Validation of a modified alcohol dehydrogenase assay for ethanol determination

Enzymatic assay, based on oxidation-reduction reaction catalyzed by alcohol dehydrogenase, is one of the methods used to determine ethanol concentration. The present study was directed to determine the exact amount of enzyme required to accomplish oxidation-reduction reaction so that the concentration of ethanol in the sample can be determined precisely and accurately. Results of the present study indicate that the lowest unit activity of the enzyme that can be used for ethanol determination is 4000 units/mL, even though longer incubation time compared to the original method was used to ensure reaction completion. Validation of the method confirmed that the assay have acceptable linearity range within 0.01 - 0.06% (v/v) of ethanol with correlation coefficient of 0.9999. Both accuracy and precision parameters fulfill the Association of Analytical Communities (AOAC) International requirement, and therefore can be accepted as a quantitative analysis method. Limit of detection and limit of quantitation for the modified method were 0.0017% (v/v) and 0.0056% (v/v), respectively.