Objective To compare magnetic beads kit,agrose gel recovery kit and heparinase I three methods to purify the micro DNA from crude heparin, then use q-PCR to identify the species origins and select the best method.Methods Using magnetic beads kit,agrose gel recovery kit and heparinase I to purify micro DNA from crude heparin and combined the porcine,bovine and ovine identification kits to identify the species origins and conformed the minimum detection limit of different percentage of ovine crude heparin in porcine crude heparin.Results Three pretreatment methods all can solve the pretreatment difficulties and we found that the haparinase was the best method; the minimum detection limit was 0.01%of ovine crude heparin in porcine crude heparin.Conclusion The heparinase method is the best pretreatment method and can successfully solve the pretreatment difficulties.Heparinase combine the porcine, bovine and ovine identification kits can identify the species origins from crude heparin.

Objective To develop and verify a method for determination of residual host cell DNA in recombinant human interferon α2b substances, which is used for the quality control of the product.Methods The residual host cell DNA was extracted by wako DNA extractor kit and determined by SYBRGreen based q-PCR using standard DNA as control.The residual host cell DNA was analyzed according to the standard curve.The developed method was verified by primer specifity, results accuracy and precision and used for determination of 3 batches of interferon substances. Results The minimum quantitative limit of residual host cell DNA by the developed method was 12 fg/μL, while the linear range was 12 fg/μL-120 ng/μL, with a correlation coefficient (r) of 0.998.The designed primers were specific to the DNA templates.The recovery rates of spiked samples with different DNA quantity were between 50%-200%.The residual host cell DNA determined by this method were not more than the limit, which were complied with the requirements for residual host cell DNA in Chinese Pharmacopeia ( volume III,2010 edition and 2015 edition) .Conclusion The wako DNA extractor kit could successfully solved the technical difficulties of sample pretreatment during residual DNA assay.The q-PCR method was simple, rapid and accurate for quantitation of residual host cell DNA in interferon substances.

Objective A high performance liquid chromatographic (HPLC) method was established for the determination of glycerol in propofol medium and long chain fat emulsion injection.MethodsThe chromatographic conditions were as follows: Kromasil 100-5-NH2 column(4.6×250mm,5μm) with the column temperature was 40℃,acetonitrile-water(8515)as mobile phase with flow rate of 1.0mL/min.Glycerol was detected by refractive index (RI) detector at 40℃.ResultsThe linear range of glycerol was 455.3916-2276.9580μg/mL(r=0.9999,n=7),the average recovery rate was 99.5%,RSD was 0.6%(n=9),the limit of detection(LOD) was 121ng and the limit of quantification(LOQ)was 364ng.ConclusionThe method was simple, rapid, strong specifity and accurate with good reproducibility, which is suitable for the content determination of glycerol in propofol medium and long chain fat emulsion injection.

To establish a high performance liquid chromatography(HPLC)method to determine the content of bacteriostats in the ocular extractives eye drops, Diamonsil C18(4. 6 mm×250 mm, 5 μm)column was used, with gradient elusion by 1% triethylamine solution(pH 3. 0)(mobile phase A)and methanol(mobile phase B). The detection wavelength was 256 nm; the column temperature was 40 °C; and the flow rate was 1. 0 mL/min. Under these conditions, the three bacteriostats of methylparaben, ethylparoben and chlorhexidine acetate showed good resolution. The bacteriostats exhibited good linear relationship between the peak area and the concentration in the concentration range of 0. 1- 80 μg/mL(r> 0. 999 1). The recoveries were from 97. 2% to 104. 1%, and the RSD was 0. 8% to 1. 2%. The content of bacteriostats in all the five batches of ocular extractives eye drops was less than 10% of the prescription amount. It was found that the activated carbon used in the production process had strong adsorption effect on the bacteriostat, and that the lower the temperature and the higher the concentration of activated carbon, the stronger the adsorption of bacteriostatic agent. The adsorption capacity of activated carbon for different bacteriostats is: chlorhexidine acetate > ethylparoben > methylparaben. The results showed that the established HPLC method was easy to operate with high sensitivity and good repeatability. It can be used to determine the content of bacteriostat in ocular extractives eye drops quickly and accurately. In addition, this study reveals for the first time the effect of impurity removal process on bacteriostat in the production of ocular extractives eye drops. It is not suitable to use activated carbon to remove impurities before adding parabens and chlorhexidine acetate bacteriostats. The current work provides a new guiding basis for the monitoring and improvement of the quality of ocular extractives eye drops.

To investigate the freshness, high molecular weight substances, the determination of polypeptide, haemolysis and agglomeration, biological activity of Cervus and Cucumis polypeptide injection; to provide the direction for improving the quality of products for enterprises; furthermore, to provide reference for the revision of the quality standards of Cervus and Cucumis polypeptide injection. Firstly, we investigated the factors affecting the freshness of the injection, including biogenic amines, aflatoxins, the acid value and peroxide value of the melon seeds. The method of dansyl chloride pre-column derivatization-HPLC was used to determine the content of 8 biogenic amines in Cervus and Cucumis polypeptide injection. The method validation results showed good specificity, precision, linearity and recovery rates, which was suitable for the determination of biogenic amines in Cervus and Cucumis polypeptide injection. The results of sample determination showed that relatively higher concentrations of cadaverine were detected in the products from company B. The results of aflatoxins, acid value and peroxide value showed that the melon seeds from some companies had rancidity, mildew and other problems, indicating that the quality standards of multi-component biochemical drugs containing animal- and plant-derived components should be controlled in terms of freshness. Secondly, the methods for the determination of high molecular weight substances and polypeptides in the quality standard were improved. Tricine-SDS-PAGE electrophoresis was used instead of gel chromatography to determine the high molecular weight substances, which improved the accuracy of determination. The kits were used instead of folin-phenol for the determination of peptide content, which is easy to operate, specific and suitable for high-throughput sample determination. Finally, the haemolysis, agglomeration, and biological activity of Cervus and Cucumis polypeptide injection were studied. The results showed that no haemolysis and agglomeration were found in all samples, and the inhibitory effect of samples on THP-1 proliferation in vitro from different companies was different to some extent. In conclusion, the optimized quality standard is more suitable for the detection of Cervus and Cucumis polypeptide injection, and can lay the foundation for improving the safety of multi-component biochemical drugs.