A method for detecting coumarin

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application particularly relates to a method for detecting coumarin. BACKGROUND

[0002] Coumarin (1,2-benzopyrone or 2H-1-benzopyran-2-one) is a phenolic compound, which is widely present in higher plants, especially in the seeds, roots and leaves of dicotyledonous plants, and is found in a small number of animals and microorganisms. Coumarin has multiple biological activities in plants, which not only affects the seed development of plants, but also affects many physiological and biochemical processes of plants, thus having broad application prospects in the field of agriculture. First, coumarin has a function similar to plant hormones and can act as a plant growth regulator to some extent. Second, coumarin has high inhibitory activity on the germination of plant seeds including alfalfa, Arabidopsis thaliana, Italian ryegrass, barnyard grass, red clover, stinkgrass, amaranthus, polygonum, bidens, lettuce, plantain, amaranthus retroflexus, sudangrass and brome, and has the potential to be developed into an environmentally friendly herbicide. In addition, coumarin can also be used as an intermediate for synthesizing other herbicides. The phenoxy pyridine compound synthesized by using coumarin as a lead compound exhibits high herbicidal activity. Field tests show that when the effective ingredient dosage is 300 g / ha, it can effectively control weeds such as barnyard grass, eclipta, crabgrass, crabgrass and amaranthus.

[0003] In addition to its application in agriculture, coumarin is also an important industrial raw material, which is widely used in cosmetics, optical whitening agents, dispersed fluorescent dyes, laser dyes and food industry and many other fields. In the medical field, the research on coumarin and its derivatives shows that it has multiple biological activities such as anti-tumor, anti-HIV, antibacterial, anti-inflammatory and anti-coagulation, and also has application potential in photochemical therapy.

[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, these existing detection methods have high detection cost. For example, the commonly used liquid chromatography method needs to use high-priced chromatographic grade methanol or acetonitrile as the mobile phase. The mobile phase is a continuous consumption reagent in the detection process. According to the calculation of 1 mL of mobile phase per minute and 20 minutes of sample analysis time, 2 L of mobile phase is consumed for analyzing 100 samples, and 2 L of organic waste liquid is formed, which needs to be further recovered and treated, thereby increasing the cost of waste liquid treatment. SUMMARY

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

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows.

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

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

[0009] (2) preparing reaction buffer solution: taking 1 volume of aqueous borax solution, adjusting the pH value of the aqueous borax solution to 11.8-12.2 with aqueous NaOH solution, and diluting with pure water to 4 volumes to obtain the reaction buffer solution;

[0010] (3) mixing the reaction buffer solution and the sample solution to be detected uniformly at a volume ratio of 2:1, and reacting under water bath heating condition for 20-30 min;

[0011] (4) adjusting the pH value of the solution after step (3) to 8.6-9.0 with aqueous boric acid solution and pure water, adding 2,6-dichloroquinone-4-chloroimine ethanol solution, shaking uniformly, and standing at room temperature for more than 1 h for color development to obtain the sample solution to be detected;

[0012] (5) preparing coumarin standard solution with known concentration, carrying out water bath heating reaction and color development treatment according to the above steps (3)-(4) to obtain the color standard solution, detecting the color standard solution with a capillary electrophoresis instrument with ultraviolet-visible light detector to obtain the standard capillary electrophoresis spectrum, determining the absorption peak of coumarin, then detecting the sample solution to be detected with the above capillary electrophoresis instrument to obtain the sample capillary electrophoresis spectrum, judging whether there is the absorption peak of coumarin in the sample capillary electrophoresis spectrum, so as to determine whether the sample solution to be detected contains coumarin.

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

[0014] The present application is suitable for detecting various sample solutions containing impurities, such as extract solutions of different tissue parts of plant seeds, roots, stems, leaves, medicine and environmental sample solutions, etc.

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

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

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

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

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

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

[0021] The blue charged compound with the absorption peak at the visible wavelength obtained by the color development reaction is separated and detected by using the capillary electrophoresis technology, so that the quantitative analysis of coumarin is realized.

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

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

[0024] The application provides a coumarin detection technical scheme based on capillary electrophoresis.

[0025] Compared with liquid chromatography, the method provided by the application does not need a mobile phase in the analysis process, and only needs a solution prepared from borax and boric acid as an electrophoresis buffer solution.

[0026] In addition, the application needs a short time for sample pretreatment, and can be completed within one and a half hours at least, greatly improving the detection efficiency of coumarin.

[0027] The method provided by the application 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. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Effects of different pH values on the absorption spectrum of a sample solution in the lactone ring opening stage of coumarin are shown;

[0029] Figure 2 Effects of different pH values on the maximum absorption value of a sample solution in the lactone ring opening stage are shown;

[0030] Figure 3 Effects of water bath heating and normal temperature treatment under different pH conditions on the absorption value of a sample solution are shown;

[0031] Figure 4 Effects of different water bath temperatures on the absorption value of a sample solution at pH 9.4 are shown;

[0032] Figure 5 Effects of different pH values on the absorption spectrum of a sample solution in the coloration stage are shown;

[0033] Figure 6 Effects of different pH values on the maximum absorption value of a sample solution in the coloration stage are shown;

[0034] Figure 7 Changes of the absorption peak of a sample solution with time under different coloration pH conditions are shown;

[0035] Figure 8 Changes of the maximum absorption value of a sample solution with time under different coloration pH conditions are shown;

[0036] Figure 9 Absorption spectrum of coumarin and negative control at color developing pH 8.8 and 11.1;

[0037] Figure 10 Standard capillary electrophoresis profile of 200 mg / L coumarin;

[0038] Figure 11 Standard capillary electrophoresis profile of 100 mg / L coumarin;

[0039] Figure 12 Standard capillary electrophoresis profile of 50 mg / L coumarin;

[0040] Figure 13 Capillary electrophoresis profile of 10 mg / L coumarin;

[0041] Figure 14 Linear regression analysis of capillary electrophoresis peak area and coumarin concentration;

[0042] Figure 15 Capillary electrophoresis profile of alfalfa seed sample;

[0043] Figure 16 Capillary electrophoresis profile of red clover seed sample;

[0044] Figure 17 Absorption spectrum of different seed samples. DETAILED DESCRIPTION

[0045] The application will be further described below in conjunction with the specific embodiments. It should be noted that the following described embodiments or technical features can be combined in any manner to form new embodiments without conflict. The equipment and materials used can be purchased from the market or are commonly used in the art. The methods in the following examples are conventional methods in the art unless otherwise specified.

[0046] Example 1 Determination of optimal pH conditions for opening of coumarin lactone ring

[0047] To determine the optimal pH conditions for opening the coumarin lactone ring, i.e., the optimal pH conditions for the reaction buffer, five 5 mL aliquots of 0.05 mol / L borax aqueous solution were prepared, and 0.6, 2.3, 3.4, 4.3, and 4.6 mL of 0.2 mol / L NaOH aqueous solution were added respectively. The solutions were then diluted to 20 mL with pure water, resulting in a series of solutions with pH values ​​of 9.4, 10.4, 11.8, 12.1, and 12.2. Two mL of each of these solutions with different pH values ​​were mixed thoroughly with 1 mL of 100 mg / L coumarin solution and incubated in a 60°C water bath for 30 minutes. After cooling to room temperature, 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. After standing for 1 hour, the absorption spectrum in the 400-800 nm range was measured using a Multiskan SkyHigh full-wavelength microplate reader. The experiment was repeated three times.

[0048] During the lactone ring opening phase, 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 exhibited an absorption peak near 610 nm. Within the pH range of 9.4 to 12.1, the absorption peak gradually increased with increasing pH; however, after reaching pH 12.1, the absorption peak showed an inflection point and subsequently began to decrease. Figure 2 The maximum absorbance of the sample solution near 610 nm is shown. Between pH 9.4 and 10.4, the absorbance increases rapidly with increasing pH; however, between pH 10.4 and 12.1, although the absorbance continues to rise, the rate of increase slows significantly. At pH 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 opening the coumarin lactone ring.

[0049] Example 2: Determining the reaction conditions for opening the coumarin lactone ring

[0050] To determine whether water bath heating is a necessary condition for the opening of the coumarin lactone ring under different pH conditions, the following experiment was performed: First, 5 portions of 5 mL of 0.05 mol / L borax aqueous solution were prepared, and 0.6, 2.3, 3.4, 4.3, and 4.6 mL of 0.2 mol / L NaOH aqueous solution was added, respectively, and then diluted 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, 2 mL of each of the above solutions with different pH values was mixed with 1 mL of 100 mg / L coumarin solution, and the mixture was divided into two groups for experiment: one group was placed at room temperature for 30 minutes, and the other group was 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 were added to the solution of each group, and the absorbance value of the solution at 610 nm was measured after 1 hour of standing. The experiment was repeated 3 times, and the results are shown in Table 1. Figure 3

[0051] The results show that, between pH values of 9.4 to 12.1, the absorbance value of the water bath treatment group is significantly higher than that of the room temperature treatment group; while at pH 12.2, the absorbance values of the two groups have little difference. Therefore, when the pH is less than 12.2, 60°C water bath heating significantly promotes the opening of the coumarin lactone ring.

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

[0053] To determine the effect of different heating temperatures on the opening of the coumarin lactone ring, the following experiment was performed:

[0054] First, 5 mL of 0.05 mol / L borax aqueous solution was prepared, and 0.6 mL of 0.2 mol / L NaOH aqueous solution was added, and then diluted to 20 mL with pure water to obtain a solution with a pH value of 9.4. Then, 3 portions of the above pH 9.4 solution, each 2 mL, were mixed with 1 mL of 100 mg / L coumarin solution, and the mixture was divided into 3 groups for experiment: one group was placed at 25°C for 30 minutes, the second group was placed in a 60°C water bath for 30 minutes, and the third group was 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, and the absorbance value of the solution at 610 nm was measured after 1 hour of standing; the experiment was repeated 3 times. The results are shown in Table 2. Figure 4

[0055] The results show that the higher the heating temperature, the higher the absorbance value of the solution, indicating that at a lower pH, increasing the heating temperature can promote the opening of the coumarin lactone ring.

[0056] ​​Example 4 Determination of the pH of the solution in the color development stage

[0057] To determine the optimal pH of the color development stage and its effect on the stability of the sample solution, the following experiment was performed. 5 mL of a 0.05 mol / L aqueous borax solution was taken, 4.3 mL of a 0.2 mol / L aqueous NaOH solution was added, and the volume was made up to 20 mL with pure water to obtain a solution with a pH of 12.1. 2 mL of this solution with a pH of 12.1 was taken in six portions and mixed thoroughly with 1 mL of a 100 mg / L coumarin solution, and heated in a 60°C water bath for 30 minutes. Next, different volumes of a 0.2 mol / L aqueous boric acid solution and pure water were added to the six mixed solutions to adjust the pH, as follows:

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

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

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

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

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

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

[0064] As shown in Figure 5 , the effect of the pH in the color development stage on the absorption spectrum of the solution is obvious. Under different pH conditions, an absorption peak appeared near 610 nm in each solution after 1 hour of adding the color developer, and the absorption peak gradually increased as the pH increased from 7.4 to 11.1. In addition, under high pH conditions (pH 11.1), an absorption peak also appeared near 450 nm in the solution.

[0065] The change in the maximum absorbance near 610 nm was further analyzed (see Figure 6), the maximum absorbance increased rapidly with the increase of pH between pH 7.4 and 8.8; while between pH 8.8 and 11.1, the maximum absorbance was still increasing but the increasing rate was reduced. The changes of the absorption peak and the maximum absorbance of the solution with the standing time under different color developing pH conditions are shown in Figure 7 and Figure 8 When the pH of the solution during the color developing stage was between 7.4 and 8.2, the absorption peak gradually shifted to longer wavelength (645 nm) with the extension of time; while at pH 8.8, the absorption peak remained around 610 nm within 24 hours; and at pH 11.1, the absorption peak gradually shifted to 600 nm. Similarly to the change of the absorption peak, when the pH was between 7.4 and 8.2, the maximum absorbance increased sharply within 1 to 10 hours after the color development, and the change was small within 10 to 24 hours; at pH 8.8, the maximum absorbance increased slowly within 1 to 10 hours, and decreased slightly within 10 to 24 hours; and at pH 11.1, the maximum absorbance remained relatively stable. In summary, the pH of 8.8 is favorable for maintaining the stability of the product in the sample solution, and the pH that is too low or too high has a significant impact on the stability of the reaction product. Therefore, pH 8.8 is determined as the optimal color developing pH condition.

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

[0067] As can be seen from the above Figure 5 , the absorption peak near 450 nm appears when the color developing pH condition is 11.1, so it is verified whether the absorption peak is caused by coumarin.

[0068] The specific operation is as follows: 5 mL of 0.05 mol / L borax aqueous solution is taken, 4.3 mL of 0.2 mol / L NaOH aqueous solution is added, and then pure water is added to 20 mL to obtain a solution with pH of 12.1. Then, 4 portions of 2 mL of the solution with pH of 12.1 are taken, among which two portions are added with 1 mL of 100 mg / L coumarin solution and mixed thoroughly, and the other two portions are added with 1 mL of pure water as negative control. All the samples are placed in a 60°C water bath for heating for 30 minutes, and then naturally cooled to room temperature. Then, 2 mL of 0.5% boric acid aqueous solution and 15 mL of pure water are added to 1 portion of the experimental solution and 1 portion of the negative control solution respectively, and the pH is adjusted to about 8.8; while 17 mL of pure water (pH about 11.1) is added to the other 1 portion of the experimental solution and 1 portion of the negative control solution respectively. Then, 0.5 mL of 0.5% 2,6-dichloroquinone-4-chloroimine ethanol solution is added, and the color is developed at room temperature for 1 hour, and finally the absorption spectrum in the range of 400-800 nm is measured. The experiment is repeated for 3 times.

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

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

[0071] Take 5 mL of 0.05 mol / L aqueous sodium borate solution, add 4.3 mL of 0.2 mol / L aqueous NaOH solution, and dilute with pure water to 20 mL to obtain a solution with pH = 12.1. Take 2 mL of the above-mentioned pH 12.1 solution in 6 portions, and mix with 1 mL of 10, 50, 100, 200 mg / L coumarin solution, respectively, and place in a 60°C water bath for heating for 30 minutes. After cooling to room temperature, add 2 mL of 0.2 mol / L aqueous boric acid solution, 15 mL of pure water to adjust the pH to about 8.8, and 0.5 mL of 0.5% 2,6-dichloroquinone-4-chloroimine ethanol solution, and stand at room temperature for 1 hour for color development to obtain color standard solutions of different concentrations. Next, use a UC7010 high-performance capillary electrophoresis instrument to analyze the samples. The electrophoresis detection conditions are as follows: use borate-boric acid buffer (0.05 mol / L aqueous sodium borate solution and 0.2 mol / L aqueous boric acid solution mixed in a ratio of 8:2, pH = 9.0), use a quartz capillary with an inner diameter of 0.75 μm and an effective length of about 30 cm. Use an automatic sampler for sampling, the sampling time is 5 seconds, the sampling pressure is 5 bar, and then perform electrophoresis at a voltage of 8.5 kV for 30 minutes; use a UV-visible light detector, and set the detection wavelength to 610 nm. The standard capillary electrophoresis spectrum of coumarin is shown in Figure 2. Figures 10 to 13 As shown in Figure 2, the retention time of coumarin is about 18 minutes. As shown in Figure 3, Figure 14 The peak area and the coumarin concentration show a good linear relationship, and the regression equation is y = 234.05x + 72.339, and the correlation coefficient R 2 = 0.9974.

[0072] Example 7 applies the method of the present application to detect the coumarin content in plant seeds

[0073] The coumarin content in the seeds of two plants, alfalfa and red clover, is detected by the method of the present application.

[0074] The experimental procedure is as follows: 0.2 g of seeds is weighed, ground into powder in liquid nitrogen, 1 mL of 50% ethanol solution is added, mixed and centrifuged at 10000 rpm for 10 minutes, and the supernatant is taken as the sample solution for coumarin concentration analysis. Each seed is repeated three times.

[0075] 5 mL of 0.05 mol / L aqueous borax solution, 4.3 mL of 0.2 mol / L aqueous NaOH solution, and pure water to make 20 mL, to obtain a reaction buffer with pH = 12.1. In 1 mL of the sample solution to be tested, 2 mL of the above reaction buffer is added, heated in a 60°C water bath for 30 minutes, cooled to room temperature, and then 2 mL of 0.2 mol / L aqueous boric acid solution, 15 mL of pure water, and 0.5 mL of 0.5% 2,6-dichloroquinone-4-chloroimine ethanol solution are added; after standing at room temperature for 1 hour, the color developing sample solution to be tested is obtained; the color developing sample solution to be tested is analyzed using a UC7010 high-performance capillary electrophoresis instrument to obtain a sample capillary electrophoresis spectrum, as shown in Figure 14 and 15 ; wherein the electrophoretic detection conditions are as follows: a borax-boric acid buffer (0.05 mol / L aqueous borax solution and 0.2 mol / L aqueous boric acid 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. An automatic sampler is used for sampling, the sampling time is 5 seconds, the sampling pressure is 5 bar, and then electrophoresis is carried out at a voltage of 8.5 kV for 25 minutes. An ultraviolet-visible light detector is used, and the detection wavelength is set to 610 nm.

[0076] As shown in Figure 15 and 16 , the capillary electrophoresis spectra of alfalfa and red clover seed samples, respectively, contain coumarin, as compared with the standard capillary electrophoresis spectrum of Figures 10 to 13 . Then the coumarin concentration in the sample solution to be tested is calculated using the peak area of coumarin in the sample capillary electrophoresis spectrum, and the coumarin content in the plant seeds is calculated according to the linear equation constructed in Example 6, and the results are shown in Table 1.

[0077] Figure 15 and Figure 16 The capillary electrophoresis spectra of alfalfa and red clover seed samples show that the target peak of coumarin is successfully separated at about 18 minutes of electrophoresis. Through calculation, the coumarin contents of alfalfa and red clover seeds are 608.2 μg / g and 26.8 μg / g, respectively (see Table 1).

[0078] In addition, the absorption spectrum of the above color developing sample solution to be tested in the range of 400-800 nm is measured using a microplate reader, and the absorption spectra of different seed samples are as shown inFigure 17 The results are shown in Figure 1. Figure 17 It is shown that the alfalfa seed sample has an absorption peak near 610 nm, similar to the coumarin standard solution. However, in the region of wavelength less than 540 nm and greater than 700 nm, the absorbance of the alfalfa seed is significantly higher than that of the coumarin standard solution, especially below 450 nm, the absorbance increases sharply with the decrease of wavelength; in contrast, the absorbance of the red clover seed shows a gradually decreasing trend in the wavelength range of 400-800 nm, and the absorption peak at 610 nm is not obvious. This indicates that the absorption spectrum of alfalfa and red clover seeds is different from that of the coumarin standard solution, indicating that there may be other interfering substances in the sample, so the direct use of colorimetry to determine the concentration of coumarin 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, Includes the following steps: (1) Prepare the following solutions: borax aqueous solution, NaOH aqueous solution, boric acid aqueous solution, and 2,6-dichloroquinone-4-chloroimine ethanol solution; (2) 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; (3) The reaction buffer solution and the sample solution to be tested are mixed evenly at a volume ratio of 2:1, and reacted under water bath heating for 20 to 30 minutes. (4) The pH of the solution after the reaction in step (3) was adjusted to 8.6-9.0 using boric acid aqueous solution and pure water, and 2,6-dichloroquinone-4-chloroimine ethanol solution was added. After shaking well, the solution was placed at room temperature for more than 1 hour to develop color and obtain the colorimetric sample solution to be tested. (5) Prepare a standard solution of coumarin of known concentration. After water bath heating reaction and color development treatment according to the above steps (3) to (4), a color development standard solution is obtained. The color development standard solution is detected by a capillary electrophoresis instrument with a UV-Vis detector to obtain a standard capillary electrophoresis pattern and determine the absorption peak of coumarin. Then, the color development sample solution to be tested is detected by the above capillary electrophoresis instrument to obtain a sample capillary electrophoresis pattern and determine whether there is an absorption peak of coumarin in it, 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. After water bath heating reaction and color development treatment according to steps (3) to (4) above, color development standard solutions of different concentrations are obtained. The color development standard solutions are detected by a capillary electrophoresis instrument with a UV-Vis 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 of ​​coumarin and its concentration is established to obtain the linear equation between the peak area of ​​coumarin and its concentration. Finally, based on the peak area of ​​coumarin in the sample capillary electrophoresis pattern, the concentration of coumarin 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 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℃.

6. The method for detecting coumarin according to claim 5, characterized in that, In step (4), the pH of the mixed solution from step (3) is adjusted to 8.8 using an aqueous boric acid 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 for the capillary electrophoresis system were as follows: borax-boric acid buffer solution was used, and a quartz capillary with an inner diameter of 0.75 μm and an effective length of 30 cm was employed; the sample introduction method was automatic, with an introduction time of 5 seconds and an introduction pressure of 5 bar; after the sample was introduced, electrophoretic separation was performed at 8.5 kV voltage and room temperature for 25 minutes; the detection wavelength of the ultraviolet-visible detector was 610–620 nm.

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

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