Simultaneous identification and quantification of tetrodotoxin in fresh pufferfish and pufferfish-based products using immunoaffinity columns and liquid chromatography/quadrupole-linear ion trap mass spectrometry  

作　　者：郭萌萌 吴海燕 江涛 谭志军 赵春霞 郑关超 李兆新 翟毓秀 

机构地区：[1]Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality,Ministry of Agriculture,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences [2]National Center for Quality Supervision and Test of Aquatic Products [3]Key Laboratory of Food Safety Risk Assessment,Ministry of Health,China National Center for Food Safety Risk Assessment

出　　处：《Chinese Journal of Oceanology and Limnology》2017年第4期883-893,共11页中国海洋湖沼学报（英文版）

基　　金：Supported by the National Natural Science Foundation of China(No.41106109);the China National Food Safety Standards Development Project(No.ZHENGHE-2015-356)

摘　　要：In this study, we established a comprehensive method for simultaneous identification and quantification of tetrodotoxin （TTX） in fresh pufferfish tissues and pufferfish-based products using liquid chromatography/quadrupole-linear ion trap mass spectrometry （LC-QqLIT-MS）. TTX was extracted by 1% acetic acid-methanol, and most of the lipids were then removed by freezing lipid precipitation, followed by purification and concentration using immunoaffinity columns （IACs）. Matrix effects were substantially reduced due to the high specificity of the IACs, and thus, background interference was avoided. Quantitation analysis was therefore performed using an external calibration curve with standards prepared in mobile phase. The method was evaluated by fortifying samples at 1, 10, and 100 ng/g, respectively, and the recoveries ranged from 75.8%--107%, with a relative standard deviation of less than 15%. The TTX calibration curves were linear over the range of 1-1 000 ~tg/L, with a detection limit of 0.3 ng/g and a quantification limit of 1 ng/g. Using this method, samples can be further analyzed using an information- dependent acquisition （IDA） experiment, in the positive mode, from a single liquid chromatography-tandem mass spectrometry injection, which can provide an extra level of confirmation by matching the full product ion spectra acquired for a standard sample with those from an enhanced product ion （EPI） library. The scheduled multiple reaction monitoring method enabled TTX to be screened for, and TTX was positively identified using the IDA and EPI spectra. This method was successfully applied to analyze a total of 206 samples of fresh pufferfish tissues and pufferfish-based products. The results from this study show that the proposed method can be used to quantify and identify TTX in a single run with excellent sensitivity and reproducibility, and is suitable for the analysis of complex matrix pufferfish samples.In this study,we established a comprehensive method for simultaneous identification and quantification of tetrodotoxin(TTX) in fresh pufferfish tissues and pufferfish-based products using liquid chromatography/quadrupole-linear ion trap mass spectrometry(LC-QqLIT-MS).TTX was extracted by 1% acetic acid-methanol,and most of the lipids were then removed by freezing lipid precipitation,followed by purification and concentration using irnmunoaffinity columns(IACs).Matrix effects were substantially reduced due to the high specificity of the IACs,and thus,background interference was avoided.Quantitation analysis was therefore performed using an external calibration curve with standards prepared in mobile phase.The method was evaluated by fortifying samples at 1,10,and 100 ng/g,respectively,and the recoveries ranged from 75.8%-107%,with a relative standard deviation of less than 15%.The TTX calibration curves were linear over the range of 1-1 000 ng/L,with a detection limit of 0.3 ng/g and a quantification limit of 1 ng/g.Using this method,samples can be further analyzed using an informationdependent acquisition(IDA) experiment,in the positive mode,from a single liquid chromatography-tandem mass spectrometry injection,which can provide an extra level of confirmation by matching the full product ion spectra acquired for a standard sample with those from an enhanced product ion(EPI) library.The scheduled multiple reaction monitoring method enabled TTX to be screened for,and TTX was positively identified using the IDA and EPI spectra.This method was successfully applied to analyze a total of 206 samples of fresh pufferfish tissues and pufferfish-based products.The results from this study show that the proposed method can be used to quantify and identify TTX in a single run with excellent sensitivity and reproducibility,and is suitable for the analysis of complex matrix pufferfish samples.

关 键 词：TETRODOTOXIN fresh pufferfish pufferfish-based product immunoaffinity column liquidchromatography/quadrupole-linear ion trap mass spectrometry 

分 类 号：O657.63[理学—分析化学] TS254.7[理学—化学]