Expert Review Panel for Kombucha Tea

No standard reference materials or certified reference materials listed in the SMPR were used. 200 proof ethanol from Sigma, listed in SMPR was diluted for the calibration curve and spike. Lack of use of standard or certified reference materials could impact the robustness of the method. Only 4 kombucha samples from 2 manufacturers were included in the study

3. Is there information demonstrating that the method performs within the SMPR Method Performance Requirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified. * There is information demonstrating the method performs to SMPR, with some gaps: 1. Range of analyte concentrations from kombucha did not bracket the required analytical range. The highest concentration of kombucha measured at 1.6%, lower than the 2% listed in the SMPR. 2. The recovery of ethanol in commercial kombucha was reported in table 5 as approximately 0.1, 0.5, 0.5 and 0.7. These values differ from those in table 8 on the same brands (0.3-1.6 % ABV). The differences conflict with the recovery data which is very tight. Request some explanation. IV. General Submission Package 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method? * Some of the controls on the assay procedure were not entirely clear (see following section) 2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. * --It was stated that ‘approximately 2 mL’ is taken and filtered and vortexed before dilution. The method indicated an ‘appropriate’ dilution should be made, in this study it was a 1:200 dilution. There was no indication of whether the 1:200 or other appropriate dilution should be made volumetrically, and the precision or calibration of the volumetric transfer. -- The controls for determining the end of the reaction lacked some clarity: Sec 1.9 of procedure reads: “Mix and read the absorbances of the solutions at the end of the reaction (Approx 10 minutes). If the reaction has not stopped after 10 minutes, continue to read the absorbances at 1 minute intervals until the absorbances increase constantly over 5 minutes**. ** If this creep rate is greater for the sample than for the blank, extrapolate the sample absorbances back to the time of addition of suspension 4.” Could we request some clarification on how the analyst is to know the endpoint of the reaction, and what does a ‘constant increase’ in absorbance look like? -- The method did not determine whether acetic acid or acetaldehyde expected to be found naturally in kombucha samples (and which are reactants in the enxymatic reaction) might interfere with the reaction or the results. --The method states that cross-reactivity with methanol is not an issue, but did not present data demonstrating this.