AOAC SPIFAN Nutrients ERP Reviewer Forms (December 7, 2022)

FOR EXPERT REVIEW PANEL USE ONLY NOT FOR DISTRIBUTION

General comments: - Inconsistency is observed in the abbreviations within the text and when the HMOs are abbreviated. The same abbreviations should be used throughout the text. - The correct use of “prime” in the HMO (and carobyhdrates generally speaking) should be used. Example: 2’FL instead of 2FL, 3’SL instead of 3SL etc but 3FL and not 3’FL. The correct abbreviations and compound names are provided in the SMPR. - LNnT and LNT: the N is officially in italics when the compound is written (e.g. lacto- N -tetraose) - LLOQ should be LOQ - There is no definition of the QC A and QC E provided. These appear in the sample preparation chapter. - Introduction: should be reworded and additional (scientific) references to be provided. The goal of the study (“… Triskelion will be able to provide this service to producers of ….” ) as presented is not corresponding to the type of document. Should be revised. Suggested would be to also avoid providing names, product names or brand names. - More details about the technology, advantages regarding other methods, as well as system suitability, adjustments etc. - The SMPR section should be updated. All SMPRs are available and published. - It seems the method was developed based on the draft 2’FL and LNnT SMPRs. Reconsideration of the requirements for the other HMO is recommended. - The validation was performed on mainly QC samples based on one blank matrices and two QC E samples. Consideration of adding more blank matrices and matrices containing HMOs? - Materials and methods: Reading the chapter was not easy. Detailed information and description to be provided, while assuring AOAC format. Support the text with tables. - The reconstitution described samples made from 2.5g milk powder and addition of 20g MQ . From the suspension 100 µ l were taken. The SMPR describes reconstitution of 25g IF. How was homogeneity assured? - Synthesis of the 13 C labeled 2-AB. Is the internal standard commercially available and other CROs can provide the same molecule? Additional information to be provided. - Equations for data calculations should be provided - Although MS and the MRM technique can be selective, there is not much mentioned about selectivity of the method. Additional information to be provided. See SMPR for details about the requirements. Interference of other components? (GOS, FOS, maltodextrin, etc.?) - Provide typical chromatograms of HMO separation, spectra etc that will help understand the method. - Review the acceptance criteria and make sure to consider the SMPR requirements for each HMO. Recommendation: Kleinnijenhuis and Elwakiel submitted a method and its single laboratory validation for the determination of Human Milk Oligosaccharides in milk and milk powders. Both the UPLC-MS/MS and the MRM technique can be seen as advantageous regarding HMO quantification, specifically because of their applicability in complex matrices as milk. After reviewing the submitted method, it was concluded that not all defined SMPR requirements have been met. Further, revision of the document is advised so the AOAC format and requirements are met. Therefore, I do not recommend moving the method for first action, before the above has been considered.

5