Development and validation of an economical uric acid-Fe3+/Fe2+-ferrozine-based colorimetric assay to estimate uric acid level of pure and biological samples

ABSTRACT

This work presents the development and validation of a simple, rapid, and cost-effective spectrophotometric method for quantitative analysis of uric acid in biological samples. The method relies upon uric acid-led reduction of Fe(III) to Fe(II) of sample/standard solutions which stoichiometrically engages ferrozine to form a magenta-colored complex. Different parameters including pH, metal and chelator concentrations, temperature, etc., were optimized for the maximum intensity and stability of the complex. The uric acid concentrations of synthetic/plasma solutions were determined by comparing the color intensity of Fe(ferrozine)₃ ²⁺ complex produced by test solution with the standard curve formed by known uric acid concentrations. The method was validated in accordance with ICH guidelines and subjected to human plasma analysis. The results obtained were compared with a reference (enzymatic) method which revealed that there was no significant difference between the two methods at 95% confidence level. The method is highly specific, precise, linear, accurate, and robust.