Characterization of currently marketed heparin products: Analysis of heparin digests by RPIP-UHPLC–QTOF-MS

Wang, Bo; Buhse, Lucinda F.; Al-Hakim, Ali; Boyne, Michael T.; Keire, David A.

doi:10.1016/j.jpba.2012.04.033

Cited by 40 publications

(37 citation statements)

References 39 publications

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“…29,35–37 Unfortunately, RPIP-LC has not been widely used because the volatile ion-pairing reagents, required in this analysis, contaminate instruments and the required mobile phase does not provide sufficient sensitivity, resolution, and mass range needed for the thorough characterization of LMWHs.…”

Section: Introductionmentioning

confidence: 99%

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

Zhang²,

et al. 2012

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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.

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Section: Introductionmentioning

confidence: 99%

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

Zhang²,

et al. 2012

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“…To circumvent complications involved in the separation and characterization of higher oligosaccharides, heparins are currently profiled in terms of their disaccharide composition, most often through analysis of exhaustive digests with a cocktail of heparinases. These digests consist mainly of unsaturated disaccharides, which are usually separated by chromatographic or electrophoretic methods and identified through comparison with authentic samples (32–36). …”

Section: Introductionmentioning

confidence: 99%

Profiling glycol-split heparins by high-performance liquid chromatography/mass spectrometry analysis of their heparinase-generated oligosaccharides

Алексеева

Casu

Torri

et al. 2013

Analytical Biochemistry

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Glycol-split (gs) heparins, obtained by periodate oxidation / borohydride reduction of heparin currently used as anticoagulant and antithrombotic drug, are arousing increasing interest in anti-cancer and anti-inflammation therapies. These new medical uses are favored by the loss of anticoagulant activity associated with glycol-splitting-induced inactivation of the antithrombin III (AT) binding site. The structure of gs-heparins has not been studied yet in detail. In this work, an ion-pair reversed-phase chromatography (IPRP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) widely used for unmodified heparin has been adapted to the analysis of oligosaccharides generated by digestion with heparinases of gs-heparins usually prepared from porcine mucosal heparin. The method has been also found very effective in analyzing glycol-split derivatives obtained from heparins of different animal and tissue origin. Besides the major 2-O-sulfated disaccharides, heparinase digests of gs-heparins mainly contain tetra- and hexasaccharides incorporating one or two gs residues, with distribution patterns typical for individual gs-heparins. A heptasulfated, mono-N-acetylated hexasaccharide with two gs residues has been shown to be a marker of the gs-modified AT binding site within heparin chains.

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“…Structural characterization of heparin species largely relies on NMR and/or LC-MS analysis, with the latter most often performed on fragments [25]. Significant advances in the analysis of heparin/heparan sulfate di- and oligosaccharides have been achieved by coupling ion-pair reversed-phase high-performance liquid chromatography (IPRP-HPLC) with electrospray ionization mass spectrometry (ESI-MS) [13,26–30]. However, as with heparins, one major problem in the analysis of gs-heparins is associated with their structural microheterogeneity, which can be unravelled only through careful cleavage of their chains and identification of the generated di- and oligosaccharide fragments.…”

Section: Introductionmentioning

confidence: 99%

Structural features of glycol-split low-molecular-weight heparins and their heparin lyase generated fragments

et al. 2013

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Periodate oxidation followed by borohydride reduction converts the well-known antithrombotics heparin and low-molecular weight heparins (LMWHs) into their “glycol-split” (gs) derivatives of the “reduced oxyheparin” (RO) type, some of which are currently being developed as potential anti-cancer and anti-inflammatory drugs. Whereas the structure of gs-heparins has been recently studied, details of the more complex and more bioavailable gs-LMWHs have not been yet reported. We obtained RO derivatives of the three most common LMWHs (tinzaparin, enoxaparin, and dalteparin) and studied their structures by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy and ion-pair reversed-phase high-performance liquid chromatography (IPRP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS). The LC-MS analysis was extended to their heparinase-generated oligosaccharides. The combined NMR/LC-MS analysis of RO-LMWHs provided evidence for glycol-splitting-induced transformations mainly involving internal nonsulfated glucuronic and iduronic acid residues (including partial hydrolysis with formation of “remnants”), and for the hydrolysis of the gs uronic acid residues when formed at the non-reducing ends (mainly, in RO-dalteparin). Evidence for minor modifications, such as ring contraction of some dalteparin internal aminosugar residues was also obtained. Unexpectedly, the N-sulfated 1,6-anhydro-mannosamine residues at the enoxaparin reducing end were found to be susceptible to the periodate oxidation. In addition, in tinzaparin and enoxaparin the borohydride reduction converts the hemiacetalic aminosugars at the reducing end to alditols. Typical LC-MS signatures of RO-derivatives of individual LMWH both before and after digestion with heparinases included oligosaccharides generated from the original antithrombin-binding and “linkage” regions.

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Characterization of currently marketed heparin products: Analysis of heparin digests by RPIP-UHPLC–QTOF-MS

Cited by 40 publications

References 39 publications

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

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

Profiling glycol-split heparins by high-performance liquid chromatography/mass spectrometry analysis of their heparinase-generated oligosaccharides

Structural features of glycol-split low-molecular-weight heparins and their heparin lyase generated fragments

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