Quantitative Analysis Study Of Major Impurity In Heparin By NMF Method And Separation OSCS From Contaminated Heparin

Heparin is an anticoagulant traditionally prepared from province small intestine,besides antithrombotic it posseses some other bology functions like regulation bloodfat, anti-inflammatory and ananaphylaxis, It is widely used in clinical for the need toachieve quickly anticoagulant effect.In early2008, the United States and Europe etc12countries happened seriousheparin sodium injection adverse event, caused hundreds of patients have a severeallergic reaction, even causing about patients died, then American FDA application1H-NMR and capillary electrophoresis in this batch of heparin sodium raw materialsfound in a kind of heparin pollutants, OSCS has and heparin sodium similar structure,the relative molecular weight range. The conventional biological chemical method isdifficult to distinguish them. In order to strengthen the supervision of the quality ofraw material, it is very important to find a method to detection them. Theconventional1H-NMR although can be used for the detection, but the LOD and LOQis very high.This thesis optimized1H-NMR method to detect the OSCS experimentconditions: the sample concentration is25mg/mL, TSP content is0.020%(W/V),sample spinning for10Hz, the Lb is1.00Hz, PULPROG is zg, D1is4.00s. TheLOD is0.01%and improve more than10times reported in the literature in thisexperiment conditions. The LOQ is0.1%and improve more than5times reported inthe literature.We also used anion exchange resin to separate heparin sodium and OSCS.According to the HP and OSCS charge is different, the single factor experimentresearch is studied. The results indicated that the resin type is FBD202, resin contentis20%, adsorption time for4h, adsorption PH is9.5, adsorption temperature is55℃and salt concentration is15%.This paper also studied the source of the OSCS. Enzymolysis and anionic resin adsorption method were used to extract the three parts of pig intestinal mucosa, casingand intestine’s skin, respectively. The products were analyzed and tested by1H-NMRmethod. The results showed that the product is HP of these three parts but don’tcontain OSCS.In addition, the alcohol fractional precipitation test was researched with organicsolvent ethanol, acetone and methanol. Qualitative analysis for alcohol precipitationproducts were tested by1H-NMR. The results show that ethanol, methanol, acetoneall can purification HP, but the solvent consumption is different and ethanol is9.1%-28.6%, methanol is33.3%-41.2%, acetone is16.7%-33.3%.In order to take more information from the1H-NMR spectra, this paper alsoquantitative analysis organic residue. The results showed that the linear regressionequation of acetone is y=0.0012x-0.4631,R~2=0.9825. The linear regressionequation for ethanol is y=0.0003x-0.0033, R~2=0.9989. The linear regressionequation of methanol is y=0.0006x+0.0293, R~2=0.9963. This method has theadvantages of rapid and accurate.