KR20220018154A - Analytical method for dithiocarbamate pesticide remaining on agricultural product - Google Patents

Abstract

The present invention relates to a method for analyzing dithiocarbamate pesticide remaining in agricultural products. Provided is an analysis method which can analyze a remaining amount of a dithiocarbamate-based pesticide substance in various agricultural products, omits a step of sucking and filtering after solvent extraction compared to the analysis method listed in the Korean Food Standards Codex (CS2 analysis method) and the conventional analysis method disclosed in Korean patent No. 1278631, and omits a step of CS2 coloring and compressed concentration after derivatization, so that analysis of remaining pesticide cannot only be rapidly conducted, but also a recovery rate is maximized, thereby ensuring an effect of lowering the limit of quantitation.

Description

Analysis method of dithiocarbamate pesticide remaining on agricultural products {Analytical method for dithiocarbamate pesticide remaining on agricultural product}

The present invention relates to a method for analyzing dithiocarbamate pesticides remaining in agricultural products.

Dithiocarbamate pesticides account for 9% of total pesticide production (43% of horticultural fungicides), and are used to control more than 70 crop diseases in countries around the world.

Dithiocarbamate pesticides are dialkyl dithiocarbamate (DDC), ethylene bis dithiocarbamate (EBDC), propylene bis dithiocarbamate (PBDC) depending on their chemical structure. ), and according to each group, DDC pesticides are known as ferbam, fermate, ziram, zerlate, and thiram, and EBDC pesticides are known as mancozeb, Maneb, zineb, metiram, and nabam are known, and propineb, a PBDC pesticide, is known.

In Korea, mancozeb, metiram, propineb, and thiram are used, and as pesticides used in the past but not used now, maneb and jineb ( zineb), etc.

Dithiocarbamate pesticides are effective against various pathogens and have low toxicity and low cost. It is a widely used crop protection agent. Since dithiocarbamate pesticides are being sprayed on various crops, a rapid, accurate and reliable residue analysis method is essential to secure safety by inspecting and evaluating the level of pesticide residues in food. In particular, dithiocarbamate-based pesticides are essential for the control of citrus sunspot disease, and there is a need for rapid analysis in order to expand exports of citrus fruits because of their high usage.

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, and a method of detecting them by methylation using CH ₃ I. A method of converting a substance into a substance and analyzing it by reading 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 making it 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 the qualitative and quantitative analysis of dithiocarbamate pesticides since Gustafsson and Thomson published it in 1981. Although it has been used for analysis and monitoring, it is not commonly used in the actual field due to its complicated operation, time and cost consuming, and inconveniences such as the use of a large amount of organic solvent and reagent preparation process. In addition, this unconventional food analysis method (CS ₂ method) is a general-purpose analytical method for analyzing a variety of agricultural products, as the derivatization process is severely disturbed in samples with high protein content, such as pulse samples such as soybeans and red beans, and the recovery rate tends to decrease rapidly. there is

On the other hand, Korean Patent No. 1278631 discloses a rapid and efficient method for analyzing dithiocarbamate-based pesticides, which is characterized by improving the speed of pretreatment by simultaneously performing extraction and methylation in one tube. The process is included, and the time required for the derivatization step is 10 minutes or more, and there is a problem that the analysis time is still long because it includes a concentration step under reduced pressure. The present invention is a method that further shortens the time required for the dithiocarbamate-based pesticide analysis method disclosed in Korean Patent Registration No. 1278631, and omits the suction filtration step after solvent extraction, and omits the process of concentration under reduced pressure after derivatization. As a method characterized by minimizing the analysis time by doing so, there has been no disclosure of a method for analyzing dithiocarbamate-based pesticides remaining in agricultural products of the present invention.

The present invention has been derived from the above needs, and the present invention provides a method for analyzing dithiocarbamate-based pesticides remaining in agricultural products, and the method for analyzing dithiocarbamate-based pesticides provided by the present invention is a conventional method for analyzing dithiocarbamate-based pesticides. Completion of the present invention by confirming that not only can the pre-treatment and analysis time be greatly shortened, but also the limit of quantitation can be very low compared to the food standard analysis method (CS ₂ analysis method) or the analysis method disclosed in Korean Patent No. 1278631 did

In order to achieve the above object, the present invention comprises the steps of: (1) pulverizing an agricultural product sample to be analyzed for residual pesticide components, and weighing the pulverized sample;

(2) adding a NaOH solution containing ethylenediaminetetraacetic acid (EDTA) and L-cysteine to the weighed sample and shaking, followed by centrifugation;

(3) adding methanol containing methyl iodide to the supernatant obtained by centrifugation in step (2), shaking, and purifying using a filter; and

(4) LC-MS/MS analysis of the solution purified in step (3); provides a method for analyzing the residual amount of dithiocarbamate-based pesticide components remaining in agricultural products, including.

In addition, the present invention provides a kit for analyzing the residual amount of dithiocarbamate-based pesticides remaining in agricultural products containing methanol containing sodium hydroxide (NaOH) solution containing ethylenediaminetetraacetic acid (EDTA), L-cysteine and methyl iodide. to provide.

The present invention relates to a method for analyzing dithiocarbamate-based pesticides remaining in agricultural products, and it is possible to analyze the residual amount of dithiocarbamate-based pesticides in various agricultural products, and the analysis method of the present invention is as shown in FIG. In contrast to the analysis method listed in the Food Codex (CS ₂ analysis method) and the analysis method disclosed in the conventional Korean Patent No. 1278631, the step of suction filtration after solvent extraction was omitted, and after derivatization, CS ₂ color development and reduced pressure concentration step It is an analysis method that has the effect of lowering the limit of quantitation by not only allowing rapid analysis of pesticide residues by omitting , but also maximizing the recovery rate.

1 is a flowchart for the analysis method of the present invention, the analysis method listed in the Food Codex, and the analysis method disclosed in Korean Patent Registration No. 1278631.
2 is an LC-MS chromatogram of an apple without pesticide (A) and an apple (B) treated with 0.5 mg/ml of mancozeb.
3 is an LC-MS chromatogram of an apple (A) without pesticides and an apple (B) treated with 0.5 mg/ml propineb.

The present invention comprises the steps of: (1) pulverizing a sample of agricultural products to be analyzed for residual pesticide components, and weighing the pulverized sample;

(2) adding a NaOH solution containing ethylenediaminetetraacetic acid (EDTA) and L-cysteine to the weighed sample and shaking, followed by centrifugation;

(3) adding methanol containing methyl iodide to the supernatant obtained by centrifugation in step (2), shaking, and purifying using a filter; and

(4) analyzing the solution purified in step (3) by liquid chromatography-mass spectrometry (LC-MS/MS); analyzing the residual amount of dithiocarbamate pesticide components remaining in agricultural products, including it's about how

Preferably, the steps of: (1) pulverizing a sample of agricultural products to be analyzed for residual pesticide components using dry ice, and weighing the pulverized sample;

(2) To 5 g of the weighed sample, 15-25 ml of 0.4-0.5 M NaOH solution containing 0.2-0.3 M EDTA (ethylenediaminetetraacetic acid) and 0.05-0.15 g of L-cysteine were added to 1 shaking for ˜3 min, followed by centrifugation for 4-6 min;

(3) To 0.5 ml of the supernatant obtained by centrifugation in step (2), 0.4 to 0.6 ml of methanol containing 0.04 to 0.06 M of methyl iodide is added, and shaking for 0.5 to 1.5 minutes ), and then purifying using a 0.1-0.3 μm scanning filter; and

(4) analyzing the solution purified in step (3) by liquid chromatography-mass spectrometry LC-MS/MS) is about

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

The sample is preferably at least one selected from grains, beans, vegetables, documents, and fruits, and more preferably at least one selected from rice, apples, tangerines, paprika, and grapes, but is not limited thereto.

In step (1), pulverization is preferably performed using dry ice or briefly pulverized (minced), but is not limited thereto. It is preferable to pulverize (shred) afterward.

In step (3), purification is preferably performed using a 0.1-0.3 μm scanning filter, but is not limited thereto. The methanol used in step (3) is well mixed with methyl iodide, and liquid chromatography ( It has the characteristic that it can be used directly as a mobile phase in LC (liquid chromatography). Therefore, instead of the methanol used in step (3) of the present invention, any organic solvent that can be used as a mobile phase of LC while being well mixed with methyl iodide can be used.

The LC is preferably HPLC or UPLC, but any LC analyzer used in the art can be applied.

In addition, the present invention is a kit for analyzing the residual amount of dithiocarbamate-based pesticides remaining in agricultural products containing methanol containing sodium hydroxide (NaOH) solution containing ethylenediaminetetraacetic acid (EDTA), L-cysteine and methyl iodide. it's about

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.

[Materials and Methods]

1. Target pesticides and agricultural products

Target pesticides are the fungicides mancozeb (mancozeb) and propineb (propineb), and the agricultural products used in the examples of the present invention are agricultural products using the mancozeb and propineb, rice, apples, tangerines, paprika and grapes was selected. The untreated samples were purchased at an eco-friendly agricultural products store, and after purchasing the untreated samples, samples in which no pesticides were detected were used by checking whether or not pesticides were detected.

2. Preparation and storage of standard solution

The extraction solution was prepared by mixing 0.25M EDTA and 0.45M NaOH and adding 0.1g L-cysteine. The standard substances Mancozeb and Propineb were prepared by adding and dissolving 7.35 ml and 7.85 ml of extraction solutions to a 10 mg sample in consideration of their respective purity to prepare a 1000 mg/L stock solution. To prepare a standard solution for preparing a calibration curve, the extraction solution was diluted to concentrations of 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 and 1 mg/L. The stock solution was stored frozen at -20℃, and the safety of the standard solution was confirmed during the experiment.

3. Pretreatment method

The sample was homogenized by pulverizing it with dry ice and stored frozen before use. A 5 g sample was weighed in a 50 ml tube, 20 ml of an extraction solution was added, shaken for 2 minutes, and centrifuged at 3000 rpm for 5 minutes.

For derivatization, take 0.5 ml of the supernatant of the sample centrifuged in a 1.5 ml tube, add 0.5 ml of 0.05M methyl iodide dissolved in methanol, shake for 1 minute, and then 0.20 μm Filtration with an injection filter was used.

4. LC-MS/MS analysis

The LC-MS/MS used for the analysis was a Shimadzu Nexera X2 ^® LC instrument equipped with an LC-8050 ^® tandem triple quadrupled mass spectrometer (MS/MS) (Shimadzu, Kyoto, Japan). For the column, a Shiseido CAPCELL CORE C ₁₈ column (2.1×150 mm, 2.7 μm), which is a reversed-phase C ₁₈ column, was used. For the mobile phase, 0.1% formic acid and 5 mM ammonium formate were added to distilled water and methanol, respectively. and then used as mobile phases of A and B.

Samples for analysis were analyzed by injecting 10 μl of each. In addition, in order to improve the selectivity and sensitivity of the test pesticides, analysis was performed in MRM (multiple reaction monitoring) mode. The final established instrumentation conditions are presented in Table 1.

LC-MS/MS analysis conditions Instrument Shimadzu Nexera X2 ^® Column Shiseido CAPCELL CORE C ₁₈ column (2.1×150mm, 2.7μm) mobile phase A: 5 mM ammonium formate, 0.1% formic acid in water B: 5 mM ammonium formate, 0.1% formic acid in methanol hours (minutes) A (%) B(%) 0.0 85 15 1.0 85 15 1.5 40 60 10.0 10 90 12.0 10 90 12.1 2 98 13.0 2 98 flow rate 0.3ml/min Injection volume 10 μl Column temperature 40℃ MS/MS Shimadzu 8050 Ionization mode Electrospray Ionization (ESI, positive) Interface Temperature 150℃ DL Temperature 300℃ Heat block Temperature 400℃ interface voltage 4000V Conversion dynode voltage 10000V Detector voltage 1920V Scan type MRM mode Run time 13min

5. Measurement of recovery rate, limit of detection and limit of quantitation

The linearity, detection limit, and quantitation limit of the analytical method of the present invention were evaluated. The recovery rate was analyzed to test the efficiency and reliability of the dithiocarbamate assay. To check the linearity, a calibration curve was prepared using each peak area for a concentration range of 0.005 to 1 mg/ℓ of a standard solution of dithiocarbamate pesticides (mancozeb, propineb), and the coefficient of determination of the calibration curve was obtained. , The detection limit and quantitation limit were based on the signal-to-noise ratio (S/N ratio) on the chromatogram. The detection limit was measured based on an S/N ratio of 3 or more, and the limit of quantification was measured based on an S/N ratio of 9 or more.

In order to evaluate the accuracy and reproducibility of the analysis method, the standard solution was added to the untreated samples of rice, apple, tangerine, paprika, and grape, which are representative crops for each food group, and then analyzed to determine the recovery rate. The treatment concentration was carried out at two concentrations, low concentration and high concentration. The low concentration was 5 to 20 times the detection limit, and the high concentration was 25 to 250 times the detection limit. All experiments were performed in 5 repetitions for each concentration and sample, and the mean and relative standard deviation were calculated.

Example 1. Validation of the assay according to the present invention

The standard mixed solution prepared to measure the limit of detection and limit of quantitation for the analysis was diluted to 0.005 to 1 mg/L, and the analysis was repeated 5 times, respectively. In LC-MS/MS analysis, the standard curves of the pesticides of Mancozeb and Propineb showed high linearity at 0.998 or higher, and the detection limits of the pesticides of Mancozeb and Propineb were 0.002 mg/ℓ and 0.001 mg/ℓ , and the limits of quantitation were 0.005 mg/L and 0.002 mg/L (Table 2).

Validation of Assays for Mancozeb and Propineb pesticide linearity (R ² ) Detection limit (mg/ℓ) Limit of Quantitation (mg/ℓ) Mancozeb 0.9988 0.002 0.005 propineb 0.9985 0.001 0.002

Example 2. Assay validation in agricultural products

The effectiveness of the analysis method of the present invention was verified with the linearity, detection limit, and quantitative limit, and the final concentration of the eco-certified rice, apple, tangerine, paprika and grape samples was diluted to 0.005~1mg/ℓ and analyzed. (Table 3). All agricultural sample standard solutions showed high linearity with a linearity of 0.98 or more, and the detection limits of mancozeb and propineb in the samples ranged from 0.001 to 0.002 mg/ℓ, and the limit of quantitation (LOQ) was from 0.001 to 0.001 mg/ℓ. It was found to be in the range of 0.005 mg/L. It was confirmed that the sensitivity was sufficient to detect the non-detection standard of 0.01 mg/ℓ of agricultural products for which the residual tolerance standard was not determined.

Validation of Mancozeb and Propineb in Agricultural Products sample pesticide Linearity (R ² ) Detection limit (mg/ℓ) Limit of Quantitation (mg/ℓ) rice Mancozeb 0.9928 0.001 0.003 propineb 0.9981 0.001 0.003 apologize Mancozeb 0.9994 0.002 0.005 propineb 0.9989 0.001 0.002 citrus Mancozeb 0.9808 0.001 0.002 propineb 0.9874 0.001 0.002 paprika Mancozeb 0.9996 0.002 0.005 propineb 0.9998 0.001 0.003 grape Mancozeb 0.9994 0.001 0.003 propineb 0.9997 0.001 0.003

Example 3. Recovery rate according to agricultural products and pesticides

The recovery rate analysis was performed by adding a standard mixture solution to the sample so that the final concentrations were 0.01 and 0.5 mg/ℓ for 5 agricultural samples, and applying LC-MS/MS to verify the recovery rate.

The results for the recovery rate for the pesticides to be analyzed are shown in Table 4. The recovery rates of mancozeb and propineb, the pesticides to be analyzed by LC-MS/MS, were 98.24 and 99.36% at low concentrations (0.01 mg/ℓ) in the rice sample, with standard deviations of 3.38 and 1.55%. At high concentration (0.5mg/ℓ), the recovery rates were 75.15 and 86.50%, and the standard deviations were 3.43 and 2.04%. In the case of the apple sample, the recovery rates were 94.82, 100.98%, with standard deviations of 5.11 and 0.97% at the low concentration (0.01 mg/ℓ) at the low concentration (0.01 mg/ℓ), and the recovery rates were 82.33, 88.45%, and standard deviations at the high concentration (0.5 mg/ℓ). was 4.41 and 3.90%. In the case of citrus samples, the recovery rates were 100.00, 98.06% and standard deviations of 2.39 and 2.43% at the low concentration (0.01mg/ℓ), and at the high concentration (0.5mg/ℓ), the recovery rates were 96.20 and 94.95%, and the standard deviations were 2.19 and 2.40%. In the case of paprika samples, the recovery rate was 92.10~111.86% at low and high concentrations of the dithiocarbamate pesticide, and the standard deviation was 2.90~5.82%. 98.72% and the standard deviation was 0.67~3.83%.

The recovery rate suggested by Korea and the European Union is 70-120% and a relative standard deviation of 20% or less, and the International Food Standards Commission stipulates a recovery rate of 60-120% and a relative standard deviation of 15-30% or less.

In the present invention, the recovery rate suggested by the dithiocarbamate-based pesticide component analysis method using LC-MS/MS was at least 70% or more and the relative standard deviation was 20% or less, thus satisfying domestic and international standards. Therefore, it was determined that the pretreatment method and analysis method applied in the present invention could be applied to analyze the pesticide component remaining in agricultural products (FIGS. 2, 3 and Table 4).

Recovery rate according to agricultural products and pesticides sample pesticide Recovery (%) Standard Deviation(%) 0.01mg/L 0.5mg/L 0.01mg/L 0.5mg/L rice Mancozeb 98.24 75.15 3.38 1.55 propineb 99.36 86.50 3.43 2.04 apologize Mancozeb 94.82 82.33 5.11 4.41 propineb 100.98 88.45 0.97 3.90 citrus Mancozeb 100.00 96.20 2.39 2.19 propineb 98.06 94.95 2.43 2.40 paprika Mancozeb 92.10 103.69 5.82 4.57 propineb 94.92 111.86 3.53 2.90 grape Mancozeb 93.72 95.51 3.83 1.25 propineb 91.73 98.72 3.22 0.67

In addition, as a result of confirming the time required for the analysis method of the present invention in comparison with the existing food standard analysis method (CS ₂ analysis method) and Korea Patent (No. 1278631), the analysis method of the present invention can significantly reduce the analysis time. was confirmed (Table 5).

Comparison of processing time and throughput according to analytical methods division Food Standards Analysis Method
(CS ₂ method) Korea registered patent
(No. 1278631) Analytical method of the present invention 1 preprocessing time 100 minutes 50 minutes 30 minutes 1 analysis time 40 minutes 10 minutes 13 minutes Analysis volume per day 4 cases 12 cases 25 cases limit of quantification 0.1ppm 0.005ppm 0.005ppm

Claims (6)

(1) pulverizing a sample of agricultural products to be analyzed for residual pesticide components, and weighing the pulverized sample;
(2) adding a NaOH solution containing ethylenediaminetetraacetic acid (EDTA) and L-cysteine to the weighed sample and shaking, followed by centrifugation;
(3) adding methanol containing methyl iodide to the supernatant obtained by centrifugation in step (2), shaking, and purifying using a filter; and
(4) analyzing the solution purified in step (3) by liquid chromatography-mass spectrometry (LC-MS/MS); analyzing the residual amount of dithiocarbamate pesticide components remaining in agricultural products including; Way. The method of claim 1, wherein the dithiocarbamate pesticide is mancozeb, propineb, ferbam, ziram, thiram, maneb, centipede. A method for analyzing the residual amount of dithiocarbamate-based pesticides remaining in agricultural products, characterized in that at least one selected from zineb, metiram, and nabam. The method of claim 1, wherein the agricultural sample is at least one selected from cereals, beans, vegetables, documents, and fruits. The method of claim 1, wherein the purification in step (3) is performed using a 0.1-0.3 μm scanning filter. According to claim 1,
(1) pulverizing a sample of agricultural products to be analyzed for residual pesticide components using dry ice, and weighing the pulverized sample;
(2) To 5 g of the weighed sample, 15-25 ml of 0.4-0.5 M NaOH solution containing 0.2-0.3 M EDTA (ethylenediaminetetraacetic acid) and 0.05-0.15 g of L-cysteine were added to 1 shaking for ˜3 min, followed by centrifugation for 4-6 min;
(3) To 0.5 ml of the supernatant obtained by centrifugation in step (2), 0.4 to 0.6 ml of methanol containing 0.04 to 0.06 M of methyl iodide is added, and shaking for 0.5 to 1.5 minutes ), and then purifying using a 0.1-0.3 μm scanning filter; and
(4) analyzing the solution purified in step (3) by liquid chromatography-mass spectrometry (LC-MS/MS); analyzing the residual amount of dithiocarbamate pesticide components remaining in agricultural products including; Way. A kit for analyzing the residual amount of dithiocarbamate pesticides remaining in agricultural products including sodium hydroxide (NaOH) solution containing ethylenediaminetetraacetic acid (EDTA), methanol containing L-cysteine and methyl iodide.

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