Quantitative 2D ¹H, ¹³C HSQC NMR Spectroscopy for the Determination of Chondroitin Sulfate and Dermatan Sulfate Content in Danaparoid Sodium

Abstract

Objective Danaparoid sodium is a biopolymeric complex drug composed of the most abundant heparan sulfate (HS) followed in descending order by dermatan sulfate (DS) and chondroitin sulfate (CS). This composite nature explains its peculiar antithrombotic and anticoagulant properties and make it particularly advantageous when the risk of heparin-induced thrombocytopenia occurs. A specific control of the danaparoid composition is required by the Ph. Eur. The monograph includes the CS and DS limit contents and describes the method for their quantification through selective enzymatic degradations.

Materials and Methods In this study, a quantitative two-dimensional nuclear magnetic resonance (NMR) method is proposed as a new method suitable for CS and DS quantification. Statistical comparison of the results provided by the analysis of a series of danaparoid samples with both NMR and enzymatic methods highlights a small systematic difference, likely derived from lyase-resistant sequences bearing oxidized terminals. Some modified structures, whose survival to the enzymatic action was confirmed by mass spectrometry, can be detected and quantified by NMR.

Conclusion and Results The proposed NMR method can serve for the determination of DS and CS contents, is an easy-to-apply method with no dependence from enzymes and standards, and provides extensive structural information on the overall glycosaminoglycans mixture.

Keywords

danaparoid sodium - chondroitinases - NMR spectroscopy - glycosaminoglycans - LC-MS analysis

Publication History

Received: 30 December 2022

Accepted: 09 March 2023

Article published online:
24 April 2023

• References

• 1 Acostamadiedo JM, Iyer UG, Owen J. Danaparoid sodium. Expert Opin Pharmacother 2000; 1 (04) 803-814
• 2 Magnani HN. Heparin-induced thrombocytopenia (HIT): an overview of 230 patients treated with orgaran (Org 10172). Thromb Haemost 1993; 70 (04) 554-561
• 3 Tardy-Poncet B, Combe M, Piot M, Chapelle C, Akrour M, Tardy B. Effects of argatroban, danaparoid, and fondaparinux on trombin generation in heparin-induced thrombocytopenia. Thromb Haemost 2013; 109 (03) 504-509
• 4 Bikdeli B, Madhavan MV, Gupta A. et al; Global COVID-19 Thrombosis Collaborative Group. Pharmacological agents targeting thromboinflammation in COVID-19: review and implications for future research. Thromb Haemost 2020; 120 (07) 1004-1024
• 5 Skoutakis VA. Danaparoid in the prevention of thromboembolic complications. Ann Pharmacother 1997; 31 (7–8): 876-887
• 6 Magnani HN, Gallus A. Heparin-induced thrombocytopenia (HIT). A report of 1,478 clinical outcomes of patients treated with danaparoid (Orgaran) from 1982 to mid-2004. Thromb Haemost 2006; 95 (06) 967-981
• 7 European Directorate for the Quality of Medicines & Healthcare. Danaparoid Sodium Monograph. 10th ed.. Vol. II. Strasbourg, France: Council of Europe; 2019
• 8 Huang W, Lunin VV, Li Y. et al. Crystal structure of Proteus vulgaris chondroitin sulfate ABC lyase I at 1.9A resolution. J Mol Biol 2003; 328 (03) 623-634
• 9 Féthière J, Eggimann B, Cygler M. Crystal structure of chondroitin AC lyase, a representative of a family of glycosaminoglycan degrading enzymes. J Mol Biol 1999; 288 (04) 635-647
• 10 Michelacci YM, Dietrich CP. A comparative study between a chondroitinase B and a chondroitinase AC from Flavobacterium heparinum: isolation of a chondroitinase AC-susceptible dodecasaccharide from chondroitin sulphate B. Biochem J 1975; 151 (01) 121-129
• 11 Fransson LÅ, Anseth A, Antonopoulos CA, Gardell S. Structure of dermatan sulfate. VI. The use of cetylpyridinium chloride-cellulose microcolumns for determination of the hybrid structure of dermatan sulfates. Carbohydr Res 1970; 15 (01) 73-89
• 12 Malmström A, Bartolini B, Thelin MA, Pacheco B, Maccarana M. Iduronic acid in chondroitin/dermatan sulfate: biosynthesis and biological function. J Histochem Cytochem 2012; 60 (12) 916-925
• 13 Gu K, Linhardt RJ, Laliberté M, Gu K, Zimmermann J. Purification, characterization and specificity of chondroitin lyases and glycuronidase from Flavobacterium heparinum. Biochem J 1995; 312 (Pt 2): 569-577
• 14 Ustün B, Sanders KB, Dani P, Kellenbach ER. Quantification of chondroitin sulfate and dermatan sulfate in danaparoid sodium by (1)H NMR spectroscopy and PLS regression. Anal Bioanal Chem 2011; 399 (02) 629-634
• 15 Guerrini M, Beccati D, Shriver Z. et al. Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events. Nat Biotechnol 2008; 26 (06) 669-675
• 16 Szajek AY, Chess E, Johansen K. et al. The US regulatory and pharmacopeia response to the global heparin contamination crisis. Nat Biotechnol 2016; 34 (06) 625-630
• 17 European Directorate for the Quality of Medicines & Healthcare. Heparin Sodium Monograph. 10 th ed.. Vol II. Strasbourg, France: Council of Europe; ; July 2019
• 18 United States Pharmacopoeia. Heparin Sodium Monograph. Rockville, MD: United States Pharmacopoeia; ; USP-NF. 2022
• 19 Vilanova E, Vairo BC, Oliveira SMCG. et al. Heparins sourced from bovine and porcine mucosa gain exclusive monographs in the Brazilian pharmacopeia. Front Med (Lausanne) 2019; 6: 16
• 20 Monakhova YB, Diehl BWK. Nuclear magnetic resonance spectroscopy as a tool for the quantitative analysis of water and ions in pharmaceuticals: example of heparin. J Pharm Biomed Anal 2018; 154: 332-338
• 21 Monakhova YB, Fareed J, Yao Y, Diehl BWK. Anticoagulant activity of porcine heparin: structural-property relationship and semi-quantitative estimation by nuclear magnetic resonance (NMR) spectrometry. J Pharm Biomed Anal 2019; 174: 639-643
• 22 Monakhova YB, Diehl BWK, Do TX, Schulze M, Witzleben S. Novel method for the determination of average molecular weight of natural polymers based on 2D DOSY NMR and chemometrics: example of heparin. J Pharm Biomed Anal 2018; 149: 128-132
• 23 Mauri L, Marinozzi M, Phatak N. et al. 1D and 2D-HSQC NMR: two methods to distinguish and characterize heparin from different animal and tissue sources. Front Med (Lausanne) 2019; 6: 142
• 24 Monakhova YB, Diehl BWK, Fareed J. Authentication of animal origin of heparin and low molecular weight heparin including ovine, porcine and bovine species using 1D NMR spectroscopy and chemometric tools. J Pharm Biomed Anal 2018; 149: 114-119
• 25 Nesměrák K, Pospíchal R. Spectrometric methods in pharmaceutical analysis of glycosaminoglycans: the state-of-the-art. Monatsh Chem 2020; 151: 1185-1192
• 26 Toby TK, Sommers CD, Keire DA. Detection of native chondroitin sulfate impurities in heparin sodium with a colorimetric micro-plate based assay. Anal Methods 2012; 4 (06) 1488-1491
• 27 Holme KR, Perlin AS. Nuclear magnetic resonance spectra of heparin in admixture with dermatan sulfate and other glycosaminoglycans. 2-D spectra of the chondroitin sulfates. Carbohydr Res 1989; 186 (02) 301-312
• 28 McEwen I, Mulloy B, Hellwig E. et al. Determination of oversulphated chondroitin sulphate and dermatan sulphate in unfractionated heparin by (1)H-NMR - Collaborative study for quantification and analytical determination of LoD. Pharmeur Bio 2008; 2008 (01) 31-39
• 29 Kishimoto TK, Viswanathan K, Ganguly T. et al. Contaminated heparin associated with adverse clinical events and activation of the contact system. N Engl J Med 2008; 358 (23) 2457-2467 [published correction appears in N Engl J Med. 2010 Mar 18;362(11):1056]
• 30 Gardini C, Urso E, Guerrini M, van Herpen R, de Wit P, Naggi A. Characterization of danaparoid complex extractive drug by an orthogonal analytical approach. Molecules 2017; 22 (07) 1116
• 31 Beccati D, Roy S, Yu F. et al. Identification of a novel structure in heparin generated by potassium permanganate oxidation. Carbohydr Polym 2010; 82 (03) 699-705
• 32 Mourier PA, Guichard OY, Herman F, Viskov C. Heparin sodium compliance to the new proposed USP monograph: elucidation of a minor structural modification responsible for a process dependent 2.10 ppm NMR signal. J Pharm Biomed Anal 2011; 54 (02) 337-344
• 33 Kellenbach E, Sanders K, Michiels PJ, Girard FC. (1)H NMR signal at 2.10 ppm in the spectrum of KMnO(4)-bleached heparin sodium: identification of the chemical origin using an NMR-only approach. Anal Bioanal Chem 2011; 399 (02) 621-628
• 34 Guerrini M, Naggi A, Guglieri S, Santarsiero R, Torri G. Complex glycosaminoglycans: profiling substitution patterns by two-dimensional nuclear magnetic resonance spectroscopy. Anal Biochem 2005; 337 (01) 35-47
• 35 Torri G, Guerrini M. Quantitative 2D NMR analysis of glycosaminoglycans. In: Holzgrabe U, Wawer I, Diehl B. eds. NMR Spectroscopy in Pharmaceutical Analysis. Amsterdam: Elsevier; 2008: 407-428
• 36 Zang Q, Keire DA, Wood RD. et al. Determination of galactosamine impurities in heparin samples by multivariate regression analysis of their (1)H NMR spectra. Anal Bioanal Chem 2011; 399 (02) 635-649
• 37 Mauri L, Boccardi G, Torri G. et al. Qualification of HSQC methods for quantitative composition of heparin and low molecular weight heparins. J Pharm Biomed Anal 2017; 136: 92-105
• 38 Mauri L, Marinozzi M, Mazzini G. et al. Combining NMR spectroscopy and chemometrics to monitor structural features of crude heparin. Molecules 2017; 22 (07) 1146
• 39 Mascellani G, Liverani L, Prete A. et al. Active sites of dermatan sulfate for heparin cofactor II. Isolation of a nonasaccharide fragment containing four disaccharide sequences [α-L-iduronic acid 2-O-sulfate (1, 3)-β-D-N-acetylgalactosamine 4-sulfate]. Carbohydr Chem 1995; 14 (08) 1165-1177
• 40 Panagos C, Thomson D, Bavington CD, Uhrín D. Structural characterisation of oligosaccharides obtained by Fenton-type radical depolymerisation of dermatan sulfate. Carbohydr Polym 2012; 87 (03) 2086-2092
• 41 Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986; 1 (8476): 307-310