A cost-effective colourimetric assay for quantifying hydrogen peroxide in honey

Honey is a natural product with many beneficial properties including antimicrobial action. Production of hydrogen peroxide (H₂O₂) in diluted honey is central to this action. Here, we describe an optimized method for measuring levels of H₂O₂ in honey. This method is based on established methods, with the level of dilution, the time between dilution and reading the assay, and aeration of the samples during the assay identified as critical points for ensuring reliability and reproducibility. The method is cost-effective and easy to perform using common laboratory equipment. Using this method, we quantified the hydrogen peroxide content of five different, unprocessed polyfloral honeys collected in NC, USA. Our results show that H₂O₂ production by these honeys varies greatly, with some samples producing negligible levels of H₂O₂. We assessed the effect of colour on the assay by measuring the recovery of spiked H₂O₂ from light and dark honey and from serially diluted dark corn syrup, and found the amount of H₂O₂ that could be detected was lower in dark corn syrup and darker honey samples.