Rapid Method for Quantitation of Seven Human Milk Oligosaccharides in Infant Formula and Premix

As the evidence supporting the beneficial effects of human milk oligosaccharides (HMOs) grows, so does the commercial interest in their inclusion in infant formula products. This also requires analytical methods capable of their quantification from finished infant formula products as well as from premixed ingredients in some cases. The objective of the present study was the development and single-laboratory validation of a method that can be used for this purpose for seven HMOs: 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), difucosyllactose (DFL), 3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT). The present method uses labeling by reductive amination, with 4-aminobenzoic acid ethyl ester (benzocaine) as the labeling reagent and picoline borane as the reducing agent, then applies HPLC separation with UV detection. The seven HMOs could be analyzed from infant formula and premix samples with recoveries between 91-108%, relative standard deviations of 4.3% or lower across all replicates, and limits of quantitation between 0.001% and 0.004% of powder sample by weight. The method was found to be rapid and reliable, with a runtime of only 14 minutes per injection, in contrast to other methods found in literature which typically use nearly or more than an hour. In addition, it uses instrumentation that's readily available in most analytical laboratories.