J. TOXICOL.

TOXIN REVIEWS, 20(1), 3550 (2001)

CURRENT METHODOLOGIES FOR DETECTION OF TETRODOTOXIN

Tamao Noguchi 1, * and Yahia Mahmud 2
1 Faculty of Fisheries and School of Marine Science and Engineering, Nagasaki University, 114, Bunkyo-machi, Nagasaki 8528521, Japan

ABSTRACT In attempt to protect the 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 a particular size of mice. These potential drawbacks and the world-wide pressure of refraining from the killing of the live animals subsequently led the scientists for developing alternative chemical methods to the mouse bioassay for TTX detection and quantication. Among them, high-performance liquid chromatography (HPLC) has largely been used in routine work. INTRODUCTION Tetrodotoxin (TTX) is one of the best-known notorious marine toxin that occasionally causes human intoxication, including fatality. Poisoning from TTX have been almost exclusively associated with the ingestion of toxic puffer, especially from the waters of the Indo-Pacic Ocean regions.
* Corresponding author. 35
Copyright 2001 by Marcel Dekker, Inc. www.dekker.com

ORDER

REPRINTS

36

NOGUCHI AND MAHMUD

The toxicosis is characterized by the onset of symptoms of the victim. The treatment of the illness is mainly based on the symptoms of the patient. More fruitful treatment can be provided if the causative toxin is identied. Detection and determination of TTX are therefore essential not only for diagnosis and treatment purposes, also for making quarantine rule and public awareness. Quantitative and/or qualitative detection of TTX in a sample is/are performed by several chemical and biological methods, as described below. MOUSE BIOASSAY Mice have been commonly used for determination of toxicity. To know the concentration of TTX in a sample of interest, the mouse bioassay is performed by intraperitoneal injection. After injection with TTX associated extracts, the mice show the characteristic signs and symptoms, like unique scratching of the shoulders/mouth by their hind limbs, weakness progressing to paralysis in hind limbs, uncoordinated movement, shallowness of breathing, convulsion, jumping, followed by respiratory failure. Toxicity of an extract is expressed in terms of mouse units (MU), where 1 MU is dened as the amount of TTX required to kill a 20 g male mouse of ddY strain in 30 min (1). Minimum detectable limit is about 0.2 g of TTX (1 MU) per assay. Mouse bioassay is also used to identify an unknown toxin extract in comparison with a TTX specic dose-death time relationship curve. A series of test solutions are prepared by diluting the unknown toxin extract with 0.1% acetic acid. Aliquots of each test solution are intraperitoneally injected into a group of mice. Using the median value of their death times at each dilution level, the dosedeath time curve is drawn, which provides the nature of the toxin since the dose death time curve is specic to toxin. For example, Hashimoto and Noguchi (2) preliminarily identied goby toxin as TTX with considerable accuracy, using the dose-death time curve (Fig. 1). Although the mouse has been the animal of choice for determination of TTX, it is not without controversy due to some potential drawbacks related to the bioassay, such as low accuracy caused by the individual variation inherent to biological system, lack of specicity, inconvenience of purchasing a particular size of mice of specic strain and recent international movement of prevention from cruelty to animals. These limitations have spawned the development of several alternative chemical methods, with the goal of obtaining comprehensive tests surpassing the mouse assay, as illustrated under the following headlines. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) HPLC methods have been explored by many researchers for both qualitative and quantitative analysis of TTX and its analogues. The uorometric HPLC

ORDER

REPRINTS

DETECTION OF TETRODOTOXIN

37

Figure 1. Dose-death time curve for goby toxin, TTX and saxitoxin (STX).

method is mainly composed of a high pressure pump with a syringe loading sample injector, a stainless steel column, reaction pump for delivering reaction reagent, uoromonitor and chromato-recorder for calculation of peak area. Toxin is rst separated from the contaminants with a buffer system on a column packed with either silica gel, C-18 or ion exchange resin and the toxin eluate is then mixed with sodium hydroxide (NaOH). The toxin is converted into uorescent compounds which passed through a tube placed in an aluminum block oven. Eventually, when the uorescent compounds passed through a uoromonitor equipped with a lamp, the retention time of the toxin and uorescence intensity are recorded, showing chromatogram in the chromatorecorder. Toxins are determined from the retention time of the authentic TTXs. In quantitative analysis of HPLC, the detection limit of TTX is about 0.03 g/10 l. Until now, several attempts have been made to detect TTX and its analogues under different conditions of HPLC, and a number of advances in our understanding of the biochemistry of TTXs are a direct result of these developments. Briey, some promising methodologies can be described here. In the early 1980s, a uorometric continuous TTX analyzer was constructed by combining HPLC and a post column reaction with NaOH to monitor puffers of potential danger (3). In this system, the toxin was rst separated from contaminants on a column of a weak cation exchange gel (Hitachi Gel 3011C) with a 0.06 M citrate buffer solution (pH 4.0), and toxin concentrations of above

ORDER

REPRINTS

38 Table 1.

NOGUCHI AND MAHMUD Operating Conditions of HPLC for the Analysis of Newt Toxins TXX : D-7480 Hitachi : Inertsil ODS-3 : 30C : 60 mM ammonium phosphate buffer (pH 5.0) containing 10 mM HSA and 2% acetonitrile : 4 N NaOH : 0.8 ml/min : 110 C : Excitation 384 nm, emission 505 nm

HPLC system Column (4.6 250 mm, GL Sci. Inc., Japan) Column temperature Mobile phase Reagent Flow rate Reaction temperature Detection

8 MU/g were detected. However, due to its poor performance in separating and detecting of TTX analogues, an improved analyzer was constructed later by which derivatives, such as 6-epi TTX, 4,9-anhydro-6-epi TTX, 4-epi-11-deoxyTTX, 11-nor TTX-6(R)-ol, 4-epi TTX, 4,9-anhydroTTX, 11-deoxyTTX, and 4,9anhydro-11-deoxyTTX isolated from puffer and newt specimens were detected (4). TTX derivatives were separated on a Develosil ODS-5 column (1.0 25

Figure 2. HPLC of authentic TTXs (left) and the Japanese newt (Cynops pyrrhogaster) toxin (right). a: TTX, b: 6-epi TTX, 4,9-anhydro-6-epiTTX, e: 4,9-anhydroTTX.

ORDER

REPRINTS

DETECTION OF TETRODOTOXIN

39

cm) with 0.06 N heptauorobutyric acid in 0.001 N ammonium acetate buffer (pH 5.0). Four normal NaOH was used to produce uorescent compounds. Separation between TTX and 6-epi TTX is, however, considered as the major achievement by this improved analyzer. This analyzer is especially useful for monitoring tropical animals which contain a considerable amount of 6-epi TTX. Reversed-phase ion-pairing HPLC method has also been the system of choice by many researchers for the fastest and efcient analysis of TTX and its analogues, where heptanesulfonic acid (HSA) is used as counterion (5, 6). In this method, the detection reagent for TTX and related substances does not react with any PSP component if present in the contaminant sample. Table 1 represents a reversed-phase HPLC condition with slight modication of the method of Nagashima et al. (5), being used to analyze partially puried Japanese newt (Cynops pyrrhogaster) poison, TTXs (Fig. 2) (7).

THIN-LAYER CHROMATOGRAPHY (TLC) In TLC analysis, TTX is spotted onto a silica gel-60 F 254 pre-coated plate (Merck). The plate is developed in pyridine-ethyl acetate-acetic acid-water (15: 5: 3 :4), 3-butanol-acetic acid-water (2:1 :1) or 1-butanol-acetic acid-water (12: 3: 5) solvent in a sealed container. 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 100C for 10 min. The toxin is visualized as a yellow uorescent spot under UV light (365 nm). In TLC analysis, the R f values of TTX are around 0.70, 0.45 and 0.20 with pyridine-ethyl acetate-acetic acid-water, 3-butanol-acetic acid-water and 1-butanol-acetic acid-water solvent, respectively. It is also possible to detect TTX on the TLC plate using the Weber reagent which gives pink spot of the toxin. In TLC, the detection limit is about 2 g of TTX (10 MU). TLC is a useful technique in those laboratories where HPLC and other costly analytical systems are not available.

ELECTROPHORESIS Electrophoresis is relatively a simple and rapid method with high resolution detection of polar compounds like TTX. When 1 l of TTX (10 MU, corresponding to 2 g) is applied onto a 5 18 cm cellulose acetate membrane (Chemetron), the ion molecules of TTX move toward the cathode with a mobility (R m) clearly smaller than that of authentic saxitoxin (STX). The analysis is performed for 30 min in an electrolytic buffer solution of 0.08 mol/l Tris-HCl (pH 8.7), under the inuence of an applied electric eld with a constant current of 0.8 mA/cm width. The toxin is visualized in the same manner as described for TLC.

ORDER

REPRINTS

40

NOGUCHI AND MAHMUD

CAPILLARY ISOTACHOPHORESIS Capillary isotachophoresis is a rapid, accurate and potential detection technique for TTX. A small amount of TTX in contaminated extracts can be determined by this method (8). It is performed using a cationic system, as TTX exists as a cation under acidic and neutral conditions. Conditions for capillary isotachophoresis comprised of 5 mM potassium acetate (pH 6.0) as an electrolyte, containing 0.2% Triton X-100 and 0.5 vol of dioxane, and 10 mM -alanine adjusted to pH 4.5 with acetic acid as a terminating electrolyte. When TTX is applied to isotachophoretic analyzer (Shimadzu IR-2A) equipped with a potential gradient detector, a new zone appears at the position with a potential unit (PU) value of 0.32, eventually monitored by the detector. PU is expressed as (PG S-PG L)/(PG TPG L), where PG S , PG L , and PG T stands for potential gradient values for sample, leading and terminating ions, respectively (9). The quantitative detection limit by this method is about 0.25 g of TTX (1.2 MU).

ULTRAVIOLET (UV) SPECTROSCOPY In UV spectroscopy, TTX is generally determined by irradiating a crude toxin with UV light. A small amount of TTX is dissolved in 2 ml of 2 N NaOH and heated in a boiling water bath for 45 min. After cooling at room temperature, the solution is examined for the UV absorption spectrum characteristic to the C 9base, 2-amino-6-hydroxymethyl-8-hydroxyquinazoline which should have been derived from TTX and/or related substances, if present (10). In the analysis, UV spectrum of the alkali decomposed compounds of TTX appears as a shoulder at around 276 nm (Fig. 3), indicating the formation of the C 9-base (Fig. 4) specic to TTX or related substances (11).

GAS-CHROMATOGRAPHYMASS SPECTROMETRY (GC-MS) GC-MS analysis is an indirect method to detect TTX in a crude extract which is difcult to purify for other advanced analysis. In this method, TTX and its derivatives (0.2 ml with 25 MU) are dissolved in 2 ml of 2 N NaOH and heated in a boiling water bath for 45 min. After being cooled at room temperature, the alkaline decomposed compounds are adjusted to pH 4.0 with 1N HCl and extracted thrice with three volumes of 1-butanol. The extracts are combined and evaporated to dryness in vacuo, and to the residue is added a mixture of N,

ORDER

REPRINTS

DETECTION OF TETRODOTOXIN

41

Figure 3. UV absorption spectra of alkaline hydrolyzates of authentic TTX (left) and horseshoe crab (Carcinoscorpius rotundicauda) toxin (right).

Figure 4.

Structure of C 9-base, formed by alkali-hydrolysis of TTX.

ORDER

REPRINTS

42

NOGUCHI AND MAHMUD

Figure 5. Mass spectra of C 9-base-(TMS) 3 derivative from a marine puffer toxin (left) and authentic TTX (right).

O-bis acetamide, trimethylchlorosilane and pyridine (2: 1 :1), in order to derive trimethylsilyl (TMS) C 9-base compounds. The derivatives are then submitted to GC/MS analysis. The column temperature is maintained from 180 to 250C at a rate of 5C /min. The ow rate of inlet helium carrier gas is maintained at 20 ml/min. The ionizing voltage is usually kept at 70 eV with the ion source temperature at 200C. An example of mass spectrometry of the selected ion-monitored chromatograms (SIM) from the TMS derivatives of alkali-decomposed puffer Takifugu oblongus poison TTX (C 9-base) is shown in Fig. 5. Sharp fragment ions appear at m / z 407 (parent peak), 392 (base peak) and 376, indicating the presence of the quinazoline skeleton in the toxin (12). It is noteworthy that each peak of SIM monitored at m / z 376, 392 and 407 appears at the same retention time of 16.0 min (data not shown).

INFRARED (IR) SPECTROMETRY IR spectrometry is the analytical technique for determination of functional groups of TTX. IR spectra of TTX are measured in a KBr pellete, using a JASCO model IR-S spectrophotometer (13). Fig. 6 represents an IR spectrum of puffer toxin, showing characteristic absorption in 3360 (OH), 3200, 1660 (guanidium), 1610 (COO ) and 1075 cm 1. Although the spectrum in IR analysis is presumed to be complex, it is a helpful tool for identication of TTX.

ORDER

REPRINTS

DETECTION OF TETRODOTOXIN

43

Figure 6.

IR spectrum of puffer toxin.

FAST ATOM BOMBARDMENT MASS SPECTROMETRY (FABMS) FAB-MS is a direct method for the qualitative conrmation of TTX. The analysis is carried out on a JEOL JMS DX-300 mass spectrometer equipped with a JEOL DA-5000 data system (14). Xenon is used to provide the primary beam of atoms, the acceleration voltage of the primary ion being 3 KV. Scanning is repeated within a mass range of m / z 100500. In this analysis, about 0.1 mg of TTX (500 MU) and glycerol as matrix are placed on the sample stage of the mass spectrometer, mixed well, and submitted to the ion chamber of the spectrometer (FAB). Both positive and negative mass spectra of TTX are then measured. As shown in Fig. 7, TTX shows (M H) and (M H H 2 O) ion

Figure 7.

Positive (right) and negative (left) FAB mass spectra of TTX.

ORDER

REPRINTS

44

NOGUCHI AND MAHMUD

peaks at m / z 320 and 302, respectively, in the positive mass spectrum, and an (M H) peak at m / z 318 in the negative spectrum. Secondary ion-mass spectrometry, performed by a Hitachi M-80B mass spectrometer equipped with a Hitachi M-0101 data system, presented essentially the same result as shown by FAB-MS. Extensively puried sample is required for the successful application of this method.

LIQUID CHROMATOGRAPHYMASS SPECTROMETRY (LC-MS) LC-MS is a recent development for the detection of TTX with considerable accuracy (15). In this method, combined HPLC-MS is performed using Hitachi M-1000 system coupled to a mass spectrometer. HPLC system is equipped with an ODS-3 (1.5 x 150 mm) column. Acetonitrile (50%; ow rate 70 l/min) is used as a mobile solvent. The efuent from the column is split to provide ow to the ion-spray interface. An example of mass spectra of a brackishwater puffer toxin in LC/MS analysis is shown in Fig. 8 (16). In the MS, a protonated molecular ion peak (M H) appeared at m/z 320 showing the molecular weight of the toxin of 319, in good accordance with that of TTX.

ELECTROSPRAY IONIZATION-TIME OF FLIGHT MASS SPECTROMETRY (ESI-TOF/MS) ESI-TOF/MS is a valuable technique for determination of TTX, although it is not widely used so far in marine toxin determination. In this analysis, a portion of puried TTX (less than 0.05 mg) is dissolved in a small amount of

Figure 8. Mass spectrum of chromatogram at 8.6 min from a brackishwater puffer (Tetraodon nigroviridis) toxin in LC/MS analysis.

ORDER

REPRINTS

DETECTION OF TETRODOTOXIN

45

Figure 9. ESI-TOF/MS analysis of a frog (Polypedates sp.) toxin. Y-axis: Relative intensity; X-axis: Mass spectrum at m / z .

1% acetic acid, and added to 50% aqueous methanol. ESI-TOF/MS is taken on a Micromass Q-Tof mass spectrometer. Recently, TTX in a tree-frog Polypedates sp. extract has been successfully analysed by ESI-TOF/MS analysis (17). As shown in Fig. 9, in the spectrum of the toxin, a protonated molecular ion peak (M H) appeared at m / z 320.1103, suggesting the molecular weight of the toxin to be 319.1025 which agrees well with that of authentic TTX (C 11 H 17 N 3 O 8 319.1016).

PROTON NUCLEAR MAGNETIC RESONANCE (1 H-NMR) SPECTROMETRY H-NMR has been playing an important role as a complementary method to determine the absolute conguration of TTX and its derivatives. At present 13 derivatives of TTX have been isolated, whose 1 H-NMR data are presented by various investigators (6, 1823). In 1 H-NMR analysis, the lyophilized powder of puried toxin is dissolved in 0.6 ml of 4% acetic-D 3 acid-D (99.5% purity) in D 2 O and placed in an NMR tube. Tetramethylsilane (Me 4 Si) is used as an external standard. The spectrum is taken on a VARIAN UNITY plus 500 spectrometer at 500 MHz. Fig. 10 exhibits the 1 H-NMR spectra for TTX, under the aforesaid condition (16). TTX exhibits signals at 2.03 (s, CH 3 COOD), 2.34 (d, J 9.6 Hz), 3.97 (s), 4.03 (s), 4.05 (s),
1

ORDER

REPRINTS

46

NOGUCHI AND MAHMUD

Figure 10.

H-NMR spectrum of a brackishwater puffer (Tetraodon nigroviridis) toxin.

4.09 (s), 4.26 (s), 4.29 (s) and 5.44 ppm (d, J 9.4 Hz). C 4 E H was coupled with C 4a E H with a spin-spin coupling constant of 9.4 and 9.6, respectively, which is a typical characteristic of TTX (Fig. 10).

CYTOTOXICITY TEST Cell bioassays are effective biological screening tools for a wide range of drugs, chemicals, and toxic compounds. The utility and design of these in vitro methods is described by the mechanism of action associated with the compounds

ORDER

REPRINTS

DETECTION OF TETRODOTOXIN

47

of interest and the availability of appropriate target cell lines. For the quantitative measurement of a sodium channel blocker TTX, even at low level of detection (approximately 3 nM) in marine bacteria, the tissue culture bioassay (TCBA) has been used (24). This assay is based on the ability of sodium channel blocking toxins to antagonize the combined effects of two chemicals, veratridine and ouabain on neuroblastoma cell lines (ATCC, CCL 131). Veratridine (0.075 mM) and ouabain (1 mM) cause the cells to round up and die subsequently. Cells continue to grow in the presence of TTX, as TTX counteracts the veratridine. The amount of toxin is estimated from the linear relationship of the relative abundance of living cells and the concentration of toxin in the samples. The results are scored by visually noting the morphology of a signicant number of cells under microscope. The sensitivity of this method is much higher than that of the mouse assay. This method is, however, not always convenient for routine bioassays, because it is time consuming, and needs some experience to distinguish dead cells and living cells during microscopic examination. To replace the time consuming and subjective cell counting procedure, TCBA in combination with a water-soluble tetrazolium salt 2-(4-iodophenyl)-3(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST1) was improved for the quantitative measurement of TTX, using a microplate reader (25). WST-1 is used as an indicator of living cells. TTX concentrations can be measured between the range of 270 nM by this method. WST-1-TCBA is also a useful assay for the detection of TTX in a supernatant of bacterial culture, and its sensitivity is several times higher than that of TCBA as reported by Kogure et al. (24). This method seems to be promising, owing to the omission of both washing and solubilizing steps which allows greater reproducibility of the assay.

IMMUNOASSAY Immunoassays are commonly used as inexpensive, sensitive and highly selective methods for the detection and quatitation of a wide variety of drugs and other substances of biomedical signicance (26). For TTX detection, several immunoassay techniques have been developed so far, without much success. Watabe et al. (27) developed an enzyme-linked immunosorbent assay (ELISA) system by raising a monoclonal antibody (MAb) against a TTX derivative, tetrodonic acid (TDA). In this approach, TDA was conjugated with bovine serum albumin (BSA) and injected intraperitoneally into BALB/c mice. Spleen cells of mice were then isolated and fused with myeloma cells X63-Ag86.5.3, and cloned. MAb was produced in ascites uid in the mouse by cloned cell, and its detection capability for TTX was 0.03100 g/well (0.31,000 g/ml). This method had, however, low sensitivity. Huot et al. (28) reported the production of two anti-

ORDER

REPRINTS

48

NOGUCHI AND MAHMUD

TTX MAbs. Binding of these antibodies was only partially inhibited (48% and 25%) by free TTX at a concentration of 50 ng/ml, showing, however, a low performance. Raybould et al. (26) developed a rapid, signicantly sensitive and specic competitive inhibition enzyme immunoassays (CIEIAs) for TTX detection and quantitation in biological samples, by using a newly developed MAb from mice. In this CIEIA approach, MAb detected TTX with sensitivities at IC 50 and IC 20 of 67 ng/ml and 23 ng/ml, respectively. Matsumura et al. (29, 30) reported the competitive enzyme-linked immunoassay (EIA) for TTX determination, although these methods required a long time for the analysis. Recently, a MAb against TTX has been developed from Balb/c mice immunized with TTXBSA conjugate, by which a rapid (generally takes 30 min) and highly sensitive EIA system has been established for quantitative analysis of TTX (31). In this promising method, MAb can detect TTX at concentrations of 2100 ng/ml. This method is considered as a useful tool for monitoring TTX in puffer sh or other sea foods. Using this highly specic MAb, a useful immunoafnity column chromatography has also been developed recently for isolation and identication of TTX from the urine samples of a poisoned patient (32). This chromatographic technique is performed in combination with uorometric high performance liquid chromatography (HPLC). The maximum recovery rate of TTX by this immunoafnity is reported to be 88 %. The detection limit of TTX is 2 ng/ml of the urine. For diagnosis of TTX-poisoning, this chromatographic method might be used as a potential tool.

REFERENCES 1. Environmental Health Bureau, Ministry of Health and Welfare, Food Hygiene Inspection Manual, Food Hygiene Association, Tokyo. pp. 232 240, 1991. 2. Hashimoto, Y., and Noguchi, T., Occurrence of tetrodotoxin-like substance in a goby Gobius criniger, Toxicon, 9: 7984, 1971. 3. Yasumoto, T., Nakamura, M., Oshima, Y., and Takahata, J., Construction of a continuous tetrodotoxin analyzer, Bull. Japan. Soc. Sci. Fish., 48: 14811483, 1982. 4. Yotsu, M., Endo, A., and Yasumoto, T., An improved tetrodotoxin analyzer. Agric. Biol. Chem., 53, 893895, 1989. 5. Nagashima, Y., Maruyama, J., Noguchi, T., and Hashimoto, K., Analysis of paralytic shellsh poison and tetrodotoxin by ion-pairing high performance liquid chromatography, Nippon Suisan Gakkaishi, 53: 819823, 1987. 6. Arakawa, O., Noguchi, T., Shida, Y., and Onoue, Y., Occurrence of 11oxotetrodotoxin and 11-nortetrodotoxin-6(R)-ol in a xanthid crab Atergatis oridus collected at Kojima, Ishigaki Island, Fish. Sci., 60: 769771, 1994.

ORDER

REPRINTS

DETECTION OF TETRODOTOXIN

49

7. Tsuruda, K., Arakawa, O., and Noguchi, T., Toxicity and toxin proles of a newt, Cynops pyrrhogaster from Western Japan, J. Nat. Toxins, 2000. (In print) 8. Shimada, K., Ohtsuru, M., Yamaguchi, T., and Nigota, K., Determination of tetrodotoxin by capillary isotachophoresis, J. Food Sci., 48, 665667, 1983. 9. Miyazaki, H., and Katoh, K., Isotachophoretic analysis of peptides. J. Chromatogr., 119: 369374, 1976. 10. Suenaga, K., and Kotoku, S., Detection of tetrodotoxin in autopsy material by gas Chromatography, Arch. Toxicol., 44, 291297, 1980. 11. Tanu, M. B., and Noguchi, T., Tetrodotoxin as a toxic principle in the horseshoe crab Carcinoscorpius rotundicauda collected from Bangladesh, J. Food. Hyg. Soc. Jpn., 40: 426430, 1999. 12. Narita, H., Noguchi, T., Maruyama, J., Ueda, Y., Hashimoto, K., Watanabe, Y., and Hida, K., Occurrence of tetrodotoxin in a trumpet shell boshubora Charonia sauliae , Nippon Suisan Gakkaishi, 47: 934941, 1981. 13. Onoue, Y., Noguchi, T., and Hashimoto, K., Tetrodotoxin determination methods. In: Seafood toxins (ed. by Ragelis, E. P.), American Chemical Society, Washington D. C. pp. 345355, 1984. 14. Noguchi, T., Ali, A. E., Arakawa, O., Miyazawa, K., Kanoh, S., Shida, Y., Nishio, S., and Hashimoto, K., Tetrodonic acid-like substance; a possible precursor of tetrodotoxin, Toxicon, 29: 845855, 1991. 15. Shida, Y., Arakawa, O., Onoue, Y., and Noguchi, T., LC/MS of marine toxin-1. In: Proceedings of the 46th annual conference on mass spectrometry, Japan, Mass Spectrometry Society, P. 137, 1998. 16. Mahmud, Y., Yamamori, Y., and Noguchi, T., Occurrence of TTX in a brackishwater puffer midorifugu Tetraodon nigroviridis, collected from Thailand, J. Food. Hyg. Soc. Jpn., 40: 473480, 1999. 17. Tanu, M. B., Mahmud, Y. Tsuruda, K., Arakawa, O., and Noguchi, T., Occurrence of tetrodotoxin in a tree-frog Polypedates sp. from Bangladesh, Toxicon, 2000. (In print) 18. Nakamura, M., and Yasumoto, T., Tetrodotoxin derivatives in puffer sh, Toxicon, 23: 271276, 1985. 19. Endo, A., Khora, S. S., Murata, M., Naoki, H., and Yasumoto, T., Isolation of 11-nortetrodotoxin-6(R)-ol and other tetrodotoxin derivatives from the puffer Fugu niphobles, Tetrahedron Letters, 29: 41274128, 1988. 20. Yasumoto, T., Yotsu, M., and Murata, M., New tetrodotoxin analogues from the newt Cynops ensicauda , J. Am. Chem. Soc., 110: 23442345, 1988. 21. Yotsu, M., Iorizzi, M., and Yasumoto, T., Distribution of tetrodotoxin, 6epitetrodotoxin and 11-deoxytetrodotoxin in newts, Toxicon, 28: 238241, 1990.

ORDER

REPRINTS

50

NOGUCHI AND MAHMUD

22. Yotsu, M., Hayashi, Y., Khora, S. S., Sato, S., Yasumoto, T., Isolation and structural assignment of 11-nortetrodotoxin-6(S)-ol from the puffer Arothron nigropunctatus, Biosci. Biotech. Biochem., 56: 370371, 1992. 23. Yotsu, M., Schimmele, B., and Yasumoto, T., Isolation and structural assignment of 5-deoxytetrodotoxin from the puffer sh Fugu poecilonotus, Biosci. Biotech. Biochem., 63: 961963, 1992. 24. Kogure, K., Tampline, M. L., Simidu, U., and Colwell, R. R., A tissue culture assay for tetrodotoxin, saxitoxin and related toxins, Toxicon, 26, 191197, 1988. 25. Hamasaki, K., Kogure, K., and Ohwada, K., A biological method for the quantitative measurement of tetrodotoxin (TTX): Tissue culture bioassay in combination with a water-soluble tetrazolium salt, Toxicon, 34: 490 495, 1996. 26. Raybould, T. J. G., Bignami, G. S., Inouye, L. K., Simpson, S. B., Byrnes, J. B. Grothaus, P. G., and Vann, D. C., A monoclonal antibody-based immunoassay for detecting tetrodotoxin in biological samples, J. Clin. Lab. Anal., 6:6572, 1992. 27. Watabe, S., Sato, Y., Nakaya, M., Hashimoto, K., Enomoto, A., Kaminogawa, S., and Yamauchi, K., Monoclonal antibody raised against tetrodonic acid, a derivative of tetrodotoxin, Toxicon, 27: 265268, 1989. 28. Huot, R. I., Armstrong, D. L., and Chanh T. C., Protection against nerve toxicity by monoclonal antibodies to the sodium channel blocker tetrodotoxin, J. Clin. Invest., 83: 18211826, 1989. 29. Matsumura, K., and Fukiya, S., Indirect competitive enzyme immunoassay for tetrodotoxin using a biotin-avidin system, J. Assoc. Off. Anal. Chem. Int., 75: 883886, 1992. 30. Matsumura, K., A monoclonal antibody against tetrodotoxin that reacts to the active group for toxicity, Europ. J. Pharmacol., 293: 4145, 1995. 31. Kawatsu, K., Hamano, Y., Yoda, T., Terano, Y., and Shibata, T., Rapid and highly sensitive enzyme immunoassay for quantitative determination of tetrodotoxin, Jpn. Med. Sci. Biol., 50: 133150, 1997. 32. Kawatsu, K., Shibata, T., and Hamano, Y., Application of immunoafnity chromatography for detection of tetrodotoxin from urine samples of poisoned patients, Toxicon, 37: 325333, 1999.

Request Permission or Order Reprints Instantly!

Interested in copying and sharing this article? In most cases, U.S. Copyright Law requires that you get permission from the articles rightsholder before using copyrighted content. All information and materials found in this article, including but not limited to text, trademarks, patents, logos, graphics and images (the "Materials"), are the copyrighted works and other forms of intellectual property of Marcel Dekker, Inc., or its licensors. All rights not expressly granted are reserved. Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly. Simply click on the "Request Permission/Reprints Here" link below and follow the instructions. Visit the U.S. Copyright Office for information on Fair Use limitations of U.S. copyright law. Please refer to The Association of American Publishers (AAP) website for guidelines on Fair Use in the Classroom. The Materials are for your personal use only and cannot be reformatted, reposted, resold or distributed by electronic means or otherwise without permission from Marcel Dekker, Inc. Marcel Dekker, Inc. grants you the limited right to display the Materials only on your personal computer or personal wireless device, and to copy and download single copies of such Materials provided that any copyright, trademark or other notice appearing on such Materials is also retained by, displayed, copied or downloaded as part of the Materials and is not removed or obscured, and provided you do not edit, modify, alter or enhance the Materials. Please refer to our Website User Agreement for more details.

Order now! Reprints of this article can also be ordered at http://www.dekker.com/servlet/product/DOI/101081TXR100102535