Journal of Shandong University (Health Sciences) ›› 2021, Vol. 59 ›› Issue (3): 41-47.doi: 10.6040/j.issn.1671-7554.0.2020.1448

Previous Articles     Next Articles

Identification of heparin and heparan sulfate by HILIC-MS/MS

LIU Dongke1, WANG Fengshan1,2   

  1. 1. Key Laboratory of Chemical Biology(Ministry of Education), Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, Shandong, China;
    2. Laboratory of Carbohydrate Chemistry and Glycobiology of Shandong Province, National Glycoengineering Research Center, Shandong University, Jinan 250012, Shandong, China
  • Published:2021-04-06

PDF (PC)

639

Abstract: Objective To establish a hydrophilic interaction chromatography-mass spectrometry(HILIC-MS/MS)method for simultaneous determination of 8 disaccharides in heparin and heparan sulfate after digestion by heparinase Ⅰ, Ⅱ and Ⅲ, and to provide method and data references for the identification of heparin and heparan sulfate. Methods A PC HILIC column(4.6 mm×250 mm, 5 μm)was used and gradiently eluted with 10 mmol/L ammonium acetate aqueous solution(A)and 10 mmol/L ammonium-90% acetonitrile(B)at the flow rate of 0.9 mL/min, column temperature of 40 ℃ and injection volume of 10 μL. The contents of the 8 disaccharides of heparin and heparan sulfate produced by enzymatic digestion were determined by electrospray ionization source and multi reaction monitoring mode and were compared with each other. Results The linear relationships of the 8 components were good in the range of measured molar concentrations(r2>0.996 0), and the average recovery were 90.34%-99.62%, RSD were 4.7%-6.7%. The results showed that the disaccharides composition and proportion of heparin and heparan sulfate were significantly different. In heparin, ΔUA2S-GlcNS6S(TriS)accounted for about 69.8% on average, while in heparan sulfate, ΔUA-GlcNAc(0S)accounted for about 51.0%. Conclusion The method established in this paper is simple, fast, accurate and reliable with high sensitivity, strong specificity, good stability, and can be used as one of the determination and identification methods for heparin and heparan sulfate.

Key words: Heparin, Heparan sulfate, Hydrophilic interaction chromatography-mass spectrometry, Disaccharides, Identification

CLC Number: 

  • R917
[1] Mulloy B, Forster MJ. Conformation and dynamics of heparin and heparan sulfate [J]. Glycobiology, 2000, 10(11): 1147-1156.
[2] Casu B, Lindahl U. Structure and biological interactions of heparin and heparan sulfate [J]. Adv Carbohydr Chem Biochem, 2001, 57: 159-206. doi: 10.1016/s0065-2318(01)57017-1.
[3] Gallagher JT, Walker A. Molecular distinctions between heparan sulphate and heparin. Analysis of sulphation patterns indicates that heparan sulphate and heparin are separate families of N-sulphated polysaccharides [J]. Biochem J, 1985, 230(3): 665-674.
[4] Rabenstein DL. Heparin and heparan sulfate: structure and function [J]. Nat Prod Rep, 2002, 19(3): 312-331.
[5] Li JP, Kusche-Gullberg M. Heparan sulfate: biosynthesis, structure, and function [J]. Int Rev Cell Mol Biol, 2016, 325: 215-273. doi: 10.1016/bs.ircmb.2016.02.009.
[6] Gandhi NS, Mancera RL. Heparin/heparan sulphate-based drugs [J]. Drug Discov Today, 2010, 15(23-24): 1058-1069.
[7] Fügedi P. The potential of the molecular diversity of heparin and heparan sulfate for drug development [J]. Mini Rev Med Chem, 2003, 3(7): 659-667.
[8] Skidmore MA, Guimond SE, Dumax-Vorzet AF, et al. Disaccharide compositional analysis of heparan sulfate and heparin polysaccharides using UV or high-sensitivity fluorescence(BODIPY)detection [J]. Nat Protoc, 2010, 5(12): 1983-1992.
[9] Viola M, Vigetti D, Karousou E, et al. New electrophoretic and chromatographic techniques for analysis of heparin and heparan sulfate [J]. Electrophoresis, 2008, 29(15): 3168-3174.
[10] Volpi N, Galeotti F, Yang B, et al. Analysis of glycosaminoglycan-derived, precolumn, 2-aminoacridone-labeled disaccharides with LC-fluorescence and LC-MS detection [J]. Nat Protoc, 2014, 9(3): 541-558.
[11] Linhardt RJ. Analysis of glycosaminoglycans with polysaccharide lyases [M] // Ausubel FM, Brent R, Kingston RE, et al. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 2001, Unit 17.13B.1-16.
[12] Galeotti F, Volpi N. 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 [J]. Anal Chem, 2011, 83(17): 6770-6777.
[13] Volpi N. High-performance liquid chromatography and on-line mass spectrometry detection for the analysis of chondroitin sulfates/hyaluronan disaccharides derivatized with 2-aminoacridone [J]. Anal Biochem, 2010, 397(1): 12-23.
[14] Wang Z, Li D, Sun X, et al. Liquid chromatography-diode array detection-mass spectrometry for compositional analysis of low molecular weight heparins [J]. Anal Biochem, 2014, 451: 35-41. doi: 10.1016/j.ab.2014.02.005.
[15] Yang B, Chang Y, Weyers AM, et al. Disaccharide analysis of glycosaminoglycan mixtures by ultra-high-performance liquid chromatography-mass spectrometry [J]. J Chromatogr A, 2012, 1225: 91-98. doi: 10.1016/j.chroma.2011.12.063.
[16] Sadowski R, Gadzaa-Kopciuch R, Kowalkowski T, et al. Characterization of low-molecular-weight heparins by strong anion-exchange chromatography [J]. J AOAC Int, 2017, 100(6): 1706-1714.
[17] Miller RL, Guimond SE, Shivkumar M, et al. Heparin isomeric oligosaccharide separation using volatile salt strong anion exchange chromatography [J]. Anal Chem, 2016, 88(23): 11542-11550.
[18] Mourier P, Anger P, Martinez C, et al. Quantitative compositional analysis of heparin using exhaustive heparinase digestion and strong anion exchange chromatography [J]. Anal Chem Res, 2015, 3: 46-53. doi: 10.1016/j.ancr.2014.12.001.
[19] 杜佳燕,黄海月,苏晓明,等.反相液相色谱-电喷雾-离子阱-飞行时间质谱法定量分析N-非取代肝素/硫酸乙酰肝素[J]. 质谱学报, 2019, 40(3): 222-232. DU Jiayan, HUANG Haiyue, SU Xiaoming, et al. Quantitative analysis of N-unsubstitued heparin/heparan sulfate by reversed-phase liquid chromatography-electrospray ionization ion trap-time-of-flight mass spectrometry [J]. J Chin Mass Spectrom Soc, 2019, 40(3): 222-232.
[20] 韩章润,邢新会,于广利,等.临床肝素类药物酶解分析二糖组成[J]. 分析化学, 2015, 43(7): 964-970. HAN Zhangrun, XING Xinhui, YU Guangli, et al. Heparinase digestion-based disaccharide analysis of clinical heparin and heparinoids drug [J]. Chin J Anal Chem, 2015, 43(7): 964-970.
[21] Galeotti F, Volpi N. Novel reverse-phase ion pair-high performance liquid chromatography separation of heparin, heparan sulfate and low molecular weight-heparins disaccharides and oligosaccharides [J]. J Chromatogr A, 2013, 1284: 141-147. doi: 10.1016/j.chroma.2013.02.013.
[22] Yang B, Weyers A, Baik JY, et al. Ultra-performance ion-pairing liquid chromatography with on-line electrospray ion trap mass spectrometry for heparin disaccharide analysis [J]. Anal Biochem, 2011, 415(1): 59-66.
[23] Brustkern AM, Buhse LF, Nasr M, et al. Characterization of currently marketed heparin products: reversed-phase ion-pairing liquid chromatography mass spectrometry of heparin digests [J]. Anal Chem, 2010, 82(23): 9865-9870.
[24] Sun X, Sheng A, Liu X, et al. Comprehensive identification and quantitation of basic building blocks for low-molecular weight heparin [J]. Anal Chem, 2016, 88(15): 7738-7744.
[25] Ouyang Y, Wu C, Sun X, et al. Development of hydrophilic interaction chromatography with quadruple time-of-flight mass spectrometry for heparin and low molecular weight heparin disaccharide analysis [J]. Rapid Commun Mass Spectrom, 2016, 30(2): 277-284.
[26] Sun X, Guo Z, Yu M, et al. Hydrophilic interaction chromatography-multiple reaction monitoring mass spectrometry method for basic building block analysis of low molecular weight heparins prepared through nitrous acid depolymerization [J]. J Chromatogr A, 2017, 1479:121-128. doi: 10.1016/j.chroma.2016.11.061.
[27] Korir AK, Limtiaco JF, Gutierrez SM, et al. Ultraperformance ion-pair liquid chromatography coupled to electrospray time-of-flight mass spectrometry for compositional profiling and quantification of heparin and heparan sulfate [J]. Anal Chem, 2008, 80(4): 1297-1306.
[28] 李春波. AB SCIEX离子淌度差分质谱技术SelexIONTM—极限提高质谱鉴别能力[J]. 现代科学仪器, 2011, 10(5): 163-165. LI Chunbo. AB SCIEX SelexIONTM Technology[J]. Mod Sci Instruments, Modern Scientific Instruments, 2011, 10(5): 163-165.
[29] Daniel G, Beach ESK, Michael A. Quilliam: selective quantitation of the neurotoxin BMAA by use of hydrophilic-interaction liquid chromatography-differential mobility spectrometry-tandem mass spectrometry(HILIC-DMS-MS/MS)[J]. Anal Bioanal Chem, 2015, 407(28): 8397-8409.
[1] CUI Wei, ZHANG Yujuan. Detection of circulating tumor cell: actualities and challenges [J]. Journal of Shandong University (Health Sciences), 2018, 56(10): 40-45.
[2] XU Youwei, ZHOU Honglei, REN Dongmei, LOU Hongxiang, SHEN Tao. Chemical constituents from the aerial parts of Litsea glutinosa(Lour.)C.B. Rob. [J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2016, 54(3): 45-49.
[3] WANG Meizhu, CAO Yu, ZHAO Liang, XU Xiaoshan, ZHANG Peifang, LV Qian, BAI Fengyan, KANG Yingqian. Composition and distribution of clinical yeasts in Guizhou region, China [J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2014, 52(8): 94-97.
[4] DUAN Yan, LIU Yuanyuan. Effect of LMWH on severe preeclampsia complicated with FGR and HO-1 expression [J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2014, 52(11): 77-80.
[5] WANG Zhi-gang， JIANG Bin， MENG Qing-hu， HAO Xiao-guang， DING Xuan， LI Ji-sen. Isolation of human glioma-derived stem cells and
establishment of an invasive glioma model [J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2012, 50(5): 60-.
[6] YAN Chun-ying1, SHI Jun1, HAO Jing-hua1, ZHANG Jie2, SUN Lin-lin1, REN Wan-hua1. Effects of low molecular weight heparin on the rat model of   alcoholic liver disease [J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2011, 49(3): 41-45.
[7] YAN Chun-ying1, SHI Jun1, HAO Jing-hua1, ZHANG Jie2, SUN Lin-lin1, REN Wan-hua1. Effects of low molecular weight heparin on the rat model of   alcoholic liver disease [J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2011, 49(3): 41-45.
[8] SUN Lin-lin, SHI Jun, HAO Jing-hua, REN Wan-hua. Low molecular weight heparin attenuating non-alcoholic steatohepatitiscaused by high-fat diet in the rat [J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2010, 48(11): 58-62.
[9] LI Dawei, JIAO Wei, WANG Tieliu, YAN Lei, REN Juchao, TIAN Sujian,XU Zhonghua, FAN Yidong, LIU Hainan. Heparan sulfate proteoglycan gene polymorphism and genetic susceptibility to calcium oxalate nephrolithiasis [J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2010, 48(1): 89-93.
[10] ZHANG Kai-shu,XU Zhong-hua,JIANG Hong,YAN Lei,ZHOU Zun-lin,WANG Rong,LIU Hai-nan,FAN Yi-dong,LIU Zhao-xu,ZHENG Bao-zhong,QI Tian-wei. Effects of the siRNA silencing heparanase gene on T24 cells′proliferation and MMP-2 expression in human bladder cancer [J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2008, 46(6): 561-565.
[11] SHI Jun,HAO Jing-hua,XU Hong-wei,REN Wan-hua,ZHU Ju-ren,KONG Xiang-hui. Heparin reverses hepatic fibrosis in rats [J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2007, 45(2): 155-159.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
[1] WANG Huan-liang,SUN Bao-zhu,DU Hong-mei,ZHOU Chang-qing,ZHANG Li. Controlling the depth of propofol anesthesia using the bispectral indexand the systolic blood pressure[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2006, 44(5): 471 -474 .
[2] HUANGQing,TIAN Hui,LI Lin,LIANG Fei,LIU Xi-xian. [J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2006, 44(6): 556 -559 .
[3] ZHU Mei-jia,HAN Ju,WANG Xin-yi,LU Wei,WANG Ai-hua,GUAN Xin-hua,CAO Xia,CAO Bing-zhen. [J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2006, 44(8): 834 -839 .
[4] SONG Hai-yan, WU Yu-ling, ZHANG Yan-ping. Protective effect of oyster extract on apoptotic cells in neural tube defect induced by hyperthermia[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2007, 45(2): 113 -116 .
[5] WANG Zhi-ang,DING Xua,SUN Peng/sup>,WANG Cheng-wei,HAO Xiao-guang,PAN Shun. Role of pre-operative cerebral angiography in cerebral artery stenosis stent angioplasty[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2007, 45(2): 146 -148 .
[6] ZENG Ji-ping,WANG Li-na,WANG Li-xiang,REN Xiao-hui,ZHANG Meng-ye,XIA Wen,CUI Xing. [J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2006, 44(5): 467 -470 .
[7] SHU Ya,QI Feng. Jaw thrust device (JTD) on obesity patients for maintaining the airway patency during general anesthesia induction[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2007, 45(10): 1072 -1074 .
[8] SONG Yong-hong,MA Chun-hong,LV Hong-juan,ZHU Chuan-fu,NIE Xiang-min,WANG Mei,LIU Yan,ZHANG Ping. HLA allele polymorphism in northern Hans of China[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2007, 45(6): 546 -553 .
[9] ZHANG Xiang-li,LIU Feng-ying. Correlation between serum TPA and sVCAM-1 and pathogenesis of pre-eclampsia[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2007, 45(7): 705 -707 .
[10] YU Hui1,2, CHEN Shaohua1, ZHAO Jiajun2, GAO Ling3
. Effects of ethanol on glycogen content, GSK3β，
and PAMPK of L02 cells[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2009, 47(04): 75 -78 .
Copyright 2018 © Editorial office of Journal of Shandong University (Health Sciences)
Supported by Beijing Magtech Co.Ltd