The Detection Of Harmful Impurities In Crude Heparin

Heparin is an acidic mucopolysaccharide which was found in1916when DagMclean was studing clotting mechanism. It is composed of different mixtures of chainlength of polysaccharide chains including uronic acid (L-aidoo uronic acid IdoA;D-glucuronic acid, GlcA) and glucosamine (α-D glucosamine GlcN). So far, no drug cancompletely replace it, which is still one of the most important biochemical substances.Heparin is widely distributed in mammalian liver, lung, intestine, heart, kidney, spleen,placenta, thymus, muscle and blood and other tissues. In2008, heparin raw materials andfinished drug products entering the United States from overseas were found to contain akind of non-native contaminants that is over-sulfated chondroitin sulfate (OSCS). Withincreasing reports about the harmful heparin products, through the collaborative researchof FDA, companies and research institutions, it has been confirmed that the allergicreactions in some patients is related to the presence of OSCS in heparin. Therefore, thedetection of OSCS in crude heparin became an important indicator of quality control. Itwas reported that the signal of N-acetyl methyl peak of the OSCS contaminated heparinwhich is different from heparin can be detected by1H-NMR in foreign litertures. Bydetecting this peak, we can determine the presence of OSCS in heparin. In addition,foreign research teams have studied the detection method of OSCS’s concentration inheparin by strong anion exchange-high performance liquid chromatography. With theemergence of bovine spongiform encephalopathy and other animal viral diseases, theexclusive porcine origin is a key requirement to guarantee the safety of heparin. Thewhole study was as follows:1.1H-NMR detection of OSCS in crude heparinThe crude heparin had a pretreatment of adjusting the pH to remove the protein crudeheparin and precipitating by an equal volume of80%ethanol; then detected the N-acetylmethyl signals of crude heparin by the scanning conditions of400MHz,298K, and64scans. After several trials, N-acetyl methyl peak signal of OSCS was not detected in thetested samples, and N-acetyl-methyl signal of DS was accompanied by signal of heparin.Therefore, these tested samples were not contaminated by artificially added OSCS, and contains a lot of DS. By adding the appropriate OSCS standard to the crude heparin, westudied the limit of detection of OSCS by1H-NMR. The signal of0.25%OSCS which isadded to the tested simple was too weak to distinguish from noise. So the limit ofdetection of OSCS was0.5%by this method.2. SAX-HPLC detection of OSCS in crude heparinReferring to the method of U.S.FDA literature, we added series amounts of OSCSUSP standards to crude heparin to determine the limit of detection of OSCS. The results ofthe two methods were consistent with NMR, the peak of0.25%OSCS added to crudeheparin was difficult to distinguish; Therefore, the limit of detection of OSCS in crudeheparin was0.5%by this method. The method from U.S. FDA literature needed consumeless time than that from Chinese Pharmacopoeia2010edition, so the method from U.S.FDA has more practical value.3Real-time fluorescent quantitative PCR detection of crude heparin in ruminant geneWe have amplicted ruminant DNA in crude heparin by using two kinds of primersand two kinds of fluorescent quantitative PCR method. Both of these two kinds of primerscould cause cross-reaction between procine DNA and ruminant DNA, but thiscross-reaction could not be a serious obstacle for the identification of ruminant DNA andporcine DNA. The limits of detection of pocrine DNA and ruminant DNA by using thesetwo kinds of primers were1.16pg/μl、0.05pg/μl and0.116pg/μl、0.05pg/μl, respectively.However, fluorescent probe reaction system had lighter effect of cross-reaction and betterspecificity for ruminant DNA detection, the limit of detection of ruminant DNA is0.05pg/μl. Therefore, with abundant test costs, using fluorescent probes to detect ruminant DNAin crude heparin could be more accurate.