Quantitative determination of human milk oligosaccharides in faecal matter

Human milk oligosaccharides (HMOs) are a major component of human milk and colostrum, yet they are non-digestible and thus not utilized directly by the infant. Nevertheless, they are important for infant health and development and have been implicated in immune development, pathogen deflection, cognitive development and the development of healthy microbiome. To understand how HMOs may be utilized it is important to be able to measure them both in milk and faeces. Many methods for the determination of HMOs in milk have been published. However, there are fewer reports of methods describing the quantitative determination of oligosaccharides in faeces. Here we report a validated method for the determination of 30 oligosaccharides in faeces. Oligosaccharides are labelled with 2-aminobenzamide and determined by liquid chromatography with fluorescence detection. The method precision determined as relative standard deviation under intermediate reproducibility conditions is below 12 % for all of the oligosaccharides. Recoveries were in the range 86.6-115 % for the 8 oligosaccharides for which quantitative standards were available, and are estimated to be in the range 81-117 % when using 2'-fucosyllactose as a universal calibrant assuming equimolar response factors of the 2-aminobenzamide labelled oligosaccharides.