JP6516092B2 - Method for preparing a sample for analysis of residual pesticides - Google Patents

Description

  The present invention relates to a method of preparing a sample for analysis of residual pesticides, and more particularly to a method of preparing a sample used to analyze residual pesticides in food.

  The so-called positive list system, which prohibits the sale of foods in which pesticides remain in excess of the specified amount in principle, was enforced in Japan in 2006, and it is clear that some of the many pesticides are not likely to harm human health. The remaining reference value has been set for all about 800 types of pesticides except pesticides of Therefore, for food to be sold, it is necessary to analyze about 800 types of pesticides at the same time, and the Ministry of Health, Labor and Welfare has notified of an analysis method therefor (Non-Patent Document 1).

  The simultaneous analysis method notified by the Ministry of Health, Labor and Welfare (hereinafter referred to as “notification method”) is basically a sample preparation process for homogenizing the food sample by grinding, extracting residual pesticide from the prepared sample And a purification step for removing contaminants from the extract obtained in the extraction step, and a measurement / analysis step for analyzing the test solution obtained in the purification step. In the extraction step, a solvent (acetonitrile) is added to the sample homogenized in the sample preparation step and homogenized, and then suction filtration is performed. Then, the filtrate is subjected to salting out, the solvent is removed, and the obtained residue is dissolved in a mixed solvent (mixed solvent of acetonitrile and toluene) to prepare an extract. In the purification step, the extract obtained in the extraction step and the mixed solvent of acetonitrile and toluene are injected in this order to a graphite carbon / aminopropylsilylated silica gel laminated mini-column to obtain an eluate from the mini-column. Then, the solvent is removed from the eluate, and the residue obtained is dissolved in a predetermined solvent to prepare a predetermined amount of a test solution. In the measurement / analysis process, the pesticides contained in the food sample are evaluated at the same time by analyzing the prepared test solution by GC / MS or GC / MS / MS or LC / MS or LC / MS / MS.

  However, the notification method requires a large amount of organic solvent because it uses a mini-column, and it requires many solvent removal operations, and it is applied to the measurement and analysis process because the operation process is complicated. It takes a long time to prepare a possible test solution.

  Therefore, a QuEChERS method (catcher's method) has been proposed in which a sample for analysis can be obtained in a short time by a simple operation (Non-Patent Document 2). The QuEChERS method is also adopted in the EU standards, and is being adopted by food companies etc. in Japan as a simultaneous analysis method of residual pesticides in place of the notification method.

  The QuEChERS method analyzes the extraction process for extracting residual pesticides from food samples, the salting-out process of the extract obtained in the extraction process, the purification process of the extract obtained through the salting-out process, and the extract obtained through the purification process It consists of measurement and analysis process. In the extraction step, acetonitrile is added to the food sample to shake and extract the residual pesticide. In the salting-out step, water and acetonitrile are separated by adding salt to the extract obtained in the extraction step and shaking to separate residual pesticides contained in the extract into the acetonitrile layer, as well as high polarity. Transfer of contaminants to the water layer. In the purification step, a powdery or granular solid phase such as ethylenediamine-N-propylsilylated silica gel or graphite carbon capable of adsorbing foreign substances contained in the extract solution subjected to the salting-out step is added and dispersed in the extract solution to shake. Then centrifuge. Then, in the measurement and analysis process, the pesticides contained in the food sample are evaluated at once by analyzing the supernatant solution obtained by centrifugation in the purification process by GC / MS or LC / MS or LC / MS / MS. .

  The QuEChERS method removes the contaminants contained in the extract by adding the solid phase to the extract, so the operation is simpler than in the notification method using a mini-column, and the analysis is performed in a short time. Sample (test solution) can be prepared, but its purification process is dispersion solid-phase extraction, so contaminating substances such as food matrix tend to remain in the test solution, which may affect analysis results There is. Also, the resulting test solution may significantly contaminate analytical instruments such as GC / MS and its columns due to contaminating substances.

Ministry of Health, Labor and Welfare, "A test method for substances that are components of agricultural chemicals, feed additives, or pharmaceuticals for animals remaining in food", [March 9, 2015 search], Internet (URL: http: //www.mhlw. go.jp/stf/seisakunitsuite/bunya/kenkou_iryou/shokuhin/zanryu/zanryu3/siken.html)

Food Analysis Development Center SUNATEC, "About the QuEChERS method", [March 9, 2015 search], Internet (URL: http://www.mac.or.jp/mail/140401/03.shtml)

  The present invention is intended to be able to analyze food pesticide residues easily and in a short time with high accuracy.

The present invention relates to a method of preparing a sample used to analyze pesticide residues in food. In this preparation method, the remaining pesticide is extracted from the food using a water-soluble organic solvent , and the obtained extract is mixed with water to prepare a mixed solution, a nanofiltration membrane, an ultrafiltration membrane or Process 2 of obtaining a filtrate using a separation membrane that can be used as a microfiltration membrane, obtaining a filtrate, a process 3 of passing the filtrate through an adsorbent capable of adsorbing residual pesticide in the filtrate, and 3 And 4) passing an extraction solvent capable of extracting residual pesticides through the adsorbent.

The mixed solution prepared in step 1 is filtered in step 2, and the mixed solution from which the solid contained in the extract is separated is obtained as a filtrate. Since a predetermined separation membrane is used in step 2, residual pesticides contained in the mixed solution pass through the separation membrane and remain in the filtrate. When the filtrate is allowed to pass through the adsorbent in step 3, the residual pesticide contained in the filtrate is adsorbed by the adsorbent, and the foreign substances contained in the filtrate pass through the adsorbent together with the filtrate. Then, when the extraction solvent is allowed to pass through the adsorbent in step 4, the residual pesticide adsorbed on the adsorbent is extracted by the extraction solvent . Therefore, the extraction solvent that has passed through the adsorbent can be used as a sample for analyzing residual pesticides in food.

The preparation method of the present invention can execute Step 2, Step 3, and Step 4 by, for example, applying centrifugal force to the mixed solution, the filtrate, and the extraction solvent .

  In this case, step 2, step 3 and step 4 can be performed efficiently, and the target sample for analysis can be prepared more easily and quickly.

  The separation membrane used in step 2 is, for example, polysulfone resin, polyethersulfone resin, polyvinylidene fluoride resin, aromatic polyamide resin, cellulose acetate resin, polytetrafluoroethylene resin, polyamide resin, polyacrylonitrile resin, polyvinyl chloride An organic film or an alumina film made of a resin material selected from the group consisting of polyacrylonitrile copolymer resin and polycarbonate resin.

  The organic film used as the separation film is preferably plasma-treated or ozone-treated. The organic film subjected to the plasma treatment or the ozone treatment is less likely to be attached with the residual pesticide because the hydrophilicity is increased, and the residual pesticide contained in the mixed solution can be passed smoothly.

  The adsorbent used in step 3 is, for example, at least one member selected from the group consisting of styrene / divinylbenzene copolymer resin, divinylbenzene copolymer resin, cyanopropylated silica gel, phenylated silica gel and octadecylated silica gel. is there.

One form of the preparation process of the present invention further comprises the step 5 for passing the extraction solvent which has passed through the adsorbent for contaminant contained in the extraction solvent which has passed through the adsorbent capable of adsorbing purification material. When the process 5 is included, since the contaminating substance remaining in the extraction solvent which has passed through the adsorbent is captured by the purified material, it is possible to prepare an analytical sample that is less susceptible to the influence of the contaminating substance.

Step 5 can be performed, for example, by applying a centrifugal force to the extraction solvent . In this case, step 5 can be performed efficiently, and the target sample for analysis can be prepared more easily and quickly.

  The purification material used in step 5 is, for example, at least one selected from the group consisting of ethylenediamine-N-propylsilylated silica gel, aminopropylsilylated silica gel, graphite carbon, octadecylated silica gel and magnesium silicate.

In the preparation method of the present invention, at least one selected from the group consisting of, for example, acetonitrile or methanol as an organic solvent in step 1 and acetone, toluene, dichloromethane and hexane as a solvent for extraction in step 4 Therefore, one having a polarity lower than that of the organic solvent used in step 1 is used.

The present invention according to another aspect relates to a kit for preparing a sample to be used to analyze residual pesticides of food by the preparation method of the present invention. The preparation kit comprises a bottomed centrifuge tube and a pesticide which is contained in a mixture of water and a water-soluble organic solvent capable of dissolving and removing pesticides which can be inserted into and withdrawn from the inside of the centrifuge tube. A nanofiltration membrane, an ultrafiltration membrane, a first unit capable of vertically separating the inside of a centrifuge tube by an adsorption layer capable of adsorbing and passing water, and an insertion / extraction to / from the inside of the first unit Alternatively, the separation unit which can be used as a microfiltration membrane is provided with a second unit capable of partitioning the inside of the centrifuge tube above and below the adsorption layer of the first unit.

  The first unit is inserted into the centrifuge tube, the mixed solution containing pesticide residue is injected into the second unit of the preparation kit with the second unit inserted into the first unit, and the centrifuge tube is applied to the centrifuge. The mixed solution is subjected to centrifugal force and passes through the separation membrane of the second unit and the adsorption layer of the first unit in this order. At this time, the solid content contained in the mixed solution is separated by the separation membrane, and the residual pesticide which has passed through the separation membrane together with the mixed solution is adsorbed to the adsorption layer.

  Next, the first unit is withdrawn together with the second unit from the centrifuge tube, and the liquid mixture accumulated at the bottom of the centrifuge tube is discarded. After washing the centrifuge tube, only the removed first unit is reinserted into the centrifuge tube, and the solvent for extracting the pesticide residue is injected into the first unit. Then, when the centrifuge tube is applied to a centrifuge, the extraction solvent receives centrifugal force and passes through the adsorption layer of the first unit. At this time, the residual pesticide adsorbed on the adsorption layer is extracted by the extraction solvent passing through the adsorption layer. Therefore, the extract of the residual pesticide by the extraction solvent is accumulated at the bottom of the centrifuge tube. When the first unit is extracted again from the centrifuge tube, the extract collected at the bottom of the centrifuge tube can be removed, and this extract can be used as a sample for analysis of residual pesticides.

  One form of the preparation kit according to the present invention is capable of adsorbing contaminants mixed in residue pesticides, which can be inserted / extracted into / from the inside of the centrifuge tube and into / from which the first unit can be inserted / extracted. And the 3rd unit which can divide the inside of a centrifuge tube up and down between the bottom part and the adsorption bed of the 1st unit by the refinement layer which has liquid permeability further is further provided.

  The third unit can be inserted into the washed centrifuge tube, and the first unit can be inserted into the third unit inserted into the centrifuge tube. The solvent for extracting the pesticide residue is injected into the first unit of the centrifuge tube into which the third unit and the first unit have been inserted in this way, and the centrifuge tube is applied to the centrifuge, and the solvent for extraction is subjected to centrifugal force. The adsorbent bed of the first unit and the purified bed of the third unit are passed in this order. At this time, the residual pesticide adsorbed on the adsorption layer is extracted by the extraction solvent which passes through the adsorption layer, and the contaminant contained in the extraction solvent from which the residual pesticide is extracted, the extraction solvent passes the purification layer When it is adsorbed to the purification layer. Therefore, the extract of the residual pesticide by the extraction solvent is accumulated at the bottom of the centrifuge tube. When the third unit and the first unit are withdrawn from the centrifuge tube, the extract collected at the bottom of the centrifuge tube can be removed, and this extract solution can be used as a sample for analysis of residual pesticides. Since this analytical sample is one from which the contaminant components have been removed by the purification layer, it is possible to enhance the analysis accuracy of the residual pesticide.

  The preparation kit according to the present invention may include two of the same centrifuge tubes. In this case, it is possible to withdraw the first unit together with the second unit from the centrifuge tube in which the mixed liquid is accumulated at the bottom, insert the withdrawn second unit into another centrifuge tube, and execute the next step. In the process of preparing a pesticide analysis sample, the washing of the centrifuge tube becomes unnecessary.

  Since the method for preparing a sample for analysis of residual pesticides according to the present invention includes the above-described steps, it is possible to easily prepare an analytical sample capable of analyzing residual pesticides in food with high accuracy in a short time. .

  The preparation kit of the sample for analysis of the pesticide residue according to the present invention is a combination of a first unit having an adsorption layer and a second unit having a separation membrane for a centrifuge tube, so the pesticide residue of food is highly accurate. An analytical sample that can be analyzed can be prepared easily and quickly in a short time.

BRIEF DESCRIPTION OF THE DRAWINGS The longitudinal cross-sectional view of one form of the preparation kit of the sample for analysis which concerns on this invention. The longitudinal cross-sectional view of the state which isolate | separated the said preparation kit in every element. The figure which shows 1 process of the preparation method of the sample for analysis using the said preparation kit. The figure which shows the other process of the preparation method of the sample for analysis using the said preparation kit. The figure which shows the further another process of the preparation method of the sample for analysis using the said preparation kit. The figure which shows the further another process of the preparation method of the sample for analysis using the said preparation kit. Sectional drawing of the plasma processing apparatus used in the Example. GC / MS SCAN chromatograms of analytical samples obtained in Example 1 and Comparative Example 1. GC / MS SCAN chromatograms of analytical trial fees obtained in Example 2 and Comparative Example 2. GC / MS SCAN chromatograms of analytical samples obtained in Example 2 and Comparative Example 3.

  One embodiment of a sample preparation kit for analysis according to the present invention will be described with reference to FIGS. 1 and 2. The sample preparation kit 1 for analysis uses residual analytical instruments such as a gas chromatograph mass spectrometer (GC / MS) and a liquid chromatograph mass spectrometer (LC / MS) capable of simultaneously analyzing multi-component-containing samples, and remains from food. An analytical sample suitable for analyzing residual pesticides in foods, particularly an analytical sample suitable for simultaneous analysis, is prepared from a mixture obtained by extracting an agrochemical, from the liquid extract described below The third embodiment is mainly provided with the centrifuge tube 10, the first unit 100, the second unit 200, and the third unit 300.

  The centrifuge tube 10 and each unit 100, 200, 300 are formed of resin having solvent resistance, for example, polypropylene resin, polyethylene resin, polytetrafluoroethylene resin, perfluoroalkoxyalkane resin or polyamide resin. is there. Moreover, the centrifuge tube 10 may be made of glass.

  The centrifuge tube 10 is a cylindrical container applicable to a centrifuge usable in a laboratory, and is closed by the entire upper end being opened and the bottom being formed conical.

  The first unit 100 is a cylindrical container which can be inserted into and withdrawn from the inside of the centrifuge tube 10, and the entire upper portion is open, and the first passage hole 101 is provided at the center of the bottom. The upper portion of the first unit 100 has a horizontally projecting first flange portion 102. Further, the adsorption layer 103 is disposed on the entire bottom of the first unit 100.

  The adsorption layer 103 is formed by filling a powdery, particulate or granular adsorbent capable of adsorbing residual pesticides contained in the mixed solution to a certain thickness at the bottom of the first unit 100. There is a liquid-permeable property that allows the mixture to pass.

  The adsorbent used in the adsorption layer 103 is capable of adsorbing the residual pesticide contained in the mixed solution, and capable of desorbing the adsorbed pesticide when it comes in contact with a low polarity solvent. As an adsorbent having such a function, for example, various carbon-based materials such as basic, neutral or acidic alumina, silica, activated carbon, graphite carbon, mesoporous carbon or activated carbon fiber, or styrene / divinyl benzene co A resin porous body or the like formed using a resin material such as a polymer based resin or a divinyl benzene copolymer based resin can be used. As the styrene / divinylbenzene copolymer resin, various resins can be used as long as they contain styrene and divinylbenzene as a structural unit, and examples thereof include styrene / divinylbenzene copolymer resin and styrene / divinylbenzene / methacrylate Copolymer resins can be used. Further, as the divinylbenzene copolymer resin, various resins can be used as long as they contain divinylbenzene as a constitutional unit. For example, divinylbenzene / methacrylate copolymer resin and divinylbenzene / vinylpyrrolidone copolymer Resin can be used.

  As the adsorbent, one having a functional group introduced by chemical modification can also be used in order to enhance the adsorptivity of the agrochemical contained in the mixed solution. For example, various aluminas and silicas used as adsorbents may be those having an aromatic ring or alkyl chain such as cyanopropyl group, phenyl group or octadecyl group introduced. The resin material may be, for example, a carboxy divinyl benzene vinyl pyrrolidone copolymer resin, a piperazine divinyl benzene vinyl pyrrolidone copolymer resin, or the like into which a carboxy group or a piperazine group is introduced.

  Particularly preferred adsorbents are styrene / divinylbenzene copolymer resins, divinylbenzene copolymer resins, cyanopropylated silica gels, phenylated silica gels or octadecylated silica gels.

  An adsorbent can be suitably selected according to the kind of solvent used in preparation of the target sample for analysis. Under the present circumstances, it can also be used together by mixing or laminating | stacking 2 or more types of things. In addition, the adsorbent may be used as a mixture with other materials to such an extent that the adsorptivity of the residual pesticide contained in the mixed liquid and the desorbability of the residual pesticide upon contact with a low polarity solvent are not impaired.

  A liquid-permeable net or sheet (not shown) is disposed on the bottom of the first unit 100 to prevent the adsorbent from leaking out of the first liquid-passing hole 101.

  The second unit 200 is a cylindrical container which can be inserted into and withdrawn from the inside of the centrifuge tube 10, and the entire upper portion is open, and the first passage hole 201 is provided at the center of the bottom. The upper portion of the second unit 200 has a second flange portion 202 that protrudes in the horizontal direction. The outer diameter of the container portion of the second unit 200 is set to be substantially the same as the inner diameter of the first unit 100 so that the second unit 200 can be inserted into the first unit 100, and the outer diameter of the second flange portion 202 is the first flange. It is set to be the same as that of the section 102.

  In the second unit 200, the separation membrane 203 is disposed on the entire bottom. The separation membrane 203 has a function as a nanofiltration membrane, an ultrafiltration membrane or a microfiltration membrane capable of separating residual pesticides and contaminating substances other than solvents from the liquid mixture, and the nonadsorbability and passage of the residual pesticides And is made of a material having solvent resistance.

  As such a separation membrane 203, an organic membrane or an inorganic membrane can be used. The organic film is a film formed using an organic material, and for example, polysulfone resin, polyether sulfone resin, vinylidene fluoride resin, aromatic polyamide resin, cellulose acetate resin, polytetrafluoroethylene resin, polyamide resin, What consists of resin materials, such as polyacrylonitrile resin, polyvinyl chloride polyacrylonitrile copolymer resin, or polycarbonate resin, is mentioned. Among them, polysulfone resins, polyethersulfone resins, vinylidene fluoride resins, aromatic polyamide resins, polytetrafluoroethylene resins or polyamide resins are particularly preferable in terms of solvent resistance.

  On the other hand, the inorganic film is a film formed using an inorganic material, and examples thereof include those made of alumina, titanium oxide or zirconia. Among these, an alumina film is preferable.

  When a nanofiltration membrane or an ultrafiltration membrane is used as the separation membrane 203, it is preferable to use one having a pore size of at least 1,000 molecular weight cut-off (MWCO), particularly one having a pore size of at least 3,000 MWCO. Although nanofiltration membranes and ultrafiltration membranes with a pore size of less than 1,000 MWCO have a high separation effect between residual pesticides and contaminants, pressure adjustment is required because higher pressures need to be applied to filter the mixture. If wrong, some solid contaminants may pass through the separation membrane 203 together with the residual pesticide.

  When a microfiltration membrane is used as the separation membrane 203, the pore size is preferably 0.1 μm or less. A microfiltration membrane with a pore size of more than 0.1 μm may allow solid contaminants to pass through along with the residual pesticide.

  In the case where an organic film is used as the separation film 203, it is preferable that the organic film, in particular, the organic film having a function as a nanofiltration film, be subjected to plasma treatment or ozone treatment. The organic film subjected to the plasma treatment or the ozone treatment is less likely to be attached with the residual pesticide because the hydrophilicity is increased, and the residual pesticide contained in the mixed solution can be passed smoothly. In addition, even if the nanofiltration membrane has a pore diameter of less than 1,000 MWCO, the residual pesticide can be passed smoothly by plasma treatment or ozone treatment, so a high pressure is required to filter the mixture. It is possible to separate residual pesticides and contaminating substances with high accuracy without adding.

  The third unit 300 is a cylindrical container that can be inserted into and withdrawn from the inside of the centrifuge tube 10, and the entire upper portion is open, and the third passage hole 301 is provided at the center of the bottom. An upper portion of the third unit 300 has a third flange portion 302 projecting in the horizontal direction. In the third unit 300, the outer diameter of the container portion is set to be substantially the same as the inner diameter of the centrifuge tube 10, and the inner diameter of the container portion is the first unit 100 so that the first unit 100 can be inserted therein. It is set to be approximately the same as the outer diameter. The outer diameter of the third flange portion 302 is set to be the same as that of the first flange portion 102.

  In the third unit 300, the purified layer 303 is disposed on the entire bottom. The refined layer 303 is a powder, particulate or granular refined material capable of adsorbing foreign substances other than residual pesticides contained in the low polarity extraction solvent described later at a bottom portion of the third unit 300, and has a certain thickness. And the solvent for extraction has a liquid-permeable property.

  The purified material used in the purified layer 303 is capable of adsorbing foreign substances contained in the solvent for extraction, while it is difficult to adsorb residual agricultural chemicals contained in the solvent for extraction, that is, by adsorbing the foreign substances. It is possible to separate contaminating substances and pesticide residues. As a purified material having such a function, for example, various carbon-based materials such as basic, neutral or acidic alumina, silica, magnesium silicate, activated carbon, graphite carbon or activated carbon fiber, or styrene / divinylbenzene The resin porous body etc. which were formed using resin materials, such as copolymer system resin or divinylbenzene copolymer system resin, can be used. As the styrene / divinylbenzene copolymer resin and the divinylbenzene copolymer resin, the same ones that can be used as the adsorbent used in the adsorption layer 103 can be used.

  The above-mentioned purified material is chemically modified by ethylenediamine N-propyl group, propylamino group, octadecyl group, octyl group, trimethylaminopropyl group, cyanopropyl group, etc. by chemical modification in order to enhance the adsorptivity of the contaminants contained in the extraction solvent What introduce functional groups, such as a phenyl group, a diol group, or an amino group, may be used.

  Particularly preferred as the purification material is ethylenediamine-N-propylsilylated silica gel, aminopropylsilylated silica gel, graphite carbon, octadecylated silica gel or magnesium silicate.

  The purification material can be appropriately selected according to the type of extraction solvent used in preparation of the target sample for analysis. Under the present circumstances, it can also be used together by mixing or laminating | stacking 2 or more types of things. In addition, the purified material may be used as a mixture with other materials to such an extent that the separability of the contaminants and the residual pesticide is not impaired.

  A liquid-permeable net or sheet (not shown) is disposed on the bottom of the third unit 300 to prevent leakage of the purified material from the third liquid-flowing hole 301.

  The sample preparation kit 1 for analysis is generally integrated by inserting the third unit 300, the first unit 100 and the second unit 200 in this order in the centrifuge tube 10 as shown in FIG. 1 and sold in this state And can be transported. When the units 100, 200, and 300 are integrated by being inserted into the centrifuge tube 10, the adsorption layer 103 of the first unit 100 divides the interior of the centrifuge tube 10 up and down. Further, the separation membrane 203 of the second unit 200 divides the inside of the centrifuge tube 10 above and below the adsorption layer 103. Furthermore, the purified layer 303 of the third unit 300 divides the interior of the centrifuge tube 10 up and down between its bottom and the adsorption layer 103.

  The integrated analysis sample preparation kit 1 may be equipped with a lid for closing the opening of the second unit 200 in order to prevent the separation membrane 203 and the like from being contaminated. As this lid, for example, a lid that can be screwed to the outer peripheral surface of the centrifuge tube 10 so as to cover the entire flange portion of the second flange portion 202, the first flange portion 102 and the third flange portion 302, or the entire flange portion What has flexibility which can be inserted so that it may cover is used.

  The sample preparation kit 1 for analysis separates each unit 100, 200, 300 from the centrifuge tube 10 individually as shown in FIG. In each step of the preparation method, necessary ones of the units 100, 200, 300 are selected and inserted into the centrifuge tube 10.

  Next, a method for preparing an analytical sample to be used for simultaneous analysis of residual pesticides in food using the above-described analytical sample preparation kit 1 will be described. The method of preparing the sample for analysis mainly comprises an extraction step of the residual pesticide, a purification step for separating the residual pesticide from the residual pesticide-containing liquid obtained by the extraction step, and a sample for analysis of the residual pesticide separated in the purification step It includes three steps of preparation process for preparing.

(Extraction process)
In this step, residual pesticides are extracted from the food to be subjected to simultaneous analysis of residual pesticides, and a predetermined residual pesticide-containing solution, that is, a mixed solution is prepared (Step 1). Foods to be subjected to extraction of residual pesticides are not particularly limited in kind, such as various agricultural products, meat, fish and shellfish, or processed products thereof, and usually the required amount is in a fine state for extraction of residual pesticides. Pulverize or crush it to make it uniform.

  Extraction of residual pesticides from food can be carried out according to the notification method or the method adopted in the QuEChERS method. That is, the solvent for extraction of residual pesticides (first solvent) is added to the required amount of the homogenized food sample, homogenized, and this is suction filtered. The extraction solvent to be used here is usually a highly polar organic solvent such as acetonitrile, methanol or acetone which can dissolve residual pesticides and is easily soluble in water.

  In the filtrate obtained by suction filtration, that is, the extract of the residual pesticide, it is usually preferable to precipitate out the contaminant by salting out with the addition of a salt. The salt used herein is sodium chloride, trisodium citrate or hydrate thereof, disodium hydrogen citrate or hydrate thereof, anhydrous magnesium sulfate, magnesium sulfate, anhydrous sodium sulfate, sodium sulfate, anhydrous sodium acetate or acetic acid It is sodium etc. These salts may be used in combination as appropriate.

  Next, the extract is centrifuged to separate into a solid and an extract. Then, water is added to the extract to prepare a mixed solution L of the extract and water.

(Purification process)
In this step, a filtrate is obtained by filtering the mixed solution L prepared in the extraction step using the separation sample 203 using the analysis sample preparation kit 1 (step 2), and the filtrate is passed through the adsorption layer 103. (Step 3).

  Here, in the analysis sample preparation kit 1 in which each unit 100, 200, 300 is separated from the centrifuge tube 10, as shown in FIG. 3, the first unit 100 is inserted into the centrifuge tube 10, and the first unit The second unit 200 is inserted into 100. Then, as shown in FIG. 4, the mixed solution L is injected into the second unit 200, and the centrifuge tube 10 is subjected to a centrifugation process with a centrifuge. By this process, the mixture L is subjected to centrifugal force and passes through the separation membrane 203 of the second unit 200 as shown by the arrow in FIG. The filtrate that has passed through the separation membrane 203 is scattered into the first unit 100 through the second passage hole 201 and is applied to the adsorption layer 103 by the application of centrifugal force, and passes through the first passage hole 101 of the centrifuge tube 10. It collects in the bottom. At this time, the mainly solid foreign substances contained in the mixture L are separated from the mixture L by the separation membrane 203. In addition, the residual pesticide contained in the filtrate that has passed through the separation membrane 203 is adsorbed to the adsorbent of the adsorption layer 103, and the contaminants dissolved in the filtrate pass through the adsorption layer 103 together with the filtrate. As a result, the residual pesticide in the mixed solution L is separated from the contaminants contained in the mixed solution L, the solvent for extraction, and the water, and purified.

  The analysis sample preparation kit 1 after the purification process is performed extracts the second unit 200 and the first unit 100 from the centrifuge tube 10 in this order or simultaneously. Then, the mixed solution L accumulated at the bottom of the second unit 200 and the centrifuge tube 10 is discarded, and the centrifuge tube 10 is washed for the next step.

(Preparation process)
In this step, the extraction solvent (second solvent) capable of extracting residual agricultural chemicals is allowed to pass through the adsorption layer 103 (step 4), and the extraction solvent (second solvent) having passed through the adsorption layer 103 is purified from the purification layer 303 Pass through (step 5) to prepare a sample for analysis of interest.

  Here, as shown in FIG. 5, the third unit 300 is inserted into the washed centrifuge tube 10 after the purification step, and the first unit 100 that has undergone the purification step is inserted into the third unit 300. Then, as shown in FIG. 6, the extraction solvent S is injected into the first unit 100, and the centrifuge tube 10 is subjected to a centrifugation process with a centrifuge. By this treatment, the extraction solvent S passes the adsorptive layer 103 of the first unit 100 as shown by the arrows in FIG. 6 by the application of centrifugal force, and extracts residual pesticides adsorbed on the adsorbent at that time. In addition, the extraction solvent S which has passed through the adsorption layer 103 is scattered into the third unit 300 through the first passage hole 101 and is applied with a centrifugal force to pass through the purification layer 303 and through the third passage hole 301. It collects in the bottom of the centrifuge tube 10. At this time, contaminant substances extracted from the adsorption layer 103 together with the residual pesticide by the extraction solvent S are adsorbed to the purified material and separated from the extraction solvent S.

  The extraction solvent S used in this step is usually an organic solvent having a polarity relatively lower than that of the extraction solvent (first solvent) used in the extraction step, and examples thereof include acetone, toluene, hexane or dichloromethane, or a mixture thereof A mixture of those selected appropriately from solvents can be used.

  The extraction solvent S accumulated at the bottom of the centrifuge tube 10 in this step can be collected from the centrifuge tube 10 when the first unit 100 and the third unit 300 are withdrawn from the centrifuge tube 10 in this order or simultaneously. Since this extraction solvent S is obtained by extracting the residual pesticide from the adsorption layer 103, it may be concentrated as it is or as necessary, and all residual pesticides may be used for GC / MS, LC / MS, etc. It can be used as a sample for analysis.

  The sample preparation kit for analysis 1 according to the above-described embodiment and the method for preparing a sample for analysis using the same use the third unit 300 provided with the purified layer 303, but contaminating components extracted from food together with residual pesticide Depending on the type and amount of the solvent, the contaminating components can be sufficiently removed by the separation membrane 203, or the contaminating components adsorbed on the adsorption layer 103 together with the residual pesticide are extracted by the extraction solvent S applied to the adsorption layer 103 in the preparation step. In the case where it is difficult or the like, the third unit 300 can be omitted, whereby the process 5 can be omitted.

  The sample preparation kit for analysis 1 may have two identical centrifuge tubes 10. In this case, there is no need to wash the centrifuge tube 10 when moving from the purification step to the preparation step, and it is possible to use the centrifuge tube 10 used in the purification step and the centrifuge tube 10 used in the preparation step.

  In the preparation of the sample for analysis, when the moisture of the mixed solution L is easily adsorbed to the adsorption layer 103 of the first unit 100, the residual agrochemical adsorbed on the adsorption layer 103 is extracted by the extraction solvent S. The adsorbed water may be mixed into the extraction solvent S, and as a result, the analysis sample may contain water. In this case, the analysis sample preparation kit 1 is located below the adsorption layer 103 in the centrifuge tube 10 (when the analysis sample preparation kit 1 includes the third unit 300, the space between the adsorption layer 103 and the purification layer 303). The 4th unit which can arrange a dehydration layer containing a dehydration material can further be included as an element. In this case, in the preparation step, the dehydration layer can be disposed below the adsorption layer 103, and the extraction solvent S that has passed through the adsorption layer 103 is removed of moisture when passing through the dehydration layer, An analytical sample with less contamination can be prepared.

  As the dehydrating material, various materials such as anhydrous sodium sulfate, anhydrous potassium carbonate, calcium chloride, potassium fluoride, silica gel, molecular sieve or zeolite can be used according to the type of the extraction solvent S and the like. These dehydrating agents may be used alone or in combination by mixing or laminating two or more. When the analysis sample preparation kit 1 includes the third unit 300, the dehydration layer may be stacked on the purified layer 303 without using the fourth unit.

  The above-mentioned method of preparing the sample for analysis using the sample preparation kit for analysis 1 executes steps 2, 3, 4 and 5 by centrifuging the centrifuge tube 10 with a centrifuge. The process can also be carried out without using a centrifuge. For example, after a mixed solution, a solvent and the like are injected into a predetermined unit inserted into the centrifuge tube 10, the internal pressure is increased by introducing nitrogen gas and air airtightly from the opening side of the unit, thereby increasing the mixture and so on. A solvent or the like can also be passed through the separation membrane 203, the adsorption layer 103, or the like.

  The method of preparing a sample for analysis according to the present invention can efficiently prepare a sample for simultaneous analysis of residual pesticide when it is carried out using the sample preparation kit for analysis 1, but the sample preparation kit for analysis 1 It can also be implemented by using a general-purpose instrument or device for each process without using it.

  The method and kit for preparing a sample for analysis according to the present invention can also be used to prepare a sample used to individually analyze residual pesticides in food.

[Plasma treatment of separation membrane]
The separation film was subjected to plasma treatment using the plasma treatment apparatus 400 shown in FIG. The plasma processing apparatus 400 includes a copper rod 410, a first glass tube 420, a second glass tube 430 and a third glass tube 440 coaxially arranged with the copper rod 410, a first glass tube 420 and a second glass. In the space between the second glass tube 430 and the third glass tube 440, the first copper plate 421 disposed between the tube 430 and the second copper plate 441 wound around the outer peripheral surface of the third glass tube 440 are provided. When an alternating voltage is applied between the copper rod 410 and the second copper plate 441 while flowing argon gas, plasma can be generated between the second glass tube 430 and the third glass tube 440.

  In a state where the separation film 500 is wound in a single layer on the outer peripheral surface of the second glass tube 430, an argon gas containing a small amount of water is flowed through the plasma processing apparatus 400 to apply an AC voltage between the copper rod 410 and the second copper plate 441. did. The surface of the separation film 500 was plasma-treated for 2 minutes by plasma generated thereby. The flow rate of argon gas was 250 mL / min.

  The separation membrane treated here is a nanofiltration membrane made of polypiperazine amide (trade name “XN45”: molecular weight cutoff (MWCO) 500) manufactured by TriSep.

[Preparation of food sample]
Food sample A:
Finely chopped commercial cucumbers were homogenized to prepare food sample A.
Food sample B:
A finely chopped spinach from the city version was homogenized to make a food sample B.

Example 1
10.0 g of food sample A and a pesticide standard solution (trade name "Pesticide standard mixture solution 31" manufactured by Kanto Chemical Co., Ltd.) were weighed into a 50 mL centrifuge tube, stirred and mixed, and allowed to stand for a while. The pesticide standard solution was added so that the concentration of the food sample A was 200 ppb.

  10 mL of acetonitrile was added to a centrifuge tube from which a food sample A or the like was weighed, and shaken manually for 1 minute. Add 1 g of trisodium citrate dihydrate, 0.5 g of disodium hydrogen citrate 1.5 hydrate, 1 g of sodium chloride and 4 g of anhydrous magnesium sulfate to this, and shake vigorously for an additional minute manually. It was analyzed. Subsequently, the centrifuge tube was attached to a centrifuge and centrifuged at 3,500 rpm for 10 minutes, and 6.0 mL of water was added to the liquid layer (4.0 mL) in the centrifuge tube to prepare a mixture.

  20 mL of a solvent in which acetonitrile and water were mixed at a volume ratio of 2: 3 was passed through the plasma-treated nanofiltration membrane to wash the nanofiltration membrane. Then, using a filtration cell (trade name "HP 4750 Stirred Cell", a trade name of STERLITE C) equipped with this nanofiltration membrane, the entire filtrate (10 mL) is filtered by pressurizing with nitrogen gas, and the first filtrate is Obtained. Moreover, 5 mL of solvents which mixed acetonitrile and water by the volume ratio of 2: 3 with the nanofiltration membrane after obtaining the 1st filtrate were filtered in the same way, and the 2nd filtrate was obtained.

  Acetonitrile and water are mixed at a volume ratio of 2: 3 into an adsorption column packed with 750 mg of a polymeric adsorbent (trade name "InertSep RP-1": styrene / divinylbenzene / methacrylate copolymer) by GL Science Inc. 6 mL of the solvent was injected, and air pressure was applied to the adsorption column from a syringe. Thus, the solvent was passed through the adsorption column, and the eluate was discarded. Next, 5 mL was taken from the mixed filtrate of the first filtrate and the second filtrate, this mixed filtrate was injected into the above adsorption column and passed in the same manner, and the eluate was discarded.

  5 mL of acetone was injected into the adsorption column after passing the mixed filtrate, and this acetone was passed in the same manner to secure an eluate. The eluate was subjected to dehydration treatment with anhydrous sodium sulfate and then concentrated appropriately to obtain an analysis sample having a sample concentration of 1 g / mL. Here, the sample concentration refers to a concentration that indicates that 1 mL of the sample for analysis is obtained from the weight of the extract of the food sample. For example, when the sample concentration is 1 g / mL, this means that the extract equivalent to 1 g of the food sample is adjusted to a solution of 1 mL by final concentration. The sample concentration can be adjusted by the degree of concentration of the extract.

  The time required for the preparation of the sample for analysis was 55 minutes from the start of the operation of adding acetonitrile to the centrifuge tube obtained by weighing the food sample A and the like.

Comparative Example 1 (Preparation of Sample for Analysis by QuEChERS Method According to European Standard EN 15662)
10.0 g of food sample A and a pesticide standard solution (trade name "Pesticide standard mixture solution 31" manufactured by Kanto Chemical Co., Ltd.) were weighed into a first centrifuge tube with a volume of 50 mL, stirred and mixed and allowed to stand for a while. The pesticide standard solution was added so that the concentration of the food sample A was 200 ppb.

  Acetonitrile 10mL was added to the 1st centrifuge tube which measured food sample A grade | etc., And it shaken for 1 minute manually. Add 1 g of trisodium citrate dihydrate, 0.5 g of disodium hydrogen citrate 1.5 hydrate, 1 g of sodium chloride, 4 g of anhydrous magnesium sulfate to this, and shake vigorously by hand for 1 minute to salt out did. Subsequently, the first centrifuge tube was attached to a centrifuge and centrifuged at 3,500 rpm for 10 minutes.

  150 mg of magnesium sulfate and 25 mg of ethylenediamine-N-propylsilylated silica gel (trade name "InertSep PSA" from GL Sciences Inc.) were added to another second centrifuge tube, and 1 mL of a liquid layer was collected from the first centrifuge tube. The solution was stirred by shaking for 30 seconds with a vortex mixer. Then, the second centrifuge tube was attached to a centrifuge and centrifuged at 13,000 rpm for 2 minutes, and 500 μL of the supernatant of the second centrifuge tube was collected. Three kinds of phenanthrene d-10, anthracene d-10 and 9-bromoanthracene were added to this supernatant liquid as an injection spike so that the concentration would be 100 ppb, respectively, to obtain an analysis sample having a sample concentration of 1 g / mL. The time required for preparation of this analytical sample was 30 minutes from the time when the operation of adding acetonitrile to the first centrifuge tube from which food sample A and the like were weighed was started.

Example 2
Conducted except that the plasma-treated nanofiltration membrane was changed to a non-plasma-treated precision filtration membrane, a polytetrafluoroethylene membrane (trade name “Preflon membrane”: Pore diameter 0.1 μm, trade name of Freon Industries Ltd.) Operating as in Example 1, a sample for analysis was prepared from food sample B. In addition, the sample for analysis was further purified.

In the purification process of the sample for analysis, a mixture of 30 mg of graphite carbon (trade name “InertSep GC” by GL Science, Inc.) and 250 mg of aminopropylsilylated silica gel (trade name “InertSep NH ₂ ” by GL Science, Inc.) is filled. The sample for analysis was injected into the resulting column, and air pressure was applied to the column from a syringe. Thus, the whole amount of the sample for analysis which has passed through the column is heated to 40 ° C. and appropriately concentrated. Then, as an injection spike, three kinds of phenanthrene d-10, anthracene d-10 and 9-bromoanthracene are each 100 ppb It added so that it might become. In this way, two types of analytical samples were prepared: one with a sample concentration of 1 g / mL and one with a sample concentration of 4 g / mL. The time required for the preparation of the sample for analysis and the entire purification process was 70 minutes from the start of the operation of adding acetonitrile to the centrifuge tube obtained by weighing the food sample B and the like.

Comparative example 2 (preparation of analytical sample by notification method)
20.0 g of food sample B was weighed out, and an agrochemical standard solution (trade name "agrochemical standard mixed solution 31" manufactured by Kanto Chemical Co., Ltd.) was added thereto to 50 ppb, and after stirring and mixing, it was allowed to stand for a while. Acetonitrile 50mL was added to this, and after homogenization, it suction-filtered and obtained the 1st filtrate. Moreover, after adding 20 mL of acetonitrile to the residue on filter paper and homogenizing, it suction-filtered and obtained the 2nd filtrate. The extract was prepared by combining the first filtrate and the second filtrate and adding acetonitrile thereto to adjust the volume to exactly 100 mL.

  10 g of sodium chloride and 20 mL of 0.5 mol / L phosphate buffer (pH 7.0) are added to 20 mL of the separated extract, which is shaken with a shaker and allowed to stand, and the separated aqueous layer is discarded did. On the other hand, the acetonitrile layer was dehydrated by adding anhydrous sodium sulfate, and the solvent was removed at 40 ° C. or less after filtering off the anhydrous sodium sulfate. To this residue, 2 mL of a solvent in which acetonitrile and toluene were mixed at a volume ratio of 3: 1 was added to obtain a sample solution.

Then, graphite carbon 500mg and mini-column formed by laminating charged with aminopropylsilylated silica gel 500mg (GL Science tradename Ltd. "InertSep GC / NH _2") acetonitrile graphite carbon side and a toluene 3: 1 by volume 10 mL of mixed solvent was injected and the effluent was discarded. Then, after injecting a sample solution from the graphite carbon side of this mini-column, 20 mL of a solvent in which acetonitrile and toluene were mixed at a volume ratio of 3: 1 was injected, and all of the eluate was secured. The eluate was concentrated to 1 mL or less at 40 ° C. or less, 10 mL of acetone was added thereto, and concentrated to 1 mL or less at 40 ° C. or less. The concentrate was dissolved again by adding 5 mL of acetone, and the solvent was removed. The residue at this time was dissolved in acetone so that the volume was exactly 1 mL, and used as an analytical sample with a sample concentration of 4 g / mL. The time required for preparation of the sample for analysis was 110 minutes from the start of the operation of adding acetonitrile after mixing of the food material B and the pesticide standard solution. In this sample for analysis, three kinds of phenanthrene d-10, anthracene d-10 and 9-bromoanthracene were added as injection spikes to a concentration of 100 ppb.

Comparative Example 3 (Preparation of an analytical sample by QuEChERS method according to European Standard EN 15662)
150 mg of magnesium sulfate, 25 mg of ethylenediamine-N-propylsilylated silica gel (trade name "InertSep PSA" by GL Sciences Inc.) and 7.5 mg of graphite carbon (trade name "Graphitized Carbon Black" by Waters) in a second centrifuge tube In addition, the same operation as in Comparative Example 1 was carried out except that 1 mL of the liquid layer collected from the first centrifuge tube was added, and a sample for analysis with a sample concentration of 1 g / mL was prepared from food sample B. The time required for preparation of the sample for analysis was 30 minutes from the start of the operation of adding acetonitrile to the first centrifuge tube from which the food sample B and the like were weighed.

The sample for analysis prepared in each example and each comparative example was analyzed under the following conditions using evaluation GC / MS (trade name "Trace GC / Polaris Q" by Thermo Fisher Scientific Co., Ltd.), and the added pesticide The recovery rate was determined. The results are shown in Tables 1A, 1B and 1C. Further, FIG. 8 shows GC / MS SCAN chromatograms of samples for analysis obtained in Example 1 and Comparative Example 1, and FIG. 9 shows GC / MS SCAN chromatography of samples for analysis obtained in Example 2 and Comparative Example 2. FIG. 10 shows the GC / MS SCAN chromatograms of the analytical samples obtained in Example 2 and Comparative Example 3 in FIG.

column:
5% phenyl-methyl silicon inner diameter 0.25 mm, length 30 m, film thickness 0.25 μm
Column temperature:
50 ° C (1 minute)-30 ° C / minute-125 ° C (0 minute)-5 ° C / minute-200 ° C (0 minute)-10 ° C / minute-300 ° C (11 minutes 30 seconds)
Inlet temperature: 220 ° C
Carrier gas: Helium ionization mode (voltage): EI (70 eV)

  Table 2 shows the results of determination of the ratio of the number of pesticide peaks with a recovery of 70 to 120% and the ratio of the number of pesticide peaks with a recovery of 70 to 120% with respect to the total number of peaks of pesticides.

  When Example 1 and Comparative Example 1 are compared, the pesticides from which a 70 to 120% recovery rate was obtained were almost the same number. However, according to FIG. 8, Example 1 has less contamination peaks than Comparative Example 1, and the purification effect is high.

  When Example 2 and Comparative Example 2 are compared, the number of pesticides from which a 70 to 120% recovery rate was obtained was almost the same. However, according to FIG. 9, Example 2 has fewer contamination peaks than Comparative Example 2, and the purification effect is high.

  When Example 2 and Comparative Example 3 are contrasted, the number of pesticides from which a 70 to 120% recovery rate was obtained was larger in Example 2. Further, according to FIG. 10, Example 2 has less contamination peaks than Comparative Example 3, and the purification effect is high.

DESCRIPTION OF SYMBOLS 1 Sample preparation kit for analysis 10 centrifuge tube 100 1st unit 103 adsorption layer 200 2nd unit 203 separation membrane 300 3rd unit 303 purification layer L mixed solution S solvent for extraction

Claims (12)

A method of preparing a sample used to analyze pesticide residues in food, comprising:
A step 1 of extracting the residual pesticide from the food using a water-soluble organic solvent , and mixing the obtained extract with water to prepare a mixture;
Step 2 of filtering the mixed solution to obtain a filtrate using a separation membrane that can be used as a nanofiltration membrane, an ultrafiltration membrane or a microfiltration membrane;
Passing the filtrate through an adsorbent capable of adsorbing the residual pesticide in the filtrate;
After the step 3, passing an extracting solvent capable of extracting the residual pesticide through the adsorbent;
A method of preparing a sample for analysis of residual pesticides, including: The method for preparing a sample for analysis of a pesticide residue according to claim 1, wherein Step 2, Step 3 and Step 4 are respectively performed by applying centrifugal force to the mixed solution, the filtrate and the solvent for extraction .   The separation membrane is made of polysulfone resin, polyethersulfone resin, polyvinylidene fluoride resin, aromatic polyamide resin, cellulose acetate resin, polytetrafluoroethylene resin, polyamide resin, polyacrylonitrile resin, polyvinyl chloride-polyacrylonitrile copolymer The method for preparing a sample for analysis of residual pesticides according to claim 1 or 2, which is an organic film or an alumina film made of a resin material selected from the group consisting of a resin and a polycarbonate resin.   The method for preparing a sample for simultaneous analysis of residual pesticides according to claim 3, wherein the organic film is plasma-treated or ozone-treated.   The adsorbent according to claim 1, wherein the adsorbent is at least one selected from the group consisting of styrene / divinylbenzene copolymer resin, divinylbenzene copolymer resin, cyanopropylated silica gel, phenylated silica gel and octadecylated silica gel. The preparation method of the sample for analysis of the residual pesticide in any one of 4. The method according to any one of claims 1 to 5, further comprising a step 5 of passing the solvent for extraction having passed through the adsorbent to a purified material capable of adsorbing a contaminant contained in the solvent for extraction having passed through the adsorbent. The preparation method of the sample for analysis of the residual pesticide of description. The method for preparing a sample for analysis of residual pesticides according to claim 6, wherein step 5 is carried out by applying centrifugal force to the extraction solvent .   8. The method according to claim 6, wherein the purified material is at least one selected from the group consisting of ethylenediamine-N-propylsilylated silica gel, aminopropylsilylated silica gel, graphitic carbon, octadecylated silica gel and magnesium silicate. Method of preparing samples for analysis of pesticide residues. The organic wherein using acetonitrile or methanol as the organic solvent in Step 1, and acetone as the extraction solvent in step 4, toluene, there is at least one selected from the group consisting of dichloromethane and hexane used in step 1 The method for preparing a sample for analysis of residual pesticides according to any one of claims 1 to 8, wherein one having a polarity lower than that of the solvent is used. A kit for preparing a sample used to analyze food pesticide residues.
A centrifuge tube whose bottom is closed,
An adsorption capable of adsorbing and passing through the residual pesticide contained in a mixture of water and a water-soluble organic solvent capable of dissolving and removing the residual pesticide which can be inserted into and withdrawn from the inside of the centrifuge tube A first unit capable of partitioning the interior of the centrifuge tube up and down by a layer;
The separation membrane which can be used as a nanofiltration membrane, an ultrafiltration membrane or a microfiltration membrane, which can be inserted into and removed from the inside of the first unit, can divide the inside of the centrifuge tube above and below the adsorption layer The second unit,
Preparation kit of samples for analysis of residual pesticides.   By means of a permeable layer which is capable of adsorbing foreign substances mixed in the residual pesticide which can be inserted / extracted into / from the inside of the centrifuge tube and into / from which the first unit can be inserted / extracted The sample preparation kit for analysis of a pesticide residue according to claim 10, further comprising a third unit capable of vertically dividing the inside of the centrifuge tube between the bottom and the adsorption layer.   The kit for preparing a sample for analysis of residual pesticides according to claim 10 or 11, wherein two centrifuge tubes are provided.

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