**8. Thin-layer chromatography**

TLC is a very commonly used technique in synthetic chemistry for identifying compounds, determining their purity. In TLC analysis, TTX is spotted onto a silica gel 60 precoated plate (Merck). The plate is developed in two different solvent systems of pyridine-ethyl acetate-AcOH -water (15:5:3:4) and 3-BuOH-AcOH-water (2:1:1) in a sealed chamber. The solvent rises by capillary action and an ascending chromatographic separation is obtained. The plate is then sprayed with 10% KOH followed by heating at 100℃ for 10 minutes. The toxin is visualized as a yellow fluorescent spot under UV light (365nm). In TLC analysis, the Rf values of TTX are around 0.71(0.65) and 0.50, respectively [10, 51]. It is also possible to detect TTX on the TLC plate using the Weber reagent that gives pink spot of the toxin. The detection limit is about 2 μg of TTX (10 MU). TLC is a useful technique in those laboratories where HPLC and other costly analytical systems are not available.

Instrumental Analysis of Tetrodotoxin 267

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.

**Author details** 

Manabu Asakawa\*

Yasuo Shida

Tamao Noguchi

**12. References** 

Corresponding Author

 \*

**Acknowledgement** 

helpful advice on ribbon worms.

*Nippon KagakuZasshi*, 71:590-592.

*Chem.Pharm. Bull.* 12:1357-1374.

*Higashi-Hiroshima, Hiroshima, Japan* 

*Tokyo Healthcare University, Setagaya, Tokyo, Japan* 

and Keisuke Miyazawa

*Department of Bioresource Science and Technology, Department of Bioresource Science and Technology, Graduate School of Biosphere Science, Hiroshima University, Kagamiyama,* 

*Clean Energy Research Center, University of Yamanashi, Takeda, Kofu, Yamanashi, Japan* 

This work was supported by a Grant-in-Aid for Scientific Research (C) (2) (No.11660205), (C)(No.18580204), (C)(No.20580220) and (B)(No.20380110) from Japan Society for the Promotion of Science. We are grateful to Dr.Hiroshi Kajihara (Hokkaido University) for his

[1] Yokoo, A. (1950) Chemical studies on pufferfish toxin (3) – separation of spheroidine.

[2] Tsuda, K., Ikuma, S., Kawamura, M., Tachikawa, R., Sakai, K., Tamura, C., Amakasu, D. (1964) Tetrodotoxin. VII. On the structures of tetrodotoxin and its derivatives.

[3] Woodward, R.B. (1964) The structure of tetrodotoxin. *Pure Appl. Chem.* 9:49-74.
