SPSFAM Allergens ERP

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.

See previous answer indicating no specific system suitability tests apparent.

The method was submitted as two separate documents, which makes it difficult for a reader to understand the final method workflow proposed by the authors. In addition, the method is not presented in a way that would be easy for an end user to read and follow. The authors should submit a more precise, stepwise explanation of the method from start to finish (e.g. including all final parameters for extraction, digestion, clean- up/SPE, LC-MS/MS, and data analysis. The method does not provide sufficient information as to how the authors conducted the data analysis or how an end user would conduct the data analysis. For example, which peptides and transitions were used for quantification? How should an end use choose those? In the transition list table, the authors indicate at least two peptides that would be used for quantification, but the supporting data only shows results from one peptide from each allergenic food. How was that selection made, and how would end users make that selection? Also, one would assume that the authors are calculating the peak areas for the monitored transitions, but no information is given about the parameters the authors used or what end users should do to derive the quantitative information. Did the authors require a minimum number of points across the peak, for example? The method also needs to have substantially more information about how calibration curves were prepared and how end users would prepare calibration curves for the method. What exact material (e.g. lyophilized whole milk or fluid whole milk) was used for the calibration curves and what were the curves prepared in (in water or in some sort of buffer or in matrix-matched extracts)? Was each concentration extracted and prepared separately or were dilutions performed following digestion and SPE cleanup? How do the authors plan to address the presence of target peptides in species other than the target species (hen’s egg peptides in turkey; hazelnut peptide h4 in peach; and Bos Taurus peptides in water buffalo and wild yak? It is possible that this submission could be the beginning of a sound method, but the lack of clarity on several key issues noted in this review, makes it difficult to actually assess the method. The final procedures for the method are unclear, in part due to two separate documents being submitted at different times and, in part, due to a general lack of precision and clarity in the writing and presentation of information. While there are a number of items that need to be clarified, the largest overarching issue with the presentation of the method is the lack of complete, accurate, and clear quantitative units and descriptions of materials used throughout presentation. Clear and accurate units must be provided not only for reviewers to evaluate the effectiveness of the method, but also so that the method delivers correct and usable data to end users. This method requires significant revision on the issue of units as well as general clarity before it should be considered again.

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

7. Any general comments about the method?