Quantitation of Tetrodotoxin and Its Analogues with a Combination of Liquid Chromatography–Tandem Mass Spectrometry and Quantitative ¹H-NMR Spectroscopy

Tetrodotoxin and its analogues are the causative toxins of pufferfish poisoning. Tetrodotoxin has been recently detected in bivalve mollusks collected in New Zealand and Europe, highlighting the need to include tetrodotoxin in monitoring programs for bivalves by instrumental methods. In the present study, tetrodotoxin and its analogues in commercially available tetrodotoxin reagents were quantitated accurately by quantitative 1 H-nuclear magnetic resonance (qNMR) spectroscopy. The results were applied to estimate relative molar responses of tetrodotoxin and its analogues in hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC/MS/MS). All four components (tetrodotoxin hemilactal form ( 1 ), tetrodotoxin 10,7-lactone form ( 2 ), 4- epi tetrodotoxin ( 3 ), and 4,9-anhydrotetrodotoxin ( 4 )) generated by equilibrating tetrodotoxin in aqueous solution were prepared as a mixture. From the HSQC spectrum of the mixture, the separated signals derived from three components, excluding 1, were selected and used for the quantitation. In addition, the relative molar responses of 3 and 4 on HILIC/MS/MS were calculated to be 0.73 and 0.46, respectively. These values could be useful for quantitation of 3 and 4 using the tetrodotoxin standard by HILIC/MS/MS. Our results also indicate that qNMR is useful for preparation of tetrodotoxin certified reference material.