KR102193402B1 - Method for analyzing residual amount of dithiocarbamate-based pesticides in agricultural products containing high level of protein - Google Patents

Abstract

The present invention relates to a method for analyzing a residual amount of dithiocarbamate pesticides remaining in agricultural products containing a high content of protein. The method for analyzing the residual amount of dithiocarbamate pesticides of the present invention is simple and maximizes a recovery rate of the residual amount of dithiocarbamate-based pesticides through optimization of a deproteinization and methylation process. Therefore, the method for analyzing the residual amount of dithiocarbamate pesticides is a very useful method for analyzing the content of dithiocarbamate-based pesticides remaining in agricultural products having a high protein content.

Description

Method for analyzing residual amount of dithiocarbamate-based pesticides in agricultural products containing high level of protein}

The present invention relates to a method for analyzing the residual amount of dithiocarbamate-based pesticides remaining in agricultural products containing a high content of protein.

Dithiocarbamate-based pesticides are effective non-penetrating organic disinfectants for a wide range of diseases, and are currently widely used not only in Korea but also in agricultural fields worldwide for disease control. 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 (FIG. 1). 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. In Korea, mancozeb ranked first (1,663 tons) and propineb fourth (355 tons) based on domestic shipments of disinfectants in 2017. Currently, in Korea, standards for safe use of pesticides have been established to control anthrax in fruit trees, including apples and pears, and to prevent downy mildew of vegetables such as tomatoes and lettuce, and allowance of residual pesticides has been established for 93 agricultural products including persimmons. have.

Most dithiocarbamate-based pesticides contain metal ions such as Fe (iron), Mn (manganese), and Zn (zinc) in their molecular structure, and are poorly soluble in water and organic solvents. It is a compound with very limited extraction and purification methods. Currently, dithiocarbamate-based pesticide residue analysis methods include a commonly used CS2 gas trapping method and a methylation method using methyl iodide (CH ₃ I). The CS2 analysis method is a method of decomposing molecules, collecting and analyzing the total amount of CS2. The qualitative and content of each component for a single pesticide cannot be known, and the reproducibility is poor due to the interference of natural CS2 or similar gases. To compensate for these shortcomings, a methylation method has been recently developed and used, which extracts the dithiocarbamate component using a weakly alkaline EDTA-2Na aqueous solution, and then uses HPLC/UVD through a series of pretreatment processes including methylation. This is how to analyze.

Physical and Chemical Properties of Dithiocarbamate Pesticides compound
Formular
Molecular Weight
Log P _ow
Solubility water
(mg/L) Solvent (g/L) Thiram
(Thiram) C ₆ H ₁₂ N ₂ S ₄ 240.4 1.73 18 Ethanol <10, acetone 80, chloroform 230, n -hexane 0.04, thichloromethane 170, toluene 18, isopropanol 0.7 Paa balm
(Ferbam) C ₉ H ₁₈ FeN ₃ S ₆ 416.5 -1.6 130 Can be soluble in chloroform, pyridine, acetonitrile, and acetone Jiram
(Ziram) 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 Mannev
(Maneb) C ₄ H ₆ MnN ₂ S ₄ 265.3 - insoluble Insoluble (soluble in chelating reagents) Mankozeb [C ₄ H ₆ MnN ₂ S ₄ ] _x Zn _y 271.2 0.26 6.2 Insoluble (soluble in chelating reagents) Metiram [C ₁₆ H ₃₃ N ₁₁ S ₁₆ Zn ₃ ] _x (1088.6) _x 0.3 insoluble Soluble in pyridine with decompositions Geneve
(Zineb) C ₄ H ₆ N ₂ S ₄ Zn 275.8 ≤1.3 10 Insoluble (soluble in chelating reagents) Night
(Nabam) C ₄ H ₆ N ₂ Na ₂ S ₄ 256.3 - 200,000 Insoluble (soluble in chelating reagents) Propineb
(Propineb) [C ₅ H ₈ N ₂ Zn] _x 289.8 -0.26 10 Toluene, hexane dichloromethane <0.1, DMF + DMSO >200 g/l

The analysis method including this methylation process was applied to agricultural product monitoring in Korea in 2010, and is being used for qualitative and quantitative analysis of the components of this series of pesticides contained in samples in countries around the world such as Poland, Japan, and the Netherlands. However, most of these analysis methods are optimized for fruits and vegetables and fruits (recovery rate> 90%), and when applied to beans containing proteins, which are biochemical macromolecules, there is a problem that the recovery rate is rapidly lowered to 20-40%. It is difficult to use as a general-purpose analysis method targeting samples. In particular, it is known that, unlike fruits and vegetables, a high content of protein contained in the bean samples interferes with the methylation process as a cause of the rapid decrease in the recovery rate of the methylation method for pulse samples.

Therefore, methylation efficiency can be improved by performing methylation after deproteinization to remove pulse protein before methylation. Physical methods of separating high-molecular proteins according to the size (molecular weight) of protein and chemical methods such as sedimenting proteins by adding organic and inorganic chemicals have been reported. The former requires expensive equipment and takes a little longer, and the latter is relatively easy to operate and has high protein removal efficiency, but dithiocarbamate pesticides are still high in bean agricultural products containing protein in high content. The method of analyzing the residual amount of is low in efficiency and accuracy, so a plan is needed to overcome this.

Daily intake and protein content of crops Crop Daily intake (g/day) Protein content (%) Soy bean 4.05 36.2 Pea, sparkle varieties (Pea, sparkle) 0.23 24.4 Red bean 0.61 19.3 Eastern (Cowpea) 0.10 11.2 Sword bean 0.02 9.0 Kidney bean 0.49 8.8 Pea 0.23 7.9

On the other hand, Korean Patent No. 1278631 discloses a method for analyzing dithiocarbamate-based pesticides of citrus quickly and efficiently, and Korean Patent No. 0430932 discloses a method for analyzing Mancozeb using HPLC. There is no method for analyzing the residual amount of dithiocarbamate-based pesticides remaining in agricultural products containing protein content.

The present invention was derived from the above requirements, and the present invention provides a method for analyzing the residual amount of dithiocarbamate-based pesticides remaining in agricultural products containing a high content of protein, and by the method of the present invention, a high protein sample The present invention was completed by confirming that it was possible to accurately analyze the residual amount of dithiocarbamate-based pesticides by performing methylation after protein removal using soybeans, red beans and peas (sparkle variety).

In order to achieve the above object, the present invention (1) after wetting an agricultural product sample to be analyzed for residual pesticide components, dissolved in EDTA-2Na (ethylenediaminetetraacetic acid-disodium) aqueous solution containing L-cysteine and sodium chloride, and pH Adjusting to 9.5 to 9.6;

(2) After the step (1), chloroform and amyl alcohol were added, stirred, and then centrifuged to separate the supernatant in the order of EDTA-aqueous solution layer, protein-gel layer and chloroform layer, and obtain a supernatant. Proteolytic step;

(3) adding TBAH (tetrabutylammonium hydrogen sulfate) to the supernatant obtained in step (2) and adjusting the pH to 7.0±0.3;

(4) adjusting the pH in step (3) and adding an organic solvent containing methyl iodide (CH ₃ I) to methylate residual pesticide components to be analyzed;

(5) after step (4), adding a dichloromethane solution containing 1,2-propanediol, and then concentrating under reduced pressure;

(6) re-dissolving the sample concentrated under reduced pressure in methanol and filtering; And

(7) After step (6), detecting the residual amount of dithiocarbamate-based pesticide; The residual amount of dithiocarbamate-based pesticide remaining in agricultural products containing a high content of protein, comprising: Provide an analysis method.

The present invention relates to a method for analyzing the residual amount of dithiocarbamate pesticides remaining in agricultural products containing a high content of protein, and the method for analyzing the residual amount of dithiocarbamate pesticides of the present invention optimizes the protein and methylation steps. It is simple and maximizes the recovery rate of the residual amount of dithiocarbamate-based pesticides. Accordingly, the method for analyzing the residual amount of dithiocarbamate-based pesticides of the present invention is a very useful method for analyzing the content of dithiocarbamate-based pesticides remaining in agricultural products having high protein content.

1 shows the chemical structure of a dithiocarbamate-based pesticide.
2 is a UV absorption spectrum of methylated propineb (A), mancozeb (B) and metiram (C).
3 is a photograph showing the separated layers for each number of times the protein-removing process was performed in order to remove the protein in the soybean sample. A: 1 time, B: 2 times, C: 3 times, D: 4 times
FIG. 4 shows the recovery rates of proteineve (A), mancozeb (B), and metiram (C) according to the number of proteins in the red bean sample.
5 is a calibration curve for methylation of propineb (A), mancozeb (B) and metiram (C).
6 is a chromatogram confirming the recovery rate of propineb from the soybean ( Glycine max Merrill) extract, A is a chromatogram of a soybean extract without propineb as a control, and B is 5.0mg/kg as a standard material. It is a chromatogram of propineb, C is a chromatogram of a soybean extract containing 5.0 mg/kg of propineb, but without any protein, D is 5.0 mg/kg of propineb, and This is a chromatogram of soybean extract with protein.
Figure 7 is peas ( Pisum sativum L.) A chromatogram confirming the recovery rate of propineb from the extract, A is a chromatogram of a pea extract without propineb, and B is a chromatogram of 5.0mg/kg propineb as a standard. Gram, C is a chromatogram of a pea extract containing 5.0 mg/kg of propineb, but not subjected to protein removal, D is a pea containing 5.0 mg/kg of propineb, and subjected to protein production It is a chromatogram of the extract.
Figure 8 is a chromatogram confirming the recovery rate of metiram from the red bean extract, A is a chromatogram of the red bean extract without metiram as a control, and B is a chromatogram of 5.0mg/kg metiram as a standard, C is the chromatogram of the red bean extract containing 5.0 mg/kg of metiram, but without the production of protein, and D is the chromatogram of the red bean extract containing 5.0 mg/kg of metiram and with the production of protein. .
9 is an SRM chromatogram of LC-MS/MS for confirming the recovery rate of propineb contained in the red bean sample according to the method of the present invention, (A) is the SRM chromatogram of the control group not treated with propineb And (B) is an SRM chromatogram of a group treated with 2.0 mg/kg of propineb in an adzuki bean sample.
10 is an SRM chromatogram of LC-MS/MS for confirming the recovery rate of mancozeb contained in the red bean sample according to the method of the present invention, (A) is the SRM chromatogram of the control group not treated with mancozeb, (B) is an SRM chromatogram of a group treated with 2.0 mg/kg of mancozeb in an adzuki bean sample.
11 is an SRM chromatogram of LC-MS/MS for confirming the recovery rate of metiram contained in a red bean sample according to the method of the present invention, (A) is an SRM chromatogram of a control group not treated with metiram, (B) is an SRM chromatogram of a group treated with 2.0 mg/kg of metiram in an adzuki bean sample.

In the present invention, 1) after wetting an agricultural product sample to be analyzed for residual pesticide components, dissolving in an aqueous solution of EDTA-2Na (ethylenediaminetetraacetic acid-disodium) containing L-cysteine and sodium chloride, and adjusting the pH to 9.5 to 9.6 ;

(2) After the step (1), chloroform and amyl alcohol were added, stirred, and then centrifuged to separate the supernatant in the order of EDTA-aqueous solution layer, protein-gel layer and chloroform layer, and obtain a supernatant. Proteolytic step;

(3) adding TBAH (tetrabutylammonium hydrogen sulfate) to the supernatant obtained in step (2) and adjusting the pH to 7.0±0.3;

(4) after step (3), methyl iodide residual pesticide component to be analyzed by adding an organic solvent containing methyl iodide (CH ₃ I);

(5) after step (4), adding a dichloromethane solution containing 1,2-propanediol, and then concentrating under reduced pressure;

(6) re-dissolving the sample concentrated under reduced pressure in methanol and filtering; And

(7) After step (6), detecting the residual amount of dithiocarbamate-based pesticide; The residual amount of dithiocarbamate-based pesticide remaining in agricultural products containing a high content of protein, comprising: It is about the analysis method.

The residual pesticide components are propineb, mancozeb, metiram, ferbam, ziram, thiram, maneb, and zineb ) And nabam (nabam) is preferably at least one selected from, but is not limited thereto. Step (2) may be performed 1 to 5 times, preferably 3 to 5 times, but is not limited thereto.

The addition of chloroform and amyl alcohol in step (2) is preferably 40 to 60 parts by volume of chloroform and 8 to 12 parts by volume of amyl alcohol, based on 5 parts by weight of the agricultural product sample of step (1), but is not limited thereto. Does not.

In the step (4), the organic solvent is preferably a mixed solution of 1:1-4 (v/v) n-hexane and dichloromethane, but is not limited thereto.

The agricultural product sample is preferably beans, and more preferably, any one selected from soybeans, peas, red beans, small peas, eastern beans and kidney beans, but is not limited thereto.

The method for analyzing the residual amount of dithiocarbamate-based pesticides remaining in agricultural products containing the high content of protein

(1) after wetting the agricultural product sample to be analyzed for residual pesticide components, dissolving in an aqueous solution of EDTA-2Na (ethylenediaminetetraacetic acid-disodium) containing L-cysteine and sodium chloride, and adjusting the pH to 9.5 to 9.6;

(2) After step (1), 40 to 60 parts by volume of chloroform and 8 to 12 parts by volume of amyl alcohol were added to 5 parts by weight of the agricultural product sample of step (1), and stirred for 8 to 12 minutes. Then, centrifuge at 2,000 to 4,000 rpm for 8 to 12 minutes to separate the supernatant in the order of EDTA-aqueous solution layer, protein-gel layer, and chloroform layer, and repeat 3 to 5 times to obtain the supernatant. Proteining;

(3) adding 0.8 to 1.2 mL of tetrabutylammonium hydrogen sulfate (TBAH) of 0.3 to 0.5 M to 10 mL of the supernatant obtained in step (2), and adjusting the pH to 7.0±0.3;

(4) After step (3), a mixed solution of 1:1 to 4 (v/v) n-hexane and dichloromethane containing 0.03 to 0.1 M methyl iodide (CH ₃ I) was added to analyze Methylating the residual pesticide component;

(5) After step (4), adding a dichloromethane solution containing 15 to 25% (w/v) 1,2-propanediol, and then concentrating under reduced pressure;

(6) re-dissolving the sample concentrated under reduced pressure in methanol and filtering; And

(7) After step (6), the step of detecting the residual amount of the dithiocarbamate-based pesticide; preferably includes, but is not limited thereto.

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.

[Materials and methods]

1. Reagents, materials and equipment

The standard product for analysis of propineb (purity 68.5%) used in the present invention was sold and used by Bayer, Inc., and the standard product for analysis of Mancozeb (purity 97.5%) and metiram (purity 90.0%) was It was purchased and used from AccuStandard (USA). EDTA-2Na (ethylenediaminetetraacetic acid-disodium) used for extraction was purchased from Daejeong (Korea) and used. NaOH is Japan's Yakuri Pure Chemicals Co., Ltd. (Japan) was purchased and used, and L-cysteine, 1,2-propanediol was manufactured by Tokyo Chemical Industry Co., Ltd. It was purchased from (Japan) and used. Methyl iodide was purchased and used from Kanto Chemical Co., Inc. (Japan), and TBAH (tetrabutylammonium hydrogen sulfate) was purchased and used from Sigma Aldrich (USA). Sodium sulfate (anhydrous) and sodium chloride (anhydrous) were purchased and used from Daejeong (Korea), and amyl alcohol was used by Junsei Chemical Co., Ltd. GR grade was purchased from (Japan) and used. n-hexane, methanol, dichloromethane, chloroform, hydrochloric acid, and distilled water (water) were used by purchasing GR grade from Merck (Germany). During pretreatment, a shaker (Lab. Companion IS-971R, Jeio Tech, Korea and BV1010, Benchmark Scientific, USA) was used to shake agricultural product samples, and a concentrator (Eyela NE-1001, Tokyo Rikakikai Co., Ltd.) was used to maintain reduced pressure. Ltd., Japan), and a centrifuge (Allegra X-15R, Beckman Culter Life Sciences, USA) was used for centrifugation.

2. Sample to be analyzed

The agricultural product samples to be analyzed used to improve the residual analysis method of dithiocarbamate-based pesticides are relatively high intake among beans known as high protein foods, and soybeans with high protein content (protein content 36.21%), peas (sparkle varieties, etc.) Protein content 24.4%) and red beans (protein content 19%) were selected. Pesticide-free cultivation samples of the bean were purchased and analyzed after processing according to the food pretreatment method for pesticide residue analysis by purchasing organic products sold at large supermarkets. Analysis samples were stored frozen at -24°C and used by taking a certain amount at each analysis point.

3. Standard calibration curve

Dithiocarbamate-based pesticides Propineb, Mancozeb, and Metiram standard solutions contain 0.5 g of L-cysteine and 0.25M of EDTA-2Na (ethylenediaminetetraacetic acid-disodium) aqueous solution (containing 0.45M NaOH, pH). 9.5 ~ 9.6) to prepare a stock solution to 100mg / kg. After diluting it to a concentration of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 2.5mg/kg, methylated and injected into HPLC/UVD by 20µl each standard based on the area of the peak. A calibration curve was prepared.

4. Analyzer

(1) HPLC/UVD analysis

Analysis of dithiocarbamate-based pesticides propineb, mancozeb, and metiram was performed using an HPLC system equipped with an ultraviolet absorption spectroscopic detector (Dionex ultimate 3000, USA), and the column was Shiseido C ₁₈ (4.6㎜ ID×250㎜, 5.0㎛, Japan) was used (Table 3).

Under the established instrument analysis conditions, the retention time of Propineb was 12.4 minutes, Mancozeb was 11.7 minutes, and Metiram was 11.5 minutes.

HPLC/UVD conditions for analysis of dithiocarbamate compounds Equipment name Dionex ultimate 3000 (Thermo Science, USA) Detector Diode array detector(DAD, wavelength: 272nm) column Shiseido-C18 (4.6mm I.D.×250mm, 5.0㎛), Japan Temperature 40℃ Mobile phase [A:B=water:acetonitrile=(v/v)]



minute A B 0.0 80 20 15.5 5 95 18.0 5 95 20.0 80 20 Flow rate 1.0ml/min Injection volume 20.0µl

(2) LC-MS/MS analysis

LC-MS/MS (TSQ Quantum Access Max, Thermo Science, USA) with excellent selectivity and resolution is used to confirm the qualitative reliability of the residuals of the representative dithiocarbamate-based fungicides detected in the sample through HPLC/UVD analysis. And analysis conditions are disclosed in Table 4.

5. Sample extraction, Protein And establishment of conditions for the methylation step

In order to improve the recovery rate in the analysis of the residual amount of dithiocarbamate-based pesticides contained in the bean agricultural product sample containing an excessive amount of protein, an appropriate pretreatment analysis method was established by comparing the recovery rate according to various factors such as protein and pH control. .

(1) sample extraction

Most agricultural products contain moisture, so polar organic solvents such as acetone, acetonitrile and methanol are used rather than non-polar organic solvents to increase extraction efficiency, but dithiocarbamate-based pesticides, which are analyte components of the present invention, are metal Since it contains ions, it is a compound that is poorly soluble in most organic solvents and water.In the present invention, 0.25M EDTA-2Na aqueous solution (containing 0.45M NaOH, pH 9.5~) is used as a solvent for extracting dithiocarbamate compounds from the target agricultural products. 9.6) was used.

When EDTA is dissolved in an aqueous solution, hydrogen ions at the edges are dissolved and act as -4 ions. At the same time, EDTA can act as a six-digit ligand and form a chelate compound with strong affinity for metal ions, so organic solvents and water Dithiocarbamate-based pesticides, which are poorly soluble in water, made it possible to become an aqueous solution.

In the present invention, 0.45M NaOH was used for pH control so that the pH of the solution was maintained at 9-10.

(2) Protein ( deproteinization ) step

In the case of agricultural product samples containing protein (for example, beans), it was determined that a high content of protein interferes with methylation. Therefore, it was intended to apply deproteinization before the methylation step.

In the present invention, 50 ml of chloroform or dichloromethane and 10 ml of amyl alcohol were added in stages to the extraction solution, followed by repeated three consecutive times.

(3) methylation step

In order to establish an optimized methylation step, 10 ml, 20 ml, and 50 ml of the aqueous protein solution were taken and the methylation process was relatively compared, and when 10 ml was used, it was judged as the most suitable. After taking 10 ml of the supernatant, 1 ml of an aqueous 0.41M TBAH (tetrabutylammonium hydrogen sulfate) solution was added as a phase transfer catalyst. Thereafter, the pH of the extract was maintained in a neutral state by using 1N HCl aqueous solution to enable methylation.

Dithiocarbamate pesticides are pesticides that require a methylation process for residual analysis, and methyl iodide is generally used as a methylation reagent. In the present invention, methylation was performed with n-hexane:dichloromethane (1:1, v/v) containing 0.05M methyl iodide (CH ₃ I).

Through the present invention, when methylated 3 times with 30 ml of the mixed solvent, the optimum recovery rate could be confirmed. Thereafter, in order to prevent the product from volatilization during concentration of the methylated dithiocarbamate-based pesticide, a dichloromethane solution containing 20% 1,2-propanediol was added to prevent the product from volatilization.

Example 1. Among bean samples containing high protein Dithiocarbamate type Pesticide residue analysis

In order to extract dithiocarbamate-based pesticides from bean samples, propineb, mancozeb, and metiram containing metal ions among the pesticides used in Korea were selected as representative pesticides.

To extract the component, 20 ml of distilled water was added to 5 g of each sample, and left to stand for 20 minutes to moisten. Thereafter, 0.5 g of L-cysteine and NaCl were added to 80 ml of 0.25 M EDTA-2Na aqueous solution (containing 0.45 M NaOH, pH 9.5 to 9.6), followed by extraction by shaking at 2,500 rpm for 10 minutes.

Thereafter, 50 ml of chloroform or dichloromethane and 10 ml of amyl alcohol were added to the obtained extract, shaken at 2,500 rpm for 10 minutes, and then centrifuged at 3000 rpm for 10 minutes to produce protein. This process was repeated several times to remove the protein as much as possible.

Thereafter, 10 ml of the centrifuged supernatant was taken, 1 ml of a 0.41 M tetrabutylammonium hydrogen sulfate aqueous solution was added, and 1 M HCl was added to quickly adjust the pH to 7.0, and then used as a methylated sample.

3 g of NaCl was added to the solution, and 30 ml of n-hexane:dichloromethane (1:4, v/v) solution containing 0.05M methyl iodide was first added, followed by the second 10 ml. Methylation was induced and extracted. Thereafter, dehydration was passed through a layer of sodium sulfate anhydrous and left at room temperature for about 10 minutes. Dichloromethane containing 20% 1,2-propanediol (1,2-propanediol) 1 ml was added thereto, and the mixture was concentrated under reduced pressure in a water bath below 30℃ using a rotary vacuum evaporator. After dissolution, it was analyzed by HPLC/UVD.

Ⓛ Limit of Detection (LOD) and Limit of Quantitation (LOQ)

The limit of detection (LOD) was the lowest concentration that could detect an analyte in the sample, and the detection limits of propineb, mancozeb, and metiram were calculated by analyzing various concentrations of standard solutions.

Limit of Quantitation (LOQ) was the lowest concentration capable of quantifying analytes in a sample, and the limits of quantitation of propineb, mancozeb, and metiram were obtained by analyzing standard solutions of various concentrations.

② Recovery rate and repeatability

A recovery rate test was performed to determine the suitability of the chloroform-gel method of three dithiocarbamate-based pesticides (propineb, mancozeb, and metiram) among bean samples.

The final concentrations of each pesticide in the sample were treated to be 0.4 and 2.0 mg/kg, and after treatment with 2 levels and 3 repetitions, the average value and the relative standard deviation were calculated according to the residual amount method of the present invention. The recovery rate test was conducted in accordance with the "Standards and Methods of Crop Persistence Test (related to Article 5, Paragraph 1, No. 5)" (Table 5).

Acceptable range for recovery rate Fortification RSD (%) Recovery rate (%) ≤0.001mg/kg 35 50-120 > 0.001mg/kg ≤0.01mg/kg 30 60-120 > 0.01mg/kg ≤0.1mg/kg 20 70-120 > 0.1mg/kg ≤1.0mg/kg 15 70-110 > 1.0mg/kg 10 70-110

③ Linearity of calibration curve

To obtain the linearity of the calibration curve, methylated propineb, mancozeb, and metiram were added to HPLC/UVD at different 7-9 levels (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 2.5mg/kg). ), the calibration standard solution was analyzed to confirm the straightness.

④ Verification of analysis method

In order to reconfirm the analysis methods of propineb, mancozeb, and metiram detected in the sample through HPLC/UVD analysis, and to confirm the qualitative reliability of the residuals, the pulse sample is at a concentration of 50 times the quantitative limit (2.0mg/kg). After treating the pesticide, the above analysis process was performed, and it was verified through instrument analysis using LC-MS/MS.

Example 2. Protein Confirmation of increase in recovery rate through step optimization

The physicochemical properties of the three pesticides propineb, mancozeb, and metiram selected as representative components among the dithiocarbamate-based pesticides to be analyzed of the present invention are disclosed in Table 6. The n-octanol/water partition coefficient of dithiocarbamate pesticides is propineb -0.26, mancozeb 0.26, and metiram 0.3, which are poorly soluble in non-polar organic solvents. Since the unshared electron pair of the sulfur or nitrogen atom in the molecule absorbs a specific wavelength band by electron transfer, it shows strong absorbance in UV, so the HPLC/UVD selected in the process analysis method was used. In the case of the methylated dithiocarbamate-based compound, all three pesticides showed the highest absorbance at 272 nm and were selected as the measurement wavelength for instrument analysis (FIG. 2).

The analysis was performed using a reversed phase column (C ₁₈ ), which is commonly used for the separation of neutral compounds, and the gradient conditions of the mobile phase that can be sufficiently separated in order to minimize interference of impurities in the analysis sample were determined by dithiocarba. It was finally selected as the analysis conditions of the mate.

Physicochemical properties of propineb, mancozeb, and metiram compound Molecular Weight Vapor pressure (mPa) LogP _ow ^{a )} Water solubility
(mg/L) Propineb 289.8 <1.6×10 ^-7 (20℃) -0.26 10 Mankozeb 271.2 <1.33×10 ^-2 (20℃) 0.26 6.2 Metiram (1088.6)x <0.010 (20℃) 0.3 insoluble

a) n-octanol/water partition coefficient

Representative high-protein foods such as beans, peas (sparkle varieties), and red beans are known to have a high protein content of about 19 to 36.1%.

In the residual analysis of dithiocarbamate-based pesticides, the present inventors hypothesized that a high content of protein reacts with a methylation reagent to inhibit methylation of dithiocarbamate-based pesticides. In order to prove this assumption, as a result of performing residual analysis through methylation reaction of dithiocarbamate-based pesticides on various pulse samples, it showed a low recovery rate of about 20 to 50%, and the reliability of the assumptions of the present inventors was high. Was judged.

Therefore, the present invention was able to propose to apply the'protein' process essentially prior to methylation for efficient analysis of dithiocarbamate-based pesticides remaining in pulse samples with high protein content.

In the present invention, 50 ml of chloroform and 10 ml of amyl alcohol were added to the extract and shaken, and then centrifuged at 3,000 rpm for 10 minutes to remove the protein in the sample extract. As a result of performing the protein-free process, it was confirmed that the lower layer was clearly divided into a transparent chloroform layer, the middle layer was a white gel layer containing protein, and the upper layer was divided into an aqueous solution layer.

At this time, if only the aqueous solution layer of the upper layer was taken with a pipette and the protein-free process was repeated several times, it was confirmed that the white gel layer containing the protein decreased with each repeated number of times (FIG. 3).

(1) red bean ( Vigna) anagularis Willd.)

Table 7 shows the recoveries of each component for the number of protein-removed proteins of a representative dithiocarbamate-based pesticide for a red bean (19% protein content) sample. In the conventional analysis method that did not apply the protein-free process, propineb, mancozeb, and metiram were calculated with a recovery rate of about 40-50% in the bean samples. On the other hand, it was confirmed that the recovery rate was rapidly improved as the number of times the protein-free process was applied increased (Table 7).

The recovery rate of propineb, mancozeb, and metiram according to the number of times the red bean sample was repeated with the second protein step (%) Pesticide No. of Deproteinization ¹⁾ Recovery (%) in red bean R1 R2 R3 Average±SD (%) ²⁾ Propineb
- 44.3 48.1 40.4 44.3±3.9 Ⅰ 74.2 70.8 71.9 72.3±1.7 Ⅱ 85.8 78.4 80.1 81.4±3.9 Ⅲ 92.9 86.9 92.2 90.9±3.3 Ⅳ 98.5 92.7 104.5 98.6±5.9 Mankozeb



- 52.0 59.1 42.7 51.3±8.2 Ⅰ 72.8 71.9 70.5 71.7±1.2 Ⅱ 80.6 80.7 80.0 80.4±0.4 Ⅲ 94.9 91.1 90.9 92.3±2.3 Ⅳ 100.2 104.4 99.9 101.5±2.5 Metiram



- 47.6 44.2 52.2 48.0±4.0 Ⅰ 73.2 70.4 70.2 71.3±1.7 Ⅱ 82.4 87.5 82.5 84.1±2.9 Ⅲ 94.3 95.5 94.5 94.8±0.6 Ⅳ 98.6 98.5 98.1 98.4±0.3

¹⁾ 50 ml of chloroform and 10 ml of amyl alcohol are used

²⁾ Mean and standard deviation for 3 replicates

Ⓛ propineb

In the case of propineb, when the adzuki beans were methylated without proceeding with the removal of protein (0 times), the recovery rate exceeded about 40%, but the recovery rate of about 72% was achieved when the red beans were carried out once. When the protein removal was performed 3 times, an average of 90% or more recovery was observed, and when the protein removal was performed 4 times, a recovery rate of about 98% was confirmed (FIG. 4A).

When the recovery rate was compared with when the protein-free process was performed and when it was not performed, it was confirmed that about 60% of the recovery rate increased. When the protein-free process was performed only once, an increase in the recovery rate of about 30% was observed, and after each increase in the number of times, the recovery rate increased. A high recovery rate of 99% was confirmed.

② Mancozeb

In the case of Mancozeb, when methylated without proceeding with the protein removal (0 times), the recovery rate of about 50% was confirmed, and when the protein was removed once, the recovery rate increased by about 20%, indicating a recovery rate of 72%. When the protein removal was performed 3 times, the average recovery rate was 90% or more, and when the protein was performed 4 times, a recovery rate of about 102% was confirmed (FIG. 4B).

When the recovery rate was compared with when the protein-free process was performed and when it was not performed, it was confirmed that the recovery effect of about 50% was increased. When the protein-free process was performed only once, an increase in the recovery rate of about 20% was observed, and after each increase in the number of times, the recovery rate increased. A high recovery rate of 102% was confirmed.

③ Metiram

In the case of metiram, when methylated without proceeding with protein removal (0 times), it was confirmed that the recovery rate was about 48%, and when the protein was performed once, the recovery rate increased by about 23%, indicating a recovery rate of 71%. . When the protein removal was performed 3 times, the average recovery rate was 90% or more, and when the protein production was performed 4 times, a recovery rate of about 98% was confirmed (FIG. 4C).

When comparing the recovery rate when the protein-removing process was performed and when it was not performed, it was confirmed that the recovery rate increased by about 50%. When the protein-free process was performed only once, an increase in the recovery rate of about 20% was observed, and after each increase in the number of times, the recovery rate increased. A high recovery rate of 98% was confirmed.

In the present invention, when considering the recovery rate data for each number of protein removals, analysis time and efficiency, etc., a good recovery rate of more than 90% on average is calculated in three treatments of the dithiocarbamate pesticides propineb, mancozeb, and metiram Became. When the protein was performed 3 times, compared to 4 times, it was determined that the number of times of protein removal was appropriate for the reason of relatively short analysis time and low solvent usage (Table 8).

Protein efficiency versus time, solvent and recovery rate No. of Deproteinization
Time (minutes)
Solvent (ml) Average recovery(%) Propineb Mankozeb Metiram One 30 60 72.3 71.7 71.3 2 60 120 81.4 80.4 84.1 3 90 180 90.9 92.3 94.8 4 120 240 98.6 101.5 98.4

(2) Soybean ( Glycine max Merrill)

As a result of applying the methylation method using methyl iodide to soybeans (protein content 36.21%) extracted through EDTA-2Na aqueous solution, propineb and mancozeb showed a low recovery rate of more than about 20%. The need to improve the overall residual analysis method for Korea was recognized. A liquid-liquid extraction method, chloroform-gel-method, was applied during the process of protein production in order to remove proteins that interfere with methylation of dithiocarbamate pesticides. In the case of the chloroform-gel-method, chloroform and amyl alcohol are used to remove proteins, and chloroform is added to the EDTA-2Na aqueous solution to denature the protein to form a white gel layer. Through this, it could be confirmed with the naked eye that the protein was removed.

For propineb residual analysis on soybean samples, the average recovery rate of 1-6 times of protein removal was 44.9% for 1 time, 52.7% for 2 times, and 74.1% for 3 times when 50 ml of chloroform was used. , 4 times showed 91.7%, 5 times 83.5%, 6 times 62.5%. When 100 ml of chloroform was used, 1 time 53.9%, 2 times 77.3%, 3 times 84.9%, 4 times Was 87.2%, 5 times 84.0%, 6 times 71.4% (Tables 9 to 11).

When the protein-removal process was performed 4 times, in the case of 50 ml of chloroform, the increase was 66.8% to 24.2-91.7%, and in the case of 100 ml of chloroform, the increase was 63.0% to 24.2 to 87.2%. When 50 ml of chloroform and 100 ml of chloroform were used, both showed the highest recovery rate in 4 times the protein production process. It was also confirmed that 4 times the protein-free process was suitable in the recovery test for mancozeb performed in parallel.

Recovery rate (%) of propineb in the soybean protein-free process (chloroform 50 ml + amyl alcohol 10 ml) No. of Deproteinization
Recovery rate (%) R1 R2 R3 Mean±standard deviation 0 18.0 22.8 31.7 24.2±6.9 One 46.9 43.3 44.3 44.9±1.9 2 53.2 52.2 52.8 52.7±0.5 3 79.8 71.6 70.9 74.1±4.9 4 92.2 92.4 90.6 91.7±1.0 5 86.8 83.3 80.4 83.5±3.2 6 63.4 61.4 62.9 62.5±1.0

Recovery rate (%) of propineb from the soybean protein production process (chloroform 100ml + amyl alcohol 10ml) No. of Deproteinization
Recovery rate (%) R1 R2 R3 Mean±standard deviation 0 18.0 22.8 31.7 24.2±6.9 One 38.7 52.6 50.7 53.9±3.9 2 71.2 77.9 79.9 77.3±2.9 3 83.0 89.3 82.4 84.9±3.8 4 91.5 88.4 81.5 87.2±5.1 5 80.9 90.2 80.8 84.0±5.4 6 80.8 67.2 75.6 71.4±5.9

Recovery rate (%) of mancozeb in the soybean protein protein process (chloroform 50 ml + amyl alcohol 10 ml) No. of Deproteinization
Recovery rate (%) R1 R2 R3 Mean±standard deviation 0 18.0 22.8 31.7 24.7±4.4 One 48.3 51.2 50.9 50.1±1.6 2 72.1 69.8 75.3 72.4±2.8 3 80.2 84.8 82.7 82.6±2.3 4 85.6 83.7 80.9 83.4±2.4 5 79.5 85.1 81.3 82.0±2.9 6 78.9 69.3 73.1 73.8±4.8

(3) Pea ( Pisum) sativum L.)

As a result of analyzing the propineb component by applying methylation to the pea (sparkle variety) sample, it showed a recovery rate of about 37.5%, confirming the difficulty of residual analysis. It was judged that the protein contained in peas (sparkle variety, protein content 24.4%) exhibited high solubility at pH 9 and thus dissolved in a weak alkaline EDTA-2Na aqueous solution. Since the dissolved pea protein acts as an interfering substance during propineb methylation, a protein-removal process using chloroform was applied to remove the protein. Chloroform reacted with the protein dissolved in the EDTA-2Na aqueous solution to permanently denature and remove the protein. The denatured protein could be confirmed through a white gel layer.

The average recovery rate of the protein-removal process from 1 to 5 times was 68.7% for once, 74.6% for 2 times, 88.6% for 3 times, 94.3% for 4 times, and 81.7 for 5 times when 50 ml of chloroform was used. % Is shown (Table 12). When four times the protein production process was performed, the highest recovery rate was shown.

Recovery rate of propineb (%) in the process of protein production of peas (50 ml chloroform + 10 ml amyl alcohol) Protein breakdown time Recovery rate (%) 1 ^st treatment 2 ^nd treatment 3 ^rd treatment Mean±standard deviation 0 38.5 36.0 38.0 37.5±1.3 One 74.8 61.3 70.0 68.7±6.8 2 70.9 75.0 77.9 74.6±3.4 3 89.9 86.9 88.9 88.6±1.4 4 94.8 90.9 97.4 94.3±3.2 5 82.9 77.1 85.0 81.7±4.1

Example 3. Verification of conformity of test methods

(1) Limit of Detection, LOD ) And Limit of Quantitation, LOQ )

The limit of detection (LOD) of the analyzer is the lowest concentration that can detect the analyte, and the accuracy of the quantified value cannot be guaranteed, but the minimum concentration that can measure the possibility of the analyte's presence in the sample. Means. In addition, it means a value of 3 to 5 times the S/N (signal/noise) ratio on the chromatogram. As a result of obtaining the S/N ratio by analyzing the standard solutions of various concentrations, the detection limits of propineb, mancozeb, and metiram were 0.2 ng.

The limit of quantitation (LOQ) is the lowest concentration that can quantify the analyte in the sample, and the S/N ratio on the chromatogram analyzed by treating the standard in an untreated sample is 6-10 times or more. At the threshold level, the relative standard deviation (RSD) of the average value of the recovery rate and the recovery rate must be able to satisfy the criteria. The Ministry of Food and Drug Safety recommends setting the limit of quantification at a level of 0.01 to 0.05 mg/kg. The quantification limits of propineb, mancozeb, and metiram calculated by Equation (1) were all at the level of 0.04mg/kg and were suitable for the residual analysis standard (Eq. (2), Equation (3), Equation (4)). .

Formula (1): LOQ(mg/kg)=A(ng)×[B(ml)/C(g)]×[D(ml)/E(ml)]×[F/G(µl)]

A: minimum detection amount, B: volume of sample after extraction, C: weight of sample, D: final volume, E: volume of sample used in derivatives and processes, F: dilution factor, G: device injection volume

Equation (2): Propineb LOQ (mg/kg) = 0.2 (ng) x [100 (ml) / 5 (g)] x [2 (ml) / 10 (ml)] x [1/20 ( Μl)]=0.04mg/kg

Equation (3): Mancozeb's LOQ (mg/kg) = 0.2 (ng) x [100 (ml)/5 (g)] x [2 (ml) / 10 (ml)] x [1/20 (µl) )]=0.04mg/kg

Formula (4): Metiram's LOQ (mg/kg)=0.2(ng)×[100(ml)/5(g)]×[2(ml)/10(ml)]×[1/20(µl) )]=0.04mg/kg

Ⓛ Propineb

The regression equation of the calibration curve obtained by injecting 20 μl of propineb standard solution (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 2.5mg/kg) into the instrument is Y=0.037617187×-0.005350334 (R ² =0.999), the R ² value was 0.999 or more, which was very good in linearity (FIG.

② Mankozeb

The regression equation of the calibration curve obtained by injecting 20 µl of Mancozeb's standard solution (0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 2.5mg/kg) into the instrument is Y=0.075119727×-0.016864242 (R ² =0.999). ^The value of ² was 0.999 or more, which was very good in linearity (FIG. 5B).

③ Metiram

The regression equation of the calibration curve obtained by injecting 20 μl of the standard solution (0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 2.5mg/kg) of metiram into the instrument is Y=0.037506763×-0.003304746 (R ² =0.999), ^The value of ² was 0.999 or more, which was very good in linearity (FIG. 5C).

(2) Recovery rate and repeatability

Ⓛ Propineb

In order to verify the recovery rate for the method of the present invention, as a result of performing a recovery rate test of propineb using soybeans and peas, as disclosed in FIGS. 6 and 7, in the chromatogram of the untreated sample of propineb Interfering substances were not observed, and it was confirmed that the recovery rate was very low when the protein-free protein was not performed.

In addition, after treating soybean and pea samples at the level of 10 times (0.4mg/kg) and 50 times (2.0mg/kg) of the limit of quantification of propineb in the soybean and pea samples in 3 repetitions, the dithiocarbamate system of the present invention As a result of analysis using the pesticide residue analysis method, the recovery rate was 85.3~93.5% (Tables 13 and 14), and the analysis error was less than 10% regardless of the treatment level.

Soybean ( Glycine max Merrill) and Pea ( Pisum) sativum L.) Recovery rate and MLOQ (method limit of quantitation) of propineb recovered from sample Crop Fortification level (mg/kg) Recovery mean±SD (%) MLOQ(mg/kg) Big head
0.4 88.3±1.1 0.04 2.0 90.7±1.3 pea 0.4 91.0±1.4 0.04
2.0 93.5±1.6

② Mankozeb

As a result of performing a recovery rate test of Mancozeb to verify the improved analysis process in the present invention, no interfering substances were observed in the chromatogram of the untreated sample of Mancozeb. Mancozeb was treated three times at the level of 10 and 50 times the limit of quantitation in a non-treatment sample of agricultural products containing the protein of Mancozeb, and the established analytical process was performed. As a result, the recovery rates were 91.5 and 91.3%, respectively. , The analysis error was less than 10% regardless of the treatment level (Table 15).

Recovery rate and LOQ of Mancozeb recovered from red bean sample Fortification
(mg kg ^-1 ) Recovery rate (%) Average±SD (%) ¹⁾ LOQ
(mg kg ^-1 ) R1 R2 R3 0.4 88.7 92.9 93.0 91.5±2.5 0.04
2.0 91.0 91.5 91.3 91.3±0.3

③ Metiram

As a result of performing a recovery rate test of metiram to verify the improved analysis process in the present invention, no interfering substances were observed in the chromatogram of the untreated sample of metiram as disclosed in FIG. 8. In the chromatogram of the untreated sample of propineb, no interfering substances were observed, and it was confirmed that the recovery rate was very low when the protein was not performed.

In addition, as a result of performing the established analysis process after treatment with metiram at the level of 10 times (0.4mg/kg) and 50 times (2.0mg/kg) of the limit of quantitation in the red bean sample, the established analysis process was performed, the average recovery rate was 85.1. % And 92.9% (Table 16), and the analysis error was less than 10% regardless of the treatment level.

Recovery rate and LOQ of metiram recovered from red bean sample Fortification
(mg kg ^-1 ) Recovery rate (%) Mean±SD (%) ¹⁾ LOQ
(mg kg ^-1 ) R1 R2 R3 0.4 91.9 82.0 81.6 85.1±5.8 0.04
2.0 95.6 96.1 87.1 92.9±5.0

(4) Verification using LC-MS/MS analysis method

In the present invention, for qualitative reconfirmation of the residual analysis method of three dithiocarbamate-based pesticides (propineb, mancozeb, and metiram) improved through HPLC/UVD, a triple quadrupole detector (LC-MS/MS) was used. The instrumental analysis method for verification was established.

As the ion source of the three pesticides, ESI ion sources were used among ESI (electrospray ionization) and APCI (atmospheric pressure chemical ionization), and detection was confirmed in the negative ion mode among the positive and negative ion modes.

Ⓛ Propineb

Propineb was detected under the ESI ion source and negative ion mode conditions. The precursor ion of propineb was selected as [M-H]+ ion, m/z=254.400, and the product ion was m/z=61.060, 206.710.

The propineb peak was not observed in the SRM (Selective reaction monitoring) chromatogram of the red bean sample, and only the residual content of propineb was accurately confirmed in the sample to which the standard was artificially added (FIG. 9). Therefore, in the case of propineb, the target pesticide of the present invention, when the LC-MS/MS SRM condition is used, it is determined that it can be used as an additional analysis method for the residuals of propineb in addition to the quantitative method using HPLC/UVD.

② Mankozeb

In the case of Mancozeb, detection was confirmed in the ESI ion source and negative ion mode. Mancozeb's precursor ion was selected as [M-H]+ ion, m/z=239.320, and product ion was selected as m/z=47.480, 58.400. Through selective reaction monitoring (SRM), the mancozeb peak was not observed in the red bean samples, and in the sample to which the standard substance was artificially added, only the residual mancozeb was accurately confirmed (FIG. 10).

Therefore, in the case of mancozeb, the target pesticide of the present invention, when using the LC-MS/MS SRM condition, it is determined that it can be used as an additional analysis method of the residuals of mancozeb in addition to the quantitative method using HPLC/UVD.

③ Metiram

In the case of Metiram, detection was confirmed in the ESI ion source and in the negative ion mode. The precursor ion of metiram was selected as [M-H]+ ion, m/z=239.249, and the product ion was m/z=58.415, 223.493. Metiram peak was not observed in the red bean sample through selective reaction monitoring (SRM), and in the sample to which the standard substance was artificially added, only the residual portion of metiram could be accurately identified (FIG. 11).

Claims (8)

(1) Ethylenediaminetetraacetic acid-disodium (EDTA-2Na) containing L-cysteine and sodium chloride after wetting any one selected from soybeans, peas, red beans, small beans, eastern and kidney beans for analysis of residual pesticide components Dissolving in an aqueous solution and adjusting the pH to 9.5 to 9.6;
(2) After step (1), to 5 g of the bean, 40-60 ml of chloroform and 8-12 ml of amyl alcohol were added, stirred, and centrifuged to separate the supernatant EDTA-aqueous solution layer, protein- Separating the gel layer and the chloroform layer in that order, and obtaining a supernatant to produce protein;
(3) adding 0.8 to 1.2 mL of tetrabutylammonium hydrogen sulfate (TBAH) of 0.3 to 0.5 M to 10 mL of the supernatant obtained in step (2), and adjusting the pH to 7.0±0.3;
(4) After step (3), a mixture solution of 1:1-4 (v/v) n-hexane and dichloromethane containing methyl iodide (CH ₃ I) was added to methylate the residual pesticide component to be analyzed. Letting go;
(5) After step (4), adding a dichloromethane solution containing 15 to 25% (w/v) 1,2-propanediol, and then concentrating under reduced pressure;
(6) re-dissolving the sample concentrated under reduced pressure in methanol and filtering; And
(7) After step (6), detecting the residual amount of dithiocarbamate-based pesticide; The residual amount of dithiocarbamate-based pesticide remaining in beans containing a high content of protein, characterized in that it comprises Analysis method. The method of claim 1, wherein the residual pesticide component is propineb, mancozeb, metiram, ferbam, ziram, thiram, maneb ), a method for analyzing the residual amount of dithiocarbamate-based pesticides remaining in beans containing a high content of protein, characterized in that at least one selected from zineb and nabam. The method of claim 1, wherein the step (2) is performed 1 to 5 times. The method for analyzing the residual amount of dithiocarbamate-based pesticides remaining in beans containing a high content of protein. delete delete delete delete The method of claim 1,
(1) Ethylenediaminetetraacetic acid-disodium (EDTA-2Na) containing L-cysteine and sodium chloride after wetting any one selected from soybeans, peas, red beans, small beans, eastern and kidney beans for analysis of residual pesticide components Dissolving in an aqueous solution and adjusting the pH to 9.5 to 9.6;
(2) After step (1), 40-60 ml of chloroform and 8-12 ml of amyl alcohol were added to 5 g of the pulses of step (1), and stirred for 8-12 minutes, Centrifuge at 2,000 to 4,000 rpm for 8 to 12 minutes to separate the supernatant of EDTA-aqueous solution layer, protein-gel layer, and chloroform layer in that order, and repeat 3 to 5 times to obtain the supernatant. step;
(3) adding 0.8 to 1.2 mL of tetrabutylammonium hydrogen sulfate (TBAH) of 0.3 to 0.5 M to 10 mL of the supernatant obtained in step (2), and adjusting the pH to 7.0±0.3;
(4) After step (3), a mixed solution of 1:1 to 4 (v/v) n-hexane and dichloromethane containing 0.03 to 0.1 M methyl iodide (CH ₃ I) was added to analyze Methylating the residual pesticide component;
(5) After step (4), adding a dichloromethane solution containing 15 to 25% (w/v) 1,2-propanediol, and then concentrating under reduced pressure;
(6) re-dissolving the sample concentrated under reduced pressure in methanol and filtering; And
(7) After step (6), detecting the residual amount of dithiocarbamate-based pesticide; The residual amount of dithiocarbamate-based pesticide remaining in beans containing a high content of protein, characterized in that it comprises Analysis method.

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