摘要
目的:建立蛇毒血凝酶纯度测定方法。方法:分别采用SDS-聚丙烯酰胺凝胶电泳法(SDS-PAGE)和等电聚焦法(IEF),对凝胶考染并扫描;采用体积排阻高效液相色谱法(SEC-HPLC),使用TSK G3000SW(7.8 mm×300 mm)凝胶柱,流动相为55 mmol.L-1柠檬酸钠-0.01%Tween 80溶液;流速0.5 mL.min-1,检测波长280 nm;采用反相高效液相色谱法(RP-HPLC),使用C8Vydac(4.6 mm×250 mm,5μm)色谱柱,流动相为0.1%三氟乙酸溶液(A)-含0.1%三氟乙酸的90%乙腈(B),梯度洗脱,流速0.8 mL.min-1,;柱温为45℃,检测波长:280 nm。结果:4种方法测得蛇毒血凝酶的纯度分别为89.3%,88.5%,95.4%,87.4%。结论:对于蛇毒血凝酶纯度测定,非还原SDS-PAGE、IEF及RP-HPLC法均可准确地反映其样品纯度。
Objective：To establish a method to determine the purity of Haemocoagulase（HC）.Methods：SDS-polyacrylamide gel electrophoresis（SDS-PAGE）,isoelectric focusing（IEF）,size exclusion column-HPLC（SEC-HPLC）and reversed phase-HPLC（RP-HPLC） were used respectively to determine the purity of HCA.TSK G3000SW（7.8 mm×300 mm） column was applied for SEC-HPLC,using 55 mmol·L-1 citric acid sodium-0.01% Tween 80 solution as the mobile phase the flow rate of 0.5 mL·min-1,the detection wavelength at 280 nm;C8 Vydac（4.6 mm×250 mm,5 μm） column was employed for RP-HPLC,using 0.1% triflouroacetic acid（TFA） as the mobile phase A,and 90% acetonitrile containing 0.1% TFA as the mobile phrase B,at the flow rate of 0.8 mL·min-1,the detection wavelength 280 nm.Results：The results of purity detected by the four methods were 89.3%,88.5%,95.4% and 87.4%,respectively.Conclusion：Except SEC-HPLC,the other three methods were feasible to determine the purity of HCA.
出处
《药物分析杂志》
CAS
CSCD
北大核心
2012年第8期1398-1401,共4页
Chinese Journal of Pharmaceutical Analysis
基金
中国食品药品检定研究院中青年基金项目资助(2010A7)
