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

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 labelled 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 in to eight matrices at three or four spike levels giving a total of 176 data points. Recoveries were in the range 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 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 which is suitable for the determination of seven HMOs in infant formula.