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

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.

[1]  Gary Benson,et al.  GlycReSoft: A Software Package for Automated Recognition of Glycans from LC/MS Data , 2012, PloS one.

[2]  D. Keire,et al.  Characterization of currently marketed heparin products: analysis of heparin digests by RPIP-UHPLC-QTOF-MS. , 2012, Journal of pharmaceutical and biomedical analysis.

[3]  R. Linhardt,et al.  Analysis of glycosaminoglycan-derived disaccharides by capillary electrophoresis using laser-induced fluorescence detection. , 2012, Analytical biochemistry.

[4]  R. Linhardt,et al.  Complete mass spectral characterization of a synthetic ultralow-molecular-weight heparin using collision-induced dissociation. , 2012, Analytical chemistry.

[5]  R. Linhardt,et al.  Electrophoresis for the analysis of heparin purity and quality , 2012, Electrophoresis.

[6]  R. Linhardt,et al.  Synthetic heparin. , 2012, Current opinion in pharmacology.

[7]  R. Linhardt,et al.  Disaccharide analysis of glycosaminoglycan mixtures by ultra-high-performance liquid chromatography-mass spectrometry. , 2012, Journal of chromatography. A.

[8]  G. Montalescot,et al.  US Food and Drug Administration approval of generic versions of complex biologics: implications for the practicing physician using low molecular weight heparins , 2012, Journal of Thrombosis and Thrombolysis.

[9]  R. Linhardt,et al.  Engineering of routes to heparin and related polysaccharides , 2011, Applied Microbiology and Biotechnology.

[10]  Joseph Zaia,et al.  Improved liquid chromatography-MS/MS of heparan sulfate oligosaccharides via chip-based pulsed makeup flow. , 2011, Analytical chemistry.

[11]  R. Linhardt,et al.  The proteoglycan bikunin has a defined sequence , 2011, Nature chemical biology.

[12]  F. Galeotti,et al.  Online reverse phase-high-performance liquid chromatography-fluorescence detection-electrospray ionization-mass spectrometry separation and characterization of heparan sulfate, heparin, and low-molecular weight-heparin disaccharides derivatized with 2-aminoacridone. , 2011, Analytical chemistry.

[13]  Christopher J. Jones,et al.  Heparin characterization: challenges and solutions. , 2011, Annual review of analytical chemistry.

[14]  Robert J Linhardt,et al.  Heparin mapping using heparin lyases and the generation of a novel low molecular weight heparin. , 2011, Journal of medicinal chemistry.

[15]  J. Turnbull,et al.  Disaccharide compositional analysis of heparan sulfate and heparin polysaccharides using UV or high-sensitivity fluorescence (BODIPY) detection , 2010, Nature Protocols.

[16]  C. Boudier,et al.  Effects on molecular conformation and anticoagulant activities of 1,6-anhydrosugars at the reducing terminal of antithrombin-binding octasaccharides isolated from low-molecular-weight heparin enoxaparin. , 2010, Journal of medicinal chemistry.

[17]  R. Linhardt,et al.  High-resolution preparative separation of glycosaminoglycan oligosaccharides by polyacrylamide gel electrophoresis. , 2010, Analytical biochemistry.

[18]  Catherine E Costello,et al.  Improved hydrophilic interaction chromatography LC/MS of heparinoids using a chip with postcolumn makeup flow. , 2010, Analytical chemistry.

[19]  J. Xie,et al.  Quantification of heparan sulfate disaccharides using ion-pairing reversed-phase microflow high-performance liquid chromatography with electrospray ionization trap mass spectrometry. , 2009, Analytical chemistry.

[20]  G. Jacobson,et al.  Effective reversed-phase ion pair high-performance liquid chromatography method for the separation and characterization of intact low-molecular-weight heparins. , 2009, Analytical biochemistry.

[21]  Haiying Liu,et al.  Lessons learned from the contamination of heparin. , 2009, Natural product reports.

[22]  Catherine E Costello,et al.  A chip‐based amide‐HILIC LC/MS platform for glycosaminoglycan glycomics profiling , 2009, Proteomics.

[23]  J. Zaia,et al.  Comparative glycomics of connective tissue glycosaminoglycans , 2008, Proteomics.

[24]  N. Leymarie,et al.  Characterization of heparin oligosaccharides binding specifically to antithrombin III using mass spectrometry. , 2008, Biochemistry.

[25]  R. Sasisekharan,et al.  Low Molecular Weight Heparins: Structural Differentiation by Bidimensional Nuclear Magnetic Resonance Spectroscopy , 2007, Seminars in thrombosis and hemostasis.

[26]  G. Torri,et al.  Characterization of di- and monosulfated, unsaturated heparin disaccharides with terminal N-sulfated 1,6-anhydro-beta-D-glucosamine or N-sulfated 1,6-anhydro-beta-D-mannosamine residues. , 2007, Carbohydrate research.

[27]  R. Linhardt,et al.  Matrix-assisted laser desorption/ionization mass spectrometric analysis of uncomplexed highly sulfated oligosaccharides using ionic liquid matrices. , 2006, Analytical chemistry.

[28]  J. Henriksen,et al.  On-line size-exclusion chromatography/mass spectrometry of low molecular mass heparin. , 2004, Journal of mass spectrometry : JMS.

[29]  Robert J Linhardt,et al.  Liquid Chromatography/Mass Spectrometry Sequencing Approach for Highly Sulfated Heparin-derived Oligosaccharides* , 2004, Journal of Biological Chemistry.

[30]  R. Linhardt.  2003 Claude S. Hudson Award address in carbohydrate chemistry. Heparin: structure and activity. , 2003, Journal of medicinal chemistry.

[31]  J. Zaia,et al.  Compositional analysis of glycosaminoglycans by electrospray mass spectrometry. , 2001, Analytical chemistry.

[32]  K. Biemann,et al.  Sequencing of 3-O sulfate containing heparin decasaccharides with a partial antithrombin III binding site. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[33]  K. Haustein.  Pharmacokinetic and Pharmacodynamic Properties of Oral Anticoagulants, Especially Phenprocoumon , 1999, Seminars in thrombosis and hemostasis.

[34]  J. Weitz.  Low-molecular-weight heparins. , 1997, The New England journal of medicine.

[35]  R. Califf,et al.  A comparison of low-molecular-weight heparin with unfractionated heparin for unstable coronary artery disease. Efficacy and Safety of Subcutaneous Enoxaparin in Non-Q-Wave Coronary Events Study Group. , 1997 .

[36]  R. Linhardt,et al.  C-2 Epimerization of N-Acetylglucosamine in an Oligosaccharide Derived From Heparan Sulfate , 1996 .

[37]  J. Hirsh,et al.  Heparin-induced thrombocytopenia in patients treated with low-molecular-weight heparin or unfractionated heparin. , 1995, The New England journal of medicine.

[38]  U. R. Desai,et al.  Molecular weight of heparin using 13C nuclear magnetic resonance spectroscopy. , 1995, Journal of pharmaceutical sciences.

[39]  J. Weiler,et al.  Gradient polyacrylamide gel electrophoresis for determination of molecular weights of heparin preparations and low-molecular-weight heparin derivatives. , 1992, Journal of pharmaceutical sciences.

[40]  E. Pacaud,et al.  On the Relationship Between Molecular Mass and Anticoagulant Activity in a Low Molecular Weight Heparin (Enoxaparin) , 1992, Thrombosis and Haemostasis.

[41]  R. Linhardt,et al.  Disaccharide compositional analysis of heparin and heparan sulfate using capillary zone electrophoresis. , 1991, Analytical biochemistry.

[42]  E. Tromborg,et al.  Development and validation of a size exclusion chromatography method for determination of molecular masses and molecular mass distribution in low molecular weight heparin. , 1991, Thrombosis research.

[43]  R. Linhardt,et al.  Fractionation of heparin-derived oligosaccharides by gradient polyacrylamide-gel electrophoresis. , 1987, The Biochemical journal.

[44]  R. Linhardt,et al.  Hyphenated techniques for the analysis of heparin and heparan sulfate , 2011, Analytical and bioanalytical chemistry.