Ohtsuka Y., Tokunaga H., Fukushima S.^*, Kudo K., Imamura T., Jitsufuchi N., Ikeda N.

Department of Forensic Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan
^*Forensic Science Laboratory of Fukuoka Prefectural Police HQ, Fukuoka, Japan

A sensitive and reliable method for the determination of tetrodotoxin (TTX) in human serum was developed using GC/MS.
Methods. TTX was extracted from serum with 2% acetic acid in methanol, purified with a Sep-Pak C₁₈ cartridge, and transformed into 2-amino-6-hydroxymethyl-8-hydroxy quinazoline (C₉-base) with 1 M potassium hydroxide. The C9-base was purified with another Sep-Pak C₁₈ cartridge, converted to TMS derivative together with ß-estradiol (IS), and applied to GC/MS. Quantitation was carried out by the SIM mode with the target ions of m/z 392 for C₉-base and m/z 285 for IS.
Results. Recovery of TTX and C₉-base from the cartridge was significantly increased by removing metallic impurities in the cartridge with EDTA and by adding TFA to the C₉-base solution. Lower limit of detection was 1 ng/ml.
Conclusion. Our method was proved to be much more sensitive than previously published methods, and should be useful in forensic toxicological examination of globefish poisoning.