Simultaneous determination of ethanol and acetaldehyde in liquor using a flow system composed of two enzyme reactors and an octadecylsilica column

Abstract

Simultaneous determination of ethanol and acetaldehyde was performed with an apparatus consisting of two enzyme reactors placed either side of an octadecylsilica column in a single flow line. The enzymes used were alcohol dehydrogenase for ethanol analysis, and aldehyde dehydrogenase for acetaldehyde analysis. The most favorable concentration of NAD+ in the carrier for the simultaneous determination of ethanol and acetaldehyde was studied in wine or sake, in which the ethanol concentration was much higher that that of acetaldehyde. An NAD+ concentration of 0.1 mM was adopted. To distinguish between NADH formed due to ethanol and that formed due to acetaldehyde, several buffers (pH 7.8) were also examined for use as the carrier medium, and triethanolamine buffer was found to be the most favorable. The calibration curve for acetaldehyde was linear (r2 = 1.000) in the range of 0.2-100 μM. With respect to ethanol, plotting the logarithm of the peak area versus that of the concentration gave a linear relationship (r 2 = 0.997) in the range of 0.04-100 mM. This method was applied to the simultaneous analysis of ethanol and acetaldehyde in several types of liquor. The results were similar to those obtained using a commercially available test-kit method, suggesting the reliability and practicality of this method in analyzing real samples.