WO2018043465A1 - Method for preparing sample for analyzing residual agricultural chemical - Google Patents

Abstract

A residual agricultural chemical is extracted from a food using a water-soluble solvent and the obtained extract is mixed with water to give a liquid mixture. In a centrifuge tube (A) the inside of which is partitioned into upper and lower parts by an adsorbent layer (110) capable of adsorbing the residual agricultural chemical, a separation membrane (130) having a pore size with a molecular weight cutoff of 10,000 or greater is disposed above the adsorbent layer (110) to form a first treatment layer. Then, the liquid mixture is poured onto the separation membrane (130) and a centrifugal force is applied to the centrifuge tube (A) so that the liquid mixture is passed through the separation membrane (130) and the adsorbent layer (110). In another centrifuge tube (B) the inside of which is partitioned into upper and lower parts by a second treatment layer (210) comprising a dehydration layer (211) and a purification layer (212), the adsorbent layer (110), through which the liquid mixture has passed, is disposed above the second treatment layer (210). Next, an extraction solvent is poured onto the adsorbent layer (110) and a centrifugal force is applied to the centrifuge tube (B). Then, the extraction solvent having passed through the second treatment layer (210) is employed as a sample for analyzing the residual solution.

Description

Method for preparing samples for analysis of pesticide residues

The present invention relates to a method for preparing a sample for analysis of residual agricultural chemicals, and more particularly to a method for preparing a sample for analyzing residual agricultural chemicals in foods using a gas chromatograph mass spectrometer. This application claims priority based on Japanese Patent Application No. 2016-167690 filed in Japan on August 30, 2016 and Japanese Patent Application No. 2017-156274 filed in Japan on August 12, 2017, and its contents Is hereby incorporated by reference.

The positive list system, which in principle prohibits the sale of foods with residual agricultural chemicals exceeding the specified amount, was enforced in Japan in 2006, and it is clear that there is no risk of harming human health among many agricultural chemicals Residual standard values have been set for all of the approximately 800 types of pesticides except for pesticides. Therefore, it is necessary to analyze about 800 kinds of agricultural chemicals at the same time for the food to be sold, and the Ministry of Health, Labor and Welfare has notified the analysis method for that purpose (Non-patent Document 1).

The simultaneous analysis method notified by the Ministry of Health, Labor and Welfare (hereinafter referred to as the “notification method”) is basically a sample preparation process for homogenizing by crushing food samples, and extracting residual pesticides from the prepared samples An extraction step, 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 by the purification step. In the extraction step, a solvent (acetonitrile) is added to the sample homogenized in the sample preparation step and homogenized, followed by suction filtration. And after salting out this filtrate, the residue obtained by removing a solvent is melt | dissolved in a mixed solvent (mixed solvent of acetonitrile and toluene), and an extract is prepared. In the purification step, the extract obtained in the extraction step and the mixed solvent of acetonitrile and toluene are injected in this order into the graphite carbon / aminopropylsilylated silica gel laminated minicolumn to obtain an eluate from the minicolumn. Then, a predetermined amount of the test solution is prepared by dissolving the residue obtained by removing the solvent from the eluate in a predetermined solvent. In the measurement / analysis step, the prepared test solution is analyzed by GC / MS, GC / MS / MS, LC / MS, or LC / MS / MS, thereby simultaneously evaluating agricultural chemicals contained in the food sample.

However, since the notification method prepares a test solution having versatility that can handle both GC / MS and LC / MS, the operation process is complicated and complicated. For example, since a large amount of an organic solvent is required due to the use of a minicolumn, several solvent removal operations are required. Moreover, since the operation in each process is complicated, not only a long time is required to prepare a required test solution, but also the reliability of the test solution can vary depending on the maturity and skill of the operator.

Therefore, a QuEChERS method (Catchers method) has been proposed in which a sample for analysis can be obtained in a relatively short time by a simple operation (Non-Patent Document 2). The QuEChERS method is also adopted in EU standards, and is being adopted by food business operators and the like in Japan as a simultaneous analysis method for residual agricultural chemicals replacing the notification method.

The QuEChERS method analyzes an extraction process for extracting residual agricultural chemicals from food samples, a salting-out process of the extract obtained in the extraction process, a purification process of the extract that has undergone the salting-out process, and an extract that has undergone the purification process. Measurement and analysis process. In the extraction process, acetonitrile is added to the food sample and the residual pesticide is extracted by shaking. In the salting out process, salt is added to the extract obtained in the extraction process and shaken to separate water and acetonitrile. The residual pesticides contained in the extract are transferred to the acetonitrile layer, and are highly polar. The contaminated substances are transferred to the water layer. In the purification process, a powdered or granular solid phase such as ethylenediamine-N-propylsilylated silica gel or graphite carbon capable of adsorbing impurities contained in the extract that has undergone the salting-out process is added to and dispersed in the extract. After centrifugation, centrifuge. In the measurement / analysis process, the supernatant solution obtained by centrifugation in the purification process is analyzed by GC / MS, GC / MS / MS, LC / MS, or LC / MS / MS, and included in the food sample. Evaluate pesticides all at once.

The QuEChERS method removes contaminants contained in the extract by adding a solid phase to the extract and is therefore easier to operate than the notification method using a mini-column. Samples (test solutions) can be prepared. However, since the purification process is a dispersed solid-phase extraction, contaminants such as food matrix are likely to remain in the test solution, which may affect the analysis results. Further, the obtained test solution may significantly contaminate an analytical instrument such as GC / MS and its column due to residual contaminants.

Ministry of Health, Labor and Welfare, “Testing methods for substances remaining in foodstuffs such as agricultural chemicals, feed additives or veterinary medicines” [searched August 3, 2016], Internet (URL: http: //www.mhlw. go.jp/stf/seisakunitsuite/bunya/kenkou_iryou/shokuhin/zanryu/zanryu3/siken.html)

Sauna Food Analysis and Development Center SUNATEC, "QuEChERS Law", [August 3, 2016 search], Internet (URL: http://www.mac.or.jp/mail/140401/03.shtml)

The present invention provides a simple and short analytical sample suitable for analyzing food pesticide residue with high accuracy using a gas chromatograph mass spectrometer such as GC / MS, GC / MS / MS or GC / TOFMS. It makes it possible to prepare in time.

The present invention relates to a method for preparing a sample for analysis of residual pesticides, particularly a sample for analyzing residual pesticides in foods using a gas chromatograph mass spectrometer such as GC / MS, GC / MS / MS, or GC / TOFMS. It is related with the preparation method of this. This preparation method includes the following steps. Step 1 of extracting a pesticide residue from food using a water-soluble first solvent and mixing the resulting extract with water to prepare a mixed solution.
-Internally by a first treatment layer having an adsorbent layer having liquid permeability and capable of adsorbing residual agricultural chemicals, and a separation membrane disposed above the adsorbent layer and having a pore size of 10,000 molecular weight cutoff or more. Step 2 injecting the mixed solution onto the separation membrane of the first centrifuge tube partitioned vertically, and passing the mixed solution through the first treatment layer by applying centrifugal force to the first centrifuge tube.
・ After disposing the adsorbent layer that has undergone step 2 above the second treatment layer of the second centrifuge tube, the interior of which is divided vertically by a second treatment layer that has liquid permeability and contains a dehydrating agent; Is injected into the adsorbent layer and a centrifugal force is applied to the second centrifuge tube to apply the second solvent to the adsorbent layer and the second treatment layer. Step 3 is passed through.

When water is mixed with the extract in Step 1, a hydrophobic substance that is a contaminant in the extract is precipitated. For this reason, in the liquid mixture prepared in Step 1, a hydrophobic substance is deposited. In step 2, when a centrifugal force is applied to the first centrifuge tube in which the mixed solution prepared in step 1 is injected onto the separation membrane, the mixed solution receives the centrifugal force, and the separation membrane of the first treatment layer, the adsorbent layer, Are passed in this order. At this time, hydrophobic substances and other contaminants precipitated in the mixed solution are captured by the separation membrane, and residual agricultural chemicals in the mixed solution are adsorbed on the adsorbent layer. As a result, the mixed solution is separated into the contaminants captured by the separation membrane, the residual agricultural chemicals adsorbed on the adsorbent layer, and the liquid that has passed through the first treatment layer.

In step 3, when a centrifugal force is applied to the second centrifuge tube in which the second solvent is injected onto the adsorbent layer, the second solvent receives the centrifugal force and passes through the adsorbent layer and the second treatment layer in this order. At this time, the second solvent passes through the adsorbent layer while dissolving the residual agricultural chemical adsorbed on the adsorbent layer. Accordingly, the second solvent becomes an extract obtained by extracting the residual agricultural chemical from the adsorbent layer and passes through the second treatment layer. When passing through the second treatment layer, moisture contained in the second solvent from which the residual pesticide is extracted is removed by the dehydration layer of the second treatment layer. Therefore, the bottom of the second centrifuge tube is an extract of residual agricultural chemicals that has been dehydrated with the second solvent. This extract can be applied to a gas chromatograph mass spectrometer as a sample for analysis of residual agricultural chemicals.

In the second treatment layer, it is preferable to dispose a purification layer capable of capturing a contaminant contained in the second solvent that has passed through the adsorbent layer, below the dehydration layer. In this case, the extract of residual agricultural chemicals by the second solvent, which has been dehydrated by the dehydration layer, is removed by trapping the contaminants remaining there in the purification layer. Therefore, in this case, it is possible to prepare an analytical sample that is less likely to contaminate the gas chromatograph mass spectrometer.

The preparation method of the present invention preferably uses acetonitrile or acetone as the first solvent. Further, it is preferable to use a separation membrane having a pore diameter of 0.05 to 0.45 μm and made of polytetrafluoroethylene resin or polyvinylidene fluoride resin. Further, as the adsorbent layer, a layer containing styrene / divinylbenzene copolymer resin, styrene / divinylbenzene / methacrylate copolymer resin, divinylbenzene / vinylpyrrolidone copolymer resin, or silica having a phenyl group or octadecyl group introduced is used. Is preferred. Further, a layer containing potassium carbonate or magnesium sulfate is used as the dehydrating layer, and a mixed solvent of at least one of acetone, ethyl acetate, acetonitrile or acetone, ethyl acetate and acetonitrile and a hydrocarbon solvent is used as the second solvent. Is preferred.

The present invention according to another aspect also relates to a method for preparing a sample for analysis of residual agricultural chemicals, and particularly to a method for preparing a sample for analyzing residual agricultural chemicals in foods using a gas chromatograph mass spectrometer. This preparation method includes the following steps.
Step 1 of extracting a pesticide residue from food using a water-soluble first solvent and mixing the resulting extract with water to prepare a mixed solution.
-The mixed solution is injected by injecting the mixed solution onto the separation membrane of the first centrifuge tube whose interior is partitioned vertically by a separation membrane having a pore size of 10,000 molecular weight cutoff or more, and applying centrifugal force to the first centrifuge tube Step 2-1 for obtaining a filtrate by passing through a separation membrane.
-The filtrate is injected onto the adsorbent layer of the second centrifuge tube, the interior of which is divided vertically by an adsorbent layer that has liquid permeability and can adsorb residual agricultural chemicals, and applies centrifugal force to the second centrifuge tube. Step 2-2 for passing the filtrate through the adsorbent layer.
・ After disposing the adsorbent layer that has undergone step 2-2 above the treatment layer of the third centrifuge tube whose interior is vertically divided by the treatment layer that has liquid permeability and contains a dehydrating agent, dissolves residual agricultural chemicals. A step of injecting a second solvent that can be applied to the gas chromatograph mass spectrometer onto the adsorbent layer and passing the second solvent through the adsorbent layer and the treatment layer by applying a centrifugal force to the third centrifuge tube 3

When water is mixed with the extract in Step 1, a hydrophobic substance that is a contaminant in the extract is precipitated. For this reason, in the liquid mixture prepared in Step 1, a hydrophobic substance is deposited. In step 2-1, when a centrifugal force is applied to the first centrifuge tube into which the mixed solution prepared in step 1 has been injected onto the separation membrane, the mixed solution receives the centrifugal force and passes through the separation membrane. At this time, the hydrophobic substance and other contaminants precipitated in the mixed solution are captured by the separation membrane. As a result, the mixed liquid accumulates at the bottom of the first centrifuge tube as a filtrate from which contaminants trapped by the separation membrane have been removed.

Next, in step 2-2, when a centrifugal force is applied to the second centrifuge tube in which the filtrate obtained in step 2-1 is injected onto the adsorbent layer, the filtrate receives the centrifugal force and passes through the adsorbent layer. To do. At this time, the residual pesticide in the filtrate is adsorbed on the adsorbent layer. As a result, the filtrate is separated into residual agricultural chemicals adsorbed on the adsorbent layer and liquid components that have passed through the adsorbent layer.

In step 3, when a centrifugal force is applied to the third centrifuge tube in which the second solvent is injected onto the adsorbent layer, the second solvent receives the centrifugal force and passes through the adsorbent layer and the treatment layer in this order. At this time, the second solvent passes through the adsorbent layer while dissolving the residual agricultural chemical adsorbed on the adsorbent layer. Accordingly, the second solvent becomes an extract obtained by extracting the residual agricultural chemical from the adsorbent layer and passes through the treatment layer. When passing through the treatment layer, moisture contained in the second solvent from which the residual pesticide is extracted is removed by the dehydration layer of the treatment layer. Therefore, the bottom of the third centrifuge tube is an extract of residual agricultural chemicals that has been dehydrated with the second solvent. This extract can be applied to a gas chromatograph mass spectrometer as a sample for analysis of residual agricultural chemicals.

In the treatment layer, it is preferable to dispose a purification layer capable of capturing the contaminant contained in the second solvent that has passed through the adsorbent layer, below the dehydration layer. In this case, the extract of residual agricultural chemicals by the second solvent, which has been dehydrated by the dehydration layer, is removed by trapping the contaminants remaining there in the purification layer. Therefore, in this case, it is possible to prepare an analytical sample that is less likely to contaminate the gas chromatograph mass spectrometer.

In the preparation method of the present invention according to this aspect, it is preferable to use acetonitrile or acetone as the first solvent. In the mixed solution prepared in step 1, the mixing ratio of the extract (A) and water (B) is set to 50:50 to 60:40 by volume ratio (A: B), and the pore size is used as a separation membrane. Is preferably 0.05 to 0.2 μm and made of polytetrafluoroethylene resin or polyvinylidene fluoride resin. Further, as the adsorbent layer, a layer containing styrene / divinylbenzene copolymer resin, styrene / divinylbenzene / methacrylate copolymer resin, divinylbenzene / vinylpyrrolidone copolymer resin or silica into which phenyl group or octadecyl group is introduced is used. And, after adjusting the mixing ratio of the first solvent (A) and water (B) in the filtrate obtained in step 2-1 to 40:60 to 50:50 by volume ratio (A: B), step 2 -2, it is preferable to inject a filtrate with a mixed ratio adjusted onto the adsorbent layer. Further, a layer containing potassium carbonate or magnesium sulfate is used as the dehydrating layer, and a mixed solvent of at least one of acetone, ethyl acetate, acetonitrile or acetone, ethyl acetate and acetonitrile and a hydrocarbon solvent is used as the second solvent. Is preferred.

In the preparation method of the present invention according to this aspect, for example, a part of the filtrate obtained in step 2-1 is collected and secured as a sample for analyzing residual agricultural chemicals using a high performance liquid chromatograph mass spectrometer. Then, the remainder of the filtrate obtained in step 2-1 can be injected onto the adsorbent layer in step 2-2.

In this case, the preparation method of the present invention comprises a method for analyzing a pesticide residue in food with an analytical sample suitable for analysis with a gas chromatograph mass spectrometer and an analytical sample suitable for analysis with a high performance liquid chromatograph mass spectrometer. Two types can be prepared.

The present invention according to still another aspect provides a sample preparation device for analysis of residual agricultural chemicals, in particular, a gas chromatograph from a mixture of an extract and water obtained by extracting residual agricultural chemicals from food using a water-soluble solvent. The present invention relates to a preparation device for preparing a sample for analyzing pesticide residues using a tomograph mass spectrometer. The analytical sample preparation device includes a first unit and a second unit.

The first unit can be inserted into and removed from the centrifuge tube A whose bottom is closed, and the inside of the centrifuge tube A. When the first unit is inserted into the centrifuge tube A, it has liquid permeability and is a mixed liquid. A cylindrical body A1 that can partition the inside of the centrifuge tube A up and down by an adsorbent layer that can adsorb residual agricultural chemicals contained in the tube, and can be inserted into and extracted from the inside of the cylindrical body A1, and the cylindrical body A1 And a cylindrical body A2 capable of disposing a separation membrane having a pore diameter equal to or greater than a 10,000 molecular weight cutoff above the adsorbent layer.

On the other hand, the second unit is separate from the centrifuge tube A and can be inserted into and removed from the centrifuge tube B with the bottom closed and the centrifuge tube B. When the second unit is inserted into the centrifuge tube B In addition, a cylindrical body B1 having liquid permeability and capable of partitioning the inside of the centrifuge tube B vertically by a treatment layer including a dehydration layer is included.

The cylindrical body A1 of the first unit can be inserted inside the cylindrical body B1 of the second unit.

When preparing the analytical sample for residual agricultural chemicals using this analytical sample preparation device of the present invention, in the first unit, the cylindrical body A1 is inserted into the centrifuge tube A, and the cylindrical body A1 is cylindrical. The centrifuge tube A, the tubular body A1, and the tubular body A2 are integrated by further inserting the tubular body A2. On the other hand, in the second unit, the cylindrical body B1 is inserted into the centrifuge tube B, and the centrifuge tube B and the cylindrical body B1 are integrated.

In the integrated first unit, when the mixed solution is injected onto the separation membrane of the cylindrical body A2 and centrifugal force is applied to the centrifuge tube A, the mixed solution receives centrifugal force, and the separation membrane of the cylindrical body A2 and It passes through the adsorbent layer of the cylindrical body A1 in this order and accumulates at the bottom of the centrifuge tube A. At this time, hydrophobic substances and other contaminants precipitated in the mixed solution are captured by the separation membrane, and residual agricultural chemicals in the mixed solution are adsorbed on the adsorbent layer. As a result, the mixed solution is separated into contaminants trapped in the separation membrane, residual agricultural chemicals adsorbed on the adsorbent layer, and liquids accumulated at the bottom of the centrifuge tube A.

Next, the cylindrical body A2 and the cylindrical body A1 are extracted from the centrifuge tube A. And the cylindrical body A1 is inserted into the cylindrical body B1 of the integrated second unit from the adsorbent layer side, and the cylindrical body A1 is integrated with the second unit. Subsequently, when an extraction solvent capable of dissolving the residual agricultural chemical and applicable to the gas chromatograph mass spectrometer is injected onto the adsorbent layer of the cylindrical body A1, and the centrifugal force is applied to the centrifuge tube B, the extraction solvent is centrifuged. Under the force, it passes through the adsorbent layer of the cylindrical body A1 and the treatment layer of the cylindrical body B1 in this order. At this time, the extraction solvent passes through the adsorbent layer while dissolving the residual agricultural chemical adsorbed on the adsorbent layer. For this reason, the extraction solvent becomes an extract obtained by extracting the residual agricultural chemical from the adsorbent layer and further passes through the treatment layer. When passing through the treatment layer, moisture contained in the extraction solvent from which the residual pesticide is extracted is removed by the dehydrating agent in the treatment layer. Therefore, the bottom part of the centrifuge tube B is an extract of residual agricultural chemicals using an extraction solvent and is dehydrated. This extract can be applied to a gas chromatograph mass spectrometer as a sample for analysis of residual agricultural chemicals.

The present invention according to still another aspect provides a sample preparation device for analysis of residual agricultural chemicals, in particular, a gas chromatograph from a mixture of an extract and water obtained by extracting residual agricultural chemicals from food using a water-soluble solvent. The present invention relates to a preparation device for preparing a sample for analyzing pesticide residues using a tomograph mass spectrometer. This analytical data preparation device includes a first unit and a second unit.

The first unit can be inserted into and removed from the centrifuge tube A whose bottom is closed, and the inside of the centrifuge tube A. When the first unit is inserted into the centrifuge tube A, it has liquid permeability and is a mixed liquid. Can be inserted / extracted into / from the inside of the centrifuge tube A, and the cylindrical body A1 ′ that can partition the inside of the centrifuge tube A vertically by an adsorbent layer that can adsorb the residual agricultural chemical contained in the centrifuge tube A. And a cylindrical body A2 ′ that can partition the inside of the centrifuge tube A up and down by a separation membrane having a pore diameter of 10,000 molecular weight cutoff or more when inserted.

On the other hand, the second unit is separate from the centrifuge tube A and can be inserted into and removed from the centrifuge tube B with the bottom closed and the centrifuge tube B. When the second unit is inserted into the centrifuge tube B In addition, a cylindrical body B1 having liquid permeability and capable of partitioning the inside of the centrifuge tube B vertically by a treatment layer including a dehydration layer is included.

The cylindrical body A1 'of the first unit can be inserted inside the cylindrical body B1 of the second unit.

When preparing the analytical sample for residual agricultural chemicals using this analytical sample preparation device of the present invention, in the first unit, the cylindrical body A2 ′ is inserted into the centrifugal tube A, and the centrifugal tube A and the cylindrical body A2 are inserted. 'Integrate with. On the other hand, in the second unit, the cylindrical body B1 is inserted into the centrifuge tube B, and the centrifuge tube B and the cylindrical body B1 are integrated.

When the mixed solution is injected onto the integrated separation membrane of the cylindrical body A2 ′ of the first unit and a centrifugal force is applied to the centrifuge tube A, the mixed solution is subjected to centrifugal force, and the separation membrane of the cylindrical body A2 ′. And accumulates at the bottom of the centrifuge tube A. At this time, the hydrophobic substance and other contaminants precipitated in the mixed solution are captured by the separation membrane. As a result, the mixed liquid is separated into the contaminants captured by the separation membrane and the filtrate accumulated at the bottom of the centrifuge tube A.

Next, the cylindrical body A2 'is extracted from the centrifuge tube A, and the filtrate collected at the bottom of the centrifuge tube A is collected. Thereafter, the cylindrical body A1 'is inserted into the centrifuge tube A, and the centrifuge tube A and the cylindrical body A1' are integrated. Then, when the filtrate previously collected from the centrifuge tube A is injected onto the adsorbent layer of the cylindrical body A2 ′ of the integrated first unit and centrifugal force is applied to the centrifuge tube A, the filtrate is subjected to centrifugal force. And passes through the adsorbent layer of the cylindrical body A1 ′ and accumulates at the bottom of the centrifuge tube A. At this time, the residual pesticide in the filtrate is adsorbed on the adsorbent layer. As a result, the filtrate is separated into the residual pesticide adsorbed on the adsorbent layer and the liquid accumulated at the bottom of the centrifuge tube A.

Next, the cylindrical body A1 'is extracted from the centrifuge tube A. Then, the cylindrical body A1 'is inserted from the adsorbent layer side into the integrated second body cylindrical body B1, and the cylindrical body A1' is integrated with the second unit. Subsequently, when an extraction solvent capable of dissolving the residual agricultural chemical and applicable to the gas chromatograph mass spectrometer is injected into the cylindrical body A1 ′ and centrifugal force is applied to the centrifuge tube B, the extraction solvent receives centrifugal force, It passes through the adsorbent layer of the cylindrical body A1 ′ and the treatment layer of the cylindrical body B1 in this order. At this time, the extraction solvent passes through the adsorbent layer while dissolving the residual agricultural chemical adsorbed on the adsorbent layer. For this reason, the extraction solvent becomes an extract obtained by extracting the residual agricultural chemical from the adsorbent layer and further passes through the treatment layer. When passing through the treatment layer, moisture contained in the extraction solvent from which the residual pesticide is extracted is removed by the dehydrating agent in the treatment layer. Therefore, the bottom part of the centrifuge tube B is an extract of residual agricultural chemicals using an extraction solvent and is dehydrated. This extract can be applied to a gas chromatograph mass spectrometer as a sample for analysis of residual agricultural chemicals.

The present invention according to still another aspect relates to a method for analyzing a pesticide residue in food. This analysis method includes the following steps.
Step 1 of extracting a pesticide residue from food using a water-soluble first solvent and mixing the resulting extract with water to prepare a mixed solution.
-The mixed solution is injected by injecting the mixed solution onto the separation membrane of the first centrifuge tube whose interior is partitioned vertically by a separation membrane having a pore size of 10,000 molecular weight cutoff or more, and applying centrifugal force to the first centrifuge tube Step 2-1 for obtaining a filtrate by passing through a separation membrane.
-Step 2-2A for separating and securing a part of the filtrate.
-Filtration of the remaining portion after separation in step 2-2A on the adsorbent layer of the second centrifuge tube, the interior of which is divided vertically by an adsorbent layer that has liquid permeability and can adsorb residual agricultural chemicals Step 2-2B of injecting the liquid and passing the filtrate through the adsorbent layer by applying centrifugal force to the second centrifuge tube.
・ After disposing the adsorbent layer after Step 2-2B above the treatment layer of the third centrifuge tube, which has liquid permeability and includes a dehydration layer, and the inside is partitioned vertically, dissolves residual agricultural chemicals. A step of injecting a second solvent that can be applied to the gas chromatograph mass spectrometer onto the adsorbent layer and passing the second solvent through the adsorbent layer and the treatment layer by applying a centrifugal force to the third centrifuge tube 3
Step 4 of analyzing the filtrate collected in step 2-2A using a high performance liquid chromatograph mass spectrometer.
Step 5 of analyzing the second solvent that has passed through the adsorbent layer and the treatment layer in Step 3 using a gas chromatograph mass spectrometer.

When water is mixed with the extract in Step 1, a hydrophobic substance that is a contaminant in the extract is precipitated. For this reason, in the liquid mixture prepared in Step 1, a hydrophobic substance is deposited. In step 2-1, when a centrifugal force is applied to the first centrifuge tube into which the mixed solution prepared in step 1 has been injected onto the separation membrane, the mixed solution receives the centrifugal force and passes through the separation membrane. At this time, the hydrophobic substance and other contaminants precipitated in the mixed solution are captured by the separation membrane. As a result, the mixed liquid accumulates at the bottom of the first centrifuge tube as a filtrate from which contaminants trapped by the separation membrane have been removed.

In Step 2-2A, the filtrate obtained by separating a part is a water-soluble solution containing residual agricultural chemicals extracted from food, and can be used as an analysis sample in Step 4.

Next, when a centrifugal force is applied to the second centrifuge tube in which the remainder of the filtrate is injected onto the adsorbent layer in Step 2-2B, the filtrate receives the centrifugal force and passes through the adsorbent layer. At this time, the residual pesticide in the filtrate is adsorbed on the adsorbent layer. As a result, the filtrate is separated into residual agricultural chemicals adsorbed on the adsorbent layer and liquid components that have passed through the adsorbent layer.

In step 3, when a centrifugal force is applied to the third centrifuge tube in which the second solvent is injected onto the adsorbent layer, the second solvent receives the centrifugal force and passes through the adsorbent layer and the treatment layer in this order. At this time, the second solvent passes through the adsorbent layer while dissolving the residual agricultural chemical adsorbed on the adsorbent layer. Accordingly, the second solvent becomes an extract obtained by extracting the residual agricultural chemical from the adsorbent layer and passes through the treatment layer. When passing through the treatment layer, moisture contained in the second solvent from which the residual pesticide is extracted is removed by the dehydration layer of the treatment layer. Therefore, the bottom of the third centrifuge tube is an extract of residual agricultural chemicals that has been dehydrated with the second solvent, and this extract can be used as a sample for analysis in step 5.

Since the method for preparing a sample for analysis of pesticide residue according to the present invention includes the above-described steps, a sample for analysis suitable for analyzing food residue pesticides with high accuracy using a gas chromatograph mass spectrometer is provided. It can be prepared easily and in a short time.

Since the sample preparation device for analyzing residual agricultural chemicals according to the present invention includes the first unit and the second unit described above, the residual agricultural chemicals in foods are analyzed with high accuracy using a gas chromatograph mass spectrometer. It is possible to easily prepare a sample for analysis suitable for a short time.

Since the method for analyzing pesticide residues according to the present invention includes the above-described steps, the pesticide residue extracted from food can be analyzed using both a high-performance liquid chromatograph mass spectrometer and a gas chromatograph mass spectrometer. More reliable analysis results on food residue pesticides can be obtained.

The longitudinal cross-sectional view of each part of form 1 of the sample preparation device for analysis concerning the present invention. The longitudinal cross-sectional view of the state which integrated the 1st unit of the said form 1. FIG. The longitudinal cross-sectional view of the state which integrated the 2nd unit of the said form 1. FIG. The figure which shows 1 process of the preparation method of the sample for an analysis using the said form 1. The figure which shows the other process of the preparation method of the sample for analysis using the said form 1. The figure which shows the other process of the preparation method of the sample for analysis using the said form 1. FIG. The longitudinal cross-sectional view of each part of the 1st unit used in the form 2 of the sample preparation device for analysis based on this invention. The figure which shows 1 process of the preparation method of the sample for an analysis using the said form 2. The figure which shows the other process of the preparation method of the sample for analysis using the said form 2. The graph which shows the relationship between the octanol / water partition coefficient (LogPow) and recovery rate of each pesticide about Example 1. FIG. The graph which shows the result of having investigated the ratio of the agrochemical with a recovery rate of 70% or more about each Example and each comparative example. GC / MS SCAN chromatogram of each analytical sample obtained in Comparative Examples 1 and 2. GC / MS SCAN chromatogram of each analytical sample obtained in Examples 1 and 2. GC / MS SCAN chromatogram of each analytical sample obtained in Examples 3 and 4.

[Analytical Sample Preparer Form 1]
With reference to FIG. 1, Embodiment 1 of the sample preparation device for analysis according to the present invention will be described. The preparation device 1 in this form is configured to remove residual pesticides of food from GC / MS, GC / MS / MS or GC / from a mixed solution described later prepared using an extract obtained by extracting the residual pesticides from food. This is for preparing an analytical sample suitable for simultaneous analysis using a gas chromatograph mass spectrometer such as TOFMS, and mainly includes a first unit 100 and a second unit 200.

The first unit 100 includes a centrifuge tube A, a cylindrical body A1, and a cylindrical body A2. These instruments are formed using a solvent-resistant resin, for example, a polypropylene resin, a polyethylene resin, a polytetrafluoroethylene resin, a perfluoroalkoxyalkane resin, or a polyamide resin. The centrifuge tube A may be made of glass.

The centrifuge tube A is a cylindrical container that can be applied to a centrifuge that can be used in a laboratory. The entire upper end portion is opened and the bottom portion is closed, for example, in a conical shape.

The cylindrical body A1 is a cylindrical instrument that can be inserted into and removed from the opening of the centrifuge tube A, and has an upper portion opened and an adsorbent layer 110 at the bottom. The upper part of cylindrical body A1 has the 1st flange part 120 protruded in the horizontal direction.

The adsorbent layer 110 is a powder, particulate, or granular adsorbent that is molded to a certain thickness and has a liquid permeability that allows a mixed liquid to pass through. The adsorbent layer 110 has a bottom portion of the cylindrical body A1. Is supported in the cylindrical body A1 by a liquid-permeable sheet or net (not shown).

The adsorbent used in the adsorbent layer 110 is capable of adsorbing the residual agricultural chemicals contained in the mixed solution, while being capable of desorbing the agricultural chemicals adsorbed to the extraction solvent described later. Examples of the adsorbent having such a function include various carbon-based materials such as basic, neutral or acidic alumina, silica such as silica gel, activated carbon, graphite carbon, mesoporous carbon, or activated carbon fiber, or styrene / A resin porous body formed using a resin material such as divinylbenzene copolymer resin or divinylbenzene copolymer resin can be used. As the styrene / divinylbenzene copolymer resin, various resins can be used as long as they contain styrene and divinylbenzene as structural units. For example, styrene / divinylbenzene copolymer resin and styrene / divinylbenzene / methacrylate. A copolymer resin can be used. As the divinylbenzene copolymer-based resin, various resins can be used as long as they contain divinylbenzene as a structural unit. For example, divinylbenzene / methacrylate copolymer resins and divinylbenzene / vinylpyrrolidone copolymers can be used. Resin can be used.

As the adsorbent, in order to enhance the adsorptivity of the agrochemical contained in the mixed solution, those having a functional group introduced by chemical modification can be used. For example, various aluminas and silicas used as adsorbents may be introduced with an aromatic ring or alkyl chain such as a cyanopropyl group, a phenyl group or an octadecyl group. In addition, the resin material may be a resin material into which a carboxy group or a piperazine group is introduced, such as a carboxydivinylbenzenevinylpyrrolidone copolymer resin or a piperazine divinylbenzenevinylpyrrolidone copolymer resin.

Particularly preferred as adsorbents are styrene / divinylbenzene copolymer resin, styrene / divinylbenzene / methacrylate copolymer resin, divinylbenzene / vinyl pyrrolidone copolymer resin, phenyl group-introduced silica gel or octadecyl group. Silica gel.

The adsorbent layer 110 may contain other materials such as excipients as long as the adsorbability of the residual agricultural chemical contained in the mixed solution and the desorption property of the residual agricultural chemical to the extraction solvent are not impaired.

The cylindrical body A2 is a cylindrical container that can be inserted into and extracted from the opening of the cylindrical body A1, and the upper part is opened and the bottom part is closed by the separation membrane 130. The upper part of the cylindrical body A2 has a second flange portion 140 that protrudes in the horizontal direction and has an outer diameter set to be the same as that of the first flange portion 120.

The separation membrane 130 is capable of trapping impurities contained in the mixed solution while allowing residual agricultural chemicals to pass therethrough, and has a pore size of at least 10,000 molecular weight cut-off (MWCO). Separation membranes with a pore size of less than 10,000 MWCO have a high ability to capture contaminants contained in the mixed solution, but it is easy to capture a part of the residual agricultural chemicals. In particular, there is a possibility that the recovery rate may be reduced to less than 70%, and the reliability of the analytical sample prepared by the preparation device 1 may be impaired. On the other hand, the separation membrane 130 preferably has a pore diameter of 0.45 μm or less. A separation membrane having a pore diameter exceeding 0.45 μm may make it difficult to separate contaminants from the mixed solution. From these viewpoints, the separation membrane 130 usually has a pore diameter of preferably 0.01 to 0.45 μm, particularly preferably 0.05 to 0.2 μm.

As the separation membrane 130, any organic or inorganic membrane having solvent resistance can be used as long as it has the above-mentioned pore size.

The organic membrane that can be used as the separation membrane 130 is formed using an organic material. For example, a polysulfone resin, a polyether sulfone resin, a polyvinylidene fluoride resin, an aromatic polyamide resin, a cellulose acetate resin, Examples thereof include those made of a resin material such as tetrafluoroethylene resin, polyamide resin, polyacrylonitrile resin, polyvinyl chloride-polyacrylonitrile copolymer resin, or polycarbonate resin. Among these, in terms of solvent resistance, polysulfone resin, polyether sulfone resin, polyvinylidene fluoride resin, aromatic polyamide resin, polytetrafluoroethylene resin or polyamide resin is preferable. In particular, it is preferable to use a polyvinylidene fluoride resin or a polytetrafluoroethylene resin that hardly adsorbs residual agricultural chemicals.

On the other hand, the inorganic membrane that can be used as the separation membrane 130 is a membrane formed using an inorganic material, and examples thereof include those made of alumina, titanium oxide, zirconia, or the like. Of these, an alumina film is preferable.

The organic membrane used as the separation membrane 130 may have a hydrophilicity or hydrophobicity enhanced by plasma treatment or ozone treatment. In particular, organic membranes that have been improved in hydrophilicity by plasma treatment or ozone treatment can smoothly pass residual agricultural chemicals contained in the mixed solution even if the pore size is small, thus impairing the recovery rate of residual agricultural chemicals. Therefore, the effect of separating contaminants can be enhanced.

Particularly preferable as the separation membrane 130 is that the permeability of the residual agricultural chemicals contained in the mixed solution is not easily impaired, and the separation ability of contaminants, particularly hydrophobic substances, and sugars and proteins having a large molecular weight is high. .05-0.45 μm polytetrafluoroethylene resin or polyvinylidene fluoride resin.

As shown in FIG. 2, the first unit 100 has a cylindrical body A1 inserted into the centrifuge tube A from the adsorbent layer 110 side, and the cylindrical body A1 is inserted into the centrifuge tube A. It can be integrated by inserting A2 from the separation membrane 130 side. In the integrated first unit 100, the adsorbent layer 110 of the cylindrical body A1 partitions the inside of the centrifuge tube A up and down. Moreover, the cylindrical body A2 arranges the separation membrane 130 above the adsorbent layer 110. As a result, the cylindrical bodies A1 and A2 inserted into the centrifuge tube A form a first treatment layer 150 having the separation membrane 130 as an upper layer and the adsorbent layer 110 as a lower layer.

The second unit 200 includes a centrifuge tube B and a cylindrical body B1. These instruments are formed using the same material as each instrument of the first unit 100.

The centrifuge tube B is a cylindrical container applicable to a centrifuge that can be used in a laboratory, and is larger than the centrifuge tube A of the first unit 100. The entire upper end portion is open and the bottom portion is, for example, It is closed by being formed in a conical shape.

The cylindrical body B1 is a cylindrical instrument that can be inserted into and removed from the opening of the centrifuge tube B, and has a second processing layer 210 that opens at the top and has liquid permeability at the bottom. Yes. The upper part of cylindrical body B1 has the 1st flange part 220 protruded in the horizontal direction. Further, the cylindrical body B1 has an inner diameter that is substantially the same as the outer diameter of the cylindrical body A1 so that the cylindrical body A1 of the first unit 100 can be inserted into and removed from the inside thereof.

The second treatment layer 210 includes a dehydrating layer 211 containing a dehydrating agent and a purifying layer 212 containing a purifying agent that is laminated below the dehydrating layer 211, and is arranged at the bottom of the cylindrical body B1. Is supported in the cylindrical body B1 by a sheet or net (not shown).

The dewatering layer 211 is formed by molding a dehydrating agent in the form of powder, particles or granules to a certain thickness. The dehydrating agent used here is capable of removing moisture contained in various solvents and solutions, such as sodium sulfate, potassium fluoride, potassium carbonate, calcium sulfate, calcium chloride, magnesium sulfate, or potassium chloride. is there. Of these, potassium carbonate or magnesium sulfate is preferred because of its high moisture absorption rate and high moisture absorption, and potassium carbonate having a high dehydrating ability is particularly preferred regardless of the type of solvent. Two or more kinds of dehydrating agents may be used in combination.

The purification layer 212 is formed by molding a powdery, particulate or granular purification agent to a certain thickness. The purification agent used here is capable of removing contaminants contained in various solutions. For example, basic, neutral or acidic alumina, silica such as silica gel, magnesium silicate, activated carbon, graphite carbon or Various carbon-based materials such as activated carbon fibers, or resin porous bodies formed using a resin material such as a styrene / divinylbenzene copolymer resin or a divinylbenzene copolymer resin can be used. As the styrene / divinylbenzene copolymer resin and the divinylbenzene copolymer resin, those that can be used as the adsorbent used in the adsorbent layer 110 can be used.

The above-mentioned purification agent has an ethylenediamine N-propyl group, aminopropyl group, octadecyl group, octyl group, trimethylaminopropyl group, cyanopropyl group, phenyl group by chemical modification in order to enhance the adsorptivity of impurities contained in the solution. In addition, a functional group such as a diol group or an amino group may be introduced.

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

Two or more purification agents may be used in combination.

The dehydration layer 211 and the purification layer 212 may contain other materials such as excipients as long as they do not impair the dehydration ability and the purification ability.

The second unit 200 can be integrated by inserting the cylindrical body B1 into the centrifuge tube B from the second processing layer 210 side, as shown in FIG. In the integrated second unit 200, the second treatment layer 210 of the cylindrical body B1 partitions the inside of the centrifuge tube B vertically.

The analytical sample preparation device 1 can usually be transported or sold as a set of the integrated first unit 100 and the integrated second unit 200. The integrated units 100 and 200 may be provided with lids (not shown) for closing the openings in order to prevent contamination of the respective parts such as the first treatment layer 150 and the second treatment layer 210. Good. As this lid, for example, a centrifuge tube A that can be spirally fastened to the outer peripheral surface of the centrifuge tube A and B so as to cover the entire flange portion of each unit 100, 220, a centrifuge tube A that covers the entire flange portion, The thing etc. which have the softness | flexibility which can be inserted in the upper end part of B are used.

Next, a method for preparing a sample for analysis for simultaneous analysis of residual agricultural chemicals in food using a gas chromatograph mass spectrometer using the preparation device 1 of the form 1 will be described. This method for preparing a sample for analysis mainly includes three steps of steps 1 to 3 described below, and is less likely to contaminate a gas chromatograph mass spectrometer and its column in a time as short as the QuEChERS method. A target analytical sample can be easily prepared.

(Process 1)
In this step, a pesticide residue is extracted from food using a solvent (first solvent), and the resulting extract is mixed with water to prepare a mixed solution. There are no particular restrictions on the type of food that is used to extract residual agricultural chemicals, such as various agricultural crops, meat, seafood, or processed products thereof. It is made uniform by crushing or chopping.

Extraction of residual agricultural chemicals from food can be performed according to the method adopted in the notification method or the QuEChERS method. That is, in these methods, first, a solvent is added to a homogenized required amount of food sample and homogenized to prepare a slurry. The solvent used here is a highly polar water-soluble organic solvent that can dissolve the residual agricultural chemical and is easily dissolved in water, such as acetonitrile, methanol, or acetone. In particular, it is preferable to use acetonitrile or acetone.

Next, in the notification method, the obtained slurry is suction filtered to obtain a filtrate, that is, an extract of residual agricultural chemicals. This extract is usually adjusted to pH by adding a phosphate buffer and sodium chloride, and salted out to separate into a water-soluble organic solvent layer and an aqueous layer. Then, the separated water-soluble organic solvent layer is made up to a constant volume, and then the necessary amount is separated and concentrated to obtain an extract for preparing a mixed solution. On the other hand, in the QuEChERS method, a salt is added to the obtained slurry to separate it into a water-soluble organic solvent layer and an aqueous layer. The salt used here is sodium chloride, trisodium citrate or its hydrate, disodium hydrogen citrate or its hydrate, anhydrous magnesium sulfate, magnesium sulfate, anhydrous sodium sulfate, sodium sulfate, anhydrous sodium acetate or acetic acid Sodium and the like. These salts can be used in combination. Then, the separated water-soluble organic solvent layer is centrifuged to separate into a solid content and a liquid content, and a necessary amount is separated from the liquid content to obtain an extract for preparing a mixed solution.

In the preparation of the mixed solution, water (usually purified water such as distilled water or pure water) is added to and mixed with the extract obtained according to the notification method or the QuEChERS method. The liquid mixture obtained in this way is in a state where it is precipitated and dispersed due to water mixed with a hydrophobic substance among the contaminating substances contained in the extract.

The mixing ratio of the extraction liquid (A) and water (B) at the time of preparing the mixed liquid is not particularly limited as long as it is a ratio that can promote precipitation of the hydrophobic substance contained in the extraction liquid. Usually, the volume ratio (A: B) is preferably set to 10:90 to 60:40.

(Process 2)
In this step, as shown in FIG. 4, the step is performed in the cylindrical body A2 of the integrated first unit 100 (the centrifuge tube A of the integrated first unit 100 corresponds to the first centrifuge tube). The liquid mixture L prepared in 1 is injected. Then, when the first unit 100 is mounted on the centrifuge and a centrifugal force is applied, the mixed liquid L injected into the cylindrical body A2 receives the centrifugal force, and the cylindrical body A2 has a centrifugal force as shown by an arrow in FIG. It passes through the separation membrane 130 and the adsorbent layer 110 of the cylindrical body A1 in this order. At this time, contaminants contained in the mixed solution L, in particular sugars and proteins having a large molecular weight, and hydrophobic materials precipitated in the mixed solution L are captured by the separation membrane 130, and residual pesticides in the mixed solution L are adsorbed. Adsorbed on the agent layer 110. As a result, the mixed liquid L passes through the contaminants trapped in the separation membrane 130, the residual agricultural chemicals adsorbed on the adsorbent layer 110, and the liquid accumulated in the bottom of the centrifuge tube A through the separation membrane 130 and the adsorbent layer 110. Separated into minutes L1.

(Process 3)
In this step, the first unit 100 that has undergone step 2 is disassembled into a centrifuge tube A, a cylindrical body A1, and a cylindrical body A2. As shown in FIG. 5, the adsorbent layer is formed in the cylindrical body B1 of the integrated second unit 200 (the centrifuge tube B of the integrated second unit 200 corresponds to the second centrifuge tube). The cylindrical body A1 is inserted from the 110 side, and the cylindrical body A1 is integrated with the second unit 200. Thereby, the adsorbent layer 110 which passed through the process 2 is arrange | positioned above the 2nd process layer 210 of cylindrical body B1.

Next, as shown in FIG. 6, a solvent S (corresponding to the second solvent) is injected into the cylindrical body A1. The solvent used here can dissolve the residual agricultural chemical adsorbed on the adsorbent layer 110 and can be applied to a gas chromatograph mass spectrometer. Examples thereof include acetone, ethyl acetate, toluene, and acetonitrile. . A mixed solvent of at least one of acetone, ethyl acetate, and acetonitrile and a hydrocarbon solvent can also be used. The hydrocarbon solvent used in this mixed solvent is usually an aliphatic hydrocarbon solvent such as pentane, hexane, heptane, octane or decane, or an aromatic hydrocarbon solvent such as toluene. Two or more hydrocarbon solvents may be used in combination. As the mixed solvent, a mixed solvent in which acetone and hexane are mixed at a volume ratio of 1: 1 or a mixed solvent in which ethyl acetate and hexane are mixed at a volume ratio of 9: 1 is particularly preferable.

Next, when the second unit 200 in which the solvent S is injected into the cylindrical body A1 is mounted on a centrifuge and a centrifugal force is applied, the solvent S injected into the cylindrical body A1 receives the centrifugal force, and FIG. Passes through the adsorbent layer 110 of the cylindrical body A1 and the treatment layer 210 of the cylindrical body B1 in this order as indicated by arrows. At this time, the solvent S passes through the adsorbent layer 110 while dissolving the residual agricultural chemical adsorbed on the adsorbent layer 110. For this reason, the solvent S becomes an extract obtained by extracting the residual agricultural chemical from the adsorbent layer 110 and further passes through the second treatment layer 210. Moisture mixed in the extract when the extract from the solvent S passes through the second treatment layer 210 (this moisture is mainly contained in the adsorbent layer 110, and the solvent S is contained in the adsorbent layer 110). Is removed by the dehydrating agent contained in the dewatering layer 211 of the treatment layer 210. In addition, contaminants mixed in the extract are captured and removed by the purification agent contained in the purification layer 212 of the treatment layer 210. Therefore, at the bottom of the centrifuge tube B, a residue pesticide extract E with the solvent S, which has been dehydrated and purified, accumulates. This extract E can be used as a sample for analysis of residual agricultural chemicals using a gas chromatograph mass spectrometer.

The solvent S used in this step has different dehydrating effects depending on the combination with the type of dehydrating agent contained in the dehydrating layer 211 of the second treatment layer 210, but when potassium carbonate or magnesium sulfate having high versatility is used as the dehydrating agent. In addition, acetone, ethyl acetate, acetonitrile, or the above-described mixed solvent (that is, a mixed solvent of at least one of acetone, ethyl acetate, and acetonitrile and a hydrocarbon solvent, which is particularly excellent in the ability to extract residual agricultural chemicals from the adsorbent layer 110). ) Is preferably used.

In this step, the residual pesticide extract E collected at the bottom of the centrifuge tube B, that is, the residual pesticide analysis sample, can be collected by extracting the cylindrical body B1 together with the cylindrical body A1 from the centrifuge tube B. . The collected extract E can be applied to a gas chromatograph mass spectrometer as it is or after being appropriately concentrated if necessary.

The method for preparing the analytical sample using the preparation device 1 according to the first aspect is to select a combination of the conditions of the mixed solution prepared in Step 1 (mixing ratio of the extract and water) and the type of the separation membrane 130. Thus, the separation accuracy between the residual agricultural chemicals and contaminants contained in the mixed solution in step 2 can be increased. For example, in the mixed solution prepared in step 1, the mixing ratio of the extract (A) and water (B) is set to 10:90 to 60:40 by volume ratio (A: B) as described above, When a separation membrane 130 made of polytetrafluoroethylene resin or polyvinylidene fluoride resin having a pore diameter of 0.05 to 0.45 μm is selected, the remaining agricultural chemicals and contaminants contained in the mixed solution are separated in step 2 In particular, the separation accuracy with respect to 130 is enhanced, and the purification layer 212 can be omitted in the second treatment layer 210 of the cylindrical body B1 of the second unit 200.

[Form 2 of sample preparation device for analysis]
With reference to FIG. 7, Embodiment 2 of the sample preparation device for analysis according to the present invention will be described. In form 2, only the first unit 100 of form 1 is changed to the following first unit 101, and the second unit 200 is the same as form 1.

The first unit 101 of this embodiment includes a centrifuge tube A, a cylindrical body A1 ', and a cylindrical body A2'. The centrifuge tube A has the same specifications as the centrifuge tube A of form 1 in terms of size, material, and the like. The tubular body A1 'and the tubular body A2' are both formed using the same material as the tubular body A1 and the tubular body A2 of the first embodiment.

The cylindrical body A1 'is a cylindrical instrument that can be inserted into and removed from the opening of the centrifuge tube A, and has an upper portion opened and an adsorbent layer 110 at the bottom. The upper part of the cylindrical body A1 'has a first flange portion 120 protruding in the horizontal direction. The adsorbent layer 110 is the same as that of the cylindrical body A1 of the first embodiment, and the cylindrical body A1 is formed by a liquid-permeable sheet or net (not shown) disposed at the bottom of the cylindrical body A1 ′. 'Supported within.

The cylindrical body A <b> 2 ′ is a cylindrical container that can be inserted into and removed from the opening of the centrifuge tube A, the top is open, and the bottom is closed by the separation membrane 130. The upper part of the cylindrical body A2 'has a second flange portion 140 protruding in the horizontal direction. The separation membrane 130 is the same as that of the cylindrical body A1 of the form 1.

In the centrifuge tube A of the first unit 101, each of the cylindrical body A1 'and the cylindrical body A2' can be inserted individually. That is, as shown in FIG. 8, the tube A2 'can be inserted into the centrifuge tube A from the separation membrane 130 side. In this case, the separation membrane 130 of the cylindrical body A2 'divides the inside of the centrifuge tube A vertically (the centrifuge tube A in this state corresponds to the first centrifuge tube). Further, as shown in FIG. 9, the tube A 1 ′ can be inserted into the centrifuge tube A from the adsorbent layer 110 side. In this case, the adsorbent layer 110 of the cylindrical body A1 'partitions the inside of the centrifuge tube A vertically (the centrifuge tube A in this state corresponds to the second centrifuge tube).

Next, a method for preparing an analytical sample for simultaneous analysis of residual agricultural chemicals in foods using a gas chromatograph mass spectrometer using the preparation device 1 of form 2 will be described. This preparation method mainly includes three steps, steps 1 to 3, which will be described below, and is used for the purpose of analyzing the gas chromatograph mass spectrometer and its column in a short time as short as the QuEChERS method. Samples can be easily prepared in a short time.

(Process 1)
In this step, a pesticide residue is extracted from food using a solvent (first solvent), and the resulting extract is mixed with water to prepare a mixed solution. The details of this step are the same as those in Step 1 of the preparation method using the preparation device 1 of Form 1.

(Process 2)
This step includes the following step 2-1 and step 2-2.
Step 2-1
In this step, as shown in FIG. 8, the cylindrical body A2 ′ is inserted into the centrifuge tube A, and the mixed liquid L prepared in step 1 is injected into the cylindrical body A2 ′. When the centrifuge tube A is attached to the centrifuge in this state and a centrifugal force is applied, the liquid mixture L injected into the cylindrical body A2 ′ receives the centrifugal force and passes through the separation membrane 130. At this time, contaminants contained in the mixed solution L, in particular, sugars and proteins having a large molecular weight and hydrophobic materials deposited in the mixed solution L are captured by the separation membrane 130, and the filtrate from which the hydrophobic materials have been removed is removed from the centrifuge tube. Accumulate at the bottom of A. As a result, the mixed solution is separated into a contaminant substance captured by the separation membrane 130 and a filtrate containing residual agricultural chemicals that has passed through the separation membrane 130. The filtrate collected at the bottom of the centrifuge tube A is collected by using a pipette or the like and used in the next step 2-2.

Step 2-2:
In this step, as shown in FIG. 9, a cylindrical body A1 ′ is inserted into the centrifuge tube A, and the filtrate collected in step 2-1 is injected into this cylindrical body A1 ′. When the centrifuge tube A is attached to the centrifuge in this state and a centrifugal force is applied, the filtrate injected into the cylindrical body A1 ′ receives the centrifugal force and passes through the adsorbent layer 110. At this time, the residual pesticide in the filtrate is adsorbed on the adsorbent layer 110, and the liquid component from which the residual pesticide is separated is collected at the bottom of the centrifuge tube A. As a result, the filtrate is separated into residual agricultural chemicals adsorbed on the adsorbent layer 110 and liquid components that have passed through the adsorbent layer 110.

(Process 3)
In this process, the cylindrical body A1 ′ that has undergone the process 2-2 is extracted from the centrifuge tube A, and this cylindrical body A1 ′ is integrated in the same manner as in the preparation method according to the first embodiment (this state 200) The second centrifuge tube B of the second unit 200 corresponds to a third centrifuge tube.) Is inserted from the adsorbent layer 110 side into the cylindrical body B1 and integrated with the second unit 200. As a result, the adsorbent layer 110 that has undergone step 2-2 is disposed above the second treatment layer 210 of the cylindrical body B1.

Hereinafter, when the residual pesticide adsorbed on the adsorbent layer 110 is extracted by injecting the solvent S into the cylindrical body A1 ′ as in Step 3 of the preparation method according to the form 1, the solvent S is placed at the bottom of the centrifuge tube B. The residue pesticide extract E obtained by the above process and dehydrated and purified is collected. This extract E can be used as a sample for analysis of residual agricultural chemicals using a gas chromatograph mass spectrometer.

In the method for preparing an analytical sample using the preparation device 1 of Embodiment 2, the conditions of Step 2-2 can be set in Step 2 separately from the conditions of Step 2-1. For example, by adding a first solvent or water to the filtrate obtained in step 2-1 (that is, a mixture of a first solvent in which residual agricultural chemicals are dissolved) and water, the filtrate is added. In Step 2-2, the volume ratio of the first solvent to water in can be set to a condition in which the residual agricultural chemical in the filtrate is easily adsorbed by the adsorbent layer 110.

For example, a styrene / divinylbenzene copolymer resin, a styrene / divinylbenzene / methacrylate copolymer resin, a divinylbenzene / vinylpyrrolidone copolymer resin, or a phenyl group or an octadecyl group is introduced as the adsorbent layer 110 of the first unit 100. When a layer containing silica is used, the mixing ratio of the first solvent (A) and water (B) in the filtrate obtained in step 2-1 is 40:60 to 50:50 by volume ratio (A: B). After the adjustment, when the filtrate is injected onto the adsorbent layer 110 of the cylindrical body A1 ′ in step 2-2, the residual pesticide that passes through the adsorbent layer 110 is reduced in step 2-2. The reliability of the analytical sample is increased.

The method for preparing an analytical sample using the preparation device 1 according to the second aspect is to select a combination of the condition of the mixed solution prepared in step 1 (mixing ratio of the extract and water) and the type of the separation membrane 130. Thus, the separation accuracy between the residual agricultural chemicals and contaminants contained in the mixed solution in step 2 can be increased. For example, in the mixed solution prepared in step 1, the mixing ratio of the extract (A) and water (B) is set to 50:50 to 60:40 by volume ratio (A: B), and the separation membrane 130 is used. When a product made of polytetrafluoroethylene resin or polyvinylidene fluoride resin having a pore size of 0.05 to 0.2 μm is selected, the remaining agricultural chemicals and contaminants contained in the mixed solution in step 2-1 are separated from the separation membrane 130. The separation accuracy of the second unit 200 can be particularly improved, and the purification layer 212 can be omitted in the second treatment layer 210 of the cylindrical body B1 of the second unit 200.

In the method for preparing a sample for analysis using the preparation device 1 according to the form 2, analysis of residual agricultural chemicals applicable to a high performance liquid chromatograph mass spectrometer such as LC / MS, LC / MS / MS or LC / TOFMS Both the sample for analysis (first analysis sample) and the residual pesticide analysis sample (second analysis sample) applicable to the gas chromatograph mass spectrometer may be prepared from the mixture prepared in step 1 it can.

In this case, a part of the filtrate collected at the bottom of the centrifuge tube A in step 2-1 is collected and secured, and this is used as a first analysis sample to be applied to a high performance liquid chromatograph mass spectrometer. Use. In the subsequent steps, except for the point that the entire amount of the remaining filtrate accumulated at the bottom of the centrifuge tube A is injected into the cylindrical body A1 ′ inserted into the centrifuge tube A in step 2-2. This is performed in the same manner as the above-described analytical sample preparation method using the preparation device 1 according to the above, and the extraction liquid E accumulated at the bottom of the centrifuge tube B in step 3 is applied to the gas chromatograph mass spectrometer. 2 Used as a sample for analysis.

In the method for analyzing pesticide residues using the first analysis sample and the second analysis sample, the first analysis sample is analyzed using a high-performance liquid chromatograph mass spectrometer, and the first analysis sample is analyzed using a gas chromatograph mass spectrometer. 2 Analyze the sample for analysis. In this way, residual pesticide species suitable for analysis with a high performance liquid chromatograph mass spectrometer are analyzed with a high performance liquid chromatograph mass spectrometer, and residual pesticides suitable for analysis with a gas chromatograph mass spectrometer. Since the seeds are analyzed by a gas chromatograph mass spectrometer, the number of residual pesticide species that can be analyzed is increased, and the analysis accuracy of each residual pesticide species is increased, thereby obtaining a highly reliable analysis result.

In this analysis method, the first analysis sample that has been diluted by adding water, a buffer solution, or an organic solvent can be applied to analysis by a high-performance liquid chromatograph mass spectrometer. In this case, in the analysis result by the high performance liquid chromatograph mass spectrometer, the influence of the impurities contained in the first analysis sample is suppressed, and a more accurate analysis result can be expected.

The analytical sample preparation method and preparation device according to the present invention can be used not only for simultaneous analysis of residual agricultural chemicals in foods but also for preparing samples for individual analysis of residual agricultural chemicals.

[Preparation of food samples]
Food sample 1:
The food version 1 was prepared by finely chopping the leek of the city version.
Food sample 2:
The apple was homogenized by finely chopping the city version of the apple to prepare food sample 2.

[Comparative Example 1] (Preparation of analytical sample by QuEChERS method according to European standard EN15662)
10.0 g of food sample 1 and pesticide standard solution (trade name “Agricultural Chemical Standard Mixture 70 (former 31)” from Kanto Chemical Co., Ltd.) are weighed into a 50 mL centrifuge tube (hereinafter referred to as centrifuge tube T1). The mixture was allowed to stand for a while after stirring and mixing. The pesticide standard solution was added so that the concentration in the food sample 1 was 200 ppb.

10 mL of acetonitrile was added to the centrifuge tube T1 from which a food sample 1 and the like were weighed and shaken manually for 1 minute. To this was added 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. did. Subsequently, the centrifuge tube T1 was attached to a centrifuge and centrifuged at 3,500 rpm for 10 minutes.

In another centrifuge tube (hereinafter referred to as centrifuge tube T2), 150 mg of magnesium sulfate, 25 mg of ethylenediamine-N-propylsilylated silica gel (trade name “InertSep PSA” from GL Sciences Inc.) and graphite carbon (trade name “Waters” (Graphitized Carbon Black)) 7.5 mg was added, and 1 mL of the liquid layer collected from the centrifuge tube T1 was added thereto, and the mixture was stirred by shaking with a vortex mixer for 30 seconds. Then, the centrifuge tube T2 was attached to a centrifuge and centrifuged at 13,000 rpm for 2 minutes, and 500 μL of the supernatant in the centrifuge tube T2 was collected. Three types of phenanthrene d-10, anthracene d-10, and 9-bromoanthracene were added to the supernatant as injection spikes so that each had a concentration of 100 ppb to obtain an analytical sample having a sample concentration of 1 g / mL. The time required for the preparation of this analysis sample was 30 minutes from the start of the work of adding acetonitrile to the centrifuge tube T1 that weighed the food sample 1 and the like.

[Comparative Example 2] (Preparation of analysis sample by notification method)
Weigh 20.0 g of food sample 1 and add to it a pesticide standard solution (trade name “Agricultural Standard Mixture 70 (former 31)” from Kanto Chemical Co., Inc.) so that it becomes 50 ppb, and after stirring and mixing for a while Left to stand. 50 mL of acetonitrile was added to this and homogenized, followed by suction filtration to obtain a first filtrate. Moreover, 20 mL of acetonitrile was added to the residue on the filter paper and homogenized, followed by suction filtration to obtain a second filtrate. The 1st filtrate and the 2nd filtrate were match | combined, and acetonitrile was added to this, and the extract was prepared by adjusting to 100 mL correctly.

To 20 mL of the collected extract, 10 g of sodium chloride and 20 mL of 0.5 mol / L phosphate buffer (pH 7.0) are added, 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 after removing anhydrous sodium sulfate by filtration, the solvent was removed at 40 ° C. or lower. To this residue, 2 mL of a solvent prepared by mixing acetonitrile and toluene in a volume ratio of 3: 1 was added to obtain a sample solution.

Next, a volume ratio of 3: 1 of acetonitrile and toluene from the graphite carbon side of a mini column (trade name “InertSep GC / NH2” of GL Science Co., Ltd.) miniaturized and packed with 500 mg of graphite carbon and 500 mg of aminopropylsilylated silica gel 10 mL of the mixed solvent was injected, and the effluent was discarded. And after inject | pouring a sample solution from the graphite carbon side of this mini column, 20 mL of solvent which mixed acetonitrile and toluene in the volume ratio of 3: 1 was inject | poured, and all the eluates were ensured. The eluate was concentrated to 1 mL or less at 40 ° C. or lower, 10 mL of acetone was added thereto, and the mixture was again concentrated to 1 mL or lower at 40 ° C. or lower. The concentrate was dissolved again by adding 5 mL of acetone, and then the solvent was removed. The residue at this time was dissolved in acetone so that the volume was exactly 1 mL, and an analytical sample having a sample concentration of 4 g / mL was obtained. The time required for the preparation of the analysis sample was 110 minutes from the time when the operation of adding acetonitrile after the mixing of the food material and the pesticide standard solution was started. In this analysis sample, three types of phenanthrene d-10, anthracene d-10 and 9-bromoanthracene were added as injection spikes so that the concentration was 100 ppb.

[Example 1]
An analytical sample preparation device 1 according to Form 2 having the following specifications was prepared.

◎ First unit 101
Centrifuge tube A:
Inner diameter 15.3mm, height 120mm, capacity 15mL
Tubular body A1 ′:
Outer diameter 15.0mm, height 61.7mm, capacity 5.5mL
Tubular body A2 ′:
Outer diameter 15.0mm, height 62.7mm, capacity 4mL
Adsorbent layer 110:
650 mg of silica gel introduced with a phenyl group set to a height of 11 mm.
Separation membrane 130:
A polytetrafluoroethylene resin film having a pore diameter of 0.1 μm and a thickness of 30 μm.

◎ Second unit 200
Centrifuge tube B:
Inner diameter 27.6mm, height 114.5mm, capacity 50mL
Cylindrical body B1:
Outer diameter 17.3mm, height 85.0mm, capacity 12mL
Second treatment layer 210:
It consists of a dehydration layer 211 in which 1,800 mg of potassium carbonate is set to a height of 7 mm, and a purification layer 212 in which a mixture of graphite and silica gel introduced with aminopropyl groups is set to a height of 2 mm.

Step 1:
Weigh 10.0 g of food sample 1 and pesticide standard solution (trade name “Pesticide Standard Mixture 70 (former 31)” of Kanto Chemical Co., Ltd.) into a 50 mL capacity centrifuge tube (hereinafter referred to as centrifuge tube T3). The mixture was allowed to stand for a while after stirring and mixing. The pesticide standard solution was added so that the concentration in the food sample 1 was 150 ppb.

10 mL of acetonitrile was added to the centrifuge tube T3 where the food sample 1 and the like were weighed and homogenized for 1 minute. To this was added 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, and the mixture was shaken vigorously for another 1 minute. Analyzed. Subsequently, the centrifuge tube T3 was attached to a centrifuge and centrifuged at 3,500 rpm for 10 minutes, and 0.82 mL of water was added to 1 mL of the supernatant liquid collected from the centrifuge tube T3 to prepare a mixed solution.

Step 2-1
As shown in FIG. 8, a cylindrical body A2 ′ was inserted into the centrifuge tube A, and the liquid mixture prepared in step 1 was injected into the cylindrical body A2 ′. The centrifuge tube A in this state was attached to a centrifuge and treated at 2,500 rpm for 10 minutes. And cylindrical body A2 'was extracted from the centrifuge tube A, and the filtrate collected on the bottom part of the centrifuge tube A was extract | collected using the pipette. The collected filtrate was adjusted so that the volume ratio of acetonitrile to water was 45:55 by adding water.

Step 2-2:
As shown in FIG. 9, a cylindrical body A1 ′ was inserted into the centrifuge tube A used in step 2-1. Then, the entire amount of the filtrate whose volume ratio of acetonitrile and water was adjusted in Step 2-1 was injected into the cylindrical body A1 ′. The centrifuge tube A in this state was attached to the same centrifuge used in step 2-1, and treated at 2,000 to 3,500 rpm for 8 minutes.

Step 3:
After step 2-2, the tubular body A1 ′ was extracted from the centrifuge tube A. Then, the cylindrical body A1 ′ is integrated with the second unit 200 by inserting the cylindrical body A1 ′ from the adsorbent layer 110 side into the cylindrical body B1 of the integrated second unit 200, and the cylindrical body. Acetone (3.5 mL) was injected into A1 ′ as an extraction solvent. The second unit 200 was attached to the same centrifuge used in step 2-1, and treated at 400 rpm for 5 minutes.

For the second unit 200 after the processing by the centrifuge, the cylindrical body A1 'and the cylindrical body B1 were extracted from the centrifuge tube B, and the acetone solution collected at the bottom of the centrifuge tube B was secured as a sample for analysis. The time required for the preparation of the analysis sample was 50 minutes from the start of adding acetonitrile to the centrifuge tube T3 from which the food sample 1 and the like were taken. This required time was the same for Examples 2 to 4.

[Example 2]
In Step 3, an analysis sample was obtained in the same manner as in Example 1 except that 3.5 mL of ethyl acetate was used as the extraction solvent.

[Example 3]
In Step 3, an analysis sample was obtained in the same manner as in Example 1 except that 3.5 mL of a mixed solvent having a volume ratio of acetone and hexane of 1: 1 was used as the extraction solvent.

[Example 4]
In Step 3, an analysis sample was obtained in the same manner as in Example 1 except that 3.5 mL of a mixed solvent having a volume ratio of ethyl acetate and hexane of 9: 1 was used as the extraction solvent.

Evaluation of Comparative Examples 1 and 2 and Examples 1 to 4 Analytical samples prepared in Examples 1 to 4 and Comparative Examples 1 and 2, respectively, were appropriately concentrated or diluted appropriately with acetone and analyzed by GC / MS. Thus, the recovery rate of the agrochemical added to the food sample 1 was obtained. Further, the analytical samples prepared in Examples 1 to 4 and Comparative Examples 1 and 2, respectively, are concentrated or diluted with acetone to adjust the concentration to 2 g / mL, and this is analyzed by GC / MS. The SCAN chromatograms were compared. The GC / MS measurement conditions are as follows.

GC / MS equipment:
Product name “Trace GC / PolarisQ” of Thermo Fisher Scientific Co., Ltd.
column:
5% Phenyl-Methylsilicon Inner Diameter 0.25mm, Length 30m, Film Thickness 0.25μm
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)

FIG. 10 shows the relationship between the octanol / water partition coefficient (LogPow) at 20 ° C. and pH 7 and the recovery rate in Example 1 for each pesticide contained in the pesticide standard solution added to food sample 1. FIG. 11 shows the results of examining the proportion of agricultural chemicals with a recovery rate of 70% or more for each of Examples 1 to 4 and Comparative Examples 1 and 2. Further, FIG. 12 shows GC / MS SCAN chromatograms of the analytical samples obtained in Comparative Examples 1 and 2, and FIGS. 13 and 14 show GC / MS of the analytical samples obtained in Examples 1 to 4. The SCAN chromatogram is shown.

According to FIG. 10, the recovery rate of many pesticides is 70% or more, and it can be seen that Example 1 is suitable as a sample preparation method for simultaneous analysis of residual pesticides in foods using a gas chromatograph mass spectrometer. . Comparing the recovery rates of pesticides for Comparative Examples 1 and 2 and Examples 1 to 4, 70% to 120%, which is the required standard of the “Guidelines for evaluating the validity of test methods for pesticides remaining in foods” by the Ministry of Health, Labor and Welfare The ratio of the agricultural chemicals at which the recovery rate satisfying the above was obtained was almost 90% in all cases. However, according to FIGS. 12 to 14, Examples 1 to 4 have fewer contamination peaks than Comparative Examples 1 and 2, and the purification effect is higher. Therefore, it is considered that the analytical samples obtained in Examples 1 to 4 are less likely to contaminate the gas chromatograph mass spectrometer and its column than those obtained in Comparative Examples 1 and 2.

As described in Examples 1 to 4 and Comparative Examples 1 and 2, the time required for the preparation of the analysis sample was 50 minutes for Examples 1 to 4, 30 minutes for Comparative Example 1 (QuEChERS method), It is 110 minutes for the comparative example 2 (notification method). In the case of preparing an analytical sample from many types of food samples, Examples 1 to 4 show many of the steps for preparing an analytical sample from another food sample during the preparation of the analytical sample of one food sample. This can be done in parallel at the same time, but in Comparative Examples 1 and 2, this is somewhat limited. For this reason, for example, the time required for preparing an analytical sample from 16 types of food samples is approximately 170 minutes for Examples 1 to 4, approximately 150 minutes for Comparative Example 1, and approximately 1 for Comparative Example 2. , 050 minutes, while Examples 1 to 4 are more effective in reducing the required time compared to Comparative Example 2, while the shortcomings in required time compared to Comparative Example 1 are improved.

[Example 5]
Except for the point that the dehydration layer 211 of the second treatment layer 210 was changed to one in which magnesium sulfate 1,100 mg was set to a height of 8 mm, the analysis sample according to Form 2 having the same specifications as those used in Example 1 was used. A preparation device 1 was prepared. Using this preparation device 1, the following steps were performed.

Step 1:
10.0 g of food sample 2 and pesticide standard solution (trade name “Pesticide Standard Mixture 70 (former 31)”, “Pesticide Mixture Standard 55” and “Pesticide Mixture 58” of Kanto Chemical Co., Inc.) Was measured in a centrifuge tube having a capacity of 50 mL (hereinafter referred to as centrifuge tube T3) and allowed to stand for a while after stirring and mixing. The concentration of the pesticide standard solution in the food sample 2 is 10 ppb for the “pesticide standard mixed solution 70 (former 31)”, and each of the “pesticide mixed standard solution 55” and the “pesticide mixed standard solution 58” is 40 ppb. It added so that it might become.

10 mL of acetonitrile was added to the centrifuge tube T3, which weighed 2 food samples, and homogenized for 1 minute. To this was added 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, and the mixture was shaken vigorously for another 1 minute. Analyzed. Subsequently, the centrifuge tube T3 is attached to a centrifuge and centrifuged at 3,500 rpm for 10 minutes, and 1.025 mL of water is added to 1.25 mL of the supernatant liquid collected from the centrifuge tube T3 to prepare a mixed solution. did.

Step 2-1
As shown in FIG. 8, a cylindrical body A2 ′ was inserted into the centrifuge tube A, and the liquid mixture prepared in step 1 was injected into the cylindrical body A2 ′. The centrifuge tube A in this state was attached to a centrifuge and treated at 2,800 rpm for 10 minutes. And cylindrical body A2 'was extracted from the centrifuge tube A, and a part (0.455 mL) of the filtrate which collected on the bottom part of the centrifuge tube A was extract | collected with the pipette, and it was set as the sample for 1st analysis. The filtrate remaining at the bottom of the centrifuge tube A was adjusted by adding water so that the volume ratio of acetonitrile to water was 45:55.

Step 2-2:
As shown in FIG. 9, a cylindrical body A1 ′ was inserted into the centrifuge tube A used in step 2-1. Then, the entire amount of the filtrate whose volume ratio of acetonitrile and water was adjusted in Step 2-1 was injected into the cylindrical body A1 ′. The centrifuge tube A in this state was attached to the same centrifuge used in step 2-1, and treated at 2,000 rpm for 5 minutes.

Step 3:
After step 2-2, the tubular body A1 ′ was extracted from the centrifuge tube A. Then, the cylindrical body A1 ′ is integrated with the second unit 200 by inserting the cylindrical body A1 ′ from the adsorbent layer 110 side into the cylindrical body B1 of the integrated second unit 200, and the cylindrical body. Acetone (3.5 mL) was injected into A1 ′ as an extraction solvent. This second unit 200 was mounted on the same centrifuge used in step 2-1, and treated at 400 rpm for 8 minutes. Subsequently, 2.5 mL of acetone was injected into the cylindrical body A1 ′, and then the second unit 200 was mounted again on the centrifuge and further processed at 400 rpm for 8 minutes.

About the 2nd unit 200 after the process by a centrifuge, cylindrical body A1 'and cylindrical body B1 are extracted from the centrifuge tube B, the acetone solution collected at the bottom of the centrifuge tube B is concentrated to 0.5 mL, Secured as a second analytical sample. The time required for the preparation of the second analysis sample was 50 minutes from the start of the operation of adding acetonitrile to the centrifuge tube T3 that weighed the food sample 2 and the like.

Evaluation of Example 5 Recovery of pesticide added to food sample 2 by analyzing the first analytical sample and the second analytical sample obtained in Example 5 by LC / MS / MS and GC / MS / MS, respectively The rate was determined. At this time, the first analysis sample was diluted by adding 0.045 mL of water, and applied to LC / MS / MS. The second analysis sample was applied to GC / MS / MS as it was. The measurement conditions for LC / MS / MS and GC / MS / MS are as follows.

LC / MS / MS measurement conditions:
LC / MS / MS equipment:
Product name “Agilent 1260 Infinity / 6460 Triple Quadrupole” of Agilent Technologies, Inc.
column:
C18 inner diameter 2.1mm, length 100mm, particle diameter 1.8μm
Column temperature:
40 ° C
Mobile phase:
A: Purified water containing 0.1% formic acid and 10 mM ammonium formate B: Acetonitrile gradient (elapsed time / composition):
0 min / A90%, B10%
20 minutes / A 10%, B 90%
30 minutes / A10%, B90%
45 minutes / A 90%, B 10%
Flow rate:
300 μL / min injection volume:
5 μL
Measurement mode:
MRM

GC / MS / MS measurement conditions:
GC / MS / MS equipment:
Product name of “Trace 1310 GC / TSQ8000Evo” of Thermo Fisher Scientific Co., Ltd.
column:
5% Phenyl-Methylsilicon Inner Diameter 0.25mm, Length 30m, Film Thickness 0.25μm
Column temperature:
100 ° 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:
260 ° C
Carrier gas:
Helium ionization voltage:
70 eV
Measurement mode:
Timed-SRM

Tables 1A and 1B show the recovery rates of individual pesticides obtained by analyzing the first analytical sample by LC / MS, and individual pesticides obtained by analyzing the second analytical sample by GC / MS. The recovery rates are shown in Tables 2A and 2B. In addition, the collection rate of each table | surface is the average value which analyzed three types of 1st samples for an analysis and 2nd sample for analysis prepared from the same food sample 2 according to the process of Example 5, respectively. *

Of the pesticides contained in the first analytical sample, thiacloprid was also detected from the food sample 2 and thus is not reflected in Tables 1A and 1B. In addition, among the pesticides contained in the second analytical sample, Trifloxystrobin and Propargite were also detected in food sample 2, and Allethrin-1,2 had its peak overlapped with the interference peak, making it difficult to quantify Therefore, they are not reflected in Tables 2A and 2B.

According to Table 1A and Table 1B, the first analysis sample has a recovery rate that satisfies 70 to 120%, which is the required standard of the “Guidelines for evaluating the validity of test methods for agricultural chemicals remaining in food” by the Ministry of Health, Labor and Welfare. The proportion of pesticide applied was 100%. Moreover, according to Table 2A and Table 2B, the ratio of the agrochemicals with which the recovery rate which satisfy | fills the said request | requirement criteria was obtained in the 2nd analysis sample was 88%.

DESCRIPTION OF SYMBOLS 1 Preparation device 100,101 1st unit 110 Adsorbent layer 130 Separation membrane 150 1st process layer 200 2nd unit 210 2nd process layer 211 Dehydration layer 212 Purification layer A, B Centrifuge tube A1, A2, A1 ', A2' , B1 Cylindrical body L Mixed liquid S Extraction solvent

Claims (16)

A method for preparing a sample for analyzing pesticide residues in foods using a gas chromatograph mass spectrometer,
Step 1 of extracting the residual pesticide from the food using a water-soluble first solvent and mixing the resulting extract with water to prepare a mixture;
A first treatment layer having a liquid permeability and capable of adsorbing the residual agricultural chemical and a separation membrane disposed above the adsorbent layer and having a pore size of 10,000 molecular weight cutoff or more. Injecting the mixed solution onto the separation membrane of the first centrifuge tube whose interior is partitioned vertically, and passing the mixed solution through the first treatment layer by applying centrifugal force to the first centrifuge tube 2 When,
After disposing the adsorbent layer that has undergone step 2 above the second treatment layer of the second centrifuge tube, the interior of which is divided vertically by a second treatment layer that has liquid permeability and includes a dehydration layer, A second solvent capable of dissolving residual pesticide and applicable to the gas chromatograph mass spectrometer is injected onto the adsorbent layer, and centrifugal force is applied to the second centrifuge tube to adsorb the second solvent. Step 3 of passing through the agent layer and the second treatment layer;
Of a sample for analysis of residual pesticides containing The analysis of residual agricultural chemicals according to claim 1, wherein a purification layer capable of capturing a contaminant contained in the second solvent that has passed through the adsorbent layer is disposed below the dehydration layer in the second treatment layer. Sample preparation method. The method for preparing a sample for analysis of residual agricultural chemicals according to claim 1 or 2, wherein acetonitrile or acetone is used as the first solvent. The residual agricultural chemical analysis sample according to any one of claims 1 to 3, wherein the separation membrane has a pore diameter of 0.05 to 0.45 µm and is made of polytetrafluoroethylene resin or polyvinylidene fluoride resin. Preparation method. As the adsorbent layer, a styrene / divinylbenzene copolymer resin, a styrene / divinylbenzene / methacrylate copolymer resin, a divinylbenzene / vinylpyrrolidone copolymer resin, or a layer containing silica into which a phenyl group or an octadecyl group is introduced is used. A method for preparing a sample for analysis of residual agricultural chemicals according to any one of claims 1 to 4. A layer containing potassium carbonate or magnesium sulfate is used as the dehydrating layer, and a mixed solvent of at least one of acetone, ethyl acetate, acetonitrile or acetone, ethyl acetate and acetonitrile and a hydrocarbon solvent is used as the second solvent. A method for preparing a sample for analysis of residual agricultural chemicals according to any one of claims 1 to 5. A method for preparing a sample for analyzing pesticide residues in foods using a gas chromatograph mass spectrometer,
Step 1 of extracting the residual pesticide from the food using a water-soluble first solvent and mixing the resulting extract with water to prepare a mixture;
By injecting the mixed solution onto the separation membrane of the first centrifuge tube whose interior is partitioned vertically by a separation membrane having a pore size of 10,000 molecular weight cutoff or more, and applying centrifugal force to the first centrifuge tube Step 2-1 for obtaining a filtrate by passing the mixed solution through the separation membrane;
The filtrate is injected onto the adsorbent layer of the second centrifuge tube, the interior of which is partitioned vertically by an adsorbent layer that has liquid permeability and can adsorb the residual agricultural chemical, and is centrifuged in the second centrifuge tube A step 2-2 for passing the filtrate through the adsorbent layer by applying force;
After the adsorbent layer that has undergone step 2-2 is disposed above the treatment layer of the third centrifuge tube whose interior is vertically divided by a treatment layer having a liquid permeability and including a dehydration layer, the residual agricultural chemical Is injected into the adsorbent layer, and centrifugal force is applied to the third centrifuge tube to apply the second solvent to the adsorbent layer. And step 3 of passing through the treatment layer,
Of a sample for analysis of residual pesticides containing The residual pesticide analysis sample according to claim 7, wherein a purification layer capable of adsorbing a contaminant contained in the second solvent that has passed through the adsorbent layer is disposed below the dehydration layer in the treatment layer. Preparation method. The method for preparing a sample for analysis of residual agricultural chemicals according to claim 7 or 8, wherein acetonitrile or acetone is used as the first solvent. In the mixed solution, the mixing ratio of the extract (A) and the water (B) is set to 50:50 to 60:40 by volume ratio (A: B), and the pore size of the separation membrane is 0.1. The method for preparing a sample for analysis of residual agricultural chemicals according to any one of claims 7 to 9, wherein the sample is made of polytetrafluoroethylene resin or polyvinylidene fluoride resin having a size of 05 to 0.2 µm. As the adsorbent layer, a styrene / divinylbenzene copolymer resin, a styrene / divinylbenzene / methacrylate copolymer resin, a divinylbenzene / vinylpyrrolidone copolymer resin, or a layer containing silica into which a phenyl group or an octadecyl group is introduced, And, after adjusting the mixing ratio of the first solvent (A) and the water (B) in the filtrate obtained in the step 2-1 by volume ratio (A: B) to 40:60 to 50:50, the step The method for preparing a sample for analysis of residual agricultural chemicals according to any one of claims 7 to 10, wherein the filtrate is injected onto the adsorbent layer in 2-2. A layer containing potassium carbonate or magnesium sulfate is used as the dehydrating layer, and a mixed solvent of at least one of acetone, ethyl acetate, acetonitrile or acetone, ethyl acetate and acetonitrile and a hydrocarbon solvent is used as the second solvent. A method for preparing a sample for analysis of residual agricultural chemicals according to any one of claims 7 to 11. A portion of the filtrate obtained in step 2-1 was collected and secured as a sample for analyzing the residual agricultural chemical using a high-performance liquid chromatograph mass spectrometer, and obtained in step 2-1. The method for preparing a sample for analysis of residual agricultural chemicals according to any one of claims 7 to 12, wherein the remainder of the filtrate is injected onto the adsorbent layer in step 2-2. Analysis for preparing a sample for analyzing the residual pesticide using a gas chromatograph mass spectrometer from a mixture of the extract obtained by extracting the residual pesticide from food with a water-soluble solvent and water A sample preparation device comprising:
Centrifuge tube A with the bottom closed;
An adsorbent that can be inserted into and removed from the inside of the centrifuge tube A, has liquid permeability when inserted into the centrifuge tube A, and can adsorb the residual pesticide contained in the mixed solution A cylindrical body A1 that can vertically divide the inside of the centrifuge tube A by a layer;
A separation membrane that can be inserted into and removed from the inside of the cylindrical body A1 and has a pore diameter of 10,000 molecular weight cutoff or more when inserted into the cylindrical body A1 is provided above the adsorbent layer. A cylindrical body A2 that can be disposed on
A first unit including:
A centrifuge tube B that is separate from the centrifuge tube A and has a closed bottom,
The centrifuge tube B can be inserted into and removed from the centrifuge tube B, and when inserted into the centrifuge tube B, the centrifuge tube B is moved up and down by a treatment layer having liquid permeability and including a dehydrating layer. A cylindrical body B1 that can be partitioned into;
A second unit including:
With
The cylindrical body A1 can be inserted into the cylindrical body B1 in an overlapping manner.
Sample preparation device for analysis of residual agricultural chemicals. Analysis for preparing a sample for analyzing the residual pesticide using a gas chromatograph mass spectrometer from a mixture of the extract obtained by extracting the residual pesticide from food with a water-soluble solvent and water A sample preparation device comprising:
Centrifuge tube A with the bottom closed;
An adsorbent that can be inserted into and removed from the inside of the centrifuge tube A, has liquid permeability when inserted into the centrifuge tube A, and can adsorb the residual pesticide contained in the mixed solution A cylindrical body A1 ′ capable of partitioning the inside of the centrifuge tube A vertically by a layer;
The centrifuge tube A can be inserted into and removed from the centrifuge tube A, and when inserted into the centrifuge tube A, the centrifuge tube A is moved up and down by a separation membrane having a pore diameter of 10,000 molecular weight cutoff or more. A cylindrical body A2 ′ that can be partitioned into
A first unit including:
A centrifuge tube B that is separate from the centrifuge tube A and has a closed bottom,
The centrifuge tube B can be inserted into and removed from the centrifuge tube B, and when inserted into the centrifuge tube B, the centrifuge tube B is moved up and down by a treatment layer having liquid permeability and including a dehydrating layer. A cylindrical body B1 that can be partitioned into;
A second unit including:
With
The cylindrical body A1 ′ can be inserted inside the cylindrical body B1 in an overlapping manner.
Sample preparation device for analysis of residual agricultural chemicals. A method for analyzing residual pesticides in foods,
Step 1 of extracting the residual pesticide from the food using a water-soluble first solvent and mixing the resulting extract with water to prepare a mixture;
By injecting the mixed solution onto the separation membrane of the first centrifuge tube whose interior is partitioned vertically by a separation membrane having a pore size of 10,000 molecular weight cutoff or more, and applying centrifugal force to the first centrifuge tube Step 2-1 for obtaining a filtrate by passing the mixed solution through the separation membrane;
Step 2-2A for separating and securing a part of the filtrate;
On the adsorbent layer of the second centrifuge tube, the interior of which is divided vertically by an adsorbent layer that is liquid-permeable and capable of adsorbing the residual agricultural chemicals, the remaining part after fractionation in step 2-2A Injecting the filtrate and applying the centrifugal force to the second centrifuge tube to pass the filtrate through the adsorbent layer, 2-2B;
After disposing the adsorbent layer that has undergone step 2-2B above the treatment layer of the third centrifuge tube, the interior of which is vertically divided by a treatment layer having a liquid permeability and including a dehydration layer, Is injected into the adsorbent layer, and centrifugal force is applied to the third centrifuge tube to apply the second solvent to the adsorbent layer. And step 3 of passing through the treatment layer,
Step 4 of analyzing the filtrate collected in Step 2-2A using a high performance liquid chromatograph mass spectrometer;
Step 5 of analyzing the second solvent that has passed through the adsorbent layer and the treatment layer in Step 3 using a gas chromatograph mass spectrometer;
For analysis of residual pesticides.

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