Simplification of extraction procedure and evaluation of extraction ratio in pufferfish toxin examination method

Abstract

We compared two extraction methods for pufferfish toxin: the method described in "Standard Methods of Analysis in Food Safety Regulation'' (official method), and an alternative in which the procedures of the official method were simplified (simple method). Mainly the livers of Takifugu rubripes were extracted with the two methods, and the amount of tetrodotoxin (TTX) in each extract was quantified by high-performance liquid chromatography with fiuorescence detection (HPLC-FL). In general, the results of the simple method were about 10-20% higher than those of the official method. A strong positive correlation between the values determined by the simple method (y) and that of the official method (x) was detected; the fitted regression line and 90% confidence interval could be expressed as y＝(1.21±0.033) x－(1.36±1.29), where the extraction ratio [ratio of (extractant＋sample) to the sample] was 3 to 6. Non-toxic cultured T. rubripes livers spiked with known concentrations of TTX were extracted by the simple method, and TTX as well as 4-epiTTX and 4,9-anhydroTTX converted from TTX were quantified by HPLC-FL. As a result, the recovery rate was 82.7 to 119%, which satisfied the reference value of the guidelines in Japan.