Determination of Tetrodotoxin in Puffer-fish Tissues, and in Serum and Urine of Intoxicated Humans by Liquid Chromatography with Tandem Mass Spectrometry

Abstract

A simple and rapid method was developed for the analysis of tetrodotoxin in puffer-fish tissues, and in serum and urine of humans poisoned after consuming puffer-fish, by means of high-performance liquid chromatography with tandem mass spectrometry (LC/MS/MS). Tetrodotoxin was extracted with 2% acetic acid. The extracted solution from puffer-fish tissues was diluted with water, and the extracted solution from human serum and urine was cleaned up by LC/MS/MS with a methacrylate-styrenedivinylbenzene cartridge. The LC separation was performed on a C18 column (50 mm x 2.1 mm i.d.) using 10 mmol/L IPCC-MS7-methanol (65 : 35) as the mobile phase at a flow rate of 0.2 mL/min. The mass spectral acquisition was done in the positive ion mode by applying selected reaction monitoring (SRM). The recoveries of tetrodotoxin were 79-90% from puffer-fish tissues fortified at 0.1 microg/g and 1 microg/g, and 93-101% from human serum and urine fortified at 0.5 ng/mL and 5 ng/mL. The detection limits of tetrodotoxin were 0.01 microg/g in puffer-fish tissues and 0.1 ng/mL in human serum and urine. Thirty samples of puffer-fish from wholesale markets, and 7 serum and 5 urine samples of humans poisoned after consuming puffer-fish were analyzed by this method. Tetrodotoxin was detected in all puffer-fish tissues, and all serum and urine samples at the levels of 0.04-140 microg/g, 0.9-1.8 ng/mL and 15-150 ng/mL, respectively.