Critical Evaluation and Validation of a High-Throughput Microplate-Based Cupric Reducing Antioxidant Capacity Method...

The cupric ion reducing antioxidant capacity (CUPRAC) assay, originally developed to measure the antioxidant capacity of nutritional products spectrophotometrically, utilized water as the solvent for Trolox. Due to the limited solubility of Trolox in aqueous solutions, the optimization of the solvent system was investigated to enhance analytical performance. Solvent combinations consisting of methanol, ethanol, and water were evaluated to identify the mixture that ensures complete dissolution and maximum absorbance signal, using a ternary plot diagram and mathematical modeling. A methanol/water ratio of 0.64:0.36 was identified as the optimal solvent composition. Under these conditions, the CUPRAC assay demonstrated a linear range of 0-50 μM, a limit of detection of 0.91 μM, and a limit of quantification of 2.75 μM. Precision, expressed as the coefficient of variation, was below 5%, and accuracy-defined as the deviation between nominal and back-calculated concentrations-remained within ±7.0%, in accordance with the variation range recommended by the International Committee on Harmonization. The estimated molar absorption coefficient at the optimized solvent ratio (ε_Trolox = 2.62 × 10⁴ L mol^-1 cm^-1) was applied to determine the antioxidant capacity of fish commercial feed ingredients containing a mixture of rosemary and olive extracts.