What is Tetrodotoxin

TTX Structure

Introduction to Tetrodotoxin (TTX) is a relatively common alkaloid contained in living organisms. It is an extremely toxic neurotoxin, approximately 1,250 times more toxic than cyanide. There are different opinions on the origin of TTX. Some think that TTX is exogenous, while others think it is endogenous. There is no accurate conclusion yet. The relative molecular mass of TTX is 319.27, and the molecular formula is C11H17N3O8. It mainly exists in the ovaries, liver and other parts of the body. The toxic content is highest in the ovaries and liver. It is stable in nature and difficult to destroy by ordinary cooking methods. There are many common homologues, mainly 5-deoxyTTX, 11-deoxy, 4,9-anhydroTTX, 5,11-dideox yTTX, 6,11-dideoxyTTX, 5,6,11-trideoxyTTX, 4,9-anhydro- 5 ,6,11-trideoxy. Pure TTX is a white crystal with excellent solubility in weakly acidic solutions and will naturally precipitate in a weakly alkaline environment. TTX can exist as a zwitterion and will be destroyed in strong acid and strong alkali. Strong alkali can destroy it into a non-toxic compound: 2-amino-8-hydroxy-6-hydroxymethyl-quinazoline.

TTX has a small molecular weight and a special structure. It is easily soluble in weakly acidic solvents, and its structure is easily destroyed in strong acid and strong alkali solvents. In the early 1960s, through in-depth research on the structure of TTX, it went through 15 years of research. After years of research, using methods such as preparing derivatives and conducting X-diffraction experiments, researchers finally discovered the TTX cage orthoester structure. All the carbon atoms in the structure are asymmetrically substituted, and they are balanced mixture exists in three forms in the free state.

Tetrodotoxin Detection Method

• Mouse Bioassay

In the 1940s, researchers began to conduct mouse bioassays. When a certain concentration of TTX is injected into mice, the mice will appear dead, and there is a certain linear relationship between the death time and the TTX content. At first, the toxicity of TTX was expressed in murine units (MU). Under experimental conditions, a 20g mouse was subcutaneously injected with 0.2mL TTX solution, and the dose that caused death in 10 minutes was 1 murine unit. This method is simple to operate and does not require special equipment, but it requires a large number of mice, is easily affected by individual differences in mice, and has poor repeatability.

• Enzyme-linked Immunoassay

In the 1980s, enzyme-linked immunoassay to measure TTX gradually became widely used. It is an analysis method derived based on the binding of antigens and antibodies, as well as the influence of bond energy such as hydrogen bonds. The researchers used the enzyme-linked immunoassay method to conduct a qualitative analysis of TTX. Since the enzyme-linked immunoassay method has high sensitivity and can ensure this specificity and quantitative relationship, when screening TTX in aquatic products, a large number of tests can be carried out at one time, but there are certain cases of false positives in enzyme-linked immunoassay.

• Fluorescence, UV Spectrophotometry

The detection principle of the fluorescence method is that TTX is hydrolyzed in an alkaline environment to produce C9, and the content of TTX is quantified through C9 alkali. This is the earliest established method for quantitative detection of TTX. Researchers use NaOH and TTX for pre-column derivatization. The TTX derivative is fluorescent at a maximum excitation wavelength of 370 nm and a maximum emission wavelength of 495 nm. When TTX is derivatized to produce C9, the by-product is also accompanied by the production of sodium oxalate of the same quality. It has obvious UV light absorption at a wavelength of 230 nm, so TTX can also be quantitatively detected using UV spectrophotometry. Both fluorescence and UV spectrophotometry can detect TTX, but the sensitivity and accuracy are still lacking compared with other detection methods.

• Thin Layer Chromatography

Thin layer chromatography is a relatively traditional identification method. It has the characteristics of simplicity, ease of operation, good separation effect, etc., and has a wide range of applications. This method is based on the characteristic of TTX having a relatively large polarity. It reacts with sodium hydroxide to produce a color-developing substance for qualitative measurement. The commonly used developing agent for spot plates is n-butanol, acetic acid, and water with a volume ratio of 4:1:2, and the minimum detection limit is 4.0×10-9 mg/L. However, this method can only be used for qualitative analysis of TTX, and application is not widespread.

• High Performance Liquid Chromatography (HPLC) Method

HPLC-fluorescence detector (FLD) and ultraviolet detector (UV) methods are the most commonly used detection methods in laboratories. They mainly use different detectors to quantify detection substances. Under alkaline conditions, TTX is derivatized, and the resulting derivative material has a visible light absorption signal that can be detected by a fluorescence detector or ultraviolet detector. Fluorescence-derived reaction mechanism: The TTX molecule has a guanidine group with a very high pKa value, which is easily protonated in aqueous solution and very unstable in alkaline solutions, and an orthocarboxylic acid group that is easily hydrolyzed in acid or alkali.

• HPLC-MS/MS Method

HPLC-MS/MS has been a research hotspot in recent years. It mainly bombards target ions to generate multiple ion fragments, fragments the product ions with the highest response again, and finally obtains qualitative and quantitative ion fragments, so as to carry out target substance analysis. Immunoaffinity column purification-HPLC-MS/MS is currently the most frequently used method for detecting TTX. The immunoaffinity column takes advantage of the specific recognition characteristics of TTX and mouse ascites in the column packing. TTX uses mouse ascites as a carrier to bind, and then removes the matrix to achieve the purification effect.