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

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Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method HMO-04

Title: Validation of an LC-MS method for the analysis of milk oligosaccharides Author: Triskeliion Reviewer Name: Reviewer 2

Summary of Method: A UPLC-MS/MS method for quantification of multiple human milk oligosaccharides (HMO) in milk powders is described. Stable isotope labeled 2-aminobenzamine (2-AB) was used for the preparation of HMO internal standards. The HMOs were derivatized by means of 2-AB labeling. The method has been validated in a single laboratory on 3 types of products (two commercially available infant formula and one unknown product). Method Scope/Applicability: Determination of human milk oligosaccharides in milk powders and milk. Seven HMO are quantified. However, it is not clear whether the method was initially designed for 2’FL and LNnT quantification and during the progress of the work additional HMOs were added in the General comments about the method: The UPLC-MS/MS approach for HMO quantification is very interesting. Especially the use of the multiple reaction monitor (MRM) technique that allows quantification of compounds within complex matrices. The highly specific MRM technique has been widely applied in absolute quantification of proteins and metabolites and - although limited – also in HMO quantification (e.g. Xu et al. J. Nutr. 2017 ). One of the advantages is that the applied technique can easily be expanded to other target compounds, if necessary. Despite this, several points should be addressed in the current method. Among other, the method needs editing to comply with AOAC format, as well as additional information regarding sample preparation, analysis steps, data calculations, supporting figures, safety precautions, additional references etc. should be provided.

Method Clarity: The method is not easy to read and to follow the work performed.

Pros/Strengths: • Determination of more than one HMO in one run

• UPLC-MS/MS and particularly the MRM technique is advantageous for complex matrices. • 2-Picoline-borane replaced sodium cyanoborohydride in the reductive amination step. • Easy to extend when additional HMOs are of interest.

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