| Heparin is a heterogeneous linear polysaccharide with high negative charge in the glycosaminoglycans(GAGs)family,which is composed of repeated disaccharide units formed by hexaluronic acid(HexA)and D-glucosamine(GlcN)through 1-4 glycoside linkage,and mainly produced in mast cells of mammals.Heparin in clinical application for the first time since 1935,until now it has 80 years of history,heparin has been used as a clinical anticoagulant drugs,widely used in the prevention and treatment of thrombotic diseases,in the development history of anticoagulant drugs,despite anticoagulant class of small molecule drugs used in the market,but the natural source of heparin drugs still plays an irreplaceable role.Originally medicinal heparin was extracted from bovine lung and porcine intestinal mucosa.Until the outbreak of Mad Cow Disease in 1990s,all bovine heparin was recalled.The heparin market worldwide was occupied by porcine intestinal mucosa.However,heparin sodium contamination occurred in 2007?2008.At that time,merchants used anti-coagulant OSCS to mix into heparin and caused nearly 100 patients to die.Therefore,people pay more and more attention to the stability and safety of heparin supply.The main problem facing the heparin market is a single animal source Its population size,production level and price are affected by disease outbreaks,and it is easily susceptible to adulteration,pollution,and mixing with heparin from other ruminants.Studies have shown that ruminants such as cattle and ovine are similar to pig heparin in anticoagulant activity,molecular weight,biological activity and neutralization characteristics.Due to regulatory regulations,these source heparins have not been widely used in clinical practice.This situation requires the development of analytical methods to effectively identify the source of heparin.At present,real-time fluorescence quantitative PCR of residual nucleic acids in heparin is an effective method for analyzing the presence or absence of heparin in other ruminants.However,a large part of the refined heparin produced by the crude heparin was not detected with nucleic acids.Therefore,it is necessary to look for structural differences in heparin itself from different animal sources to distinguish heparin animal sources.This paper studies the structural differences between porcine and ovine,A reliable method for the identification of heparin from ovine and porcine was established.This study analyzed heparin oligosaccharides by anion exchange chromatography and combined with principal component analysis to predict heparin sources.First,heparin was extracted and purified directly from the small intestine of pigs and ovine through the heparin extraction process.The pH,DS content,and absorbance of the samples were tested in accordance with the Chinese Pharmacopoeia,which all complied with the Pharmacopoeia regulations.Then,the specificity of heparin was completely degraded and partially degraded by the specificity of heparinase.Hydrophilic interaction liquid chromatography-mass spectrometry was used to quantitatively analyze the fully digested sample of heparin.It was found that the content of four sulfate-modified trisaccharides in ovine heparin was much higher than that of pig source,The specific cause of trisaccharide production needs further study and remains to be strong evidence to distinguish pig and ovine-derived heparin.Further SAX separation and analysis of heparin oligosaccharide hexasaccharide produced by specific degradation of heparinase I proved that the structural contents of the heparin oligosaccharides from the two sources were significantly different.Combining PCA with SAX analysis can identify more than 5%ovine heparin mixed samples.This study provides a new method for the evaluation and identification of heparin animal sources.Compared with the fluorescence quantitative PCR method,this method can realize the source evaluation of various purified refined heparin,promote the comprehensive supervision of heparin source,and guarantee the drug safety. |
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