JP2013253790A - Analytical method of nivalenol - Google Patents

Abstract

【Task】
In chemical analysis methods for quantifying nivalenol, a kind of mycotoxin (mold poison) in foods and feeds, nivalenol present in a trace amount in a test sample can be accurately, quickly and easily without complicated operations. Provides a pretreatment method that can be extracted and purified.
[Solution]
Take the sample in a centrifuge tube, add water, swell well, extract with shaking with acetonitrile, add the adsorbent for dye removal and salting-out agent to the supernatant after centrifugation, and shake and centrifuge again. The supernatant is passed through a solid-phase extraction cartridge filled with a solid-phase extraction agent having a weak ionic zwitterionic functional group to capture nivalenol, and then the solid-phase extraction cartridge is washed with an appropriate washing solution. The sample solution obtained by the pretreatment of eluting nivalenol captured by the solid phase extraction cartridge with water is quantified using high performance liquid chromatography-ultraviolet absorption detection.
[Selection] Figure 1

Description

The present invention relates to a chemical analysis method for quantifying nivalenol in food or feed.

Mycotoxin is a poison produced as a secondary metabolite of mold, and more than 300 types are currently known. Typical mycotoxins include trichothecenes such as aflatoxin, ochratoxin, nivalenol (hereinafter referred to as “NIV”) and deoxynivalenol (hereinafter referred to as “DON”), patulin, and fumonisin. When humans or animals ingest mycotoxins, serious health damage may occur in the liver, kidneys, gastrointestinal tract, etc., and in severe cases, death may occur. These mycotoxins are harmful factors that contaminate agricultural products, and fungi that are phytopathogenic fungi contaminate stored grains and the like during production and storage. NIV and DON are produced by Fusarium fungi, which are pathogens of red mold disease that cause deterioration in the quality of wheat and wheat (wheat and barley). Japan is in an environment where there is a lot of rainfall during the late stage of wheat growth and red mold disease is likely to occur. In Japan, acute red mold intoxication has frequently occurred among people and livestock who have eaten rice and wheat damaged by red mold disease in the 1950s. At present, the damage of red mold disease is decreasing. However, when red mold disease spreads due to weather conditions, there is a possibility that pollution or health problems due to NIV or DON may occur. Since it is difficult to completely remove and decompose NIV and DON even in processing and cooking processes, it is important to prevent contamination at the production stage. In 2002, the Ministry of Health, Labor and Welfare established a DON provisional standard (1.1 mg / kg) for wheat (Non-patent Document 1). In 2002, the Ministry of Agriculture, Forestry and Fisheries established the provisional allowance of DON for feed (feed fed to cattle over 3 months of age: 4 ppm, feed fed to livestock excluding cats over 3 months of age: 1 ppm) In 2008 (Non-patent Document 2), an implementation standard “Guidelines for reducing deoxynivalenol / nivalenol contamination of wheat” was formulated (Non-patent Document 3). Although provisional reference values have been set for DON, NIV exposure in Japan is below the tolerable daily intake (TDI) set by the Food Safety Commission. No standard value has been set from the viewpoint that the intake of NIV from food in general Japanese is considered unlikely to adversely affect health. However, considering that NIV toxicity is 10 times higher than that of DON and red mold disease is likely to occur in Japan's climate of high temperature and high humidity, strict management and regulation for NIV is necessary.

A review (non-patent document 4) has been published regarding the analysis of mycotoxins, and analysis methods using gas chromatography (hereinafter referred to as “GC”) and liquid chromatography (hereinafter referred to as “HPLC”) are mainly used. Yes. For DON, the Ministry of Health, Labor and Welfare has issued a notification test method (hereinafter referred to as “notification method”) and is widely used (Non-Patent Document 1 and Non-Patent Document 5). In the notification method (Non-Patent Document 1), a high-performance liquid chromatograph with an ultraviolet spectrophotometric detector (hereinafter referred to as “HPLC-UV method”) is used as a qualitative and quantitative test, and an HPLC-mass spectrometer (hereinafter referred to as a confirmation test). , "HPLC-MS method") or GC-mass spectrometer (hereinafter referred to as "GC-MS method"). FIG. 5 shows an outline of a notification method related to DON. In the notification method, a multi-functional column is used to remove contaminant components. However, since the contaminant components are not sufficiently removed even in wheat, the HPLC-UV method (detection detection wavelength 220 nm) also contains contaminant components around DON. It will be detected. For this reason, the HPLC-MS method or the GC-MS method is used as a confirmation test, but it is not only complicated to perform two tests, but there are many problems in terms of equipment, maintenance cost, and analysis time. . On the other hand, for NIV, there is no official test method such as the DON notification method. However, when NIV is measured according to the DON notification method, the NIV is accurately determined because the removal of contaminant components is insufficient. It is difficult to quantify. It can be quantified by using an HPLC-tandem mass spectrometer (MS / MS) (Non-Patent Document 4). However, such an instrument is very expensive and has a high maintenance cost. It is not an effective technique for mycotoxins that require on-site analysis and simple and rapid analysis.

By the way, the QuEChERS method has attracted attention as a technique for eliminating the complexity of pretreatment in the analysis of pesticide residues in foods. The QuEChERS method is a combination of the initial letters of Quick, Easy, Cheap, Effective, Rugged, and Safe (Non-patent Document 6). In this method, a disposable centrifuge tube is filled with a sample and acetonitrile as an extraction solvent (basically the same amount as the sample), magnesium sulfate for salting-out dehydration, and an adsorbent for removing impurities. It is a very simplified pretreatment method that involves mixing, shaking, centrifuging, and using the supernatant as a test solution, and it is said that many pesticides can be extracted by this method. By using this method, the pretreatment for residual pesticide analysis, which has conventionally taken 1 to 2 hours, is completed in about 30 minutes, which is promising not only for simplification of pretreatment but also for labor saving. . Although simplification of such pretreatment is also expected in the pretreatment of mycotoxins, there is no report on a pretreatment technique for mycotoxin analysis using the QuEChERS method. In addition, the QuEChERS method has been pointed out to be problematic in that the extraction and recovery rate of highly polar pesticides is low, and many contaminants are eluted around the elution position of polar pesticides, which hinders measurement. The QuEChERS method was developed for a HPLC-MS or GC-MS method capable of selective detection, and when this method is applied to a general-purpose HPLC-UV method, interference by contaminating components is extremely high. To be serious. Therefore. Further ingenuity is required to apply to the analysis of highly water-soluble NIV.

Immunoaffinity columns packed with adsorbents with selective reactive antibodies are commercially available for pretreatment methods for the analysis of aflatoxins and ochratoxins, which are a kind of mycotoxins. For pretreatment of NIV and DON These immunoaffinity columns are also commercially available. This is a method in which a measurement sample is passed through an immunoaffinity column, contaminated components are removed by washing a buffer solution, and then eluted with methanol and separated and quantified by the HPLC-UV method. Although the pretreatment is a simple method, since NIV and DON can be extracted only from an aqueous solution, it cannot be applied to a pretreatment of a sample solution obtained by organic solvent extraction as in the notification method. However, since it is difficult to efficiently extract NIV from a food sample or the like using an extraction solvent as an aqueous solution, there is a problem that a new extraction method that is completely different from existing methods must be developed. In addition, this immunoaffinity column is disposable for only one sample, and there is a problem that its cost is very high.

As another method for detecting NIV and DON, a method for detecting and quantifying trichothecene mycotoxins using a monoclonal antibody having high affinity for trichothecene mycotoxins or an antibody fragment thereof (Patent Document 1), T cells and specimens There is a method for detecting and quantifying trichothecene-based mycotoxins by inducing apoptosis in the T cells by contacting them and measuring the induced apoptosis (Patent Document 2). Since these methods are detected by a recognition mechanism having high specificity for trichothecene-based mycotoxins, they are characterized by being less susceptible to interference by contaminating components. However, since trichothecene mycotoxins are comprehensively detected and quantified, individual quantitative values cannot be obtained.

Japanese Patent Publication No. 2005-245211 Table 01/018196

Notification of Food Safety Department No.0521001, Food Safety Department, Ministry of Health, Labor and Welfare, dated May 21, 2002 July 5, 2002, 14 Livestock No. 2267 Notice of Director of Feed Division, Ministry of Agriculture, Forestry and Fisheries Date of December 17, 2008, No. 20 Yasushi No. 8915, No. 20 Production No. 5731, Ministry of Agriculture, Forestry and Fisheries Nobuko Tabata, “Analysis Method of Mold Toxins”, Bunseki, 2008, No. 10, p. 531 (2008). Date of July 17, 2003, Food Safety No. 071001 Notice from the Ministry of Health, Labor and Welfare M.M. Anastasides, S.A. J. et al. Lehotay, Journal of AOAC International, vol. 86, no. 2, p. 412 (2003) Y. Inoue, W.H. Kamichitani, M.M. Saito, Y .; Kobayashi, A.A. Yamamoto, Chromatographia, vol. 73, no. 9-10, p. 849 (2011). Yasuyuki Kobayashi, Jun Yamamoto, Waka Kamachaya, Yoshinori Inoue, Analytical Chemistry, vol. 60, no. 8, p. 635 (2011).

The present invention has been made in view of the above-described problems. In a chemical analysis method for quantifying NIV, which is a trichothecene mycotoxin in food or feed, a trace amount can be contained in a test sample without performing complicated operations. It is an object of the present invention to provide a pretreatment method and an analytical method using an HPLC-UV method, which can extract and purify NIV present in the plant with high accuracy, quickly and easily.

As a result of earnest research to provide an analytical method capable of quantifying NIV, which is a trichothecene-based mycotoxin, quickly, inexpensively and easily, the present inventors have developed a novel pre-extraction process and made the pre-extraction liquid hydrophilic. Purification by using a solid phase extractant with a weakly ionic zwitterionic functional group introduced into an ionic substrate (hereinafter referred to as “zwitterion type solid phase extractant”), which is disturbed by contaminant components The present inventors have found that NIV can be quantified with high accuracy and easily by the HPLC-UV method, and have completed the present invention.

In the chemical analysis of nivalenol in food or feed, the present invention uses a solid-phase extraction cartridge filled with a solid-phase extraction agent having a weakly ionic zwitterionic functional group, so that This is an analytical method that extracts and purifies nivalenol and analyzes it. Take a sample in a centrifuge tube, add water, swell well, extract with shaking with acetonitrile, and remove the pigment in the extract after centrifugation. The pre-extraction step of adding the adsorbent and salting-out agent and shaking and centrifuging again to obtain the supernatant, and passing the supernatant obtained in the pre-extraction step through the solid-phase extraction cartridge After capturing nivalenol, the purification step of washing the solid phase extraction cartridge with a washing solution to remove contaminant components and eluting nivalenol captured by the solid phase extraction cartridge with water; The sample solution obtained by pretreatment with, to quantify the nivalenol as determined by HPLC-UV method, an analysis of nivalenol called.

In the present invention, one form of the zwitterionic solid phase extractant is a solid phase in which a weak ionic zwitterionic polymer represented by the following formula (1) is introduced into a porous hydrophilic substrate: It is an extractant.

In the present invention, another form of the zwitterionic solid phase extractant is that a weak ionic zwitterionic polymer represented by the following formula (2) is introduced into a porous hydrophilic substrate. It is a solid phase extraction agent.

In the present invention, the hydrophilic base material of the zwitterionic solid phase extractant is a porous non-aromatic hydrophilic polymer having a functional group into which the compounds of the formulas (1) to (2) can be introduced. Gels or silica gels can be used.

In the present invention, activated carbon or carbon-based adsorbent can be used as the dye removing adsorbent.

In the present invention, a salting-out agent composed of sodium chloride and magnesium sulfate can be used as the salting-out agent.

The analysis method of the present invention is a combination of a simple extraction method and a zwitterionic solid-phase extraction agent having a high affinity for a highly water-soluble compound, whereby NIV in food or feed can be rapidly and inexpensively obtained with an inexpensive instrument. NIV can be quantified with high accuracy by a general-purpose HPLC-UV method because it can be easily extracted with high efficiency and the removal rate of impurities is high. By using this method, it is possible to improve the analysis accuracy of NIV, improve the analysis efficiency, and reduce the cost. In addition, since the measurement solution containing NIV obtained by this pretreatment method is an aqueous solution, it is easy to perform quantitative analysis using not only the HPLC-UV method but also other analysis methods. For example, the present invention can be applied to the HPLC-MS method described as the confirmation test method in the DON notification method. Furthermore, application to an immunoassay method is also possible.

FIG. 1 is a diagram showing an operation flow in the analysis method of the present invention. FIG. 2 is an explanatory diagram showing an example of the configuration of a solid phase extraction cartridge filled with a zwitterionic solid phase extraction agent used in the present invention. FIG. 3 is a chromatogram of the bran extract obtained by the analysis method and notification method of the present invention. FIG. 2a) is a chromatogram of NIV 0.5 ppm standard. FIG. 2b) is a chromatogram of brown wheat measured by pretreatment according to the analysis method of the present invention. FIG. 2c) is a chromatogram of brown wheat added with 0.5 ppm of NIV measured by pretreatment according to the analysis method of the present invention. FIG. 2d) is a chromatogram of brown wheat measured by pretreatment by the notification method. FIG. 2e) is a chromatogram of brown wheat added with 0.5 ppm of NIV measured by pretreatment according to the notification method. FIG. 4 is a chromatogram of the bran extract obtained by the analysis method and notification method of the present invention. 4a) is a chromatogram of NIV 1 ppm standard solution. FIG. 4b) is a bran chromatogram measured by pretreatment by the analysis method of the present invention. FIG. 4c) is measured by pretreatment by the analysis method of the present invention. Chromatogram of bran added with 1 ppm of NIV Fig. 4d) is a chromatogram of bran measured by pretreatment by the notification method. FIG. 5 is a diagram showing an operation flow of the notification method.

The present invention combines a pre-extraction step for simple extraction using a centrifuge tube and a purification step for removing contaminant components using a solid-phase extraction cartridge filled with a zwitterionic solid-phase extraction agent. The most characteristic configuration is that the sample solution for measurement is separated and quantified using the HPLC-UV method.

The operation flow of the present embodiment is shown in FIG. The embodiment of the present invention will be described in accordance with the operation flow of FIG.

The pretreatment of the present invention comprises two steps, a preliminary extraction step and a purification step. In the pre-extraction step, after extracting NIV from food or feed, impurities such as pigments are removed to some extent, water is removed, and a solution ready for the next purification step is prepared. In the purification step, NIV and DON are captured and concentrated using a zwitterionic solid-phase extraction agent, and the remaining contaminants that interfere with HPLC analysis are removed.

In the pre-extraction process, the supernatant to be subjected to the purification process is obtained through the steps of swelling the sample to be measured, shaking extraction, centrifuging, removing impurities such as pigments with a salting-out agent / adsorbent, and centrifuging.
In the pre-extraction step, a predetermined amount of food or feed to be measured is collected in a centrifuge tube after being crushed or shredded. The centrifuge tube used here may be made of glass or resin. To the collected sample, add the same amount of water as the sample and shake vigorously, then leave it for 20-30 minutes. Then add twice the amount of water added acetonitrile and immediately shake vigorously for 1 minute. In the notification method, extraction is directly performed with acetonitrile: water (85:15), but when the sample is not sufficiently swollen, a high extraction rate cannot be obtained. Therefore, by adding water in advance and leaving it to shake, the sample can be sufficiently swollen and easily extracted. Moreover, since NIV is very water-soluble, in this invention, it extracts on the conditions used as water: acetonitrile (1: 2). After extraction with shaking with acetonitrile, the mixture is immediately centrifuged to obtain a supernatant. Centrifugation conditions are not particularly specified, but are performed at 3000 to 10000 rpm for 2 to 5 minutes. The obtained supernatant is transferred to another centrifuge tube, adsorbent for dye removal and a salting-out agent are added, shaken again, and centrifuged to obtain a supernatant. As the dye adsorbent, activated carbon or carbon-based adsorbent can be used. These can use what is marketed. The amount of the activated carbon or carbon-based adsorbent used is 0.5 to 1 g with respect to 20 mL of the obtained supernatant. Moreover, although sodium chloride and magnesium sulfate are used as a salting-out agent, these sodium chloride and magnesium sulfate are 1-3g with respect to 20 mL of obtained supernatants.
Further, the salting-out agent added in this step is added to increase the degree of migration (solubility) of NIV to the extraction solvent (acetonitrile in the present invention) due to the salting-out and dehydration effects. Anhydrous sodium sulfate is widely used as a material that brings about such an effect. However, when anhydrous sodium sulfate is used, there is a problem in that NIV is adsorbed and taken into anhydrous sodium sulfate and the extraction recovery rate decreases. Arise.

1 mL is collected from the supernatant obtained in the preliminary extraction step, and 2 mL of acetonitrile is added thereto, stirred and mixed, and subjected to a purification step using a zwitterionic solid phase extractant. In the purification process, the sample solution to be subjected to the HPLC-UV method through the following steps: supernatant obtained in the pre-extraction process ⇒ capture of NIV with zwitterionic solid phase extraction agent ⇒ removal of contaminating components by washing solution ⇒ elution with water Get.
3 mL of a solution prepared by adding acetonitrile is passed through a solid phase extraction cartridge filled with 100 mg of zwitterionic solid phase extraction agent at 0.5 to 2 mL / min to capture NIV. After passing through the entire volume, 1 mL of an appropriate cleaning solution is passed through to elute and remove the load solution and hydrophobic contaminants remaining in the packed bed. Thereafter, 0.5 mL of water is used to elute the NIV trapped in the zwitterionic solid phase extractant.
Here, the use of a zwitterionic solid-phase extraction agent not only has high affinity for NIV and can be efficiently captured and concentrated, but also obtains an eluate from a solid-phase extraction cartridge as a test solution as an aqueous solution. It is important in that it can be removed without retaining highly hydrophobic components. Separation of NIV by HPLC is performed in a reverse phase distribution mode using octadecylsilyl silica gel as a stationary phase and acetonitrile: water (for example, 5:95) as a mobile phase. In this separation mode, when the liquidity of the sample solution is stronger than the mobile phase, the retention and separation fluctuations increase. Therefore, it is desirable to use an aqueous solution containing no organic solvent as a sample. Further, when the sample contains a highly hydrophobic contaminant component, the dissolution of the contaminant component continues after the measurement target component elutes, resulting in a problem that the analysis time becomes abnormally long. Therefore, it is desirable that the measurement sample does not contain a highly hydrophobic contaminant component.
From these facts, it is preferable to use a zwitterionic solid phase extractant that can capture and concentrate NIV, remove hydrophobic contaminants, and obtain a measurement sample in an aqueous solution.
In the present invention, acetonitrile is added to the supernatant obtained in the preliminary extraction step to adjust the liquidity. Although the present invention incorporates a dehydration step, the supernatant after centrifugation contains a small amount of water. The capture rate of the zwitterionic solid-phase extraction agent varies greatly depending on the water concentration in the sample loading solution. Therefore, acetonitrile is added to increase the acetonitrile concentration, that is, reduce the water concentration. In the present invention, 2 mL of acetonitrile is added to 1 mL of the supernatant to lower the residual water concentration. The washing solution also serves to push out the sample loading solution remaining in the solid phase extraction cartridge, so it must be miscible with the sample loading solution, and the NIV trapped in the zwitterionic solid phase extraction agent is eluted. Must not be. Therefore, the same acetonitrile as the sample loading solution is used. After washing the solid phase extraction cartridge, 0.5 mL of water is used to elute the NIV trapped in the zwitterionic solid phase extraction agent. This eluate becomes a measurement sample by the HPLC-UV method. The zwitterionic solid phase extractant used in the present invention can elute highly water-soluble components such as NIV with only water. Therefore, operations such as drying and re-dissolution (transfer) as used in the notification method are not necessary, and a sample solution having a sample liquid property suitable for the HPLC-UV method as described above can be obtained. It becomes possible. Since the measurement solution containing NIV obtained by this pretreatment method is an aqueous solution as described above, it can be easily applied to quantitative methods other than the HPLC-UV method. For example, it can be quantified by the HPLC-MS method described as a confirmation test method in the DON notification method, and can also be applied to an immunoassay method.

In the present invention, NIV is captured by a zwitterionic solid-phase extraction agent having high affinity for a water-soluble compound for purification. In the present embodiment, the zwitterionic solid phase extractant is a solid phase extractant in which a weak ionic zwitterionic polymer is introduced into a porous hydrophilic substrate.

As the first and second forms of the zwitterionic polymer introduced into the hydrophilic substrate in the present invention, a diallylamine derivative-maleic acid copolymer represented by the following formula (1) or the following formula (2) Are allylamine derivatives-maleic acid copolymers, which are introduced into a hydrophilic substrate. Further, these copolymers may be mixed and introduced into the hydrophilic substrate. These zwitterionic polymers can be obtained, for example, by hydrolyzing a diallylamine derivative or a copolymer obtained by copolymerization of an allylamine derivative and maleic anhydride. The composition ratio (ratio of m and n in Formula (1) and Formula (2)) of diallylamine derivative or allylamine derivative and maleic acid is 1-2: 1. Although it is difficult to accurately determine the molecular weight of these zwitterionic polymers, those having an average molecular weight of 5,000 to 100,000 are used in the present invention.

Since the zwitterionic polymer is water-soluble, and a large number of water molecules are hydrated around each ionic group, the solid phase extractant in which these polymers are introduced into a hydrophilic substrate is High water retention. There has already been a report on the water retention of a solid-phase extraction agent into which a diallylamine-maleic acid copolymer has been introduced, and it is said that hydrophilic compounds are captured based on hydrophilic interactions (Non-patent Documents 7 and Non-patent Documents). 8). In the present invention, highly water-soluble NIV is trapped by hydrophilic interaction in the water retention layer formed on the surface of the solid phase extractant based on the hydration power of the zwitterionic polymer. A hydrophilic interaction is a complex interaction such as an interaction based on the hydrophilic group of the stationary phase, such as polarity and hydrogen bonding, and distribution to a hydrated phase formed on the surface of the stationary phase. Used for chromatographic separation. This separation system is composed of a polar stationary phase and a polar mobile phase such as acetonitrile, and separates polar compounds using affinity for the polar stationary phase.

In the present invention, in order to clearly express the hydrophilic interaction, a hydrophilic material is also used as the base material into which the zwitterionic polymer is introduced. As one form of the hydrophilic substrate, it is possible to use a porous hydrophilic polymer gel obtained by copolymerization of a known hydrophilic monomer and a hydrophilic crosslinkable monomer. In order to chemically introduce the ionic polymer, it is necessary to have a reactive functional group. Such a reactive substrate can be obtained by copolymerizing a reactive monomer having a reactive functional group and a crosslinkable monomer, or by introducing a reactive functional group into a hydrophilic substrate by post-reaction. The introduction of the zwitterionic polymer can be carried out by reaction with an ammonium group (hereinafter referred to as “ammonium group” including an imino group) or a reaction with a carboxyl group. In view of the stability and stability of the reaction product, it is advantageous to utilize a reaction with an ammonium group. Therefore, in the present invention, a porous hydrophilic polymer gel or silica gel having a functional group such as an epoxy group capable of reacting with an ammonium group or a halogenated alkyl group as a reactive functional group is used as a base material.

One form of hydrophilic polymer gel having a functional group capable of reacting with an ammonium group is a hydrophilic vinyl monomer having a functional group capable of reacting with an ammonium group and a hydrophilic cross-linking having two or more vinyl groups. It is a crosslinkable polymer obtained by suspension copolymerization with a polymerizable monomer. Examples of the functional group of the hydrophilic vinyl monomer having a functional group that reacts with an ammonium group include a halogenated alkyl 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. Other examples include functional methacrylate monomers, crosslinkable monomers having a cyanuric acid skeleton such as triallyl isocyanurate and trimethallyl isocyanurate. In addition, a third vinyl monomer can be added in order to improve physical properties such as hydrophilicity and hydrogen bonding property of the hydrophilic polymer gel. 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. In the hydrophilic polymer gel of the present invention, the amount of the vinyl monomer having a functional group that reacts with an ammonium group is 20 to 80% by weight, preferably 30 to 80% by weight, based on the total vinyl monomer, and has sufficient hardness. In order to ensure the above, at least 20% by weight or more of the hydrophilic crosslinkable monomer having two or more vinyl groups is used. Moreover, it is preferable to use 0-20 weight% of other vinyl monomers added in order to improve the physical properties, such as hydrophilic property and hydrogen bonding property of a hydrophilic base material.

Another form of the hydrophilic polymer gel having a functional group capable of reacting with an ammonium group is obtained by introducing a functional group capable of reacting with an ammonium group into a hydrophilic carrier having many hydroxyl groups by a chemical reaction. Examples of the hydrophilic carrier having a large number of hydroxyl groups include hydrophilic hydrophilic carriers made of synthetic polymers such as polyvinyl alcohol-based crosslinkable resins and hydroxymethacrylate-based crosslinkable resins, and hydrophilic carriers made from polysaccharides such as cellulose and dextran. Etc. can be used. A functional group capable of reacting with an ammonium group such as a halogenated alkyl group or an epoxy group can be introduced into the hydroxyl group of these hydrophilic carriers by a known method.

A further form of the hydrophilic substrate used in the present invention is porous silica gel obtained by a known method. In order to chemically introduce the zwitterionic polymer, it is necessary to introduce a functional group capable of reacting with an ammonium group in advance. As a method for introducing a reactive functional group capable of reacting with an ammonium group, a silane coupling agent having a reactive functional group may be reacted with a silanol group on the silica gel surface by a known method. For example, it is possible to introduce an epoxy group capable of reacting with an ammonium group on the silica gel surface by placing silica gel in a toluene solution of γ-glycidoxypropyltrimethoxysilane and reacting under reflux conditions.

In the present invention, since a high adsorption capacity is required, a porous hydrophilic substrate having a sufficient specific surface area is required. Therefore, in the present invention, it is preferable to use those having an average pore diameter of 6 to 50 nm and a specific surface area of 10 to 800 m ² / g as non-swelling pore physical properties. Moreover, the particle diameter of the hydrophilic base material used in the present invention can be arbitrarily set, but generally the average particle diameter is 25 to 200 μm. Moreover, regarding the shape of the hydrophilic substrate, it may be an irregular crushing type or a spherical shape. A zwitterionic functional group is introduced into the reactive functional group of such a hydrophilic substrate.

The solid phase extraction agent into which the zwitterionic functional group is introduced is used by being filled in an empty cartridge for solid phase extraction. Various types of empty cartridges are available, but are not particularly defined in the present invention. In addition, there is no special provision for the material of the empty cartridge or frit, but it must be resistant to the acetonitrile used in this analysis method, and is usually made of inexpensive polypropylene or polyethylene. It is done. FIG. 2 shows an example of the structure of a solid phase extraction cartridge used in the present invention. The solid-phase extraction agent 12 into which the zwitterionic functional group has been introduced is filled into an empty cartridge (syringe type in FIG. 2) 11 in which the lower frit 13 is inserted. The upper frit 14 is inserted to fix the packed bed of solid phase extraction agent. The solid-phase extraction cartridge 10 produced in this way is used for the purification process of NIV pretreatment.

The sample solution containing NIV prepared through the above-described pre-extraction step and purification step is separated and detected and quantified by high performance liquid chromatography-ultraviolet absorption detection method (HPLC-UV method). For the separation conditions, a column packed with octadecylsilane-bonded silica gel is used in accordance with what is prescribed in the DON notification method. The mobile phase may be specified in the DON notification method, but the NIV retention is small and the organic solvent concentration is reduced from that specified in the DON notification method in order to improve separation from the remaining contaminant components. It is preferable. For example, water containing 5% acetonitrile is used as the mobile phase. This separation mechanism is called a reverse phase distribution mode, eluting from a highly water-soluble one, and the one having a higher hydrophobicity is more strongly held in the column. However, when the manufacturer and the manufacturing method of the separation column are different, even if the mobile phase is used, the same separation is not necessarily obtained. That is, it is necessary to change the mobile phase conditions according to the separation column. Therefore, in the present invention, no separation condition is defined, and a condition for obtaining optimum separation can be set as appropriate with reference to the DON notification method. In addition, although the HPLC-MS method is described as a confirmation test method in the DON notification method, when a confirmation test is required even when the analysis method of the present NIV is used, the HPLC-MS according to the DON notification method is used. A confirmation test using the method may be performed.

As described above, by using the NIV analysis method of the present invention, NIV remaining in food, feed, food raw materials, etc. can be quantified easily and rapidly. EXAMPLES Next, the present invention will be described with reference to examples, but the present invention is not limited to the examples.

Example 1
(1) Synthesis of zwitterionic polymer-introduced solid-phase extraction agent: Synthesis of hydrophilic substrate The hydrophilic substrate was synthesized by an aqueous suspension polymerization method. A mixture prepared by dissolving 2 g of 2,2′-azobisisobutyronitrile in a mixed solution of 160 g of glycidyl methacrylate, 40 g of ethylene dimethacrylate, and 200 g of butyl acetate was added to 1,000 mL of a 0.1% aqueous solution of methylcellulose, and a stirrer was used. The stirring blade was rotated at 400 rpm to disperse the oil layer. 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 75 g of a hydrophilic base material of 45 to 90 μm. When the average particle size and dispersity (d75 / d25) of this hydrophilic substrate were measured with a Beckman Coulter Multisizer 3 Coulter Counter, the average particle size was 64.5 μm and the dispersity was 1.26. Moreover, when the specific surface area and average pore diameter were measured with a Beckman Coulter SA3100 Surface Area Analyzer, the specific surface area was 31 m ² / g and the average pore diameter was 20 nm.

(2) Synthesis of zwitterionic polymer-introduced solid-phase extraction agent: zwitterionic polymer-introduced diallylamine-maleic acid copolymer (Nittobo PAS-410C, concentration 40%) 50 mL, water 250 mL, 2- After adding to a mixed solvent of 100 mL of propanol to obtain a uniform solution, 50 g of the hydrophilic substrate obtained in (1) of Example 1 was added and stirred to obtain a uniform slurry. Further, 50 mL of 5M NaOH was added and reacted at 60 ° C. for 20 hours with stirring. After completion of the reaction, washing with 2-propanol, water, and methanol was performed in this order, followed by air drying overnight to obtain a zwitterionic solid phase extractant. In order to determine the amount of the introduced zwitterionic polymer, the amount of nitrogen was 0.64 N% as measured by Perkin Elmar 2400 Series II CHNS / O Elemental Analyzer. 100 mg of this zwitterionic solid phase extraction agent was filled into an empty cartridge for solid phase extraction (volume: 3 mL).

(3) Brown barley pretreatment by the method of the present invention 10 g of ground barley was collected in a polyethylene centrifuge tube (capacity: 50 mL) with a screw-type lid, added with 10 mL of water, shaken vigorously for 1 minute, and left for 20 minutes. . Thereafter, 20 mL of acetonitrile was added, and after vigorous shaking for 1 minute, the mixture was centrifuged at 5000 rpm for 3 minutes, and the supernatant was collected in another polyethylene centrifuge tube (volume: 25 mL). To the resulting supernatant, 0.5 g of activated carbon, 1 g of sodium chloride, and 1 g of magnesium sulfate were added, shaken for 1 minute, and then centrifuged under the same conditions as above to obtain a supernatant. 1 mL of the supernatant was collected in a polyethylene centrifuge tube (volume: 10 mL), 2 mL of acetonitrile was added, and the mixture was lightly stirred and mixed. The total amount of this solution was passed through the zwitterionic solid phase extraction agent-packed solid phase extraction cartridge prepared in Example 1 (2) at a flow rate of 1 mL / min. Next, 1 mL of acetonitrile was passed at a flow rate of 1 mL / min to wash the zwitterionic solid phase extractant. After sufficiently extruding acetonitrile, 0.5 mL of water was passed through to elute NIV trapped by the zwitterionic solid phase extractant to obtain a sample solution for measurement. Moreover, NIV was added so that it might become 0.5 ppm at the time of swelling with the water of brown wheat, and the standard addition sample solution pre-processed by the same method was also prepared.

(4) Comparative Example 1: Pretreatment of bran by notification method 50 g of crushed brown barley was collected in a 500 mL Erlenmeyer flask, 200 mL of acetonitrile: water (85:15) was added, and the mixture was shaken vigorously for 30 minutes. It was. The contents of the flask were filtered through a glass filter (Whatman GF / B). 10 mL of the filtrate was passed through a multifunctional column (Romer Lab, MultiSep 227) at a flow rate of 0.5 mL / min. About 3 mL of the first effluent was discarded, and then 5 mL of the effluent was collected in a test tube with a stopper. did. 4.0 mL of this effluent was accurately taken into a test tube with a stopper and heated in a 40 ° C. water bath to slowly remove the solvent. Next, 1.0 mL of acetonitrile: water: methanol (5: 90: 5) was added to the residue and dissolved, and then centrifuged at 10,000 rpm for 5 minutes to obtain a measurement sample solution. Moreover, NIV was added so that it might become 0.5 ppm at the time of swelling with the water of brown wheat, and the standard addition sample solution pre-processed by the same method was also prepared.

(5) Analysis of NIV in brown barley extract by HPLC-UV method The sample solution for measurement obtained in Example 2 and Comparative Example 1 was measured by HPLC-UV method. The measurement conditions of the HPLC-VU method are as follows. Column: CapcellPak C18 (5 μm, 4.6 × 250 mm), mobile phase: water: acetonitrile = 95: 5, flow rate: 1.0 mL, column temperature: 40 ° C., detection: ultraviolet absorption detector, 220 nm. The obtained chromatogram is shown in FIG. In the notification method, the peak of the contaminating component is detected in the vicinity of NIV. However, it can be seen that the method of the present invention can quantify NIV without being disturbed by the contaminating component. In addition, when the addition recovery rate is calculated from the NIV added at this time, it is 66.5% in the notification method and 92.3% in the analysis method of the present invention, and the analysis method of the present invention can obtain a higher recovery rate. did it.

(Example 2) Analysis of NIV in bran extract by HPLC-UV method NIV in bran was measured using the analysis method and the notification method of the present invention. The pretreatment is performed in the same procedure as in Example 2 and Comparative Example 1, and the measurement method is the same as in Comparative Test 1. However, the amount of NIV added was 1 ppm. The results are shown in FIG. As is clear from the figure, in the notification method, a peak based on a contaminant component is detected at the elution position of NIV, and it is difficult to accurately quantify NIV. In such a case, a confirmation test by HPLC-MS is required, and a complicated double measurement must be performed. On the other hand, in the method of the present invention, it was found that a peak based on a contaminating component was not detected at the elution position of NIV, and NIV could be accurately quantified. Incidentally, when the addition recovery rate is determined from the NIV added at this time, it is 67.4% in the notification method and 89.2% in the analysis method of the present invention, and the analysis method of the present invention can obtain a higher recovery rate. did it.

According to the present invention, by combining a simple extraction method and a zwitterionic solid-phase extraction agent having high affinity for a highly water-soluble compound, NIV in food or feed can be rapidly and inexpensively obtained with an inexpensive instrument. It becomes possible to extract easily with high efficiency. In the analysis method of the present invention, the analysis method of the present invention can efficiently extract NIV from foods or feeds, and removes contaminating components that interfere with the separation and detection of NIV in the measurement by the HPLC-UV method. Since it can be removed by a zwitterionic solid phase extractant, NIV can be simultaneously quantified with high accuracy. In addition, the time required for pretreatment is short and simple, and multiple sample simultaneous processing is possible. Furthermore, the removal rate of hydrophobic contaminant components is high, and the throughput in the HPLC-UV method is also high. By using this method, not only the accuracy of NIV analysis can be improved, but also analysis efficiency can be improved and costs can be reduced. Moreover, since the measurement solution containing NIV obtained by this pretreatment method is an aqueous solution, it can be applied to a quantification method other than the HPLC-UV method, and NIV can be quantified using an immunoassay method. is there. Furthermore, when a detailed confirmation test is required, although the convenience is impaired, the confirmation test can be performed using an HPLC-MS method according to the DON notification method. As described above, the spread of the analysis method of the present invention can contribute to ensuring food safety.

10: Column (Zwitterionic solid phase extraction agent-packed solid phase extraction cartridge)
11: Empty cartridge (syringe type)
12: Zwitterionic solid phase extractant 13: Lower frit 14: Upper frit

Claims (6)

In chemical analysis of nivalenol in food or feed, extract nivalenol in food or feed to be measured using a solid-phase extraction cartridge filled with a solid-phase extractant with weak ionic zwitterionic functional group An analytical method for performing purification and analysis,
Take the sample in a centrifuge tube, add water to swell sufficiently, extract with shaking with acetonitrile, add the dye-removing adsorbent and salting-out agent to the extract after centrifugation, and shake and centrifuge again. And get the supernatant,
A preliminary extraction process,
The supernatant obtained in the preliminary extraction step is passed through the solid-phase extraction cartridge to capture nivalenol, and then the solid-phase extraction cartridge is washed with a washing solution to remove impurities, and the solid-phase extraction cartridge To elute nivalenol trapped in water with water,
A method for analyzing nivalenol, wherein a sample solution obtained by a pretreatment comprising a purification step is measured by high performance liquid chromatography-ultraviolet absorption detection to quantify nivalenol. The solid phase extractant having the weak ionic zwitterionic functional group is a solid phase in which a weak ionic zwitterionic polymer represented by the following formula (1) is introduced into a porous hydrophilic substrate. The method for analyzing nivalenol according to claim 1, which is an extractant.

The solid phase extractant having a weak ionic zwitterionic functional group is a solid phase in which a weak ionic zwitterionic polymer represented by the following formula (2) is introduced into a porous hydrophilic substrate: The method for analyzing nivalenol according to claim 1, which is an extractant.

The said hydrophilic base material consists of the porous non-aromatic hydrophilic polymer gel or silica gel which has a functional group which can introduce | transduce the compound of said Formula (1) thru | or Formula (2). The analysis method of nivalenol of Claim 2 or Claim 3. The method for analyzing nivalenol according to any one of claims 1 to 4, wherein the adsorbent for removing the dye is activated carbon or a carbon-based adsorbent. The method for analyzing nivalenol according to any one of claims 1 to 5, wherein the salting-out agent comprises sodium chloride and magnesium sulfate.

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