Method for detecting coumarin

Abstract

The invention discloses a method for detecting coumarin, which comprises the following steps: (1) preparing the following solutions: a borax aqueous solution, a NaOH aqueous solution, a boric acid aqueous solution and a 2, 6-dichloroquinone-4-chloroimine ethanol solution; (2) adjusting the pH value of the borax aqueous solution to 11.8-12.2 by using a NaOH aqueous solution to obtain a reaction buffer solution; (3) uniformly mixing the reaction buffer solution and the to-be-detected sample solution according to a volume ratio of 2: 1, and reacting under a water bath heating condition; (4) adjusting the pH value of the solution to 8.6-9.0 by adopting a boric acid aqueous solution and pure water, adding a 2, 6-dichloroquinone-4-chloroimine ethanol solution, uniformly shaking, and placing at room temperature for color development to obtain a to-be-detected color development sample solution; and (5) detecting the to-be-detected chromogenic sample solution by using the capillary electrophoresis apparatus to obtain a sample capillary electrophoresis pattern, and judging whether the absorption peak of coumarin exists in the sample capillary electrophoresis pattern so as to determine whether the to-be-detected sample solution contains coumarin.

Description

Technical Field

[0001] The present invention particularly relates to a method for detecting coumarin. Background Art

[0002] Coumarin (1,2-benzopyranone or 2H-1-benzopyran-2-one) is a phenolic compound that is widely found in higher plants, especially in the seeds, roots and leaves of dicotyledons. A few are found in animals and microorganisms. Coumarin has multiple biological activities in plants. It can not only affect the seed development of plants, but also affect many physiological and biochemical processes of plants. Therefore, it has broad application prospects in the agricultural field. First, coumarin has a function similar to that of plant hormones and can be used as a plant growth regulator to a certain extent. Secondly, coumarin has high inhibitory activity on the germination of plant seeds including alfalfa, Arabidopsis, multiflora ryegrass, barnyard grass, red clover, stinkweed, grain amaranth, Polygonum multiflorum, Bidens pilosa, lettuce, plantain, Amaranthus retroflexus, Sudan grass and Aegilops tauschii, and has the potential to be developed into an environmentally friendly herbicide. In addition, coumarin can also be used as an intermediate for the synthesis of other herbicides. Phenoxypyridine compounds synthesized with coumarin as the lead compound show high herbicidal activity. Field tests have shown that when the active ingredient is used at a dosage of 300g / ha, it can effectively control weeds such as barnyard grass, channa striata, foxtail grass, crabgrass and Amaranthus retroflexus.

[0003] In addition to its application in agriculture, coumarin is also an important industrial raw material, widely used in cosmetics, optical brighteners, disperse fluorescent dyes, laser dyes, food industry and other fields. In medicine, the study of coumarin and its derivatives has shown that they have multiple biological activities such as anti-tumor, anti-HIV, antibacterial, anti-inflammatory and anticoagulant, and also show potential for application in photochemotherapy.

[0004] At present, the detection methods of coumarin mainly include liquid chromatography, liquid chromatography-mass spectrometry, gas chromatography, gas chromatography-mass spectrometry, and ultra-high performance liquid chromatography-tandem mass spectrometry. However, the detection costs of these existing detection methods are relatively high; taking the commonly used liquid chromatography method as an example, this method requires the use of expensive chromatographic grade methanol or acetonitrile as the mobile phase, which is a reagent that is continuously consumed during the detection process. Based on the mobile phase flow rate of 1 mL per minute and the analysis time of each sample of 20 minutes, the analysis of 100 samples requires the consumption of 2L of mobile phase and the formation of 2L of organic waste liquid that needs to be further recycled and processed, thereby increasing the cost of waste liquid treatment. Summary of the invention

[0005] The present invention aims to provide a method for detecting coumarin, which has high accuracy, reduces detection cost and improves detection efficiency.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions.

[0007] A method for detecting coumarin comprises the following steps:

[0008] (1) Prepare the following solutions: borax aqueous solution, NaOH aqueous solution, boric acid aqueous solution, and 2,6-dichloroquinone-4-chloroimine ethanol solution;

[0009] (2) Preparation of reaction buffer: Take 1 volume of borax aqueous solution, adjust the pH value of the borax aqueous solution to 11.8-12.2 with NaOH aqueous solution, and make up to 4 volumes with pure water to obtain a reaction buffer solution;

[0010] (3) The reaction buffer solution and the sample solution to be tested are mixed evenly in a volume ratio of 2:1, and reacted in a water bath for 20 to 30 minutes;

[0011] (4) adjusting the pH value of the solution after the reaction in step (3) to 8.6-9.0 using a boric acid aqueous solution and pure water, adding a 2,6-dichloroquinone-4-chloroimine ethanol solution, shaking well, and placing at room temperature for more than 1 hour for color development to obtain a color sample solution to be tested;

[0012] (5) preparing a coumarin standard solution of known concentration, performing water bath heating reaction and color development treatment according to the above steps (3) to (4) to obtain a color development standard solution, using a capillary electrophoresis instrument with an ultraviolet-visible light detector to detect the color development standard solution to obtain a standard capillary electrophoresis spectrum, and determining the absorption peak of coumarin; then using the above capillary electrophoresis instrument to detect the color development sample solution to be tested to obtain a sample capillary electrophoresis spectrum, judging whether there is an absorption peak of coumarin therein, thereby determining whether the sample solution to be tested contains coumarin.

[0013] The core of the invention is to use borax and NaOH to provide stable alkaline conditions, then open the lactone ring of coumarin by heating in a water bath to generate cis-o-hydroxycinnamate; then use boric acid to adjust the pH value of the solution to a suitable range, and then add 2,6-dichloroquinone-4-chloroimine to carry out a color development reaction to generate a blue compound, which has an absorption peak in the wavelength range of visible light and is charged, so that it is easy to separate it from impurities by capillary electrophoresis, thereby accurately detecting coumarin.

[0014] The invention is suitable for detecting various sample solutions containing impurities, such as extracts from different tissue parts such as plant seeds, roots, stems, leaves, etc., medicines and environmental sample solutions, etc.

[0015] Preferably, the following solutions are prepared in step (1): 0.05 mol / L borax aqueous solution, 0.1-0.2 mol / L NaOH aqueous solution, 0.05-0.2 mol / L boric acid aqueous solution, and 0.5 wt% 2,6-dichloroquinone-4-chloroimine ethanol solution.

[0016] Preferably, in step (2), the pH value of the borax aqueous solution is adjusted to 12.1 using a NaOH aqueous solution.

[0017] Preferably, the temperature of the water bath heating in step (3) is 60-70°C.

[0018] Preferably, in step (4), the pH value of the mixed solution of step (3) is adjusted to 8.8 using a boric acid aqueous solution; and the amount of 2,6-dichloroquinone-4-chloroimine ethanol solution added is 1 to 2.5% by volume.

[0019] Preferably, the detection conditions of the capillary electrophoresis instrument are: using borax-boric acid buffer (0.05 mol / L borax aqueous solution and 0.2 mol / L boric acid aqueous solution mixed in a ratio of 8:2, pH = 9.0), using a quartz capillary with an inner diameter of 0.75 μm and an effective length of 30 cm; the injection method is automatic injection, the injection time is 5 seconds, and the injection pressure is 5 bar; after injection, electrophoresis separation is performed at 8.5 kV voltage and room temperature, and the electrophoresis time is 25 minutes; the detection wavelength of the UV-visible detector is 610-620 nm.

[0020] Furthermore, in step (5), coumarin standard solutions of different concentrations are prepared, and after water bath heating reaction and color development treatment according to the above steps (3) to (4), color development standard solutions of different concentrations are obtained, and the color development standard solutions are detected by a capillary electrophoresis instrument with an ultraviolet-visible light detector to obtain standard capillary electrophoresis patterns of different coumarin concentrations; then, based on the standard capillary electrophoresis patterns of different coumarin concentrations, a standard curve between the peak area and the concentration of coumarin is established to obtain a linear equation between the peak area and the concentration of coumarin; finally, based on the peak area of ​​coumarin in the sample capillary electrophoresis pattern, the coumarin concentration in the sample solution to be tested is calculated using the above linear equation.

[0021] The present invention adopts the capillary electrophoresis technology to separate and detect the blue charged compound with absorption peak at visible wavelength obtained by the above color development reaction, so as to realize the quantitative analysis of coumarin.

[0022] Preferably, in step (5), 10 mg / L, 50 mg / L, 100 mg / L, and 200 mg / L coumarin standard solutions are used to establish a standard curve between the peak area and concentration of coumarin, and a good linear relationship can be obtained.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a technical solution for detecting coumarin based on capillary electrophoresis. Since capillary electrophoresis analysis itself has the advantages of high resolution, good reproducibility, accurate quantitative analysis and low reagent consumption, the method of the present invention also has the above advantages.

[0025] Compared with liquid chromatography, the method of the present invention does not require a mobile phase during the analysis process, and only requires a solution prepared by borax and boric acid as an electrophoresis buffer. Borax and boric acid are conventional reagents with low prices. It takes about 20 mL to add a buffer once during the capillary analysis process, and more than 30 samples can be analyzed continuously. The analysis of 100 samples consumes about 60 mL of buffer; therefore, the analysis cost of the method of the present invention is low, and no organic waste liquid is generated.

[0026] Furthermore, the present invention requires a relatively short time for sample pretreatment, which can be completed within at least one and a half hours, thereby greatly improving the detection efficiency of coumarin.

[0027] The method of the present invention not only reduces the detection cost and improves the detection efficiency, but also provides a more convenient detection means for the wide application of coumarin. In addition, the method of the present invention can also be prepared into a special kit for coumarin detection, which is convenient for practical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Shows the effect of different pH values ​​on the absorption spectrum of sample solutions during the opening stage of the coumarin lactone ring;

[0029] Figure 2 It shows the effect of different pH values ​​on the maximum absorbance of the sample solution during the lactone ring opening stage;

[0030] Figure 3 Show the effect of water bath heating and room temperature treatment on the absorbance of sample solutions under different pH conditions;

[0031] Figure 4 Shows the effect of different water bath temperatures on the absorbance of the sample solution at pH 9.4;

[0032] Figure 5 Shows the effect of different pH values ​​on the absorption spectrum of the sample solution during the color development stage;

[0033] Figure 6 Shows the effect of different pH values ​​on the maximum absorbance of the sample solution during the color development stage;

[0034] Figure 7 Shows the changes of the absorption peak of the sample solution over time under different color development pH conditions;

[0035] Figure 8 Display the change of maximum absorbance of sample solution over time under different color development pH conditions;

[0036] Fig. 9 Comparison of the absorption spectra of coumarin and negative control under the color development pH conditions of 8.8 and 11.1;

[0037] Fig.10 This is the standard capillary electrophoresis pattern of 200 mg / L coumarin;

[0038] Fig.11 This is the standard capillary electrophoresis pattern of 100 mg / L coumarin;

[0039] Fig.12 This is the standard capillary electrophoresis pattern of 50 mg / L coumarin;

[0040] Fig.13 This is the capillary electrophoresis spectrum of 10 mg / L coumarin;

[0041] Fig.14 It is the linear regression analysis between capillary electrophoresis peak area and coumarin concentration;

[0042] Fig.15 This is the capillary electrophoresis pattern of alfalfa seed sample;

[0043] Fig.16 This is the capillary electrophoresis spectrum of red clover seed samples;

[0044] Fig.17 Absorption spectra of different seed samples. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with specific embodiments. It should be noted that, under the premise of no conflict, the embodiments or technical features described below can be arbitrarily combined to form new embodiments. The equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0046] Example 1 Determination of the Optimal pH Conditions for Opening the Coumarin Lactone Ring

[0047] In order to clarify the optimal pH condition for the opening of the coumarin lactone ring, that is, the optimal pH condition of the reaction buffer, 5 portions of 5mL 0.05mol / L borax aqueous solution were taken, and 0.6, 2.3, 3.4, 4.3 and 4.6mL 0.2mol / L NaOH aqueous solution were added, and then the volume was adjusted to 20mL with pure water to obtain a series of solutions with pH values ​​of 9.4, 10.4, 11.8, 12.1 and 12.2, respectively. 2mL of the above solutions with different pH values ​​were mixed with 1mL 100mg / L coumarin solution and placed in a 60℃ water bath for 30 minutes. After the solution was cooled to room temperature, 17mL 0.2mol / L boric acid aqueous solution and 0.5mL 0.5% 2,6-dichloroquinone-4-chloroimine ethanol solution were added. After standing for 1 hour, the absorption spectrum in the range of 400-800nm ​​was measured using a Multiskan SkyHigh full-wavelength microplate reader. The experiment was repeated 3 times.

[0048] In the lactone ring opening stage, the effect of pH on the absorption spectrum of the sample solution is as follows: Figure 1 As shown. Under different pH conditions, the sample solution showed an absorption peak near 610nm. In the pH range of 9.4 to 12.1, the absorption peak gradually increased with the increase of pH; and after the pH value reached 12.1, the absorption peak had an inflection point and then turned to a downward trend. Figure 2 The maximum absorbance of the sample solution at around 610 nm is shown. Between pH 9.4 and 10.4, the absorbance increases rapidly with the increase of pH; while at pH 10.4 to 12.1, although the absorbance is still increasing, the rate of increase slows down significantly. When the pH reaches 12.2, the maximum absorbance of the sample solution is lower than that at pH 12.1. These results indicate that pH 12.1 is the optimal condition for the opening of the coumarin lactone ring.

[0049] Example 2 Determination of reaction conditions for opening the coumarin lactone ring

[0050] In order to clarify whether water bath heating under different pH conditions is a necessary condition for the opening of the coumarin lactone ring, the following experiment was conducted: First, take 5 portions of 5 mL of 0.05 mol / L borax aqueous solution, add 0.6, 2.3, 3.4, 4.3 and 4.6 mL of 0.2 mol / L NaOH aqueous solution, and then dilute to 20 mL with pure water to obtain solutions with pH values ​​of 9.4, 10.4, 11.8, 12.1 and 12.2, respectively. Then, take 2 mL of each of the above solutions with different pH values ​​and mix them thoroughly with 1 mL of 100 mg / L coumarin solution, and divide them into two groups for experiments: one group is placed at room temperature for 30 minutes, and the other group is placed in a 60°C water bath for 30 minutes. Subsequently, 17 mL of 0.2 mol / L boric acid aqueous solution and 0.5 mL of 0.5% 2,6-dichloroquinone-4-chloroimine ethanol solution are added to the solution of each group of experiments. After standing for 1 hour, the absorbance change of the solution at 610 nm is measured. The experiment was repeated 3 times, and the results are shown in Figure 3 shown.

[0051] The results showed that when the pH value was between 9.4 and 12.1, the absorbance of the water bath treatment group was significantly higher than that of the room temperature treatment group; while at pH 12.2, the absorbance of the two groups was not much different. Therefore, when the pH was less than 12.2, 60°C water bath heating significantly promoted the opening of the coumarin lactone ring.

[0052] Example 3 Determination of water bath heating temperature for opening the coumarin lactone ring

[0053] In order to clarify the effect of different heating temperatures on the opening of the coumarin lactone ring, the following experiments were conducted:

[0054] First, take 5 mL of 0.05 mol / L borax aqueous solution, add 0.6 mL of 0.2 mol / L NaOH aqueous solution, and dilute to 20 mL with pure water to obtain solutions with pH values ​​of 9.4. Next, take 3 portions of the above pH 9.4 solution, each portion is 2 mL, and mix them thoroughly with 1 mL of 100 mg / L coumarin solution, and divide them into 3 groups for experiments: one group is placed at 25°C for 30 minutes, the second group is placed in a 60°C water bath for 30 minutes, and the third group is placed in a 90°C water bath for 30 minutes. Subsequently, 17 mL of 0.2 mol / L boric acid aqueous solution and 0.5 mL of 0.5% 2,6-dichloroquinone-4-chloroimine ethanol solution were added to the solution of each group of experiments. After standing for 1 hour, the absorbance change of the solution at 610 nm was measured; the experiment was repeated 3 times. The results are as follows Figure 4 shown.

[0055] The results showed that the higher the heating temperature, the higher the absorbance of the solution, indicating that when the pH is low, increasing the heating temperature can promote the opening of the coumarin lactone ring.

[0056] Example 4 Determining the pH value of the solution during the color development stage

[0057] In order to clarify the optimal pH value in the color development stage and its effect on the stability of the sample solution, the following experiment was conducted. Take 5 mL of 0.05 mol / L borax aqueous solution, add 4.3 mL of 0.2 mol / L NaOH aqueous solution, and dilute to 20 mL with pure water to obtain a solution with a pH value of 12.1. Take 6 portions of 2 mL of the solution with a pH value of 12.1, mix them thoroughly with 1 mL of 100 mg / L coumarin solution, and heat them in a 60°C water bath for 30 minutes. Next, add different volumes of 0.2 mol / L boric acid aqueous solution and pure water to the 6 mixed solutions to adjust the pH value, as follows:

[0058] ① Add 17mL of 0.2mol / L boric acid aqueous solution and 0mL of pure water to obtain a solution with a pH of about 7.4.

[0059] ② Add 14 mL of 0.2 mol / L boric acid aqueous solution and 3 mL of pure water to obtain a solution with a pH of about 7.6.

[0060] ③ Add 10mL of 0.2mol / L boric acid aqueous solution and 7mL of pure water to obtain a solution with a pH of about 7.9.

[0061] ④ Add 6 mL of 0.2 mol / L boric acid aqueous solution and 11 mL of pure water to obtain a solution with a pH of about 8.2.

[0062] ⑤ Add 2mL of 0.2mol / L boric acid aqueous solution and 15mL of pure water to obtain a solution with a pH of about 8.8.

[0063] ⑥ Add 0mL of 0.2mol / L boric acid aqueous solution and 17mL of pure water to obtain a solution with a pH of about 11.1. Subsequently, add 0.5mL of 0.5% 2,6-dichloroquinone-4-chloroimine ethanol solution to the above solution for color development, and measure the absorption spectrum in the range of 400-800nm ​​at room temperature after 1, 2, 3, 4, 6, 8, 10, 12 and 24 hours. Repeat the experiment 3 times.

[0064] like Figure 5 As shown in the figure, the pH value in the color development stage has a significant effect on the absorption spectrum of the solution. Under different pH conditions, after adding the color developer for 1 hour, each solution showed an absorption peak near 610nm, and when the pH value increased from 7.4 to 11.1, the absorption peak gradually increased. In addition, under high pH conditions (pH 11.1), the solution also showed an absorption peak near 450nm.

[0065] The changes in the maximum absorbance around 610 nm were further analyzed (see Figure 6), between pH 7.4 and 8.8, the maximum absorbance value increases rapidly with the increase of pH; while between pH 8.8 and 11.1, although the maximum absorbance value still increases, the increase rate decreases. Under different color development pH conditions, the changes of solution absorption peak and maximum absorbance value with storage time are shown in the figure. Figure 7 and Figure 8 As shown. When the pH value of the solution in the color development stage is between 7.4 and 8.2, the absorption peak gradually shifts to the long wavelength (645nm) over time; at pH 8.8, the absorption peak remains near 610nm within 24 hours; at pH 11.1, the absorption peak gradually shifts to 600nm. Similar to the change in the absorption peak, when the pH value is between 7.4 and 8.2, the maximum absorbance value rises sharply within 1 to 10 hours after color development, and the change range is small within 10 to 24 hours; at pH 8.8, the maximum absorbance value rises slowly within 1 to 10 hours, and decreases slightly from 10 to 24 hours; at pH 11.1, the maximum absorbance value remains relatively stable. In summary, when the pH value is 8.8, it is conducive to maintaining the stability of the product in the sample solution, while too low or too high pH values ​​have a significant effect on the stability of the reaction product. Therefore, pH 8.8 was determined to be the optimal pH condition for color development.

[0066] Example 5 Verification of whether the absorption peak near 450nm is caused by coumarin

[0067] From the above Figure 5 It can be seen that when the color development pH condition is 11.1, an absorption peak appears near 450nm, so it is verified whether the absorption peak is caused by coumarin.

[0068] The specific operation is as follows: take 5mL of 0.05mol / L borax aqueous solution, add 4.3mL of 0.2 mol / L NaOH aqueous solution, and then dilute to 20mL with pure water to obtain a solution with a pH of 12.1. Then, take out 4 portions of 2mL each from the solution with a pH of 12.1, add 1mL of 100mg / L coumarin solution to two of them and mix thoroughly, and add 1mL of pure water to the other two as negative controls. Heat all samples in a 60℃ water bath for 30 minutes and cool naturally to room temperature. Then, add 2mL of 0.5% boric acid aqueous solution and 15mL of pure water to each of the experimental solution and the negative control solution to adjust the pH to about 8.8; and add 17mL of pure water (pH is about 11.1) to each of the other experimental solution and the negative control solution. Next, 0.5 mL of 0.5% 2,6-dichloroquinone-4-chloroimine ethanol solution was added, color was developed at room temperature for 1 hour, and finally the absorption spectrum in the range of 400-800 nm was measured. The experiment was repeated 3 times.

[0069] The experimental results are as follows Figure 8As shown. At pH 8.8 and 12.1, the negative control group showed absorption peaks near 450nm, and at pH 12.1, the absorption peak of the negative control group was significantly higher than that at pH 8.8. This indicates that in the experimental group with a pH of 12.1, the absorption peak near 450nm may be caused by 2,6-dichloroquinone-4-chloroimide, rather than coumarin.

[0070] Example 6 Determination of the linear equation for peak area and coumarin concentration

[0071] Take 5mL of 0.05mol / L biological borax aqueous solution, add 4.3mL of 0.2mol / L NaOH aqueous solution, and dilute to 20mL with pure water to obtain a pH = 12.1 solution. Take 2mL of the above pH 12.1 solution for a total of 6 portions, mix with 1mL of 10, 50, 100, and 200mg / L coumarin solution, respectively, place in a 60℃ water bath and heat for 30 minutes. After cooling to room temperature, add 2mL of 0.2mol / L boric acid aqueous solution and 15mL of pure water to adjust the pH to about 8.8, and add 0.5mL of 0.5% 2,6-dichloroquinone-4-chloroimine ethanol solution, let stand at room temperature for 1 hour for color development, and obtain color standard solutions of different concentrations. Next, use UC7010 high-performance capillary electrophoresis instrument for sample analysis. The electrophoresis detection conditions are as follows: Borax-boric acid buffer (0.05 mol / L borax aqueous solution and 0.2 mol / L boric acid aqueous solution mixed in a ratio of 8:2, pH = 9.0) is used, and a quartz capillary with an inner diameter of 0.75 μm and an effective length of about 30 cm is used. The sample is injected using an automatic sampler with an injection time of 5 seconds and an injection pressure of 5 bar, followed by electrophoresis at a voltage of 8.5 kV for 30 minutes; an ultraviolet-visible light detector is used, and the detection wavelength is set to 610 nm. The standard capillary electrophoresis pattern of coumarin is Figures 10 to 13 As shown in Figure 2, the retention time of coumarin is about 18 minutes. Fig.14 As shown, there is a good linear relationship between the peak area and the coumarin concentration, the regression equation is y = 234.05x + 72.339, and the correlation coefficient R 2 =0.9974.

[0072] Example 7: Detection of coumarin content in plant seeds using the method of the present invention

[0073] The method of the present invention is used to detect the coumarin content in seeds of two plants, alfalfa and red clover.

[0074] The experimental steps are as follows: weigh 0.2 grams of seeds, grind them into powder in liquid nitrogen, add 1 mL of 50% ethanol solution, mix well, centrifuge at 10,000 rpm for 10 minutes, and take the supernatant as the sample solution for coumarin concentration analysis. Each seed is repeated three times.

[0075] Take 5mL of 0.05mol / L borax aqueous solution, add 4.3mL of 0.2 mol / L NaOH aqueous solution, and dilute to 20mL with pure water to obtain a reaction buffer solution with pH = 12.1. Add 2mL of the above reaction buffer solution to 1mL of the sample solution to be tested, place it in a 60℃ water bath and heat it for 30 minutes. After cooling to room temperature, add 2mL of 0.2mol / L boric acid aqueous solution, 15mL of pure water and 0.5mL of 0.5% 2,6-dichloroquinone-4-chloroimine ethanol solution; after standing at room temperature for 1 hour, obtain the color sample solution to be tested; use UC7010 high-efficiency capillary electrophoresis instrument to analyze the color sample solution to be tested, and obtain the sample capillary electrophoresis spectrum, see Fig.14 and 15 ; The electrophoresis detection conditions are as follows: Borax-boric acid buffer (0.05 mol / L borax aqueous solution and 0.2 mol / L boric acid aqueous solution are mixed in a ratio of 8:2, pH = 9.0) is used, and a quartz capillary with an inner diameter of 0.75 μm and an effective length of about 30 cm is used. The sample is injected using an automatic sampler, the injection time is 5 seconds, the injection pressure is 5 bar, and then the electrophoresis is performed at a voltage of 8.5 kV for 25 minutes. A UV-visible light detector is used, and the detection wavelength is set to 610 nm.

[0076] like Fig.15 and 16 The capillary electrophoresis patterns of alfalfa and red clover seed samples are shown. Figures 10 to 13 The absorption peak position was compared with the standard capillary electrophoresis pattern of to determine that both seed samples contained coumarin. Then, the peak area of ​​coumarin in the sample capillary electrophoresis pattern was used to calculate the coumarin concentration in the sample solution according to the linear equation constructed in Example 6, thereby calculating the coumarin content in the plant seeds. The results are shown in Table 1.

[0077] Fig.15 and Fig.16 The capillary electrophoresis patterns showed that the target peak of coumarin was successfully separated in the samples of alfalfa and red clover seeds at about 18 minutes of electrophoresis. The coumarin contents of alfalfa and red clover seeds were calculated to be 608.2 μg / g and 26.8 μg / g, respectively (see Table 1).

[0078] In addition, the absorption spectrum of the above-mentioned colorimetric sample solution to be tested in the range of 400-800nm ​​was measured by an enzyme marker. The absorption spectra of different seed samples were as follows: Fig.17 shown. Fig.17 The results showed that the alfalfa seed sample had an absorption peak near 610nm, which was similar to the coumarin standard solution. However, in the region with wavelengths less than 540nm and greater than 700nm, the absorbance of alfalfa seeds was significantly higher than that of the coumarin standard solution, especially below 450nm, where the absorbance increased sharply with decreasing wavelength; in contrast, the absorbance of red clover seeds showed a gradual downward trend in the wavelength range of 400-800nm, and the absorption peak at 610nm was not obvious. This indicates that the absorption spectra of alfalfa and red clover seeds are different from those of the coumarin standard solution, indicating that there may be other interfering substances in the samples, so the direct use of colorimetry to determine the coumarin concentration in plant samples may not be accurate enough.

[0079] Table 1 Determination results of coumarin content in alfalfa and red clover seeds

[0080]

Claims

1. A method for detecting coumarin, characterized in that, The following steps are involved: (1) Prepare the following solutions: borax aqueous solution, NaOH aqueous solution, boric acid aqueous solution, and 2,6-dichloroquinone-4-chloroimine ethanol solution; (2) taking 1 volume of borax aqueous solution, adjusting the pH value of the borax aqueous solution to 11.8-12.2 with a NaOH aqueous solution, and making the volume to 4 volumes with pure water to obtain a reaction buffer solution; (3) The reaction buffer solution and the sample solution to be tested are mixed evenly in a volume ratio of 2:1, and reacted in a water bath for 20 to 30 minutes; (4) adjusting the pH value of the solution after the reaction in step (3) to 8.6-9.0 using a boric acid aqueous solution and pure water, adding a 2,6-dichloroquinone-4-chloroimine ethanol solution, shaking well, and placing at room temperature for more than 1 hour for color development to obtain a color sample solution to be tested; (5) preparing a coumarin standard solution of known concentration, performing water bath heating reaction and color development treatment according to the above steps (3) to (4) to obtain a color development standard solution, using a capillary electrophoresis instrument with an ultraviolet-visible light detector to detect the color development standard solution to obtain a standard capillary electrophoresis spectrum, and determining the absorption peak of coumarin; then using the above capillary electrophoresis instrument to detect the color development sample solution to be tested to obtain a sample capillary electrophoresis spectrum, judging whether there is an absorption peak of coumarin therein, thereby determining whether the sample solution to be tested contains coumarin.

2. The method for detecting coumarin according to claim 1, characterized in that In step (5), coumarin standard solutions of different concentrations are prepared, and after water bath heating reaction and color development treatment according to the above steps (3) to (4), color development standard solutions of different concentrations are obtained, and the color development standard solutions are detected by a capillary electrophoresis instrument with an ultraviolet-visible light detector to obtain standard capillary electrophoresis patterns of different coumarin concentrations; then, according to the standard capillary electrophoresis patterns of different coumarin concentrations, a standard curve between the peak area of ​​coumarin and its concentration is established to obtain a linear equation between the peak area of ​​coumarin and its concentration; finally, according to the peak area of ​​coumarin in the sample capillary electrophoresis pattern, the coumarin concentration in the sample solution to be tested is calculated using the above linear equation.

3. The method for detecting coumarin according to claim 2, characterized in that In step (1), the following solutions are prepared: 0.05 mol / L borax aqueous solution, 0.1-0.2 mol / L NaOH aqueous solution, 0.05-0.2 mol / L boric acid aqueous solution, and 0.5 wt% 2,6-dichloroquinone-4-chloroimine ethanol solution.

4. The method for detecting coumarin according to claim 3, characterized in that In step (2), the pH value of the borax aqueous solution is adjusted to 12.1 using a NaOH aqueous solution.

5. The method for detecting coumarin according to claim 4, characterized in that In step (3), the water bath heating temperature is 60-70°C.

6. The method for detecting coumarin according to claim 5, characterized in that: In step (4), the pH value of the mixed solution of step (3) is adjusted to 8.8 using a boric acid aqueous solution; the amount of 2,6-dichloroquinone-4-chloroimine ethanol solution added is 1 to 2.5% by volume.

7. The method for detecting coumarin according to claim 6, characterized in that: The detection conditions of the capillary electrophoresis instrument are as follows: borax-boric acid buffer solution is used, and a quartz capillary with an inner diameter of 0.75 μm and an effective length of 30 cm is used; the injection method is automatic injection, the injection time is 5 seconds, and the injection pressure is 5 bar; after injection, electrophoresis separation is performed at a voltage of 8.5 kV and room temperature for 25 minutes; the detection wavelength of the UV-visible detector is 610-620 nm.

8. The method for detecting coumarin according to claim 7, characterized in that: In step (5), 10 mg / L, 50 mg / L, 100 mg / L, and 200 mg / L coumarin standard solutions are used to establish a standard curve between the peak area and concentration of coumarin.

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