JP2011058846A - Method and device for screening agricultural chemical and additive - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a screening method excellent in selectivity capable of rapidly and simply measuring a measuring target compound present in a sample to be inspected of a minute amount with high sensitivity without using a high-degree analyzing device in a chemical analyzing method for measuring a concentration of agricultural chemicals or additives in food or an environmental sample, and a portable measuring instrument using the same. <P>SOLUTION: A solution to be inspected is made to pass through a small volume column filled with an adsorbent showing selectivity with respect to the measuring target substance and the measuring target substance is adsorbed on the surface of the adsorbent to be concentrated and fixed. After an unnecessary component is cleaned off, the measuring target substance is detected while being adsorbed and fixed on the surface of the adsorbent by a fluorescence or chemiluminescence method to perform the screening (semi-quantification) of the agricultural chemicals or additives remaining in or added to food. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

The present invention relates to a chemical analysis method for measuring the concentration of agricultural chemicals and additives in food, feed, or environmental samples, and increases the amount of a measurement target compound present in a trace amount in a test sample without using an advanced analytical instrument. The present invention relates to a screening method that can be quickly and easily measured with sensitivity, and a portable measuring device for using the method.

In recent years, “food safety / reliability” has been screamed, but problems such as mixing of agrochemicals, high residue, excessive addition of food additives, etc. are unending. Since 2006, a system related to pesticides remaining in food (positive list system) has been implemented, and the number of pesticides to be measured has greatly increased along with the increase in the number of samples to be measured. Although technologies and equipment related to food analysis have made significant progress, there is a high level of contaminants other than the target compound in food. Is difficult to measure stably. Therefore, in food analysis, how to remove impurities in the test sample is an important issue.

With the implementation of the positive list system, “Testing methods for substances that are pesticide residues, feed additives, or veterinary pharmaceutical ingredients remaining in foods” was issued by the Ministry of Health, Labor and Welfare as 01-24001 on January 24, 2005. (Non-Patent Document 1), then, “Regarding the test method for substances that are pesticides, feed additives or veterinary pharmaceutical ingredients remaining in food (partially revised)” It was notified on November 29, 2005 (Non-Patent Document 2). The test method related to the positive list system is “About the Use of Rapid Analysis Method for Residual Pesticides” notified on April 8, 1997 during the former Ministry of Health and Welfare (Heisei 43, Non-patent Document 3) Is based. Fig. 1 shows the main steps in the sample pretreatment shown in Hygienic No. 43, but the pretreatment operation for removing contaminants present in the food is very complicated, and analysis is performed according to this step. In some cases, it takes several days until the analysis result is obtained. For this reason, it is difficult not only to cope with the increasing number of specimens but also to adapt to the distribution cycle of products. Furthermore, the pretreatment operation for performing such precise instrument analysis has many problems to be improved, such as requiring skill and using a large amount of organic solvents and chemicals.

Various simple methods and rapid methods have been proposed to deal with such problems, such as changing the extraction method of the compound to be measured, using a selective detector, and using a high-resolution apparatus. A part of the process is omitted or simplified, and advanced and expensive analytical instruments such as gas chromatograph mass spectrometer and liquid chromatograph mass spectrometer are required for qualitative and quantitative determination of the compounds to be measured. (Patent Document 1 to Patent Document 4). These techniques are effective for precise analysis and simultaneous analysis of multiple components, and can obtain highly reliable results. However, in addition to requiring specialized knowledge, the running cost is high and the analysis time including pretreatment is long, so it is not suitable for processing a huge number of samples. Also, since it is not a portable device in nature, it is not suitable for on-site analysis and cannot be adapted for monitoring at the water's edge.

In order to solve the problems in the instrumental analysis method as described above, there are infrared spectroscopy and immunoassay as inexpensive and simple measurement methods that do not use expensive analytical instruments. Infrared spectroscopy is an effective method for on-site analysis, is simple, and the equipment used is not very expensive. Patent Document 5 discloses an example of a sorter and an agrochemical determination device used in a crop collection place. In this method, determination is made by computer analysis based on the accumulated infrared absorption spectrum. However, infrared spectroscopy has poor selectivity, and as described in Patent Document 4, the absorption of lipids and proteins is strong, and there are interferences due to these. Therefore, it is considered effective when the pesticide to be used is specified, but interference due to impurities cannot be ignored in screening for unspecified pesticides.

On the other hand, immunoassay is an analysis method using antigen-antibody reaction. In general, an antigen labeled with a specific enzyme and an unlabeled antigen in a sample are subjected to a competitive reaction with an antibody, an antigen-antibody complex is precipitated to separate an unbound antigen, and the amount of labeled antigen bound to the antibody is determined. A method is employed in which the amount of antigenic substance in the sample is determined by measurement. This method is used for microanalysis because of its high specificity and high detection sensitivity. In addition, it is an effective analytical method as a screening method because it is easy to operate, requires no specialized knowledge, and has a measurement time of several hours. Examples applied to harmful substances such as agricultural chemicals are disclosed in Patent Documents 6 to 9. Several types of immunoassay kits for pesticide residue analysis are already on the market and are also used for on-site analysis. However, since there are few appropriate types of antigens, the types of pesticides to be measured are limited. In particular, for low molecular weight compounds, there are few appropriate antigens and it is difficult to cope with them. Therefore, there is a need for a new screening method for pesticides that are difficult to handle by immunoassay methods.

JP 2007-155657 A JP 2006-242825 A JP 2006-78419 A JP 2004-340627 A JP 2007-101186 A JP 2005-194238 A JP 2004-333130 A Japanese Patent Application Laid-Open No. 2007-68531 Special table 2007-531822 publication

Ministry of Health, Labor and Welfare, Food Safety No. 012001, dated January 24, 2005 Ministry of Health, Labor and Welfare, Food Safety No. 1129002, Notification dated November 29, 2005 Ministry of Health, Welfare No. 43, April 8, 1997 notice

The present invention has been made in view of the above problems, and extracts, concentrates and immobilizes agrochemicals in a test solution on an adsorbent that is selective for the measurement target compound, and immobilizes the agrochemical on the adsorbent surface. It is an object of the present invention to provide a screening method for agrochemicals and additives, and a measuring apparatus for the same, which is simple and quick, has a low running cost, and has a low environmental impact, such as detection by a fluorescence method or a chemiluminescence method in the as-stained state.

As a result of intensive studies by the inventor of the present invention, a small-capacity column of 2 mL or less (generally referred to as a “solid-phase extraction cartridge”) filled with an adsorbent exhibiting selectivity for the measurement target compound is used. After passing the test solution and adsorbing and concentrating the measurement target compound in the test solution on the surface of the adsorbent, the measurement target compound is fixed to the adsorbent surface without desorbing the measurement target compound from the adsorbent. In addition, the present inventors have found that it is possible to screen for agricultural chemicals and additives simply and quickly by emitting light by a fluorescence method or a chemiluminescence method and determining the concentration of the compound to be measured from the light emission amount.

In the present invention, the measurement target compound is a compound used as a fluorescent or chemiluminescent pesticide and an additive, and light is emitted by a fluorescence method or a chemiluminescence method with the measurement target compound fixed on the adsorbent surface. The concentration of the measurement target compound is determined from the amount of luminescence.

In the present invention, the compound to be measured is a non-fluorescent or non-chemiluminescent pesticide and a compound used as an additive, and is made a fluorescent or chemiluminescent compound by reaction with an appropriate derivatization reagent. In the same way, after the compound to be measured is converted to a fluorescent or chemiluminescent derivative while adsorbed and immobilized on the adsorbent surface, the resulting derivative remains adsorbed and immobilized on the adsorbent surface. Without desorbing from the adsorbent, light is emitted by the fluorescence method or chemiluminescence method, and the concentration of the compound to be measured is determined from the amount of light emitted.

The adsorbent for adsorption / concentration used in the present invention is an adsorbent having both a hydrophobic functional group and an ion exchange group, and selectively adsorbs / concentrates the measurement target compound by a combined adsorption mechanism.

Further, the adsorbent for adsorption / concentration used in the present invention is an adsorbent having both a hydrophilic functional group capable of developing a hydrophilic interaction and an ion exchange group, and is to be measured by a combined adsorption mechanism. Selective adsorption and concentration of compounds.

In the present invention, the portion filled with the adsorbent of the small-capacity column is made of a material that is light transmissive, non-fluorescent, chemiluminescent, or extremely low, and emits light emitted by the fluorescent method or chemiluminescent method. In addition, the shape is cylindrical or rectangular, and has a liquid inlet / outlet at the top and bottom.

The apparatus that enables easy screening of the agricultural chemicals and additives of the present invention is as follows. 1) A small-capacity column of 2 mL or less filled with an adsorbent for adsorbing and concentrating the measurement target compound can be installed. A sample chamber, 2) a pump system for delivering a derivatization reagent and a cleaning solution, 3) a light source and optical system for irradiating excitation light, or a pump system for delivering a chemiluminescent reagent, and 4) a small volume column Portable pesticides and additives comprising a detector for detecting the amount of fluorescence or chemiluminescence emitted from the light source and 5) an arithmetic circuit for calculating the concentration of the compound to be measured from the amount of fluorescence or chemiluminescence obtained Screening device.

According to the present invention, it is possible to easily and quickly perform highly sensitive measurement of food, feed, environmental pesticides and additives, and the like. By using the screening method and the portable screening apparatus of the present invention, it is possible to measure a small amount of residual agricultural chemicals and additives / contaminants on-site, so that measurement adapted to the distribution cycle is possible. Although the screening method of the present invention is not applicable to all existing pesticides, etc., it can be applied to the screening of many pesticides and additives because it obtains the results grouped by the structure and functional group of the compound to be measured. Is possible.

FIG. 1 is an explanatory diagram showing an operation flow of a rapid analysis method for residual agricultural chemicals in food. FIG. 2 is an explanatory view showing an example of the configuration of a small-capacity column (solid phase extraction cartridge). FIG. 3 is an explanatory view showing an example of a different configuration of a small capacity column (solid phase extraction cartridge). FIG. 4 is an explanatory diagram showing the basic configuration of the sample unit and the detection unit used in the fluorescence detection method in the screening apparatus of the present invention. FIG. 5 is an explanatory diagram showing the basic configuration of the sample unit and the detection unit used in the chemiluminescence detection method in the screening apparatus of the present invention. FIG. 6 is an explanatory diagram showing a pre-processing operation flow for using the method of the present invention. FIG. 7 is a diagram showing an example of a manual pretreatment operation using a syringe for using the screening method of the present invention. FIG. 8 is a diagram showing an example of a pretreatment operation using a liquid feed pump for using the screening method of the present invention. FIG. 9 is a view showing an example of a pretreatment operation using a suction manifold for using the screening method of the present invention. FIG. 10 is an explanatory diagram showing a detection operation flow in the screening method of the present invention. FIG. 11 is an explanatory diagram showing a basic operation procedure when fluorescence detection is used. FIG. 12 is an explanatory diagram showing a basic operation procedure when chemiluminescence detection is used. FIG. 13 shows a chemiluminescence profile when an amino acid derivative captured using an adsorbent having a hydrophobic interaction and an anion exchange interaction is detected by a chemiluminescence method. FIG. 14 shows a fluorescence profile when an antibiotic captured using an adsorbent having a hydrophilic interaction and an anion exchange interaction is detected by a fluorescence method.

In the present invention, a test solution subjected to pre-extraction treatment is passed through a small-capacity column filled with an adsorbent exhibiting selectivity for a measurement target compound, and the measurement target compound in the test solution is adsorbed on the surface of the adsorbent. After adsorbing and concentrating the compound, the measurement target compound is immobilized on the surface of the adsorbent without causing the measurement target compound to be desorbed from the adsorbent. This is a simple and rapid screening method for pesticides and additives for determining the concentration.

In the present invention, the measurement target compound adsorbed by the adsorbent is detected by the fluorescence method or the chemiluminescence method without being eluted from the adsorbent while being adsorbed and fixed on the adsorbent surface. In the fluorescence method, a compound to be measured or its derivative is excited by light, and light (fluorescence) having a wavelength specific to a substance generated in the process of returning to the ground state is detected. On the other hand, the chemiluminescence method detects a light emitted when a measurement target compound or a derivative thereof is excited by a chemical reaction and returns to a ground state, and is generally excited using an oxidation reaction or the like. Since the fluorescence method and the chemiluminescence method are highly selective detection methods, they can be detected without being affected by impurities. Further, these detection methods are luminescence detection methods, and high sensitivity detection is possible because the amount of luminescence from a place where there is no background is detected.

In the present invention, the measurement target compound that can be directly measured is a fluorescent or chemiluminescent compound. In general, a fluorescent compound can be measured by a chemiluminescence method. Examples of fluorescent pesticides and additives include benzimidazole fungicides and fungicides such as benomyl, carbendazim, thiabendazole, and fuberidazole, oxolinic acid, paraquat, orthophenylphenol (OPP), diphenyl, Examples thereof include butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), vitamin E and the like. By adsorbing and fixing these compounds on the surface of the adsorbent selective for the compound to be measured and detecting them by fluorescence or chemiluminescence methods, selectivity can be obtained in the two steps of extraction and detection. As a result, it is possible to perform high-sensitivity detection that is not easily affected by impurities.

In the present invention, even a non-fluorescent or non-chemiluminescent compound can be detected as long as it is a compound that generates a fluorescent or chemiluminescent compound by reaction with an appropriate derivatization reagent. Is possible. After a compound that becomes a fluorescent compound by reaction with a derivatization reagent is adsorbed and fixed on the surface of the adsorbent, after being guided to the fluorescent compound using an appropriate derivatization reagent while being fixed on the surface of the adsorbent, In the same manner as described above, detection is carried out by fluorescence or chemiluminescence. In general, fluorescent derivatization is carried out using amino groups (imino groups), thiol groups, hydroxyl groups, carbonyl groups, etc., but since there are many compounds having these functional groups in agricultural chemicals, these functional groups are used. Derivatization is performed using Examples of agricultural chemicals and additives that have amino groups (imino groups) that become fluorescent or chemiluminescent compounds by reaction with derivatizing reagents include dichlorane, ashram, ibotenic acid, pendimethalin, amino acids, etc. can give. An example of an agrochemical / additive having a thiol group that becomes a fluorescent or chemiluminescent compound by reaction with a derivatizing reagent is L-cysteine. In addition, examples of agricultural chemicals and additives having hydroxyl groups that become fluorescent compounds by reaction with derivatizing reagents include delphinin, chloropropylate, chloretone, calciferol, etc. Examples of such products include clofenac, dinoben, MDBA, cloprop and the like.

A well-known thing can be used as a derivatization reagent. Since the derivatization reagent is selected according to the characteristics of the compound to be measured, it is not particularly specified. In general, however, reactions such as amino groups (imino groups), thiol groups, hydroxyl groups, carbonyl groups, etc. React with highly functional groups. Among these functional groups, many reagents have been developed that use an amino group (imino group) to form a fluorescent derivative. However, in the present invention, it is not possible to use a derivatization reagent that produces a derivative that extremely reduces the interaction with the adsorbent after derivatization. For example, in the case of using an adsorbent having a hydrophobic interaction and an ion exchange interaction, if an extremely low hydrophobicity or ionicity occurs, the derivatization reaction or the cleaning of excess reagents, and further chemical During the feeding of the luminescent reagent, it becomes detached from the adsorbent and cannot be measured. In addition, when a derivatizing reagent that is soluble only in an organic solvent is used, the hydrophobic interaction between the adsorbent and the measurement target compound is weakened, and thus such a derivatizing reagent cannot be used. This is basically the same in the case of using an adsorbent having hydrophilic interaction and ion exchange interaction. If the polar or ionic property is extremely lowered by derivatization, the adsorbent is desorbed. Therefore, the compound to be measured cannot be measured by the method of the present invention.

Examples of derivatization reagents utilizing amino groups that can be used in the present invention include orthophthalaldehyde, fluoresamine, dansyl chloride, dub silk chloride, 4-fluoro-7-nitrobenzofurazan (NBD-F), 4-chloro- 7-nitrobenzofurazane (NBD-Cl), and the like. In addition, as a derivatization reagent utilizing a thiol group that can be used in the present invention, N-pyrroloisomaleimide, N- (4-anilinophenyl) maleimide, N- (7-dimethylamino-4-methylcoumarinyl) ) Reagents having an N-substituted maleimide group such as maleimide. Examples of the derivatizing reagent using a hydroxyl group include reagents having a carbonyl cyanide group such as pyrene-1-carbonylcyanide and anthracene-1-carbonylcyanide, and carbonyl azide groups such as pyrene-1-carbonylazide. The reagent which has can be used. Furthermore, examples of the derivatization reagent using a carbonyl group that can be used in the present invention include a reagent having a bidrazino group such as dansylhydrazine and 2-amino-4,5-dimethoxythiophenol.

The adsorbent used in the present invention is basically the same as a so-called solid phase extractant. The solid-phase extraction method is a physical extraction method that uses the affinity between the measurement target compound and the adsorbent serving as a solid phase. Compared to the conventional solvent extraction method (liquid-liquid extraction method) that uses distribution to organic solvent, it has features such as high extraction efficiency, good reproducibility, safety, and low running cost. At the same time, it has become widespread in recent years because it is an environmentally friendly method that does not use halogen-based solvents, which are considered as environmental pollutants, and uses a small amount of organic solvent. In addition, the solvent extraction method cannot be extracted using a solvent miscible with water (for example, alcohols). However, since the solid phase extractant is insoluble, an organic solvent in an aqueous solution can be obtained using a highly polar adsorbent. Extraction of compounds is possible. As the extraction mechanism in the solid phase extraction method, normal phase distribution, reverse phase distribution, ion exchange, complex formation and the like are used, but the most widely used extraction mechanism is reverse phase distribution. As a solid phase extraction agent for reverse phase distribution, there is an alkyl group chemically bonded silica gel in which an alkyl group is introduced into silica gel using an alkylsilane compound. A typical example is octadecyl silica (ODS) into which an octadecyl group is introduced. Polymeric solid phase extraction agents such as polystyrene gel and polymethacrylate gel are also widely used. These solid-phase extraction agents extract organic compounds in a test solution based on hydrophobic interaction with an alkyl group or an aromatic ring. Solid phase extraction agents using these reverse phase distribution mechanisms are widely used for pretreatment of agricultural chemicals and additives, but are not necessarily effective in extracting highly polar compounds. In particular, when extracting an organic compound having an ionic functional group, a decrease in recovery rate and reproducibility is a problem.

In the present invention, the compound to be measured is extracted from the test solution using the same extraction function as that of the solid phase extraction agent, but it is not necessary to elute from the solid phase extraction agent as in the solid phase extraction method. Therefore, an adsorbent having higher adsorption characteristics for the measurement target compound than a general solid phase extractant is used. However, since it is necessary to carry out the derivatization reaction while the compound to be measured is adsorbed and immobilized on the adsorbent surface, it is not possible to use an adsorption / concentration method in which the functional group to be derivatized is the main adsorption mechanism. Since functional groups to be derivatized include amino groups (imino groups), thiol groups, hydroxyl groups, carbonyl groups, etc., an adsorption mechanism that does not inhibit these functional groups is used. In general, when a derivatization reagent relating to an amino group (imino group) is applied, an adsorption mechanism that recognizes the amino group (imino group), for example, cation exchange interaction is not preferable. However, in the case of a compound having a plurality of amino groups (imino groups), not all amino groups (imino groups) are involved in the adsorption mechanism, so that cation exchange interaction can also be used.

One form of the adsorbent used in the present invention is an adsorbent having both a hydrophobic functional group and an ion exchange group. In general, many agricultural chemicals and additives have an ion exchange group or a polar group introduced into a hydrophobic skeleton. When such a compound is extracted only by the reverse phase distribution mechanism, it may be eluted in the step of washing unnecessary impurities. Therefore, in the present invention, the compound to be measured is held by a composite adsorption mechanism in which a plurality of interactions are combined. Although various combinations of adsorption mechanisms are possible, the most effective combination is a combination of hydrophobic interaction and ion exchange interaction. In general, when a compound having an ionic functional group is adsorbed by ion exchange interaction, the hydrophilicity of the measurement target compound is lowered and the apparent hydrophobicity is increased. At this time, if there is a functional group clearly showing the hydrophobic interaction in the adsorbent, the adsorbent can be strongly held by the combined action with the hydrophobic interaction. Therefore, in this invention, the adsorbent which shows the adsorption mechanism which combined the hydrophobic interaction and the ion exchange interaction is used. By using such a combination, even if the adsorbent from which the measurement target compound is extracted and adsorbed is washed with an organic solvent or a buffer solution, the measurement target compound is not easily eluted. It remains fixed to the adsorbent. As the ion exchange interaction combined with the hydrophobic interaction, either a cation exchange interaction or an anion exchange interaction can be used. Pesticides and additives have many compounds with amino groups (imino groups), so cation exchange interaction is effective, but considering derivatization, an adsorption mechanism that does not inhibit amino groups (imino groups) is used. There must be. Therefore, it is preferable to perform adsorption by anion exchange interaction using an anionic functional group such as a carboxyl group, a sulfo group, or a phospho group.

An example of an adsorbent (solid phase extractant) having both a hydrophobic functional group and an ion-exchange functional group is disclosed in JP-T-2002-517574. In this patent document, it is composed of a polymer of divinylbenzene. After introducing a chloromethyl group into the aromatic ring of the hydrophobic resin, it is reacted with an amine to introduce an ion exchange group. In the present invention, since the hydrophobic functional group and the ionic functional group of the measurement target compound are recognized and adsorbed and fixed, it is considered that the adsorbent described in the patent document can also be used. However, since many water molecules are bound around the ionic functional group and show high hydrophilicity, the ion exchange group of the adsorbent is bound to a site showing hydrophilicity rather than to a hydrophobic site. This clearly expresses the ion exchange interaction and makes it easier to recognize the ionic functional group of the compound to be measured. Therefore, in the present invention, pores that are compatible with a hydrophilic vinyl monomer having a reactive functional group and a hydrophobic cross-linkable monomer having two or more vinyl groups are compatible with these vinyl monomers and do not contribute to the polymerization reaction. In the presence of a solvent serving as a regulator, a porous cross-linkable polymer carrier obtained by an aqueous suspension polymerization method into which an ion exchange group is introduced by a secondary reaction is used.

When making an adsorbent having both hydrophobic interaction and anion exchange interaction, a hydrophilic vinyl monomer having a functional group that reacts with an amine introduced for the ion exchange group and a hydrophobic property having at least two vinyl groups A crosslinkable polymer carrier can be obtained by copolymerization with a crosslinkable monomer. Examples of the functional group of the hydrophilic vinyl monomer having a functional group that reacts with an amine include an alkyl halide group and an epoxy group. Examples of the hydrophilic reactive monomer having a halogenated alkyl group include 3-chloro-2-hydroxypropyl methacrylate, 3-chloro-2-hydroxypropyl acrylate, 2-chloroethyl methacrylate, 2-chloroethyl acrylate, and the like. can give. Examples of the hydrophilic reactive monomer having an epoxy group include glycidyl methacrylate and glycidyl acrylate. As the hydrophobic crosslinkable monomer having two or more vinyl groups copolymerizable with these monomers, aromatic crosslinkable monomers such as divinylbenzene and divinylnaphthalene can be used. The hydrophobicity and properties of the resulting crosslinkable polymer carrier depend on the amount of the hydrophobic crosslinkable monomer, but if the crosslinkable polymer carrier has sufficient hardness, the hydrophobicity having two or more vinyl groups It is possible to replace a part of the crosslinkable monomer with a hydrophobic vinyl monomer having one vinyl group. Examples of the hydrophobic vinyl monomer having one vinyl group include stearyl methacrylate, lauryl methacrylate, octyl methacrylate, and phenyl methacrylate. In order to improve the physical properties of the crosslinkable polymer carrier such as wettability and hydrogen bondability, it is possible to add other vinyl monomers.

The most important point in the adsorbent having both the hydrophobic interaction and the anion exchange interaction of the present invention is the amount of the hydrophobic functional group, the amount of the ion exchange group introduced into the hydrophilic vinyl monomer, and the balance thereof. . The amount of the vinyl monomer exhibiting hydrophobicity is 60 to 90% by weight, preferably 70 to 90% by weight, based on the total vinyl monomers. In order to ensure sufficient hardness as an adsorbent, at least 30% by weight or more of a hydrophobic crosslinkable monomer having two or more vinyl groups is used. The ion exchange capacity depends on the amount of the hydrophilic vinyl monomer having a functional group that reacts with an amine and the reactivity thereof. In the present invention, the final capacity is 0.1 to 1 meq / g, preferably 0.2. Requires an adsorbent with an ion exchange capacity of ~ 0.8 meq / g. Therefore, the amount of the hydrophilic vinyl monomer having a functional group that reacts with an amine is 40 to 10% by weight, preferably 30 to 10% by weight, based on the total vinyl monomers. Other vinyl monomers added to improve the wettability and hydrogen bonding property of the crosslinkable polymer carrier include vinyl monomers having a hydroxyl group, vinyl monomers having an amide group, vinyl monomers having a cyanuric acid group, etc. However, when these vinyl monomers are mixed in a large amount, the hydrophobicity of the adsorbent is lowered. Therefore, the addition amount is 10% by weight or less based on the total vinyl monomers.

In the present invention, since a high adsorbing ability is required, a porous polymer carrier having a sufficient specific surface area is required. Therefore, at the time of copolymerization of the vinyl monomer, the copolymerization is carried out in the presence of a solvent serving as a pore regulator that is compatible with the vinyl monomer and does not contribute to the polymerization reaction. The pore regulator is necessary for making the produced polymer porous and increasing the specific surface area, and the pore diameter and the specific surface area are adjusted by the kind and amount of the pore regulator. The pore regulator is appropriately selected depending on the physical properties of the monomer used, but in general, aromatic hydrocarbons such as toluene and xylene, esters such as butyl acetate and dimethyl phthalate, amyl alcohol, octyl Alcohols that are not miscible with water such as alcohol, and paraffins such as octane and dodecane are used. If the amount of these pore regulators used is too small, sufficient pore diameter and specific surface area cannot be obtained. On the other hand, when the amount of the pore regulator is too large, the pore diameter becomes too large, the specific surface area is reduced and the mechanical strength is lowered, and further, the particles are not formed during the aqueous suspension polymerization. Occurs. Therefore, it is used in the range of 30 to 200 parts by weight, preferably 80 to 200 parts by weight, based on 100 parts by weight of the polymerizable monomer. Although the pore diameter and specific surface area of the porous crosslinkable polymer carrier depend on the properties of the adsorption target component and the coexisting component, in the present invention, as the pore properties at the time of non-swelling, an average pore diameter of 4 to 50 nm, A specific surface area of 100 to 1000 m ² / g is preferably used.

The introduction of an anion exchange group into the porous crosslinkable polymer carrier having a reactive functional group is carried out by dissolving the amine to be introduced in water, alcohol, dimethylformamide or a mixed solvent thereof and reacting it. An anion exchange group can be introduced by a reaction for 3 to 24 hours at room temperature to 80 ° C. while dispersing a porous crosslinkable polymer carrier having a functional group and stirring. The amine to be introduced is not particularly defined, but considering the dissociation property, it is preferable to use a secondary or tertiary low molecular amine. For example, secondary amines such as dimethylamine, diethylamine, dipropylamine, dibutylamine, N-methyl-2-aminoethanol, tritylamine, triethylamine, dimethylethylamine, dimethylpropylamine, dimethylbutylamine, N, N-dimethyl-2 -Tertiary amines such as aminoethanol are used.

Another form of the adsorbent used in the present invention is an adsorbent having both a hydrophilic functional group capable of developing a hydrophilic interaction and an ion exchange group. Hydrophilic interaction is the total of multiple interactions based on the polarity and hydrogen bond between the hydrophilic group of the adsorbent and the compound to be measured, and the distribution to the water phase retained by the adsorbent. It is a simple expression. The hydrophilic interaction is called HILIC, and in recent years, a separating agent for high performance liquid chromatography using this retention mechanism is commercially available. With these separating agents, it is possible to retain and separate polar compounds present in polar organic solvents. By using this retention mechanism, it is possible to extract a highly polar measurement target compound pre-extracted with a polar organic solvent. However, in the case where a high-polarity measurement target component is retained using hydrophilic interaction, there is a problem that the retention becomes weak when water is contained in the test solution. Therefore, in the present invention, it is combined with ion exchange interaction. Adsorption mechanism is used.

As the ion exchange interaction combined with the hydrophilic interaction, either a cation exchange interaction or an anion exchange interaction can be used. As described above, the anion exchange interaction is more preferable. The carboxyl group, sulfo group, phospho group, etc. present in the hydrophilic compound are functional groups that enhance retention to the adsorbent by hydrophilic interaction and that clearly express ion exchange interaction. The adsorbent can be strongly held by a combined action with a hydrophilic interaction. Therefore, in the present invention, a combination of hydrophilic interaction and anion exchange interaction is used.

Adsorbents that have both hydrophilic interaction and anion exchange interaction are hydrophilic vinyl monomers having a functional group that reacts with amines and hydrophilic crosslinkability having two or more vinyl groups. A compound capable of introducing a sulfo group by synthesizing a crosslinkable polymer carrier by copolymerization with a monomer, reacting with the introduced tertiary ammonium group after introducing an appropriate secondary amine into the crosslinkable polymer carrier. By reacting, an adsorbent having a highly hydratable betaine structure is produced. At this time, since the reaction of the compound capable of reacting with a tertiary ammonium group and introducing a sulfo group is not an equivalent reaction, the amount of hydrophilic interaction and the reaction can be controlled by adjusting the amount of the reaction reagent and the reaction conditions to control the introduction amount. An adsorbent having both anion exchange interaction can be produced.

Examples of the functional group of the hydrophilic vinyl monomer having a functional group that reacts with an amine include an alkyl halide group and an epoxy group. Examples of the reactive monomer having a halogenated alkyl group include 3-chloro-2-hydroxypropyl methacrylate, 3-chloro-2-hydroxypropyl acrylate, 2-chloroethyl methacrylate, 2-chloroethyl acrylate, and the like. Examples of the functional monomer having an epoxy group include glycidyl methacrylate and glycidyl acrylate. Examples of hydrophilic crosslinkable monomers having two or more vinyl groups copolymerizable with these monomers include ethylene dimethacrylate, diethylene glycol dimethacrylate, glycerin dimethacrylate, trimethylolpropane trimethacrylate, and neopentyl glycol trimethacrylate. Functional methacrylate monomers, ethylene diacrylate, diethylene glycol diacrylate, glycerin diacrylate, trimethylol propane triacrylate, neopentyl glycol triacrylate, pentaerythritol triacrylate, pentaerythritol tetraacrylate, and other polyfunctional acrylate monomers, Has a cyanuric acid skeleton such as allyl isocyanurate or trimethallyl isocyanurate. Such as cross-linking monomer, and the like. In addition, a third vinyl monomer can be added in order to improve physical properties such as hydrophilicity and hydrogen bonding property of the crosslinkable polymer carrier. Examples of vinyl monomers for improving physical properties include amide monomers such as N, N-dimethylacrylamide, N, N-diethylacrylamide, and morpholinoacrylamide, 2-hydroxyethyl methacrylate, glycerin methacrylate, neopentyl glycol methacrylate, and trimethylolpropane methacrylate. And methacrylate monomers such as N-vinylpyrrolidone. It is also possible to add a vinyl monomer exhibiting anion exchange properties such as N, N-diethylaminoethyl methacrylate.

In the adsorbent having both the hydrophilic interaction and the anion exchange interaction of the present invention, the amount of the vinyl monomer having a functional group that reacts with the amine is 20 to 70% by weight, preferably 30 to The hydrophilic crosslinkable monomer having 2 or more vinyl groups is used at least 30% by weight or more in order to ensure sufficient hardness. Moreover, it is preferable to use 0-20 weight% of other vinyl monomers added in order to improve physical properties, such as a hydrophilic property and hydrogen bonding property of a crosslinkable polymer carrier.

In the present invention, since a high adsorbing ability is required, a porous polymer carrier having a sufficient specific surface area is required. Therefore, at the time of copolymerization of the vinyl monomer, the copolymerization is carried out in the presence of a solvent serving as a pore regulator that is compatible with the vinyl monomer and does not contribute to the polymerization reaction. The pore regulator is appropriately selected depending on the physical properties of the monomer used, but in general, aromatic hydrocarbons such as toluene and xylene, esters such as butyl acetate and dimethyl phthalate, amyl alcohol, octyl Alcohols that are not miscible with water such as alcohol, and paraffins such as octane and dodecane are used, and they are used in an amount of 30 to 200 parts by weight, preferably 80 to 200 parts by weight, based on 100 parts by weight of the polymerizable monomer. Although the pore diameter and specific surface area of the porous crosslinkable polymer carrier depend on the properties of the adsorption target component and the coexisting component, in the present invention, as the pore properties at the time of non-swelling, an average pore diameter of 4 to 50 nm, A specific surface area of 100 to 1000 m ² / g is preferably used.

The reaction between the porous crosslinkable polymer carrier having the reactive functional group and the secondary amine is carried out by the same method as the introduction of the anion exchange group described above. That is, the secondary amine to be introduced is dissolved in water, alcohol, dimethylformamide or the like or a mixed solvent thereof, and the porous crosslinkable polymer carrier having a reactive functional group is dispersed and stirred, A tertiary ammonium group can be introduced by a reaction at room temperature to 80 ° C. for 3 to 24 hours. The secondary amine to be introduced is not particularly defined, but considering its hydrophobicity and reactivity, it is preferable to use a secondary amine having a small alkyl group such as dimethylamine or diethylamine.

Introduction of the sulfo group into the tertiary ammonium group introduced into the crosslinkable polymer carrier is performed by any of the following methods.
(I) A crosslinkable polymer carrier having a tertiary ammonium group introduced therein is dispersed in a suitable alkaline solution, a halogenated alkylsulfonic acid such as 2-bromoethanesulfonic acid is added, and the mixture is stirred at 40-80 ° C. React for 3-8 hours.
(Ii) After sufficiently drying the crosslinkable polymer carrier having the tertiary ammonium group introduced therein, disperse it in a suitable solvent such as dehydrated acetone, add propane sultone, and stir at room temperature to 50 ° C. React for 3-8 hours.
By the above reaction, it is possible to synthesize an adsorbent having a hydrophilic interaction with a highly hydrated sulfobetaine group. At this time, since the reaction for introducing the sulfo group is not an equivalent reaction, an unreacted tertiary ammonium group remains in the crosslinkable polymer carrier into which the tertiary ammonium group has been introduced. And an anion exchange interaction can be obtained. The introduction amount of the sulfo group, that is, the remaining amount of the tertiary ammonium group is adjusted by the introduction reaction conditions (reagent amount, reagent concentration, reaction temperature, etc.) of the sulfo group.

The adsorbent used in the present invention is used by being packed into a cylindrical or square cylindrical small capacity column. FIG. 2 shows the structure of the small capacity column 1 of the present invention. The adsorbent 1a of the present invention is filled in the empty column body 1b in which the lower frit 1e is inserted, and after the upper frit 1d is inserted, the empty column cap 1c is inserted and fixed. The empty column body 1b is required to transmit light emitted from the measurement target compound extracted and fixed to the adsorbent 1a, and therefore must be a light-transmitting material. The material itself is not particularly limited, but usable materials include glass, polystyrene (PS), polymethyl methacrylate (PMMA), transparent vinyl chloride (PVC), cycloolefin polymer (COP), cycloolefin copolymer (COC), Polypropylene (PP), polyethylene terephthalate (PET), fluororesin, etc. can be used. Actually, it is selected in consideration of the wavelength of the light to be detected and the resistance to the solvent used. If light transmittance is selected, a material having a light transmittance at 300 nm of 75% or more and a total light transmittance of 85% or more is preferable. For example, polymethyl methacrylate (PMMA), cycloolefin polymer (COP) and cycloolefin copolymer (COC) are preferred. In addition, when it is difficult to manufacture the empty column body 1b integrally with the lower connection portion with a material having high light transmittance, the body 2b (FIG. 3) is separated as shown in FIG. You may produce using a material with high light transmittance. In FIG. 3, 2a to 2g correspond to 1a to 1g in FIG. 2, and 2h denotes an empty column lower cap.
Here, the “small volume” indicates that the volume of the space that can be filled with the adsorbent for adsorbing the measurement target compound is 2 mL or less. This method uses a highly selective adsorbent to adsorb and concentrate on the tip of the adsorbent packed bed, and then detects fluorescence or chemiluminescence, so that the target component can be reliably adsorbed without breaking through. A column having a volume capable of being packed with a possible amount of adsorbent may be used. When the volume of the adsorbent-filled portion is 2 mL, a maximum of about 1 g of a polymer-based solid phase extractant can be filled, but even if an adsorbent amount larger than this is used, only the adsorbent that is not involved in the adsorption is increased. It is. Furthermore, since the volume of the washing solution, derivatization, and chemiluminescence reagent is increased as the column volume increases, the volume is preferably 2 mL or less. If a selective adsorbent capable of tip adsorption / concentration is used, it is possible to adsorb the measurement target compound in an amount necessary for detection at an adsorbent amount of 20 to 200 mg. In this range, the volume of the adsorbent packed portion of the small capacity column is 0.05 to 0.5 mL.

The present invention provides a screening method capable of easily measuring agricultural chemicals and additives, and also provides a portable screening apparatus for performing screening easily and accurately. The screening apparatus includes: 1) a sample chamber in which a column of 2 mL or less filled with an adsorbent for adsorbing and concentrating a compound to be measured can be mounted; and 2) a pump system for feeding a derivatization reagent and a cleaning solution. 3) a light source and an optical system for irradiating excitation light, or a pump system for feeding a chemiluminescent reagent, 4) a detector for detecting the amount of fluorescence or chemiluminescence from a small volume column, and 5) a measurement object A portable pesticide and additive screening apparatus comprising an arithmetic circuit that calculates the concentration of a measurement target compound from the amount of light emitted from the compound.

4 and 5 show the basic configuration of the sample unit and the detection unit of the screening apparatus of the present invention. FIG. 4 shows the configuration of the sample part and the detection part in the fluorescence detection method, and FIG. 5 shows the chemiluminescence detection method. A small-capacity column 1 in which the compound to be measured is fixed on the adsorbent surface is set in the sample chamber 11 of the apparatus shown in FIG. 4 or FIG. 5, and the amount of light emitted by the fluorescence method or the chemiluminescence method is detected. A small-capacity column 1 in which a measurement target compound is fixed to the adsorbent surface is set between an upper connector 21 and a lower connector 23 of a sample chamber 11. An optical fiber 33 is inserted into the upper portion of the small-capacity column 1 and irradiated with light (excitation light) from the light source 31. In general, light of 200 to 400 nm is used as excitation light in fluorescence detection, and a xenon lamp or a mercury lamp is used as a light source lamp. In the present invention, it is desirable to use an LED or a mercury lamp which is a portable device and is easy to handle. In particular, in order to reduce various problems relating to the light source power source, it is preferable to use LEDs. However, since the LED emits a wide range of light, a cut-off filter for cutting the upper and lower light may be attached to the light source 31 as necessary. Simultaneously with the irradiation of the excitation light, the shutter 14 is opened by the shutter switch 15, and the generated fluorescence passes through the cutoff filter 13 for cutting the excitation light and is composed of a photodiode or a photomultiplier and is held by the detector holder 17. Detected by the detector 16 and sent to a calculator (not shown) via the detection signal line 18 and converted into a concentration. In the figure, 12 is a light receiving chamber, 22 is a connection joint, and 32 is a condenser lens. An example of the configuration of the apparatus in the case of detecting by the chemiluminescence method is shown in FIG. In the case of the chemiluminescence method, the configuration of the apparatus is almost the same. However, in order to remove background light emission due to only the chemiluminescence reagent, the cut-off filter 13 may be necessary. Further, since it is not photoexcitation, the light source 31 and the optical fiber 33 are not necessary. Instead, a chemiluminescent reagent is fed through a reagent introduction tube 34 by a liquid feed pump (not shown), and the amount of light emitted at that time is fluorescent. Detect as in the method and convert to concentration.

Here, a specific procedure of the screening method in the present invention will be described. First, the basic pretreatment procedure is as follows. Since this method is a screening method, pretreatment can be simplified compared to the instrumental analysis method and at a level that can be handled at the measurement site. It can be simplified. FIG. 6 shows a flow of a basic pretreatment operation procedure of this method. Basically, there are four steps of pre-extraction → deproteinization → centrifugation → loading a small volume column. If necessary, dilute and filter after pre-extraction, and filter and adjust liquidity after centrifugation.

As shown in FIG. 7, the test solution can be loaded onto the small-capacity column filled with the adsorbent using an injection cylinder 41 having an appropriate capacity. By passing the test solution through a small-capacity column filled with an adsorbent, the compound to be measured is adsorbed and fixed on the adsorbent, and then unnecessary impurities are washed. Any cleaning liquid can be used as long as the compound to be measured does not elute from the adsorbent. In general, alcohols such as methanol and acetonitrile can be used to remove organic substances. When salts remain, washing is performed using pure water. However, in the case of an extraction mechanism based on hydrophilic interaction, when the pure water is 100%, the retained measurement target compound is likely to elute, so a mixture of polar organic solvents such as alcohols and acetonitrile and water. Use solvent. As shown in FIG. 7, the cleaning liquid is also passed through the syringe barrel 41 or the like. These sample loading and washing steps may be performed using an appropriate sample feed pump 42 such as a peristaltic pump as shown in FIG. 8, or using a suction manifold 47 as shown in FIG. Is possible. In FIG. 8, 43 is a sample solution, 44 is a cleaning solution, 45 is a pipe, and 46 is a connection joint. In FIG. 9, 48 is a pressure reducing gauge, 49 is a pressure adjusting valve, 50 is a suction joint, 51 is a receiver, 52 is a cock, and 53 is a reservoir. It can also be automated by using an existing automatic extraction device, automatic solid phase extraction device, or the like.

In the present invention, a compound to be measured in a test solution is extracted, concentrated, and fixed on a small-capacity column, and then mounted on the screening apparatus of the present invention to detect fluorescence or chemiluminescence. A flow of a basic operation procedure is shown in FIG. The cut-off filter is selected according to the selection of the measurement target compound and the selection of the fluorescence method or the chemiluminescence method. Further, whether to perform the derivatization reaction is selected depending on whether the measurement target compound is fluorescent (or chemiluminescent). When the measurement target compound is detected by the fluorescence method, excitation light is irradiated to a small-capacity column, and when it is detected by the chemiluminescence method, the amount of luminescence emitted through the chemiluminescence reagent is detected. On the other hand, if it is not fluorescent (or chemiluminescent), the derivatization reagent is passed through for derivatization, and then the excess derivatization reagent is washed, and the amount of luminescence based on fluorescence or chemiluminescence is similarly detected. To do.

FIG. 11 is a diagram showing an apparatus configuration in the case of measuring by a fluorescence method requiring derivatization. A small-capacity column 1 on which a compound to be measured is extracted, concentrated and fixed is sandwiched and set between an upper connector 21 and a lower connector 23 of the sample chamber 11. The reagent introduction tube 34 is inserted into the upper part of the small volume column 1, the derivatization reagent 62 is fed by the liquid feed pump 61, and the derivatization reaction of the measurement target compound is performed in the small volume column 1. After completion of the derivatization reaction, the excess reagent cleaning solution 63 is switched to and fed by the feed pump 61 to wash the excess derivatization reagent in the small-capacity column 1. After cleaning, the waste liquid port 24 is closed to prevent liquid outflow and light incidence. Thereafter, the reagent introduction tube 34 is removed from the upper portion of the small-capacity column 1, the optical fiber 33 is attached, and the light from the excitation light source 31 is irradiated. At the same time, the shutter 14 is opened, and the amount of emitted fluorescence is changed to a photodiode or photo. Detection is performed by a detector 16 made of a multiplier, and the detected electric signal is sent to a calculation unit (not shown) to obtain the concentration. At this time, if there is interference with the excitation light, the light enters the detector 16 through the cutoff filter 13.

FIG. 12 is a diagram showing an apparatus configuration in the case of measuring by a chemiluminescence method requiring derivatization. The basic operations for derivatization and washing of the overderivatized reagent are the same as in the fluorescence method. After the compound to be measured is derivatized, the chemiluminescent reagent 64 is fed to the small-capacity column 1 by the feed pump 61, and at the same time the shutter 14 is opened, and the amount of emitted chemiluminescence is detected by a photodiode or photomultiplier. The detected electric signal is sent to a calculation unit (not shown) to obtain the concentration. In the case of the chemiluminescence method, since the feeding of the chemiluminescent reagent 64 is continued during detection, the waste liquid port 24 is not closed.

As described above, by using the screening method and screening apparatus of the present invention, it becomes possible to easily screen for agricultural chemicals and additives in food, feed, or environmental samples. Next, the present invention will be described with reference to examples, but the present invention is not limited to the examples.

Chemiluminescence detection of amino acid derivatives using an adsorbent having hydrophobic interaction and anion exchange interaction (1) Production of small-capacity column A packed with an adsorbent having hydrophobic interaction and anion exchange interaction The crosslinkable polymer carrier was synthesized by an aqueous suspension polymerization method. A mixture obtained by dissolving 2 g of 2,2′-azobisisobutyronitrile in a mixed solution of 30 g of glycidyl methacrylate, 10 g of N, N-dimethylacrylamide, 160 g of divinylbenzene, 170 g of toluene and 30 g of 1-dodecanol was added to 0.1% polyvinyl. In addition to 2,000 mL of alcohol aqueous solution, the stirring blade was rotated at 450 rpm using the stirrer, and the oil layer was disperse | distributed. Thereafter, the dispersion (suspension) was heated, and a polymerization reaction was performed at 80 ° C. for 6 hours. The produced copolymer particles were collected by filtration and washed with water and methanol in this order. After air drying for one day, classification was performed to obtain 80 g of a crosslinkable polymer carrier having a size of 32 to 90 μm. 70 g of the obtained crosslinkable polymer carrier was added to a solution of 30 g of N, N-dimethylethylamine dissolved in 200 mL of isopropyl alcohol and 800 mL of water, and reacted at 40 ° C. for 6 hours to introduce an anion exchange group. After completion of the reaction, the mixture was washed with water, methanol, and water in this order, the counter ion was exchanged with 0.1N hydrochloric acid, and then washed thoroughly with water. After that, it was replaced with methanol and dried to obtain an adsorbent having hydrophobic interaction and anion exchange interaction. The anion exchange capacity of this adsorbent was determined by a back titration method and found to be 0.34 meq / g. The specific surface area and the average pore diameter were 645 m ² / g and 5.2 nm, respectively. A polyethylene filter is inserted into the inside of an empty column body made of cycloolefin polymer (COP) having an inner diameter of 6 mm and a length of 25 mm with a connecting portion at the bottom, and the obtained adsorbent is filled with 100 mg, and the upper part of the packed bed is also made of polyethylene. A filter was inserted, and a connection part was attached to the upper part to produce a small-capacity column A.

(2) Chemiluminescence detection of dansylated glycine Using the small-capacity column A prepared in (1), chemiluminescence detection of dansyl glycine was attempted. Conditioning was performed by passing water 10 mL, methanol 10 mL, water 10 mL, 0.1 M aqueous sodium hydroxide solution 10 mL, and water 10 mL through the small column A prepared in (1) in this order. Thereafter, 5 mL of a 1 μg / mL dansyl glycine solution prepared with water was passed therethrough, and the dansyl glycine was adsorbed to the adsorbent of the small volume column A. Thereafter, 5 mL of water was passed through the small-capacity column A for washing. Add 0.1 mL of 0.05 M bis (2,4,6-trichlorophenyl) oxalate (TCPO) in ethyl acetate to column A with a small volume adsorbed with dansylglycine, and then add 30% hydrogen peroxide solution. Chemiluminescence detection of dansylglycine adsorbed on a small volume of column A was performed. After a signal based on chemiluminescence was detected, the TCPO mixed solution was injected again. The obtained chemiluminescence profile is shown in FIG. Immediately after injecting the TCPO solution, chemiluminescence was detected, and it was confirmed that chemiluminescence was generated on the adsorbent surface. Even when the TCPO solution was subsequently injected, chemiluminescence with the same intensity was detected, and it was confirmed that measurement could be performed with good reproducibility. At this time, the fluorescence of the passing liquid and the washing liquid when the dansyl glycine solution was passed was measured, but dansyl glycine was not detected, and the adsorbent having the hydrophobic interaction and the anion exchange interaction prepared in (1) was used. It was confirmed that it was strongly captured.

Fluorescence detection of antibiotics using an adsorbent having hydrophilic interaction and anion exchange interaction (3) Production of small capacity column B packed with adsorbent having hydrophilic interaction and anion exchange interaction Glycidyl A mixture obtained by dissolving 2 g of 2,2′-azobisisobutyronitrile in a mixed solution of 100 g of methacrylate, 80 g of ethylene dimethacrylate, 20 g of N, N-dimethylacrylamide and 120 g of butyl acetate, In addition to 000 mL, the stirring blade was rotated at 230 rpm using a stirrer to disperse the oil layer. Thereafter, the dispersion (suspension) was heated, and a polymerization reaction was carried out at 70 ° C. for 7 hours. The produced copolymer particles were collected by filtration and washed with water and methanol in this order. After air drying for one day, classification was performed to obtain 75 g of a crosslinkable polymer carrier having a thickness of 45 to 90 μm. 70 g of the resulting crosslinkable polymer carrier was placed in a solution obtained by adding 200 mL of isopropyl alcohol and 40 mL of water to 40 mL of a dimethylamine solution (approximately 50% aqueous solution), and reacted at room temperature for 6 hours to introduce a tertiary ammonium group. . After completion of the reaction, it was washed with water and methanol in that order and dried. 10 g of the obtained resin particles were added to a 50 M solution of 0.5 M sodium hydroxide in which 15 g of sodium 2-bromoethanesulfonate was dissolved, and a polymerization reaction was performed at 50 ° C. for 6 hours. After completion of the reaction, the mixture was washed with water, methanol and water in this order, the counter ion was exchanged with 0.1N hydrochloric acid, and the mixture was thoroughly washed with water. Then, after substituting with methanol, it was dried to obtain an adsorbent having hydrophilic interaction and anion exchange interaction. It was 0.27 mmol / g when the amount of sulfone groups of this adsorbent was determined by a titration method. Moreover, the specific surface area and the average pore diameter were 180 m ² / g and 12.1 nm, respectively. With this adsorbent, 100 mg of the same empty column as in Example 1 was packed to produce a small column B.

(4) Fluorescence detection of tetracycline antibiotics Using the small-capacity column B prepared in (3), tetracycline fluorescence detection was attempted. Conditioning was performed by passing 10 mL of water and 10 mL of 95% acetonitrile-water through the small-volume column B prepared in (3) in this order. Thereafter, 1 mL of a 0.01 mg / mL tetracycline solution prepared with 95% acetonitrile-water was passed therethrough, and the tetracycline was adsorbed to the adsorbent of the small volume column A. Thereafter, washing was performed by passing 5 mL of 95% acetonitrile-water through column B having a small volume. The column B having a small capacity on which tetracycline was adsorbed was irradiated with LED light (wavelength: 360 nm) to detect tetracycline fluorescence from a right angle direction. At this time, a cut-off filter for cutting light of 400 nm or less and 600 nm or more was attached to the excitation light of the LED. In addition, a cut-off filter that cuts light of 400 nm or less was attached to the fluorescent side, and fluorescence was detected. The excitation light was irradiated for 15 seconds, 10 seconds, and 10 seconds, and the same column was irradiated three times. The obtained fluorescence profile is shown in FIG. Fluorescence was detected for a time corresponding to the excitation light irradiation time, and it was confirmed that fluorescence was generated on the adsorbent surface. Even when the excitation light was subsequently irradiated, fluorescence having the same intensity was detected for a time corresponding to the irradiation time. Moreover, the fluorescence measurement was performed using the fluorescence spectrophotometer of the passing liquid and the cleaning liquid when the tetracycline solution was passed, but tetracycline was not detected, and the hydrophilic interaction and the anion exchange interaction prepared in (3) were observed. It was confirmed that the adsorbent had a strong trap.

(5) Detection of tetracycline in honey Tetracycline in honey was measured based on the result of (4). 10 mL of acetonitrile was added to 5 g of honey and stirred vigorously for 5 minutes, and then allowed to stand for 10 minutes. The supernatant was taken and tetracycline remaining in honey was measured by the same method as in (4). At this time, a sample in which 1 μg of tetracycline was added to honey was also prepared, and the addition recovery rate was examined by the same method as in (4). Tetracycline was not detected in the honey used here, but the recovery rate of addition was 75 to 100%, giving good results. From this result, it was found that tetracycline can be selectively detected without being affected by impurities in the test sample.

According to the present invention, it is possible to measure food, feed, environmental pesticides, additives, and the like with high sensitivity by a simple and quick method without using an expensive analytical instrument that requires specialized knowledge. is there. By using the screening method and screening device of the present invention, it is possible to measure trace amounts of residual pesticides and additives / contaminants on-site, so multiple samples can be measured in an analysis time adapted to the distribution cycle. It becomes. Although the screening method of the present invention is not applicable to all existing pesticides, etc., it can be applied to the screening of many pesticides and additives because it obtains the results grouped by the structure and functional group of the compound to be measured. Is possible. Furthermore, the compound to be measured can be expanded by the combined use with existing immunoassay methods. In addition, since the measurement method of the present invention is a screening method and may contain some positive errors, it is an analysis method that can be positioned as a preliminary test method for precision analysis, and shortens the overall analysis time. Along with cost reduction, it can contribute to ensuring food safety.

1: Column (solid phase extraction cartridge: body integrated type)
1a: Adsorbent (solid phase extraction agent)
1b: Empty column body (integrated type)
1c: Empty column cap 1d: Upper frit 1e: Lower frit 1f: Upper connection (solution inlet)
1g: Lower connection (solution outlet)
2: Column (solid phase extraction cartridge: body independent type)
2a: Adsorbent (solid phase extractant)
2b: Empty column body (independent type)
2c: Empty column upper cap 2d: Upper frit 2e: Lower frit 2f: Upper connection (solution inlet)
2g: Lower connection part (solution outlet)
2h: Empty column lower cap 11: Sample chamber 12: Light receiving chamber 13: Cut-off filter 14: Shutter 15: Shutter switch 16: Detector (photodiode, photomultiplier)
17: Detector holder 18: Detection signal line 21: Upper connector 22: Connection joint 23: Lower connector 24: Waste liquid port 31: Light source (LED or mercury lamp)
32: Condensing lens 33: Optical fiber 34: Reagent introduction tube 41: Injection cylinder 42: Sample feeding pump 43: Sample solution 44: Cleaning solution 45: Pipe 46: Connection joint 47: Suction manifold 48: Decompression gauge 49: Pressure Adjustment valve 50: suction part joint 51: receiver 52: cock 53: reservoir 61: liquid feed pump 62: derivatization reagent solution 63: cleaning solution 64: chemiluminescence solution

Claims (7)

Measurement is performed after adsorbing and concentrating the target compound in the test solution on the surface of the adsorbent packed in a small-capacity column with a volume of 2 mL or less that can be filled with an adsorbent for adsorbing the target compound. An agrochemical characterized in that the concentration of a measurement target compound is obtained from the amount of luminescence by emitting light by a fluorescence method or chemiluminescence method with the measurement target compound fixed on the surface of the adsorbent without detaching the target compound from the adsorbent. And additive screening methods. 2. The measurement target compound is a fluorescent or chemiluminescent pesticide and additive, or an agrochemical and additive that becomes fluorescent or chemiluminescent by reaction with a derivatizing reagent. Screening methods for pesticides and additives. After a non-fluorescent or non-chemiluminescent compound to be measured is adsorbed and immobilized on the surface of the adsorbent, it is induced to a fluorescent or chemiluminescent compound by a chemical reaction with a derivatization reagent suitable for the compound to be measured. The pesticide according to claim 1 or 2, wherein the produced derivative is caused to emit light by a fluorescence method or a chemiluminescence method without being desorbed from the adsorbent while adsorbed and immobilized on the surface of the adsorbent. Additive screening method. The adsorbent is an adsorbent having both a hydrophobic functional group capable of developing a hydrophobic interaction and an ion exchange group capable of developing an ion exchange interaction. The screening method of the agrochemical and additive as described in any one of thru | or 3. The adsorbent is an adsorbent having both a hydrophilic functional group capable of developing a hydrophilic interaction and an ion exchange group capable of developing an ion exchange interaction. The screening method of the agrochemical and additive as described in any one of thru | or 3. The portion filled with the adsorbent of the small-capacity column is made of a light-transmitting material, and the shape thereof is a cylindrical shape or a rectangular tube shape, and has a liquid inlet / outlet at the top and bottom. The screening method of the agrochemical and additive as described in any one of Claim 1 thru | or 5. A sample chamber that can be equipped with a small-capacity column of 2 mL or less filled with an adsorbent that adsorbs and concentrates the compound to be measured, a pump system that delivers a derivatization reagent and a cleaning solution to the small-capacity column, and excitation Light source and optical system for irradiating light, or pump system for feeding a chemiluminescent reagent, detector for detecting fluorescence or chemiluminescence from a small-capacity column, and measurement target based on the amount of detected fluorescence or chemiluminescence And an arithmetic circuit for calculating the concentration of the compound, wherein the screening method for agricultural chemicals and additives according to any one of claims 1 to 6 can be applied, and is configured to be portable. A screening device for pesticides and additives.

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