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A method for extracting neosaxitoxin from poisonous shells

A new saxitoxin and shell technology, applied in organic chemistry, instruments, measuring devices, etc., can solve the problems of poisoning, high neoSTX positive rate, and no extraction of neoSTX, etc., and achieve the effect of alleviating the demand

Active Publication Date: 2022-03-29
EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, in the daily monitoring of paralytic shellfish toxins, the positive rate of neoSTX is high, which is more likely to cause poisoning incidents
At present, there is no relevant report on the extraction of neoSTX from poisonous scallops

Method used

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  • A method for extracting neosaxitoxin from poisonous shells
  • A method for extracting neosaxitoxin from poisonous shells
  • A method for extracting neosaxitoxin from poisonous shells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 New Saxitoxin (neoSTX) Extraction and Separation and Purification

[0034] A. Preparation of hepatopancreas powder

[0035] Wash the surface of the poisonous scallop shell with clean water, cut off the adductor muscle, rinse with distilled water, carefully take out the liver and pancreas, spread the water on a sieve for 5 minutes, freeze-dry, and then use a tissue homogenizer to homogenize at 12000r / min. Mix well. Add 500mL of petroleum ether to every 200g of hepatopancreas powder, mix well, ultrasonicate for 10min, filter, dry and homogenize the hepatopancreas powder.

[0036] B. Crude extraction of toxins

[0037] Add 200mL of water to every 100g of hepatopancreas powder, vortex for 60s, let stand for 60min, remove the supernatant; add 250mL of 1% acetic acid or 0.5% formic acid solution to the precipitate, vortex for 60s, then place in 100℃ water bath and heat for 20min , after ultrasonic extraction for 10 min, centrifuge at 3500 r / min for 6 min, take ou...

Embodiment 2

[0045] Embodiment 2: Qualitative detection

[0046] Using liquid chromatography-quadrupole / ion trap composite mass spectrometry to extract ion spectrograms and secondary fragmentation spectrograms, the pure neoSTX product prepared in Example 1 is matched with the multilevel mass spectrometry information of the Canadian National Oceanographic Institute standard material; Liquid chromatography-high resolution mass spectrometry determines the accurate mass, and qualitative determination is performed by these three methods. Among them, the standard product of the National Oceanographic Research Institute of Canada was purchased in 2019, and the product batch number is LOT#20170411.

[0047] The test method of liquid chromatography-quadrupole / ion trap composite mass spectrometry is as follows:

[0048] 1) Chromatographic column: TSK-GelAmide-80, 2.1mmI.D.×10cm, 5μm.

[0049] 2) Flow rate: 0.5mL / min.

[0050] 3) Column temperature: 35°C.

[0051] 4) Injection volume: 5 μL.

[0...

Embodiment 3

[0078] Embodiment 3: quantitative test

[0079] Accurately take 50 μL of the collected solution, add 950 μL of 75% acetonitrile water (containing 0.25% formic acid), mix thoroughly, and then perform liquid chromatography-tandem mass spectrometry to determine the concentration of the collected solution.

[0080] refer to Figure 7 The standard curve plot of neoSTX in, and Figure 8 Quantitative spectra of neoSTX are shown. The results showed that the content of neoSTX was 64.671ng / mL under the condition of linear range of 6.5~180ng / mL, linear factor of 0.99983, and standard addition recovery rate of 108%, which met the purity requirements of the standard.

[0081] In the above examples, poisonous scallops are used as raw materials for toxin extraction, but in fact other poisoned bivalve molluscs, such as poisoned mussels, can be extracted from them. In addition, if the poisonous shellfish contains other toxins with high content, this preparation technology can also be used t...

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Abstract

The present invention relates to a method for extracting saxitoxin from poisonous shells, comprising A, preparation of hepatopancreas powder; B, rough extraction of toxin: first mixing hepatopancreas powder with water and vortexing, adding the volume of liver and pancreas after standing The acid extraction solution with 2.5 times the mass of pancreas powder was vortexed, heated in a water bath, ultrasonically extracted, centrifuged to obtain the supernatant, and rotated in a water bath at 50-60°C to obtain a crude toxin extraction solution; C. Purification: using dextran gel The chromatographic column was purified, the toxin crude extraction solution was centrifuged and the supernatant was passed through the gel column, first eluted with 200 times the volume of deionized water at a flow rate of 0.9mL / min, then eluted with 0.1mol / L acetic acid solution, and collected Fractions containing paralytic shellfish toxin components are then combined, concentrated and nearly dry; D, separation and purification: dissolve the sample collected in step C, separate and purify by liquid chromatography, collect chromatographic peaks according to the retention time of the target, and combine The fractions were concentrated, dissolved in 75% acetonitrile in water containing 0.1% formic acid, and stored in a -40°C refrigerator.

Description

technical field [0001] The invention belongs to the technical field of marine biotoxin preparation, and in particular relates to a method for extracting saxitoxin from poisonous shells. Background technique [0002] The paralytic shellfish toxin consists of saxiotixn (STX) and its derivatives. STX is one of the most toxic non-protein organic small molecules found so far, and its toxicity is 1500 times that of potassium cyanide. Due to the high toxicity, unpredictability and wide distribution of paralytic shellfish toxins in the world, it has repeatedly caused poisoning and even death among consumers. Therefore, many countries or regions such as the European Union, the United States, and Canada have established strict limit standards and monitored them. However, toxin detection technology, as a necessary means of monitoring, is affected by the lack of standard paralytic shellfish toxins, and is subject to many limitations. In addition, paralytic shellfish toxin has potentia...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D487/20G01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72
CPCC07D487/20G01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72G01N2030/324
Inventor 汤云瑜沈晓盛娄晓祎方长玲张璇杨光昕孔聪
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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