Analysis of tetrodotoxin in puffer-fish by LC/MS

Abstract

A simple and reliable method using liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) for the analysis of tetrodotoxin in puffer-fish was developed. Tetrodotoxin in puffer-fish was extracted with 0.1% acetic acid by heating in a boiling water bath, and the extracts were cleaned up on a Bond Elut C18 (500 mg) cartridge. The LC separation was performed on a TSK-gel ODS 80Ts column (25 cmx2 mm i.d.) using 5 mmol/L heptafluoro-n-butyric acid (HFBA)-methanol (99: 1) as the mobile phase at a flow rate of 0.2 mL/min. Positive ionization produced a typical [M+H]+ molecular ion of tetrodotoxin (m/z 320.1). The calibration graph for tetrodotoxin was rectilinear from 0.1 to 1 μg/mL with selected ion monitoring (SIM). The detection limit of tetrodotoxin in puffer-fish was 1 μg/g (equivalent to ca. 5 MU/g).