JP2952359B1 - Detection method and detection kit for organophosphorus pesticides taken into living body - Google Patents

Abstract

An object of the present invention is to provide a method capable of selectively detecting only an organophosphorus pesticide taken into a living body selectively, quickly, simply and without using specialized techniques, and with high sensitivity. SOLUTION: The following steps: a first step of adding 4- (4-nitrobenzyl) pyridine to a urine sample; a second step of heating the reaction solution obtained in the first step; A third step of making the reaction solution obtained in the second step alkaline, a fourth step of adding an organic solvent to the reaction solution obtained in the third step, and a reaction obtained in the fourth step A method for detecting an organophosphorus pesticide taken into a living body, comprising a fifth step of measuring a coloring amount of an organic phase of a solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for detecting an organophosphorus pesticide taken into a living body.

[0002]

2. Description of the Related Art The treatment of poisoned patients with drugs and poisons
It is important to identify the causative agent of the poisoning and to promptly take a treatment appropriate for the causative agent.

[0003] One of the methods conventionally used for estimating substances causing poisoning by drugs, poisons and the like is a cholinesterase measurement method. The cholinesterase measurement method can detect organophosphorus and carbamate insecticides that have been taken into the body due to accidental ingestion or the like.

Some methods for measuring cholinesterase use several types of methods. For example, the serum of an addicted patient is collected, a substrate reagent for cholinesterase is added, and the mixture is allowed to react for a certain period of time.The absorbance of the reaction solution is measured, and the inhibitory activity of cholinesterase is measured. There is a method for estimating the presence or absence of intake. However, this measurement method is not a reaction specific to the pesticides, but may give a result as if the pesticides were ingested due to other physiological factors or chemical substances. In addition, a special technique is required for sample preparation and operation of an analytical instrument, and several hours are required until a result is obtained, which is not practical for use in emergency treatment.

[0005] Further, all of the above-mentioned methods for measuring cholinesterase are detection methods for both organic phosphorus-based and carbamate-based pesticide poisoning. Therefore, when this cholinesterase measurement method is used, there is also a problem that it is not possible to determine whether the poisoning is caused by an organic phosphorus-based or carbamate-based pesticide. Both organophosphorus pesticides and carbamate pesticides inhibit cholinesterase activity in vivo and cause similar clinical symptoms such as miosis and confusion, but the appropriate treatment methods for these addictions are different. Poisoning by these pesticides can lead to death depending on the delay in treatment, and there is a need for a method that can selectively and quickly determine the poisoning of either organophosphorus or carbamate pesticides. .

[0006]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above circumstances, and selectively uses only organophosphorus pesticides taken into a living body quickly and without using specialized techniques. It is an object of the present invention to provide a method that can be detected easily and with high sensitivity, and a detection kit for performing such a method.

[0007]

Means for Solving the Problems The present inventors have found that the above-mentioned object can be achieved by the following method, and have completed the present invention.

That is, the present invention provides a method for detecting an organophosphorus pesticide taken into a living body, comprising the following steps: a first step of adding 4- (4-nitrobenzyl) pyridine to a urine sample; A second step of heating the reaction solution obtained in the first step, a third step of making the reaction solution obtained in the second step alkaline, and a reaction step of heating the reaction solution obtained in the third step. Provided is a method including a fourth step of adding an organic solvent, and a fifth step of measuring a coloring amount of an organic phase of the reaction solution obtained in the fourth step. The organic solvent used in the fourth step is selected from the group consisting of diethyl ether, hexane, and ethyl acetate.

Further, the present invention comprises a container containing an effective amount of 4- (4-nitrobenzyl) pyridine, a container containing a reagent for making it alkaline, and a container containing an effective amount of an organic solvent. Also provided is a detection kit for detecting an organophosphorus pesticide taken into a living body.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the method of the present invention for detecting an organophosphorus pesticide taken into a living body (hereinafter, simply referred to as “the detection method of the present invention”) will be described in detail.

The organophosphorus pesticides to which the detection method of the present invention can be applied include any organophosphorus compounds known as control substances such as insecticides. Specifically, phosphorous acid, phosphoric acid, thiophosphoric acid, dithiophosphoric acid,
Organic compounds containing any phosphorus derived from inorganic phosphoric acids such as pyrophosphoric acid and phosphonic acid are included.

The detection method of the present invention can determine whether or not the above-mentioned organophosphorus pesticide has been taken into a living body. That is, most of the above organophosphorus pesticides are ingested into a living body, and at least a part thereof undergoes an action such as metabolism in the living body, so that the chemical structure changes.
Organophosphorus pesticides ingested into the living body have a
It is said that 60% is metabolized and excreted in urine. The detection method of the present invention can detect these organophosphorus pesticides present in urine and products derived therefrom (hereinafter, also referred to as “metabolites and the like”).

Each step of the detection method of the present invention will be described in detail.

According to the detection method of the present invention, 4- (4
-Nitrobenzyl) pyridine, a first step,
A second step of heating the reaction solution obtained in the first step, a third step of making the reaction solution obtained in the second step alkaline, and a reaction step of heating the reaction solution obtained in the third step. The method includes a fourth step of adding an organic solvent, and a fifth step of measuring the coloring amount of the organic phase of the reaction solution obtained in the fourth step.

First, in the first step of the detection method of the present invention, 4- (4-nitrobenzyl) pyridine is added to a urine sample.

As a urine sample to which the detection method of the present invention can be applied, urine collected from a patient or the like suspected of being poisoned by an organophosphorus pesticide can be used without any treatment. Urine, even immediately after collection,
It may be one that has passed a certain time after being collected.

The above-mentioned 4- (4-nitrobenzyl) pyridine to be added to the urine sample can be added as a solid, but from the viewpoint of miscibility with the urine sample and reactivity with the organophosphorus pesticide, an organic solvent is used. It is preferable to dissolve and add it.

The organic solvent for dissolving 4- (4-nitrobenzyl) pyridine is an organic solvent capable of dissolving 4- (4-nitrobenzyl) pyridine at room temperature (10 to 35 ° C.). There is no particular limitation as long as it does not adversely affect the step. As an example, as the organic solvent, acetone, alcohol having 1 to 20 carbon atoms, dimethyl sulfoxide, ethylene glycol and the like can be used. Among these organic solvents, acetone is preferred from the viewpoint of miscibility with urine samples and minimization of interference with biological substances.

The amount of the organic solvent is not particularly limited as long as it can dissolve 4- (4-nitrobenzyl) pyridine.
It can be appropriately set according to the amount of the urine sample, the type of the organic solvent to be used, the type (material) of the container to be used, and the like. As an example, 4- (4) having a concentration of 0.1 to 0.45 g / mL (hereinafter, the milliliter is also referred to as “mL”), preferably 0.3 to 0.45 g / mL.
-Nitrobenzyl) pyridine in acetone. 0.45 g / mL is the saturation amount to 4- (4-nitrobenzyl) pyridine acetone.

The amount of 4- (4-nitrobenzyl) pyridine added depends on the amount of the urine sample, the heating time in the second step,
It can be set appropriately according to the amount of alkali added in the third step. As an example, the amount of 4- (4-nitrobenzyl) pyridine to be added can be set to 10 to 200 mg, preferably 30 to 80 mg, per 1 mL of a urine sample.

Next, in the second step of the detection method of the present invention, the reaction solution obtained in the first step is heated.

The heating temperature and time depend on the amount of urine sample, 4-
It can be set appropriately according to the amount of (4-nitrobenzyl) pyridine added, the type (material) of the container used, the type of the heater used for heating, the type of the organic solvent used, and the like. As an example, heating can be performed at 30 to 100 ° C. for 5 to 40 minutes.

In the detection method of the present invention, after the second heating step, it is preferable that the reaction solution is allowed to cool to around room temperature from the viewpoint of safety.

Next, in the third step of the detection method of the present invention, the reaction solution obtained in the second step is made alkaline. Preferably, the pH value of the reaction solution is 9-14,
Preferably, it can be adjusted to 13 or more.

There are no particular restrictions on the alkalinity reagent that can be used in the third step, as long as it does not adversely affect the subsequent steps. Hydroxides, inorganic salts, organic amines, and the like can be used from the viewpoints of the effects of interfering substances, handleability, economy, and the like.

As the hydroxide, an alkali metal hydroxide or the like, for example, sodium hydroxide can be used.
As the inorganic salt, an alkali metal carbonate or the like, for example, potassium carbonate can be used. Inorganic salts, which are inferior to organic amines in color developability, are non-volatile and therefore do not emit a bad smell, and are preferred from the viewpoint that the operator does not feel discomfort.

Examples of the organic amines include diethylamine,
Examples include diisopropylethylamine, triethylamine, tri-n-propylamine, and tetraethylenepentamine. Many organic amines have excellent coloring properties,
In particular, short-chain alkylamines such as triethylamine and the like are volatile and generate an unpleasant odor, which may cause discomfort to the operator.

As a reagent for making alkaline, tetraethylenepentamine is preferable from the viewpoint of the operability of the reagent, the coloring intensity and the stability of the coloring substance.

Next, in the fourth step of the detection method of the present invention, an organic solvent is added to the reaction solution obtained in the third step.

The reaction solution used in the fourth step contains a product obtained by a reaction between the organophosphorus pesticide taken into the living body and a product derived therefrom and 4- (4-nitrobenzyl) pyridine. As the organic solvent to be added in the fourth step, a reaction product that dissolves the reaction product more easily than urine is used, whereby the reaction product can be transferred into the added organic solvent. Furthermore, if the organic solvent to be added is immiscible with urine, it can be separated into two phases: an organic phase in which the reaction product is present, and an aqueous phase derived from urine. By measuring the color of the organic phase thus obtained in the following fifth step, the detection method of the present invention can achieve high sensitivity and accuracy. Since the urine sample to be subjected to the detection method of the present invention is itself colored, if the color of the reaction solution obtained in the third step was measured without being separated into an organic phase and an aqueous phase, This is because the color originally possessed by the sample is also measured, and only the product of the reaction cannot be measured.

As the organic solvent to be added in the fourth step, as described above, the organic phosphorus-based pesticide taken into the living body and the product derived therefrom are combined with 4- (4-nitrobenzyl) pyridine. Products that dissolve the product of the reaction more than urine dissolves, are immiscible with urine, and do not adversely affect subsequent steps can be used. Examples of the organic solvent that can be added include diethyl ether, hexane, and ethyl acetate, and diethyl ether is preferable from the viewpoint of separation from a urine sample.

The amount of the organic solvent added in the fourth step can be appropriately set according to the amount of a urine sample, the amount of a 4- (4-nitrobenzyl) pyridine solution, the amount of an alkali, and the like. As an example, the organic solvent can be added in an amount of 0.1 to 3 times, preferably 1 to 2 times the volume of the sample solution to which the organic solvent is to be added.

After the addition of the organic solvent in the fourth step, it is preferable to carry out stirring in order to sufficiently separate the aqueous phase and the organic phase.

Next, in the fifth step of the detection method of the present invention, the coloring amount of the organic phase of the reaction solution obtained in the fourth step is measured.

The measurement of the coloring amount can be performed by a method according to the purpose of the measurement such as the degree of urgency and accuracy. For example, an absorbance measuring method using a known spectrophotometer or a colorimetric method based on a color table can be used.

When a colorimetric method based on a color table is used, the presence or absence of the intake of an organic phosphorus-based pesticide can be detected by visually comparing the coloring of the organic phase with the color table. In this case, the colorimetric method can be directly performed without separating the organic phase and the aqueous phase obtained in the fourth step. The colorimetric method based on the color table may be inferior to the case of measuring absorbance in terms of sensitivity and accuracy, but special measuring equipment,
There is an advantage that the presence or absence of ingestion of an organophosphorus pesticide can be detected extremely easily without requiring special skills.

When higher sensitivity and higher precision are required, the measurement can be performed by measuring the absorbance with a spectrophotometer. In this case, it is preferable to separate the aqueous phase and the organic phase obtained in the fourth step and measure the absorbance of only the organic phase. The wavelength of the spectrophotometer can be set to 520 nm.

The order of performing each step of the detection method of the present invention described above is one embodiment, and the detection method of the present invention
It is not limited to this.

The present inventors have found that in the analysis method of the present invention, 4- (4-nitrobenzyl) pyridine reacts with a phosphoric acid moiety of an organophosphorus pesticide, and the reaction product has a structure in an alkaline solution. I think it will change and develop color. This structural change is considered to be a kind of redox reaction. Therefore, the detection method of the present invention relates to the method of 4- (4)
As long as the reaction product with (-nitrobenzyl) pyridine develops color, the order of each step can be changed, or a plurality of steps can be performed simultaneously.

Another example of the detection method of the present invention is to add a 4- (4-nitrobenzyl) pyridine and an alkaline solution to a urine sample simultaneously (or sequentially) and then heat the reaction solution. Can be.

The present invention also provides a container containing an effective amount of 4- (4-nitrobenzyl) pyridine, a container containing an effective amount of a reagent for making it alkaline, and a container containing an effective amount of an organic solvent. Provided is a kit for detecting an organophosphorus pesticide taken into a living body comprising a container (hereinafter, also referred to as “kit of the present invention”).

The kit of the present invention can be suitably used for carrying out the above-described detection method of the present invention.

In the kit of the present invention, 4- (4-nitrobenzyl) pyridine is contained in a container in a state of being dissolved in the organic solvent described in relation to the first step of the above-mentioned detection method of the present invention. Can be.

As the reagent for making the kit alkaline according to the present invention, the third reagent of the above-described detection method of the present invention is used.
What was explained in relation to the step can be used.

As the organic solvent included in the kit of the present invention, those described in relation to the fourth step of the above-described detection method of the present invention can be used.

The material, shape, size, etc., of the container for accommodating the above-mentioned reaction reagents can be appropriately selected as long as they are resistant to the respective reagents. In addition, the kit of the present invention can further include, for example, a reaction container for storing a urine sample to be tested, in addition to the container for storing the above-described reaction reagents. The additional container may be resistant to the urine sample, reagents, etc. used in the method of the present invention, and may have any shape, if it can withstand the heating conditions described in connection with the second step of the present invention. The size, material, and the like can be appropriately selected. For example, a heat-resistant glass test tube having a capacity of about 10 mL can be used.

[0047]

EXAMPLES The present invention will be described by way of examples, but the method of the present invention is not limited to these examples.

Example 1 About 1 mL of urine collected from a patient suspected of being poisoned by an organophosphorus pesticide was placed in a heat-resistant test tube, and 0.45 g / m
An acetone solution of 4- (4-nitrobenzyl) pyridine adjusted to a concentration of L was added so as to be 45 mg per 1 mL of a urine sample. After heating this solution at 100 ° C. for 20 minutes, the reaction solution was allowed to cool to room temperature. After cooling, 0.1 mL of tetraethylenepentamine was added to the reaction solution. Then, 1.0 mL of diethyl ether was added to the reaction solution.
Was added. As a result of comparing the organic phase of the obtained solution with a color table prepared in advance, the reaction of the patient ingesting the organophosphorus pesticide was recognized.

Example 2 The minimum detection was carried out in the same manner as in Example 1 by using, as a urine sample, 15 kinds of typical organophosphorus pesticides which cause poisoning adjusted to several concentrations and added to urine of a normal person. The concentration was determined. However, the measurement of the coloring amount measured the light absorbency in 520 nm using the spectrophotometer (Shimadzu Corporation, UV-200S).

On the other hand, for the same urine sample, the attached ampule was divided in the urine sample according to the operation manual of a commercially available kit for detecting pesticides in environmental water (using the cholinesterase method), and the urine and the reagent were mixed well. Left. The aforementioned reaction solution is immersed in the substrate part of the pesticide detection kit, and the substrate part and the enzyme part soaked with the reaction solution are overlapped by hand and reacted.
Three minutes later, the presence or absence of coloring of the enzyme part disk was visually determined.

The 15 types of organophosphorus pesticides used in Example 2 are as follows. Isoxathion (O, O-diethyl
O-5-phenyl-1,1-oxazol-3-yl phosphorothioate (trade name carphos)), dimethoate (O, O-dimethyl S-methylcarbamoylmethyl phosphorodithioate), diazinon (0,0- Diethyl O-2-isopropyl-
6-methylpyrimidin-4-yl phosphorothioate), disulfoton (O, O-diethyl S-2-ethylthioethyl phosphorodithioate (trade name Dyciston)),
Fenitrothion (or MEP, O, O-dimethyl O-4-nitro-
m-toluyl phosphorothioate (trade name Sumithion)), porothiophos (O-2,4-dichlorophenyl O-ethyl-S-propyl phosphorodithioate (trade name Toction)), formotion (S-formyl (methyl) carbamoyl) Methyl O, O-dimethylphosphorodithioate (trade name Antio)), malathion (diethyl (dimethoxyphosphinothioylthio) succinate (trade name marathon)), BRP (or Naled, 1,2-dibromo-2,2) -Dichloroethyl dimethyl phosphate (trade name: dibrom)), DDVP
(Or dichlorvos, 2,2-dichlorovinyldimethylfespate (trade name Death)), DEP (or trichlorfon,
Dimethyl 2,2,2-trichloro-1-hydroxyethyl phosphate (trade name: Dipterex)), DMTP (or methidathion, S-2,3-dihydro-5-methoxy-2-oxo-1,3,4-thiadiazole) -3-ylmethyl O, O-dimethylphosphorodithioate (trade name Supraside)), EDDP (or edifenphos, O-ethyl S, S-diphenylphosphorodithioate, (trade name Hinozan)), EPN ( O-ethyl O-4-nitrophenylphenyl phosphorothioate), ESP
(S- (2-ethylsulfinylisopropyl) O, O-dimethyl phosphorothioate (trade name: Estocks)).

As a result of the measurement, the method of the present invention was able to detect the concentration up to 3 to 5 times lower than that when using a commercially available kit.

When a commercially available kit was used, 20 mL of the sample was required for the measurement, whereas when the detection method of the present invention was used, the amount of the sample required for the measurement was significantly reduced to 1 mL. Could be reduced.

[0054]

When the conventional cholinesterase method is used, not only carbamate-based pesticides but also all compounds in the sample that act on the active site of the enzyme (cholinesterase) are detected. May show such test results even though you have not taken any. This is because there are many compounds in the living body that can act on the active site of cholinesterase, and they are excreted as urine. On the other hand, according to the method of the present invention, it is possible to selectively detect whether or not only organic phosphorus-based pesticides are ingested.

Further, in the conventional cholinesterase method, since the measurement is carried out by using the enzyme inhibitory activity, an organophosphorus pesticide which cannot act on the active site of cholinesterase, and metabolism, etc., cannot act on the active site. It is not possible to detect the metabolites that have been lost. On the other hand, in the method of the present invention, since the reactivity of the phosphoric acid moiety of the organic phosphorus is used, even those which cannot act on the active site as described above can be detected. For example, organophosphorus pesticides and their metabolites that can be detected by the detection method of the present invention but cannot be detected by the conventional cholinesterase method include dithiophosphoric acid, dimethyl phosphite, and trimethyl phosphate.

Further, by using the detection method of the present invention, it is possible to determine the presence or absence of ingestion of an organophosphorus pesticide with a remarkably small amount of sample as compared with the conventional method.

As described above, according to the detection method of the present invention,
Only organophosphorus pesticides taken into a living body can be detected selectively, quickly, simply and without using specialized techniques, and with high sensitivity. Therefore, by using the detection method of the present invention, it is possible to quickly and accurately determine whether or not an organic phosphorus-based pesticide has been ingested, and it is particularly useful for treatment and treatment of a patient or the like at an emergency site. It is expected.

Continuation of the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) G01N 31/00 G01N 33/15 G01N 33/493 G01N 33/52 JICST file (JOIS)

Claims (3)

(57) [Claims] 1. A method for detecting an organophosphorus pesticide taken into a living body, comprising the following steps: a first step of adding 4- (4-nitrobenzyl) pyridine to a urine sample, and a first step A second step of heating the reaction solution obtained in the second step, a third step of making the reaction solution obtained in the second step alkaline, and adding an organic solvent to the reaction solution obtained in the third step A fourth step of measuring the amount of coloration of the organic phase of the reaction solution obtained in the fourth step. 2. The organic solvent to be added in the fourth step,
The method according to claim 1, wherein the method is selected from the group consisting of diethyl ether, hexane, and ethyl acetate. 3. An in vivo living body comprising a container containing an effective amount of 4- (4-nitrobenzyl) pyridine, a container containing a reagent for making it alkaline, and a container containing an effective amount of an organic solvent. Kit for detecting organophosphorus pesticides ingested.