KR101278631B1 - Fast and efficient dithiocarbamate pesticide analytical methods for citrus - Google Patents

Abstract

The present invention relates to a dithiocarbamate-based pesticide analysis method of citrus fruits, and more particularly, to analyze the pesticide, characterized in that to improve the rapidity of pre-treatment by simultaneously performing extraction and methylation in one tube (One Tube) It is about a method.
The present invention recognizes the beneficial effect of improving the speed and efficiency in analyzing pesticides of citrus fruits. In addition, the detection limit in the sample was lowered to meet the importing country standard, which raised the level of safety management for export agricultural products.
As a result, the amount of Analytical Dithiocarbamate-based pesticides used in export tangerines, such as the United States, has been tripled, contributing to the export of Jeju tangerines, and by using analytical methods for post-management of eco-friendly agricultural products, safe agricultural products are supplied to consumers. Will contribute to promotion.

Description

Fast and efficient dithiocarbamate pesticide analytical methods for citrus

A technique for analyzing a dithiocarbamate-based pesticide analysis method of citrus fruits, and more particularly, in a pesticide analysis method characterized by improving the rapidity of pre-treatment by simultaneously performing extraction and methylation in one tube. Technology.

Dithiocarbamate pesticides are organic sulfur non-invasive fungicides and are preferred among farmers because they are relatively inexpensive and effective in controlling anthrax, rot and other diseases of vegetables and fruits. Except for pharmaceuticals (mechanical emulsions), most are produced.

Among them, mancozeb (Daisen M-45) accounts for about 7.6% of the total pesticide production (Korea Crop Protection Association, 2009), and is particularly used for controlling tangerine spots on citrus. In recent years, the demand for safety investigation by importing country is increasing due to the expansion of export agricultural products, and the rapid analysis and precise analysis of dithiocarbamate pesticides are required in citrus fruits.

Hereinafter, the patent document related to this invention is described.

First, KR 10-2003-0010550 exists as a patent document. The present invention relates to a method for extracting residual pesticides, which significantly reduces the extraction time of residual pesticides in food, and is substantially different from the present invention.

Second, KR 10-2001-0075500 exists as a patent document. The present invention relates to a detection method for simultaneously detecting acidic pesticides in water or soil samples, and does not include the problems and solutions of the prior art proposed by the present invention.

Dithiocarbamate pesticides cannot qualitatively and quantitate components divided into three groups according to the chemical structure by the conventional CS2-spectrophotometry (Keppel) method, and pretreatment using methylation analysis requires a lot of time and technology. In order to solve these problems and to quickly process customized safety surveys for each country of export agricultural products, it is required to establish an analytical method that is easy to pretreat and excellent in separation.

Accordingly, the present invention provides a method for analyzing pesticides of dithiocarbamate-based pesticides, by which extraction and methylation are simultaneously performed in one tube to improve the rapidity of pre-treatment. The purpose.

However, the technical problems to be achieved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned may be clearly understood by those skilled in the art from the description of the present invention. There will be.

The present invention relates to a dithiocarbamate-based pesticide analysis method of citrus fruits, and provides a pesticide analysis method characterized by improving the rapidity of pre-treatment by simultaneously performing extraction and methylation in one tube. do.

Preferably, the pretreatment comprises: (i) a first step of adding L-cysteine to the cut specimen and then shaking with EDTA solution; (ii) a second step of additionally administering tetrabutylammonium bisulfate solution and NaCl after shaking and then mixing and adjusting the pH to 7.0; (iii) a third step of further administering a dichloromethane: n-hexane (volume ratio = 1: 1) mixed solution containing methyl iodide after the pH adjustment and mixing and methylating; (iv) after the methylation, extracting the solvent layer by centrifugation and then adding 20% 1,2-propanediol dissolved in dichloromethane; (v) a fifth step of adding the 1,2-propanediol, concentrating until only 1,2-propanediol remains, re-dissolving with Methanol and filtering; It may be characterized in that it comprises a.

More preferably, the first step may be characterized in that after the addition of 0.1 g L-cysteine to the cut sample, 20mL of 0.25M EDTA (0.45M NaOH) solution and shake for 10 minutes.

More preferably, the second step may be characterized by adjusting the pH to 7.0 using 2M HCl by further mixing 1.5mL and 0.4g of 0.41M tetrabutylammonium bisulfate solution and then adding NaCl 3g.

More preferably, the third step may additionally administer 15 ml of a dichloromethane: n-hexane (volume ratio 1: 1) mixture containing 0.05 M methyl iodide, and mix and methylate the mixture.

More preferably, the centrifugation of the fourth step may be performed by centrifuging for 3 minutes at 3000 rpm.

More preferably, the fourth step of adding 1,2-propanediol may be characterized in that 6 ml of the upper solvent layer obtained by centrifugation is added and 0.2 ml of 20% 1,2-propanediol dissolved in dichloromethane.

More preferably, the concentration of the fifth step is concentrated until only 1,2-propanediol remains at 30 ° C., and the filtration of the fifth step is redissolved with 2 ml of Methanol and filtered with a 0.2um syringe filter. Can be.

On the other hand the present invention is a sixth step of analyzing the filtrate obtained after the fifth step by HPLC / UVD or UPLC / MS / MS; It may be characterized in that it further comprises.

The present invention recognizes the beneficial effect of improving the speed and efficiency in analyzing pesticides of citrus fruits. More specifically, as shown in Table 1 below, there is an advantage that a result about two times faster than the conventional method may be obtained.

The present invention extracts a complex process of dithiocarbamate-based pesticides in citrus fruits, which are recently extracted in a food processing method, filtered with a glass fiber filter, transferred to a separatory funnel, methylated derivatives, and then dehydrated and filtered through two distribution processes. It is simplified to non-stop treatment process after centrifugation and methylation, and it has been improved by a method that can be analyzed quickly and easily (One Tube Pre-treatment Method), which shortens the analysis time, saves reagents, etc. The limit of detection was also lowered to increase the level of safety management for export agricultural products (see table below).

Furthermore, another aspect of the advantageous effects of the technical aspects of the present invention is disclosed. In the pretreatment process, the complex process of methylation (derivative) after extraction and transfer to the separatory funnel is simplified so that it can be quickly analyzed in one step of extraction and methylation (derivative) in one tube to increase the analysis volume and analysis time and manpower. Can reduce the cost. In other words, after eliminating the suction filtration process, after two liquid-liquid distributions, the dehydration process was replaced by centrifugation, which increased the amount of analyte available three times from four to twelve. In addition, it is possible to reduce the cost of purchasing a glassware such as a separatory funnel (for dispensing liquid) and a boolean funnel (suction filtration), and to reduce the amount of reagent used by one third. In particular, the analysis time can be greatly reduced from 80 minutes to 50 minutes, thereby reducing labor costs and shortening the exposure time of the analyst from harmful substances.

Dithiocarbamate pesticides, mancozeb (Daisen M-45), account for about 7.6% of the total pesticide production (Korea Crop Protection Association, 2009). I am doing it.

By increasing the amount of analytical quantities of dithiocarbamate-based pesticides used for exporting citrus fruits, such as the United States, to contribute to the expansion of Jeju citrus exports, and by using analytical methods for post-management of eco-friendly agricultural products, safe agricultural products are supplied to consumers to promote national health. Will contribute.

1 relates to chromatograms and spectra of standard solutions (1.0 mg / l).
2 relates to chromatograms and spectra of blank and recovery (1.0 mg / l) samples.
3 relates to standard calibration curves and tables
a: DDC (thiram)
b: EBDC (mancozeb)
c: PBDC (propineb)
4 relates to chromatograms in standards and samples in LC / MS / MS.
a: DDC (thiram (10ug / L) _recovery (100ug / L) chromatogram)
b: EBDC_PBDC (mancozeb_propineb (10ug / L) chromatogram)
c: Recovery (mancozeb_propineb (100ug / L) chromatogram)
Figure 5 relates to the calibration curve and the recovery result table of the standard solution in LC / MS / MS.
a: DDC (calibration curve, recovery table)
b: EBDC_PBDC (calibration curve, recovery table)
Figure 6 is a photograph showing the process of 'dithiocarbamate-based component extraction' and 'treatment with one tube until centrifugation' in the One Tube Pre-treatment method of the present invention.
Figure 7 schematically shows the pretreatment of the invention.

The present invention provides a method that can be quickly and easily analyzed by improving the methylation pretreatment carried out according to the food processing method in analyzing dithiocarbamate-based pesticides in citrus fruits (One Tube Pre-treatment Assay).

This One Tube Pre-treatment method is a non-stop process that extracts and methylates (derivatives) from one tube and transfers the methylation (derivatives) to the funnel after extraction. HPLC / UVD and MS / MS optimal assays for DDC, EBDC, PBDC.

Hereinafter, the present invention will be described in detail based on a process referring to the drawings.

[1] preparation of reagents and laboratory materials

For the experiment of the present invention, Thiram (99%, Dr.ehrenstorfer), Mancozeb (73.5%, Dr.ehrenstorfer), Propineb (78%, Dr.ehrenstorfer), Metiram (84%, Dr.ehrenstorfer), methyl iodide, L-cysteine, sodium hydroxide, Hydrochloric acid, tetrabuthylammonium bisulfate, ethylenediaminetetraacetic acid disodium salt dihydrate, 1,2-propanediol (Sigma), ammonium formate (Fluka), sodium chloride (Junsei), dichloromethane, n-hexane, methanol, Acetonitrile ( 99.99%, HPLC grade), LC / UVD 1200 series, MS 6130 (Agilent), API 3200 MS / MS (AB SCIEX) were prepared.

Specifically, the following conditions were given.

 □ 0.25M EDTA (0.45M NaOH) solution: pH 10.0 ~ 10.5

 □ 0.41M tetrabuthylammonium bisulfate solution

 □ 0.05M methyl iodide (dichloromethane: n-hexane (1: 1, v: v))

 □ Preparation of standard material (Adjusted to pH 7.0 after adding L-cysteine and 1-1 solution)

 □ Thiram is prepared by dissolving a small amount of organic solvent (acetone) in about 1ml.

□ Expiry date: Refrigerated 15 days

[2] pretreatment method (see FIGS. 6 to 7)

[2_1] sample weighing

5 g of the citrus cut samples were placed in a 50 mL tube.

[2_2] solvent extraction

After adding 0.1 g of L-cysteine to the weighed sample, 20 mL of 0.25M EDTA (0.45M NaOH) solution was added and shaken for 10 minutes.

After the shaking, 1.5 mL of 0.41 M tetrabutylammonium bisulfate solution and 3 g of NaCl were further administered, shaken, and the pH was rapidly adjusted to 7.0 using 2 M HCl.

[2_3] derivatization (methylation)

Next, 15 ml of a mixture of dichloromethane: n-hexane (volume ratio 1: 1) containing 0.05M methyl iodide was additionally administered, followed by shaking for 5 minutes, and then left for 10 minutes.

[2_4] Centrifugation

After centrifugation at 3000 rpm for 3 minutes, 6 ml of the solvent layer (upper layer) was extracted, and 0.2 ml of 20% 1,2-propanediol dissolved in dichloromethane was added.

[2_5] concentration and filtration

Concentrated until only 1,2-propanediol remained at 30 ℃ and redissolved in 2ml of Methanol and filtered with a 0.2um syringe filter and analyzed.

[3] HPLC / UVD conditions

[3_1] Instrumental Analysis Using HPLC

In the instrument analysis using HPLC (HP-1200 series) was carried out under the following conditions specifically.

 □ Column: Agilent Zorbax SB-C18 (3.5u, 150X4.6 mm)

 □ Injection volume: 10 ul

 □ Detector: Wavelength (UV): 272 nm, Reference 360 nm

 □ Mobile Phase: Gradient mode

   -ACN: MeOH: DDW (10:40:50), 0.8ml / min (0-1min)

   -ACN: MeOH: DDW (10:60:30), 0.8ml / min (1-3min)

   ACN: MeOH: DDW (15:65:20), 0.8ml / min (3-5min)

   ACN: MeOH: DDW (15:65:20), 0.8ml / min (5-7min)

   ACN: MeOH: DDW (15:75:10), 0.8ml / min (7-10min)

 □ Column temperature: 35 ℃

 □ Retention time

   -thiram 5.41min

   mancozeb (metiram) 6.16 min

propineb 6.65min

[3_2] Device Analysis using UPLC / MS / MS

In the instrumental analysis, specific conditions of LC were set as follows.

 □ UPLC: Waters ACQUITY

 □ Column: Kinetex C18 (2.1x100 mm, particle size 2.6 um, 100A)

 □ Injection volume: 10 ul

 □ Mobile Phase: MPA-MeOH (2mM ammonium formate 0.1% formic acid)

 □ MPB-H2O (2mM ammonium formate 0.1% formic acid)

 □ Column temperature: 35 ℃

 □ Retention time

   -thiram 2.34min

   mancozeb (metiram) 2.60 min

-propineb 2.74min

In the instrument analysis, specific conditions of MS / MS were set as follows.

[4] results and discussion

Standard solution (100ppm) was added at concentrations of 0.05, 0.1, 0.5, 1.0, 5.0, and 10ppm, pretreated by the same method as the sample, derivatized and re-dissolved with methanol to prepare calibration curves. And the quantitative limit is Table 1 and the results of the recovery in the sample was shown in Table 2 (see FIGS. 1 to 5).

Specifically, Table 1 relates to the linearity, detection limit and quantitative limit of the standard, Table 2 relates to the results of the recovery by concentration in the citrus samples, and Table 3 relates to the molecular weight and MS analysis conditions for each component. .

In this method, the pre-treatment process is facilitated by quickly separating the tube from the filter, the distribution in the separatory funnel, methylation, extraction, concentration, and analysis. The extraction solution has a long extraction time, which reduces the recovery rate. LC / MS / MS also enabled detection of lower concentration limits.

The detection limit of HPLC in citrus fruits was 0.02 mg / L, the quantification limit was 0.05 mg / L, and the average recovery rate was in the range of 0.5 ~ 5.0 mg / L, 77.4 ~ 102.4%, RSD 8.75%, and in LC / MS / MS The detection limit was 0.002 mg / L, the quantification limit was 0.005 mg / L, the average recovery was 86-101.2%, and the RSD was less than 7.25%. The results of the instrument safety test were repeated six times in a row. The coefficient of variation was 1.23% for DDC, 1.48% for EBDC, and 1.71% for PBDC.

Although the present invention has been described in connection with the specific embodiments of the present invention, it is to be understood that the present invention is not limited thereto. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. In addition, the materials of each component described herein can be readily selected and substituted for various materials known to those skilled in the art. Those skilled in the art will also appreciate that some of the components described herein can be omitted without degrading performance or adding components to improve performance. In addition, those skilled in the art may change the order of the method steps described herein depending on the process environment or equipment. Therefore, the scope of the present invention should be determined by the appended claims and equivalents thereof, not by the embodiments described.

Claims (9)

In the dithiocarbamate-based pesticide analysis method of citrus fruits,
Simultaneous extraction and methylation of the citrus sample from one tube improves the rapidity of pre-treatment in steps (1) to (6) below, thereby improving DDC (thiram) and EBDC ( mancozeb), PBDC (propineb) to analyze any one or more selected from the pesticide analysis method.
(1) a first step of adding L-cysteine to the cleaved sample and then shaking with EDTA solution;
(2) a second step of additionally administering tetrabutylammonium bisulfate solution and NaCl after shaking and then mixing and adjusting the pH to 7.0;
(3) after adjusting the pH, a third step of further administering a dichloromethane: n-hexane (volume ratio = 1: 1) mixture containing methyl iodide, and mixing and methylating;
(4) after the methylation, extracting the solvent layer by centrifugation and then adding 20% 1,2-propanediol dissolved in dichloromethane;
(5) a fifth step of adding the 1,2-propanediol, concentrating until only 1,2-propanediol remains, re-dissolving with Methanol and filtering;
(6) a sixth step of analyzing the filtrate obtained in the fifth step by HPLC / UVD or UPLC / MS / MS.
delete The method of claim 1, wherein the first step comprises adding 0.1 g of L-cysteine to the cleaved sample, adding 20 mL of 0.25M EDTA (0.45M NaOH) solution and shaking for 10 minutes.
The method of claim 1, wherein the second step is agitation analysis method, characterized in that after the addition of 1.5ml 0.41M tetrabutylammonium bisulfate solution and 3g of NaCl and then mixed and mixed to adjust the pH to 7.0 using 2M HCl.
The method of claim 1, wherein the third step is a pesticide analysis method characterized in that additional 15ml dichloromethane: n-hexane (volume ratio = 1: 1) mixed solution containing 0.05M methyl iodide, mixed and methylated.
The method of claim 1, wherein the centrifugation of the fourth step is a pesticide analysis method characterized in that the centrifugation for 3 minutes at 3000rpm.
According to claim 1, wherein the fourth step of the addition of 1,2-propanediol pesticide, characterized in that the extraction of 6 ml of the upper solvent layer obtained by centrifugation and adding 0.2 ml of 20% 1,2-propanediol dissolved in dichloromethane Analytical Method.
The method of claim 1, wherein the concentration of the fifth step is concentrated until only 1,2-propanediol remains at 30 ℃, the filtration of the fifth step is redissolved with 2 ml of Methanol and filtered with a 0.2 ㎛ syringe filter Pesticide analysis method. delete

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