KR100430932B1 - A method for analyzing mancozeb using hplc - Google Patents

Abstract

PURPOSE: A method for analyzing Mancozeb using HPLC is provided, thereby rapidly and accurately analyzing Mancozeb which is agricultural chemical remained in fruits or leaf vegetables. CONSTITUTION: The method for analyzing Mancozeb using HPLC comprises the steps of: extracting fruits or leaf vegetables containing Mancozeb with 100 ml of 0.03M EDTA disodium salt solution having pH 9.5 to 10.5 for 5 minutes; absorbing the extracted solution with GF/C filter paper, and washing the remained solution with distilled water; regulating the extracted solution to pH 7.2 to 8.0, adding 7 ml of 0.41M tetrabutylammoniumhydrogensulfate and 60 ml of 0.05M chloroform of iodomethane/hexane(5:1) into the extracted solution, stirring them and stationing it for 30 minutes to methylate Mancozeb in the extracted solution; separating the extracted solution into a water-soluble layer and organic solvent layer, adding 0.05M chloroform of iodomethane/hexane(5:1) into the water-soluble layer, separating the organic solvent layer, and mixing the organic solvent layer with the pre-separated organic solvent layer; and adding 4.0 ml of 20 wt.% 1,2-propanediol into the organic solvent layer, concentrating the organic solvent layer, treating methylated Mancozeb containing concentrated solution with acetonitrile and subjecting the acetonitrile treated concentrated solution to HPLC.

Description

A method for analyzing mancozeb using HPC {A METHOD FOR ANALYZING MANCOZEB USING HPLC}

The present invention relates to a method for analyzing the fungicide mancozeb remaining in foods such as fruit trees, leafy vegetables and the like.

Dithiocarbamic acid compounds were first reported to be bactericidal by Tisdal and Williams in 1934. Later on, by Goldworthy in 1943, its analogous compound,N, N 'Ethylenebis (dithiocarbamate) (EBDC) family compounds have also been reported to be bactericidal. Dithiocarbamate-based pesticides are used in nematicides such as metham-Na (metham-Na, sodiumNMethyldithio carbamate) and very few are used as herbicides, except for herbicides (Hayes, W. Jr. and Laws, ER Jr. (1991) Class of pesticide: Handbook of pesticide toxicology ( vol. 3) Academic Press, Inc. California., p. 1436). Dithiocarbamate-based pesticides have been used to control pests for more than 70 crops in countries around the world since the bactericidal power was discovered.

Dithiocarbamate-based fungicides include dialkyl dithiocarbamate type, ethylenebis (dithiocarbamate) type (EBDC type) and propylenebis (dithiocarbamate) type (PBDC type). Among them, the EBDC type is a complex compound of ethylene bis (dithiocarbamate) and a metal, and the disinfectants belonging to it are maneb (maneb: waste paper), zinc, zinc (zineb: waste paper), and manganese. Mancozeb etc. which contain both zinc and zinc are mentioned. In other words, mancozeb is a complex of zinc with a manganese ethylene bis (dithiocarbamate) polymer. In Korea, the output of mancozeb was 1,958 tons of the total pesticide 26,087 tons (M / T) in 2000, accounting for 7.5% of the total pesticides (Pesticide Industry Association (2001), Pesticide Annual Report, 50-51). Mancozeb is a dithiocarbamate-based protective fungicide, which is a non-invasive pesticide, and has been widely used for controlling diseases such as fruit trees and leafy vegetables.

However, although dithiocarbamate-based pesticides have excellent bactericidal properties, in the late 1960s, ethylene thiourea (ETU), a metabolite from the metabolism of EBDC pesticides, was found to be carcinogenic and mutagenic. (Kenneth A. Hassll. (1990) The chemistry of pesticide Their metabolism mode of action and crop protection (2nd edition) Macmillan Press Ltd, Hong kong, 288-290; Geroge Yip. (1971) Residues of maned and ethylenethiourea on field-sprayed Lettuce and Kale.Journal of the AOAC. 54. 1373-1375; and Innes, JRM (1969) J. Nat Cancer Inst. 42. 1101-1114), accordingly, fruit, leafy vegetables It is necessary to accurately qualitatively and quantify the pesticides remaining in the foods. Since the analysis method of mancozeb is not known until now, mancozeb is not listed in the pesticide acceptance standards published in the Korean Food Code, it is only announced as dithiocarbamate.

The dithiocarbamate-based pesticide assays currently in use are based on the Keppel method in 1969, based on the Cullen method, which Cullen published in the early 1960s, captures CS ₂ gas and measures the absorbance using a coloring reagent. (George. E. Keppel. (1971) Modification of the carbon disulfide evolution method for dithiocarbamate residues.Journal of the AOAC. 52. 1, 162-167; and Keppel, GE (1971) Cooaborative study) of determination of dithiocarbamate residues by a modified carbon disulfide evolution method.Journal of the AOAC. 54. 162-167). Meanwhile, as a method of applying the same, a method of quantifying CS ₂ gas using gas-liquid chromatography (GLC) (Ministry of Agriculture, Fisheries and Food. (1980) Determination of residues of dithiocarbamate pesticides in foodstuffs by headspace method. Analyst. 106. 782-787) is also used.

The CS ₂ method is a method in which a compound containing dithiocarbamic acid is decomposed into an alkylenediamine and a CS ₂ gas when heated with a strong acid, and has an advantage of easily analyzing the dithiocarbamic acid. It is impossible to accurately analyze the quality of each component and its content (co-research method of pesticide residues (1991) et al., Dong-A Lee et al., Dong-A Technology, 108-114), as well as a time-consuming disadvantage.

Gustav Davidson (Gustafsson) and Thomson (Thomson) has announced the assay by HPLC dithiocarbamate mate Pesticides (Gustafsson, KH, Thompson, RA (1981) High-pressure liquid chromatographic determination of fungicidal dithiocarbamates. J. Agric.Food Chem. 29. 729-732) Although this paper reports the use of 0.25M EDTA and 0.45M sodium hydroxide as extraction solutions, no recovery test results were reported for mancozeb.

On the other hand, as a result of testing by the present inventors, not only EDTA was not completely dissolved in the aqueous solution at the concentration described in the above paper, but the solution was acidic, and there was a possibility that the mancozeb was decomposed, so that the accurate quantification of mancozeb was impossible. As a result of comparing and testing this method and the method established by the inventors, the method of Gustafson et al. Showed that the recovery rate of mancozeb was only about 52% compared to the method according to the present invention. It was found that it could not be applied to the accurate analysis of mancozeb.

Fruits, leafy vegetables and the like are foods, so for safety, not only the type and content of residual pesticides must be accurately identified before they are delivered to consumers, but it is also very important to maintain freshness. Accordingly, Seoul Metropolitan Agricultural & Fishery Corporation requires close inspection for the samples that are positive for the analysis of residual pesticide (EBDC) properties in the distribution stage for safety, and the time required for the close inspection according to the CS ₂ method currently used. Is about 48 hours, which is very long, and as a result, the product having a positive reaction is deteriorated in commercial value and is discarded entirely.

Therefore, there is an urgent need to provide a method for accurately detecting residual pesticides in a short time in order to secure food safety and shorten the distribution time delivered from the producer to the consumer.

Accordingly, an object of the present invention is to improve the problems of the conventional HPLC method, which cannot be applied due to problems in the limitation of the CS ₂ method, which is impossible for qualitative and quantitative determination of components, and takes much time for analysis, and solubility and recovery rate. By providing an optimized method for derivatizing mancozeb remaining in fruits, leafy vegetables and the like, and analyzing the derivatized mancozeb using HPLC.

1 is a flowchart of a method for analyzing mancozeb.

Figure 2 shows the results of testing the optimal conditions of the EDTA-2Na concentration.

Figure 3 shows the results of the test to find the optimal extraction solvent.

Figure 4 shows the results of the test to optimize the amount of TBAH addition and derivatives and time.

Figure 5 shows the results of the test to optimize the addition amount of 20% by weight 1,2-propanediol.

Figure 6 shows the results of testing the degree of degradation by derivatization of 10ppm mancozeb.

Figure 7 shows the calibration curve of mancozeb.

8 is an HPLC chromatogram of mancozeb.

Mancozeb is a macromolecule containing metal ions, poorly soluble in water, and insoluble in organic solvents. Therefore, for accurate analysis of mancozeb it is necessary to dissolve it completely in EDTA disodium salt and then derivatize it to be suitable for analysis using HPLC. To this end, the present invention provides the following method.

An aqueous solution of 0.03 M EDTA-disodium salt is added to the sample containing mancozeb to convert the mancozeb to an EBDC-Na salt that can be dissolved in water. Mancozeb degrades under acidic conditions, so the pH of the solution must be maintained in the range of 9.5-10.5. It is preferable to use 2N NaOH aqueous solution for pH adjustment, but is not necessarily limited thereto, and it is also possible to use all other bases commonly used in the art. The pH adjusted sample is extracted for 5 minutes using a shaker and then suction filtered using GF / C filter paper.

Then, the EBDC-Na salt solution in chloroform / hexane (5: 1) of CH ₃ I was added to the solution, the aqueous layer is EBDC-Na salt, an organic solvent layer, so as to create a CH ₃ I exist. This is to derivatize the EBDC so that it can be analyzed by HPLC, and the iodomethane is a methylating agent. Then, an appropriate acid is added to adjust the pH of the aqueous solution in which the EBDC-Na salt is dissolved to neutral (pH 7.2-8.0). Adjusting the pH to neutral is to prevent the OH ⁻ which may be present in the basic solution from reacting with iodomethane to form CH ₃ OH. At this time, since iodomethane is not dissolved in an aqueous solution, tetrabutylammonium hydrogensulfate (hereinafter referred to as "TBAH") is added as a phase transfer catalyst so that the sodium ion of the EBDC-Na salt of the aqueous layer is Bu ₄ N. as substituted with ⁺ such that the formation of the EBDC-Bu ₄ N ⁺ then the salt is dimethyl ethylene bis (dithiocarbamate) is produced by reaction with CH ₃ I go to the organic solvent layer.

Since the produced dimethyl ethylene bis (dithiocarbamate) is dissolved in the organic solvent layer, the solvent must be removed. Since the product is highly volatile and can be evaporated during the solvent removal process, the concentration of 20% by weight in the organic solvent layer A 1,2-propanediol chloroform solution is added to allow the product to produce hydrogen bonds with alcohol to prevent evaporation of the product.

In this way, the methylated mancozeb derivatives are analyzed using HPLC. The analysis method of the mancozeb of the present invention described above may be represented by a flowchart as shown in FIG. 1.

Mancozeb analysis method of the present invention as described above was optimized according to the procedure as described below, samples, reagents and analytical instrument for the practice of the present invention are as follows.

1. Sample

For mancozeb analysis, grapes and pears, which are domestic agricultural products, were selected, 100 ml of 0.03M EDTA disodium salt solution was added to 50 g of the sample, extracted with a stirrer for 5 minutes, and derivatized mancozeb in the extract. The concentration was analyzed by a vacuum concentrator.

As a standard mancozeb solution, 13.5 mg of mancozeb (purity 74%, Dr. Ehrenstorfer. Co) was dissolved in 100 mL of 0.03 M EDTA disodium salt aqueous solution of pH 9.50-10.50.

2. Reagents and Instruments

EDTA disodium salt, CH ₃ I, Na ₂ SO ₄ was purchased from Junsei Chemical, TBAH is from Sigma, 1,2-propanediol from Aldrich The other solvent was used for HPLC or the express.

The instrument for HPLC was used HP-1100 series, the column was purchased from Waters (ODS (2) 4.6 × 50mm, particle size 5㎛), the mobile phase was H ₂ O / CH ₃ CN (30/70), flow rate was 1.0 ml / min, sample injection amount was 10 μl, methylated derivative was detected at UV 272 nm, and analysis time was 15 minutes.

3. Establishment of analytical methods

(1) EDTA-disodium salt aqueous solution concentration

10 μg / ml of mancozeb was dissolved in 100 ml of EDTA-disodium salt aqueous solution at 0.01 M, 0.02 M, 0.03 M, 0.04 M, 0.05 M, 0.1 M and 0.25 M, respectively, to form an aqueous solution of EBDC-Na salt by concentration. . In order to prevent decomposition of the mancozeb, the pH was adjusted to a range of 9.5-10.5 using a 2N NaOH aqueous solution.

Then, after methylation of the mancozeb, the optimum conditions of the aqueous solution of EDTA-disodium salt were determined by the peak (mAU) height of the methylated mancozeb using HPLC.

As shown in FIG. 2, when the concentration of the EDTA disodium salt aqueous solution is 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.1M and 0.25M, respectively, the height (mAU) of the peak of methylated EBDC is 18, 42, 60, 56, 57, 55 and 50 mAU, respectively, thus confirming that the optimal conditions for the analysis of mancozeb are when the chelating reagent is an aqueous 0.03 M EDTA disodium salt solution.

(2) extraction solvent

11 g / ml of mancozeb was dissolved in 100 ml of 0.03 M EDTA disodium salt aqueous solution, and chloroform / hexane (5/1), chloroform, hexane, dichloromethane, A chloroform / hexane (3/1) solution was added to identify the solvent from which the derivatized mancozeb was best extracted.

As can be seen in Figure 3, the HPLC peak height of the derivatized mancozeb according to the extraction solvent is 0mAU when the extraction solvent is hexane, 61.09mAU when dichloromethane, 62.92mAU when chloroform, chloroform / hexane (3/1 ) Was 62.29 mAU, and chloroform / hexane (5/1) was 66.11 mAU. Accordingly, it was confirmed that the optimum extraction solvent was chloroform / hexane (5/1) of 0.05M CH ₃ I.

(3) TBAH addition amount and derivatization time

In order to optimize the amount of TBAH, a phase transfer catalyst, and the time required for derivatization, the pH of the aqueous solution of EBDC-Na salt was adjusted to neutral with 0.5 N hydrochloric acid (pH 7.2-8.0), and 5 ml and 7 ml of 0.41 M TBAH were added. After the addition, it was derivatized for 30, 40, 50 and 60 minutes for each TBAH addition amount. At this time, the amount of mancozeb was 10 ㎍ / ㎖, 0.05M CH ₃ I chloroform / hexane (5/1) solution was added twice in 60ml each derivatized twice.

As can be seen in Figure 4, the concentration peak (mAU) of the derivatized product is 56.88 for TBAH addition-derivatization time of 5 ml-30 minutes, 54.89 for 5 ml-40 minutes, 59.08 for 5 ml-50 minutes , 60.02 for 5ml-60min, 61.50 for 7ml-30min, 52.00 for 7ml-40min, 58.73 for 7ml-50min and 57.88 for 7ml-60min. Thus, the optimum amount of addition-derivatization time of TBAH was found to be 7 ml-30 minutes.

(4) 20 weight% 1,2-propanediol solution addition amount

In order to prevent volatilization of the product upon concentration of methylated EBDC-CH ₃ , a 20-wt% 1,2-propanediol chloroform solution is used, as shown in FIG. 5. When the addition amounts of propanediol were 1 ml, 2 ml, 3 ml, 4 ml and 5 ml, the product peaks (mAU) were 54.75, 58.22, 58.71, 60.23 and 58.27, respectively. Therefore, when the addition amount of 20% by weight of 1,2-propanediol was 4ml it was confirmed that the recovery of the product is the best.

(5) established analytical methods

Mancozeb analysis method of the present invention established according to the above procedure is as follows.

To 50 g of the sample containing mancozeb, 100 ml of 0.03 M EDTA disodium salt aqueous solution was added, followed by stirring for 5 minutes to extract. At this time, the pH of the extraction solution should be adjusted in the range of 9.5-10.5. As the base for adjusting the pH, any base commonly used in the art to which the present invention pertains may be used. In the present invention, 2M aqueous sodium hydroxide solution was used, but the concentration does not have a special meaning. The extract is suction filtered with GF / C filter paper and the residue is washed and filtered with 50 ml of distilled water.

Transfer the sample solution to the separatory funnel and adjust the pH to neutral (pH 7.2-8.0) by adding acid. In this case, any acid commonly used in the technical field to which the present invention pertains may be used, and the concentration thereof is also irrelevant.

60 ml of 0.41 M TBAH and 60 ml of 0.05M CH ₃ I chloroform / hexane (5/1, volume / volume) solution are added to the sample solution, stirred at room temperature for 5 minutes, and then left to stand for 30 minutes to methylate. . The organic solvent layer is then separated and dried over anhydrous sodium sulfate. 60 ml of a 0.05M CH ₃ I chloroform / hexane (5/1, volume / volume) solution was added to the aqueous layer for further methylation, and the organic solvent layer was separated and combined with the previous organic solvent layer, and then 20% by weight. 4 ml of 1,2-propanediol chloroform solution is added and concentrated, and the derivatized mancozeb is detected by HPLC using 10 ml of acetonitrile.

(6) stability

In order to confirm the degree of degradation over time of the derivatized EBDC, it was tested at time intervals while refrigerated (-5 ° C). After derivatization of 10 ppm of mancozeb, the degradation was tested for at least 6 days, but after 7 days, 27% decomposition began to occur and 46% was degraded on 8 days (see FIG. 6).

4. Application test of analytical methods

Hereinafter, an example and results of applying the analysis method of mancozeb established according to the present invention on grapes, pears and lettuce will be described. However, the mancozeb assay according to the present invention can be applied to fruits and leafy vegetables in which mancozeb is commonly used, in addition to the fruits and leafy vegetables exemplified herein.

(1) Application test examples for grapes and pears

In order to confirm the usefulness of the analytical method established above, the minimum detection amount and the detection limit were tested using standard materials, and the applicability of the present invention was examined by testing the recovery rate of mancozeb by selecting domestic grapes and pears. .

13.5 mg of the standard substance Mancozeb (74% purity) was taken and dissolved in 100 ml of an aqueous 0.03 M EDTA disodium salt solution at pH 9.5-10.5 to prepare a 100 ppm standard solution. Then, 1 ml of 100 ppm of standard substance was added to 100 ml of 0.03 M EDTA disodium salt aqueous solution (pH 9.5-10.5) to derivatize mancozeb according to the optimized conditions described above, and the organic solvent layer was collected and 35 The solvent is removed by depressurizingly in a water bath of < RTI ID = 0.0 > The residue is taken up in 9.2 ml of acetonitrile and used as a test solution (10 ppm). The minimum detectable amount of mancozeb was 2.5 ng, the detection limit was 0.05 ppm (2.5 ng x ㎖ / 10 ul x 1/50 g = 0.05 ng / μl), and 10 ppm of standard was 10, 5, 2.5, 1.0, 0.5 and Diluted to 0.25ppm,Linearity r as a result of drawing calibration curve²It showed = 0.9999 to obtain a good result (Fig. 8).

In the application method of the analytical method according to the present invention, 3ppm of Mancozeb standard solution was added to grapes and pears in consideration of the residual acceptance criteria (grape: 5ppm, pear: 3ppm) in the food industry, and the recovery rate was tested three times. The results are shown in Table 1 below.

TABLE 1

^* Average over 3 replicates

As can be seen in Table 1, the recovery of mancozeb was 70.61% ± 5.99 (n = 3) in grapes, 80.99% ± 1.85 (n = 3) in pears, and the percent variation between repetitions was 8.48 in grapes. , 2.28 in the embryo, it was confirmed that quantitative analysis is possible.

(2) Example of application test for lettuce

In order to test whether mancozeb in samples actually distributed in the market can be analyzed by applying the present invention, the sample and mancozeb which showed positive reaction in the property test on the sample (lettuce) at the distribution stage from the Seoul Agricultural Products Corporation Obtained the sample was sprayed was applied to the analysis method according to the present invention, the results are as follows.

According to the present invention, a method capable of accurately qualitatively and quantifying mancozeb remaining in a fruit or a leaf vegetable in a short time is provided. Therefore, the mancozeb assay according to the present invention is expected to be applicable to the analysis of the mancozeb remaining in the marketed fruit and leafy vegetables.

Claims (1)

Extracting a fruit or leaf vegetable sample containing mancozeb for 5 minutes by adding 100 ml of 0.03 M EDTA disodium salt aqueous solution in the pH 9.5-10.5 range, Suction filtering the extract with GF / C filter paper, washing the residue with distilled water, After adjusting the pH of the extract to a range of 7.2-8.0, 7 ml of 0.41M tetrabutylammoniumhydrogensulfate and 60 ml of chloroform / hexane (5/1 (volume ratio)) solution of 0.05M iodomethane were added and stirred. Left to stand for the next 30 minutes to methylate the mancozeb in the sample, The aqueous layer and the organic solvent layer were separated, and 0.05M CH ₃ I chloroform / hexane (5/1, volume / volume) solution was added to the aqueous layer to further methylation, and then the organic solvent layer was separated to separate the organic solvent. Combining with layers, and 4.0 ml of 20 wt% 1,2-propanediol was added to the organic solvent layer, concentrated, and then derivatized mancozeb was detected by HPLC using acetonitrile. Method of analyzing mancozeb remaining in fruits and leafy vegetables.