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. 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) 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%. 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 information dependent (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 actually identified using the IDA and EPI spectra. 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 that 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.