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Background: Butaphosphan (BTP) has recently been introduced into the Korean aquaculture sector as a stressattenuating agent. In this study, a sensitive chemical analytical method was established for the detection of BTP in the olive flounder (Paralichthys olivaceus) tissues. Methods: Utilizing a method employing liquid chromatography coupled with tandem mass spectrometry (LC-MS/ MS), detection sensitivity, specificity, and precision were satisfactorily established. Temporal changes in the BTP plasma and muscle concentrations were assessed after a single intramuscular injection of BTP (50 and 150 mg/kg) to the olive flounder maintained at 13 °C or 22 °C. Results: High BTP plasma levels were achieved immediately after the injection, and the drug was rapidly eliminated. Additionally, plasma BTP levels were markedly dependent on the elimination rate, which, in turn, seemed dependent on the water temperature, with the drug elimination half-life and mean residence time significantly shorter at 22 °C than 13 °C. Overall, muscle BTP levels were markedly lower than the plasma levels. Notably, muscle levels were not influenced by water temperatures. Muscle BTP concentrations were used to estimate the necessary withdrawal period for drugs used in food fish, with BTP levels maintained far below the possible hazardous limit. Conclusions: In conclusion, the established LC-MS/MS method can be used for BTP residue detection with high sensitivity and reproducibility.

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