title: Comprehensive Identification and Quantitation of Basic Building Blocks for Low-Molecular Weight Heparin.
abstract: Low-molecular weight heparins (LMWHs) are widely used anticoagulant drugs. They inherit the heterogeneous backbone sequences of the parent heparin, while the chemical depolymerization process modifies the nonreducing end (NRE) and reducing end (RE) of their sugar chains. Some side reactions may also occur and increase the structural complexity of LMWHs. It is important to precisely characterize the structures of LMWHs, especially their chemical modifications, to ensure drug quality and safety. Compositional analysis provides a powerful approach to reveal the building blocks that make up the LMWHs, which are the mutual consequence of the heparin starting materials and the manufacturing process. Here, we introduce a comprehensive analytical method to recover the most basic building blocks of LMWHs. A strategy of combining both enzymatic digestion and oxidative degradation of LMWH was used to make the NRE, RE, and backbone structures differentiable from one another. Satisfactory separation, identification, and quantitation were achieved by coupling hydrophilic interaction chromatography with a triple quadrupole mass spectrometer operating under the multiple reaction monitoring mode. After enzymatic digestion, over 30 species were detected, with both natural and chemically modified heparin basic building blocks. Two novel structures, including a trisaccharide containing two glucosamine residues and a tetrasaccharide containing a 3-O-sulfated uronic acid residue, were discovered. Reduced and oxidatively degraded samples were analyzed to provide the complementary information on both termini of LMWHs. The reproducibility of this method was evaluated, and enoxaparin injections were analyzed to demonstrate the application of this method for evaluating the sameness of LMWH products.
authors.affiliation: National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, and State Key Laboratory of Microbial Technology, Shandong University , Jinan, Shandong 250100, China.|Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, and Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Sun
authors.affiliation: National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, and State Key Laboratory of Microbial Technology, Shandong University , Jinan, Shandong 250100, China.
authors.forename: Anran
authors.initials: A
authors.lastname: Sheng
authors.affiliation: National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, and State Key Laboratory of Microbial Technology, Shandong University , Jinan, Shandong 250100, China.|Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, and Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors.forename: Xinyue
authors.initials: X
authors.lastname: Liu
authors.affiliation: Scientific Research Division, Shandong Institute for Food and Drug Control , Jinan, Shandong 250101, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Shi
authors.affiliation: National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, and State Key Laboratory of Microbial Technology, Shandong University , Jinan, Shandong 250100, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Jin
authors.affiliation: National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, and State Key Laboratory of Microbial Technology, Shandong University , Jinan, Shandong 250100, China.
authors.forename: Shaoshuai
authors.initials: S
authors.lastname: Xie
authors.affiliation: Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, and Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors.forename: Fuming
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, and Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
authors.affiliation: National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, and State Key Laboratory of Microbial Technology, Shandong University , Jinan, Shandong 250100, China.
authors.forename: Lianli
authors.initials: L
authors.lastname: Chi
mesh_terms: D001894:Borohydrides / Q000737:chemistry; D002850:Chromatography, Gel; D019763:Heparin Lyase / Q000378:metabolism; D006495:Heparin, Low-Molecular-Weight / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008970:Molecular Weight; D010084:Oxidation-Reduction; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D001894:Borohydrides; D006495:Heparin, Low-Molecular-Weight; C025364:sodium borohydride; D019763:Heparin Lyase