pH-switched single-assay spectrophotometric determination of xanthine and hypoxanthine based on urate-mediated...

Enzymatic assays based on oxidase/peroxidase coupling usually assume quantitative conversion of enzymatically generated hydrogen peroxide into the optical product. Here, we show that this assumption fails in the xanthine oxidase-horseradish peroxidase-Amplex Red system because urate, the final product of xanthine and hypoxanthine oxidation, is not a silent product. Urate competes with Amplex Red in the peroxidase cycle, decreasing the apparent yield of resorufin formation; for xanthine, this yield was only 0.40 ± 0.01, independently of pH. A mechanistic mathematical model was developed to relate substrate oxidation, hydrogen peroxide generation, radical reactions, and final absorbance. The model explains the reduced sensitivity observed for xanthine, the different response regimes found for hypoxanthine, and the residual urate concentration after reaction completion. This understanding was then used to design a sequential single-assay spectrophotometric method for the individual determination of xanthine and hypoxanthine, two metabolites that are commonly measured together in enzymatic freshness assays. Xanthine is quantified first at pH 6, where hypoxanthine oxidation is negligible, and hypoxanthine is subsequently determined in the same recording after increasing pH and xanthine oxidase concentration. Application to refrigerated cod extracts gave recoveries of 104 ± 13% for xanthine and 90 ± 12% for hypoxanthine, supporting the feasibility of the approach for freshness-related analysis and highlighting the broader need to consider product-driven effects in oxidase/peroxidase-coupled assays.