SPE-HPLC法测定马破伤风免疫球蛋白F(ab')_2中Triton X-100残留量

Determination of Triton X-100 residues in the equine antitetanus immunoglobulin F(ab')_2 by SPE-HPLC

目的：建立固相萃取高效液相色谱（SPE-HPLC）法测定马破伤风免疫球蛋白F（ab’）2中灭活剂Triton X-100的残留量。方法：采用Oasis固相萃取小柱对样品进行预处理,以水和5%甲醇为淋洗剂,以甲醇为洗脱剂,收集甲醇洗脱物用于HPLC分析。采用ZORBAX Extend-C18色谱柱（4.6 mm×150 mm,5μm）,流动相为甲醇-水（80∶20）,流速1.0 mL·min-1,检测波长230 nm,柱温30℃,进样量10μL。结果：Triton X-100浓度在0.56～55.34μg·mL-1范围内,与峰面积线性关系良好（r=0.9998）;检测限（S/N=3）为2.03 ng,定量限（S/N=10）为4.06 ng;提取回收率为91.8%。结论：本方法操作简便、准确、重复性好,可用于Triton X-100的残留量测定。

Objective： To establish a SPE- HPLC method for the determination of Triton X- 100 residues in equine antitetanus immunoglobulin F（ ab＇）2. Methods： Samples were pre- treated on Oasis HLB SPE column,then cleaned- up by water and 5% methanol,and finally extracted by methanol. The analysis of extracts was performed on a ZORBAX Extend- C18column（ 4. 6 mm × 150 mm,5 μm） with a mobile phase consisting of methanol- water（ 80∶20,v/v） at a flow rate of 1.0 mL·min- 1. The column temperature was set at 30 ℃ and the injection volume was 10 μL. Triton X- 100 was detected at the wavelength of 230 nm with a DAD detection. Results： The established HPLC method resulted in sharp and symmetric peaks of Triton X- 100,whereas the calibration curve of Triton X- 100 displayed a good linearity within the concentration of 0. 56- 55. 34 μg·mL- 1（ r =0. 9998）. The LOD and LOQ were 2. 03 ng and 4. 06 ng,respectively. Specificity,precision,accuracy,stability and robustness were acceptable. The extraction recovery was 91. 8%. Conclusion： The results indicate that the established method is convenient,accurate and reproducible,which can be used in the analysis of Triton X- 100 residues in equine antitetanus immunoglobulin F（ ab＇）2.