Abstract
Heparin is a complex mixture of heterogeneous sulfated polysaccharidic chains. Its physico-chemical characterization is based on the contribution of several methods, but advantages of the use of complementary techniques have not been fully investigated yet. Strong-Anion-Exchange HPLC after enzymatic digestion and quantitative bidimensional 1H-13C NMR (HSQC) are the most used methods for the determination of heparin structure, providing the composition of its building blocks. The SAX-HPLC method is based on a complete enzymatic digestion of the sample with a mixture of heparinases I, II and III, followed by the separation of the resulting di- and oligo-saccharides by liquid chromatography. The NMR-HSQC analysis is performed on the intact sample and provides the percentage of mono- and di-saccharides by integration of diagnostic peaks. Since, for both methods, accuracy cannot be proved with the standard procedures, it is interesting to compare these techniques, highlighting their capabilities and drawbacks. In the present work, more than 30 batches of porcine mucosa heparin, from 8 manufacturers, have been analyzed with the two methods, and the corresponding results are discussed, based on similarities and differences of the outcomes. The critical comparison of both common and complementary information from the two methods can be used to identify which structural features are best evaluated by each method, and to verify from the concordance of the results the accuracy of the two methods, providing a powerful tool for the regular characterization of single, commercial preparations of Heparin.
Highlights
Heparin is the most important anticoagulant drug and has been used in clinical practice since 1939
The most evident differences that can be observed among heparin from different sources are related to the sulfation pattern: e.g., porcine heparin is largely sulfated in position 6 of glucosamine, in comparison to bovine mucosa heparin, whereas the 2-O sulfation of iduronic acid is higher in bovine mucosa heparin than in porcine [4]
A SAX-HPLC chromatogram, as an example, is shown in Figure 1, while a table with individual results can be found in Supplementary Table 4: as previously reported, 19 oligosaccharide structures have been identified with the SAXHPLC method of the present study (Table 2)
Summary
Heparin is the most important anticoagulant drug and has been used in clinical practice since 1939. The structure of heparin chains is based on disaccharide building blocks, all made up of a uronic acid and a glucosamine. Heparin Characterization by SAX-HPLC and NMR-HSQC of heparin include distinct uronic acid content, i.e., different glucuronic to iduronic ratios (GlcA/IdoA), different levels of Nacetylation/N-sulfation of the glucosamine and of 2-O- and 6O-sulfation (in iduronate and glucosamine, respectively), as well as variable degrees of 3-O sulfation in glucosamine [2,3,4]. The industrial processes of extraction and purification of heparin can cause further variability, affecting its structure in many points, and at different levels (“process signatures”), causing partial chemical modifications such as: depolymerization, N-desulfation [5], O-desulfation with epoxidation and/or epimerization on C2C3 of sulfated iduronic acids [6], oxidation and/or disruption of the linkage region (LR) [7, 8] and other minor defects [9,10,11,12,13]. The most evident differences that can be observed among heparin from different sources are related to the sulfation pattern: e.g., porcine heparin is largely sulfated in position 6 of glucosamine, in comparison to bovine mucosa heparin, whereas the 2-O sulfation of iduronic acid is higher in bovine mucosa heparin than in porcine [4]
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