Quantification of Human Milk Oligosaccharides

fucose, 2′-FL, and 3-FL were quantified from the simulation units to which HMOs were added as per Salli et al. [26 (link)] with modifications. Standard solutions of fucose (Sigma-Aldrich, St. Louis, MO, USA), 2′-FL, and 3-FL were prepared in water to concentrations of 80, 60, 40, 20, and 10 mg/l and stored at +4 °C. Sample solutions were centrifuged at 16,000× g for 5 min; then, 50 μL of the supernatant and 200 μL of ethanol were mixed in a microcentrifuge tube and incubated at +4 °C for 30 min. After centrifugation at 16,000× g for 5 min, 200 μL of the supernatant was evaporated to dryness at 30 °C under stream of nitrogen, and the solid residue was dissolved in 2000 μL of water and filtered. Separation and detection of the analytes were performed using high-performance anion-exchange chromatography as previously described [26 (link)]. Retention times of fucose, 2′-FL, and 3-FL were 6.3 min, 26 min, and 18 min, respectively.