Determination of Ethanol Content in Water Kefir Using Headspace Gas Chromatography With Mass Spectrometry Detection: Matrix Extension and Methanol Characterization.

BACKGROUND: Water kefir is a fermented beverage using water, sugar, and cultured microorganism grains as the primary ingredients. Ethanol may be present at varying levels within the final product due to the fermentation process, so it is vital to have a validated method to meet regulatory, quality, and safety requirements. OBJECTIVE: This study describes using water kefir as a matrix for the evaluation of the previously validated method employing headspace gas chromatography mass spectrometry (HS-GCMS) detection for ethanol in kombucha. The study objective is to demonstrate the method originally using kombucha is also fit for the analysis of water kefir. This method will also evaluate the determination of methanol within the water kefir samples. METHOD: The matrix extension study was performed as per the AOAC INTERNATIONAL guidance documents outlined in Appendix K: Guidelines for Dietary Supplements and Botanicals using HS-GCMS for ethanol determination. Ethanol determination in each water kefir sample is quantified against an external standard calibration curve. The same instrumentation is used for methanol characterization. RESULTS: RSDr and HorRat values obtained for from the study demonstrated acceptable precision with RSDr values of 1.03 to 6.68% and HorRat values determined to be between 0.23 and 1.52 for ethanol determination within kefir samples. Similarly, acceptable values of RSDr ranging from 1.45 to 3.39% and HorRat ranging from 0.25 to 0.49 were observed with methanol determination. For methanol determination, the limit of detection (LOD) and limit of quantification (LOQ) determined for the method in this study to be 16 and 21 ppm, respectively. The methanol spike recovery study gave overall recoveries ranging from 89 to 91%, demonstrating acceptable method accuracy. CONCLUSIONS: The results of this study demonstrate the previously validated HS-GCMS method for ethanol determination in kombucha can also be used to quantify ethanol in water kefir samples. The method is also suitable for the determination of methanol within water kefir samples. HIGHLIGHTS: A straightforward method has been adapted to include the the quantification of ethanol and methanol in fermented beverages such as Water Kefir samples.

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

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.

Determination of Ethanol Content in Kombucha Products by Gas Chromatography with Flame Ionization Detection: A Multilaboratory Study.

Background: Kombucha is a nonalcoholic, fermented tea beverage that has recently received negative attention because of documented concentrations of ethanol in excess of allowable limits of ≥0.5% alcohol by volume (ABV). Objective: Our previously reported headspace GC with flame-ionization detection (GC-FID) method was adopted by the AOAC Expert Review Panel as First Action Official MethodSM 2016.12 in September 2016 based on published single-laboratory validation study results. This paper describes the corresponding multilaboratory study using this method to further validate its performance parameters. Methods: Four laboratories participated in the study and received practice samples, test samples, reference standards, and detailed protocols. Eight kombucha samples sent out to laboratories were randomly assigned sample numbers and were blinded in terms of content and identity. Each laboratory analyzed all samples using the GC-FID method and reported their results. Results: Cochran's C-test and single and double Grubbs' tests were used to identify and remove outliers. Horwitz ratio values for all samples were between 0.5 and 1.7. As per the Standard Method Performance Requirements (SMPRs®), all samples within the analytical range of 0.1-2.0 ABV% had RSDR values <6%. Conclusions: The results from this study demonstrate the evaluated GC-FID method meets the SMPR requirements and is fit for purpose for detecting ethanol in kombucha products. Highlights: Kombucha, a nonalcoholic, fermented beverage, has been found to contain ≥0.5% ABV. First Action Official Method 2016.12, a headspace GC-FID method for determining ethanol in kombucha, is supported by a multilaboratory study.