A reagent and method for detecting paraquat in herbicide formulations

Abstract

The present disclosure provides a reagent and a method for detecting paraquat in herbicide preparation, the reagent comprising reagent A, reagent B and reagent C, the reagent A being a solution prepared from dipotassium hydrogen phosphate and an alkaline buffer, the reagent B being methanol, and the reagent C being a solution of acetylphenylhydrazine. The method comprises mixing the herbicide preparation to be tested with the reagent A to form a uniform solution A, then adding the reagent B to the uniform solution A, shaking and waiting for phase separation, taking the supernatant B and adding it to the reagent C, immediately visually comparing the color or using a spectrophotometer to compare the color, and determining whether the herbicide preparation to be tested contains paraquat according to the color change. The present disclosure uses acetylphenylhydrazine as a reducing agent combined with solvent to control the redox potential of the reducing agent, develops a detection system specific for reducing paraquat, and applies it to the detection of latent ingredients in herbicide preparations, which has the characteristics of high detection sensitivity, convenient operation and obvious phenomenon, and is suitable for on-site detection.

Description

Technical Field

[0001] This disclosure relates to the field of pesticide formulation testing, and in particular to a reagent and method for detecting paraquat in herbicide formulations. Background Technology

[0002] Paraquat is a bipyridine-based non-selective herbicide that has been banned in China due to its painful and prolonged course of illness after ingestion. However, according to pesticide sampling results released by the Ministry of Agriculture and Rural Affairs, it has been found for many years as a hidden ingredient in herbicides such as diquat, glyphosate, and glufosinate, still present in herbicide products on the market, posing a significant risk to public health.

[0003] Paraquat free radicals have a specific blue color, and colorimetric detection methods should ideally possess good specificity. However, diquat and paraquat are both bipyridine herbicides with similar structures. In clinical practice, the alkaline sodium dithionite colorimetric method is used to distinguish paraquat and diquat in biological samples such as blood and urine. The principle is that paraquat and diquat can be reduced to blue paraquat free radicals and green diquat free radicals, respectively. However, when the concentrations of diquat and paraquat are further increased to the formulation concentrations, the concentrations become too high to compare. See also... Figure 1 In (a), when the concentration of the sample to be tested is low, such as at 50 mg / L, diquat and paraquat, after reduction with alkaline sodium dithionite, appear green and blue, respectively, and can be clearly distinguished by colorimetry; see also Figure 1 In (b), however, as the concentration of the tested sample increases, such as when the concentration reaches 20 g / L, the colors of diquat and paraquat after reduction with alkaline sodium dithionite are almost identical, making it impossible to distinguish between the two by colorimetric methods. Therefore, developing a reducing agent specific to paraquat to reduce interference from high concentrations of diquat and other substances is key to detecting the occult addition of paraquat in herbicide formulations. Summary of the Invention

[0004] This disclosure aims to address at least one of the technical problems existing in the prior art.

[0005] Therefore, this disclosure provides a reagent and method for detecting paraquat in herbicide formulations. By using acetylphenylhydrazine as a reducing agent and combining it with a solvent to regulate the redox potential of the reducing agent, a specific reduction paraquat detection system has been developed. This system enables rapid colorimetric detection of paraquat in herbicide formulations, with high detection sensitivity and convenient operation.

[0006] To achieve the above objectives, the present disclosure adopts the following technical solution:

[0007] The first aspect of this disclosure provides a reagent for detecting paraquat in herbicide formulations, which is inexpensive and has good stability.

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

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

[0010] Dissolve dipotassium hydrogen phosphate in an alkaline buffer solution.

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

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

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

[0014] Dissolve acetylphenylhydrazine in a solvent.

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

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

[0017] The second aspect of this disclosure provides a method for rapid detection of paraquat in herbicide formulations using the reagents described in any embodiment of the first aspect of this invention. This method is convenient to operate, highly sensitive, and suitable for on-site detection.

[0018] The second aspect of this disclosure provides a colorimetric detection method for paraquat in herbicide formulations, comprising:

[0019] Mix the herbicide formulation to be tested with reagent A to form a homogeneous solution A. Then add reagent B to the homogeneous solution A, shake well and wait for phase separation. Take the supernatant B and add it to reagent C. Immediately observe the colorimetric analysis or use a spectrophotometer to analyze the color. Determine whether the herbicide formulation to be tested contains paraquat based on the color change.

[0020] In some embodiments, the colorimetric detection method further includes: 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 reagent A is 1:10-1000.

[0022] In some embodiments, the volume ratio of homogeneous solution A to 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, visual colorimetric analysis is used to determine whether the formulation containing paraquat is green or blue.

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

[0026] The reagent provided in the first aspect of this disclosure, when used in the detection of paraquat in herbicide formulations, has the following characteristics and beneficial effects:

[0027] This disclosure utilizes acetylphenylhydrazine as a reducing agent combined with solvent to regulate the redox potential of the reducing agent, developing a specific detection system for paraquat reduction. This system is then applied to the detection of latent components in herbicide formulations. This method has high detection sensitivity, can perform rapid detection even under interference from herbicides such as diquat, is easy to operate, and produces obvious results, making it suitable for on-site testing. Attached Figure Description

[0028] Figure 1 In the middle (a) and (b), respectively, the effects of reducing paraquat and diquat at different concentrations with sodium dithionite are shown. They are distinguishable at low concentrations, but not at high concentrations.

[0029] Figure 2 This is a schematic diagram illustrating the effect of the reagent used in Example 1 of this disclosure to detect the hidden component paraquat in the diquat preparation (diquat mass concentration 200 g / L). 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 the diquat preparation without paraquat adulteration).

[0030] Figure 3 A standard curve for the quantitative determination of the mass concentration of paraquat adulteration in the diquat formulation using Example 1 of this disclosure.

[0031] Figure 4The image shows the effect of reducing pure paraquat and diquat with acetylphenylhydrazine, which still has distinguishability at high concentrations.

[0032] Figure 5 The UV-Vis absorption spectra of paraquat and diquat after reduction with acetylphenylhydrazine in this embodiment of the present disclosure are shown. The absorption peak of diquat in the figure does not significantly affect the absorption of paraquat free radicals at 605 nm.

[0033] Figure 6 The emission spectrum of diquat free radicals extracted with dichloromethane for fluorescence detection shows that, compared with the use of sodium dithionite as a reducing agent, the reduction of diquat with acetylphenylhydrazine produces almost no green diquat free radicals.

[0034] Figure 7 The structure of the reduction product of acetylphenylhydrazine after reducing diquat is inferred from the mass spectrometry results. Compared with diquat, the ethylene structure is destroyed and the degree of conjugation is reduced, thus the color shift is blue.

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

[0036] Figure 9 This is a schematic diagram illustrating the effect of using reagents to detect paraquat, a hidden component in glyphosate formulations, in Example 2 of this disclosure.

[0037] Figure 10 This is a schematic diagram illustrating the effect of using reagents to detect paraquat, a hidden component in glufosinate formulations, in Example 3 of this disclosure. Detailed Implementation

[0038] The embodiments of this application are described in detail below, with examples of the embodiments illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application. Furthermore, unless otherwise stated, all reagents used in the following embodiments are commercially available or can be synthesized according to methods described herein or known to the art, and reaction conditions not listed are readily available to those skilled in the art.

[0039] The technical solutions disclosed herein are not limited to the specific embodiments listed below, but should also include various applications of using acetylphenylhydrazine as a reducing agent for the identification of paraquat under interference from herbicides such as diquat.

[0040] Example 1:

[0041] This embodiment describes a method for preparing a reagent for the rapid detection of paraquat in herbicide formulations and its application in detecting the elusive component paraquat in diquat formulations. This embodiment is carried out at room temperature and is specifically completed according to the following steps:

[0042] I. Preparation of Reagents: Dissolve 60g of dipotassium hydrogen phosphate in 100mL of water, and then dissolve again using a BR buffer solution with a pH of 10 to prepare reagent A. Reagent B is methanol. Dissolve 0.5g of acetylphenylhydrazine in 100mL of N,N-dimethylformamide to obtain reagent C.

[0043] II. Detection of paraquat in actual diquat herbicide formulations: Take 10 μL of the diquat formulation to be tested and add it to 1 mL of reagent A, then shake well. Next, add 1 mL of reagent B, shake thoroughly, and wait 30 seconds for phase separation. Take 10 μL of the supernatant and add it to 1 mL of reagent C. Immediately observe the colorimetric result. If a blue or green color appears, it indicates that paraquat has been added to the diquat formulation to be tested.

[0044] See Figure 2 The diagrams, from left to right, show the colorimetric effects of paraquat concentrations of 200 g / L, 20 g / L, 2 g / L, and 0 g / L in the diquat reagent to be tested (with a paraquat concentration of 0 g / L serving as the control group, i.e., diquat preparations without paraquat adulteration). The diquat preparations contain warning color components, which combine with the red free radicals of the diquat reagent without paraquat after reduction with acetylphenylhydrazine to form a pale yellow solution. The detection limit of this method is the addition of 2 g / L paraquat to the diquat preparation (active ingredient content approximately 200 g / L).

[0045] III. Quantitative Detection of Paraquat Concentration in Actual Diquat Herbicide Formulations:

[0046] During colorimetric observation, a mobile phone or camera is used to capture photos of the mixed system from step two, and the color values ​​in its RGB three channels (red, green, and blue) are identified. The color values ​​of the photos are then calculated using the formula y = 8 × R - 5 × G (excluding the blue value) to obtain a linear regression value for quantification. R and G represent the color values ​​of the red and green channels in the captured photos, respectively. This linear regression value is then substituted into... Figure 3 The standard curve shown is used to detect the concentration of paraquat in diquat formulation: y = -183.78x - 127.79 (R²) 2 =0.9872), the concentration of paraquat in the test glyphosate formulation can be obtained. Figure 3 In the diagram, the horizontal axis x represents the mass concentration (g / L) of paraquat in the diquat formulation, and the vertical axis y represents the color value of the captured image.

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

[0048] After calculation and screening, acetylphenylhydrazine was found to be a good reducing agent. It possesses a suitable redox potential and, in the presence of alkali and oxygen, can reduce paraquat to blue free radicals (see [link to relevant documentation]). Figure 4(See right side of the image), and when encountering enemy grass, it quickly turns red (see image below). Figure 4 (Left side of the image). This allows the operator to better distinguish the colors of the two herbicides even when they coexist at high concentrations. Furthermore, this embodiment utilizes the characteristic that paraquat's solubility in methanol (143 g / L) is much greater than that of diquat (25 g / L). Methanol (as extraction solvent) and dipotassium hydrogen phosphate (as phase-separating salt) were used to extract paraquat from the herbicide formulation being tested. This altered the concentrations and ratios of paraquat and diquat in the detection system, thereby adjusting their relative redox potentials, allowing paraquat to be preferentially reduced to blue free radicals before diquat. Based on this principle, a detection method and reagent were designed and developed that can specifically identify paraquat at a concentration of 200 g / L in the herbicide formulation being tested. It exhibits strong anti-interference capabilities, good detection limits, and ease of operation.

[0049] Figure 5 The images show the UV-Vis absorption spectra of paraquat free radicals and diquat after reduction by acetylphenylhydrazine. It can be seen that the absorption peak of diquat at 425 nm after reduction has almost no effect on the colorimetric analysis of paraquat free radicals (605 nm). Figure 6 The experimental group used acetylphenylhydrazine-reduced diquat as a control group, while the control group used sodium dithionite-reduced diquat. The concentration of diquat free radicals after reduction was determined using fluorescence spectroscopy. Figure 6 It is known that using acetylphenylhydrazine as a reducing agent produces almost no colored diquat free radicals, thus effectively avoiding the colorimetric interference of diquat on paraquat free radicals. Figure 7 The structure and molecular formula of the reduced product obtained by reducing paraquat with acetylphenylhydrazine are inferred from the mass spectrometry detection results. Compared with the green paraquat free radical, the reduced product of paraquat after reduction by acetylphenylhydrazine 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, thus achieving specific recognition of the color of paraquat free radical.

[0050] When using RGB color analysis to quantitatively detect paraquat concentration, the coefficients of each RGB channel are selected based on the linearity of the standard curve. The physical meaning is as follows: the maximum absorption wavelength of the diquat reduction product is 425 nm, which is expressed as (230, 23, 19) in RGB color, while the blue paraquat free radical is (0, 70, 254). It can be seen that the changes in the R (red) channel and G (green) channel are opposite, with the R channel showing a larger change. The blue channel is not used because warning colorants and other components in the formulation would give the B (blue) channel a larger value, resulting in poor linearity.

[0051] Example 2:

[0052] This embodiment describes a method for preparing a reagent for the rapid detection of paraquat in herbicide formulations and its application in detecting paraquat, a lurking component in glyphosate formulations. This embodiment is carried out at room temperature and is specifically completed according to the following steps:

[0053] I. Preparation of reagents: Dissolve 130g of dipotassium hydrogen phosphate in 1L of water, and adjust the pH of the solution to 13 using potassium hydroxide to prepare reagent A. Reagent B is methanol. Dissolve 0.05g of acetylphenylhydrazine in 1L of dimethyl sulfoxide to obtain reagent C.

[0054] II. Detection of paraquat in actual glyphosate herbicide formulations: Take 10 μL of the glyphosate formulation to be tested and add it to 10 mL of reagent A, then shake well. Next, add 2 mL of reagent B, shake thoroughly, and wait 30 seconds for phase separation. Take 10 μL of the supernatant and add it to 10 mL of reagent C. Immediately observe the colorimetric analysis. If a blue or green color appears, it indicates that paraquat has been added to the glyphosate formulation to be tested. (See also...) Figure 8 The pale yellow glyphosate formulation on the left is glyphosate formulation without paraquat, while the blue glyphosate formulation on the right contains 2 g / L paraquat.

[0055] III. Quantitative Detection of Paraquat Concentration in Actual Glyphosate Reagent:

[0056] The absorbance γ of the mixed system observed in step two at 605 nm was measured using a spectrophotometer, and this absorbance was then substituted into the following... Figure 9 The standard curve shown is used to detect the concentration of paraquat in glyphosate formulations: y = 0.0164x + 0.1565(R) 2 The concentration of paraquat in the glyphosate formulation to be tested can be obtained by using (=0.9981). Figure 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 of the mixed system observed in step two using a spectrophotometer. The detection limit in this embodiment is 0.1 g / L.

[0057] Example 3:

[0058] This embodiment describes a method for preparing a reagent for the rapid detection of paraquat in herbicide formulations and its application in detecting paraquat, a lurking component in glufosinate formulations. This embodiment is carried out at room temperature and is specifically completed according to the following steps:

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

[0060] II. Detection of paraquat in actual glufosinate herbicide formulations: Take 100 μL of the glufosinate formulation to be tested and add it to 1 mL of reagent A, then shake well. Next, add 5 mL of reagent B, shake thoroughly, and wait 30 seconds for phase separation. Take 1 mL of the upper layer solution and add it to 10 μL of reagent C. Immediately observe the colorimetric result. If a blue or green color appears, it indicates that paraquat has been added to the glufosinate formulation to be tested. (See also...) Figure 10 The pale yellow one on the left is glufosinate formulation without paraquat, while the green one on the right is glufosinate formulation mixed with 2g / L paraquat.

[0061] This disclosure utilizes acetylphenylhydrazine as a reducing agent combined with solvent to regulate the redox potential of the reducing agent, and develops a specific detection system for paraquat reduction. It is understood that the reagents and detection methods of this disclosure are applicable not only to glyphosate, glyphosate and glufosinate formulations, but also to the detection of paraquat in other herbicide formulations.

[0062] Furthermore, in the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions 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 suitable manner in one or more embodiments or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0063] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A reagent for detecting paraquat in herbicide formulations, characterized in that, The reagents include reagent A, reagent B, and reagent C. Reagent A is a solution prepared from dipotassium hydrogen phosphate and alkaline buffer solution. Reagent B is methanol. Reagent C is a solution of acetylphenylhydrazine. The solvent in reagent C is any one or more mixtures with a molar average molecular dipole moment between 2.8 D and 4.0 D.

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

3. The reagent according to claim 1, characterized in that, The mass concentration of acetylphenylhydrazine in reagent C is not less than 0.05 g / L.

4. The reagent according to claim 1, characterized in that, The solvent in reagent C is any one or a mixture of several of the following: acetone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, and acetonitrile.

5. A method for detecting paraquat in herbicide formulations using the reagent described in any one of claims 1 to 4, characterized in that, include: Mix the herbicide formulation to be tested with reagent A to form a homogeneous solution A. Then add reagent B to the homogeneous solution A, shake well and wait for phase separation. Take the supernatant B and add it to reagent C. Immediately visually compare the color or use a spectrophotometer to compare the color. Determine whether the herbicide formulation to be tested contains paraquat based on the color change.

6. The method according to claim 5, characterized in that, The method further includes: using a spectrophotometer to determine the concentration of paraquat in the herbicide formulation to be tested by colorimetry or using image color analysis to determine the concentration of paraquat in the herbicide formulation to be tested.

7. The method according to claim 5 or 6, characterized in that, The volume ratio of the herbicide formulation to be tested to reagent A is 1:10-1000; The volume ratio of the homogeneous solution A to 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 herbicide formulation being adulterated with paraquat was determined by visual colorimetry to be either green or blue.

9. The method according to claim 5 or 6, characterized in that, The maximum absorption wavelength of paraquat concentration determined by colorimetric analysis using the aforementioned spectrophotometer was 605 nm.

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

Images