RP-HPLC法测定含保护剂重组人睫状神经营养因子含量

RP-HPLC Determination of Content of rhCNTF with Protection

目的:建立含人血白蛋白(HSA)的重组人睫状神经营养因子(rhCNTF)蛋白含量的 RP-HPLC 测定方法。方法:采用Symmetry300反相 C_4色谱柱(150mm×3.9mm,5μm);以0.1%三氟乙酸(TFA)水-0.1%TFA 乙腈分别组成流动相 A 和 B,以初始浓度比70:30进行终浓度比为10:90的31min 线性梯度洗脱,流速为0.6 mL·min^(-1),检测波长为280nm,柱温35℃。rhCNTF 主峰保留时间在27.7～28.4 min,HSA 保留时间在17.0～17.5min。结果:在线性范围为0.061～0.735 mg·mL^(-1)有良好的线性关系(r=0.9995)。结论:本方法简便、快捷,可用于含 HSA 的 rhCNTF 纯品的纯度检测及含量测定。

Objective：To establish RP -HPLC method for determining the content of rhCNTF with protective agent HSA. Methods：The separation was achieved in a Symmetry300 C4column （150 mm ×3.9 mm,5μm）. within 31 minutes. The solution of 0. 1% TFA water and 0. 1% TFA acetonitrile were used as the mobile phase in lined grade of 70：30 at the beginning and 10：90 at the end. The flow rate is 0. 6 mL · min^-1. The detection wavelength is 280nm. The column＇ s temperature is 35℃. The retention time of rhCNTF is 27.7 -28. 4 min and that of HSA is 17. 0 - 17. 5 min. Results： The data of rhCNTF showed a linear trendline at the range of 0. 061 -0. 735 mg · mL^-1 （r =0. 9995）. Conclusion ：The method is simple, quick and suitable for the detection of the purity and content of rhCNTF.