SPSFAM Allergens ERP

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. 4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions. 5. Based on the supporting information, what are the pros/strengths of the method?

In my opinion there is no need.

YES: There are.

YES: There are information demonstrating that the method system suitability tests and control as specified in the SMPR worked appropriately and expected.

The method is well described and substantively prepared. The project of the method is well integrated and includes a clear and concise description.

The developed method was evaluated following the definitions of AOAC SMPR 2016.002 with respect to Method quantitation limit (MQL), Method detection limit (MDL), Linearity, Repeatability, Reproducibility, Recovery. Analytical data was collected for allergen/matrix combinations listed in AOAC SMPR 2016.002. Specificity is another important analytical parameter. Characteristic signature peptides were chosen for each allergen, and MRM transitions for each signature peptide were determined based on their uniqueness compared to background proteins and their sensitivity of detection. For each allergen, multiple unique peptides were chosen out of unique proteins, and two MRM transitions per peptide were chosen.

6. Based on the supporting information, what are the cons/weaknesses of the method?

The cons/weakness of the method may be costs. But I think it is inevitable.

7. Any general comments about the method?

My remarks to the AOAC Candidate Method #ALL-01 are as follows: Page 13, LC-MS Separation: The authors should answer the following question: How long stationary phase was conditioned by mobile phase? Moreover, they should add details about the condition of the stationary phase: - before the analysis, - between each of the analyses in gradient elution mode (if the additional conditioning was applied for balancing of chromatographic system, despite the fact that concentrations of both components of mobile phase on the start and the end of gradient are the same).

I have no additional comments.