注射用辅酶A效价测定研究

Research of Potency Assay of Coenzyme A for Injection

目的:通过比较多种辅酶A测定方法总结出适合注射用辅酶A的效价测定方法。方法:对多批注射用辅酶A制剂进行3种方法的效价测定,分别为《卫生部颁标准二部六册》上的磷酸转乙酰化酶法、高效液相色谱法及辅酶A测定试剂盒法,其中高效液相色谱法以磷酸盐缓冲液(pH7.0)-甲醇(90:10)为流动相,采用Phenomenex C18色谱柱,流速1.0ml/min,259nm为检测波长同时利用二极管阵列检测器对200nm-400nm波段进行监视,并利用磷酸盐缓冲液(pH5.8)对样品进行溶解后进行测定。辅酶A试剂盒法是采用BioVision的Coenzyme A Assay Kit,BioVision公司开发了一种简单快捷的检测多种生物样本中辅酶A水平的方法,利用探针技术捕获游离的辅酶A,生成的产物在可见光波段有吸收。最后对3种方法的结果进行比较。结果:高效液相色谱法中辅酶A在1U/ml-100U/ml的范围内,峰形及线形关系良好(r2=1),样品溶液室温放置长时间稳定。试剂盒法中辅酶A在每孔0.032U-3.2U范围内线形关系良好(r2=0.9976)。结论:高效液相色谱法与辅酶A测定试剂盒法要优于磷酸转乙酰化酶法。实现了室温条件下稳定测定多批注射用辅酶A,改良后的高效液相色谱法可以作为辅酶A效价测定的裁定方法,试剂盒法可以作为快速筛查与检验的方法。

Objective:To summarize the assay method of coenzyme A for injection by the compare of multiple assay methods of coenzyme A.Methods:Assay the potency of coenzyme A for injection from different batches,the first method is the phosphotransacetylase method on Drug Specifications Promulgated by the Ministry of Public Health,P R China.Part 2,Vol 6,the others are HPLC and Coenzyme A Assay Kit.The HPLC method take the pH7.0 phosphate buffer-mathanol (90:10) as mobile phase at a flow rate of 1ml/min and Phenomenex C18 (250mm4.6mm,5m) as column.The detection wavelength was 259nm and oversee the spectrum and the chromatogram with Photodiode Array Detector from 200nm to 400nm.Detective samples after soluted by the pH5.8 phosphate buffer. The Coenzyme A Assay Kit from BioVision . BioVision has developed an easy, convenient assay to measure the CoA level in variety biolo gical samples. In the assay, free CoA is specifically utilized to generate products which react with OxiRed Probe to generate color.At last,compare the assay results by these methods.Results:In the HPLC experiment,the regression was linear over the concentration range of 1U/ml-100U/ml (r2=1),the sample solution keep stable at room temperature. In the Coenzyme A Assay Kit experiment ,the regression was linear over the concentration range of 0.032U/well-3.2U/well(r2=0.9976).Conclusion:The methods of HPLC and Coenzyme A Assay Kit were both better in potency assay than the phosphotransacetylase method.We can assay simultaneously couples of coenzyme A for injection at room temperature steadily,the HPLC method modified could be the referee method,the Coenzyme A Assay Kit could be the rapid method for screening and inspect.