KR102061600B1 - Method of analyzing pesticide residues of imminoctadine in agricultural products - Google Patents

Abstract

The present invention comprises the steps of (a) weighing the agricultural product sample, shaking, (b) adding a solvent to the shaken agricultural product sample, shaking and extracting the solvent, followed by centrifugation, (c) the supernatant centrifuged After taking a certain amount in the calibration of the pH, and filtering, and (d) an analysis method of the residual imine octadine pesticides in the agricultural product comprising the step of analyzing the instrument with the filtered filtrate as an analysis target, According to the present invention, it is possible to analyze an imine octadine pesticide ingredient remaining in agricultural products quickly and efficiently, and to lower the quantitative limit in the sample to meet the importing country standard and the PLS standard.

Description

Method of analyzing pesticide residues of imminoctadine in agricultural products}

The present invention relates to a method for analyzing an imine octadine residual pesticide in agricultural products, and more specifically, (a) weighing the agricultural product sample, followed by shaking, (b) adding a solvent to the shaken agricultural product sample and shaking the resultant product. After solvent extraction, centrifuging, (c) taking a certain amount of the centrifuged supernatant, correcting pH, filtering, and (d) analyzing the instrument using the filtered filtrate as an analysis target. It relates to a method for analyzing the residual pesticide imine octadine in agricultural products containing.

Iminoctadine pesticides are a wide range of protective fungicides, which are very effective in the control of anthrax and fungal diseases of vegetables and fruits, and are particularly preferred among farmers as drugs that increase shelf life in citrus fruits.

Currently, the imine octadine drug is sold as iminoctadine tris (albesilate), iminoctadine triacetate and a combination containing these components. Pesticides are one of the most widely used fungicides due to their good efficacy against pathogens that are resistant to other drugs.

In addition, recently, no suitable spraying pesticides have been registered for mangoes produced in Korea, so the process of preparing pesticide safety use standards, safety investigations on export agricultural products (citrus fruits, apples, pears, tomatoes) and strengthening pesticide acceptance criteria (unregistered drugs) Application of batch standard; 0.01ppm) requires rapid analysis and precise analysis of imine octadine pesticides.

Conventional techniques for the analysis of imine octadine include food processing assays (Non-Patent Document 1), residual imine octadine component analysis methods (Non-Patent Document 2), RDA analysis method (Non-Patent Document 3), and fungicide immigration in soil. Analysis of the half-life and residual concentration of octadine triacetate (Non-patent document 4), etc. are known.

However, the non-Patent Document 1, as shown in Figure 1, as a pretreatment process after two decompression filtration, liquid-liquid distribution and concentration process, after the derivatization process, the analysis for a long time (10 hours or more) It takes time and has a limit of quantification of 0.05ppm, which is higher than 0.01ppm, which is applied to the unregistered pesticide batch standard of PLS, which has been fully implemented since 2019, and cannot be quantified by this method.

As shown in FIG. 2, the non-patent document 2 also performs a liquid-liquid distribution three times using chloroform, and takes a large amount of reagent and time in the process of concentration under reduced pressure, and the same analyzer (LC-MS / MS ), There is a problem that the limit of quantification is 0.03 ppm, which is still higher than the application of the PEST standard of the pesticide substance list management system (PLS).

On the other hand, the non-patent document 3 and the non-patent document 4 is an analysis method of the residual imine octadine pesticide in the soil, the analysis target is different from the present invention, it is possible to quickly and efficiently analyze the residual imine octadine pesticide in the agricultural products The development of the method is urgently required.

 Food Code, Korea Food and Drug Administration, “7.1.4.44 Iminoctadine”, March 20, 2018 (No. 2018-18)  Proceedings of the Korean Society of Pesticide Science 2014 Spring Conference, pp.148-148 (2014. 4)  (Research of Pesticide Analysis on Pesticide Information Service, Rural Development Administration: http://pis.rda.go.kr)  Analytical Science & Technology Vol. 13, No. 1, pp. 34-40 (2000)

The present invention was derived to solve the problems of the prior art as described above, an object of the present invention is to extract and filter the extraction of the imine octadine pesticides in the fruit and vegetable extracts used in the recent food processing method and separating funnel funnel separation After several times, the complex process of concentration under reduced pressure, derivatization for more than 10 hours, and purification was replaced with a simple process of extraction, centrifugation and brief pH correction in a 50 mL tube. The aim is to provide a method for the analysis of pesticide residues of imine octadine in agricultural products that may lower the quantitative limit in the sample to meet the importing country and PLS standards.

In order to achieve the above object, the present invention comprises the steps of: (a) weighing the agricultural product sample, followed by shaking; (b) adding a solvent to the shaken agricultural product sample, extracting the solvent by shaking, and then centrifuging; (c) taking a certain amount of the centrifuged supernatant, correcting the pH, and filtering; and (d) retaining imineoctadine in the agricultural product, including analyzing the filtered filtrate as an analysis target. Provide an analysis method for pesticides.

According to the analysis method of the residual imine octadine pesticides of the present invention, the complicated and difficult pretreatment of the conventional food processing method, according to the characteristics of each sample is simplified to significantly reduce the analysis time, increase the processing volume to reduce the cost of analysis can do.

In other words, by simplifying the two times of pressure filtration, liquid-liquid distribution and concentration process, eliminating the derivatization process which is difficult and takes more than 10 hours, it was simply replaced by pH correction for each sample characteristic. It is possible to increase the amount of analytical materials that can be processed by supplementing the recovery rate by using a plastic choza, which is 20 times from 2 to 40 cases.

In addition, it is possible to reduce the cost of purchase of glassware such as separating funnel (for dispensing liquid) and concentrated flask (pressure-reducing filtration), and to reduce the amount of reagent used to 1/30. In particular, the analysis time is 900 minutes. It can be significantly reduced to 30 minutes, reducing labor costs and shortening the analyst's exposure time from hazardous substances.

On the other hand, the analysis method of the imine octadine pesticide residues of the agricultural products of the present invention is an improved iminotadine pesticide rapid analysis method, the analysis result (PLS) suitable for the pesticide substances list management system (PLS) implemented from 2019 full Application: 0.01ppm), can contribute to the expansion of export by increasing the level of safety management for export agricultural products in particular, by using the analysis method of the present invention for environmentally friendly agricultural products follow-up management, by ensuring that safe agricultural products are supplied to consumers It can contribute to health promotion.

1 contrasts the conventional food retardation analysis method (Non Patent Literature 1) and the analysis method of the present invention.
2 contrasts the conventional nonpatent literature 2 and the analysis method of this invention.
Figure 3 shows the pretreatment process in the analysis method of the residual imine octadine pesticide in the agricultural products according to the present invention.
Figure 4 is a photograph showing a process of pH correction during the pretreatment process of the analysis method of the imine octadine residual pesticides in the agricultural products according to the present invention.
Figure 5 shows the imineoctadine tris (albesylate) matrix matched standard (MMSTD) calibration curve graph (2.5, 5, 10, 25, 50 μg / L) of mango.
Figure 6 shows the chromatogram of the recovery of mangoimine octadine tris (albesylate), A is 0.05mg / kg imineoctadine tris (albesylate), B is 0.25mg / kg imineoctadine tris (albesil Rate).
7 is a photograph showing a process of taking a sample of agricultural products (mango) in the analysis method of the residual imine octadine pesticide in the agricultural product according to the present invention, A is the foreground of the test packaging, B is the residual test drug treatment process, C is the residual The test sampling process, D, represents the sample taken.

The present invention comprises the steps of (a) weighing the agricultural product sample, shaking, (b) adding a solvent to the shaken agricultural product sample, shaking and extracting the solvent, followed by centrifugation, (c) the supernatant centrifuged After taking a certain amount of the medium to correct the pH, and filtration, and (d) an analysis method for the residual pesticide imine octadine pesticide in the agricultural product comprising the step of analyzing the filtered filtrate as an analysis target.

In the method for analyzing residual imine octadine pesticide in the agricultural product of the present invention, step (a) is carried out by weighing 5 g of the homogenized sample using dry ice in a 50 mL tube, and then adding 3 g of guanidine hydrochloride. , 1,100 ~ 1300, preferably may be a step of shaking for 1 minute at 1,200rpm, but is not limited thereto.

In the analysis method of the residual imine octadine pesticide in the agricultural product of the present invention, the step (b) is put 10mL of methanol in the shaken agricultural product sample and shaken for 1 minute at 1,100 ~ 1300, preferably 1,200rpm, 2M NaOH 10 mL of the aqueous solution is added, followed by shaking for 1 minute at 1,100 to 1300, preferably 1,200 rpm, followed by centrifugation at 4,000 rpm for 5 minutes, but is not limited thereto.

In the method for analyzing an imine octadine residual pesticide in the agricultural product of the present invention, the step (c) takes 0.5 mL of the supernatant, and then adds a mixture of 5% formic acid and methanol or 5% formic acid, The pH of the liquid may be adjusted to 8.5 ± 0.5, preferably 8.5 ± 0.2, more preferably 8.5, and then filtered with a syringe filter, preferably a 0.2 μm syringe filter, but is not limited thereto.

In the analysis method of the imine octadine residual pesticide in the agricultural product of the present invention, the mixture of 5% formic acid and methanol or the amount of 5% formic acid may be 0.5mL, but is not limited thereto.

In the analysis method of the residual imine octadine pesticide in the agricultural product of the present invention, the device analysis of step (d) may be performed by a liquid chromatography-tandem mass spectrometer (LC / MS / MS), but is not limited thereto. .

In the analysis method of the imine octadine residual pesticide in the agricultural products of the present invention, the agricultural products may be fruits, vegetables or mixtures thereof.

In the analysis method of the imine octadine residual pesticides of the agricultural products of the present invention, the agricultural products may be selected from the group consisting of mango, tangerines, apples, pears and tomatoes, but is not limited thereto.

In the analysis method of the residual imine octadine pesticide in the agricultural product of the present invention, the quantitative limit of the agricultural product sample may be 0.006 mg / kg or less, preferably 0.001 ~ 0.006 mg / kg.

In the analysis method of the imine octadine residual pesticide in the agricultural product of the present invention, the average recovery rate in the range of 0.05 ~ 0.25 mg / kg addition concentration of the agricultural product sample may be 73.1 ~ 104.8%.

In the analysis method of the imine octadine residual pesticides of the agricultural products of the present invention, the agricultural products may be selected from the group consisting of mango, tangerines, apples, pears and tomatoes.

Analysis method of the imine octadine residual pesticide in the agricultural products of the present invention is extracted and filtered the remaining imine octadine pesticide in the fruit and vegetables, and then transferred to the separatory funnel several times to separate the extraction solvent layer and concentrated under reduced pressure and the derivative more than 10 hours The complex process in the food processing method (Non-Patent Document 1), which is purified after purification, can be analyzed quickly and easily through a simple process of centrifugation and brief pH correction after extraction in a 50 mL tube (pH Pre- By improving the treatment method, the analysis time can be shortened and the reagents can be saved, and the quantitative limit in the sample can be lowered to meet the importing country standard and the PLS standard. Can be.

Table 1 below is in contrast to the analysis method according to the present invention and the food method.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the scope of the present invention is not limited thereto.

<Example>

Structural formula and physicochemical properties of the standard used in the following examples are as follows.

In addition, the reagents and experimental materials used in the following examples are as follows.

1) Immunoctadine Tris (Albesylate) Analysis Standard: Ehrenstorfer GmbH, C14284950 (Germany), 99.0%

2) acetonitrile: Merck, HPLC grade

Methanol: Tedia, HPLC grade

4) Water: Merck, HPLC grade

5) Formic acid: Sigma-Aldrich, 95%

6) Sodium Hydroxide: Merck, GR grade

7) Guanidine Hydrochloride: Acros, 98%

8) Ammonium formate: Fluka, HPLC grade 99%

9) Syringe Filter: Minisart RC 15, 0.2㎛, satorius stedim (Germany)

10) Sample shaker: Spex, MiniG 1600 (USA)

11) Blender: Blixer 5 plus, Robot coupe (France)

12) Centrifuge: Hanil, Combi 408 (Korea)

13) Cryogenic Freezer: Wisecryo WUF-300 (Korea)

14) UPLC-MS / MS: ABSCIEX 4500 & Waters Acquity Ι class (USA)

Example 1 Pretreatment and Analyzer Conditions

1. Pretreatment Method

As shown in FIG. 3, 5 g of the homogenized sample using dry ice was weighed into a 50 mL tube, and then 3 g of guanidine hydrochloride was added, followed by shaking at 1,200 rpm for 1 minute. In this case, mango, citrus fruits, apples, pears, and tomatoes were used as the sample.

10 mL of methanol was added to the shaken sample, followed by shaking for 1 minute at 1,200 rpm. Then, 10 mL of 2M NaOH aqueous solution was added and shaken at 1,200 rpm for 1 minute, followed by centrifugation at 4,000 rpm for 5 minutes.

Then, after taking 0.5 mL of the supernatant, adding a mixture of 5% formic acid and methanol or 5% formic acid under the conditions shown in Table 2 for each sample, correcting the pH of the supernatant taken above to 8.5, Filtration with a 0.2 μm syringe filter.

At this time, all the processes were pretreated using plastic porcelain in consideration of adsorption to glass.

2. Analyzer condition

The pretreated sample was analyzed using an analyzer (LC / MS / MS), and the conditions of the analyzer are shown in Table 3 below.

Example 2 Calibration curve and detection limit (LOD) conditions

The imineoctadine standard product is Dr. Ehrmintorfer company's imineoctadine tris (albesylate) raw material was purchased and 10 mg was dissolved in 9.9 mL methanol to prepare a 1,000 mg / L stock solution.

The 1000 mg / L stock solution of imineoctadine tris (albesylate) was diluted with methanol and used as a 10 mg / L recovery test stock solution, which was prepared as a working solution by concentration.

The composition of the used solution used for the calibration curve preparation was mixed with the same ratio as the composition of the treatment sample by methanol containing 5% formic acid and the extract solution supernatant of the mango-free sample using a matrix matched calibration method (20mL ), And used solutions (concentrations: 25, 50, 100, 250, 500 µg / L) were prepared as follows.

For preparing the calibration curve, the matrix matched standard (MMSTD) concentration range was diluted and prepared with a mixed solution as shown in Table 4, and used for the analyzer.

Then, the imineoctadine tris (albesylate) standard solution was analyzed by injecting 10 μl under UPLC-MS / MS analysis conditions, and then a calibration curve was prepared and quantitatively analyzed based on the peak area on the chromatogram.

At this time, the detection limit (LOD) and quantitative limit (LOQ) of the assay were calculated according to the KOLAS Guidelines for Validation of Chemical Test Methods (KOLAS-G-015: 2012, Korea Accreditation Body). For example, LOD was calculated to be three times the 2.5ng / L × 10 repeated standard deviations = 0.00067 mg / kg.

FIG. 5 shows the imineoctadine tris (albesylate) limit level (MMSTD) calibration curve graph (2.5, 5, 10, 25, 50 μg / L) of mango.

Example 3 Analysis of Matrix Effect for Each Sample

Although the effect of the medium is not the component to be analyzed, the influence of the medium was investigated for accurate quantitative analysis because the effect of the component present in the sample affects the reproducibility and accuracy.

Table 5 shows the results of comparing the sensitivity of the standard material dissolved in pure solvent and the sample medium dissolved together.

As shown in Table 5, the mango, apples, pears slightly out of the ± 20% range classified as weak, citrus and tomatoes were found to have a difference in the medium medium ± ± 50% or more.

Example 4 Recovery Rate Test by Samples

To 5 g of the homogenized control sample homogenized with dry ice, 0.025 mL and 0.125 mL of 10 mg / L imine octadine standard solution were added to each solution accurately and then mixed uniformly, and left for 2 hours, followed by the pretreatment method. The results obtained by performing a sample pretreatment process such as extraction and filtration were shown in Table 6 below, and the chromatogram of the recovery rate of mangoimine octadine tris (albesylate) was shown in FIG. 6.

At this time, as the sample was used mango, tangerines, apples, pears and tomatoes.

Example 5 Residual Experimental Results Applying Pesticide Safety Standards in Mangoes

From July 24, 2017, by time (0, 7, 14, 21, 30), to set the safety standards for pesticide use in mangoes treated with imine octadine tris (albesylate) pesticides three times every seven days. Mango 12 families (3.8 ~ 4.8kg) were collected per repetition group for the remaining amount experiment.

The collected mango sample was removed from the seeds, homogenized by taking a certain amount (1 kg) including the shell, and stored in a sub-50 ° C. cryogenic freezer until the sample analysis, followed by the pretreatment process of Example 1 to analyze the residual pesticide. It was.

7 is a photograph showing a sampling process of the pesticide safety use standards applied to mango in the mango, A is the foreground of the test packaging, B is the residual test drug treatment process, C is the residual test sampling process, D is a sample taken will be.

Table 7 shows the results of the residual amount analysis of the imine octadine tris (albesylate), Table 8 shows the results of the residual amount converted to the imine octadine molecular weight.

As shown in the above examples, the analytical method of the present invention facilitates the pretreatment process by simply correcting an appropriate pH for each sample characteristic by omitting the derivatization process that takes 10 hours or more during pretreatment in the conventional analytical method. The entire process was used as a plastic choji to compensate for the drop in recovery rate.

In addition, it is possible to analyze up to the quantitative limit of trace concentration in LC-MS / MS by supplementing the condition of poor reproducibility in the polar column, and the quantitative limit among all samples used in the recovery test is 0.006 ppm or less, which is applied from 2019. PLS batch standard 0.01ppm can be fully analyzed, the average recovery in the range of 0.05 ~ 0.25mg / kg is 73.1 ~ 104.8%, the standard deviation is 5.0% or less can be seen.

In addition, the instrument safety results were repeated 10 times at 0.005ppm, the variation coefficient value was 4.1% showed a low variation rate.

Although the present invention has been described in detail with reference to specific embodiments, the present invention is not limited to the above-described embodiments, and many modifications are made by those skilled in the art to which the present invention pertains within the scope of the technical idea of the present invention. This possibility will be self-evident.

Claims (11)

(a) weighing 5 g of the agricultural product homogenized using dry ice into a 50 mL tube, then adding 3 g of guanidine hydrochloride and shaking for 1 minute at 1,100 to 1300 rpm;
(b) adding 10 mL of methanol to the shaken agricultural product sample, shaking for 1 minute at 1,100 to 1300 rpm, adding 10 mL of 2M NaOH aqueous solution , shaking for 1 minute at 1,100 to 1300 rpm, and centrifuging at 4,000 rpm for 5 minutes;
(c) After taking 0.5 mL of the centrifuged supernatant, adding a mixture of 5% formic acid and methanol or 5% formic acid to calibrate the pH of the supernatant taken above to 8.5 ± 0.5, and then filtration with a syringe filter. Making; And
(d) subjecting the filtered filtrate to an analyte, analyzing by means of a liquid chromatography-tandem mass spectrometer (LC / MS / MS),
Step (a) to (c) is characterized in that using the plastic material of the choja, an analysis method of the residual pesticide imine octadine in agricultural products. delete delete delete The method of claim 1, wherein the amount of the mixture of 5% formic acid and methanol is 0.3-0.5 mL. delete The method of claim 1, wherein the agricultural product is selected from fruits and vegetables. The method of claim 1, wherein the agricultural product is selected from the group consisting of mangoes, tangerines, apples, pears, and tomatoes. The method of claim 1, wherein the quantitative limit of the agricultural product sample is 0.006 mg / kg or less. The method of claim 1, wherein the average recovery rate in the range of 0.05 ~ 0.25 mg / kg addition of the agricultural product sample is 73.1 ~ 104.8%. 11. The method of claim 10, wherein the agricultural product is selected from the group consisting of mango, citrus fruits, apples, pears and tomatoes.

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