SPE-HPLC法测定马破伤风免疫球蛋白F（ab’）₂中TritonX-100残留量

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，5 μm），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10 μL。 结果：Triton X-100浓度在0.56~55.34 μg·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-C₁₈ column(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.