尿激酶中分子组分比2种测定方法的建立和比较研究

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(7.8 mm ×300 mm,5 μm ),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 ?...

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

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 content range of high molecular mass and low molecular mass urokinase was 87.0%-96.4%and 3.6%-13.0%,respectively.Conclusion:Both methods are suitable for the determination of molecular fractions in urokinase and complement for each other.