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Danaparoid sodium salt, is the active component of ORGARAN, an anticoagulant and antithrombotic drug constituted of three glycosaminoglycans (GAGs) obtained from porcine intestinal mucosa extracts. Heparan sulfate is the major component, dermatan sulfate and chondroitin sulfate being the minor ones. Currently dermatan sulfate and chondroitin sulfate are quantified by UV detection of their unsaturated disaccharides obtained by enzymatic depolymerization. Due to the complexity of danaparoid biopolymers and the presence of shared components, an orthogonal approach has been applied using more advanced tools and methods. To integrate the analytical profile, 2D heteronuclear single quantum coherence (HSQC) NMR spectroscopy was applied and found effective to identify and quantify GAG component signals as well as those of some process signatures of danaparoid active pharmaceutical ingredient (API) batches. Analyses of components of both API samples and size separated fractions proceeded through the determination and distribution of the molecular weight (Mw) by high performance size exclusion chromatographic triple detector array (HP-SEC-TDA), chain mapping by LC/MS, and mono- (1H and 13C) and bi-dimensional (HSQC) NMR spectroscopy. Finally, large scale chromatographic isolation and depolymerization of each GAG followed by LC/MS and 2D-NMR analysis, allowed the sequences to be defined and components to be evaluated of each GAG including oxidized residues of hexosamines and uronic acids at the reducing ends.

Risks of contamination of the major clinical anticoagulant heparin can arise from deliberate adulteration with unnatural or natural polysaccharides, including heparin from other animal sources, other natural products, or artifacts of manufacture, and these can escape detection by conventional means. Currently, there is no generally applicable, objective test recommended by regulators that can detect these in pharmaceutical heparin, and this continues to leave heparin exposed to contamination risks. Two-dimensional correlation spectroscopic-filtering with iterative random sampling (2D-COS-firs) is reported. It employs a difference covariance matrix with iterative random sampling, and is capable of revealing contamination in pharmaceutical heparin to a high level of sensitivity irrespective of the nature of those features. The technique is suitable to any situation in which a comparison of a single entity to a family of heterogeneous entities, particularly natural products and biosimilars, needs to be made, and will find application in pharmaceutical monitoring, manufacturing quality control, materials science, biotechnology, and metabolomic investigations.

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