KR20200117311A - Simultaneous analytical method for multi component of dithiocarbamate-based pesticide - Google Patents

Abstract

The present invention relates to a simultaneous analysis method for a residual amount of a dithiocarbamate-based multi-pesticide component, and more specifically, to a method for analyzing a dithiocarbamate-based multi-pesticide component for various samples such as various agricultural products, food, environmental samples and the like. In particular, a method of the present invention is capable of stably analyzing residual pesticide even in a sample such as pulse crops and the like, containing a large amount of proteins that interfere with methylation, and according to the present invention, it is possible to accurately and quickly analyze a dithiocarbamate-based multi-pesticide component through a simple kit that can be applied to the analysis method, by optimization using a solid reagent.

Description

Simultaneous analytical method for multi component of dithiocarbamate-based pesticide}

The present invention relates to a method for simultaneous analysis of multiple components of a dithiocarbamate-based pesticide.

Dithiocarbamate-based pesticides are a kind of organic sulfur-based pesticides that are known to have a relatively low range of efficacy and resistance, and are classified as non-penetrating fungicides according to their penetrating properties. Dithiocarbamate pesticides are dialkyl dithiocarbamate (DDC), ethylene bis dithiocarbamate (EBDC), propylene bis dithiocarbamate (propylene bis), depending on the chemical structure. Dithiocarbamate; PBDC) is classified into three groups, and according to each group, DDC pesticides are known as ferbam, fermate, ziram, zerlate, and thiram, and EBDC pesticides are mancozeb ( mancozeb), maneb, zineb, metiram and nabam are known, and propineb, a PBDC-based pesticide, is known.

In Korea, mancozeb, metiram, propineb, and thiram are used, and pesticides that were used in the past but not used now include maneb and geneb. zineb), etc. Currently, in Korea, persimmons and 93 other agricultural products have been managed by setting and managing the allowable standards for residual pesticides, and control of anthrax in fruit trees including apples and pears; To prevent downy mildew of vegetables such as tomatoes and lettuce, the standards for safe use of pesticides have been registered.

Dithiocarbamate-based pesticides are effective against various pathogens, have low toxicity, and low cost, and have been used to control more than 70 crop diseases including apples, tomatoes and pears in countries around the world such as the United States, France, and Japan. It is a useful crop protection agent.

Since dithiocarbamate-based pesticides are sprayed on various crops, a quick, accurate and reliable residual analysis method is essential to ensure safety by inspecting and evaluating the level of pesticide residues in food. Most of the pesticides are non-polar compounds and are extracted, distributed, and purified in organic solvents and finally analyzed by instrumentation. However, dithiocarbamate-based pesticides are aqueous and organic due to the chemical structure characteristics that contain metal ions. Since it is poorly soluble in a solvent, the method that can be used for extraction and purification of the residue is very limited.

Therefore, as an analysis method used internationally so far, it is a method of destroying dithiocarbamate molecules to generate CS ₂ and collecting them in a color developing reagent, or by methylation using CH ₃ I to detect spectroscopy. It is known to convert it into a photometric substance and read the absorbance for analysis or to use chromatography. However, the CS ₂ analysis method has a disadvantage that it is not only difficult to accurately analyze the qualitative and quantitative analysis of the pesticide components contained in the sample, but also is a highly flammable and toxic substance, because the generated CS ₂ is highly volatile and thus a high risk of loss. Analysis of dithiocarbamate-based pesticides by methylation using CH ₃ I and then HPLC analysis has been published by Gustafsson and Thomson in 1981. Although it has been used for analysis and monitoring, it is not commonly used in actual field due to complicated operation, time-consuming and costly, as well as inconvenience such as the use of a large amount of organic solvent and reagent preparation process. In addition, in the case of samples with high protein content, such as bean samples such as soybeans and red beans, the orbital phase analysis method has a tendency to sharply lower the recovery rate due to severe interference with the derivatization process, so there is a limitation as a general-purpose analysis method for analyzing various agricultural products.

The quick, easy, cheap, effective, Rugged, and Safe (QuEChERS) method, which is currently the most widely used method for agricultural products or foods around the world, including the United States and Europe, is the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method. In Korea, it is also announced as a rapid analysis method that simultaneously analyzes 320 pesticide residues, centered on the National Agricultural Products Quality Management Service. Meanwhile, the Ministry of Food and Drug Safety recently introduced a new method for analyzing pesticide residues of various pesticides to expand and analyze the existing 320 ingredients to 470 ingredients with the introduction of the PLS (Positive List System) system, which is fully implemented in Korea from 2019. It is in the process of notifying the revision. However, due to the unique physicochemical properties of the dithiocarbamate-based pesticide component, that is, the chemical structure containing metal, and the difficulty of the analysis conditions, all of the series components in the analysis component list. It is a reality that serious blind spots exist in securing the safety of pesticide residues in national food. In order to secure the safety of dithiocarbamate-based pesticides, which are one of the relatively high use and frequency of pesticides not only in Korea, but also in the world, and the residual allowance standard for 93 agricultural products has been established in Korea alone. It can be said that there is an urgent need to develop a method for analyzing residues of single and multiple pesticides that can quickly, simply and accurately analyze the residual pesticides of the relevant family.

On the other hand, Korean Patent No. 1278631 discloses a fast and efficient method for analyzing dithiocarbanate-based pesticides of mandarin, and Korean Patent No. 0430932 discloses a mancozeb analysis method using HPLC, but the dithiocarbamate of the present invention There has not been disclosed a method for simultaneous analysis of multiple components of pesticides.

The present invention was derived from the above requirements, and the present invention provides a method for simultaneous analysis of residual amounts of various dithiocarbamate-based pesticide components, and the analysis method of the present invention provides a variety of dithiocarbamate-based various samples. In analyzing the pesticide component of, the present invention was completed by confirming that it is an analysis method that can be easily analyzed by using solid reagents, and that it is an analysis method that can maximize the recovery rate by establishing optimal conditions.

In order to achieve the above object, the present invention

(1) weighing a sample to be analyzed for residual pesticide components;

(2) adding the weighed sample to an ethylenediaminetetraacetic acid (EDTA) solution;

(3) After step (2), L-cysteine, tetrabutylammonium hydrogen sulfate (TBAH), sodium chloride, a solid pH adjuster and a protein precipitant are added and mixed to the EDTA solution in which the sample is dissolved to prepare a mixed solution. ;

(4) after step (3), adding an organic solvent containing methyl iodide to the mixed solution and stirring to methylate residual pesticide components;

(5) after step (4), centrifuging to take a supernatant of the mixed solution and filtering; And

(6) LC-MS/MS analysis of the solution filtered in step (5); a method for simultaneous analysis of the residual amount of dithiocarbamate-based multi-pesticide components is provided.

In addition, the present invention EDTA (ethylenediaminetetraacetic acid) solution; L-cysteine, tetrabutylammonium hydrogen sulfate (TBAH), sodium chloride, solid pH adjuster and protein precipitant; It provides a kit for simultaneous analysis of the residual amount of dithiocarbamate-based various pesticide components comprising; and an organic solvent containing methyl iodide.

The present invention relates to a method for simultaneous analysis of the residual amount of a variety of dithiocarbamate-based pesticide components, and it is possible to determine the residual amount of a variety of dithiocarbamate-based pesticide components for various analysis targets, and the reagent used in the analysis of the present invention is a solid By using a reagent, it was confirmed that it was not only quick, but also simple, and had an effect of improving the recovery rate of residual pesticides. In particular, in the case of a protein-rich sample, by using a protein reagent such as ammonium sulfate and trichloracetic acid, It is an analysis method that has the effect of maximizing the recovery rate.

1 is an analysis flow chart for the analysis method of the residual amount of dithiocarbamate-based pesticides of the present invention.
Figure 2 shows the chemical structure of a dithiocarbamate-based pesticide.
3 is a result of comparing the recovery rate (%) according to the amount of TBAH used.
4 is a chromatogram of brown rice analyzed by the method for analyzing the residual amount of dithiocarbamate pesticides of the present invention.
5 is a chromatogram of mandarin analyzed by the method for analyzing the residual amount of dithiocarbamate-based pesticides of the present invention.
6 is a chromatogram of potatoes analyzed by the method for analyzing residual amounts of dithiocarbamate pesticides of the present invention.
7 is a chromatogram of blue pepper analyzed by the method for analyzing the residual amount of dithiocarbamate-based pesticides of the present invention.

The present invention

(1) weighing a sample to be analyzed for residual pesticide components;

(2) adding the weighed sample to an ethylenediaminetetraacetic acid (EDTA) solution;

(3) After step (2), L-cysteine, tetrabutylammonium hydrogen sulfate (TBAH), sodium chloride, a solid pH adjuster and a protein precipitant are added and mixed to the EDTA solution in which the sample is dissolved to prepare a mixed solution. ;

(4) after step (3), adding an organic solvent containing methyl iodide to the mixed solution and stirring to methylate residual pesticide components;

(5) after step (4), centrifuging to take a supernatant of the mixed solution and filtering; And

(6) LC-MS/MS analysis of the solution filtered in step (5); and a method for simultaneous analysis of the residual amount of dithiocarbamate-based various pesticide components including (FIG. 1).

The dithiocarbamate-based pesticides are ferbam, ziram, thiram, mancozeb, maneb, zineb, metiram, nabam ( nabam) and propineb (propineb) is preferably at least one selected from, but is not limited thereto (Fig. 2).

The sample is preferably one or more selected from grains, beans, vegetables, documents and fruits, and the grains are brown rice, beans are soybeans or red beans, vegetables are green peppers, documents are potatoes, or fruits are mandarin. It is not limited to this. The sample may be analyzed as long as it may contain dithiocarbamate, such as agricultural products, food, or environmental samples.

In step (3), the solid pH adjusting agent is preferably boric acid, phosphoric acid, or citric acid, but is not limited thereto.

In step (3), the protein precipitating agent is preferably ammonium sulfate or trichloracetic acid, but is not limited thereto.

In the step (4), the organic solvent contains methyl iodide and is capable of transferring a variety of dithiocarbamate-based pesticide components from an aqueous solution to a supernatant, and includes acetonitrile, methylene dichloride, chloroform, hexane, ethyl acetate, and acetone. And dimethyl sulfoxide (dimethyl sulfoxide; DMSO) is preferably at least one selected from, but is not limited thereto.

The method for simultaneous analysis of the residual amount of the dithiocarbamate-based various pesticide components is preferably

(1) weighing a sample to be analyzed for residual pesticide components to be 5 to 20 g;

(2) adding the weighed 5 to 20 g of the sample to 9 to 11 ml of EDTA solution;

(3) After step (2), 0.1 to 0.5 g of L-cysteine, 0.1 to 0.7 g of TBAH, 0.5 to 2.0 g of sodium chloride, 0.1 to 0.4 g of boric acid and 0.1 in the EDTA solution in which the sample is dissolved Mixing by adding ~1.5g of ammonium sulfate;

(4) After step (3), 9 to 11 ml of acetonitrile containing 0.01 to 0.1 M of methyl iodide was added to the mixed solution, followed by stirring at 1000 to 2000 rpm for 1 to 3 minutes to remain. Methylating the pesticide component;

(5) after the step (4), centrifuging at 8 to 12°C for 8 to 12 minutes at 2500 to 3500 rmp to take the supernatant of the mixed solution and filtering; And

(6) mass spectrometry (LC-MS/MS) of the solution filtered in step (5), but is not limited thereto.

In addition, the present invention EDTA (ethylenediaminetetraacetic acid) solution; L-cysteine, tetrabutylammonium hydrogen sulfate (TBAH), sodium chloride, solid pH adjuster and protein precipitant; It provides a kit for simultaneous analysis of the residual amount of dithiocarbamate-based various pesticide components comprising; and an organic solvent containing methyl iodide.

In the kit, the solid pH adjusting agent, protein precipitating agent and organic solvent are as described above.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for describing the present invention in more detail, and it is apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

1. Reagents, materials and equipment

Standards for analysis such as Mancozeb (purity 97.8%) used in an example of the present invention were purchased and used from Accustandard. EDTA-2Na (ethylenediaminetetraacetic acid-disodium) used for extraction was purchased from Dae Jung (Korea). NaOH and ammonium sulfate were purchased from Yakuri Pure Chemicals Co., Ltd. (Japan). And L-cysteine was purchased from Tokyo Chemical Industry Co., Ltd. (Japan), and Methyl iodide was purchased from Kanto Chemical Co., Inc. (Japan). Tetrabutylammonium hydrogen sulfate (TBAH), boric acid, formic acid, and ammonium formate were purchased from Sigma Aldrich (USA) and used. Jung) was purchased from Korea, and acetonitrile and water solvent were purchased from Merck (Germany), and GR grade was used.. A shaker for shaking agricultural product samples during pretreatment (Genogrinder 2010, SPEX SamplePrep, USA) was used, and a centrifuge (Allegra X-15R, Beckman Culter Life Sciences, USA) was used for centrifugation.

The chemical structural formula and physicochemical properties of the dithiocarbamate-based pesticides used in the examples of the present invention are disclosed in Table 1 below.

Chemical formula and physicochemical properties of dithiocarbamate pesticides Pesticide ingredients
Chemical formula
Molecular Weight
Log P _ow
Solubility Water (mg/ℓ) Organic solvent (g/ℓ) Thiram C ₆ H ₁₂ N ₂ S ₄ 240.4 1.73 18 Ethanol <10, acetone 80, chloroform 230, n -hexane 0.04, dichloromethane 170, toluene 18, isopropanol 0.7 Paa balm C ₉ H ₁₈ FeN ₃ S ₆ 416.5 -1.6 130 Soluble in chloroform, pyridine, acetonitrile, acetone Jiram C ₆ H ₁₂ N ₂ S ₄ Zn 305.8 1.23 1.58~18.3 Acetone 2.88, methanol 0.22, toluene 2.33, n- hexane 0.07 Maneb C ₄ H ₆ MnN ₂ S ₄ 265.3 - insoluble insoluble (soluble in chelating agents) Mankozeb [C ₄ H ₆ MnN ₂ S ₄ ] _x Zn _y 271.2 0.26 6.2 insoluble (soluble in chelating agents) Metiram [C ₁₆ H ₃₃ N ₁₁ S ₁₆ Zn ₃ ] _x (1088.6) _x 0.3 insoluble insoluble (soluble in pyridine with decompositions) Geneve C ₄ H ₆ N ₂ S ₄ Zn 275.8 ≤1.3 10 insoluble (soluble in chelating agents) Night C ₄ H ₆ N ₂ Na ₂ S ₄ 256.3 - 200,000 insoluble (soluble in chelating agents) Propineb [C ₅ H ₈ N ₂ Zn] _x 289.8 -0.26 10 Toluene, hexane, dichloromethane <0.1, DMF + DMSO >200 g/ℓ

2. Sample to be analyzed

Agricultural products to be analyzed for rapid analysis of dithiocarbamate pesticides are brown rice for grains, soybeans for beans, peppers for vegetables, mandarin for fruits, and documents according to the representative sample classification criteria for verification of the residual analysis method in the food industry. Is potatoes, and a total of 5 representative agricultural products samples were selected. Pesticide-free cultivation samples of brown rice, pepper, mandarin, potatoes and soybeans selected as representative agricultural products were analyzed after processing organic products sold at local hypermarkets according to the food pretreatment method for pesticide residue analysis. Each analysis sample was stored frozen at -20°C or lower, and a certain amount was used for analysis.

3. Sample preparation step

In order to analyze dithiocarbamate-based pesticides from the agricultural product samples, a certain amount of samples (soybean: 5g, brown rice, red pepper, mandarin and potato: 10g) were weighed in a 50ml PE tube, and an aqueous solution containing 0.25M EDTA (0.45 M NaOH containing, pH 9-10) 10 ml was added. In the case of soybeans and brown rice, an aqueous solution was added and left for 20 minutes to moisten. L-cysteine, sodium chloride, TBAH (tetrabutylammonium hydrogen sulfate), boric acid, and ammonium sulfate were added to the extract in a certain amount, and then shaken at 1,500 rpm for 2 minutes.

The present invention intends to replace all added reagents with solid reagents for quick and easy pretreatment, and for this purpose, L-cysteine, sodium chloride, TBAH including EDTA are directly mixed in a pretreatment system without dilution in aqueous solution and used. I did. In order to optimize the new pretreatment process using solid reagents, the recovery rate was calculated by selecting a solid reagent that can be used for each step and adjusting the amount of use step by step.

4. Protein step

In order to confirm an efficient derivatization reaction for a bean sample containing high protein such as soybeans, a protein-free step was added to the extract. In general, an organic solvent such as chloroform and methylene dichloride is used for the protein removal step, but in the present invention, the protein removal is performed using ammonium sulfate, a solid reagent. In order to optimize the protein production step, the recovery rate was calculated while adjusting the amount of reagent used step by step.

5. Derivatization step

To the pre-treated supernatant, 10 ml of acetonitrile containing 0.05M methyl iodide was added and shaken at 1,500 rpm for 2 minutes to induce methylation sufficiently. After centrifugation at 3,000 rpm for 10 minutes, the supernatant It was filtered and confirmed with an analyzer such as LC-MS/MS.

6. Preparation of standard calibration curve

Dithiocarbamate-based pesticide standard solutions such as Mancozeb are dissolved in 0.5g of L-cysteine and 0.25M EDTA-2Na aqueous solution (containing 0.45M NaOH, pH 9-10) to make 100mg/kg So that it was prepared as a stock solution.

In the case of soybeans, the stock solution was diluted to a concentration of 0.005, 0.01, 0.05, 0.1 and 0.5mg/kg, and in the case of 4 agricultural product samples excluding soybeans. It was diluted to concentrations of 0.01, 0.05, 0.1, 0.5 and 1.0 mg/kg. After inducing methylation, each of them was matrix matched and injected into LC-MS/MS at a time of 5 μl to prepare a standard calibration curve based on the area of the peak.

Matrix matched method of standard material for LC-MS/MS analysis of soybean target
density Standard substance
sample
One 2 3 4 5 5㎍/kg 10㎍/kg 50㎍/kg 100㎍/kg 500㎍/kg Way

STD
10㎍/kg
500µl STD
20㎍/kg
500µl STD
100㎍/kg
500µl STD
200㎍/kg
500µl STD
1000㎍/kg
500µl Acetonitrile
500µl Blank metrics
500µl Blank metrics
500µl Blank metrics
500µl Blank metrics
500µl Blank metrics
500µl Extracted sample
500µl

Matrix matched method of standard substances for LC-MS/MS analysis of brown rice, blue pepper, mandarin and potato target
density Standard substance
sample
One 2 3 4 5 10㎍/kg 50㎍/kg 100㎍/kg 500㎍/kg 1000㎍/kg
Way

STD
20㎍/kg
500µl STD
100㎍/kg
500µl STD
200㎍/kg
500µl STD
1000㎍/kg
500µl STD
2000㎍/kg
500µl Acetonitrile
500µl Blank metrics
500µl Blank metrics
500µl Blank metrics
500µl Blank metrics
500µl Blank metrics
500µl Extracted sample
500µl

LC-MS/MS analysis conditions for Mancozeb analysis Device Dionex ultimate 3000 (Thermo Science, USA) Detector TSQ Quantum Access Max (Thermo Science, USA) column Capcell core-C ₁₈ (2.1 mm ID x 150 mm, 2.7 ㎛) Column temperature 40℃ Flow rate 0.4ml/min Mobile phase





A: 0.1% formic acid + 5mM ammonium formate in water
B: Acetonitrile containing 0.1% formic acid Time (minutes) A B 1.5 90 10 3.0 10 90 6.0 10 90 6.5 90 10 10.0 90 10 Spray voltage 3,000 V Ionization Electrospray ionization negative mode Capillary temperature 250℃ Vacuum tube temperature 270℃ Gas pressure (N ₂ ) 1.0 unit (Ion sweep), 35 unit (Sheath), 15 unit (Aux)

Examples of selected reaction monitering (SRM) conditions for Mancozeb Precursor ion Product ion CE Q1 PW Q3 PW 239.320
47.480 58 0.7 0.7 58.400 15 0.7 0.7

7. Recovery rate test

In order to determine the suitability of each agricultural product sample and the analysis method of pesticide residues, a recovery rate test was performed on untreated samples. Each target agricultural product untreated sample was treated with a standard solution of dithiocarbamate pesticide at the level of 0.1 mg/kg and 0.5 mg/kg, and the analysis process was repeated 3 times, and then the average value and the relative standard deviation were determined. Obtained.

Example 1. Selection and optimization of solid reagents for rapid analysis method development

(1) L-cysteine addition

It is known that the thiol group of L-cysteine helps to bind with metal ions, thereby increasing the recovery rate (%). In the Food Code, 0.5 g of L-cysteine per 100 ml of the extraction solution was used. However, since a solid reagent was used in the present invention, only 10 ml of the extraction solution was used. For optimization, the amount of L-cysteine added was 0.1 and 0.3. And the recovery rate was measured under the conditions of 0.5g.

As a result, as disclosed in Table 6 below, even if the addition amount of L-cysteine is increased from 0.1 g to 0.5 g, the difference in the recovery rate does not change significantly, so the addition amount of L-cysteine was selected as 0.1 g.

Recovery rate according to the amount of L-cysteine added (%) L-cysteine addition amount (g) 0.0 0.1 0.3 0.5 Recovery rate (%) 82.4 101.4 99.2 100.0

(2) TBAH adding

TBAH is known to act as a phase transfer catalyst to move dithiocarbamate-based pesticide components to the organic solvent layer. In the existing method, 5 ml of 0.41M TBAH per 100 ml of the extraction solution was used, and when converted to solid, it is converted to 0.69 g. In this analysis method using a solid reagent, after a certain amount, the effect rapidly decreased and 0.1g was used. Compared to the case where this reagent was not used, the effect of increasing the recovery rate by about 40% was confirmed when 0.1 g was used (FIG. 3).

(3) sodium chloride addition

The amount of sodium chloride added is as disclosed in Table 7 below. After optimization, 1.0 g was added as a solid.

Recovery rate (%) according to the amount of sodium chloride (NaCl) added NaCl addition amount (g) 0.0 0.5 1.0 1.5 2.0 Recovery rate (%) 87.9 93.0 99.6 93.1 94.1

(4) Sodium sulfate , Anhydride addition

Water/acetonitrile (0.05M methyl ioidie in acetonitrile) extraction/derivation method. Due to the nature of the analysis method, due to the concern of moisture interference, anhydrous sodium sulfate was added to confirm the result.As disclosed in Table 8 below, the addition of anhydrous sodium sulfate is the recovery rate ( %) was excluded.

Recovery rate according to the amount of sodium sulfate added (%) Addition amount of sodium sulfate (g) 0.0 0.5 1.0 1.5 2.0 Recovery rate (%) 100.4 97.7 98.3 99.0 96.5

( 5) Selection and optimization of ammonium sulfate as a solid reagent for the protein production process

Ⓛ Blank( Negligence )in, Mankozeb's For methylation Ammonium sulfate effect

It was found that the addition of ammonium sulfate did not affect the methylation of the typical dithiocarbamate-based pesticide mancozeb, indicating that there was no problem in using the solid protein reagent for the development of this rapid assay method.

Addition amount of ammonium sulfate (g) 0.0 0.1 0.5 1.0 1.5 Recovery rate (%) 100.0 103.8 96.3 98.1 97.1

② For soybean samples containing high protein Ammonium sulfate Protein effect

For soybean samples containing high protein, the effect of the solid reagent ammonium sulfate for protein production increased to 85.5% when 0.5g was added, compared to 47.4% that was found without addition. Appeared. The effective recovery rate of the process assay is specified at 70%. On the other hand, even if ammonium sulfate is added to general samples such as brown rice, blue pepper, mandarin, and potatoes, all of them show a high recovery rate that exceeds the effective recovery rate, allowing the addition of the solid reagent to most samples without discriminating between low and high protein-containing samples. Was confirmed.

Addition amount of ammonium sulfate (g) 0.0 0.1 0.5 1.0 1.5 Recovery rate (%) 47.4 80.0 85.5 74.8 72.4

(6) Improvement of methylated derivative process to establish rapid analysis method

Ⓛ Selection of the optimal organic solvent used for methylation reagents

Methyl iodide, which is a methylation reagent for the derivatization reaction, was used as a mixed solvent of methylene chloride/hexane in the conventional analysis method, but acetonitrile was selected in the present invention. This means that the derivatization proceeds smoothly, as well as the fast and clear liquid-liquid distribution after the reaction, and the ease of post-operation that the component to be analyzed is distributed in acetonitrile (supernatant) to quickly take the solution from the top of the reaction vessel. There was an advantage to be able to perform quickly and easily.

② Selection of solid reagents for acidity control

In the derivatization process, in order for the derivatization reagent (CH ₃ I) to react effectively with the thiol group of the dithiocarbamate-based pesticide in the aqueous solution, the acidity of the aqueous solution needs to be maintained at pH 7-8. For this purpose, the acidity is usually adjusted with an acidic solution such as HCl immediately before derivatization, but in the present invention, the acidity is adjusted by adding boric acid, an acidic solid reagent. In order to maintain the required pH, the amount of boric acid added was optimized to 0.4 g. Unlike when using an acidic aqueous solution, that is, a liquid, this solid reagent can be pre-prepared in a solid form in the pretreatment step, enabling rapid analysis kit production, and enhancing pretreatment operation and ease of use.

(7) Dithiocarbamate type Establish a rapid residual analysis process for pesticides

The simple and convenient method for analyzing the residues of various pesticide components for the developed dithiocarbamate pesticide consists of three steps.

Each step consists of (a) a pre-treatment (extraction, proteolysis) step consisting of a solid reagent, (b) a derivatization step, and (c) an instrument analysis step. Steps (a) and (b) up to and including steps can be completed within 30 minutes.

The configuration of the QuEChERS kit, which is currently developed in Europe and the United States and used worldwide, consists of (1) an extraction kit and (2) a purification kit, so there are hundreds of pesticides in each country with ease of use, convenience, and quick and accurate methods. It is fully established as a rapid analysis method that can analyze components simultaneously. In Korea, it was developed as a simultaneous multi-component pesticide residue analysis method of 320 components using the QuEChERS kit, centered on the National Agricultural Products Quality Management Service, so far, a process for analyzing pesticide residues among agricultural products by national testing and research institutes, agricultural product safety inspection agencies, and agricultural product safety testing agencies. As an analysis method, it is notified in the Agricultural Products Quality Control Act. However, since it is impossible to analyze dithiocarbamate-based pesticides with the simultaneous multi-component pesticide residue analysis method using such a QuEChERS kit, the value of the rapid analysis method established through this research and development can be said to be higher.

For dithiocarbamate-based pesticides developed through this study, (1) a dedicated pretreatment kit prepared with a solid reagent, and (2) a derivatization kit with improved speed and convenience are used, which is recognized as a blind spot for analysis. It is considered to be the best way to enable residual analysis of dithiocarbamate-based pesticides.

Example 2. Experiment to verify the effectiveness of the developed multi-component pesticide rapid analysis method through the recovery rate of each sample

A recovery rate test was performed on the field samples using a new simple rapid analysis method that was converted to a pretreatment method using a solid reagent and undergoes an optimization step. Among the dithiocarbamate-based pesticides, mancozeb, which is the most used in Korea, was used (3rd in 2017, 1,778 M/T), propineb (6th in 2017, 203 M). /T) and metiram were first selected and analyzed for brown rice, green pepper, mandarin, and potato, and the samples were treated to contain 0.5mg/kg of pesticides in each pesticide-free sample, and then the recovery rate As a result of analyzing, as disclosed in Tables 11 to 13 below, it was confirmed that the average recovery rate was all 70% or more. Therefore, it was found that the simultaneous analysis method for dithiocarbamate-based pesticides met the efficacy criteria and was effective. The chromatogram results for each sample are shown in FIGS. 4 to 7.

Mancozeb recovery rates for various crops crops Fortification
(mg kg ^-1 ) Recovery rate (%) Mean(%)±SD LOQ (mg kg ^-1 ) One 2 3 Brown rice 0.1 85.0 83.3 88.5 85.6±2.6
0.01





0.5 74.6 85.2 90.0 83.3±7.9 Green pepper
0.1 79.6 100.3 93.6 91.2±10.5 0.5 86.5 94.2 91.0 90.6±3.8 Mandarin
0.1 98.6 82.8 102.5 94.6±10.4 0.5 105.1 103.1 103.3 103.8±1.1 potato
0.1 88.8 97.7 90.3 92.3±4.7 0.5 108.2 108.7 99.9 105.6±5.0

Metiram recovery rates for various crops crops Fortification
(mg kg ^-1 ) Recovery rate (%) Mean(%)±SD LOQ (mg kg ^-1 ) One 2 3 Brown rice 0.1 94.4 95.9 87.2 92.5±4.6
0.01





0.5 102.3 107.9 103.2 104.5±3.0 Green pepper
0.1 92.2 107.7 80.7 93.5±13.5 0.5 98.8 86.3 101.3 95.4±8.0 Mandarin
0.1 96.9 80.7 97.1 91.6±9.4 0.5 93.3 84.3 93.5 90.4±5.3 potato
0.1 102.4 103.9 106.8 104.4±2.3 0.5 98.8 108.3 88.6 98.5±9.9

Recovery rate of propineb for various crops crops Fortification
(mg kg ^-1 ) Recovery rate (%) Mean(%)±SD LOQ (mg kg ^-1 ) One 2 3 Brown rice 0.1 75.5 90.5 93.9 86.6±9.8
0.01





0.5 93.5 97.3 103.3 98.0±4.9 Green pepper
0.1 81.1 84.7 85.6 83.8±2.3 0.5 96.5 101.3 109.5 102.5±6.6 Mandarin
0.1 100.0 93.2 96.1 96.4±3.4 0.5 100.0 98.2 97.3 95.2±6.2 potato
0.1 88.9 87.5 80.3 85.6±4.6 0.5 92.2 104.4 86.3 94.3±9.2

The selected dithiocarbamate pesticides were confirmed that all four crops (brown rice, green pepper, mandarin, and potato) satisfied the criteria for effective recovery and coefficient of variation, and the analytical quantitative limit (LOQ) was 0.01mg/kg. I did. Through this, it was determined that a new method for analyzing residual amounts of various pesticide components using solid reagents according to the present invention could simultaneously analyze dithiocarbamate-based pesticide components for various samples.

Example 3. Application of rapid residual analysis method for soybeans containing high protein

Soybean (protein content 36.21%) was used among representative high protein bean samples, and the effect of the new rapid residue analysis method was confirmed. When applying the analysis method notified in the Food Code, the recovery rate for soybeans is within 30%, and in other bean samples such as peas (37.5) and red beans (19.0%) with high protein content, the recovery rate is very low within 30-50%. Showed that the orbital analysis method could not be used for actual analysis.

In order to secure the possibility of the analysis method of the present invention, mancozeb, a representative dithiocarbamate-based pesticide, was selected and the recovery rate was compared with a method without/including the protein-free process.

First, the mancozeb recovery rate was as low as 47.4% in the soybean sample that was subjected to the pretreatment process without the addition of ammonium sulfate, a protein-free solid reagent. However, when ammonium sulfate was added to the protein-producing step and the methylation step, the recovery rate increased by 38% to 85.5%, confirming that the addition of ammonium sulfate is very useful when analyzing beans such as soybeans with high protein content.

Meanwhile, as shown in the above results, even if ammonium sulfate is added to general samples with low protein content such as brown rice, pepper, mandarin, potatoes, etc. It was confirmed that it was possible to add ammonium sulfate, the protein-free solid reagent, to most samples without it. This can be referred to in consideration of the composition of the reagent recipe of the pretreatment kit prepared as a solid reagent.

Recovery rate of mancozeb based on the amount of ammonium sulfate added to soybean samples containing high protein Addition amount of ammonium sulfate (g) 0.0 0.1 0.5 1.0 1.5 Recovery rate (%) 47.4 80.0 85.5 74.8 72.4

Example 4. Dithiocarbamate type For rapid analysis of pesticide residues Kit

By applying the pretreatment technology for rapid residual analysis of dithiocarbamate-based pesticides of Examples 1 to 3, a dedicated kit for rapid residual analysis of dithiocarbamate-based pesticides was developed.

The kit of the present invention consists of ⓛ a PE tube containing an aqueous EDTA solution for pretreatment, ② a packaging pack (pre-treatment + protein) and ③ a solution in which methyl iodide, a derivatization reagent, is dissolved in acetonitrile. It was composed of a cap tube (derivative) containing.

Claims (8)

(1) weighing a sample to be analyzed for residual pesticide components;
(2) adding the weighed sample to an ethylenediaminetetraacetic acid (EDTA) solution;
(3) After step (2), L-cysteine, tetrabutylammonium hydrogen sulfate (TBAH), sodium chloride, a solid pH adjuster and a protein precipitant are added and mixed to the EDTA solution in which the sample is dissolved to prepare a mixed solution. ;
(4) after step (3), adding an organic solvent containing methyl iodide to the mixed solution and stirring to methylate residual pesticide components;
(5) after step (4), centrifuging to take a supernatant of the mixed solution and filtering; And
(6) LC-MS/MS analysis of the solution filtered in step (5); Simultaneous analysis of the residual amount of dithiocarbamate-based multi-pesticide components. The method of claim 1, wherein the dithiocarbamate-based pesticides are ferbam, ziram, thiram, mancozeb, maneb, zineb, and metiram. (metiram), nabam (nabam) and propineb (propineb) at least one selected from the method of simultaneous analysis of the residual amount of the dithiocarbamate-based various pesticides, characterized in that at least one. The method of claim 1, wherein the sample is at least one selected from grains, beans, vegetables, documents, and fruits. The method of claim 1, wherein the solid pH adjusting agent in step (3) is boric acid, phosphoric acid, or citric acid. The method of claim 1, wherein the protein precipitating agent in step (3) is ammonium sulfate or trichloracetic acid. The method of claim 1, wherein the organic solvent in step (4) is at least one selected from acetonitrile, methylene dichloride, chloroform, hexane, ethyl acetate, acetone, and dimethyl sulfoxide (DMSO). Simultaneous analysis method of residual amount of various kinds of okabamate pesticides. The method of claim 1,
(1) weighing a sample to be analyzed for residual pesticide components to be 5 to 20 g;
(2) adding the weighed 5 to 20 g of the sample to 9 to 11 ml of EDTA solution;
(3) After step (2), 0.1 to 0.5 g of L-cysteine, 0.1 to 0.7 g of TBAH, 0.5 to 2.0 g of sodium chloride, 0.1 to 0.4 g of boric acid and 0.1 in the EDTA solution in which the sample is dissolved Mixing by adding ~1.5g of ammonium sulfate;
(4) After step (3), 9 to 11 ml of acetonitrile containing 0.01 to 0.1 M of methyl iodide was added to the mixed solution, followed by stirring at 1000 to 2000 rpm for 1 to 3 minutes to remain. Methylating the pesticide component;
(5) after the step (4), centrifuging at 8 to 12°C for 8 to 12 minutes at 2500 to 3500 rmp to take the supernatant of the mixed solution and filtering; And
(6) LC-MS/MS analysis of the solution filtered in step (5); Simultaneous analysis of the residual amount of dithiocarbamate-based multi-pesticide components. Ethylenediaminetetraacetic acid (EDTA) solution; L-cysteine, tetrabutylammonium hydrogen sulfate (TBAH), sodium chloride, solid pH adjuster and protein precipitant; And an organic solvent containing methyl iodide; a kit for simultaneous analysis of the residual amount of dithiocarbamate-based various pesticide components comprising.

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