Tetrodotoxin (TTX) is a strong marine neurotoxin acting by blocking sodium channels of the neuron cell membrane. This toxin is a water-soluble heterocyclic guanidine compound, which is stable in neutral and weak acidic solutions and cannot be inactivated by heat treatment (3, 15, 24). TTX was isolated for the first time in 1909 by the Japanese researcher Yoshizumi Tahara from ovaries of globefish (5, 15). Thirty naturally occurring analogues of tetrodotoxin have been detected and many of them have also been shown to have toxic potential in humans and experimental animals. The main mechanism of tetrodotoxin accumulation in fish is the food chain, which begins with toxin-synthesising bacteria, including Pseudoalteromonas haloplanktis tetraodonis, marine microorganisms belonging to the genera of Vibrio (e.g. Vibrio alginolyticus) and Shewanella, (e.g. Shewanella algae, and Shewanella putrefaciens) or Alteromonas tetraodoni (3, 26, 47, 51). The main TTX sources for humans are fish from the Tetraodontidae family (puffer fish), such as Takifugu spp., Lagocephalus spp., Tetraodon alboreticulatus, Chelonodon patoca, Arothron firmamentum, and Canthigaster rivulata, which are a prized delicacy in Japanese cuisine (3). TTX content is different in various parts of the fish body – the highest level of the toxin being found in the skin and some internal organs like the liver and ovaries. This toxin also occurs in the body of marine gastropods (e.g. Nassarius glans, Nassarius papillosus, Zeuxis scalaris, Zeuxis samiplicutus, Zeuxis siquijorensis, Niotha clathrata, Charonia sauliae, Charonia lampas, Babylonia japonica, and Tutufa lissostoma), oysters, mussels, fish other than puffer fish (e.g. the members of the Gobiidae subfamily Yongeichthys nebulosus, Parachaeturichthys polynema, and Radigobius Caninus and the sillaginid Sillago japonica), and the horseshoe crab (Carcinoscorpius rotundicauda) (3, 14, 44, 47, 55, 57, 59, 61, 62). TTX intoxication caused by the consumption of marine gastropods has been observed not only in Japan, but also in Taiwan, China, New Zealand, and Europe, indicating the further spread of organisms containing this toxin around the world (8, 19, 38, 39, 55).
Chemistry of TTX
Tetrodotoxin has the chemical formula C11H17N3O8 and a molecular mass of 319.1 g/M. Its structural formula is shown in Fig. 1.
To date 30 structural analogues of TTX have been studied and it has been proved that their toxicity is connected with their structure (4). Previous studies indicated that the hydroxyl substituents cause that the analogues are more toxic than TTX, while the analogues with deoxy substituents are less harmful. The toxicity of analogues depends on the number and position of hydroxyl substituents in their structure (4). Yotsu-Yamashita et al. (67) performed studies assessing the effect of the position of the hydroxyl group in the TTX molecule on the ability of this neurotoxin to penetrate rat meninges. The obtained results revealed that the location of the hydroxyl groups at the C-6 and C-11 carbon atoms has a significant impact on the binding of TTX analogues to the sodium channels through their participation in hydrogen binding. Moreover, Pires et al. (52) proved that analogue 11-oxoTTX is 4–5 times more toxic than tetrodotoxin. There are still many analogues which have not been studied for their toxicity, so it is important that further analysis of TTX derivatives be carried out. Such studies are very important in assessment of the risk posed by this kind of toxin to public health.
TTX analogues found in puffer fish can be categorised into following families (65):
- analogues chemically equivalent to TTX (4-epiTTX and 4,9-anhydroTTX);
- deoxy analogues (5-deoxyTTX, 11-deoxyTTX, 5,11-dideoxyTTX, 6,11-dideoxyTTX and 5,6,11-trideoxyTTX);
- 11-CH2OH oxidised analogue (11-oxoTTX);
- C11-lacking analogues (11-norTTX-6(S)-ol and 11-norTTX-6(R)-ol).
Such structures as have been found and analysed for several natural TTX analogues in puffer fish and amphibians are shown in Fig. 2.
Tetrodotoxin is one of the most toxic substances to humans known. Its lethal dose is 275 times lower than cyanides and 50 times lower than strychnine and curare (15). In mice, the 50% lethal dose (LD50) of TTX administered orally is 334 μg/kg while intravenously it is 8 μg/kg (10). The toxic dose for humans has not been established, but a single dose of 1–2 mg of purified TTX can be lethal (15).
TTX inhibits neuronal firing of action potentials by binding to the voltage-gated sodium channels in nerve cell membranes, and as a result blocks the flow of sodium ions into the neurons (24). For this reason tetrodotoxin is called a sodium channel blocker. The toxin affects action potential generation and impulse conduction, resulting in blockade of the neuron and muscle paralysis. It leads to the following acute symptoms: tingling (paraesthesia) of the tongue and lips, motor paralysis, incoordination, slurred speech, aphonia, hypotension, bradycardia, cardiac dysrhythmia, and unconsciousness (18). The clinical symptoms of TTX intoxication appear very quickly and depend on the toxin amount consumed. In severe cases, death is caused by respiratory and heart failure. The treatment is symptomatic and supportive because there is no specific antidote against TTX, and is based on careful observation and repeated neurological assessment for the early implementation of measures to counter respiratory failure and cardiovascular disorders (45).
Until the very recent past, TTX poisoning was considered a problem confined to Japan and other Asian countries. Currently, tetrodotoxin poisoning is seen in regions that were previously recognised as safe. It is thought that the rapid growth of the geographical extent of this toxin is due to rising water temperatures around the world (9). The first case of TTX poisoning in Europe was reported in Malaga, Spain, after the consumption of a trumpet shell by a 49-year-old man (54). Table 1 shows the reported cases and outbreaks of TTX poisoning in various parts of the world.
Cases of TTX poisoning
|Country||Source of TTX||Number of cases||Reference|
|Thailand||Eggs of horseshoe crab (Carcinoscorpius rotundicauda)||71||(31)|
|Japan||Puffer fish, Thread-sail filefish, Marine snail||3||(3)|
|South Korea||Puffer fish||3||(6)|
Commission Regulation (EC) No 853/2004 establishes detailed regulations for hygiene in foodstuffs in the European Union. Section VIII, chapter V indicates that fishery products derived from poisonous fish of the Tetraodontidae, Molidae, Diodontidae, and Canthigasteridae families are banned from the market (7). In Japan, a regulatory limit of 2 mg equivalent of TTX/kg was laid down for tetrodotoxin, while in Europe appropriate criteria have not been determined (4, 19).
Detection of TTX
Bioassays. There are several methods for TTX detection. The first methods used were biological tests, including the mouse bioassay (MBA), tissue culture bioassay, and ELISA, available as commercial kits (63). Bioassays allow the toxicity of the sample to be assessed, but it is not possible to identify individual toxins.
The mouse bioassay was used for the first time as a method for the analysis of TTX by Hashimoto and Migita (16) in 1951 in puffer fish sample examination. Currently, because of limitations in using the MBA due to ethical reasons, its continued use for some marine toxin groups is only as the reference method (46). In mouse bioassays, seafood extracts are given to laboratory animals and then symptoms and time to death are monitored. In addition to the ethical concerns around the use of the MBA method prompted by the killing of experimental animals, other numerous disadvantages of this technique exist which are technical, such as low sensitivity and accuracy. For this reason, in recent years, a serious attempt has been made to introduce other techniques for detection of TTX.
A tissue culture bioassay may be used as an alternative method to the MBA (32, 33). The mechanism of action of TTX is based on the same principle as that of another neurotoxin – STX (saxitoxin). Therefore a cell-based assay is able to detect both TTX and STX. Kogure et al. (32) noticed that neuroblastoma Neuro-2a cells can be applied in the identification of TTX. Ouabain or veratridine are added to the cell cultures, reducing their viability by increasing the flow of sodium ions into the cells, and TTX, which acts as a sodium channel blocker, will nullify the response enabling cell growth to be continued.
Antibody-based techniques like ELISA have been widely used for TTX detection, despite difficulties in toxin-specific antibody production because of the insufficient amount of these compounds available in the world in pure form (43, 58). They were considered to offer large-scale screening capability because of their sensitivity, specificity, rapidity, simplicity, and cost-effectiveness. However, these methods are not useful for conventional screening because they may not be able to detect the majority of TTX analogues.
Chemical methods. High performance liquid chromatography with fluorescence detection (HPLC– FLD) and liquid chromatography–mass spectrometry (LC–MS) are typically used for TTX quantification; however, other techniques such as gas-chromatography–mass spectrometry (GC–MS), infrared (IR) spectrometry, and nuclear magnetic resonance (NMR) spectrometry may be suitable for qualitative determination of TTX. Thin-layer chromatography (TLC) or electrophoresis may be used additionally for TTX detection.
The first analysis of TTX was performed using the GC–MS method by derivatisation of TTX to the C9 base structure and then to a trimethylsilane (42). However, the GC–MS method should not be applied for quantitative analysis because TTX is a non-volatile compound (4). In the NMR technique, clean samples are required to avoid interfering with matrix components.
Reversed-phase (RP) chromatography with a C18 column was applied for a long time in the detection of TTX and its analogues. However, not all of them could be separated using the RP–HPLC method. Other researchers applied normal phase chromatography for the analysis of the TTXs, mainly using hydrophilic interaction liquid chromatography (HILIC) (9, 14, 40, 65). TTX is a polar molecule and it is flushed rapidly from reversed-phase columns but much more slowly in normal phases, which improves separation of its analogues, lowers noise, and heightens sensitivity.
Development of a postcolumn HPLC method with fluorescence detection was intended to obviate the need for the traditional mouse bioassay, and it provides qualitative and quantitative analysis of TTX and its analogues. This method is based on the TTX derivatisation reaction in an alkaline environment, the result of which is a fluorescent compound with excitation and emission wavelengths of 384 and 505 nm, respectively. In the early 1980s, a chromatography technique with a fluorometric measurement of TTX was developed by connecting HPLC and a postcolumn reaction with NaOH to analyse this toxic compound. The most important achievement of this improved analyser was the separation of TTX and 6-epiTTX. So far, a lot of modifications have been made to detect TTX and its analogues under variable HPLC conditions. The LC–FLD method is effective at analysing TTX and many TTX analogues such as 4-epiTTX, 11-oxoTTX, and 4,9-anhydroTTX (4, 28, 34, 35, 46). However, it has the complication of quite large differences in intensity of fluorescence between analogues, e.g. 6-epi TTX is 20 times more fluorescent than TTX, while 11-deoxy TTX is 100 times less fluorescent (53). This problem is solved when the LC– MS technique for TTX determination is used.
LC–MS techniques using atmospheric pressure ionisation with an electrospray-ionisation (ESI) in positive ion mode have become a powerful tool for TTX investigation (30, 40, 54, 56, 59, 60). The separation is usually achieved with reversed-phase columns using solvents with an added ion pair reagent, such as ammonium heptafluorobutyrate. The best-known positive ionisation produces a TTX ion of 320 mass-to-charge ratio (m/z) (38). Different TTX analogues can generate the same ion, and therefore liquid chromatography–electrospray ionisation– multiple reaction monitoring mass spectrometry (LC– ESI–MRM-MS) is commonly used to identify them (27, 38, 56, 62, 66).
In 2011, Leung et al. (36) analysed urine and plasma of Asian patients and determined the level of TTX by the LC–MS method. This technique, for which an Atlantics dC18 (2.1 mm × 150 mm, 5 μm) column and flow rate of 200 μL/min were specified, allowed the run time to be shortened to 5.5 min. The developed method was validated and applied to determination of TTX in human urine and blood samples (12). In this study the effect of an ion pair reagent such as heptafluorobutyric acid and the optimisation of its concentration at 5 mM was proved. Table 2 provides an overview of LC–MS methods which were used to analyse TTX and its analogues in different matrices.
Various LC–MS studies on the determination of TTX and its analogues
|Mobile phase||Column||Linear range||LOD and LOQ||Recovery||Matrix||Reference|
|1% acetonitrile, 20 mM ammonium 10 mM ammonium heptafluorobutyrate, formate (pH 4.0)||Reversed (250 × 4.6 phase mm)||0.05–1 nM||LOD LOQ – – 0.7 NR pM||NR||Puffer fish||(56)|
|20 mM ammonium acetate, methanol (75/25, v/v)||Reversed phase (150 × 4.6 mm)||0.01–1 μg/mL||LOD – 0.1 μg/g LOQ – NR||77.7– 80.7%||Puffer fish||(17)|
|30 mM heptafluorobutyric acid, 1 mM ammonium acetate (pH 5)||Reversed phase (250 × 4.6 mm)||NR||NR||NR||Japanese fire belly newt (Cynops pyrrhogaster)||(41)|
|A: 0.1% formic acid in water B: methanol||HILIC (150 × 4.6 mm)||1–100 ng/mL||LOD – 0.1 ng/mL LOQ – 1 ng/mL||> 95%||Human blood serum||(30)|
|16 mM ammonium formate buffer (pH 5.5), acetonitrile (3/7, v/v)||HILIC (5 μm, 150 × 2 mm)||NR||LOD – 0.5 nM/g LOQ – NR||NR||Puffer fish||(27)|
|10 mM/L ammonium formate, formic acid (95/5, v/v), 5 mM heptafluorobutyric acid, 2% acetonitrile||Reversed phase (5 μm, 2.1 × 15 mm)||0–500 ng/mL in urine samples 0–20 ng/mL for plasma samples||LOD – 0.13 ng/mL LOQ – 2.5 ng/mL||75–81%||Human urine and plasma||(12)|
|A: 10 mM ammonium formate, 10 mM formic acid in water B: acetonitrile, water (95/5, v/v), 5 mM ammonium formate, 2 mM formic acid||HILIC (3.5 μm, 150 × 2.1 mm)||62.5–2,000 ng/mL||LOD – 16 ng/mL LOQ – 63 ng/mL||NR||Puffer fish||(54)|
|A: 5% acetonitrile B: 95% acetonitrile, 1% acetic acid (pH 3.5)||HILIC (1.7 μm, 100 × 2.1 mm)||5–500 ng/mL||LOD – 0.074 ng/mL LOQ – 0.123 ng/mL||80–92%||Gastropod||(48)|
The European Food Safety Authority (EFSA) Panel on Contaminants in the Food Chain (CONTAM Panel) have disseminated the opinion that LC–MS-MS methods are the most useful for quantitative and qualitative analysis of TTX (10).
Risk to public health
In 2017, the EFSA CONTAM Panel issued a detailed report regarding the influence of tetrodotoxin and its analogues in marine bivalves and gastropods on human health. Consumption of a dose of 2 mg of TTX (which corresponds to 40 μg/kg body weight in a 50-kg Japanese adult) is considered to be dangerous and can cause serious symptoms in humans, which was described in some case reports of poisoning. However, after reviewing the available literature, the Panel did not find adequate evidence to support a minimum lethal dose for humans of 2 mg, which had been indicated in various publications.
The estimated harmfulness for analogues is lower than that set for TTX, but these results are highly doubtful since the current results and determination methods are imprecisely described. After intraperitoneal (i.p.) injection of TTX analogues in mice, most of them cause the same symptoms as TTX, however, no data are available that are precise enough to enable the no observed adverse effect level (NOAEL) or lowest observed adverse effect level (LOAEL) parameters to be quantified. Nevertheless, LD50 and LD99 doses after i.p. injection in mice have been determined and on the basis of these values, the approximate effect of analogues in bivalves and gastropods was reported.
In the EFSA report 8,268 analytical results for 1,677 samples of oysters, clams, cockles, mussels, razor clams, and scallops were included (10). These samples were collected between 2006 and 2016 in Greece, the Netherlands, and the UK. The results for TTX were obtained for all of them, disclosing 13.74% and 13.73% occurrence rates for the tetrodotoxin analogues 4-epiTTX and 4,9-anhydroTTX, respectively, and 13.06% for 5,6,11-trideoxyTTX, 11-oxoTTX, mono-deoxyTTX, and 11-norTTX-6-ol. No data were obtained regarding the occurrence of TTX in marine gastropods.
The acute reference dose (ARfD) was used to assess exposure risk for TTX. The average value obtained on the basis of reported consumption of toxin-bearing shellfish with no adverse outcome was not above 0.25 μg TTX/kg except for instances of the intake of large quantities of oysters, which may suggest that marine bivalves do not pose a threat for consumers. The EFSA CONTAM Panel established that a concentration below 44 μg of TTX and/or the same amount of TTX analogues per kg of shellfish meat should not cause adverse effects in humans, in conditions consistent with 400g maximum bivalve consumption, 70kg average adult body weight, and 0.25 μg/kg ARfD. Unfortunately, this value could not be determined for marine gastropods, and neither could the risk of exposure to TTX in consumption of these organisms be characterised, due to the limited amount of data on such consumption and the paucity of data on the poisonings.
There are still many unexplained issues regarding the influence of TTX and its analogues on human health. Taking into account that no accurate data on the toxicity of analogues are available, further studies are necessary to assess their potentially harmful effects, especially after oral administration. Further research is also required on the relationship between TTX and STX neurotoxins regarding the similarity of their mechanisms of action and induction of similar toxic effects. Detection and quantification of TTX and its analogues should be carried out by EU-accepted and validated methods using reference materials and the highest quality standards to ensure reliable results. It is suggested that further information on the occurrence and factors conducive to the accumulation of TTX in marine organisms is needed. It is also important to provide more results regarding the toxicokinetics of TTX and its analogues. Furthermore, despite the belief that tetrodotoxins cause acute intoxication without chronic effects, this issue requires more research.
Conflict of interest
Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article.
Financial Disclosure Statement: The review was financially supported by the reference laboratory activity of the National Veterinary Research Institute in Puławy, Poland.
Ahmed S.: Puffer fish tragedy in Bangladesh: an incident of Takifugu oblongus poisoning in Degholia, Khulna. Afr J Mar Sci 2006, 28, 457–458.
Akaki K., Hatano K.: Determination of tetrodotoxin in puffer-fish tissues, and in serum and urine of intoxicated humans by liquid chromatography with tandem mass spectrometry. J Food Hyg Safe Sci Jpn 2006, 47, 46–50.
Arakawa O., Hwang D.F., Taniyama S., Takatani T.: Toxins of pufferfish that cause human intoxications. In: Coastal Environmental and Ecosystem Issues of the East China Sea edited by A. Ishimatsu, H.J. Lie, Terrapub and Nagasaki University, Tokyo, 2010, pp. 227–244.
Bane V., Lehane M., Dikshit M., O’Riordan A., Furey A.: Tetrodotoxin: chemistry, toxicity, source, distribution, and detection. Toxins 2014, 6, 693–755.
Chamandi S.C., Kallab K., Mattar H., Nader E.: Human poisoning after ingestion of puffer fish caught from Mediterranean Sea. Middle East J Anesthesiol 2009, 20, 285–288.
Cho H.E., Ahn S.Y., Son I.S., In S., Hong R.S., Kim D.W., Woo S.H., Moon D.C., Kim S.: Determination and validation of tetrodotoxin in human whole blood using hydrophilic interaction liquid chromatography-tandem mass spectroscopy and its application. Forensic Sci Int 2012, 217, 76–80.
Commission Regulation (EC) No. 853/2004 of 29 April 2004 laying down specific hygiene rules for the hygiene of foodstuffs. OJ, L 139, 55–205.
Danovaro R., Fonda Umani S., Pusceddu A.: Climate change and the potential spreading of marine mucilage and microbial pathogens in the Mediterranean sea. PLoS ONE 2009, 4, 1–8.
Diener M., Christian B., Ahmed M.S., Luckas B.: Determination of tetrodotoxin and its analogues in the puffer fish Takifugu oblongus from Bangladesh by hydrophilic interaction chromatography and mass spectrometric detection. Anal Bioanal Chem 2007, 389, 1997–2002.
European Food Safety Authority (EFSA): Risks for public health related to the presence of tetrodotoxin (TTX) and TTX analogues in marine bivalves and gastropods. EFSA J 2017, 15, 4752.
Fernandez-Ortega J.F., Santos J.M., Herrera-Gutierrez M.E., Fernandez-Sanchez V., Loureo P.R., Alfonso-Rancano A., Tellez-Andrade A.: Seafood intoxication by tetrodotoxin: first case in Europe. J Emerg Med 2010, 39, 612–617.
Fong B.M.W., Tam S., Tsui S.H., Leung K.S.Y.: Development and validation of a high-throughput double solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma. Talanta 2011, 83, 1030–1036.
Fukushima S.: Examination of the poisoning level of tetrodotoxin in body fluids. Jpn J Forensic Toxicol 1991, 9, 126–127.
Gerssen A., Mulder P.P., Boer J.: Screening of lipophilic marine toxins in shellfish and algae: development of a library using liquid chromatography coupled to orbitrap mass spectrometry. Anal Chim Acta 2011, 685, 176–185.
Haque M.A., Islam Q.T., Ekram A.R.M.S.: Puffer fish poisoning. J Teachers Assoc 2008, 20, 199–202.
Hashimoto Y., Migita M.: On the assay method of puffer poison. Bull Jap Soc Sci Fish 1951, 16, 341–345.
Horie M., Kobayashi S., Shimizu N., Nakazawa H.: Determination of tetrodotoxin in puffer-fish by liquid chromatography-electrospray ionization mass spectrometry. Analyst 2002, 127, 755–759.
How C.K., Chern C.H., Huang Y.C., Wang L.M., Lee C.H.: Tetrodotoxin poisoning. Am J Emerg Med 2003, 21, 51–54.
Hungerford J.M.: Committee on natural toxins and food allergens. Marine and freshwater toxins. J AOAC Int 2006, 89, 248–269.
Hwang D.F., Noguchi T.: Tetrodotoxin poisoning. Adv Food Nutr Res 2007, 52, 141–236.
Hwang D.F., Shiu Y.C., Hwang P.A., Lu Y.H.: Tetrodotoxin in gastropods (snails) implicated in food poisoning in Northern Taiwan. J Food Prot 2002, 65, 1341–1344.
Hwang P.A., Tsai Y.H., Deng J.F., Cheng C.A., Ho P.H., Hwang D.F.: Identification of tetrodotoxin in a marine gastropod Nassarius glans responsible for human morbidity and mortality in Taiwan. J Food Prot 2005, 68, 1696–1701.
Hwang P.A., Tsai Y.H., Lu Y.H., Hwang D.F.: Paralytic toxins in three new gastropod Olividae species implicated in food poisoning in southern Taiwan. Toxicon 2003, 41, 529–533.
Isbister G.K., Kiernan M.C.: Neurotoxic marine poisoning. Lancet Neurol 2005, 4, 219–228.
Isbister G.K., Son J., Wang F., Maclean C.J., Lin C.S., Ujma J., Balit C.R., Smith B., Milder D.G., Kiernan M.C.: Puffer fish poisoning: a potentially life-threatening condition. Med J Aust 2002, 177, 650–653.
Islam Q.T., Razzak M.A., Islam M.A., Bari M.I., Basher A., Chowdhury F.R., Sayeduzzaman A.B.M., Ahasan H.A.M.N., Faiz M.A., Arakawa O., Yotsu-Yamashita M., Kuch U., Mebs D.: Puffer fish poisoning in Bangladesh: clinical and toxicological results from large outbreaks in 2008. Trans R Soc Trop Med Hyg 2011, 105, 74–80.
Jang J.H., Lee J.S., Yotsu-Yamashita M.: LC/MS analysis of tetrodotoxin and its deoxy analogues in the marine puffer fish Fugu niphobles from the southern coast of Korea, and in the brackishwater puffer fishes Tetraodon nigroviridis and Tetraodon biocellatus from Southeast Asia. Mar Drugs 2010, 8, 1049–1058.
Jang J., Yotsu-Yamashita M.: Distribution of tetrodotoxin, saxitoxin, and their analogs among tissues of the puffer fish Fugu pardalis Toxicon 2006, 48, 980–987.
Jen H.C., Lin S.J., Lin S.Y., Huang Y.W., Liao I.C., Arakawa O., Hwang D.F.: Occurrence of tetrodotoxin and paralytic shellfish poisons in a gastropod implicated in food poisoning in southern Taiwan. Food Addit Contam 2007, 8, 902–909.
Jen H.C., Lin S.J., Tsai Y.H., Chen C.H., Lin Z.C., Hwang D.F.: Tetrodotoxin poisoning evidenced by solid-phase extraction combining with liquid chromatography-tandem mass spectrometry. J Chromatogr B 2008, 871, 95–100.
Kanchanapongkul J., Krittayapoositpot P.: An epidemic of tetrodotoxin poisoning following ingestion of the horseshoe crab Carcinoscorpius rotundicauda Southeast Asian J Trop Med Public Health 1995, 26, 364–367.
Kogure K., Do H.K., Thuesen E.V., Nanba K., Ohwada K., Simidu U.: Accumulation of tetrodotoxin in marine sediment. Mar Ecol Prog Ser 1988, 45, 303–305.
Kogure K., Tampline M., Simidu U., Colwell R.R.: A tissue culture assay for tetrodotoxin, saxitoxin, and related toxins. Toxicon 1988, 26, 191–197.
Kono M., Matsui T., Furukawa K., Takase T., Yamamori K., Kaneda H., Aoki D., Jang J.H., Yotsu-Yamashita M.: Examination of transformation among tetrodotoxin and its analogs in the living cultured juvenile puffer fish, kusafugu, Fugu niphobles by intramuscular administration. Toxicon 2008, 52, 714–720.
Kono M., Matsui T., Furukawa K., Yotsu-Yamashita M., Yamamori K.: Accumulation of tetrodotoxin and 4,9-anhydrotetrodotoxin in cultured juvenile kusafugu Fugu niphobles by dietary administration of natural toxic komonfugu Fugu poecilonotus liver. Toxicon 2008, 51, 1269–1273.
Leung K.S.Y., Fong B.M.W., Tsoi Y.K.: Analytical challenges: determination of tetrodotoxin in human urine and plasma by LC-MS/MS. Mar Drugs 2011, 9, 2291–2303.
Mahmud Y., Tanu M.B., Noguchi T.: First occurrence of a food poisoning incident due to ingestion of Takifugu oblongus along with a toxicological report on three marine puffer species in Bangladesh. J Food Hyg Soc Jpn 1999, 40, 473–480.
McNabb P., Selwood A.I., Munday R., Wood S.A., Taylor D.I., MacK enzie L.A., van Ginkel R., Rhodes L.L., Cornelisen C., Heasman K., Holland P.T., King C.: Detection of tetrodotoxin from the grey side-gilled sea slug -Pleurobranchaea maculata and associated dog neurotoxicosis on beaches adjacent to the Hauraki Gulf, Auckland, New Zealand. Toxicon 2010, 56, 466–473.
Miyazawa K., Noguchi T.: Distribution and origin of tetrodotoxin. J Toxicol Toxin Rev 2001, 20, 11–33.
Nakagawa T., Jang J., Yotsu-Yamashita M.: Hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry of tetrodotoxin and its analogues. Anal Biochem 2006, 352, 142–144.
Nakashima K., Arakawa O., Taniyama S., Nonaka M., Takatani T., Yamamori K., Fuchi Y., Noguchi T.: Occurrence of saxitoxins as a major toxin in the ovary of a marine puffer Arothron firmamentum Toxicon 2004, 43, 207–212.
Naritia H., Noguchi T., Maruyama J., Ueda Y., Hashimoto K., Watanabe Y., Hida K.: Occurrence of tetrodotoxin in a trumpet shellfish “boshubora” Charonia sauliae Nippon Suisan Gakkai Shi 1981, 47, 934–941.
Neagu D., Micheli L., Palleschi G.: Study of a toxin-alkaline phosphatase conjugate for the development of an immunosensor for tetrodotoxin determination. Anal Bioanal Chem 2006, 385, 1068–1074.
Ngy L., Tada K., Yu C.F., Takatani T., Arakawa O.: Occurrence of paralytic shellfish toxins in Cambodian Mekong pufferfish Tetraodon turgidus selective toxin accumulation in the skin. Toxicon 2008, 51, 280–288.
Noguchi T., Ebesu J.S.M.: Puffer poisoning: epidemiology and treatment. Toxin Rev 2001, 20, 1–10.
Noguchi T., Mahmud Y.: Current methodologies for detection of tetrodotoxin. J Toxicol 2001, 20, 35–50.
Noguchi T., Onuki K., Arakawa O.: Tetrodotoxin poisoning due to pufferfish and gastropods, and their intoxication mechanism. Toxicology 2011, 1–10.
Nzoughet J.K., Campbell K., Barnes P., Cooper K.M., Chevallier O.P., Elliott C.T.: Comparison of sample preparation methods, validation of an UPLC-MS/MS procedure for the quantification of tetrodotoxin present in marine gastropods and analysis of pufferfish. Food Chem 2013, 136, 1584–1589.
Oda K., Araki K., Totoki T., Shibasaki H.: Nerve conduction study of human tetrodotoxication. Neurology 1989, 39, 743–745.
O’Leary M.A., Schneider J.J., Isbister G.K.: Use of high performance liquid chromatography to measure tetrodotoxin in serum and urine of poisoned patients. Toxicon 2004, 44, 549–553.
Osek J., Wieczorek K., Tatarczak M.: Seafood as potential source of poisoning by marine biotoxins. Med Weter 2006, 62, 370–373.
Pires O.R., Sebben A., Schwartz E.F., Bloch C., Morales R.A.V., Schwartz C.A.: The occurrence of 11-oxotetrodotoxin, a rare tetrodotoxin analogue, in the brachycephalidae frog Brachycephalus ephippium Toxicon 2003, 42, 563–566.
Pires O.R., Sebben A., Schwartz E.F., Morales R.A.V., Bloch C., Schwartz C.A.: Further report of the occurrence of tetrodotoxin and new analogues in the Anuran family Brachycephalidae Toxicon 2005, 45, 73–79.
Rodrıguez P., Alfonso A., Otero P., Katikou P., Georgantelis D., Botana L.M.: Liquid chromatography-mass spectrometry method to detect tetrodotoxin and its analogues in the puffer fish Lagocephalus sceleratus (Gmelin, 1789) from European waters. Food Chem 2012, 132, 1103–1111.
Rodriguez P., Alfonso A., Vale C., Alfonso C., Vale P., Tellez A., Botana L.M.: First toxicity report of tetrodotoxin and 5,6,11-trideoxyTTX in the trumpet shell Charonia lampas lampas in Europe. Anal Chem 2008, 80, 5622–5629.
Shoji Y., Yotsu-Yamashita M., Miyazawa T., Yasumoto T.: Electrospray ionization mass spectrometry of tetrodotoxin and its analogues: liquid chromatography/mass spectrometry, tandem mass spectrometry, and liquid chromatography/tandem mass spectrometry. Anal Biochem 2001, 290, 10–17.
Silva M., Azevedo J., Rodriguez P., Alfonso A., Botana L.M., Vasconcelos V.: New gastropod vectors and tetrodotoxin potential expansion in temperate waters of the Atlantic Ocean. Mar Drugs 2012, 10, 712–726.
Stokes A.N., Williams B.L., French S.S.: An improved competitive inhibition enzymatic immunoassay method for tetrodotoxin quantification. Biol Proced Online 2012, 14, 1–5.
Sui L.M., Chen K., Hwang P.A., Hwang D.F.: Identification of tetrodotoxin in marine gastropods implicated in food poisoning. J Nat Toxins 2002, 11, 213–220.
Tsai Y.H., Hwang D.F., Cheng C.A., Hwang C.C., Deng J.F.: Determination of tetrodotoxin in human urine and blood using C18 cartridge column, ultrafiltration and LC-MS. J Chromatogr B 2006, 832, 75–80.
Turner A., Powell A., Schofield A., Lees D., Baker-Austin C.: Detection of the pufferfish toxin tetrodotoxin in European bivalves, England, 2013 to 2014. Euro Surveill 2015, 20, 1–7.
Vlamis A., Katikou P., Rodriguez I., Rey V., Alfonso A., Papazachariou A., Zacharaki T., Botana A.M., Botana L.M.: First detection of tetrodotoxin in Greek shellfish by UPLC-MS/MS potentially linked to the presence of the Dinoflagellate prorocentrum minimum. Toxins 2015, 7, 1779–1807.
Watabe S., Sato Y., Nakaya M., Hashimoto K., Enomoto A., Kaminogawa S., Yamauchi K.: Monoclonal antibody raised against tetrodonic acid, a derivative of tetrodotoxin. Toxicon 1989, 27, 265–268.
Yamazaki M., Shibuya N.: Motor nerve conduction velocity is useful for patients with tetrodotoxin. Anesth Analg 1995, 80, 848–857.
Yotsu-Yamashita M., Abe Y., Kudo Y., Ritson-Williams R., Paul V.J., Konoki K., Cho Y., Adachi M., Imazu T., Nishikawa T., Isobe M.: First identification of 5,11-dideoxytetrodotoxin in marine animals, and characterization of major fragment ions of tetrodotoxin and its analogs by high resolution ESI-MS/MS. Mar Drugs 2013, 11, 2799–2813.
Yotsu-Yamashita M., Jang J.H., Cho Y., Konoki K.: Optimisation of simultaneous analysis of tetrodotoxin, 4-epitetrodotoxin, 4,9-anhydrotetrodotoxin and 5,6,11-trideoxytetrodotoxin by hydrophilic interaction liquid chromatography-tandem mass spectrometry. Forensic Toxicol 2011, 29, 61–64.
Yotsu-Yamashita M., Sugimoto A., Takai A., Yasumoto T.: Effects of specific modifications of several hydroxyls of tetrodotoxin on its affinity to rat brain membrane. J Pharmacol Exp Ther 1999, 289, 1688–1696.
You J., Yue Y., Xing F., Xia W., Lai S., Zhang F.: Tetrodotoxin poisoning caused by goby fish consumption in southeast China: a retrospective case series analysis. Clinics 2015, 70, 24–29.