Validated Multimethod Approach for Full Characterization of 2′-Fucosyl-d-lactose as an Industrially Produced Human Milk Oligosaccharide

Human milk oligosaccharides are of high interest as active ingredients in infant formulas and dietary food supplements. Full characterization of members of this compound class is challenging due to the intrinsic complexity of byproducts during synthesis by fermentation. Moreover, when method validation is targeted for a regulated environment, a robust chromatographic separation of the highly polar oligosaccharides needs to be addressed, including isomers and compounds relevant for potential product adulteration. We present a combined approach of validated chromatography and NMR spectroscopy, which allows for full mass balancing of industrially produced 2'-fucosyl-d-lactose. A combination of NMR spectroscopy, mass spectrometry, and action IR spectroscopy tackles structural elucidation of monoacetylated species as a new class of byproducts.