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ISSN : 1229-1153(Print)
ISSN : 2465-9223(Online)
Journal of Food Hygiene and Safety Vol.36 No.2 pp.105-117

Tetrodotoxin and Its Analogs: A Review of Analysis Methods and Levels in Pufferfish

Bong Ki Park1, Woo Jin Jang1, Ki-Hwan Park1, Hee-Seok Lee1, Wan-Ok Lee2, Kwang-Sik Choi2, Jihyun Lee1*
1Department of Food Science and Technology, Chung-Ang University, Anseong, Korea
2Department of Marine Life Science (BK21 FOUR), JeJu National University, Jeju, Korea
* Correspondence to: Jihyun Lee, Department of Food Science and Technology, Chung-Ang University, Anseong 17546, Korea Phone: +82-31-670-3266, Fax: +82-31-675-3108 E-mail:
January 20, 2021 January 31, 2021 February 25, 2021


Pufferfish is a major source of tetrodotoxin (TTX), a potent neurotoxin that can lead to death in humans if consumed. The content of TTX and its analogs in different pufferfish species and the distribution in various fish organs are poorly understood. In this study, we compared TTX analysis methods, including mouse bioassay, enzyme-linked immunosorbent assay, high-pressure liquid chromatography (HPLC) coupled with a fluorescence detector, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry (LC-MS) methods. Different HPLC columns (e.g., amide, hydrophilic interaction liquid chromatography, and C18 columns) and mobile phases were compared for TTX analysis using LC-MS. Additionally, published extraction methods were compared for LC-MS analysis. The content of TTX and its analogs in different pufferfish species and their distribution in various organs (e.g., liver, muscle, intestine, testis, ovary, and skin) were summarized. This information can help understand the available TTX analysis methods, variation in TTX contents in pufferfish species from different regions, and TTX distribution in various organs.


    Ministry of Food and Drug Safety(MFDS)

    Pufferfish belong to the order Tetraodontiformes, and they mostly reside in the oceans of tropical and subtropical areas. They are also found in freshwater environments and large river estuaries. Many pufferfish species contain tetrodotoxin (TTX), an extremely potent and dangerous neurotoxin that acts on sodium channels in the muscle membranes and nervous tissue1). Pufferfish has been consumed since historical times in worldwide. In Korea, pufferfish were possibly consumed in the Neolithic period approximately 5000 years ago. This tradition has also been preserved in ancient Chinese and Egyptian murals and records2). Most pufferfish contain the toxic compound TTX, and the high consumption of pufferfish in Asia has led to TTX poisoning incidents. Poisoning incidents have typically been noted in Asian countries, such as China, Taiwan, Bangladesh, Malaysia, and the United States and Mexico3-6). Japan, in particular, has had a long history of pufferfish poisoning. Potent TTX toxicity has also been reported in invasive pufferfish species entering the Mediterranean Sea due to the “Lessepsian migration” caused by creating the artificial Suez Canal7). Problems due to pufferfish consumption occur in many countries along the Mediterranean coast8-11). Besides, the increase in seawater temperature due to global warming is a rising concern as warming may create suitable habitats for pufferfish globally; thus, TTX poisoning incidents may become a worldwide concern12). The globally found pufferfish species containing TTX are shown in Table 1. Recently, TTX and its analogs have been identified13,14); however, their extraction and analysis methods used in various studies differ. Thus, it is challenging to compare the contents of TTX and its analogs in different pufferfish species. TTX contents in pufferfish may vary by species, season, and habitat7,9,11,15-17).

    Tetrodonic acid (TDA), 4-epiTTX, and anhydroTTX were first identified as pufferfish TTX analogs in 198518). Since then, many studies have been performed for identifying TTX analogs. Moreover, toxicity caused TTX analogs may vary, with few analogs exhibiting higher toxicity than TTX. Additionally, there is a lack of systemic approaches comparing TTX content in pufferfish species found worldwide.

    To ensure safe consumption of pufferfish, it is necessary to establish an analytical method with high accuracy and sensitivity to monitor the toxins found in pufferfish species (i.e., TTX analogs). TTX content has conventionally been measured using mouse bioassay (MBA)19). However, MBA can only measure the effects of pufferfish toxins and cannot determine TTX content alone. Also, MBA has ethical issues. To address this issue, instrumental analysis methods such as high-performance liquid chromatography coupled with a fluorescence detector (HPLC-FLD) have been proposed for TTX analysis20). Liquid chromatography-mass spectrometry (LC-MS) is the commonly used method for analyzing TTX analogs recently21,22). However, the currently available studies have reported variations of sample preparation and analysis methods13,16,17,23-26).

    We investigated different sample preparation methods for LC-MS analysis of TTX and its analogs to address these issues. In this study, we also reviewed various LC-MS methods applied in TTX analysis. TTX contents in different pufferfish species and the difference in TTX content depending on the habitats and seasonal variations have been reported.

    TTX overview

    TTX is a potent and old marine toxin27). In 1910, Dr. Tahara successfully isolated TTX from pufferfish ovaries and named it “tetrodotoxin” after the order Tetraodontiformes28). TTX structure was discovered by several research groups in the 1960s29-31). TTX is a colorless and odorless non-protein molecule with a low molecular weight of 319.27 g/mol. Structurally, it has a highly oxygenated carbon backbone comprising a 2,4-dioxaadamantane structure containing five hydroxyl groups that are connected to a single guanidinium moiety32). The structures of TTX and its analogs are shown in Table 2.

    TTX strongly and selectively blocks voltage-gated sodium channels without affecting other receptors and ion channel systems33). It blocks these channels outside the nerve membranes, causing paralysis, and high exposure to TTX may lead to death33). In humans, the median lethal dose (or MDL50) of TTX is predicted to be about 10,000 MU34), which is equivalent to about 2 mg of TTX35). Additionally, TTX is thermostable and generally does not degrade by cooking, and hence, TTX poisoning incidents have been occurring historically27). Approximately 30 TTX analogs have been reported to date, and the degree of toxicity of these analogs depends on their structures. For example, hydroxyl analogs are more toxic than TTX; however, deoxy analogs are less toxic than TTX23,36-38). Additionally, the effective dose (ED50) of 11-oxo TTX was smaller than the ED50 of TTX38). Thus, TTX analog composition can also considerably affect toxicity. The type and severity of poisoning symptoms in humans depend on toxin amounts consumed, age, and health. The initial symptoms start with paresthesia of the lips, tongue, and pharynx as well as gastrointestinal symptoms such as vomiting and diarrhea, followed by systemic sensory abnormalities and quadriplegia. In severe cases, extreme hypotension and seizures, cranial nerve dysfunction, and respiratory failure can be observed, potentially leading to death39). Death usually occurs 4-6 hrs after exposure to TTX. Currently, there is no fundamental cure or antidote for TTX40). TTX mediates its toxic effects by blocking the voltage-gated sodium channels in neurons and causing paralysis. Thus, it can be expected to function as a painkiller. Research is being conducted to assess TTX for treating pain, such as cancer pain41-43).

    Traditional TTX analysis methods in pufferfish

    Mouse bioassay (MBA) is the first and most frequently used method to analyze TTX in marine organisms44). However, MBA alone cannot identify TTX analogs. Other marine toxins, such as saxitoxin (STX), may block voltagegated sodium channels and exhibit symptoms similar to TTX poisoning. Owing to this similarity, TTX poisoning cannot be differentiated from STX poisoning using MBA21). Moreover, MBA is expensive and labor-intensive and is associated with ethical problems. To address these issues, immunological methods using enzyme-linked immunosorbent assay (ELISA) and competitive inhibition enzymatic immunoassay (CIEIA) have been proposed for TTX analysis45,46). However, the use of ELISA has several issues such as variability. Although CIEIA has better accuracy and repeatability than ELISA, the antibody used is too expensive21,47). Additionally, neither assay can detect TTX analogs. The initial instrumental analysis of TTX was performed using gas chromatographymass spectrometry (GC-MS) with good sensitivy48,49). As TTX is non-volatile, it needs to be derivatized for use in GC-MS. This proves to be a major disadvantage, as derivatization is time-consuming21).

    The analysis of TTX analogs in pufferfish samples using HPLC-FLD was first developed in 198550). This method helps identify TTX and its analogs (i.e., 11-oxoTTX, 4- epiTTX, and 4, 9-anhydroTTX). However, the HPLC-FLD method is not suitable for routine sample analysis because of changes in the fluorescence intensity of TTX analogs and the interference of background signals of the matrices. The fluorescence intensity of 6-epiTTX and 11-norTTX-6 (R)-ol was 20 and 100 times higher than that of TTX, while that of 11-deoxyTTX were 100 times lower than that of TTX51,52).

    LC-MS methods for TTX analog analysis

    Recently, LC-MS has been widely used for TTX analysis in pufferfish samples, as TTX analogs can also be identified and quantified with high accuracy and specificity using the method22). Table 3 shows various sample preparation methods for LC-MS analysis. For sample preparation in most cases, pufferfish samples are homogenized and extracted using 0.1−2% acetic acid-containing heated water 7,16,17,20,23,36). Then, solid-phase extraction (SPE) using C18 cartridges and filtration are performed together for further clean-up before LC-MS analysis. Hydrophilic interaction liquid chromatography (HILIC) SPE must be performed in gravity flow as vacuum interferes with the TTX recovery rate53). Recently, accelerated solvent extraction has been used in the extraction of TTX from pufferfish54). This method has a high recovery rate, but it is associated with long prepration time because of an added lyophilization process54).

    Various LC-MS methods used for TTX analysis are shown in Table 4. Reverse-phase chromatography has been the conventionally used method for TTX analysis9,17,25,26,53,55,56). However, this method cannot separate all analogs of TTX. In addition, TTX analogs are highly polar in nature and are rapidly eluted in reverse-phase separation, resulting in poor separation of TTX analogs. Thus, normal-phase separation is a preferred option for TTX analogs to ensure better separation21). HILIC columns have a hydrophilic stationary phase that can separate hydrophilic substances such as TTX analogs and are reported to have a lower background noise. These columns also have better resolution and sensitivity for TTX analog analysis than C18 columns24,57). Ammonium formate or ammonium acetate in water with organic phases (i.e., acetonitrile or methanol) is widely used as the mobile phase for TTX analysis. Moreover, both acetonitrile and methanol facilitate similar separation of TTX analogs; however, owing to the low cost of methanol, the latter is a preferred choice for the process53).

    TTX contents in pufferfish analyzed by LC-MS

    TTX is the most characteristic toxin among pufferfish toxins. Although there are many studies on the accumulation of TTX in pufferfish, little is known about the biosynthesis of TTX. Interestingly, pufferfish lose their TTX capacity when cultured58,59). Therefore, it has been hypothesized that TTX in pufferfish is derived from the food chain and not from an endogenous factor58).

    Recent studies have used LC-MS for identifying and quantifying TTX in pufferfish13,17,23,24). For identification, the fragmentation pattern of TTX has been propsed in positive ionization mode of LC-MS. The precursor ion of TTX ion at m/z 320 is fragmented to m/z 302 (loss of one water molecule), m/z 284 (loss of three water molecules), m/z 256, m/z 178, and m/z 162 fragment ions21).

    Table 5 shows the distribution of TTX in various organs of pufferfish samples analyzed by LC-MS. TTX contents in pufferfish differ greatly by organs. The liver and ovaries of pufferfish have the highest TTX contents, followed by the intestine and skin11,20,24). The muscles and testicles usually contain trace amounts or do not contain TTX, except for those in Lagocephalus lunaris and Chelonodon patoca58). During the spawning period, TTX is transferred from the liver to the ovaries, which results in increased TTX in the ovaries of female pufferfish60,61). In contrast, TTX in male pufferfish is distributed from the liver to the skin during spawning period, and hence, the skin in male pufferfish has high TTX content61). Additionally, TTX contents are generally higher in females and male pufferfish during the maturation/spawning period than usual contents61).

    TTX analog contents in pufferfish analyzed by LC-MS

    Table 6 shows TTX analog levels in pufferfish determined using LC-MS. TTX analogs can be identified and quantified using ultra-HPLC coupled with triple quadrupole or ion-trap mass spectrometry11,16,23,25,36). Few TTX analogs that have been identified in pufferfish include tetodonic acid (TDA); 4-epiTTX; anhydroTTX; 11-deoxyTTX; 11-norTTX-6,6- diol; 11-norTTX-6(R)-ol; 11-norTTX-6(S)-ol; 5-deoxyTTX; and 6,11-dideoxyTTX-5,6,11-trideoxyTTX18,62-64). In 2001, Shoji et al. identified TTX analogs in pufferfish by LC-MS and obtained characteristic fragmentation ion spectra of TTX and its analogs in positive ionization mode of electrospray ionization tandem mass spectrometry (MS/ MS)25). Authentic TTX, TTX analogs, and semi-purified TTX mixture, have been used to quantify TTX and analogs in LC-MS/MS15-17,23,36).

    Major TTX analogs found in pufferfish and their composition vary across different species. In all tissues of Fugu niphobles and two Tetraodon spp. in Korea and F. niphobles in Japan, 5,6,11-trideoxyTTX and 6,11-dideoxyTTX were identified as the major TTX analogs, and 5-deoxyTTX and 11-deoxyTTX were the minor analogs16). Up to 8 TTX analogs have been found in Lagocephalus sceleratus in the Aegean Sea, with 5,6,11-trideoxyTTX being the major TTX analog17). Moreover, L. sceleratus in the Aegean Sea had a higher level of 11-deoxyTTX and 11-norTTX-6(S)-ol than TTX11,17). In the Atlantic Sphoeroides marmoratus, TTX analogs such as 4,9-anhydroTTX and 4-epiTTX and three deoxy analogs 5-deoxyTTX, 6,11-dideoxyTTX, and 11- deoxyTTX were identified36).


    TTX is one of the oldest and most potent marine toxins as well as a non-protein, low molecular weight neurotoxin that blocks sodium channels in humans. Many methods have been used for TTX analysis; however, LC-MS is being studied for identifying and quantifying TTX analogs due to their respective disadvantages. In LC-MS analysis, HILIC column use, in particular, results in better separation of TTX analogs than reverse-phase column (e.g., C18 column) use. In pufferfish, LC-MS using HILIC columns revealed TTX, with the liver and ovary having the highest toxin levels, followed by the intestine and skin, muscle, and testis. Differences in TTX levels were noted based on species, seasonal variations, and organ. TTX analog levels varies with species and region. And certain TTX analogs were present in high levels, with few having higher toxicity than TTX. However, little is known about the TTX and its analog composition various species pufferfish. In terms of food safety, further research is required monitor TTX and its analog composition in pufferfish.


    테트로도톡신(Tetrodotoxin)은 나트륨 통로를 차단하는 신경독소로 가장 오래되고 강력한 해양독소 중 하나이다. 전통적으로 아시아에서 복어 섭취로 인한 테트로도톡신 중독사고가 많이 발생했었다. 최근 아열대 복어 종과 그 잡종 섭취로 인한 테트로도톡신 중독사고가 아시아 외 지 역에서도 산발적으로 발생하고 있다. 그러나 복어 종별 테 트로도톡신 및 유사체의 함량과 기관 내 분포에 대해서는 잘 알려져 있지 않다. 이 총설에서는 식품공전 복어독(테 트로도톡신) 분석법인 쥐를 이용한 생물학적 정량법, 효소 면역측정법, 고압 액체 크로마토그래피-형광검출기, 가스 크로마토그래피-질량분석기를 이용한 분석법의 장단점을 비교하였다. 또한 최근 주로 사용되는 액체 크로마토그래 피-질량분석기를 이용한 테트로도톡신과 테트로도톡신 유 사체 분석법을 소개하고 이에 사용된 전처리법, 컬럼 및 이동상을 비교하였다. 또한 복어 종별 테트로도톡신과 그 유사체 함량과 다양한 기관에서의 분포를 정리하였다. 이 총설은 테트로도톡신 분석법과 다양한 복어 종의 테트로 도톡신과 유사체 함량 및 기관에서의 분포를 이해하는데 도움을 줄 수 있을 것이다.


    This research was supported by a grant (20163MFDS641) from the Ministry of Food and Drug Safety.



    Global report of tetrodotoxin content in pufferfish

    Structures of tetrodotoxin (TTX) and TTX analogs found in pufferfish

    Different pufferfish sample preparation techniques for the analysis of tetrodotoxin and its analogs

    Different LC-MS methods for tetrodotoxin analysis in pufferfish

    Tetrodotoxin contents in pufferfish samples

    Tetrodotoxin analog contents in pufferfish samples


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