Determination of tetrodotoxin in puffer fish by ion-paired reagent-assisted dispersive solid-phase extraction (ion-paired-dSPE) in combination with hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS)

Tetrodotoxin (TTX), one of the most potent neurotoxins, poses a significant threat to consumer health. This study presents an advanced analytical method combining ion-paired reagent-assisted dispersive solid-phase extraction (ion-paired-dSPE) with hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS), for the quantification of TTX residues in puffer fish. Edible tissue samples were extracted twice using a 1 % formic acid–methanol solution and purified via optimized ion-paired-dSPE procedures. TTX adsorbed onto the magnetic nano-adsorbents was subsequently eluted with an acetonitrile–water–formic acid solution (60:40:1, v/v/v). Detection was achieved through electrospray ionization in positive mode, with quantification performed in multiple reaction monitoring (MRM) mode. Correlation coefficients ( r ) in matrix-matched external standard curves ranged from 0.9995 to 0.9997. The limits of detection (LOD) and quantification (LOQ) in puffer fish tissues were 0.1 μg/kg and 0.3 μg/kg, respectively. Spiked recoveries at three concentrations in negative tissue samples ranged from 73.4 % to 87.5 %, with precision under 10.6 %. The method was validated in accordance with 2002/657/EC guidelines and successfully applied to TTX detection in cultured puffer fish samples.