Quantitative determination of trace hydrogen peroxide in the presence of sulfide using the Amplex Red/horseradish peroxidase assay

Abstract

The Amplex Red/horseradish peroxidase (AR/HRP) assay for H₂O₂ is one of the most sensitive and simple approaches for H₂O₂ quantification, which is effected by measuring the highly-fluorescent resorufin formed from oxidation of AR by the oxidizing intermediates generated by reaction of HRP and H₂O₂. The direct reactions of S(-II) with both H₂O₂ and resorufin are too slow to be of relevance on analytical timescales, however, the reaction between S(-II) and the HRP/H₂O₂ oxidizing intermediates is rapid enough to compete with the desired reaction of these oxidizing intermediates with AR, suppressing formation of the resorufin analyte. As this mode of interference can be considered simply a competition between the AR reagent and S(-II) for the intermediate oxidizing species, a simple equation is derived in this work enabling one to correct for this interference and obtain a good estimate of the true H₂O₂ concentration after measuring the apparent H₂O₂ concentration and the S(-II) concentration. This mode of interference is general to any compound that can act as a HRP substrate even if not directly reactive with H₂O₂. As such, the approach described is widely applicable to many potential reducing interferents and opens up the use of the AR/HRP assay to a much wider range of conditions, as well as demonstrating the utility of explicitly considering the mechanism of the analytical process.

Keywords: Amplex Red; Horseradish peroxidase; Hydrogen peroxide; Sulfide.