免疫亲和柱净化-液相色谱-串联质谱法测定海洋生物中河豚毒素  

Determination of Tetrodotoxin in Marine Organisms by High Performance Liquid Chromatography- Mass Spectrometry and Cleaned up by Immunoaffinity Column

作　　者：严忠雍[1,2] 张小军[1,2] 李奇富 王莹 柳家鹏 龙举[1,2] 祝银[1,2] 杨会成 

机构地区：[1]浙江省海洋水产研究所,舟山316021 [2]浙江省海洋渔业资源可持续利用技术研究重点实验室,舟山316021 [3]江苏美正生物科技有限公司,无锡214135 [4]浙江省海洋开发研究院,舟山316021

出　　处：《分析化学》2015年第2期277-281,共5页Chinese Journal of Analytical Chemistry

基　　金：国家十二五科技支撑项目(No.2012BAD29B06);浙江省自然科学基金项目(No.LQ13C200004)资助~~

摘　　要：建立了海洋生物中河豚毒素的免疫亲和柱净化-液相色谱-串联质谱分析方法。采用1%乙酸-甲醇提取样品,提取液通过磷酸盐缓冲液稀释,调节至pH 7~8,经免疫亲和柱富集和净化后,LC-MS/MS测定,外标法定量。在ACQUITY UPLC BEH Amide亲水柱上进行分离,流动相为乙腈和含有0.1%甲酸的5 mmol/L乙酸铵溶液,梯度洗脱,电喷雾正离子多反应模式监测。河豚毒素在0.3~20.0μg/L线性范围内,相关系数大于0.997,定量限为0.3μg/kg,回收率为88.7%~102.3%,相对标准偏差2.0%~6.4%。本方法重现性好、灵敏度高,适用于海洋生物中河豚毒素的测定。A method was developed for the determination of tetrodotoxin in marine organisms by high perfor-mance liquid chromatography-mass spectrometry with immunoaffinity column. The samples were extracted with 1% acetic acid methanol solution and diluted with phosphate buffer at pH 7-8. After cleaned up by immuno-affinity column, the samples were analyzed by LC-MS/MS and quantitatively determined by external standard method. The chromatographic separation was performed on an ACQUITY UPLC BEH Amide column with gradient elution by using acetonitrile and 5 mol/L ammonium acetate solution containing 0. 1% formic acid as mobile phase. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple reaction monitoring mode. Linear ranges of TTX was in the range of 0. 3 -20. 0 μg/L with correlation coeffi-cient more than 0. 997. The quantification limit of the method was 0. 3 μg/kg. The recoveries of standard addition for tetrodotoxin were 88. 7%-102. 3%, and the relative standard deviation was 2. 0%-6. 4%. The method could be used to identify and quantify tetrodotoxin in marine organisms with satisfactory reproducibility and sensitivity.

关 键 词：免疫亲和柱 液相色谱-串联质谱 海洋生物 河豚毒素 

分 类 号：O657.63[理学—分析化学]