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A new 4D analytical method using all-ion fragmentation (AIF) coupled with LC-ESI-IM-MS enables robust identification and quantification of diverse human milk oligosaccharides (HMOs), advancing infant health research.

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Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Human Milk Research

Background:

  • Human milk oligosaccharides (HMOs) are vital for infant development, but their complex structures pose analytical challenges.
  • Existing methods combining ion mobility (IM) with LC-MS lack robustness for analyzing large human milk (HM) cohorts.

Purpose of the Study:

  • To introduce a novel, robust all-ion fragmentation (AIF) LC-ESI-IM-MS method for comprehensive HMO characterization.
  • To enable efficient resolution and quantification of diverse HMO structural isomers, including novel compounds.

Main Methods:

  • Developed a four-dimensional (4D) analytical approach integrating high-resolution LC, IM drift time, accurate mass precursor, and fragment ion measurements.
  • Implemented two strategies: one for in-depth structural elucidation and another for high-throughput analysis of HM samples.

Main Results:

  • Simultaneously identified and quantified up to 200 HMOs with a degree of polymerization up to 13.
  • Discovered five trifucosyl-lacto-N-tetraose (TF-LNT) isomers, four previously undetected in HM, and elucidated their structures.
  • Differentiated HM types and revealed variations in HMOs up to DP 11 across lactational stages using the high-throughput method.

Conclusions:

  • The robust AIF LC-ESI-IM-MS approach facilitates in-depth monitoring and confident identification of a wide range of HMOs.
  • This method advances HMO research by enabling characterization of numerous HMOs in large sample sets, aiding understanding of structure-function relationships.