SPSFAM Allergens ERP

7. Any general comments about the method?

The following aspects of the method (summary, presentation of the topic, definition of research objectives, presentation of research methodology and detailed presentation of method) are described in a satisfactory manner. My remarks to the AOAC Candidate Method #ALL-02 are as follows: Page 9, LC-MS/MS Analysis: 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 (balancing of the chromatographic system). Page 47, AOAC LC/MS allergen detection supplemental data for ImmunogenX (Aug 18/2016), Table 1 Procedure followed for the SPE of the PTC digested food samples, line specifications/procedure: ‘rotoevaporate to dryness’ What was the duration of this step of the procedure? Please add the information, e.g., no more than 2 minutes. In my opinion: Too long drying is not recommended on the grounds that it may cause the loss of analytes. Page 58, AOAC LC/MS allergen detection supplemental data for ImmunogenX (Aug 18/2016), Figure 6 Peptide recovery for the listed allergen marker peptides from water and a chocolate matrix using a strong anion exchange (SAX), C18 and strong cation exchange (SCX) SPE cartridge: In the figure, ranges of RSD values should also be marked (in the same manner as in Figure 8 on p. 60) Additional remarks: Page 7, Apparatus, pH Meter/pH paper: ‘pH indicator strips’ The authors should elaborate on this phrase. In my opinion, the phrase ‘pH indicator strips’ is not precise enough. Page 8, Sample and Test Portion Preparation: ‘The ground material is frozen at -20°C and then placed in a cryogenic grinding chamber with a magnetic bar.’ If possible, please add the details about the magnetic bar (e.g., the kind, the manufacturer). Page 9, Sample and Test Portion Preparation, The third sentence from the end (before the next part ‘Determination’): ‘Transfer 200 µL to a 96 well taking only the upper layer avoiding transfer of any solids.’ What was the pipette used – what was the maximum value of the volume? Alternatively, in your answer you can include words such as : 'using a pipette, eg. 200 ul' (add the manufacturer). Analytical methodology can be implemented by specialist inspectorates and other services dealing with the routine check of allergens at their trace level. Innovative procedure of separating and quantitative analysis of hidden allergenic compounds can be used for the correct identification and quantitative analysis of the allergens, also by the European Food Safety Agency (EFSA).