Expert Review Panel for Kombucha Tea

AOAC Kombucha ERP Reviewer: Blake Ebersole KOM-05 Stenerson method review – HP-SPME-GC-MS

Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing. * The method is a headspace GC-MS method with a solid phase microextraction step. The method is expected to be applicable for the SMPR. Headspace sampling for volatile substances like ethanol is a proven way to reduce matrix interference versus direct injection methods. Solid Phase Microextraction is a newer way to reduce the likelihood of matrix interferences. GC-MS is highly sensitive in its separation and detection of pure compounds from complex mixtures. Potential limitations of the method include a potential for incomplete extraction of analyte through the microextraction step, and the saturation and non-linearity of the MS detector at higher concentrations. 2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR. * The instrumentation used would be expected to meet these SMPR for ethanol in kombucha. The method includes a number of separation and purification steps that are advanced compared to what has been traditionally used for alcohol in beverages in terms of sensitivity and eliminating potential matrix interferences. The authors also used deuterated ethanol as an internal reference standard to more accurately determine recovery of spiked ethanol in fermented samples. A dilution factor of 10:1 was applied across all standards and samples to correct for potential linearity issues due to detector saturation at higher concentrations. The study materials included certified reference materials for beer and ethanol/water. The study materials used to calculate method repeatability (%RSD) did not include kombucha that contains naturally high amounts of alcohol, but did use a low-alcohol kombucha spiked with known amounts of pure ethanol. It is possible that the matrices represented in the repeatability analysis do not reflect the range of possible types of kombucha as defined in the SMPR. For example, a higher alcohol, unfiltered kombucha may have different amounts of solids like yeast and bacteria which can be matrix interferences. The authors did perform duplicate analysis on 13 commercial kombucha samples, of which most were high alcohol. Percent differences on duplicate measures of these samples were calculated, that can be used to support the repeatability data. There were also several ruggedness tests performed that are not required by many validation guidelines. The use of different microextraction columns, GC column temperatures, and diluents were studied to determine their impact on linearity and precision. The authors also used a deuterated ethanol internal standard which can help to distinguish between naturally occurring alcohol and a reference spike. 3. Terminology –