Top-Down Approach for the Direct Characterization of Low Molecular Weight Heparins Using LC-FT-MS

Abstract

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 by 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 (GlycerSoft 1.0) was used to automatically assign structures within 5-ppm mass accuracy.

Glycosaminoglycan Glycomics Using Mass Spectrometry

“…More highly sulfated oligosaccharides are likely to irreversibly bind to the stationary phase. Hydrophilic interaction chromatography MS has been used to profile both CS and HS/heparin oligosaccharides (14,(53)(54)(55)(56). The hydrophilic interaction chromatography dimension separates oligosaccharides according to size and polarity.…”

Sequence Analysis and Domain Motifs in the Porcine Skin Decorin Glycosaminoglycan Chain

“…on similar conditions to those used for the analysis of intact chains of low molecular weight heparin (46,47). Briefly, a Luna HILIC column (2.0 ϫ 150 mm, 200 A, Phenomenex, Torrance, CA) was used to separate the partially or fully digested decorin GAG.…”

USP compendial methods for analysis of heparin: chromatographic determination of molecular weight distributions for heparin sodium

“…Liquid chromatography with mass spectrometry has been used to profile heparin preparations [8][9][10]. The technique is capable of resolving up to about 20-mers; for larger oligomers, overlapping MW patterns prevent interpretation of the data.…”

“…mass spectrometry [9], or the UV/refractive index ratio of a sample prepared by beta-elimination [18,19]). For unfractionated heparin, the most widely implemented method is SEC with refractive index and light scattering detection [20,21].…”