# Combining NMR Spectroscopy and Chemometrics to Monitor Structural Features of Crude Hep-arin
## 1 Introduction  
In spite of its 100 year history, heparin and its lower molecular weight versions remain the most used anticoagulant and antithrombotic drug [1]. Heparin is a sulfated polysaccharide composed of alternating disaccharide sequences of uronic acid (either D-glucuron or L-iduronic acid) and glucosamine. The glucosamine residue can be \(N\)-acetylated or \(N\)-sulfated in position 2, while both uronic acid and glucosamine can be sulfated in position 2 and 6, respectively. More rarely, the glucosamine residue can be sulfated in position 3, and found in the pentasaccharide sequence GlcNS/Ac-GlcNAc-GlcNAcNS,3S,6S/OH-IdoA2S-GlcNAcNS,6S, corresponding to the heparin binding site for antithrombin (AT). The degree of sulfation and distribution within the heparin chains depends on the animal or organ source; however, the detailed monosaccharide sequence is still largely undisclosed [2]. Until the late 1980s and early 1990s, when cases of bovine spongiform encephalopathy (BSE) were reported in United Kingdom and other countries, bovine heparin products were widely used together with those extracted from porcine intestine. As a result of BSE, bovine heparin was withdrawn from both the U.S. and European markets and entirely replaced by porcine mucosa heparin, limiting bovineheparin use to South America and some Islamic countries. The removal of bovine heparin from the market increased the market demand for porcine heparin. As a result, at present more than 50% of worldwide heparin production originates from China [3]. In addition, a shortage in the heparin supply (due also to a pig disease outbreak) was presumably the reason for the heparin contamination with the semisynthetic over-sulfated chondro sulfate (OSCS), an inexpensive adulterant with anticoagulant activity, which occurred in late 2007 in the US and Europe [4]. This so called heparin crisis, in addition to causing many deaths and severe side effects, demonstrated the vulnerability of the global heparin supply chain. Fortunately, the subsequent introduction of new tests (e.g., NMR and HPLC tests) to the heparin monographs of several pharmacopoeias around the world increased the degree of quality control of this drug, decreasing the risk of product adulteration [3]. However, the complexity of the heparin supply chain provides other opportunities for intentional adulteration, such as contamination with foreign substances or the addition of non-porcine sources of crude heparin (i.e., bovine or ovine heparin), which might not be detected by the current monograph test methods. Heparin is produced from tissues of food animals, and for medical use the drug has been primarily obtained from pig or beef intestine, and can be sourced from sheep as well. Typically, the pig intestine is collected and processed in slaughterhouses approved by regulatory authorities and then transported to crude heparin processing facilities. In these facilities, the intestinal mucosa is removed and the dissociated mucosa is treated with proteolytic enzymes under alkaline conditions. After multiple steps, the highly anionic glycosaminoglycans (GAGs) can be adsorbed onto an anion exchange resin, which is washed. Then, the partially purified heparin is eluted, filtered, precipitated, and vacuum dried to form a substance known as crude heparin, where the glycosaminoglycan (GAG) fraction is concentrated. Crude heparin is then further purified at the level of active pharmaceutical ingredient (API) to remove impurities such as dermatan and chondroitas, nucleic acids, residual proteins, metal ions, and unwanted counterions. In addition, the purification involves oxidation (KMnO\({}_{4}\) or H\({}_{2}\)O\({}_{2}\)) and alkaline treatments to remove color and inactivate endotoxins and viruses that might be present in the product [5; 6]. Whereas the purification of crude heparin to heparin sodium API is always conducted under cGMP conditions, only a few manufacturers can establish traceability back to the living animals.