Characterizing Oligosaccharides in Breast Milk with Liquid Chromatography

In a new study led by researchers at Danone Research & Innovation in Utrecht, The Netherlands, they explored a new system to help better characterize and profile up to 200 human milk oligosaccharides (HMOs) in breast milk. Their research, published in Analytical Chemistry, is based around an all-ion fragmentation (AIF) liquid chromatography-electrospray ionization-IM separation-mass spectrometry (LC-ESI-IM-MS) system.

Breast milk, or human milk, is a standard choice for infant nutrition, since it contains the components needed for optimal infant health and development. The World Health Organization (WHO) says breastfeeding is one of the most effective ways to ensure child health and survival, as it is safe, clean, and contains antibodies that can help protect against several common childhood illnesses (2). Breast milk can provide the energy and nutrients that an infant needs for the first months of life, provide up to half or more of a child’s nutritional needs during the second half of the first year, and up to one-third during the second year of life. Its composition typically adapts to the nutritional, immunological, and developmental needs of the infant throughout lactation.

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After lipids and lactose, human milk oligosaccharides (HMOs) make up the third largest biomolecular fraction of breast milk, carrying out many vital functions for infant health and development. HMOs are largely indigestible, so they typically reach the large intestine, where they carry on three main functions: promoting healthy microbiome development through their probiotic role, modulating the neonatal immune system, and contributing to fighting infections by preventing the adherence of pathogenic bacteria and viruses. Additionally, they can contribute to an infant’s cognitive development.

Many contemporary analytical methods that aim for more detailed HMO characterization combine ion mobility (IM) with liquid chromatography–mass spectrometry (LC–MS); while this enhances structural resolution, these approaches typically lack the robustness needed for being applied to HM cohorts, which can contain hundreds of samples at a time.

In this study, the scientists introduced a novel all-ion fragmentation (AIF) liquid chromatography-electrospray ionization-IM separation-mass spectrometry (LC-ESI-IM-MS) method to better analyze and characterize HMOs. Four analytical dimensions were integrated: high-resolution LC separation, IM drift time, accurate mass precursor, and fragment ion measurements. This 4D analytical characterization proved sufficient for resolving various HMO structural isomers in efficient ways. With this approach, up to 200 HMO compounds, with a maximum degree of polymerization of 13, could be simultaneously identified and relatively quantified.

Two methods were created using this 4D analytical approach; one was intended for in-depth characterization of multiple known (yet novel) HMO structures, and the other designed for more robust, increased-throughput analyses. Using the first approach, five trifucosyl-lacto-N-tetraose isomers (TF-LNTs), four of which were never detected before in HM, as well as additional difucosyl-lacto-N-heaose isomers (DF-LNHs), were revealed, their structures being fully elucidated through AIF and IM. This showed the method’s potential for in-depth characterization of novel complex HMO structures.

The increased-throughput method, which featured a shorter LC gradient, was also applied to real-world HM samples. Using this approach, the scientists could differentiate the HM types I–IV based on a broader range of partly new marker HMOs. They could also derive new insights into variations of multiple and rare HMOs up to DP 11 across lactational stages.

Overall, the AIF LC-ESI-IM-MS approach was able to facilitate in-depth monitoring and confident identification of various distinct HMOs, which range from simple to very complex. This study can help advance HMO research through facilitating characterization of a broad range of HMOs in high numbers of HM samples.

References

Gonsalves, J.; Bauzá-Martinez, J.; Stahl, B.; Dingess, K. A.; Mank, M. Robust and High-Resolution All-Ion Fragmentation LC-ESI-IM-MS Analysis for In-Depth Characterization or Profiling of Up to 200 Human Milk Oligosaccharides. Anal. Chem. 2025. DOI: 10.1021/acs.analchem.4c06081

Breastfeeding. World Health Organization 2024. https://www.who.int/health-topics/breastfeeding#tab=tab_1 (accessed 2025-3-19)