KR102308572B1 - Simultaneous analytical method for multi component of dithiocarbamate-based pesticide - Google Patents

Abstract

The present invention relates to a method for simultaneous analysis of residual amounts of dithiocarbamate-based pesticide components, and to a method for analyzing dithiocarbamate-based pesticide components in various samples such as agricultural products, food, and environmental samples. In particular, it is a method that can stably analyze residual pesticides even in samples such as pulses containing a large amount of protein that interferes with methylation. It is possible to analyze various pesticide components of the okabamate system.

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 dithiocarbamate pesticides.

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

In Korea, mancozeb, metiram, propineb, and thiram are used. Pesticides used in the past but not used now include maneb and jineb (maneb). zineb), etc. Currently, domestic pesticide residues are established and managed for 93 agricultural products other than persimmons, and control of anthrax in fruit trees including apples and pears; To prevent downy mildew disease of vegetables such as tomatoes and lettuce, safety standards for pesticide use are registered.

Dithiocarbamate pesticides are effective against various pathogens and have low toxicity and low cost. It is a useful crop protection agent.

As dithiocarbamate pesticides are being sprayed on various crops, a fast, accurate and reliable residue analysis method is essential to secure safety by inspecting and evaluating the level of pesticide residues in food. Most pesticides are non-polar compounds, which are extracted, distributed, and purified in an organic solvent for final instrumentation analysis. Due to poor solubility in solvents, methods available for extraction and purification of residues are very limited.

Therefore, the analysis methods used internationally so far include a method of destroying dithiocarbamate molecules _{to generate CS 2} and collecting them in a color reagent, or a method of detecting them by methylation using _{CH 3 I.} A method of converting a photometric detection material to reading and analyzing the absorbance or using chromatography is known. However, the CS ₂ analysis method has the disadvantage of being a highly flammable and toxic substance, as well as difficult to accurately analyze the quality and quantity of pesticides contained in the sample because the _{generated CS 2 is highly volatile and has a high risk of loss.} The analysis method of analyzing by HPLC after methylation of dithiocarbamate pesticides using CH ₃ I has been published by Gustafsson and Thomson in 1981. Although it has been used for analysis and monitoring, it is not commonly used in the actual field because the operation is complicated, takes a lot of time and money, and there are inconveniences such as the use of a large amount of organic solvent and the reagent preparation process. Moreover, the static phase analysis method has a limitation as a general-purpose analysis method for analyzing various agricultural products, as the recovery rate tends to decrease sharply due to severe interference with the derivatization process in samples with high protein content, such as soybean and red bean samples.

Currently, the fastest, multi-pesticide residue analysis method used the most for agricultural products or food worldwide, including the United States and Europe, is the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method. In Korea, it has been announced as a rapid analysis method that simultaneously analyzes 320 pesticide residues, centered on the National Agricultural Products Quality Control Center. Meanwhile, the Ministry of Food and Drug Safety (MFDS) recently developed a new pesticide residue analysis method to analyze the existing 320 ingredients and 470 ingredients with the introduction of the PLS (Positive List System) system, which is fully implemented in Korea from 2019. We are in the process of making a revision announcement. However, due to the unique physicochemical characteristics of the chemical structure containing dithiocarbamate, that is, the chemical structure containing metal, and the difficult analysis conditions, all of the series components in the analysis component list are is excluded, the reality is that serious blind spots exist in securing the safety of pesticide residues in the national food. In order to secure the safety of dithiocarbamate pesticides, which are one of the agrochemicals with relatively high usage and frequency not only in Korea but also worldwide, and for which residue tolerance standards have been established for 93 agricultural products in Korea alone. It can be said that there is an urgent need to develop a single-component and multi-component pesticide residue analysis method that can quickly, simply, and accurately analyze the pesticide residues of the relevant series.

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

The present invention has been derived from the above needs, and the present invention provides a method for simultaneous analysis of residual amounts of dithiocarbamate-based multiple pesticide components, and the analysis method of the present invention provides a dithiocarbamate-based multi-species 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 can maximize recovery 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 to the EDTA solution in which the sample is dissolved and mixed 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 the supernatant of the mixed solution and filtering; and

(6) LC-MS/MS analysis of the solution filtered in step (5); provides a method for simultaneous analysis of residual amounts of dithiocarbamate-based pesticide components, including.

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

The present invention relates to a method for simultaneous analysis of residual amounts of dithiocarbamate-based multiple pesticide components, and it is possible to separate the residual amount of dithiocarbamate-based multiple pesticide components for various analyses, and the reagent used in the analysis of the present invention is solid. By using the reagent, it was confirmed that there is an effect of increasing the recovery rate of pesticide residues quickly and conveniently. 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 analytical method that has the effect of maximizing the recovery rate.

1 is an analysis flow chart for the method for analyzing the residual amount of dithiocarbamate-based pesticides of the present invention.
Figure 2 shows the chemical structure of dithiocarbamate-based pesticides.
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-based pesticides of the present invention.
5 is a chromatogram of mandarin analyzed by the residual amount analysis method of dithiocarbamate pesticides of the present invention.
6 is a chromatogram of potatoes analyzed by the method for analyzing the residual amount of dithiocarbamate pesticides of the present invention.
7 is a chromatogram of green pepper analyzed by the residual amount analysis method of dithiocarbamate 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 to the EDTA solution in which the sample is dissolved and mixed 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 the supernatant of the mixed solution and filtering; and

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

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

Preferably, the sample is at least one selected from cereals, beans, vegetables, documents and fruits, the grains are brown rice, the beans are soybeans or red beans, the vegetables are green peppers, the documents are potatoes, or the fruits are mandarin, but It is not limited thereto. The sample may be freely analyzed as long as it can contain dithiocarbamate, such as agricultural products, food, or environmental samples.

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

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

In step (4), the organic solvent contains methyl iodide, and is capable of transferring dithiocarbamate-based pesticide components from an aqueous solution to a supernatant, acetonitrile, methylene dichloride, chloroform, hexane, ethyl acetate, acetone. and at least one selected from the group consisting of dimethyl sulfoxide (DMSO), 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), 0.1-0.5 g of L-cysteine, 0.1-0.7 g of TBAH, 0.5-2.0 g of sodium chloride, 0.1-0.4 g of boric acid and 0.1 adding -1.5 g of ammonium sulfate and mixing;

(4) After step (3), 9 to 11 ml of acetonitrile containing 0.01 to 0.1 M of methyl iodide is added to the mixed solution, and stirred at 1000 to 2000 rpm for 1 to 3 minutes to remain methylating the pesticide component;

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

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

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

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

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

1. Reagents, Materials and Instruments

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

Chemical structural formulas and physicochemical properties of the dithiocarbamate pesticides used in Examples of the present invention are shown 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) tiram 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 parbam 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) Mancozeb [C ₄ H ₆ MnN ₂ S ₄ ] _x Zn _y 271.2 0.26 6.2 insoluble (soluble in chelating agents) methiram [C ₁₆ H ₃₃ N ₁₁ S ₁₆ Zn ₃ ] _x (1088.6) _x 0.3 insoluble insoluble (soluble in pyridine with decompositions) Zineb C ₄ H ₆ N ₂ S ₄ Zn 275.8 ≤1.3 10 insoluble (soluble in chelating agents) Nabam 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 subject to analysis for rapid analysis of dithiocarbamate pesticides are brown rice for cereals, soybeans for beans, red pepper for vegetables, mandarin for fruits, and paper is potato, and a total of five representative agricultural samples were selected. Pesticide-free cultivation samples of brown rice, red pepper, mandarin, potato and soybean selected as representative agricultural products were analyzed after processing according to the food pretreatment method for pesticide residue analysis after purchasing organic products sold at local hypermarkets. Each analysis sample was stored frozen at -20°C or lower, and a certain amount was taken and used during analysis.

3. Sample preparation steps

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

The present invention was to replace all reagents added with solid reagents for quick and convenient pretreatment. For this purpose, L-cysteine, sodium chloride, and TBAH including EDTA were directly mixed in a pretreatment system without dilution in an aqueous solution and used. did. In order to optimize the new pre-treatment process using solid reagents, usable solid reagents were selected for each step, and the recovery rate was calculated by controlling the amount used step by step.

4. Altar protein step

In order to confirm an efficient derivatization reaction for a high protein-containing pulse sample such as soybean, a whole protein step was added to the extract. Although it is common to use organic solvents such as chloroform and methylene dichloride for the protein reduction step, in the present invention, protein reduction was performed using ammonium sulfate, a solid reagent. For the optimization of the protein step, the recovery rate was calculated while adjusting the reagent usage step by step.

5. derivatization step

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

6. Creation of standard calibration curve

A standard solution of dithiocarbamate pesticides such as Mancozeb is 100 mg/kg by dissolving a certain amount of each standard in 0.5 g of L-cysteine and 0.25 M EDTA-2Na aqueous solution (containing 0.45 M NaOH, pH 9-10). As possible, it was prepared as a stock solution.

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

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

STD
10 μg/kg
500 μl STD
20 μg/kg
500 μl STD
100 μg/kg
500 μl STD
200 μg/kg
500 μl STD
1000 μg/kg
500 μl acetonitrile
500 μl blank matrix
500 μl blank matrix
500 μl blank matrix
500 μl blank matrix
500 μl blank matrix
500 μl extracted sample
500 μl

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

STD
20 μg/kg
500 μl STD
100 μg/kg
500 μl STD
200 μg/kg
500 μl STD
1000 μg/kg
500 μl STD
2000㎍/kg
500 μl acetonitrile
500 μl blank matrix
500 μl blank matrix
500 μl blank matrix
500 μl blank matrix
500 μl blank matrix
500 μl extracted sample
500 μl

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 hours (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)

Example of Selected reaction monitoring (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 test

For each agricultural sample, a recovery rate test was performed on untreated samples to determine the suitability of the residual pesticide analysis method. The standard solution of dithiocarbamate pesticide was treated to 0.1mg/kg and 0.5mg/kg level in each untreated sample of the target agricultural product, and after repeating the above analysis process 3 times, the average value and the relative standard deviation were calculated saved

Example 1. Selection and optimization of solid reagents for rapid analytical pretreatment development

(1) L-cysteine addition

It is known that the thiol group of L-cysteine helps to bind metal ions, thereby increasing the recovery rate (%). In the Food Standards Code, 0.5 g of L-cysteine per 100 ml of the extraction solution was used, but since a solid reagent is 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, 0.3 And the recovery rate was measured under the conditions of 0.5 g.

As a result, as shown in Table 6 below, even when the amount of L-cysteine added was increased from 0.1 g to 0.5 g, the difference in recovery did not change significantly, so the amount of L-cysteine added 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 (%) 82.4 101.4 99.2 100.0

(2) TBAH adding

TBAH is known to act as a phase transfer catalyst to move the dithiocarbamate-based pesticide component to the organic solvent layer. In the existing method, 5 ml of 0.41M TBAH per 100 ml of the extraction solution was used, which is converted to 0.69 g when converted to a solid. In this assay using a solid reagent, the effect rapidly decreased after a certain amount, so 0.1 g was used. Compared to the case where this reagent was not used, an 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 was as shown in Table 7 below. After the optimization process, 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 (%) 87.9 93.0 99.6 93.1 94.1

(4) sodium sulfate , added anhydride

Water/acetonitrile (0.05M methyl ioidie in acetonitrile) was used for extraction/derivatization due to the nature of the analysis method, so moisture interference was concerned. As a result, anhydrous sodium sulfate was added and confirmed as shown in Table 8. %) was excluded as it did not appear to have an effect.

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 (%) 100.4 97.7 98.3 99.0 96.5

( 5) Selection and Optimization of Solid Reagent Ammonium Sulfate for the Process of Protein

ⓛ Blank( no charge )at, Mancozeb's for methylation of ammonium sulfate effect

It was confirmed that the addition of ammonium sulfate did not affect the methylation of Mancozeb, a representative dithiocarbamate pesticide, so there was no problem in using the solid protein reagent for the development of this rapid assay.

Ammonium sulfate addition amount (g) 0.0 0.1 0.5 1.0 1.5 Recovery (%) 100.0 103.8 96.3 98.1 97.1

② For high protein-containing soybean samples of ammonium sulfate Altar protein effect

For high protein-containing soybean samples, the effect of ammonium sulfate, a solid reagent for total protein, was increased to 85.5% when 0.5 g was added, compared to 47.4% in the case of no addition, resulting in about 38% derivatization synergistic effect. appeared to be The effective recovery of the process method is specified as 70%. On the other hand, even when ammonium sulfate is added to general samples such as brown rice, green pepper, mandarin, and potato, all of them show a high recovery rate exceeding the effective recovery rate. was confirmed to be

Ammonium sulfate addition amount (g) 0.0 0.1 0.5 1.0 1.5 Recovery (%) 47.4 80.0 85.5 74.8 72.4

(6) Improvement of methylation derivation process for establishment of rapid analysis method

ⓛ Selection of the optimal organic solvent used for the methylation reagent

For methyl iodide, a methylation reagent for the derivatization reaction, a methylene chloride/hexane mixed solvent is used in the conventional analysis method, but acetonitrile is selected in the present invention. This is the analysis process after derivatization, such as not only the derivatization proceeds smoothly, but also the quick and clear liquid-liquid distribution after the reaction and the ease of post-operation that the analyte component is distributed in acetonitrile (supernatant) to quickly take the solution from the top of the reaction vessel. It had the advantage of being able to perform it quickly and easily.

② Selection of solid reagent for acidity control

In the derivatization process, it is _{necessary to maintain the pH of the aqueous solution at pH 7-8 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. For this purpose, the acidity is usually adjusted with an acidic solution such as HCl just before derivatization, but in the present invention, boric acid, an acidic solid reagent, was added to adjust the acidity. In order to maintain the required pH, the amount of boric acid added was optimized to 0.4 g. Unlike the case of using an acidic aqueous solution, that is, a liquid, this solid reagent can be pre-prepared in a solid form in the pre-treatment step, enabling the rapid production of an analysis kit, and has the advantage of improving the pre-treatment operation and ease of use.

(7) dithiocarbamate Establishment of rapid residue analysis process for pesticides

The simple and convenient multi-pesticide residue analysis method for the developed dithiocarbamate pesticide consists of three steps.

Each step consists of (a) pretreatment (extraction, proteinization) step consisting of solid reagents, (b) derivatization step, and (c) instrument analysis step. Steps (a) and (b) can be completed within 30 minutes.

The composition diagram of the QuEChERS kit, which has been developed in Europe and the United States and is used worldwide, is composed of (1) an extraction kit and (2) a purification kit. It is fully established as a rapid analysis method that can analyze components at the same time. In Korea, 320 component simultaneous multi-component pesticide residue analysis method using the QuEChERS kit was developed by the National Agricultural Product Quality Management Service, and so far, it is a process for analyzing pesticide residues in agricultural products at national testing and research institutes, agricultural product safety inspection institutes, and agricultural product safety inspection institutes. As an analysis method, it is announced in the Agricultural Products Quality Control Act. However, since it is impossible to analyze dithiocarbamate pesticides with the simultaneous multi-component pesticide residue analysis method using the QuEChERS kit, the value of the rapid analysis method established through this research and development is higher.

For dithiocarbamate pesticides developed through this study, if (1) a dedicated pretreatment kit prepared with a solid reagent and (2) a derivatization kit with improved speed and convenience are used, it is recognized as a blind spot for analysis. It is considered to be the optimal method to enable residue analysis for dithiocarbamate pesticides.

Example 2. Effect verification experiment of the developed rapid analysis method for multi-component pesticide residues through the recovery rate for each sample

A recovery rate test was performed on field samples using a new simple rapid analysis method that was converted to a pretreatment method using a solid reagent and went through an optimization step. Among the dithiocarbamate pesticides, mancozeb, the most used in Korea (3rd in domestic sales in 2017, 1,778 M/T), propineb (6th in use in 2017, 203 M) /T) and metiram were first selected and analysis was performed on brown rice, green pepper, mandarin, and potato. was analyzed, and as a result, as shown in Tables 11 to 13 below, it was confirmed that the average recovery rates were all 70% or more. Therefore, it was found that the simultaneous analysis method for dithiocarbamate pesticides was effective because it met the efficacy criteria. Chromatogram results for each sample are shown in FIGS. 4 to 7 .

Recovery rate of mancozeb on various crops crops Fortification
(mg kg ^-1 ) Recovery (%) 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

Recovery rate of metiram for various crops crops Fortification
(mg kg ^-1 ) Recovery (%) 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 (%) 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

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

Example 3. Application of rapid residue analysis method to high protein-containing soybeans

The effect of the new rapid residue analysis method was confirmed using soybean (protein content 36.21%) among representative high protein-containing pulse samples. When the analytical method announced in the Food Codex was applied, the recovery rate for soybeans was less than 30%, and the recovery rate for other beans with high protein content, such as peas (37.5) and red beans (19.0%), was also very low, within 30-50%. Therefore, the static analysis method could not be used for actual analysis.

In order to secure the possibility of the assay of the present invention, mancozeb, a representative dithiocarbamate-based pesticide, was selected and the recovery rates were compared in a method without/including the protein process.

First, in the soybean sample that was pretreated without the addition of ammonium sulfate, a solid protein reagent, the recovery rate of mancozeb was 47.4%, which was as low as expected. However, when ammonium sulfate was added and the protein-reducing step and methylation step were performed, the recovery rate increased by 38% to 85.5%, confirming that the addition of ammonium sulfate was quite useful when analyzing pulse samples such as soybeans with high protein content.

On the other hand, as seen in the above results, even when ammonium sulfate is added to general samples with low protein content such as brown rice, red pepper, mandarin, and potato, all of them show a high recovery rate that is effective without disturbing derivatization, thereby distinguishing low/high protein-containing samples. It was confirmed that the addition of ammonium sulfate, the corresponding protein solid reagent, was possible for most of the samples without it. This may be referred to in consideration of the composition of the reagent recipe for the pretreatment kit prepared as a solid reagent.

Recovery rate according to the addition amount of ammonium sulfate of Mancozeb to soybean samples containing high protein Ammonium sulfate addition amount (g) 0.0 0.1 0.5 1.0 1.5 Recovery (%) 47.4 80.0 85.5 74.8 72.4

Example 4. dithiocarbamate For rapid analysis of pesticide residues kit

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

The kit of the present invention consists of ⓛ a PE tube containing an aqueous solution of EDTA for pretreatment, ② a packaging pack containing each solid reagent precisely weighed (pretreatment + full protein), and ③ a solution in which methyl idide, a derivatization reagent, is dissolved in acetonitrile. It was composed of a cap tube (derivatized) containing

Claims (8)

(1) weighing at least one sample selected from cereals, beans, and documents to be analyzed for residual pesticide components to be 5-20 g;
(2) adding the weighed sample to 9-11 ml of EDTA (ethylenediaminetetraacetic acid) solution;
(3) After step (2), 0.1-0.5 g of L-cysteine, 0.1-0.7 g of TBAH (tetrabutylammonium hydrogen sulfate), 0.5-2.0 g of sodium chloride, 0.1-0.4 in the EDTA solution in which the sample is dissolved preparing a mixed solution by adding and mixing g of boric acid, phosphoric acid, and citric acid;
(4) After step (3), at least one selected from acetonitrile, methylene dichloride, chloroform, hexane, ethyl acetate, acetone and dimethyl sulfoxide (DMSO) containing 0.01 to 0.1M methyl iodide adding 9 to 11 ml of an organic solvent to the mixed solution and stirring at 1000 to 2000 rpm for 1 to 3 minutes to methylate residual pesticide components;
(5) after step (4), centrifuging at 2500 to 3500 rmp at 8 to 12° C. for 8 to 12 minutes, and filtering the supernatant of the mixed solution; and
(6) LC-MS/MS analysis of the solution filtered in step (5); a method for simultaneous analysis of residual amounts of dithiocarbamate-based pesticide components, including. The method of claim 1, wherein the dithiocarbamate pesticide is ferbam, ziram, thiram, mancozeb, maneb, zineb, methiram (metiram), nabam (nabam) and propineb (propineb), characterized in that at least one selected from the dithiocarbamate-based method for simultaneous analysis of residual amounts of various pesticide components. delete delete delete delete delete delete

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