CN104820033A - Detection method of tetrodotoxin - Google Patents
Detection method of tetrodotoxin Download PDFInfo
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- CN104820033A CN104820033A CN201510220110.3A CN201510220110A CN104820033A CN 104820033 A CN104820033 A CN 104820033A CN 201510220110 A CN201510220110 A CN 201510220110A CN 104820033 A CN104820033 A CN 104820033A
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- tetraodotoxin
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Abstract
The invention relates to a detection method of tetrodotoxin in tachypleus tridentatus and carcinoscorpius rotundicauda, which includes the following steps: (1) preparing a standard stock solution; (2) pre-treating a sample; and (3) performing liquid chromatographic test. In the invention, a high performance liquid chromatographic method is used for detecting the tetrodotoxin in tachypleus tridentatus and carcinoscorpius rotundicauda for determining whether toxin in horseshoe crabs belongs to tetrodotoxin or not, thereby knowing what kinds of the horseshoe crab causing a person to be suffered from horseshoe crab poisoning and what the content is, thereby providing reference to prevention and treatment of horseshoe crab poisoning.
Description
Technical field
The invention belongs to biological technical field, relate to the detection method of the tetraodotoxin in a kind of Tachypleus tridentatus and rounded tail king crab body.
Background technology
Tachypleus tridentatus is mainly distributed in Seto Island Sea and north, the Kyushu Island bank of Japan, Vietnam, western Filipine island, Su Mendala and Java Island, Malay North Borneo and Dong Andeng marine site, Kalimantan, mainly be distributed in the coastal marine sites such as Zhejiang on the south the Changjiang river, Fujian, Guangdong, Guangxi, Hainan and Taiwan in China, existing is many with Fujian, Guangdong, Guangxi, Hainan.Rounded tail king crab is distributed in Hong-Kong, the North Sea, gulf, Leizhou, marine site to the north of the bank of north, Indonesia Java Island, to the east of to the west of Philippine south and river mouth, Henghe Northeastern India.Mainly be distributed in the West Coast in the North Sea, West Coast, the Lezhou Peninsula and Hainan China rounded tail king crab, discovered in recent years Lezhou Peninsula south coast and to the east of the littoral distribution all having rounded tail king crab, seabeaches as many in the ground such as Zhanjiang, Hong Kong are also distributed with many rounded tail king crabs.Our people have the custom of eating king crab, and king crab meat is like crab, and just mouthfeel is thicker.Often there is the poisoning event of food king crab to occur in the Guangdong of China, Guangxi.
Tetraodotoxin (tetrodotoxin, TTX) is the nerve toxin that a kind of toxicity is very strong, and it separates the earliest from globe fish.Also find that tetraodotoxin does not exist only in the fish of Fugu, is also present in goby, octopus, starfish, crab class and gastropod, in the sea life that king crab etc. are numerous even in addition in recent years.They not only itself have stronger toxicity but also by approach such as food chain enrichments, can pollute other aquatic products, eat these aquatic products form very big potential danger to people.Become a difficult problem Southeast Asia tetraodotoxin is poisoning, be all generally because intake of that globe fish causes poisoning.Because Japanese globe fish by as one delicacies, so most tetrodotoxism event is all occur in Japan.In addition, eating not clear fingerling by mistake is also the major reason that this type of poisoning occurs, and thus strengthens seeming particularly necessary to the monitoring of tetraodotoxin in aquatic products, many province local Fishery Bureau using tetraodotoxin in aquatic products as conventional sense project.The detection method of tetraodotoxin is of a great variety, has several large classes such as bioassay method, Physico-chemical tests method, immunochemistry assay method.Mouse method was the most frequently used and the easiest method, and Liao Yongyan etc. also to have detected the tetraodotoxin in Tachypleus tridentatus and rounded tail king crab body by small white mouse method in 2000, but this method has quantitatively inaccurate and shortcomings such as poor repeatability.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a kind of method of the tetraodotoxin detected in Tachypleus tridentatus and rounded tail king crab body.
Its technical scheme is as follows:
A detection method for tetraodotoxin in Tachypleus tridentatus and rounded tail king crab body, comprises the following steps:
(1) preparation of Standard Reserving Solution
Precision weighing TTX 1.0mg is placed in 5ml centrifuge tube, and the concentration drawing 1.0mL with 1ml liquid-transfering gun is 0.2% acetic acid, namely obtains the TTX stock solution of 1.0mg/mL, be placed in refrigerator and preserve after making it dissolve;
(2) pre-treatment of sample
Get Tachypleus tridentatus or rounded tail king crab is to be measured organizes 10g, acetic acid homogenate in glass homogenizer that 10ml pH=3.5 concentration is 0.1% is added after shredding, after abundant homogenate, sample is placed in flask, add the acetic acid that 50ml concentration is 0.1%, then boiling water bath 10-20 minute in water-bath, heat while stirring, rotating speed controls at 150-200r/min, with 0.45 μm of filter membrane vacuum filtration after cooling, filtrate is put into evaporating dish, the water-bath of 60-70 DEG C carries out evaporation and concentration, until 10ml, then filter and be dissolved to 10ml, concentrated filtrate 10ml is placed in centrifuge tube, through the speed of 10000r/min, centrifugal 15 minutes, get supernatant after the filtering with microporous membrane of 0.22 μm, put into clean centrifuge tube, as liquid chromatography liquid to be measured,
(3) liquid chromatography test
Acetic acid mobile phase chromatographic condition Stationary liquid: SunFire
tMc18 chromatographic column, diameter 4.6mm × length 150mm, sample size is 20 μ; Mobile phase: 0.2% acetic acid; Flow velocity: 0.8mL/min; UV detect wavelength: 210nm; Column temperature: 25 DEG C; Each sampling volume is 60 μ L; PH is 3.0;
Phosphoric acid mobile phase chromatographic condition Stationary liquid: SunFire
tMc18 chromatographic column, is of a size of 4.6mm × 150mm, and sample size is 20 μ l; Mobile phase: 0.05mol/L NaH
2pO
4-0.05mol/L Na
2hPO
4, pH=6.8, sodium heptanesulfonate ion pair 5mM; Flow velocity: 1mL/min; UV detect wavelength is 200nm; Column temperature: 25 DEG C; Each sampling volume is 60 μ L; PH is 6.8.
Compared with prior art, beneficial effect of the present invention:
1, the present invention adopt high performance liquid chromatography detect in Tachypleus tridentatus and rounded tail king crab body tetraodotoxin, to prove whether the toxin in king crab body contains tetraodotoxin on earth, thus know that what king crab of food causes the poisoning and content of food king crab how many, the prevention and therapy poisoning for food king crab provides reference.
2, in inventive samples pre-treatment step, boiling water bath 10-20 minute in water-bath, heat while stirring, rotating speed controls at 150-200r/min, within the scope of this, material viscosity can not increase along with the carrying out stirred, if rotating speed is too high, after viscosity increases, rotating speed can reduce the stripping that can affect tetraodotoxin gradually.
Accompanying drawing explanation
Fig. 1 be 0.2% acetic acid (pH=3.0) do the tetraodotoxin standard sample liquid chromatogram of mobile phase.
Fig. 2 is 0.05mol/LNaH
2pO
4-0.05mol/L Na
2hPO
4(pH=6.8) and sodium heptanesulfonate ion pair 5mM do the tetraodotoxin standard sample liquid chromatogram of mobile phase.
Fig. 3 is the tetraodotoxin standard sample liquid chromatogram that phosphoric acid does mobile phase.
Fig. 4 be 0.2% acetic acid (pH=3.0) do the dissolved matter liquid chromatogram of mobile phase.
Fig. 5 be with phosphate buffer be mobile phase as titer, all band scanning ultraviolet spectrogram.
Fig. 6 is dissolved as mobile phase as titer using the acetum of 0.2%, the ultraviolet spectrogram of all band scanning.
Fig. 7 is the tetraodotoxin standard sample with 0.2% acetate dissolution, the ultraviolet spectrogram of all band scanning.
Fig. 8 is with the muscle samples solution of Tachypleus tridentatus for representative, the sample of extracting is carried out to the ultraviolet spectrogram of all band scanning.
Fig. 9 is with the muscle samples solution of rounded tail king crab for representative, the sample of extracting is carried out to the ultraviolet spectrogram of all band scanning.
Figure 10 be 194 selected respectively to standard items, 198,200,204,5 kinds of wavelength such as 214nm carry out chromatogram detection.
Figure 11 is with under phosphoric acid mobile phase, 200nm ultraviolet wavelength condition, and the chromatogram of carrying out 0.2% acetic acid and tetraodotoxin standard sample detects.
Tetraodotoxin standard sample, Tachypleus tridentatus muscle, rounded tail king crab muscle chromatogram compare by Figure 12.
Tetraodotoxin standard sample, Tachypleus tridentatus yellow connective tissue, rounded tail king crab yellow connective tissue chromatogram compare by Figure 13.
The height of tetraodotoxin standard sample, Tachypleus tridentatus muscle, rounded tail king crab muscle chromatogram chromatographic peak compares by Figure 14.
Tetraodotoxin standard sample, Tachypleus tridentatus yellow connective tissue, rounded tail king crab yellow connective tissue chromatogram are carried out the comparison of chromatographic peak height by Figure 15.
Embodiment:
With embodiment, the invention will be further described below, but the present invention is not limited to these embodiments.
Materials and methods
The one-tenth king crab of Tachypleus tridentatus is purchased from seafood wholesale market, Zhanjiang; Rounded tail king crab becomes king crab to pick up from the East Sea, Zhanjiang islander to pacify sea area.
Instrument
Japan Shimadzu EL-120HA electronic balance (sensibility reciprocal 0.01g), Japan Shimadzu AY120 electronic balance (sensibility reciprocal 0.0001g), Tianjin Jin Teng experimental facilities company limited GM-0.33 II vacuum diaphragm suction filtration machine, Keda Innovation Co., Ltd KDC-160H height refrigerated centrifuge.Japan's Shimadzu LC-20AT high performance liquid chromatograph, Japanese Shimadzu SPD-20A is visible-UV-detector, SunFire
tMc18 chromatographic column (4.6mm × 150mm), Italian Hana HI8424 pH meter, Beijing Pu Xi all purpose instrument company limited TU1901 ultraviolet-visible spectrophotometer.
Reagent
Tetraodotoxin, wins biological company limited through Beijing bit, and from the import of ENZO company of the U.S., purity is greater than 99%.Sodium heptanesulfonate, glacial acetic acid, NaH
2pO
4and Na
2hPO
4be analysis pure, methyl alcohol is chromatographically pure, purchased from Zhanjiang Linda company.Ultrapure water is made by oneself.
Method
The preparation of Standard Reserving Solution
Precision weighing TTX 1.0mg is placed in 5ml centrifuge tube, draws the acetic acid (0.2%) of 1.0mL, namely obtain the TTX stock solution of 1.0mg/mL, be placed in refrigerator and preserve after making it dissolve with 1ml liquid-transfering gun.
The pre-treatment of sample
The tetraodotoxin extraction process carrying out improveing with reference to the method for Zhou Xin etc. carries out sample preparation: get Tachypleus tridentatus or the rounded tail king crab 10g that organizes to be measured, add acetic acid (pH=3.5) homogenate in glass homogenizer of 10ml 0.1% after shredding.After abundant homogenate, sample is placed in flask, adds 50ml acetic acid (0.1%), then boiling water bath 10-20 minute in water-bath, heat while stirring, rotating speed controls at 150-200r/min.With 0.45 μm of filter membrane vacuum filtration after cooling, filtrate is put into evaporating dish, the water-bath of 60-70 DEG C carries out evaporation and concentration, until 10ml, then filter and be dissolved to 10ml.Concentrated filtrate 10ml is placed in centrifuge tube, through the speed of 10000r/min, centrifugal 15 minutes.Get supernatant after the filtering with microporous membrane of 0.22 μm, put into clean centrifuge tube, as liquid chromatography liquid to be measured.Tachypleus tridentatus, rounded tail king crab are organized and are carried out 2 sub-sampling extractings altogether.
Chromatographic condition
Acetic acid mobile phase chromatographic condition Stationary liquid: SunFire
tMc18 chromatographic column, diameter 4.6mm × length 150mm, sample size is 20 μ; Mobile phase: 0.2% acetic acid; Flow velocity: 0.8mL/min; UV detect wavelength: 210nm; Column temperature: 25 DEG C; Each sampling volume is 60 μ L; PH is 3.0;
Phosphoric acid mobile phase chromatographic condition Stationary liquid: SunFire
tMc18 chromatographic column, is of a size of 4.6mm × 150mm, and sample size is 20 μ l; Mobile phase: 0.05mol/L NaH
2pO
4-0.05mol/L Na
2hPO
4, pH=6.8, sodium heptanesulfonate ion pair 5mM; Flow velocity: 1mL/min; UV detect wavelength is 200nm; Column temperature: 25 DEG C; Each sampling volume is 60 μ L; PH is 6.8.
Results and analysis
The determination of mobile phase
First select the acetic acid (pH=3.0) of 0.2% to do mobile phase in an experiment, carry out liquid-phase chromatographic analysis to tetraodotoxin standard sample, result is as Fig. 1.
As seen from Figure 1, when taking acetic acid as mobile phase, standard sample chromatogram is undesirable.
After select 0.05mol/LNaH
2pO
4-0.05mol/L Na
2hPO
4(pH=6.8) and sodium heptanesulfonate ion pair 5mM do mobile phase, result is as Fig. 2.
As seen from Figure 2, standard sample baseline straightening, can demonstrate 2 peaks well, chromatogram is more satisfactory.
In order to determine whether mobile phase has interference to the peak in the standard sample of Fig. 2, and carry out liquid-phase chromatographic analysis to phosphoric acid mobile phase, result is as Fig. 3.
As seen from Figure 3, phosphoric acid mobile phase is quite little to the interference of liquid chromatography, 2 peaks in Fig. 2 Plays sample, should not be that the interference of phosphoric acid mobile phase causes.
Because tetraodotoxin standard sample 0.2% acetate dissolution, so carry out stratographic analysis to 0.2% acetic acid, result is as Fig. 4.
As seen from Figure 4, except having a peak between 2.30, other position baseline straightening, can not cause interference to dissolved matter.This also illustrates, is to flow to be on good terms to be separated (Fig. 2) by the tetraodotoxin of acetate dissolution preferably with phosphoric acid.
The determination of determined wavelength
Using mobile phase as titer, carry out all band scanning to the flowing liquid that mixes, result is as Fig. 5.
As seen from Figure 5, phosphate buffer mobile phase does not almost have absorption peak under ultraviolet spectrophotometer, can not produce interference to liquid chromatography results.
Because the tetraodotoxin acetum of 0.2% dissolves, Tachypleus tridentatus and rounded tail king crab are organized all to carry with dilute acetic acid solution and take out, so the acetum to 0.2% dissolves and carries out all band scanning, the results are shown in Figure 6.
As seen from Figure 6,0.2% acetic acid maximum absorption peak is 210.00nm.
Because tetraodotoxin standard sample 0.2% acetate dissolution, so, then all band scanning is carried out to tetraodotoxin standard solution, the results are shown in Figure 7.
As seen from Figure 7, the tetraodotoxin standard sample maximum absorption peak of 0.2% acetate dissolution is 208.00nm.
Again with the muscle samples solution of Tachypleus tridentatus and rounded tail king crab for representative, all band scanning is carried out to the sample of extracting, the results are shown in Figure 8,9.
As seen from Figure 8, Tachypleus tridentatus muscle extracting sample maximum absorption peak is 230.00nm.As seen from Figure 9, rounded tail king crab muscle extracting sample maximum absorption peak is 212.00nm.
The ultraviolet absorption peak of comprehensive analysis acetic acid, tetraodotoxin and Tachypleus tridentatus and rounded tail king crab sample is known, and 194-214nm should be the desired wavelength interval detecting tetraodotoxin.So, 194 selected respectively to standard items, 198,200,204,5 kinds of wavelength such as 214nm carry out chromatogram detection, the results are shown in Figure 10.
As seen from Figure 10, the absorption peak of 198nm (Figure 10 b) and 200nm (Figure 10 c) TTX is the highest, and both are more or less the same.It is ideal that this illustrates that 198nm, 200nm ultraviolet wavelength detects tetraodotoxin.200nm is chosen to be the determined wavelength of this experiment subsequent experimental.
The determination of tetraodotoxin peak position
After determining mobile phase and determined wavelength, the peak position of carrying out standard sample tetraodotoxin is determined.Under phosphoric acid mobile phase, 200nm ultraviolet wavelength condition, carry out the stratographic analysis of 0.2% acetic acid and tetraodotoxin standard sample, the results are shown in Figure 11.
From Figure 11, in the 0.2% acetic acid chromatogram of a, only there is an absorption peak in 2.3.0 position; And in the chromatogram of the tetraodotoxin standard sample of 0.2% acetate dissolution of b, except having an absorption peak in 2.30 positions, near 8.3, have another obvious absorption peak.This illustrates, first peak in Fig. 2 standard sample chromatogram should be solvent acetic acid, and second peak is tetraodotoxin.
Tachypleus tridentatus compares with rounded tail king crab tetraodotoxin
The comparison of Tachypleus tridentatus and rounded tail king crab muscle tetraodotoxin
Tetraodotoxin standard sample, Tachypleus tridentatus muscle, rounded tail king crab muscle chromatogram are compared, result is as Figure 12.
From Figure 12, near 8.5, (Figure 12 a) has an obvious absorption peak to rounded tail king crab muscle (Figure 12 c), and Tachypleus tridentatus muscle (Figure 12 b) this peak is not obvious with tetraodotoxin standard sample.This illustrates, containing tetraodotoxin in rounded tail king crab muscle, and hardly containing tetraodotoxin in Tachypleus tridentatus muscle.
The comparison of Tachypleus tridentatus and rounded tail king crab yellow connective tissue tetraodotoxin
Tetraodotoxin standard sample, Tachypleus tridentatus yellow connective tissue, rounded tail king crab yellow connective tissue chromatogram are compared, result is as Figure 13.
From Figure 13, near 8.5, (Figure 13 a) has an obvious absorption peak to rounded tail king crab yellow connective tissue (Figure 13 c), and Tachypleus tridentatus yellow connective tissue (Figure 13 b) this peak is not obvious with tetraodotoxin standard sample.This illustrates, containing tetraodotoxin in rounded tail king crab yellow connective tissue, and hardly containing tetraodotoxin in Tachypleus tridentatus yellow connective tissue.
Compare the size of tetraodotoxin amount in Tachypleus tridentatus and rounded tail king crab body
The height of tetraodotoxin standard sample, Tachypleus tridentatus muscle, rounded tail king crab muscle chromatogram chromatographic peak is compared, the results are shown in Figure 14.
From Figure 14, near 8.5, the absorption peak (Figure 14 c) of rounded tail king crab muscle is the highest, and the absorption peak of standard sample (Figure 14 a) secondly, and Tachypleus tridentatus muscle (Figure 14 b) is not obvious at the absorption peak of this point.This illustrates, many containing tetraodotoxin in rounded tail king crab muscle, and hardly containing tetraodotoxin in Tachypleus tridentatus muscle.
Tetraodotoxin standard sample, Tachypleus tridentatus yellow connective tissue, rounded tail king crab yellow connective tissue chromatogram are carried out the comparison of chromatographic peak height, result is as Figure 15.
From Figure 15, near 8.5, the absorption peak (Figure 15 c) of rounded tail king crab yellow connective tissue is the highest, and the absorption peak of standard sample (Figure 15 a) secondly, and Chinese yellow connective tissue (Figure 15 b) is not obvious at the absorption peak of this point.This illustrates, many containing tetraodotoxin in rounded tail king crab yellow connective tissue, and hardly containing tetraodotoxin in Tachypleus tridentatus yellow connective tissue.
The above, be only best mode for carrying out the invention, is anyly familiar with those skilled in the art in the technical scope that the present invention discloses, and the simple change of the technical scheme that can obtain apparently or equivalence are replaced and all fallen within the scope of protection of the present invention.
Claims (1)
1. a detection method for the tetraodotoxin in Tachypleus tridentatus and rounded tail king crab body, is characterized in that: comprise the following steps:
(1) preparation of Standard Reserving Solution
Precision weighing TTX 1.0mg is placed in 5ml centrifuge tube, and the concentration drawing 1.0mL with 1ml liquid-transfering gun is 0.2% acetic acid, namely obtains the TTX stock solution of 1.0mg/mL, be placed in refrigerator and preserve after making it dissolve;
(2) pre-treatment of sample
Get Tachypleus tridentatus or rounded tail king crab is to be measured organizes 10g, acetic acid homogenate in glass homogenizer that 10ml pH=3.5 concentration is 0.1% is added after shredding, after abundant homogenate, sample is placed in flask, add the acetic acid that 50ml concentration is 0.1%, then boiling water bath 10-20 minute in water-bath, heat while stirring, rotating speed controls at 150-200r/min, with 0.45 μm of filter membrane vacuum filtration after cooling, filtrate is put into evaporating dish, the water-bath of 60-70 DEG C carries out evaporation and concentration, until 10ml, then filter and be dissolved to 10ml, concentrated filtrate 10ml is placed in centrifuge tube, through the speed of 10000r/min, centrifugal 15 minutes, get supernatant after the filtering with microporous membrane of 0.22 μm, put into clean centrifuge tube, as liquid chromatography liquid to be measured,
(3) liquid chromatography test
Acetic acid mobile phase chromatographic condition Stationary liquid: SunFire
tMc18 chromatographic column, diameter 4.6mm × length 150mm, sample size is 20 μ; Mobile phase: 0.2% acetic acid; Flow velocity: 0.8mL/min; UV detect wavelength: 210nm; Column temperature: 25 DEG C; Each sampling volume is 60 μ L; PH is 3.0;
Phosphoric acid mobile phase chromatographic condition Stationary liquid: SunFire
tMc18 chromatographic column, is of a size of 4.6mm × 150mm, and sample size is 20 μ l; Mobile phase: 0.05mol/L NaH
2pO
4-0.05mol/L Na
2hPO
4, pH=6.8, sodium heptanesulfonate ion pair 5mM; Flow velocity: 1mL/min; UV detect wavelength is 200nm; Column temperature: 25 DEG C; Each sampling volume is 60 μ L; PH is 6.8.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106596790A (en) * | 2016-12-28 | 2017-04-26 | 山东出入境检验检疫局检验检疫技术中心 | Method for rapidly detecting content of tetrodotoxin in fish flesh through liquid chromatography-mass spectrometry |
| CN107957473A (en) * | 2018-01-12 | 2018-04-24 | 杨洁 | A kind of quick determination method of tetraodotoxin |
| CN108008134A (en) * | 2017-11-30 | 2018-05-08 | 上海海洋大学 | A kind of tetraodotoxin Rapid detection test strip and its application |
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| CN104391061A (en) * | 2014-10-31 | 2015-03-04 | 浙江省海洋水产研究所 | Method for determining tetrodotoxin in marine organisms by utilizing immunoaffinity column purification-liquid phase chromatography-tandem mass spectrometry |
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| CN104391061A (en) * | 2014-10-31 | 2015-03-04 | 浙江省海洋水产研究所 | Method for determining tetrodotoxin in marine organisms by utilizing immunoaffinity column purification-liquid phase chromatography-tandem mass spectrometry |
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| ATTAYA KUNGSUWAN等: "Tetrodotoxin in the Horseshoe Crab Carcinoscorpius rotundicauda Inhabiting Thailand", 《NIPPONSUISANGAKKAISHI》, vol. 53, no. 2, 31 December 1987 (1987-12-31), pages 261 - 266 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106596790A (en) * | 2016-12-28 | 2017-04-26 | 山东出入境检验检疫局检验检疫技术中心 | Method for rapidly detecting content of tetrodotoxin in fish flesh through liquid chromatography-mass spectrometry |
| CN108008134A (en) * | 2017-11-30 | 2018-05-08 | 上海海洋大学 | A kind of tetraodotoxin Rapid detection test strip and its application |
| CN107957473A (en) * | 2018-01-12 | 2018-04-24 | 杨洁 | A kind of quick determination method of tetraodotoxin |
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