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

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.