摘要
通过高效液相色谱法(HPLC)建立一种测定CN10冻干品中蛋白含量的方法。以0.1 mol/L TrisHCl、0.1 moL/L NaCl、pH 7.2缓冲液作为流动相,色谱柱为TSK2000SWxl(4.6 mm×250 mm)凝胶过滤色谱柱,体积流量为0.65 mL/min,进样体积50μL,检测波长选择280 nm,依据HPLC外标法原理建立CN10含量测定方法,并根据《生物制品质量控制分析方法验证技术审评一般原则》对方法进行相关验证。结果:在该条件下,CN10和人血白蛋白之间有良好的分离度,CN10浓度在0.1~0.6 mg/mL的范围内与其峰面积呈良好的线形关系(R2=0.998 1),最低检出限和最低定量限分别0.01 mg/mL和0.04 mg/mL。方法的重复性RSD(%)为0.38,平均回收率为(100.40±2.28)%。用该方法检测CN10冻干制剂中的CN10蛋白含量时,不受人血白蛋白的影响,具有较高的准确性和专属性。
To establish a high performance liquid chromatography( HPLC) method for detecting the content of CN10 protein in CN10 lyophilized preparation,the mobile phase consisted of 0. 1 mol /L Tris-HCl,0. 2 mol /L NaCl( adjusted pH value to 7. 2 with HCl),TSK2000 SWxl column( 4. 6 mm × 250 mm) was used to separate CN10 and some related substances. The detection wavelength was280 nm and the flow rate was 0. 65 mL /min. CN10 content was calculated by external standard method. The method was validated according to'The general principles of technical review of quality control analysis method of biological products'. Under this chromatography conditions,an excellent separation was achieved for CN10 and human serum albumin. It showed a good linear relationship between the peak area and the concentration of CN10 in the range of 0. 1 ~ 0. 6 mg /mL( R2= 0. 998 1). The lowest detection limit( LOD) and the lowest quantitation limit( LOQ) were 0. 01mg/mL and 0. 04 mg/mL respectively. The average recovery was( 100.40 ±2.28) % and the precision( RSD) was 0.38. When using this method to detect CN10 content in CN10 lyophilized preparation,this method was highly specific and accurate. It also was not affected by human albumin.
出处
《药物生物技术》
CAS
2014年第3期246-249,共4页
Pharmaceutical Biotechnology
关键词
冻干品
SEC-HPLC
外标法
含量测定
CNTF
人血白蛋白
SEC-HPLC,Lyophilized formulation,External standard method,Quantitative determination,CNTF,Human serum albumin
