Determination Standardization and Validation of Optimized Spectrophotometric Assays for Total Antioxidant, Polyphenol, and Flavonoid Determination: A Kinetic and Methodological Framework
This study addresses persistent methodological inconsistencies in phytochemical assays by developing a harmonized, high-sensitivity framework for TAC, TPC, and TFC. Using the Ferric Bipyridyl Reducing Capacity (FBRC), Folin-Ciocalteu, and Aluminum Chloride methods, critical parameters—including maximum wavelength (λmax), temperature, kinetics, pH, and stoichiometry—were systematically re-evaluated and optimized. The novelty of this work lies in the transition from empirical "snapshot" measurements to kinetically optimized and AOAC-validated protocols that ensure inter-laboratory reproducibility. Optimized conditions for TAC were established at 520 nm, pH 4, and 30 minutes at 50°C. TPC was optimized at 750 nm (20 min at 50°C), while TFC achieved stability at 430 nm after 10 minutes at ambient temperature. Unlike conventional methods that often suffer from baseline drift, these refined protocols achieved superior sensitivity. Comprehensive validation in accordance with AOAC guidelines confirmed high precision, with repeatability (%RSD) ranges of 0.82–5.68% (TAC), 0.85–6.22% (TPC), and 0.29–5.09% (TFC). Linearity was established over concentration ranges of 0.2–5 ppm for TAC (ascorbic acid equivalent), 1–10 ppm for TPC (gallic acid equivalent), and 2–20 ppm for TFC (quercetin equivalent). All methods demonstrated excellent linearity (R2 = 0.9978) and significantly lower limits of detection (LODs: 0.027, 0.164, and 0.065 ppm) than those reported in the literature. These protocols offer a rapid, cost-effective, standardized alternative to expensive chromatography. By resolving the kinetic and chemical variables that typically cause discrepancies in research, this study provides a validated "gold-standard" approach that ensures the global comparability of phytochemical data across diverse plant matrices.
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