Simultaneous screening and quantitation of human milk oligosaccharides by liquid chromatography-Mass spectrometry

Human milk oligosaccharides (HMOs) are the third most abundant solid component in human milk (HM) and play a vital role in shaping the gut-microbiota and modulating neonatal immunity. Despite identifying 162 HMOs, clinical research primarily focused on a few. High throughput methods that encompass a broader range of HMOs for clinical studies remain limited. This study aimed to develop a novel method for simultaneously quantifying seven key HMOs (i.e., 2 '-fucosyllactose, 3 '-sialyllactose, 6 '-sialyllactose, lacto-N-tetraose, lacto-Nfucopentaose I, lacto-N-difucohexaose I, disialyllacto-N-tetraose), and screening a broad HMO panel. The method utilizes hydrophilic interaction liquid chromatography coupled to mass spectrometry in polarity switching mode, and MS/MS annotation against the NIST Milk Oligosaccharide MS Library. Analysis of 40 HM samples pre- and post-pasteurization identified 110 HMOs. Additionally, the use of Feature-Based Molecular Networking for novel HMO structure identification is introduced. However, higher MSn fragmentation dimensions and complementary analytical techniques are necessary to enhance annotation confidence, structural assignments, and standardize HMO isolation. These findings highlight the diversity of HMOs and suggest implications for HM banks, where HMO profiles and maternal secretor phenotypes of donors are often unexamined.