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

FOR EXPERT REVIEW PANEL USE ONLY NOT FOR DISTRIBUTION

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: Triskelion Reviewer Name: Reviewer 1

Summary of Method: Powdered, or liquid samples are derivatized with 2-AB and separated by reversed phase chromatography. The separated derivatized HMOs are quantified by MS utilizing a custom synthesized 13 C labeled SIL-2AB IS. Method Scope/Applicability: Determination of 7 HMOs from 2.5-500 mg/100g (as-fed basis) in milk and milk powders. General comments about the method: Not clear how purity of HMO standards is addressed. Materials sourced from Carbosynth are anticipated to range from ~90-98% purity. 6’-SL and 3’-SL are supplied in the form of sodium salts, these details need to be included in the method. This becomes critical when assessing samples rather than spikes derived from the same material used to calibrate the instrumentation. The method needs some editing to comply with AOAC format as well as clarification of exact procedures in order to setup/replicate. Editorial suggestions: • Please note, 3’-FL is not equivalent to 3-FL. The ‘ designation indicates which end of lactose the fucose is attached to. The correct designation is 3-FL.

• Units misspelled in the Figure 01 text • Please provide example chromatograms

Method Clarity:

Some additional detail would be helpful to setup/run this method.

Pros/Strengths: • Method meets SMPR criteria for accuracy within the ranges studied.

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