Top-down approach for the direct characterization of low molecular weight heparins using LC-FT-MS

Anal Chem. 2012 Oct 16;84(20):8822-9. doi: 10.1021/ac302232c. Epub 2012 Sep 26.


Low molecular heparins (LMWHs) are structurally complex, heterogeneous, polydisperse, and highly negatively charged mixtures of polysaccharides. The direct characterization of LMWH is a major challenge for currently available analytical technologies. Electrospray ionization (ESI) liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for the characterization complex biological samples in the fields of proteomics, metabolomics, and glycomics. LC-MS has been applied to the analysis of heparin oligosaccharides, separated by size exclusion, reversed phase ion-pairing chromatography, and chip-based amide hydrophilic interaction chromatography (HILIC). However, there have been limited applications of ESI-LC-MS for the direct characterization of intact LMWHs (top-down analysis) due to their structural complexity, low ionization efficiency, and sulfate loss. Here we present a simple and reliable HILIC-Fourier transform (FT)-ESI-MS platform to characterize and compare two currently marketed LMWH products using the top-down approach requiring no special sample preparation steps. This HILIC system relies on cross-linked diol rather than amide chemistry, affording highly resolved chromatographic separations using a relatively high percentage of acetonitrile in the mobile phase, resulting in stable and high efficiency ionization. Bioinformatics software (GlycReSoft 1.0) was used to automatically assign structures within 5-ppm mass accuracy.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Anticoagulants / analysis*
  • Carbohydrate Sequence
  • Chromatography, Liquid / methods
  • Computational Biology / methods
  • Enoxaparin / analysis
  • Fourier Analysis
  • Heparin, Low-Molecular-Weight / analysis*
  • Hydrophobic and Hydrophilic Interactions
  • Molecular Sequence Data
  • Software
  • Spectrometry, Mass, Electrospray Ionization / methods
  • Swine


  • Anticoagulants
  • Enoxaparin
  • Heparin, Low-Molecular-Weight