Determination of Seven Human Milk Oligosaccharides (HMOs) in Infant Formula and Adult Nutritionals: First Action 2022.07

Abstract

Background: Human milk oligosaccharides (HMOs) are important components of breast milk and may be responsible for some of the benefits of breastfeeding, including resistance to infections and the development of a healthy gut microbiota. Selected HMOs are now available for addition to infant formula, and suitable methods to control the dosing rate are needed.

Objective: To develop and validate a suitable method for the analysis of HMOs in infant formula.

Method: A method was developed for the determination of seven human milk oligosaccharides (2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose (6'SL), 2',3-difucosyllactose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT)) in infant formula and adult nutritionals. The oligosaccharides are labeled at their reducing end with 2-aminobenzamide, separated by liquid chromatography and detected using a fluorescence detector. Maltodextrins are enzymatically hydrolyzed before analysis to prevent potential interference; likewise, an optional β-galactosidase treatment can be used to remove β-galactooligosaccharides. Fructooligosaccharides or polydextrose do not generally interfere with the analysis.

Results: The method has been validated in a single laboratory on infant formula and adult nutritionals. The seven HMOs were spiked into eight matrixes at three or four spike levels, giving a total of 176 data points. Recoveries were in the range of 90.9-109% in all cases except at the lowest spike level in one matrix (elemental formula), where the LNT recovery was 113%, the LNnT recovery was 111%, and the 6'SL recovery was 121%. Relative repeatabilities (RSD(r)) were in the range of 0.1-4.2%. The performance is generally within the requirements outlined in the Standard Method Performance Requirements (SMPR®) published by AOAC INTERNATIONAL.

Conclusions: The method developed is suitable for the determination of seven HMOs in infant formula and demonstrated good performance during single-laboratory validation.

Highlights: A method has been developed that is suitable for the determination of seven HMOs in infant formula.