| Summary: | Tetrodotoxin (TTX) inhibits neurotransmission in animals, and there is no specific antidote. In clinical practice in China, <i>Althaea rosea</i> (<i>A. rosea</i> flower) extract has been used to treat TTX poisoning. In this work, the efficacy of the ethyl acetate fraction extract of <i>A. rosea</i> flower in treating TTX poisoning in rats was investigated. A high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed to determine nine neurotransmitters in rat brain tissue, including γ-aminobutyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT), noradrenaline (NE), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindole-3-acetic acid (5-HIAA), epinephrine (E), and tyramine (Tyn). The detoxifying effect of <i>A. rosea</i> flower was verified by comparing the changes in neurotransmitters’ content in brain tissue before and after poisoning in rats. The assay was performed in multiple reaction monitoring mode. The quantification method was performed by plotting an internal-standard working curve with good linearity (R<sup>2</sup> > 0.9941) and sensitivity. Analyte recoveries were 94.04–107.53% (RSD < 4.21%). Results indicated that the levels of 5-HT, DA, E, and NE in the brains of TTX-intoxicated rats decreased, whereas the levels of GABA, Tyn, and 5-HIAA showed an opposite trend, and HVA and DOPAC were not detected. The levels of all seven neurotransmitters returned to normal after the gavage administration of ethyl acetate extract of <i>A. rosea</i> flower to prove that the ethyl acetate extract of <i>A. rosea</i> flower had a therapeutic effect on TTX poisoning. The work provided new ideas for studies on TTX detoxification.
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