrics (ComplexGAPI, AGREE, AGREEprep, Eco-Scale, BAGI).

Main Results:

• Optimized parameters (35% acetonitrile, pH 2.8, 0.6 mL/min) yielded well-resolved peaks with short retention times (GA: 2.039 min, FA: 3.077 min, CA: 3.990 min).
• Method demonstrated excellent linearity (R² > 0.999) and sensitivity (LOD/LOQ: 0.151-1.236 μg/mL and 0.453-3.70 μg/mL).
• High accuracy, precision, recovery, analyte stability, and negligible matrix effects were confirmed, meeting regulatory standards.

Conclusions:

• The developed RP-HPLC method is analytically efficient and environmentally compatible, confirmed by chemometric optimization and multi-tool environmental evaluation.
• This sustainable workflow significantly reduces solvent consumption and hazardous waste compared to conventional methods.
• The validated, robust, and eco-friendly method is suitable for routine quantification of phenolic acids in human plasma for clinical and pharmacokinetic research.