响应面法优化检测己二酸二肼衍化后破伤风类毒素纯度的排阻型高效液相法  

Optimization of size-exclusion high performance liquid chromatography for determination of purity of adipate hydrazine derivatized tetanus toxoid by response surface methodology

作　　者：蔡峰[1] 马诚[1] 蓝天[1] 陈思[1] 周觉非 赵祥宇[1] 杨溢尧 崔长法[1] 

机构地区：[1]成都生物制品研究所有限责任公司,四川成都610023

出　　处：《中国生物制品学杂志》2016年第10期1094-1098,1103,共6页Chinese Journal of Biologicals

摘　　要：目的响应面法优化检测己二酸二肼(adipate hydrazine,ADH)衍化后破伤风类毒素(tetanus toxoid,TT)纯度的排阻型高效液相(SEC-HPLC)法。方法 SEC-HPLC层析柱Superdex 200 increase 3.2/300流动相为:20 mmol/L Na_2CO_3/NaHCO_3,150 mmol/L NaCl,pH 9.0,柱温25℃,单针检测时间60 min。采用响应面法及中心复合表面设计模型(Central Composite Face-centred,CCF)对层析柱的流速、进样量、样品浓度进行优化,通过分离度、峰对称性、峰理论塔板数、单针进样时间等评价指标,确定最优色谱参数,并在最优色谱参数下连续进样100针,验证其分离效果。结果色谱柱的最优色谱条件为:流速50μl/min,进样量5μl,样品浓度1.0 mg/ml,连续进样100针各响应量均满足优化目标。结论成功采用响应面法优化了检测ADH衍化后TT纯度的SEC-HPLC法,且优化后的方法具有良好的稳定性。Objective To optimize the method for size-exclusion high performance liquid chromatography（SEC-HPLC）for determination of purity of adipate hydrazine（ADH）derivatized tetanus toxoid（TT-AH）by response surface methodology（RSM）. Methods SEC-HPLC column Superdex 200 increase 3.2/300 was adopted, using a mobile phase consisting of 20 mmol/L sodium carbonate/ sodium hydrogen carbonate and 150 mmol/ L sodium chloride at pH 9. 0 and a temperature of 25 ℃. The time for detection of a single injection was 60 min. The flow rate, injection volume and sample concentration were optimized using CCF design based on RSM. The chromatographic parameters were optimized by evaluation on resolution, symmetry and theoretical plate number peak of as well as time for a single injection. A total of 100 samples were loaded consecutively under the optimized condition to verify the resolutin effect. Results The optimal flow rate, injection volume and sample concentration for SEC-HPLC were 50 μl/ min, 5 μl and 1. 0 mg/ml respectively.All the response values of samples in 100 consecutive injections met the requirements for optimization. Conclusion The SEC-HPLC method for determination of purity of TT-AH was successfully optimized by surface response methodology,which showed high stability.

关 键 词：响应面法 己二酸二肼 破伤风类毒素 纯度 排阻型高效液相法 

分 类 号：R392-33[医药卫生—免疫学] O657.72[医药卫生—基础医学]