JP2002330800A - Method for measuring pesticide residue - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring pesticide residues by which an amount of a comprehensive enzyme inhibition of a specimen in which two or more kinds of pesticides remain by pesticides can be known and the maximum residual concentration of each the pesticide into the specimen can be known by specifying pesticides naturally having a possibility of remaining in the specimen and reading out and calculating a previously input constant (inhibition constant) denoting a degree of the enzyme inhibition of the single body of the pesticide. SOLUTION: This method for measuring the pesticide residues allows measuring cholinesterase activities by detecting hydrogen peroxide generated by conjugating cholinesterase and comprises estimating the risk of pollution by the pesticide residues by calculating the residual concentration of each the pesticide having a possibility of being included at the maximum from a measured value obtained by measuring deterioration of the cholinesterase activities in a sample containing a plurality of the known pesticides and previously input cholinesterase inhibition activity of each the pesticide and comparing the concentration with a reference value of the residue of each the pesticide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting an organophosphorus pesticide or a carbamate pesticide remaining in food or environmental water with minimum influence of measurement interference, and a method for detecting a plurality of pesticides. The present invention relates to a method for performing determination based on pesticide residue reference values when coexisting.

[0002]

BACKGROUND OF THE INVENTION Organophosphorus pesticides and carbamate pesticides are widely used as pesticides, fungicides, and herbicides because they are more easily metabolized and degraded in the environment than organochlorine pesticides. However, organophosphorus insecticides, carbamate insecticides and their metabolites, when accumulated in the animal body, significantly harm the nervous system. In recent years, opportunities for conducting these pesticide tests have increased. In general, for detection of pesticides in such a field, a method that can easily and quickly measure near the site is desired.

Conventionally, detection of pesticides such as insecticides has been carried out using a large-sized precision analyzer such as a gas chromatograph, a gas chromatograph mass spectrometer, and a high-performance liquid chromatograph. However, such a method for detecting pesticides using a precision analyzer can comprehensively and accurately detect the components contained in a sample, but the measuring device is expensive, the measuring operation is complicated, and the skill is high. It takes time,
There are problems such as the necessity of using pesticides for device calibration every measurement.

[0004] On the other hand, as a simple method for detecting pesticides, there is a method of indirectly measuring the amount of a pesticide from a change in enzyme catalytic activity caused by the coexistence of a pesticide using a pesticide inhibiting enzyme as an element. For example,
Organophosphorus insecticides and carbamate insecticides are typical cholinesterase inhibitors. Based on this principle, the change in the enzyme catalyzing ability (hydrolysis rate) of cholinesterase due to the coexistence of pesticides is determined. There is a method of calculating the amount.

[0005] A simple pesticide detection device suitable for on-site measurement disclosed in Japanese Patent No. 2927221 corresponds to this. In addition, a simple measurement test paper (trade name: AT-10, Chisso Corporation) having the same principle and capable of discrimination with a certain concentration threshold is also commercially available.

For the measurement of cholinesterase activity at this time, the decomposition product of the specific substrate is subjected to an appropriate coloring method (for example, thiocholine generated by hydrolysis of acetylthiocholine is colored by a thiol-functional coloring reagent DTNB). Color development is generally performed using a colorimeter.

[0007]

However, in the method for detecting pesticides by cholinesterase inhibition, a large number of pesticides exhibiting inhibitory effects on the element (enzyme) exist, and the degree of inhibition differs for each pesticide. When two or more pesticides coexist, it is impossible to know the amount of each pesticide (there is no selectivity for each pesticide such as an antigen-antibody reaction). In addition, the pesticides actually sprayed are often a mixture of a plurality of pesticides, and it is difficult to make a determination based on the residual reference value.

Further, regarding the measurement of cholinesterase activity by an absorption spectrophotometry using various oxidative coloring systems, the influence of various measurement disturbances (for example, the coexistence of a coloring substance, an oxidizing agent, and a reducing agent in a sample). However, there is a problem that a signal indicating the presence or absence of the pesticide is apparently obtained although the signal is not caused by the presence of the pesticide.

In view of the above problems, the present invention makes it possible to know the overall amount of enzyme inhibition by a pesticide in a sample in which a plurality of types of pesticides remain, and to determine the amount of pesticide that may originally remain in the sample. A residual pesticide measurement method that reads out and calculates a constant (inhibition constant) indicating the degree of enzyme inhibition of a pesticide alone, which is specified and input in advance, and thereby knows the maximum residual concentration of each pesticide in the sample. To provide a technical issue.

[0010]

In order to solve the above-mentioned problems, the present invention provides a method for measuring residual pesticides in a sample from the degree of inhibition of cholinesterase activity by pesticides, the method comprising conjugated cholinesterase and choline oxidase. Cholinesterase activity can be accurately measured by detecting the hydrogen peroxide generated by the measurement, and the measured value of the decrease in cholinesterase activity in a sample containing a plurality of known pesticides is compared with the cholinesterase inhibitory ability of each pesticide stored in advance. Therefore, the technical means of calculating the residual concentration of each pesticide that may be contained at the maximum, and evaluating the risk due to residual pesticide contamination by comparing with the residual reference value of each pesticide, is taken. Was.

As a result, the effect of interfering substances on the measurement of cholinesterase activity in a real sample can be reduced.
The measurement accuracy of the inhibition measurement is improved. Then, when enzyme inhibition observation is performed in a sample in which multiple pesticides coexist, it is necessary to know the maximum residual concentration of all pesticides of multiple unknown concentrations contained in one measurement result (relative activity). By comparing this concentration with the residual reference value for each pesticide, it is possible to roughly determine the safety and danger due to residual pesticide contamination of the sample.

[0012]

Embodiments of the present invention will be described below in detail with reference to the drawings.

First, inhibition measurement (cholinesterase activity measurement) in a sample in which a plurality of pesticides are mixed is performed by using an amperometric sensor of an electrochemical device which is superior in selectivity to the spectrophotometric method and has less measurement interference. It is.
For example, an electrode described in JP-A-2000-321234 and having high sensitivity and high selective response to hydrogen peroxide applied as a polyphenol sensor can be used. In the method of detecting a signal due to pesticide inhibition as hydrogen peroxide, first, a cholinesterase is brought into contact with a specimen containing a pesticide, and this is dropped into an electrolyte containing choline oxidase and a cholinesterase substrate (acetylcholine, benzoylcholine, etc.) for a certain period of time. After the reaction for producing hydrogen peroxide, the reaction is stopped (in this case, quinidine sulfate or the like can be used as a reaction terminator). Thereafter, the electrode, the reference electrode, and the counter electrode as described above are inserted into the electrolytic cell, and an amperometric measurement is performed according to a standard method. The obtained steady-state reduction current value (ii) indicates the degree of inhibition by the pesticide. It is recorded as a signal (υ _i ) (however, Vi = ii / kt, k: proportionality constant, t: time) (see equation (2)).

[0014]

(Equation 1) The same measurement is performed for a sample containing no pesticide, and the signal obtained from the measurement is _expressed as (υ _n ) (where Vi = in / k
(t, in: steady-state reduction current value of a sample not containing pesticides, k: proportionality constant, t: time) are recorded (see equation (1)).

[0015]

(Equation 2) From the above formulas (1) and (2), the degree of enzyme inhibition by the pesticide is determined by the ratio of the activity in the presence of the pesticide to the activity in the absence of the pesticide, that is, the relative activity (RA: relative) of the formula (3).
activity) is represented as (υ _i / υ _n ).

[0016]

(Equation 3) Expanding equation (3), the reciprocal of the relative activity responds linearly to the pesticide concentration ([I]).

[0017]

(Equation 4) The Michaelis constant of acetylcholinesterase with respect to acetylthiocholine is Km = 1 × 10 ⁻⁴ , and when the substrate concentration is [S] = 2 × 10 ⁻⁴ M, the equation is as shown in equation (5).

[0018]

(Equation 5) Then, by measuring the relative activity, the concentration of the pesticide can be known from equation (6).

[0019]

(Equation 6)

For example, when the inhibition constant (ki) is 1 × 10 ⁻⁷
When the inhibition of pesticides is observed, the relationship between the semi-residual activity concentration, the lower limit of quantification, and the upper limit of quantification is shown in FIG.
become that way.

Inhibition constants for enzymes differ from each other.
The enzyme activity (Vi _{(A + B + C)} ) in the coexistence of seed pesticides (pesticide A, pesticide B, pesticide C) can be obtained from equation (7).

[0022]

(Equation 7) The relative activity (RA) relative to the activity in the absence of the pesticide is represented by the formula (8).

[0023]

(Equation 8) Expanding equation (8) yields the following.

[0024]

(Equation 9) The Michaelis constant of acetylcholinesterase with respect to acetylthiocholine is Km = 1 × 10 ⁻⁴ , and when the substrate concentration is [S] = 2 × 10 ⁻⁴ M, it becomes as shown in the formula (10). Is expressed as the sum of the amounts of enzyme inhibition by each pesticide.

[0025]

(Equation 10) For example, the degree of inhibition on the enzyme is determined by the inhibition constants Ki _A = 1 × 10 ⁻⁶ M and Ki _B = 1 × 10 ⁻⁷ M, K
pesticide A, pesticide B, pesticide C different from i _C = 1 × 10 ⁻⁸ M
The pesticide concentration dependence of the relative activity of each pesticide is as shown in FIG. 2. When these three pesticides coexist, the degree of inhibition as a whole is calculated from the equation (10) according to the amount inhibited by each pesticide. Therefore, it is possible to determine the maximum amount of each pesticide, even if the amount of each pesticide cannot be calculated.

For example, referring to FIG. 2, in a sample in which pesticides A, B, and C at unknown concentrations coexist, if the relative activity obtained by performing inhibition observation is 0.7, For A, it can be determined that a maximum of 1.3 × 10 ⁻⁶ M pesticide B, a maximum of 1.3 × 10 ⁻⁷ M pesticide C, and a maximum of 1.3 × 10 ⁻⁸ M are contained. All pesticides are safe because they are below the residue standard.

[0027]

EXAMPLES In order to show the validity of the formula (10), the response of acetylcholinesterase to two coexisting pesticides will be examined below.

Test pesticides: bendiocarb (carbamate pesticide), malathion (organophosphorus pesticide) Theoretical inhibition formula in the presence of two pesticides

[Equation 11] Conditions: Inhibition constant of bendiocarb (carbamate-based pesticide) K _iA = 1.67 × 10 ⁻⁷ M Inhibition constant of malathion (organophosphorus-based pesticide) K _iB = 1.67 × 10 ⁻⁴ M Km = 1 × 10 ⁻⁴ M, [S] = 2 × 10 ⁻⁴ M The theoretical value of the relative activity and the measured value of the relative activity under the above conditions were plotted as shown in FIG.

As described above, since the cholinesterase activity measurement can be performed with little influence of interfering substances even in the actual sample, the relative activity (RA) can be calculated accurately. When the relative activity (RA) is calculated, the maximum residual concentration of each of a plurality of pesticides to be determined can be known by the following pesticide concentration determination method.

The enzyme activity (V _n ) in the absence of the pesticide and the enzyme activity (V _i ) in the presence of the pesticide, that is, the activity of cholinesterase in the presence and absence of the pesticide are determined by the substrate acetylthiocholine (ATC). ), The thiocholine (TC) produced by the decomposition was colored by the thiol functional reagent DTNB, and the TNB thus colored was converted to an absorption wavelength of 4.
It is obtained by measuring the absorbance at 12 nm (see FIG. 3).

However, it can be said that in the case of coexistence of a plurality of kinds of pesticides and one kind of pesticides, when the inhibition measurement by this method is carried out in an actual sample (for example, vegetables), it is not possible to use the pesticide. In the case of the enzyme activity (V _n ) in the presence of coexistence, it is performed under the condition that the sample extract does not coexist.
In the case of the enzyme activity (Vi) in the coexistence of the pesticide, the measurement is performed under the coexistence condition of the sample extract. In this case, the inhibition is stronger in the sample extract than in the absence of the extract. As a typical example, the results of inhibition measurement in tomato extracts are shown in FIG.

As a method for measuring cholinesterase activity,
Free choline produced by the enzyme-catalyzed reaction of cholinesterase is converted to hydrogen peroxide using choline oxidase, which is a kind of oxidoreductase.
Since the measurement is performed using the electrochemical device sensor showing specificity to hydrogen peroxide described in JP-A-000-32234, the influence of interfering substances in the actual sample is reduced much more than the DTNB method, It is expected that the accuracy of the enzyme inhibition measurement will be improved (see FIGS. 5 and 6).

The basic configuration of the measuring instrument is a potentiostat for giving a low potential, a measuring cell for conducting a conjugate enzyme reaction and an electrochemical measurement, a connector for connecting each electrode, and an amplifying electric signal from the electrode. An electrical amplification unit; and a data processing unit for calculating an obtained signal to make it a relative activity (RA) and storing it.

Further, a data processing section for storing and reading out the inhibition constant of the pesticide which inhibits a given cholinesterase, data of the pesticide which is sprayed in advance and which may be detected, and which reads the inhibition constant of the pesticide. The processing unit may include a data processing unit and a data display unit for performing safety / danger determination based on the relationship between the maximum residual amount of each of the plurality of pesticides to be determined and the respective residual pesticide reference values.

Thus, by comparing the residual reference values of all the pesticides to be determined with each other, it is possible to roughly determine the safety / risk due to the residual pesticide contamination of the sample. The discrimination method is to judge as dangerous if the maximum residual concentration of each of a plurality of pesticides to be determined is greater than the respective residual reference values by one pesticide.

[0036]

According to the method for measuring residual pesticide residues in a sample from the degree of inhibition of cholinesterase activity by pesticides, cholinesterase activity can be measured by detecting hydrogen peroxide produced by coupling cholinesterase. From the measured value of the decrease in cholinesterase activity in a sample containing a plurality of pesticides, and the cholinesterase inhibitory ability of individual pesticides stored in advance,
Calculate the residual concentration of each pesticide that may be contained at the maximum, and evaluate the risk of residual pesticide contamination by comparing with the residual standard value of each pesticide. Therefore, measure cholinesterase activity in actual samples. The influence of interfering substances can be reduced, and the measurement accuracy of the inhibition measurement is improved. And
When enzyme inhibition observation is performed in a sample in which multiple pesticides coexist, the maximum residual concentration of all pesticides of multiple unknown concentrations contained in one measurement result (relative activity) can be known. By comparing this concentration with the residual reference value for each pesticide, it is possible to roughly determine the safety and danger of a sample due to residual pesticide contamination.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing a relationship between a half residual activity concentration, a lower limit of quantification, and a upper limit of quantification when observing inhibition of a pesticide having a specific inhibition constant.

FIG. 2 is a graph showing a method for determining the concentration of each pesticide in the presence of a plurality of pesticides.

FIG. 3 is a reaction formula when the cholinesterase activity is expressed by the rate of hydrolysis of the substrate acetylthiocholine (ATC) and the color of thiocholine (TC) produced by decomposition with the thiol functional reagent DTNB.

FIG. 4 is a graph showing an example in which inhibition is stronger in a sample extract than in the absence of an extract.

FIG. 5 is a diagram showing a measurement principle in which a hydrogen peroxide sensor of an electrochemical device is applied to a sensor for measuring cholinesterase activity.

FIG. 6 is a diagram showing a method for measuring cholinesterase activity.

FIG. 7 is a diagram in which a theoretical value of relative activity and an actually measured value of relative activity are plotted.

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[Procedure amendment]

[Submission date] April 17, 2002 (2002.4.1.
7)

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] Claim 1

[Correction method] Change

[Correction contents]

 ──────────────────────────────────────────────────続 き Continued on front page F term (reference) 4B063 QA01 QQ20 QQ32 QQ61 QQ89 QQ91 QR03 QR41 QR57 QS28 QX04

Claims (1)

[Claims] 1. A method for measuring residual pesticide residues in a sample from the degree of inhibition of cholinesterase activity by pesticides, wherein the cholinesterase activity can be measured by detecting hydrogen peroxide generated by coupling cholinesterase. From the measured value of the decrease in cholinesterase activity in a sample containing a plurality of pesticides, and the cholinesterase inhibitory ability of individual pesticides stored in advance,
A method for measuring residual pesticides, in which the residual concentration of each pesticide which may be contained at the maximum is calculated and the risk due to residual pesticide contamination is evaluated by comparing with the residual reference value of each pesticide.