Expert Review Panel for Kombucha Tea

3. Is there information demonstrating that the

2.2 Analytical Range and limit of quantification (LOQ) The authors have nicely demonstrated linearity of the assay. LOQ Calculations by the authors: 1. The authors may clarify their calculations of LOQ systematically and clearly. Also back calculate the expected mcg of analyte in assay with the calculated LOQ . 2. Table 6 provides data for calibration for ethanol stock solution of 0.065% but not for 0.0395% claimed LOQ of the method. This probably requires further clarification. Recovery Data: 1. The authors have presented their spike recovery data in Tables 1-4, for 4 different Kombucha sample studied. Can results in Tables 1-4 presented as % Ethanol (v/v) so that it’s easy to compare the data across the study. 2. The assay is designed to run a standard (0.05 mg/mL) in batch of the analysis. The authors may clarify the purpose of running standard in the assay. It’s not used in calculation. The calculation employs NADH extinction coefficient in calculation of ethanol depending on number of molecules of NADH generated in the assay. Is it possible demonstrate accuracy of the assay by calculation of ethanol using ΔA values obtained for the standard. 3. The assay analyzed standard in every batch. Is its purpose to monitor the assay performance for the batch? The range of ΔA values (0.5 + ??) may be helpful if the purpose of running standard mentioned in the earlier sentence is correct. 4. It may be pointed out the that ΔA values observed for two of the samples in the study is lower than that obtained for the corresponding standard in the batch. 5. The authors have demonstrated a satisfactory recovery of ethanol in four of the studied Kombucha samples based on a single spike level (Kombucha + Ethanol standard 0.05 mg/mL). Listed % spike Recovery is satisfactory 97.7-101.6%. It will be nice to know that the assay provide satisfactory spike recovery at multiple spike concentrations also. 3.3 Precision/ Repeatability Evaluation: 1. The precision (RSDr) was determined by analysis of 3 replicates in a single day and was found to be satisfactory % RSDr = 0.14-1.63%. 2. The precision of the assay performed over two days by two analyst (total number of replicates n = 4) provided % RSD in the range of 3.1 - 5.2%. This value is higher than Repeatability (RSDr) requirement of the SPMPR (2016.001) which is < 4% and lower than Reproducibility (RSDR) requirements of the SMPR which is < 6%. Precision of the assay as %RSD by different technicians on two different days increased 2-3 fold in comparison when it’s performed by a single analyst in a single day. It is likely that the reproducibility % RSD of the assay observed by the method developers within lab will increase further when the assay is performed by different labs. Thus based on the current data there is a good possibility that the method reproducibility across different laboratories is likely to be above SMPR requirements of < 6%.

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.

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? 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. 3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify.

No

Not very clearly

System suitably not clearly defined