Reagent and method for detecting paraquat in herbicide preparation

Abstract

The invention provides a reagent and a method for detecting paraquat in a herbicide preparation, the reagent comprises a reagent A, a reagent B and a reagent C, the reagent A is a solution prepared from dipotassium phosphate and an alkaline buffer solution, the reagent B is methanol, and the reagent C is a solution of acetylphenylhydrazine. The method comprises the following steps: mixing a to-be-detected herbicide preparation with a reagent A to form a uniform solution A, then adding a reagent B into the uniform solution A, uniformly shaking, waiting for phase separation, taking a supernatant B, adding the supernatant B into a reagent C, immediately carrying out visual colorimetry or colorimetry by using a spectrophotometer, and determining whether the to-be-detected herbicide preparation contains paraquat or not according to color change. Acetylphenylhydrazine is used as a reducing agent and combined with a solvent to regulate and control the oxidation-reduction potential of the reducing agent, a detection system for specifically reducing paraquat is developed, the detection system is applied to detection of recessive components of herbicide preparations, and the detection system has the advantages of being high in detection sensitivity, convenient and fast to operate and obvious in phenomenon and facilitates field detection and use.

Description

Technical Field

[0001] The present invention relates to the field of pesticide preparation detection, and in particular to a reagent and method for detecting paraquat in a herbicide preparation. Background Art

[0002] Paraquat is a bipyridine-type herbicide that is banned by the state due to its painful and long course of disease after entering the human body. However, according to the results of pesticide spot checks published by the Ministry of Agriculture and Rural Affairs, it has been a hidden ingredient in herbicides such as diquat, glyphosate and glufosinate ammonium for many years and still exists in herbicide products on the market, posing a great risk to public health.

[0003] Paraquat free radical has a specific blue color, and the colorimetric detection method should have good specificity. However, diquat and paraquat are both bipyridine herbicides with similar structures. In the colorimetric detection method, the alkaline sodium dithionite colorimetric method is used clinically to identify paraquat and diquat in biological samples such as blood and urine. The specific principle is that paraquat and diquat can be reduced to blue paraquat free radical and green diquat free radical. However, when the concentration of diquat and paraquat is further increased to the content of the preparation, it will be impossible to compare because the concentration is too high. See Figure 1 In (a), when the concentration of the sample to be tested is low, such as 50 mg / L, diquat and paraquat after reduction with alkaline sodium dithionite appear green and blue respectively, and the two can be clearly distinguished by colorimetry; see Figure 1 (b), but as the concentration of the test sample increases, such as when the test sample concentration reaches 20g / L, the colors of diquat and paraquat after reduction with alkaline sodium dithionite are almost the same, and the two cannot be distinguished by colorimetric method. Therefore, developing a reducing agent specific to paraquat to reduce the interference caused by high concentrations of diquat and other substances is the key to detecting the hidden addition of paraquat in herbicide formulations. Summary of the invention

[0004] The present disclosure aims to solve at least one of the technical problems existing in the prior art.

[0005] To this end, the present invention provides a reagent and method for detecting paraquat in herbicide preparations, which utilizes acetophenylhydrazine as a reducing agent in combination with a solvent to regulate the redox potential of the reducing agent, and develops a detection system for specific reduction of paraquat, which can realize rapid colorimetric detection of paraquat in herbicide preparations, has high detection sensitivity and is easy to operate.

[0006] In order to achieve the above objectives, the present disclosure adopts the following technical solutions:

[0007] A first aspect of the present disclosure provides a reagent for detecting paraquat in a herbicide preparation, which has low cost and good stability.

[0008] The reagents include reagents A, B and C, wherein reagent A is a solution prepared from dipotassium hydrogen phosphate and alkaline buffer solution, reagent B is methanol, and reagent C is a solution of acetophenylhydrazine.

[0009] The reagent A is prepared according to the following steps:

[0010] Dissolve potassium phosphate dibasic in alkaline buffer.

[0011] In some embodiments, the mass concentration of dipotassium hydrogen phosphate in the reagent A is not less than 130 g / L, and the upper limit of the mass concentration of dipotassium hydrogen phosphate in the reagent A is the mass concentration of dipotassium hydrogen phosphate corresponding to when the reagent A reaches a saturated state.

[0012] In some embodiments, the pH range of the alkaline buffer in the reagent A is 8-13.

[0013] In some embodiments, the reagent C is prepared according to the following steps:

[0014] Acetylphenylhydrazine is dissolved in a solvent.

[0015] In some embodiments, the mass concentration of acetophenylhydrazine in the reagent C is not less than 0.05 g / L, and the upper limit of the mass concentration of acetophenylhydrazine in the reagent C is the mass concentration of acetophenylhydrazine corresponding to when the reagent C reaches a saturated state.

[0016] In some embodiments, the solvent in the reagent C is selected from any one or more mixtures having a molar average molecular dipole moment between 2.8D and 4.0D, such as any one solution of acetone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide and acetonitrile or a mixture of multiple solutions.

[0017] The second aspect of the present disclosure provides a method for rapidly detecting paraquat in a herbicide preparation using the reagent described in any embodiment of the first aspect of the present disclosure. The method is easy to operate, highly sensitive, and suitable for on-site detection.

[0018] A second aspect of the present disclosure provides a colorimetric detection method for paraquat in a herbicide formulation, comprising:

[0019] The herbicide preparation to be tested is mixed with reagent A to form a uniform solution A, and then reagent B is added to the uniform solution A. After shaking, wait for phase separation, take the supernatant B and add it to reagent C. Immediately compare the color visually or using a spectrophotometer to determine whether the herbicide preparation to be tested contains paraquat based on the color change.

[0020] In some embodiments, the colorimetric detection method further comprises: using a spectrophotometer to colorimetrically determine the concentration of paraquat in the herbicide formulation to be tested or using image color analysis to determine the concentration of paraquat in the herbicide formulation to be tested, thereby achieving quantitative detection of the paraquat concentration in the herbicide formulation to be tested.

[0021] In some embodiments, the volume ratio of the herbicide formulation to be tested to reagent A is 1:10-1000.

[0022] In some embodiments, the volume ratio of the homogeneous solution A to the reagent B is 1:0.2-5.

[0023] In some embodiments, the volume ratio of supernatant B to reagent C is 1:0.01-100.

[0024] In some embodiments, the color of the paraquat spiked into the formulation is determined visually to be green or blue.

[0025] In some embodiments, the maximum absorption wavelength of the spectrophotometer for colorimetric determination of paraquat concentration is 605 nm.

[0026] The application of the reagent provided in the first aspect of the present disclosure in the detection of paraquat in herbicide preparations has the following characteristics and beneficial effects:

[0027] The disclosed embodiment uses acetophenylhydrazine as a reducing agent in combination with a solvent to regulate the redox potential of the reducing agent, develops a detection system for specific reduction of paraquat, and applies it to the detection of hidden components of herbicide preparations. The method has high detection sensitivity, can perform rapid detection under interference such as diquat, is easy to operate, has obvious phenomena, and is conducive to on-site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 (a) and (b) are the effects of reducing paraquat and diquat at different concentrations using sodium dithionite. There is a certain degree of differentiation at low concentrations, but no differentiation at high concentrations.

[0029] Figure 2 This is a schematic diagram of the effect of using reagents to detect the invisible ingredient paraquat in a diquat preparation (diquat mass concentration of 200 g / L) according to Example 1 of the present disclosure. From left to right, the paraquat mass concentrations are 200 g / L, 20 g / L, 2 g / L and 0 (i.e., the control group is a diquat preparation without paraquat adulteration).

[0030] Figure 3 A standard curve for quantitatively detecting the adulterated mass concentration of paraquat in a diquat preparation using Example 1 of the present disclosure.

[0031] Figure 4This is a diagram showing the effect of using acetophenylhydrazine to reduce pure paraquat and diquat in the present disclosure, which still has discrimination at high concentrations.

[0032] Figure 5 This is a UV-visible absorption spectrum of paraquat and diquat after reduction with acetophenylhydrazine in the embodiment of the present disclosure. In the figure, the absorption peak of diquat does not substantially affect the absorption of paraquat free radical at 605 nm.

[0033] Figure 6 The emission spectra of diquat radicals extracted with dichloromethane for fluorescence detection show that the reduction of diquat with acetophenylhydrazine produces almost no green diquat radicals compared with the reduction with sodium dithionite.

[0034] Figure 7 This is the structural formula of the reduction product after acetophenylhydrazine reduces diquat, which is inferred based on the mass spectrometry results. Compared with diquat, the ethylene structure of this structure is destroyed and the degree of conjugation is reduced, so the color is blue-shifted.

[0035] Figure 8 This is the standard curve for quantifying paraquat adulteration in glyphosate formulations using a spectrophotometer in Example 2 of the present disclosure.

[0036] Fig. 9 This is a schematic diagram of the effect of using reagents to detect paraquat, an invisible ingredient in glyphosate preparations, according to Example 2 of the present disclosure.

[0037] Fig.10 This is a schematic diagram of the effect of using reagents to detect paraquat, an invisible ingredient in glufosinate preparations in Example 3 of the present disclosure. DETAILED DESCRIPTION

[0038] Embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application. In addition, if not explicitly stated, all reagents used in the following embodiments are commercially available, or can be synthesized according to this article or known methods, and for the reaction conditions not listed, they are also easily available to those skilled in the art.

[0039] The technical solution disclosed in the present invention is not limited to the specific implementation methods listed below, but should also include various applications of using acetophenylhydrazine as a reducing agent for identifying paraquat under the interference of herbicides such as diquat.

[0040] Embodiment 1:

[0041] This embodiment is a method for preparing a reagent for rapid detection of paraquat in herbicide preparations and the use of the reagent in detecting the invisible component paraquat in diquat preparations. This embodiment is carried out at room temperature and is specifically completed in the following steps:

[0042] 1. Preparation method of reagents: Dissolve 60g of potassium dihydrogen phosphate in 100mL of water and dissolve it in BR buffer solution with a pH of 10 to prepare reagent A. Reagent B is methanol. Dissolve 0.5g of acetophenylhydrazine in 100mL of N,N-dimethylformamide to obtain reagent C.

[0043] 2. Detection of paraquat in actual herbicide diquat preparations: Take 10μL of the diquat preparation to be tested, add it to 1mL of reagent A, and shake well. Then add 1mL of reagent B, shake well and wait for 30s for the solution to separate, take 10μL of the upper layer solution and add it to 1mL of reagent C, and immediately observe the color. If blue or green appears, it means that paraquat is mixed in the diquat preparation to be tested.

[0044] See also Figure 2 , from left to right are schematic diagrams of the colorimetric effects when the paraquat mass concentrations in the diquat reagent to be tested are 200 g / L, 20 g / L, 2 g / L and 0 (the mass concentration of paraquat is 0 as the control group, i.e., the diquat preparation without paraquat adulteration). Since the diquat preparation contains components such as warning color, it combines with the red free radicals of the diquat reagent not adulterated with paraquat after reduction with acetophenylhydrazine to form a light yellow solution. The detection limit of this method is 2 g / L paraquat adulterated in the diquat preparation (active ingredient content is about 200 g / L).

[0045] 3. Quantitative detection of paraquat concentration in actual herbicide diquat preparations:

[0046] During colorimetric observation, use a mobile phone or camera to collect photos of the mixed system in step 2, and identify the color values ​​of the three RGB channels (red, green, and blue). Calculate the color value of the photo according to y=8×R-5×G (excluding the blue value) to obtain the linear regression value for quantification. R and G represent the color values ​​of the red channel and the green channel in the collected photo, respectively. Substitute the linear regression value into Figure 3 The standard curve for detecting the concentration of paraquat in diquat preparations is shown as follows: y = -183.78x -127.79 (R 2 =0.9872), the paraquat concentration in the diquat preparation to be tested can be obtained. Figure 3 In the figure, the abscissa x represents the mass concentration of paraquat in the diquat preparation (g / L), and the ordinate y represents the color value of the collected photo.

[0047] The detection principle of this embodiment is as follows:

[0048] After calculation and screening, acetophenylhydrazine is a better reducing agent with a suitable redox potential. It can reduce paraquat to blue free radicals in the presence of alkali and oxygen (see Figure 4middle right figure), and red when encountering diquat (see Figure 4 (left figure in the middle). This enables the operator to better distinguish the colors of the two when they coexist at high concentrations. Furthermore, the disclosed embodiment also utilizes the characteristic that the solubility of paraquat in methanol (143g / L) is much greater than the solubility of diquat (25g / L), and uses methanol (as an extraction solvent)-potassium hydrogen phosphate (as a phase separation salt) to extract paraquat in the herbicide preparation to be tested, thereby changing the concentration and ratio of paraquat and diquat in the detection system, and then adjusting the relative redox potential of the two, so that paraquat can be reduced to a blue free radical before diquat. Based on this principle, a detection method and reagent were designed and developed, which can specifically identify paraquat at the concentration of the herbicide preparation to be tested (200g / L), have strong anti-interference ability, and have the characteristics of good detection limit and convenient operation.

[0049] Figure 5 This is the UV-visible absorption spectra of paraquat free radical and diquat after being reduced by acetophenylhydrazine. It can be seen that the absorption peak of diquat after reduction at 425nm hardly affects the colorimetry of paraquat free radical (605nm). Figure 6 The experimental group was treated with acetophenylhydrazine to reduce diquat, and the control group was treated with sodium dithionite to reduce diquat. The concentration of diquat free radicals after reduction was determined by fluorescence spectroscopy. Figure 6 It can be seen that the use of acetophenylhydrazine as a reducing agent produces almost no colored diquat free radical, thereby effectively avoiding the colorimetric interference of diquat on paraquat free radical. Figure 7 It is the structural molecular formula of the reduction product obtained after acetophenylhydrazine reduces diquat, which is inferred based on the results of mass spectrometry. Compared with the green diquat free radical, the diquat reduction product after acetophenylhydrazine reduction has a low degree of conjugation due to the destruction of the ethylene structure, and the absorption wavelength is blue-shifted, avoiding the color wavelength of paraquat free radical, thereby achieving specific recognition of the color of paraquat free radical.

[0050] When using RGB color analysis to quantitatively detect the concentration of paraquat, the coefficients of each RGB channel are selected based on the linearity of the standard curve. Its physical meaning is: the maximum absorption wavelength of the diquat reduction product is 425nm, which is converted into RGB color expression as (230, 23, 19), while the blue paraquat free radical is (0, 70, 254). It can be seen that the changes of the two in the R red channel and the G green channel are opposite, and the change amplitude of the R channel is larger. The reason for not using the blue channel is that the components such as the warning color added to the preparation will give the B blue channel a larger value, making the linearity poor.

[0051] Embodiment 2:

[0052] This embodiment is a method for preparing a reagent for rapid detection of paraquat in herbicide preparations and the application of the reagent in detecting the invisible component paraquat in glyphosate preparations. This embodiment is carried out at room temperature and is specifically completed in the following steps:

[0053] 1. Preparation method of reagents: 130 g of dipotassium hydrogen phosphate was dissolved in 1 L of water, and potassium hydroxide was used to adjust the pH of the solution to 13 to prepare reagent A. Reagent B was methanol. 0.05 g of acetophenylhydrazine was dissolved in 1 L of dimethyl sulfoxide to obtain reagent C.

[0054] 2. Detection of paraquat in actual herbicide glyphosate preparations: Take 10μL of the glyphosate preparation to be tested, add it to 10mL of reagent A, and shake well. Then add 2mL of reagent B, shake thoroughly and wait for 30s for the solution to separate, take 10μL of the upper layer solution and add it to 10mL of reagent C, and immediately observe the color. If blue or green appears, it means that paraquat is mixed into the glyphosate preparation to be tested. See Figure 8 The light yellow one on the left is the glyphosate preparation without paraquat, and the blue one on the right is the glyphosate preparation mixed with 2g / L paraquat.

[0055] 3. Quantitative detection of paraquat concentration in actual herbicide glyphosate reagent:

[0056] The mixed system in step 2 is observed by colorimetry using a spectrophotometer to measure the absorbance y at 605 nm, and the absorbance is substituted into Fig. 9 The standard curve for detecting the concentration of paraquat in glyphosate formulations is shown as follows: y = 0.0164x + 0.1565 (R 2 =0.9981), the paraquat concentration in the glyphosate preparation to be tested can be obtained. Fig. 9 In the figure, the horizontal axis x represents the mass concentration of paraquat in the glyphosate formulation, and the vertical axis y represents the absorbance at 605 nm measured by a spectrophotometer for the mixed system during the colorimetric observation in step 2. The detection limit of this embodiment is 0.1 g / L.

[0057] Embodiment 3:

[0058] This embodiment is a method for preparing a reagent for rapid detection of paraquat in herbicide preparations and the application of the reagent in detecting the invisible component paraquat in glufosinate preparations. This embodiment is carried out at room temperature and is specifically completed in the following steps:

[0059] 1. Preparation method of reagents: Dissolve 500g of dipotassium hydrogen phosphate in 1L of water, and use potassium hydroxide to adjust the pH of the solution to 8 to prepare reagent A. Reagent B is methanol. Dissolve 5g of acetophenylhydrazine in 1L of acetone to obtain reagent C.

[0060] 2. Detection of paraquat in actual herbicide glufosinate preparations: Take 100μL of the glufosinate preparation to be tested, add it to 1mL of reagent A, and shake well. Then add 5mL of reagent B, shake thoroughly and wait for 30s for the solution to separate, take 1mL of the upper layer solution and add it to 10μL of reagent C, and immediately observe the color. If blue or green appears, it means that paraquat is mixed into the glufosinate preparation to be tested. See Fig.10 The light yellow one on the left is the glufosinate preparation without paraquat, and the green one on the right is the glufosinate preparation mixed with 2g / L paraquat.

[0061] The present invention utilizes acetophenylhydrazine as a reducing agent in combination with a solvent to regulate the redox potential of the reducing agent, and develops a detection system for specific reduction of paraquat. It can be understood that the reagents and detection methods of the present invention are applicable not only to diquat, glyphosate and glufosinate preparations, but also to the detection of paraquat in other herbicide preparations.

[0062] In addition, in the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0063] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A reagent for detecting paraquat in a herbicide preparation, characterized in that: The reagents include reagent A, reagent B and reagent C, wherein the reagent A is a solution prepared from dipotassium hydrogen phosphate and alkaline buffer solution, the reagent B is methanol, and the reagent C is a solution of acetophenylhydrazine.

2. The reagent according to claim 1, characterized in that In the reagent A, the mass concentration of dipotassium hydrogen phosphate is not less than 130 g / L, and the pH range of the alkaline buffer is 8-13.

3. The reagent according to claim 1, characterized in that The mass concentration of acetophenylhydrazine in the reagent C is not less than 0.05 g / L, and the solvent in the reagent C is selected from any one or more mixtures having a molar average molecular dipole moment between 2.8D and 4.0D.

4. The reagent according to claim 1, characterized in that The solvent in the reagent C is selected from any one solution or a mixed solution of multiple solutions of acetone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide and acetonitrile.

5. A method for detecting paraquat in a herbicide preparation using the reagent according to any one of claims 1 to 4, characterized in that: include: The herbicide preparation to be tested is mixed with the reagent A to form a uniform solution A, and then the reagent B is added to the uniform solution A. After shaking, wait for phase separation, take the supernatant B and add it to the reagent C, and immediately compare the color visually or using a spectrophotometer to determine whether the herbicide preparation to be tested contains paraquat based on the color change.

6. The method according to claim 5, characterized in that The method further comprises: colorimetrically determining the concentration of paraquat in the tested herbicide formulation using a spectrophotometer or determining the concentration of paraquat in the tested herbicide formulation using image color analysis.

7. The method according to claim 5 or 6, characterized in that: The volume ratio of the herbicide preparation to be tested to the reagent A is 1:10-1000; The volume ratio of the uniform solution A to the reagent B is 1:0.2-5; The volume ratio of the supernatant B to the reagent C is 1:0.01-100.

8. The method according to claim 5 or 6, characterized in that: The color of the paraquat added to the herbicide formulation to be tested is determined to be green or blue by visual colorimetry.

9. The method according to claim 5 or 6, characterized in that: The maximum absorption wavelength of paraquat concentration measured by colorimetric determination using the spectrophotometer is 605 nm.

10. The method according to claim 5 or 6, characterized in that: The herbicide preparation to be tested is diquat, glyphosate or glufosinate ammonium.

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