SPSFAM Allergens ERP

1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. 2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method. 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. 3. Is there information demonstrating that the

YES: The definitions specified in the SMPR were used and applied appropriately in the supporting documentation (manuscripts, method studies, etc.).

YES: There is a piece of information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Material stated in the SMPR.

YES: There is a piece of information demonstrating that the method performs within the SMPR Method Performance Requirements table specifications for all analyses in the SPMR applicability statement. In the method proposed by the authors, the specification of the following data according to the SMPR Method Performance Requirements table specifications is as following: Calibrations were performed over a range of 1-1000 ppm for each allergen. The calibration linearity was greater than 0.9978 with calibration residuals within 13% of the actual value over the calibration range. Table 1 presents the MQL and MDL for the selected allergen marker peptides. The MQL met the Method Performance Requirements and equalled 1 on the basis of the calibration residual results. Going to 0.3 ppm resulted in calibration residuals above 20% difference from the actual level. The MDL’s ranged from 0.1 to 0.05 based on a signal to noise of 3:1 extrapolated from the 1 ppm calibration level. Spike recoveries for egg, hazelnut, milk and peanut in the allergen matrix combinations listed in the AOAC SMPR 2016.002 method performance document was performed a 5 ppm and 50 ppm. The Tables 2 and 3 list the recovery results for each specified allergen matrix combination. The 5 ppm spike recovery data ranged from 75-101% with a standard deviation of 15% or less. The 50 ppm spike recovery data ranged from 90-104% with a standard deviation of less than 12%. The repeatability of the method was tested on seven separate extractions-digestions and LC/MS/MS analysis of the food allergens in a cookie at 5 ppm. Table 5 presents the results for one peptide for each allergen. The repeatability ranged from 4.5-9.4 percent standard deviation for the four allergens.

I provided my additional remarks in the part ‘7. Any general comments about the method?’

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?

In my opinion, there is no need to implement any additional steps in the method evaluated.