The Study On Quality Control Of Deproteinized Hemoderivative Of Calf Blood And Calf Serum Injection

Deproteinized hemoderivative of calf blood or calf serum injection produces an organ-unrelated increase of the cellular energy metabolism.It can promote the energy-dependent processes of the functional metabolism and of the conservation metabolism,which makes recoverery of organism.It has very effective therapeutic value in cerebral nerves injury and cerbral infarction.The foreign representative product is Actovegin,which is produced be Nycomed in Denmark.It has tens million dollars amount of imports every year,ranked top list of import drugs.Because of its clinical value,it has aroused doctor's and patient's increasing concerns.However,domestic drug manufactories started to replicate in succession in recent years, which led to a series of problems,such as overplus productive capacity,vicious price competition and negligence of quality control.These problems made people worry about the situation of injection's quality.Recently,a series of drug accidents had happened,such as "Qi Er-Yao", "Houttuynia cordata injection" and "Xin Fu".For the safty of drug's medication,it's necessary to find new way of quality control to achieve the aim of better quality control.This injection has very complicate contents,present drug standard could not illustrate quality problems any more.It is necessary to make comparation to establish new standard for this injection.Small molecular peptide is one of effective contents in this injection.This paper established the analysis method of RP-HPLC,CZE and LC-MS,which were not be listed in this injection's drug standard,for actovegin and five domestic representative products.The study on small peptides by HPLC had optimized some conditions such as mobile phase rate and detection wavelength.This method adopted gradiant elution:the mobile phase A consisted of acetonitrile-water-TFA solution(7:93:0.1),the mobile phase B consisted of acetonitrile- water(4:1). UV-detection was accomplished at 210nm,flow rate was accomplished at 0.3ml/min.The study on small peptides by CZE had optimized some conditions such as concentration and pH of buffer solution,voltage,etc.This method adopted NaH₂PO₄ with pH2.5.voltage is 18kV.The separation temperature is 25℃.The results showed that there are 10 and 8 material in actovegin's RP-HPLC and CZE maps,domestic products had less than actovegin.To resort to LC-MS to make out the moleculars of different peptides.The results showed that all products have the same kinds of 5 peptides,actovegin and two domestic products have 2 other possible peptides.The molecular of these peptides are between 300 and 1000Da.They are mostly consist of several amino acids,from bipeptide,tripeptide to nonapeptide.According to the conclution of MS analysis,to set up a programme to calculate the possible amino acids that small peptides could contain,which would be preparation for MS-MS analysis.The analytical method had good accuracy,reproducibility,and high recovery rate,which could be considered as a quality guidance for domestic injection manufactories.Inorganic salts has great influence to body's normal function,which is very necessary to the normal metabolism.This experiment had a study on 8 normal inorganic metal ions of Deproteinized Hemoderivative of Calf Blood or Calf serum Injection.To determine the content of Kalium and Natrium,0.2%CsCl was added as deionizer.To establish the best GFAAS method, three condition were optimized,including matrix modifier,ashing temperature,and atomization temperature.The calcination temperature of Cu,Fe,Zn,Pb are 1000,1400,700 and 800℃.the atomization temperature of Cu,Fe,Zn,Pb are 2300,2500,1800,1800℃.Matrix modifiers were added when determining the content of Fe,Zn,Pb,such as Mg(NO₃)₂,NH₄H₄PO₄,etc.The results showed that actovegin and domestic products had large contrast between each other.One domestic product had very high content in Ca,Mg and Fe.The standard of this medicine doesn't have any requirement for the content of inorganic metal ions,this paper provided some analysis data for the standard revise of this medicine.In addition,this paper applied gel chromatography method,pre-column derivative method, Folin-phenol method,and Warburg's test to determine the content of macromolecular material, free amino acid,small active peptides and respiratory activity.The results showed that domestic representative products have less content of free amino acid,small active peptides and respiratory activity.This paper analyzed the reason for these differences,and gave some suggestion which could improve quality of domestic products as a guidance.