Quality by Design‐Driven Development, Greenness, and Whiteness Assessment of a Robust RP‐HPLC Method for Simultaneous Quantification of Ellagic, Sinapic, and Syringic Acids

Abstract

ABSTRACT A robust, environmentally responsible reversed‐phase high‐performance liquid chromatography (RP‐HPLC) method was developed and validated for the simultaneous quantification of three phenolic acids—ellagic acid (EGA), sinapic acid (SPA), and syringic acid (SGA)—using a Quality by Design (QbD) framework. Preliminary method development and risk assessment identified organic phase ratio, flow rate, and pH as critical method parameters (CMPs) significantly influencing chromatographic attributes such as retention time (RT), tailing factor (TF), and theoretical plates (TPs). A Taguchi orthogonal array facilitated initial screening, while response surface methodology (RSM) employing a Box–Behnken design enabled multivariate optimization of CMPs. Optimal separation was achieved using a mobile phase comprising methanol and 0.1% formic acid (60:40 v/v), delivered at 1.0 mL/min with detection at 275 nm and a column temperature of 30°C. The method was validated per ICH Q2(R1) guidelines and demonstrated excellent linearity ( R 2 > 0.999), sensitivity (LOD: 0.451–1.14 µg/mL), precision (%RSD < 2%), and recovery (98%–101%). Environmental and operational sustainability were assessed using ComplexGAPI, AGREE, AGREEprep, Analytical Eco‐Scale, and the Blue Applicability Grade Index (BAGI). The method exhibited high ecological compatibility (Eco‐Scale score: 77; AGREE: 0.75; BAGI: 75.0), substantiating its greenness and whiteness. This integrated analytical strategy underscores a modern paradigm for method development that harmonizes scientific rigor with green chemistry principles. The proposed method is thus suitable for routine quantification of EGA, SPA, and SGA in biological or food matrices and offers a scalable platform for future high‐throughput applications in pharmaceutical and nutraceutical analysis.

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