Schmidt Madsen_2026_ACS.Omega_11_43526

Formaldehyde and methanol are important compounds in enzymatic reactions, food products, and biological processes. However, sensitive and accurate detection of these compounds remains a critical challenge. The chromotropic acid method can detect formaldehyde and methanol after prior oxidation via formation of a purple chromogen, which is formed when chromotropic acid reacts with formaldehyde in sulfuric acid. The method allows fast analysis within a few minutes and enables accurate quantification of formaldehyde down to low micromolar concentrations. The addition of sodium bisulfite can further enhance the sensitivity of the method by stabilizing formaldehyde. Although the method is highly sensitive and specific for formaldehyde detection, it is not routinely applied in enzyme assay analysis. We demonstrate that the chromotropic acid method is suitable for sensitive quantification of formaldehyde and methanol through oxidation to formaldehyde (catalyzed by alcohol oxidase) in two enzyme assays comprising methanol dehydrogenase and glucuronoyl esterase. Most importantly, we show how the method can be tailored to variable experimental conditions, potentially enabling detection in various sample matrices. We successfully implemented the method for versatile, fast, and sensitive detection of both methanol and formaldehyde.