Enzymic ethanol assay: a new colorimetric method based on measurement of hydrogen peroxide

We describe a new colorimetric method for measuring ethanol in plasma by use of a peroxidase-coupled assay system and alcohol oxidase (EC 1.1.3.13) from Pichia species. Absorptivity is low enough to give useful results without sample dilution. The procedure is also applicable to saliva samples and utilizes only one working reagent. The absorbance of the blue dye that is formed is measured at 600 nm. The standard curve for the method is linear for ethanol concentrations up to 4 g/L. Average analytical recovery of ethanol in human plasma exceeded 99%. Within-run and between-run CVs were less than 2.45% and less than 1.92%, respectively. Results correlate very well with those by gas chromatography (r = 0.9977). The method is adaptable to automation.