Tetrodotoxin (TTX) is a marine biotoxin that has high scientific value. However, the lack of efficient TTX extraction and preparation methods has led to a scarcity of TTX samples for clinical application. In this study, TTX from the liver of Takifugu pseudommus was ultrasound-assisted extracted with acidified organic solvents. The extraction process was analyzed and optimized by single factor method and response surface methodology (RSM). The optimal extraction conditions predicted by a response surface model were as follows: liquid:material ratio, 2.8:1; extraction temperature, 60 °C; extraction time, 23.3 min. Under these conditions, the extraction of TTX had a yield of 89.65%, and the results were further verified by experimental extraction, and analyzed by ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). It was found that the extracts of T. pseudommus liver contained TTX and its four analogues at certain proportions (TTX: 10.4%; 5,6,11-trideoxyTTX: 83.3%; 5,11-dideoxyTTX:2.4%; 4,9-anhydro TTX:2.6%; 5-deoxyTTX:1.3%). This study demonstrates a stable and efficient extraction process of TTX from pufferfish liver, which can be helpful for further research and analysis, as well as the utilization of TTX from pufferfish.

Methyl-3-quinoxaline-2-carboxylic acid (MQCA) is the last major remaining detectable metabolite of Olaquindox in animal tissue. A rapid, sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the detection and quantification of MQCA in fish tissue using deuterated quinoxaline-2-carboxylic acid (d(4)-QCA) as internal standard. Various parameters affecting sample preparation, LC separation and MS/MS detection were investigated, and the optimal conditions concerned were determined. Fish tissue samples were subject to hydrochloric acid hydrolysis followed by Oasis MAX solid-phase extraction clean-up; analysis was performed using UPLC coupled to electrospray MS/MS. The chromatographic separation was achieved in less than 5 min. The limit of detection and the limit of quantification were 0.1 and 0.25 ng/g, respectively. The average recoveries of MQCA, spiked at levels of 0.25-50.0 ng/g, were from 92.7 to 104.3%. The relative standard deviation values were <6%. The validated method was successfully applied to analyze 60 batch samples collected from the local market.

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