Abstract: In this study, a heart-cutting two-dimensional ultra-high performance liquid chromatography-triple quadrupole/linear ion trap mass spectrometry method was developed and successfully validated for the determination of tetrodotoxin (TTX) in fresh and processed aquatic products. This method was based on the use of two different stationary phase columns (Hypercarb PGC and Acquity BEH Amide), which were connected through a six-port two-position switching valve. Samples were extracted with 0.2% (V/V) acetic acid aqueous solution, and then the extract was diluted with the solvent, filtered and directly injected for analysis. The separation of TTX was carried out on a Hypercarb PGC (2.1 mm × 100 mm, 3 μm) as the 1st dimension through elution with 0.2% (V/V) formic acid aqueous solution. The fraction containing TTX was switched into a trap column (XBridge BEH Amide guard column (4.6?mm × 10?mm, 2.5?μm)), and acetonitrile was simultaneously merged into the fraction by a dilution pump through a tee. After the trap column retained the TTX completely, the valve switched it into the 2nd dimensional stream, and then the TTX was separated on an Acquity BEH Amide (2.1 mm × 100 mm, 1.7 μm) with gradient elution using acetonitrile-H2O both containing 0.1% (V/V) formic acid, detected by positive electrospray ionization tandem mass spectrometry in the multiple reaction monitoring information-dependent acquisition enhanced product ion scanning (MRM-IDA-EPI) mode, and quantified by standard solvent external standard method. The average spiked recoveries were 82.3%–95.4% with relative standard deviations (RSDs) of 2.1%–14% (n = 6). The limit of quantitation (RSN = 10) was 0.002 mg/kg. This simple, selective and sensitive method has been successfully applied to the determination of TTX in fresh and processed aquatic products.

Key words: tetrodotoxin; fresh and processed aquatic products; heart-cutting; two-dimensional ultra-high performance liquid chromatography-triple quadrupole/linear ion trap mass spectrometry