Determination of Seven Human Milk Oligosaccharides (HMOs) in Infant Formula and Adult Nutritionals, First Action: 2025.08

BACKGROUND: The introduction of human milk oligosaccharides (HMOs) as ingredients in infant formula requires that suitable methods are available for their determination. Three methods were proposed to AOAC INTERNATIONAL for this analysis. Of the three, two methods (AOAC 2022.02 and 2022.07) were essentially based on the same approach. OBJECTIVE: To conduct multi-laboratory testing (MLT) with the consolidated method from AOAC methods 2022.02 and 2022.07 for analysis of Human Milk Oligosaccharides (HMOs) in Infant Formula and Adult Nutritionals. METHODS: The optimal procedural steps and settings were taken from each method to provide a method that uses ultra-high performance liquid chromatography (UHPLC) with 2-aminobenzamide (2AB) derivatization followed by hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection for quantitative analysis of seven HMOs in infant formula and adult nutritionals. RESULTS: A summary of the method parameters selected from each method along with reasoning is provided, as well as a comparison study of the previous methods. The data demonstrated that results do not differ from those of the previous methods, supporting appropriate trueness. The data also suggests the precision of the new method is likely comparable to the parent methods. CONCLUSION: The method was reviewed by the AOAC Expert Review Panel on Nutrients and determined that the method provided trueness to the previous methods, and additional data will be collected within the MLT to further support precision. HIGHLIGHTS: The method was granted AOAC First Action Official MethodsSM status.