Determination of Ethanol in Kombucha, Juices, and Alcohol-Free Beer by Enzytec Ethanol: Single-Laboratory Validation, First Action 2017.07.

EnzytecTM Liquid Ethanol is an enzymatic test for the determination of ethanol in kombucha, juices, and alcohol-free beer. The kit contains two components in a ready-to-use format. Quantification is based on the catalytic activity of alcohol dehydrogenase, which oxidizes ethanol to acetaldehyde and converts NAD+ to NADH. Measurement is performed in 3 mL cuvettes at 340 nm within 20 min. Samples with alcohol contents around 0.5% alcohol by volume need to be diluted 1:20 or 1:50 with water before measurement. Acetaldehyde interferes at concentrations higher than 3000 mg/L, whereas sulfite interferes at concentrations higher than 300 mg/L. The linear measurement range is from 0.03 up to 0.5 g/L ethanol, whereas LOD and LOQ are 1.9 and 3.3 mg/L ethanol, respectively. Kombucha with concentrations between 2.85 and 5.82 g/L showed relative repeatability standard deviation around 1%, whereas juices were below 2%. Results from a reproducibility experiment revealed that at a concentration of 0.1 g/L, the RSDR was at 2.5%, whereas at higher concentrations between 0.2 and 0.3 g/L, coefficients around 1% were obtained. Trueness was checked by using Cerilliant aqueous ethanol solutions and beer with concentration of 0.4 and 4 g/L (BCR-651 and BCR-652). Spiking of kombucha and juice samples resulted in recoveries between 95% and 104%. Acceptable stability was found for the whole test kit under accelerated conditions at 37°C for 2 weeks. The kit is also not susceptible to short freezing-thawing cycles and harsh transport conditions.