尿激酶中分子组分比2种测定方法的建立和比较研究  

Establishment and comparative study of two methods for the determination of molecular fractions in urokinase

作　　者：严翠霞[1] 徐明明[1] 程菁 孙珍[1] 郑璐侠[1] 陈钢[1] 邵泓[1] YAN Cui-xia;XU Ming-ming;CHENG Jing;SUN Zhen;ZHENG Lu-xia;CHEN Gang;SHAO Hong(NMPA Key Laboratory of Quality Control of Therapeutic Monoclonal Antibodies,Shanghai Institute for Food and Drug Control,Shanghai 201203,China)

机构地区：[1]上海市食品药品检验所国家药监局治疗类单抗质量控制重点实验室,上海201203

出　　处：《药物分析杂志》2021年第1期104-110,共7页Chinese Journal of Pharmaceutical Analysis

摘　　要：目的:建立尿激酶中分子组分比测定的分子排阻色谱法与十二烷基硫酸钠毛细管电泳法,并对2种方法的方法学验证与样品测定结果比较。方法:分子排阻色谱法采用TSKgel G2000SWXL色谱柱(7.8mm×300 mm,5μm),以0.1 mol·L^(-1)磷酸二氢钠缓冲液(pH 3.0)为流动相,流速0.5 mL·min^(-1),柱温35℃,检测波长280 nm,进样量50μL;十二烷基硫酸钠毛细管电泳法采用未涂层熔融石英毛细管(50μm×30.2cm,有效长度20.2 cm),分离电压为15 kV,柱温25℃,检测波长214 nm,进样端为正极,5 kV压力进样20 s。结果:通过对分子排阻色谱法与十二烷基硫酸钠毛细管电泳法的方法学验证与样品测定结果的比较,证明两种方法测定结果基本一致。分子排阻色谱法:高分子量与低分子量尿激酶质量浓度在0.05~5.1mg·mL^(-1)范围内呈良好的线性关系;检测下限分别为1.5μg·mL^(-1)与5.1μg·mL^(-1),定量下限分别为5.1μg·mL^(-1)与16.9μg·m L^(-1);8批样品中高分子量与低分子量尿激酶相对含量范围分别为87.0%~96.4%与3.6%~13.0%。十二烷基硫酸钠毛细管电泳法:高分子量与低分子量尿激酶质量浓度在0.1~5.2 mg·mL^(-1)范围内呈良好的线性关系;检测下限分别为2.5μg·mL^(-1)与51.8μg·mL^(-1),定量下限分别为7.5μg·mL^(-1)与155.4μg·m L^(-1);8批样品中高分子量与低分子量尿激酶相对含量范围分别为88.2%~97.3%与2.7%~11.8%。结论:分子排阻色谱法与十二烷基硫酸钠毛细管电泳法都适用于尿激酶中分子组分比的测定并互为补充。Objective:To establish size exclusion chromatography(SEC)and sodium dodecyl sulfate capillary electrophoresis(CE-SDS)for the determination of the molecular fractions in urokinase,and the methodological validation and sample determination results of the two methods were compared.Methods:SEC was performed on a TSKgel G2000 SWXL column(7.8 mm×300 mm,5μm)with 40.1 mol·L^(-1) sodium dihydrogen phosphate buffer(pH 3.0)mobile phase as the mobile phase,the flow rate was 0.5 mL·min^(-1),the column temperature was 35℃,the detection wavelength was 280 nm and the injection volume was 50μL.CE-SDS was performed on a uncoated fused quartz capillary tube(50μm×30.2 cm,effective length 20.2 cm),the separation voltage was 15 kV,the column temperature was 25℃,the detection wavelength was 214 nm,the injection end was positive electrode and the injection pressure was 5 kV for 20 s.Results:By comparing the results of the methodological validation and sample determination results of the two methods,it was proved that the results of the two methods were almost the same.The SEC method results showed the linear relationship of high molecular mass and low molecular mass urokinase was good when the concentration of urokinase was in the range of 0.05-5.1 mg·mL^(-1).The detection limit of high molecular mass and low molecular mass urokinase was 1.5μg·mL^(-1) and 5.1μg·mL^(-1),respectively,and the lower limit of quantification was 5.1μg·mL^(-1) and 16.9μg·mL^(-1),respectively.The relative content range of high molecular mass and low molecular mass urokinase was 87.0%-96.4%and 3.6%-13.0%,respectively.The CE-SDS method results showed the linear relationship of high molecular mass and low molecular mass urokinase was good when the concentration of urokinase was in the range of 0.1-5.2 mg·mL^(-1).The detection limit of high molecular mass and low molecular mass urokinase was 2.5μg·mL^(-1) and 51.8μg·mL^(-1),respectively,and the lower limit of quantification was 7.5μg·mL^(-1) and 155.4μg·mL^(-1) respectively.The relative

关 键 词：尿激酶 分子组分比 高分子量 低分子量 分子排阻色谱法 十二烷基硫酸钠毛细管电泳法 

分 类 号：R917[医药卫生—药物分析学]