**10. Capillary isotachophoresis**

Capillary isotachophoresis proved to be a very effective technique for the analyses of organic acids, carbohydrates, drugs and amino acids. It is a rapid and accurate detection technique for TTX [52]. It is performed using a cationic system, as TTX exists as cation under acidic and neutral conditions. Conditions for capillary isotachophoresis composed of a leading electrolyte of 5 mM potassium acetate (pH6.0), containing 0.2% Triton X-100 and 0.5 volume of dioxane, and a terminating electrolyte of 10mM β-alanine adjusted to pH 4.5 with acetic acid. When TTX is applied to isotachophoretic analyzer (Shimadzu IR-2A) equipped with a potential gradient 0.32, it is eventually monitored by the detector. The quantitative detection limit by this method is about 0.25μg of TTX. It was possible to quantify TTX content of contaminated puffer extracts without any pretreatment.

## **11. Conclusion**

In an attempt to protect consumers from TTX-intoxication, the mouse bioassay has historically been the most universally applied tool to determine the toxicity level in monitoring programs. This bioassay, however, shows low precision and requires a continuous supply of mice of a specific size. These potential drawbacks and world-wide pressure to refrain from the unnecessary killing of live animals subsequently led scientists to develop alternative chemical methods to the mouse bioassay for TTX detection and quantification. TTX levels in pufferfish are normally estimated using the mouse bioassay. However, this assay and other techniques such as TLC, electrophoresis, LC, spectrophotometry, and the enzyme-linked immunosorbent assay (ELISA) pose ethical concerns, are not specific and lack sensitivity and precision at low concentrations. HPLC-FLID and LC-MS/GC-MS are sensitive techniques for identification of TTX. However, due to the complexity of sample matrices and insolubility of TTX in organic solvents, HPLC-FLD and LC-MS (or LC-MS/MS) are more preferred methods than GC-MS. MS spectrometry is a powerful technique that also has an important future for the analysis of marine toxins. In addition to high sensitivity and selectivity, MS can provide structural information useful for the confirmation of toxin identity and the identification of new toxins. The drawback of LC-MS and LC-MS/MS analyses is that they involve the use of expensive instruments, which require higher maintenance compared to GC-MS. Nevertheless, for routine analysis of TTXs, HPLC-FID and LC-MS (LC-MS/MS) are expected to replace the conventional mouse bioassay.
