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 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.

Figure 1

Synthesis and structure of LMWH (enoxaparin). It is produced by optimized cleavage of the of Heparin from porcine intestinal mucosa is converted to its benzathonium salt, then to its benzyl ester and is chemically β-eliminated by alkaline treatment affording enoxaparin as a sodium salt with a mean molecular weight of 4000–4500 Da with approximately 90% of the oligosaccharides are within the range of 2000–8000 Da. The structures of a major and a minor enoxaparin chain are shown.

Figure 2

A. Total ion chromatogram TIC of intact LMWH (triplicate) using ESI HILIC LC-FT MS. Heparin oligomer dp3-dp28 can be separated and FT MS resolved by HILIC LC MS corresponded to the size and charge. B. Selected extracted ion chromatograms (EIC) for the major LMWH structures from dp3 to dp28 demonstrated the highly efficient separation of LMWH and sensitive detection using this HILIC LC-FT MS platform. C. Selected mass spectra and identified major structures. Table 1S in the supplemental materials showed the detailed identification information.

Figure 3

Quantitative comparison of identified anhydro-heparin oligosaccharides from two commercialized LMWH products. Oligosaccharide compositions are given as [ΔHexA, HexA, GlcN, Ac, SO₃].

Figure 4

Quantitative comparison of identified heparin oligosaccharides from two commercialized LMWH products. Oligosaccharide compositions are given as A. [ΔHexA = 1, HexA, GlcN, Ac, SO₃] or B. [ΔHexA = 0, HexA, GlcN, Ac, SO₃].

Figure 5

Relative amount (percentage) of identified 3 major LMWH components.