JP4433758B2 - Pesticide residue measurement method - Google Patents

Description

  The present invention relates to a method for measuring residual pesticides in agricultural products. Specifically, quantitative analysis of cholinesterase-inhibiting pesticides is carried out easily and quickly without using a test pesticide standard product.

  Organophosphorus pesticides and carbamate pesticides have been widely used as pesticides, fungicides, and herbicides for a long time because they are more easily decomposed in nature than organochlorine pesticides. However, among these, organophosphorus insecticides, carbamate insecticides, and their metabolites are typical enzyme poisons that cause cholinesterase activity inhibition. When accumulated in animals, they significantly damage the nervous system. From the viewpoint of risk management or environmental pollution management, opportunities for testing these pesticides have been increasing in recent years to ensure safety.

  Conventionally, the above insecticides and the like are detected using a precision analyzer such as a gas chromatograph or a gas chromatograph mass spectrometer. However, the detection method using such an analytical device can comprehensively and accurately detect the components contained in the sample, but the measurement device is expensive, the measurement operation is complicated and requires skill, take time. In addition, there is a problem that agrochemicals must be used for instrument calibration for each measurement.

  On the other hand, as mentioned above, organophosphorus insecticides and carbamate insecticides are typical cholinesterase inhibitors, so it is possible to detect the indirect activity of the enzyme activity (hydrolysis rate) of cholinesterase due to the coexistence of agricultural chemicals. In particular, there is a simple pesticide inspection method for calculating the coexistence amount of these pesticides (for example, see Non-Patent Document 1 and Non-Patent Document 2).

Masanori Onishi, “Biochemical Experimental Method 21, Introduction to Enzyme Reaction Kinetics Experiment” Society Publishing Center, (1991) Hirokoshi Horikoshi et al., “Enzyme Science and Engineering” Kodansha Scientific, (1992)

Cholinesterase, a Michaelis-Mentens enzyme, can be used in a free state or in an immobilized state. Michaelis-Mentens kinetic calculation is easier when the former, that is, when cholinesterase coexists with a substrate that shows specificity for cholinesterase (hereinafter also simply referred to as substrate) in the measurement solution, The rate (v _n ) of the enzyme reaction in the absence of coexistence can be obtained from equation (1).

On the other hand, the inhibition of cholinesterase activity by organophosphorus insecticides and carbamate insecticides, in the case of organophosphorus agents, phosphorylates the hydroxyl group of the ester bond site serine of cholinesterase, and in the case of carbamate agents, this is carbamoylated and inactivated. This results from the formation of a complex enzyme-inhibitor complex (EI) and a decrease in enzyme catalytic ability. In addition, since the pesticide exhibits a competitive inhibition characteristic that does not bind to the enzyme-substrate complex, the rate (v _i ) of the enzyme reaction in the presence of the pesticide can be obtained from the formula (2). (Here, Ki representing the dissociation constant of the enzyme-inhibitor complex (EI) is called an inhibition constant, and the value varies depending on the inhibitor under the same enzyme reaction conditions.)

As can be seen by comparing the formulas (1) and (2), competitive inhibition of the Michaelis-Mentens type enzyme affects the reaction rate as a change in the Michaelis constant (Km) due to the coexistence of the inhibitor. In other words, if the enzyme concentration and substrate concentration added to the reaction system are the same with and without the presence of pesticides, v _i /
v Ratio of enzyme activity represented by _n : Reciprocal number of RA (Relative activity): RA ⁻¹ can be expressed by equation (3), and the concentration of an unknown pesticide can be expressed by equation (4) developed from equation (3). [I] can be known.

However, the cholinesterase activity in the extraction solution, which is the molecular term of RA represented by v _i , cannot obtain sufficient accuracy of activity measurement due to the influence of components other than agricultural chemicals contained in the extraction solution. The main factor is that cholinesterase is non-specifically inactivated or activated by components other than agricultural chemicals contained in the measurement solution (in many cases, it is inactivated, and apparently the concentration of agricultural chemicals is high. Is calculated).

  For the reasons described above, the comparison value of cholinesterase activity in the sample extraction solution and the ideal cholinesterase activity in the absence of pesticides: RA is used to calculate the pesticide concentration by the dynamic treatment of Michaelis-Mentens. It was not easy to do.

  In view of the above problems, the present invention is a method for measuring an agrochemical that exhibits cholinesterase activity inhibition properties contained in crops and the like, in a measurement solution in which components are extracted from crops by a relatively easy pretreatment method. It is an object of the present invention to provide a simple calculation method for quantitatively treating the output signal obtained in step 1 as the concentration of a cholinesterase-inhibiting pesticide.

  In order to solve the above problems, the present invention has taken the following measures. First, a crop containing no cholinesterase-inhibiting pesticide was prepared, and a measurement solution was prepared by extracting and purifying the crop components by a relatively easy method compared to the official inspection method and the like. In this measurement solution, cholinesterase activity was measured and analyzed by the Lineweaver-Burk plot method, and both the maximum reaction rate and the Michaelis constant that determine the catalytic properties of cholinesterase due to the influence of components other than agricultural chemicals derived from crops were found. I found it to change. The present inventors also found that the maximum reaction rate and the rate of change of the Michaelis constant are unique to the belonging crop.

  Furthermore, the present inventors have provided a database for correcting factors that vary depending on the belonging crop, and a pesticide to be tested for a measurement solution obtained by purifying and extracting agrochemical components from the crop by a relatively simple cleanup process. A database for correcting factors that change every time, a database on cholinesterase inhibition ability for each test pesticide, and a database on the catalytic properties of cholinesterase, measurement results of cholinesterase activity measurement, and in an ideal solution From the comparison value with the measurement result, it was found that the quantitative calculation of the agrochemical component based on the dynamic treatment of Michaelis Mentens can be performed.

That is, the present invention has the following configuration.
(1) First, the cholinesterase activity is measured in the measurement solution in which the extracted components of the crop to be inspected are dissolved and in the ideal solution, and the degree of change in cholinesterase activity due to the test pesticide or components other than the test pesticide is measured. Relative activity (RA) is determined from the activity comparison in each measurement. Preliminarily measure the activity determined by the ratio of the maximum reaction rate and Michaelis constant of cholinesterase in the crop extract solution containing no pesticide and in the ideal solution, and from the analysis results by Lineweaver-Burk plot method, The ratio of the maximum reaction rate in the solution is determined as the first correction coefficient: α and the second correction coefficient: β determined by the ratio of the Michaelis constant in each solution at that time. Next, by substituting the constant (inhibition constant: Ki) indicating the cholinesterase inhibitory ability of the test pesticide and the substrate concentration in the reaction solution into the calculation formula based on the reaction rate equation of Michaelis-Mentens-type enzyme antagonist inhibition The concentration of the pesticide contained in the measurement solution is temporarily calculated. Further, the third correction coefficient related to the recovery rate of the test pesticide from the crop crop to the measurement solution: γ and the dilution factor from the crop crop to the measurement solution are used for the inspection. Calculate the concentration of cholinesterase-inhibiting pesticides contained in the crop.
(2) Strictly speaking, the cholinesterase is acetylcholinesterase or butyrylcholinesterase, and the origin of the enzyme is not particularly limited, but preferably an eel-derived acetylcholinesterase is preferably used.
(3) Strictly speaking, the substance to be tested is an N-methyl carbamate insecticide, a phosphate-type organophosphorus insecticide, a thiono-form organophosphorus insecticide that has a high inhibitory ability against acetylcholinesterase or butyrylcholinesterase. And a cholinesterase-inhibiting pesticide which is an oxidation product of a dithio-type organophosphorus insecticide.
(4) The method for extracting and purifying the cholinesterase-inhibiting pesticide from agricultural crops is such that after extraction with an organic solvent, filtration and separation of large contaminants, normal layer chromatography, and reduced-pressure drying of the passing product are performed. A relatively simple pre-processing method may be provided.
(5) The ideal solution should contain only a buffer solution for adjusting the pH in cholinesterase activity measurement to the optimum pH for cholinesterase activity and an appropriate amount of electrolyte.
(6) The measurement solution may be prepared by dissolving the vacuum dried component obtained by the pretreatment method with the ideal solution.
(7) The first correction coefficient that is obtained from the analysis result by the Lineweaver-Burk plot method and is different for each belonging crop, related to the component that changes the maximum reaction rate of cholinesterase derived from the belonging crop contained in the measurement solution: α And it is better to build a database of α created for each belonging crop.
(8) Regarding the components other than the test pesticide that change the Michaelis constant of the cholinesterase derived from the assigned crop contained in the measurement solution, the second second which is obtained from the analysis result by the Lineweaver-Burk plot method and is different for each assigned crop It is better to construct a database of correction coefficient: β and β created for each belonging crop.
(9) A constant indicating the cholinesterase inhibitory ability of the test pesticide obtained from the analysis result by the Dixon plots method in the ideal solution to which the pesticide is added and measuring the cholinesterase activity by the above method: It is better to build the Ki database created in
(10) Applied to correct the concentration of the pesticide in the measurement solution temporarily calculated to the concentration in the original body, and from the crop to be inspected by the pretreatment method to the measurement solution It is preferable to construct a third correction coefficient related to the recovery rate of the test pesticide until it is collected: γ and a database of γ created for each test pesticide.

  As described above, according to the present invention, a measurement pretreatment method that can be performed easily and quickly as compared with an official inspection method or the like is performed, and the influence of factors that vary depending on the test pesticide and the belonging crop is removed. It is possible to easily calculate the concentration and quantitative analysis of the cholinesterase-inhibiting pesticide by creating a database of three types of correction coefficients, a constant relating to the cholinesterase-inhibiting ability for each test pesticide, and a constant relating to the catalytic ability of cholinesterase Become. In addition, according to the present invention, it is a simple and excellent measurement method that can overcome the conventional problem that a test pesticide must be used for instrument calibration.

  Hereinafter, embodiments of pesticide measurement according to the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a flow diagram showing the steps from extracting and purifying the test pesticide component from a crop to be inspected for carrying out pesticide measurement according to the present invention to obtain a measurement solution. Step 1 is the first step of pulverizing or grinding the crop to increase the extraction rate and extraction efficiency of the test pesticide component from the crop, and step 2 is methanol or the like for the test pesticide component in the crop. Step 3 is a third step of shaking to dissolve and extract in the organic solvent, and Step 3 is a third step of filtering to remove large contaminants remaining in the organic solvent layer. Uses solid-phase adsorption to extract the crop extract components other than the test pesticide component mixed in the organic solvent layer after the third step, and collects only the test pesticide component in the organic solvent layer as much as possible. This is the fourth step to be performed. The solid-liquid separation method may be performed by normal layer column chromatography using magnesium silicate (trade name: Florisil) or the like as a filler. Step 5 is the fifth step in which the extracted components of the crop are collected as dry solids from the effluent collected in the fourth step using a rotary evaporator or the like. Step 6 is the dry solids collected in the fifth step. Is the sixth step in which the solution is dissolved in the ideal solution. By the above method, the measurement solution which extracted the test agrochemical component from the agricultural crop used as test object is prepared. In this example, per 1 ml of the measurement solution, 0.1 g of the extracted component of the attributed crop is included.

  Next, one method for measuring cholinesterase activity will be described, but this is an example, and other known methods may be used. The quickest means of measuring the hydrolysis rate of cholinesterase is to select acetylthiocholine or butyrylthiocholine as the substrate, and use the hydrolysis product thiocholine as a thiol-functional reagent: 5,5'-dithio-bis (2 -Nitrobenzoic acid) (DTNB) is converted into yellow TNB, and there is a method for detecting and measuring the change in absorbance over time. For details, see JP-A-2002-62265 and JP-A-2003-298.

The method for measuring the cholinesterase activity in the presence of the test pesticide is as follows. First, a cholinesterase inhibition reaction by the test pesticide is performed by adding a cholinesterase solution to the measurement solution and incubating for 30 minutes or more. Next, simultaneously with the addition of the substrate and the color former, measurement of change in absorbance with time is started. In this step, by adding a substrate to the measurement solution, a competitive reaction between inhibition by the test pesticide contained in the measurement solution and decomposition of the substrate starts at the cholinesterase catalytic active site, and the Since the competitive reaction reaches equilibrium, the rate of change in absorbance (time derivative of absorbance, ΔAbs. _Si ) is stable. In addition, since the degradation of the cholinesterase-specific substrate is slower than the color development reaction and is the rate-determining step in the entire reaction, the cholinesterase activity in the presence of the test pesticide (from the time differential value of the absorbance change ( v _Si ) can be calculated.

In the step for measuring cholinesterase activity, when a commercially available acetylcholinesterase is selected as the enzyme, it is optimal to select acetylthiocholine as the substrate to be added, and the final concentration is in the range of 0.1 to 0.3 mM. , Preferably 0.2
It is good to add so that it may become mM. Also, the aqueous solution of acetylthiocholine slowly hydrolyzes to form thiocholine even at room temperature and under irradiation, and naturally develops color when coexisting with the color former. Therefore, store it in a light-shielding container and store it in a cooling bath at 5 ° C or lower. Is preferred.

  On the other hand, in the step for measuring cholinesterase activity, when a commercially available butyrylcholinesterase product is selected as the enzyme, it is optimal to select butyrylthiocholine as the substrate to be added, and the final concentration is 0.1 to 0.3 mM. Range. Preferably, it is good to add so that it may become 0.2 mM. An aqueous solution of butyrylthiocholine also slowly hydrolyzes to form thiocholine at room temperature and under irradiation, and naturally develops color by coexistence with the color former. Therefore, place it in a light-shielding container and store it in a cooling bath at 5 ° C or lower. It is preferable to do.

In addition, when the ideal solution is initially placed in the measurement cell instead of the measurement solution, and measurement is performed in the same process as the above process, the measurement value that can be compared with the above-described measurement value: ΔAbs. _Si ( ΔAbs. _Rn ), that is, an output signal in response to cholinesterase activity (v _Rn ) not affected by the test pesticide. As described above, the relative activity value RA representing the degree of cholinesterase activity inhibition by the test pesticide is temporarily calculated by the two measurements.

  By the way, preparing a cucumber that does not contain the cholinesterase-inhibiting pesticide at all, in the measurement solution prepared by the pretreatment method, changing the concentration of the substrate to be added, and performing the measurement by the cholinesterase activity measurement method, the Lineweaver-Burk The result of analysis by the plot method is shown in Fig. 2- (a). On the other hand, the result of the same analysis in the ideal solution is shown in FIG. It was found that the former and the latter differed in both the maximum reaction rate that determines the catalytic properties of cholinesterase and the Michaelis constant.

Moreover, in order to correct the cholinesterase activity change in the attributed crop extract solution and the ideal solution in the absence of the pesticide, the ratio of the maximum reaction rate in each reaction condition is the ratio of α and Michaelis constant in equation (5). Was expressed as β in equation (6). The values of α and β were slightly different depending on the production area and harvest time, but as shown in Table 1, it was found that the values were almost the same for each belonging crop.

Further, using the ideal solution, while changing the cholinesterase-inhibiting pesticide concentration and the acetylthiocholine concentration, measurement was performed by the cholinesterase activity measurement method, and Dixon
Table 2 shows the inhibition constants Ki for various pesticides against the acetylcholinesterase obtained from the results of analysis by the plot method.

より、

従って

Next, an agrochemical concentration calculation formula using the α and β correction coefficients will be described. The reaction rate (v _Rn ) in an ideal solution in the absence of pesticides is expressed by equation (7) and responds to the absorbance change rate (ΔAbs. _Rn ).

Than,

Therefore

Α, β values, Km _Rn values for each belonging crop to be measured, constants indicating cholinesterase inhibitory ability that differ for each pesticide: Ki is stored in a database and assigned to the above formula (12). It is possible to calculate the concentration of pesticide by canceling the influence on the cholinesterase activity caused by components other than the pesticide contained therein, so that accurate measurement can be realized.

  Furthermore, after preparing various crops not containing the cholinesterase-inhibiting pesticide, adding various cholinesterase-inhibiting pesticides at known concentrations, preparing a measurement solution by the pretreatment method, and measuring by the cholinesterase activity measurement method The concentration of the agrochemical was calculated according to the equation (12). As an example of the relationship between the added pesticide concentration and the calculated pesticide concentration obtained by measurement, FIG. 3 shows the results when fenocarb is added to Hiroshima tomatoes that hardly contain the cholinesterase-inhibiting pesticide.

It was found that the combined slopes of all crops and pesticides were 1 or less, which was presumed to be due to the recovery rate in the pretreatment process up to the measurement. Further, it was found that the correlation coefficient (hereinafter referred to as correction coefficient: γ) is an eigenvalue for almost every agricultural chemical. Table 3 shows γ for the various agricultural chemicals.

From the above, the concentration in the measurement solution calculated by substituting RA determined by the pretreatment method and the cholinesterase activity measurement method into the equation (12) is further multiplied by the dilution rate up to the measurement (or concentration rate) It was found that by dividing by the correction coefficient: γ (that is, equation (13)), it is possible to calculate the concentration of pesticide in the original crop.

It is a flowchart which shows the process until it extracts and refine | purifies the said test agricultural chemical component from the agricultural crop used as the test object for implementing the agricultural chemical measurement of this invention, and makes it a measurement solution. It is the figure which plotted the relationship between each substrate density | concentration in the ideal solution in the absence of an agricultural chemical, and the attribution crop extract solution in the absence of an agricultural chemical, and the light absorbency change rate at the substrate concentration. The X axis (horizontal axis) is the reciprocal of the substrate concentration, and the Y axis (vertical axis) is the reciprocal of the absorbance change rate. It is a figure which shows the relationship between an addition pesticide density | concentration and the calculation density | concentration obtained by measurement.

Explanation of symbols

1 1st process 2 2nd process 3 3rd process 4 4th process 5 5th process 6 6th process

Claims (1)

In a method for measuring a cholinesterase-inhibiting pesticide contained in crops from the degree of cholinesterase activity decrease,
(1) After crushing crops and extracting with an organic solvent, the dried product that has been subjected to normal layer chromatography and vacuum drying is dissolved in a buffer that adjusts the pH in the measurement system to the optimum pH for cholinesterase activity. A pretreatment step for obtaining a measurement solution;
(2) The cholinesterase activity is measured in the measurement solution obtained in the pretreatment step, the cholinesterase activity is measured in the buffer used as a standard for comparison with the measurement solution, and the comparison is made. A measuring step for measuring the degree of inhibition of cholinesterase activity,
(3) A correction coefficient for activity inhibition that differs depending on the type and production area of the crop, that is, the ratio of the maximum reaction rate (α) and the ratio of the Michaelis constant (β) are preliminarily databased, and the measured values obtained in the measurement step are A correction step of correcting with the correction coefficients (α) and (β);
(4) extracting a constant (Ki) indicating a cholinesterase inhibitory ability that differs for each pesticide from a database in advance;
(5) calculating in advance a constant (Km) relating to the catalytic properties of cholinesterase;
(6) In the kinetic calculation formula based on the competitive inhibition reaction of Michaelis maintenance type enzyme, the correction value of the activity inhibition calculated by the correction step, the constant indicating the cholinesterase inhibitory ability (Ki), the constant relating to the catalytic properties of cholinesterase (Km) and a temporary calculation step of substituting the concentration of the substrate in the measurement solution and temporarily calculating the concentration of pesticide,
(7) The recovery rate (γ) in which the cholinesterase-inhibiting pesticide is recovered in the pretreatment step is stored in a database for each pesticide in advance, and the concentration of the pesticide obtained in the temporary calculation step is corrected with the recovery rate (γ). A quantitative process for calculating the concentration of pesticides,
A method for measuring pesticide residues, comprising:

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