Determination of Ethanol Content in Kombucha Using Headspace Gas Chromatography with Mass Spectrometry Detection: Single-Laboratory Validation

J AOAC Int. 2021 Mar 5;104(1):122-128. doi: 10.1093/jaoacint/qsaa094.


Background: Kombucha is a fermented beverage made with tea, sugar, and a symbiotic colony of bacteria and yeast that is usually marketed as a non-alcoholic beverage. Products must contain <0.5% and <1.1% alcohol by volume in the United States and Canada respectively to be classified as non-alcoholic products. Prior studies have found that Kombucha beverages can become very acidic and may contain levels of alcohol above 1% which can be a potential health risk to children and the developing fetus during pregnancy.

Objective: Given the public safety concerns and legal requirements associated with the level of alcohol within Kombucha beverages, there is a need for accurate and reliable methods. Herein we describe the validation of a sensitive, rapid, and simple Headspace Gas Chromatographic method with mass spectrometric detection for determining ethanol in Kombucha.

Methods: Method performance characteristics measured included linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ) as per AOAC International guideline Appendix K Part 1. Performance was evaluated against the AOAC Standard Method Performance Requirements 2016.001 for determination of ethanol in Kombucha.

Results: The linear dynamic range for this method was confirmed over the range of 0.025 to 2.47% ABV. The LOD and LOQ were determined to be 0.0002% and 0.002% ABV, respectively. With a spike recovery of 102% for accuracy and precision of RSDr ≤ 4% the method met the SMPR requirements within the analytical range.

Conclusions: The results of this validation study demonstrated the method is fit for the purpose of quantifying ethanol in Kombucha and is suitable for rapid and easy integration by laboratories to ensure that regulatory requirements are met.

MeSH terms

  • Canada
  • Child
  • Ethanol* / analysis
  • Gas Chromatography-Mass Spectrometry
  • Humans
  • Laboratories*
  • Mass Spectrometry


  • Ethanol