**Instrumental Analysis of Tetrodotoxin**

Manabu Asakawa, Yasuo Shida, Keisuke Miyazawa and Tamao Noguchi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/48668

## **1. Introduction**

244 Chromatography – The Most Versatile Method of Chemical Analysis

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Tetrodotoxin (TTX), a pufferfish ("fugu" in Japanese) toxin named after its order name Tetraodontiformes, is the toxic principle of puffer fish poisoning. This toxin (C11H17N3O8; a molecular weight of 319) is one of the most potent nonproteinaceous toxins as well as the best-known marine natural toxins (Figure 1). In Japan, pufferfish have been a traditional food for many years, and since people have become accustomed to eating them, cases of TTX poisoning are frequent. It poses a serious hazard to public health. These cases have occurred on a regular basis not only in Japan but also in Asia for a number of years, sporadically resulting in severe poisoning or even death. On the other hand, the Japanese are aware of the its toxicity and have devised methods to reduce TTX levels especially in the liver. However, TTX poisoning incidents continue to occur in Japan. Since there is no antidote for the toxin, patient mortality is very high. Judging from statistics provided by the Japanese Ministry of Health, Labour and Welfare, the number of deaths due to puffer poisoning has steadily declined, from more than 10 cases every year between 1960 and 1981 to less than 10 cases with low mortality every year since 1982, generally with low mortality. This decline is probably due to not only strict adherence to government regulations but also an increase in cultured puffer rather than a decrease in wild puffer. The toxicosis is characterized by the onset of symptoms in the victim. Treatment of the illness is mainly based on the symptoms of the patient. More fruitful treatment can be provided if the causative toxin is identified. In 1950, TTX was isolated for the first time as a crystalline prism from toxic pufferfish ovaries by Yokoo [1]. Its structure was elucidated by three groups in 1964 [2-4]. TTX is a powerful and specific sodium channel blocker [5]. When ingested by humans, it acts to block the sodium channels in the nerve cells and skeletal muscles, and to thereby block excitatory conduction, resulting in the occurrence of typical symptoms and signs such as respiratory paralysis and even death in severe cases. The lethal potency is 5000 to 6000 MU/mg. One MU (mouse unit) is defined as the amount of toxin required to kill a 20g male mouse within 30 min after intraperitoneal administration, and the minimum lethal dose (MLD) for humans is estimated to be approximately 10,000 MU

© 2012 Asakawa et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Asakawa et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

equivalent to 2 mg of pure TTX crystals [6]. Many derivatives of TTX have been found, although their toxicities vary widely. As seen in Figures 1, 2 TTX is a heterocyclic guanide compound whose chemical structure has been characterized. Various TTX derivatives from pufferfish and other TTX-bearing organisms have been identified to date as a result of recent progress in the instrumental analysis of TTX (Figure 3). In marine pufferfish species, toxicity is generally highest in the liver and ovary, whereas in brackish and freshwater species, toxicity is higher in the skin [7-13]. TTX was long believed to be present only in the pufferfish. In 1964, Mosher *et al.* detected TTX in California newt *Taricha torosa*, which was the first TTX-containing organism other than pufferfish [14]. Since then, the distribution of TTX has spread to animals other than pufferfish. The toxin has been detected in a tropical goby *Yongeichthys criniger* [15], atelopid frogs of Costa Rica *Atelopus* spp.[16], blue-ringed octopus *Hapalochlaena maculosus* [17], and several species of carnivorous gastropods such as trumpet shell *Charonia sauliae*[18], ivory shell *Babylonia japonica* [19], frog shell *Tutufa lissostoma* [20] as shown in Table 1*.* In addition, some species of starfish on which these gastropods prefer to feed also contain TTX [21]. The trumpet shell *Charonia sauliae* accumulates TTX by ingesting toxic starfish, supporting the hypothesis that the TTX of pufferfish is not endogenous, but is introduced via the food chain. The exact origin of TTX in the food chain, however, remains unknown. Because the ecologic environments of TTXbearing animals apparently have no common factor other than being closely related to an aquatic system, bacteria (omnipresent organisms that commonly inhabit the aquatic system), are implicated as the primary source of TTX. Toxic crabs, flatworms, horseshoe crabs, ribbon worms and arrow worms were also added to the list of TTX-bearing animals. In Japan, TTX is assayed by the official method using mice [22]. It requires ddY strain male mice, but no special instrumentation. This method is simple and convenient but not so sufficiently accurate, and provides no information on the composition of the toxin, nor is it able to distinguish TTX from other neurotoxins such as paralytic shellfish poison (PSP). In addition, animal rights activists across the world are strongly opposed to bioassays using live animals, including mice. Thin layer chromatography (TLC) and electrophoresis are useful means for TTX detection, but they are not suitable for TTX determination. With this background, attempts have been made to develop analytical methods using high performance liquid chromatography (HPLC) in Japan. Detection and determination of TTX are essential not only for diagnosis and treatment purposes, but also for issuing quarantines and public awareness. Quantitative and/or qualitative detection of TTX in a sample is/are performed by a few instrumental analysis methods. The toxin has long been believed to occur exclusively in pufferfish. However, owing to the recent outstanding progress in instrumental methods for the analysis of TTX, its distribution and accumulation in various aquatic organisms have been established. In addition, this toxin has been detected in many other vertebrates and invertebrates. A few intestinal bacteria of TTX-bearing animals were found to produce TTX. This suggested that the accumulated TTX in these animals was being passed along the food web having been acquired from such TTX-producing bacteria. In this section, although there are many aspects of TTX research with respect to treatment and prevention, biologic distribution, sources, infestation mechanism, detection methods, Instrumental Analysis of Tetrodotoxin 247

chemistry and pharmacology, the focus is to provide an overview of the instrumental analysis of TTX and present chromatographic methods for the isolation of TTX. Rapid and accurate analysis of a mixture and its analogs occurring in a variety of marine organisms is becoming increasingly important from the standpoint of public health, since food poisoning from the ingestion of these toxins is often fatal to a human. There are also increasing demands for chemical assays of TTX for the study of its biosynthetic and metabolic pathways, which remain unknown. In an attempt to protect consumers from TTXintoxication, the mouse bioassay has historically been the 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 the scientists to develop chemical-based alternatives to the mouse bioassay for TTX detection and quantification. In addition, the mouse assay can neither provide any information on toxin composition, nor distinguish TTX from other neurotoxins such as paralytic shellfish poison (PSP). A few marine animals have been found to contain both TTX and PSP simultaneously. Many detection methods for TTX have been developed. A few methods including the mouse bioassay, high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) are typically used to qualitatively and quantitatively detect TTX, but other methods including gas-chromatography-mass spectrometry (GC-MS), infrared (IR) spectrometry and nuclear magnetic resonance (1H-NMR) spectrometry are often used to qualitatively detect TTX. Among them, HPLC and LC-MS are the most powerful and sensitive tool for qualitatively and quantitatively detecting TTX. In addition these methods, TTX can also be identified by thin-layer chromatography (TLC) or electrophoresis. Though these methods are not instrumental analysis, these methods are simpler and more practical. In this section, an attempt has been made to review

the current information and the recent progress on instrumental analysis of TTX.

**Figure 1.** The tautomer of TTX

chemistry and pharmacology, the focus is to provide an overview of the instrumental analysis of TTX and present chromatographic methods for the isolation of TTX. Rapid and accurate analysis of a mixture and its analogs occurring in a variety of marine organisms is becoming increasingly important from the standpoint of public health, since food poisoning from the ingestion of these toxins is often fatal to a human. There are also increasing demands for chemical assays of TTX for the study of its biosynthetic and metabolic pathways, which remain unknown. In an attempt to protect consumers from TTXintoxication, the mouse bioassay has historically been the 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 the scientists to develop chemical-based alternatives to the mouse bioassay for TTX detection and quantification. In addition, the mouse assay can neither provide any information on toxin composition, nor distinguish TTX from other neurotoxins such as paralytic shellfish poison (PSP). A few marine animals have been found to contain both TTX and PSP simultaneously. Many detection methods for TTX have been developed. A few methods including the mouse bioassay, high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) are typically used to qualitatively and quantitatively detect TTX, but other methods including gas-chromatography-mass spectrometry (GC-MS), infrared (IR) spectrometry and nuclear magnetic resonance (1H-NMR) spectrometry are often used to qualitatively detect TTX. Among them, HPLC and LC-MS are the most powerful and sensitive tool for qualitatively and quantitatively detecting TTX. In addition these methods, TTX can also be identified by thin-layer chromatography (TLC) or electrophoresis. Though these methods are not instrumental analysis, these methods are simpler and more practical. In this section, an attempt has been made to review the current information and the recent progress on instrumental analysis of TTX.

**Figure 1.** The tautomer of TTX

246 Chromatography – The Most Versatile Method of Chemical Analysis

equivalent to 2 mg of pure TTX crystals [6]. Many derivatives of TTX have been found, although their toxicities vary widely. As seen in Figures 1, 2 TTX is a heterocyclic guanide compound whose chemical structure has been characterized. Various TTX derivatives from pufferfish and other TTX-bearing organisms have been identified to date as a result of recent progress in the instrumental analysis of TTX (Figure 3). In marine pufferfish species, toxicity is generally highest in the liver and ovary, whereas in brackish and freshwater species, toxicity is higher in the skin [7-13]. TTX was long believed to be present only in the pufferfish. In 1964, Mosher *et al.* detected TTX in California newt *Taricha torosa*, which was the first TTX-containing organism other than pufferfish [14]. Since then, the distribution of TTX has spread to animals other than pufferfish. The toxin has been detected in a tropical goby *Yongeichthys criniger* [15], atelopid frogs of Costa Rica *Atelopus* spp.[16], blue-ringed octopus *Hapalochlaena maculosus* [17], and several species of carnivorous gastropods such as trumpet shell *Charonia sauliae*[18], ivory shell *Babylonia japonica* [19], frog shell *Tutufa lissostoma* [20] as shown in Table 1*.* In addition, some species of starfish on which these gastropods prefer to feed also contain TTX [21]. The trumpet shell *Charonia sauliae* accumulates TTX by ingesting toxic starfish, supporting the hypothesis that the TTX of pufferfish is not endogenous, but is introduced via the food chain. The exact origin of TTX in the food chain, however, remains unknown. Because the ecologic environments of TTXbearing animals apparently have no common factor other than being closely related to an aquatic system, bacteria (omnipresent organisms that commonly inhabit the aquatic system), are implicated as the primary source of TTX. Toxic crabs, flatworms, horseshoe crabs, ribbon worms and arrow worms were also added to the list of TTX-bearing animals. In Japan, TTX is assayed by the official method using mice [22]. It requires ddY strain male mice, but no special instrumentation. This method is simple and convenient but not so sufficiently accurate, and provides no information on the composition of the toxin, nor is it able to distinguish TTX from other neurotoxins such as paralytic shellfish poison (PSP). In addition, animal rights activists across the world are strongly opposed to bioassays using live animals, including mice. Thin layer chromatography (TLC) and electrophoresis are useful means for TTX detection, but they are not suitable for TTX determination. With this background, attempts have been made to develop analytical methods using high performance liquid chromatography (HPLC) in Japan. Detection and determination of TTX are essential not only for diagnosis and treatment purposes, but also for issuing quarantines and public awareness. Quantitative and/or qualitative detection of TTX in a sample is/are performed by a few instrumental analysis methods. The toxin has long been believed to occur exclusively in pufferfish. However, owing to the recent outstanding progress in instrumental methods for the analysis of TTX, its distribution and accumulation in various aquatic organisms have been established. In addition, this toxin has been detected in many other vertebrates and invertebrates. A few intestinal bacteria of TTX-bearing animals were found to produce TTX. This suggested that the accumulated TTX in these animals was being passed along the food web having been acquired from such TTX-producing bacteria. In this section, although there are many aspects of TTX research with respect to treatment and prevention, biologic distribution, sources, infestation mechanism, detection methods,


Instrumental Analysis of Tetrodotoxin 249

**Figure 2.** Chemically equilibrium of TTX, 4-*epi*TTX and 4, 9-anhydroTTX

**Table 1.** Distribution of TTX in animals

**Table 1.** Distribution of TTX in animals

**Figure 2.** Chemically equilibrium of TTX, 4-*epi*TTX and 4, 9-anhydroTTX



Instrumental Analysis of Tetrodotoxin 251

adsorbed toxin was eluted with 1% AcOH in 20% EtOH after washing the column with dist. H2O. The water eluate (Fr.I) and the eluate with 1% AcOH in 20% EtOH (Fr.II) was isolated (Figure 6). The main toxic fraction (Fr.II) was evaporated to dryness *in vacuo*. The resulting residue (total toxicity 2,433,000 MU; specific toxicity 99 MU/mg) was dissolved in a small amount of water, and the pH was adjusted to 5.5 with 1N NaOH. This solution was applied to a Bio-Gel P-2 column (φ 3.5 × 100cm). The column was washed with 3000 ml of water and then developed with 2,000 ml of 0.03 M AcOH. The toxicity was detected exclusively in the 0.03 M AcOH fraction. This fraction was concentrated to dryness under reduced pressure and the residue (3,300 MU/mg) was dissolved in a small volume of water. The resulting solution was chromatographed on a Bio-Rex 70 (H+ form, φ1.0 x 100cm) column using a linear gradient of H2O and 0.03 M AcOH at the flow rate of 0.5 ml /min. The toxic fractions were monitored for TTX via the mouse bioassay and high performance liquid chromatography (HPLC) as described later. The main toxic fractions (fr. 85–100; Fr.I) and minor fractions (fr.50-84) were obtained, and rechromatographed in the same manner (Figure 7). The toxic fraction (Fr.I) thus obtained were freeze-dried, and then dissolved in 0.5 ml of 1% AcOH. Approximately 2.0 ml of MeOH and 5.0 ml of diethylether were added to this solution, and the mixture was stored in the refrigerator overnight. During storage, stratified plate-like crystals appeared (Figure. 8). The crystals were isolated by decantation, and recrystallized by method same as that described above. Approximately, Bio-Gel P-2 column chromatography was very effective, as the specific toxicity sharply increased from 99 to 3,300 MU/mg. After recrystallization, the specific toxicity of this toxin ultimately increased to 3,520 MU/mg. From the combined homogenates with the toxicity of roughly 7,400 MU/g, approximately 25 mg of the stratified plate-like crystalline TTX was obtained. Generally, the ribbon worm has a simple structure. Since pure crystals of TTX could be obtained from this highly toxic ribbon worm efficiently by the above-described series of chromatographies, the ribbon worm is a promising source of TTX for use as a reagent in the fields of medicine and pharmacology. Previously, authentic specimens of TTX were typically

prepared from pufferfish ovaries for use as reference standards, as reported in [4].

(A): Arrow indicates the ribbon worm (scale bar = 10 cm) on the surface of the cultured oyster shell.

*Crassostrea gigas* hanging onto floating culture rafts in Hiroshima Bay.

**Figure 4.** Ribbon worms *Cephalothrix* sp. ("himomushi" in Japanese) adherent to the cultured oyster

(B): Ribbon worm removed from the cultured oyster shell

(A) Hemilactal type, (B) Lacton type, (C) 4,9-Anhydro type **Figure 3.** The structure of three types of TTX analogues
