JPH09133669A - Method for determining paralytic shell poison - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a high-precision liquid chromatographic analysis method by which all paralytic shell poisons can be highly specifically determined with high sensitivity by improving the fluorescence generating reactions of gonyautoxins and saxitoxins having a hydroxyl group at the first unit for determining paralytic shell poisons by using a post-column derivative detecting method. SOLUTION: After a paralytic shell poison is separated through a column 4, a reagent for causing a fluorescence generating reaction is supplied to the elute from a first reagent feeding pump 5 so that the concentration of an oxidizing agent (iodine peroxide, etc.) after mixture can become >=1mM and the pH of the mixed solution after mixture can become >=8 and a reactor 7 is maintained at 90-110 deg.C so that the reaction can take place. Then an acidic solution is fed to the reacted solution from a second reagent feeding pump 8 so as to improve the intensity of fluorescence and the intensity of fluorescence is measured with a fluorescence detector 10.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for analyzing paralytic shellfish toxins such as goniotoxins or saxitoxins.

[0002]

2. Description of the Related Art Paralytic shellfish poison is a general term for compounds showing neurotoxicity such as goniotoxins and saxitoxins and is produced by some dinoflagellates and poisons bivalves that feed on these algae. Eating the bivalve causes food poisoning with a high mortality rate, which is a major social problem in fisheries and food hygiene.

Conventionally, the following methods are known for the quantitative analysis of paralytic shellfish poison.

Mouse toxicity test method: A biological test method based on the life and death of mice, which is an official method.

High Performance Liquid Chromatography Method: A sample containing paralytic shellfish poison was subjected to high performance liquid chromatography,
The paralytic shellfish poison to be analyzed is separated, and then the eluate from the column is mixed with a buffer solution containing orthoperiodic acid, which is an oxidizing agent, after the pH and orthoperiodic acid concentration are mixed,
Mix to pH 9 and 3.5 mM, respectively, and react with paralytic shellfish toxin at a temperature of 65 ° C, then mix with an aqueous acid solution and use the fluorometer to measure the fluorogenic substance generated by the reaction. How to measure and analyze.

[0006]

However, in the method (mouse toxicity test method) of the above-mentioned conventional techniques, there are problems of analytical accuracy and sensitivity due to individual differences of mice and test technology, and the breeding management of mice. Is complicated, and these are obstacles in the examination.

In the above-mentioned conventional method (high-performance liquid chromatograph method), the operation is simple and the accuracy is high, but the sensitivity varies depending on the type of paralytic shellfish poisoning.
Goniotoxins having a hydroxyl group at position (GTX-1, GTX-4
However, there was a problem that high sensitivity could not be expected.

In order to solve the above-mentioned problems of the prior art, the present invention has further improved the method, which has a feature that the method has a simple operation and a high precision, and has increased sensitivity and specificity. It is an object to provide a method for analyzing paralytic shellfish poison.

[0009]

In order to achieve the above object, the present invention provides a sample containing a paralytic shellfish poison by high performance liquid chromatography to separate the paralytic shellfish poison to be analyzed by a column. Separation step, (b) a mixing step in which the eluate from the column is mixed with an oxidant, (c) a reaction step in which the mixed solution is heated to react paralytic shellfish poison, (d) an aqueous acid solution or a buffer solution is then added to the reaction solution. In the method for the analysis of paralytic shellfish toxins, in which the fluorescent substance generated by the reaction is measured using a fluorometer, the eluent from the column is mixed in the mixing process. A buffer solution containing an oxidizing agent to be mixed,
The concentration of the oxidizing agent after mixing is 1 mM or less, and the concentration of the oxidizing agent is such that the pH value of the mixed solution after mixing is 8 or more, and a buffer solution having such a pH buffering capacity, The reaction step is characterized in that the mixed solution is heated under an arbitrary temperature condition between 90 ° C. and 110 ° C. so as to react with the paralytic shellfish poison.

In the present invention, the oxidizing agent is orthoperiodic acid H ₅ IO ₆ , metaperiodic acid HIO ₄ or a salt of periodic acid (a hydrate of periodate or a salt of polyacid,
Ortho acid salt, dimesoic acid salt, meso acid salt, diortho acid salt, and meta acid salt) or lead (IV) acetate Pb (CH ₃ CO ₃
O) refers to ₄ . In the present invention, the oxidant concentration at which the concentration of the oxidant after mixing is 1 mM or less is
For example, if the column eluent and the oxidant-containing reaction solution are mixed in equal amounts, the oxidant concentration in the oxidant-containing reaction solution should be 2 mM.
When the periodate salt is used as the oxidant, it means the concentration converted into the periodate amount.
Further, in the present invention, it is desirable to use a borate buffer solution, a carbonate buffer solution, or the like as the buffer solution having a pH buffering ability such that the pH value of the mixed solution after mixing becomes 8 or more.

The above-described constitution of the present invention is similar to the above-mentioned prior art method in that the paralytic shellfish toxin is separated by high performance liquid chromatography and the postcolumn derivatization detection for converting the paralytic shellfish toxin into a fluorescent compound is performed. Become. However, the conventional post-column derivatization detection method has a problem that the sensitivity to goniotoxins having a hydroxyl group at the 1-position is low, and therefore the inventors of the present invention have solved it by solving the problems such as reagent concentration, reaction time and reaction temperature. We conducted basic experiments such as. As a result, 1
Goniotoxins having a hydroxyl group at the position show fluorescence even if they are reacted with basic acid at high temperature without the action of periodic acid, and the action concentration of periodic acid is low. I found that the sensitivity was improved. In addition, 1
For goniotoxins that do not have a hydroxyl group at the position, it was necessary to act periodic acid in order to lead to a fluorescent compound, but lead tetraacetate also has similar effects in addition to periodic acid. I found it. These facts are not publicly known, and can be found for the first time by a series of studies by the inventors. The present invention applies the results of these studies to post-column derivatization detection to determine the optimal reaction conditions.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS According to the configuration of the present invention described above,
A sample containing paralytic shellfish poison is subjected to high performance liquid chromatography to separate the paralytic shellfish poison to be analyzed, and then the eluate from the column is mixed with a buffer solution containing an oxidizing agent. Oxidizer concentration and pH are 1 mM or less and pH 8 or more, respectively, and the paralytic shellfish poison is allowed to react at any temperature between 90 ° C and 110 ° C, and then acid aqueous solution or buffer is added. After mixing the solutions to pH 5 or less, measure the fluorogenic substances generated by the reaction using a fluorimeter and analyze. By this post-column derivatization reaction, goniotoxins, including goniotoxins having a hydroxyl group at the 1-position, are converted into substances exhibiting strong fluorescence, which can be easily detected and measured by a fluorometric method.

Further, the fluorescent substance forming reaction according to the present invention can be used for the purpose of analyzing paralytic shellfish poisoning without preliminarily applying it to a high performance liquid chromatograph. For example,
Goniotoxins having a hydroxyl group at the 1-position are
There is a method of analyzing goniotoxins by reacting in the absence of an oxidant and at a temperature condition of 90 ° C to 110 ° C, and then measuring fluorescence under acidic conditions.

[0014]

The present invention will be described in more detail with reference to the following examples. In addition, this invention is not limited to a following example.

First, the analyzer will be described.

FIG. 1 is a flow chart of a high performance liquid chromatograph used in the present invention. In FIG. 1, 1 is a mobile phase liquid feeding pump, 2 is a sample introducer, 3 is a column thermostatic bath, 4 is a column, 5 is a first reagent liquid feeding pump, 6 is a reaction thermostatic bath, 7 is a reactor, and 8 is The second reagent delivery pump, 9 is a mixer, 10 is a fluorescence detector, and 11 is a data processor.

Next, the operation of one embodiment of the present invention using the analyzer shown in FIG. 1 will be described.

First, the mobile phase is sent by the solution sending pump 1, a sample containing paralytic shellfish poison is introduced from the sample introducer 2 using a microsyringe, etc., and the paralytic shellfish poison to be analyzed is stored in the column. Separation is performed in the column 4 kept at a constant temperature by the constant temperature bath 3. The reaction reagent containing the oxidant was fed by the first reagent feed pump 5 to the mobile phase which was continuously eluted from the column, and mixed, and the mixture was mixed in the reaction thermostat 6 at 90 ° C to
It is sent to the reactor 7 placed at an arbitrary temperature for 0 ° C. Here, the paralytic shellfish poison is led to a fluorescent compound. Then the second
An acid aqueous solution or a buffer solution is sent by the reagent sending pump 8 and mixed with the reaction solution by the mixer 9, so that the pH value of the mixed solution becomes 5 or less. By this treatment, the fluorescence intensity of the fluorescent compound is increased and then the fluorescent compound is detected by the fluorescence detector 1.
0 is measured and this is processed by the data processor 11.

The column 4 includes, for example, a column for reverse phase chromatography “STR ODS-II” (inner diameter 4 mm × length 150 mm).
mm) is used. However, column 4 may be of any type as long as it can separate the paralytic shellfish to be analyzed.

As the mobile phase, for example, 8 mM sodium phosphate buffer <pH 7.0> containing 4 mM sodium 1-heptanesulfonate is used. However, there is no restriction on the type and concentration of the mobile phase as long as it can separate the paralytic shellfish to be analyzed.

As a reaction reagent for converting paralytic shellfish poison to a fluorescent compound, for example, boric acid (sodium) buffer solution <pH9.2> containing periodic acid is used and mixed with the column eluate. Incubate at 90 ℃ -110 ℃ for 10-60 seconds. Periodic acid is preferably used at a concentration such that the concentration after mixing with the column eluate is 1 mM or less. Further, the buffer solution may be of any type and concentration as long as the pH after mixing with the column eluate is 8 or more. Besides the borate buffer solution, a carbonate buffer solution or the like may be used. It can also be used.

As the reagent for acidifying the reaction solution, for example, an aqueous phosphoric acid solution is used and mixed with the reaction solution for 5 to 20 seconds. However, the reagent may be of any type and concentration as long as it can bring the pH of the reaction solution to 5 or less.In addition to the phosphoric acid aqueous solution, an acetic acid aqueous solution, a phosphate buffer solution, an acetate buffer solution, A liquid or the like can also be used.

The detection wavelength is excitation wavelength 330 nm, fluorescence wavelength 39
The wavelength is preferably 0 nm, but any wavelength may be used as long as it can detect the fluorescent compound produced by the reaction.

Next, a specific analysis example will be shown.

(1) Separation Conditions Column: STR ODS-II (internal diameter 4 mm × length 150 mm) Mobile phase: 8 mM phosphate (sodium) buffer solution containing 4 mM sodium 1-heptanesulfonate <pH 7.0> Flow rate: 0.6 mL / min Temperature: 40 ° C (3) Detection conditions [Fluorescent derivatization reagent] 80 mM boric acid (sodium) buffer containing 1 mM periodic acid <pH9.2> Flow rate: 0.3 mL / min [acidic Reagent] 120 mM phosphoric acid aqueous solution Flow rate: 0.3 mL / min Reaction constant temperature bath temperature: 100 ° C Detection: Fluorescence intensity Ex: 330nm, Em: 390nm (4) Analytical results Figure 2 shows the analytical results. FIG. 2 is a chromatogram of a standard sample of goniotoxins obtained by the above-mentioned specific analysis. It is an analysis result obtained by diluting a standard sample solution of goniotoxins 4 times and introducing 20 μL of the diluted solution into the column. is there. 2, GTX-1, GTX-2, GTX
-3 and GTX-4 represent goniotoxin 1, goniotoxin 2, goniotoxin 3 and goniotoxin 4, respectively. As a result, four paralytic shellfish poison components in the test sample were separated and detected. Also, goniotoxin 1 and goniotoxin 4 having a hydroxyl group at the 1-position have been detected with high sensitivity.

[0026]

EFFECTS OF THE INVENTION According to the method for analyzing paralytic shellfish toxins of the present invention, paralytic shellfish poisons containing goniotoxins having a hydroxyl group at the 1-position can be analyzed with high sensitivity and specificity regardless of the type. You can In addition, the features of the conventional high-performance liquid chromatogram method, which are easy to operate and highly accurate, are not impaired.Furthermore, fluorescent derivatization that does not contain an oxidizing agent can save analysis cost. Therefore, it is possible to save the labor and time for preparing the reaction solution.

[Brief description of the drawings]

FIG. 1 is a diagram showing an analyzer (high-performance liquid chromatograph) used in one embodiment of the present invention.

FIG. 2 is a chromatogram showing the analysis results of goniotoxins, which is an example of the present invention.

[Explanation of symbols]

 1: Liquid delivery pump 2: Sample introduction device 3: Column constant temperature bath 4: Column 5: First reagent liquid feed pump 6: Reaction constant temperature bath 7: Reactor 8: Second reagent liquid feed pump 9: Mixer 10: Fluorescence Detector 11: Data processor

Claims (1)

[Claims] 1. A separation process in which a sample containing paralytic shellfish poison is subjected to high performance liquid chromatography to separate the paralytic shellfish poison to be analyzed by a column, and (2) an eluent from the column is added with an oxidizing agent. Mixing process, c) a reaction process in which the mixed liquid is heated to react with paralytic shellfish toxin, d. Next, the reaction liquid is mixed with an aqueous acid solution or buffer solution to a pH of 5 or less, and fluorescence generated by the reaction In the method for analysis of paralytic shellfish toxin, which comprises a fluorescence measurement step of measuring a volatile substance using a fluorometer, a buffer solution containing an oxidant mixed with the eluate from the column in the mixing step is oxidized after mixing. The concentration of the agent is 1 mM or less, the concentration of the oxidizing agent is such that the pH value of the mixed solution after mixing is 8 or more, and the buffer solution has such a pH buffering capacity. 90 ° C for the mixture The analysis method of paralytic shellfish poisons, characterized in that the optional heating at a temperature reaction step of reacting a paralytic shellfish poison between 110 ° C..