Method for rapidly detecting hesperidin modified derivative

Abstract

The invention relates to the technical field of drug detection, in particular to a method for rapidly detecting hesperidin modified derivatives, which comprises the following steps: reacting a sample with a reaction reagent, performing ultraviolet-visible light detection, and determining the proportion of each hesperidin modified derivative in the sample, the reaction reagent comprises an aluminum salt reagent. The invention innovatively provides a method for rapidly detecting the ratio of 3 ', 5-dimethyl hesperidin to 3'-methyl hesperidin in hesperidin modification derivatization, and the method is simple and convenient to operate and good in safety, and can be mastered by general technicians. According to the method, the sample treatment process is simple, the detection target specificity is high, the hesperidin modified derivative can be rapidly detected, the problem that in the hesperidin modified derivative composition preparation process, due to different methylation degrees, the monomer proportion batch fluctuation range is large is solved, and the detection accuracy is high. The preparation method of the hesperidin modified derivatization composition can be matched with the hesperidin modified derivatization composition to prepare the hesperidin modified derivatization composition.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and particularly relates to a method for rapidly detecting modified derivatives of hesperidin. Background Art

[0002] Citrus flavonoids are a class of benzene-γ-pyrone derivatives widely present in citrus plants. Their basic structure is a "flavan nucleus" skeleton (C6-C3-C6), which is composed of two aromatic rings connected by a linear 3-carbon chain. Among them, polymethoxyflavones are a class of highly methylated flavonoid compounds unique to the citrus genus, with relatively high contents in the peels of Chinese sweet oranges and citrus fruits. Methoxy groups are mostly present at positions 6, 7, 8, 3', and 4', and they have pharmacological activities such as anti-pathogenic microorganisms, anti-mutagenesis, anti-platelet aggregation, and anti-cancer. Hesperidin and its methylated modified derivatives belong to this category and have characteristics such as low polarity, planar structure, and significant biological activity.

[0003] The methylation modification of hesperidin is generally carried out under alkaline conditions. The γ-pyrone ring in the molecule opens to form hesperidin chalcone, which has unstable properties and regenerates hesperidin after acidification. Therefore, the methylation products of hesperidin are a mixture composed of various flavanone and chalcone compounds, and their methylation degrees are also different. In previous studies by the inventors, various flavanone and chalcone modified derivatives of hesperidin were isolated from this mixture. Moreover, the primary and secondary methylation products showed a synergistic effect in the tyrosinase activity influence test. The combination of the two showed better enzyme inhibitory activity than the single use of monomers, and the enzyme inhibitory activity also changed with the change of the ratio of the two in the combination. Therefore, a method for stably detecting the ratio of the two is worthy of exploration.

[0004] Currently, the methods for detecting hesperidin disclosed in patents or literatures mainly include high-performance liquid chromatography and capillary electrophoresis. The detection objects are mostly traditional Chinese medicine compound preparations, which have problems such as long detection time, the need to use relatively expensive instruments, complex sample pretreatment methods, and inapplicability to dynamic detection in industrial production. Moreover, there are no publicly disclosed patents or literatures for simultaneously detecting two modified derivatives of hesperidin; in the preparation process of the composition, the methylation degree of hesperidin cannot be precisely controlled, resulting in a large batch fluctuation range of the ratio of the two main monomers in the composition product. Therefore, there is an urgent need for a method that can rapidly detect the component ratio between these two products in the modified derivatization of hesperidin. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for rapidly detecting the ratio between 3'-methylhesperidin and 3',5-dimethylhesperidin in the modified derivatization of hesperidin, aiming at the blank of the existing technology, and solving the problem of large batch fluctuation range of the monomer ratio caused by different methylation degrees in the preparation process of the composition of the modified derivatives of hesperidin.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for rapidly detecting hesperidin modified derivatives, comprising reacting a sample with a reaction reagent, followed by ultraviolet-visible light detection to determine the proportion of each hesperidin modified derivative in the sample; the reaction reagent includes an aluminum salt reagent.

[0008] Preferably, the reaction reagent includes a methanol solution of aluminum chloride.

[0009] Preferably, methanol is used as the solvent, which has good solubility for both hesperidin derivatives and aluminum chloride.

[0010] Preferably, the concentration of the reaction reagent is 0.5 - 1.5 mg / ml.

[0011] Preferably, the reaction temperature of the reaction is 40 - 60 °C, and the reaction time is 3 - 10 minutes.

[0012] Preferably, the hesperidin modified derivatives include one or more of 3'-methyl hesperidin and 3',5-dimethyl hesperidin.

[0013] Preferably, it includes the following steps:

[0014] A. Dissolve the sample to prepare a sample solution for standby;

[0015] B. Add anhydrous aluminum chloride to methanol to prepare the reaction reagent for standby;

[0016] C. Set the detection parameters of the ultraviolet-visible light detection, and zero with the reaction reagent as the blank solvent;

[0017] D. After adding the reaction reagent to the sample solution, carry out the reaction, and after the reaction is completed, carry out the ultraviolet-visible light detection to obtain the absorbance, substitute it into the standard linear regression formula, and determine the proportion of the hesperidin modified derivative in the sample.

[0018] Preferably, the standard linear regression formula is obtained by carrying out the ultraviolet-visible light detection on the standard curve solution under the same conditions; the standard curve solution includes a standard reference substance solution and a standard test solution; the preparation of the standard curve solution includes:

[0019] a. Weigh the 3'-methyl hesperidin reference substance and dissolve it to prepare a standard reference substance solution A;

[0020] b. Weigh the 3',5-dimethyl hesperidin reference substance and dissolve it to prepare a standard reference substance solution B;

[0021] c. Prepare a standard test solution by mixing the standard reference solution A and the standard reference solution B at a volume ratio of 0.5 - 1.5.

[0022] Preferably, the standard linear regression formula includes:

[0023] S1. Obtain the absorbance by subjecting the standard curve solution to ultraviolet - visible light detection.

[0024] S2. Obtain the peak area by subjecting the standard curve solution to liquid chromatography detection.

[0025] S3. Perform linear regression analysis on the absorbance obtained in S1 and the peak area obtained in S2 to obtain the standard linear regression formula.

[0026] The standard linear regression formula can be a relationship between absorbance and concentration, or a relationship between absorbance and peak area. After obtaining the peak area, it is then converted back to concentration. The advantage of doing this is that there will be systematic errors during the preparation of the sample solution, such as experimental operator errors, balance instrument errors, etc. Therefore, using the liquid chromatography peak area to characterize the actual concentration can reduce errors and make the linear regression formula more accurate.

[0027] Preferably, in step D, the volume ratio of the sample solution to the reaction reagent is 1:30; the ultraviolet - visible light detection includes one or more of ultraviolet - visible light spectral scanning and dual - wavelength detection; the concentrations of the sample solution, the standard reference solution A, and the standard reference solution B are the same.

[0028] When the sample solution and the reaction reagent are mixed in step D, in actual operation, affected by the detection limit and linear range of the ultraviolet instrument, generally, the concentration of the sample solution is first adjusted to below the detection limit of the instrument, and this adjustment ratio is recorded. Then, during the reaction, the reaction reagent is used for adjustment. In practice, the ratio is approximately around 1:30.

[0029] Preferably, the ultraviolet - visible light detection is ultraviolet - visible light spectral scanning, and the parameter settings include: the scanning range is 600 - 190 nm, the photometric mode is Abs, the scanning speed is fast, and the scanning interval is 0.2 nm; the concentrations of the sample solution, the standard reference solution A, and the standard reference solution B are all 1 mg / ml; the reaction is carried out in a thermostatic heating and stirring collector.

[0030] In actual operation, the sample solution, the standard reference solution A, the standard reference solution B, etc. are all initially prepared at a concentration of 1 mg / ml during the test, and then diluted proportionally to below the detection limit of the instrument later.

[0031] Compared with the prior art, implementing the present invention has the following beneficial effects:

[0032] (1) Innovatively provide a method for quickly detecting the ratio between 3’,5-dimethylhesperidin and 3’-methylhesperidin in the modification and derivatization of hesperidin. This method is easy to operate, has good safety, and can be mastered by ordinary technicians.

[0033] (2) The method has a simple sample treatment process, strong specificity for the detection target, can quickly detect the modified derivatives of hesperidin, solves the problem of large batch fluctuations in the monomer ratio due to different methylation degrees in the preparation process of the composition of the modified derivatives of hesperidin, and can be used in the preparation of this composition in combination with the preparation method of the modified derivatives of hesperidin. Description of the Drawings

[0034] Figure 1 It is the spectral scanning detection result diagram of the API solution;

[0035] Figure 2 It is the spectral scanning detection result diagram of the 3’-methylhesperidin solution;

[0036] Figure 3 It is the spectral scanning detection result diagram of the 3’,5-dimethylhesperidin solution;

[0037] Figure 4 It is the spectral scanning detection result diagram of the 3’-methylhesperidin solution after reacting with the reaction reagent;

[0038] Figure 5 It is the spectral scanning detection result diagram of the 3’,5-dimethylhesperidin solution after reacting with the reaction reagent;

[0039] Figure 6 It is the spectral scanning detection result diagram of the test sample I after heating reaction in Example 2;

[0040] Figure 7 It is the spectral scanning detection result diagram of the test sample II after standing at room temperature in Example 2;

[0041] Figure 8 It is the spectral scanning detection result diagram of the test sample III after heating reaction in Example 2;

[0042] Figure 9 It is the spectral scanning detection result diagram of the test sample IV after heating reaction in Example 2;

[0043] Figure 10 It is the spectral scanning detection result diagram of Example 3 at the 1st minute of the reaction;

[0044] Figure 11 It is the spectral scanning detection result diagram of Example 3 at the 3rd minute of the reaction;

[0045] Figure 12Spectral scan detection result graph for the 5th minute of the reaction in Example 3;

[0046] Figure 13 Spectral scan detection result graph for the 7.5th minute of the reaction in Example 3;

[0047] Figure 14 Spectral scan detection result graph for the 10th minute of the reaction in Example 3;

[0048] Figure 15 Graph of the linear regression analysis result. Detailed implementation manners

[0049] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments.

[0050] For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0051] The present discovery adopts the following technical solutions:

[0052] A method for rapidly detecting hesperidin modified derivatives, comprising the following steps:

[0053] 1. Establishment of the linear relationship formula:

[0054] (1) Turn on the ultraviolet-visible spectrophotometer. After the instrument self-checks, enter the spectral scan function and adjust the parameter settings as follows: the scanning range is 600 - 190 nm; the photometric mode is Abs; the scanning speed is fast; the scanning interval is 0.2 nm.

[0055] (2) Weigh anhydrous aluminum chloride and prepare a reaction reagent with methanol at a concentration of 0.5 mg / ml; weigh the reference substances of 3'-methylhesperidin and 3',5-dimethylhesperidin respectively, dissolve and prepare reference substance solutions A and B respectively, and then quantitatively prepare test sample solutions with different ratios (reference substance solution A / reference substance solution B: 0.5, 0.75, 1, 1.25, 1.5).

[0056] (3) After adding the reaction reagent to the reference substance solution A, reference substance solution B and each test sample solution, immediately place them in a thermostatic heating and stirring device for reaction. The water bath temperature is 50 °C and the reaction time is 3 minutes.

[0057] (4) Place the reaction reagent in a clean cuvette as the blank solvent to zero the baseline of the ultraviolet-visible spectrophotometer. After the test solution reaction is completed, immediately conduct the detection. Based on the ratio of the absorbances at the maximum absorption wavelengths of 3’,5-dimethylhesperidin and 3’-methylhesperidin and the actually prepared ratio, plot a curve and perform linear regression analysis to obtain the linear relationship formula between the ultraviolet absorbance ratio and the actual ratio.

[0058] 2. Rapid detection of modified and derivatized hesperidin samples:

[0059] (1) Take the modified and derivatized hesperidin sample and prepare a 1 mg / ml sample solution for standby.

[0060] (2) Enter the photometric measurement function of the ultraviolet-visible spectrophotometer, set dual-wavelength detection (the dual wavelengths are the maximum absorption wavelengths of 3’,5-dimethylhesperidin and 3’-methylhesperidin respectively), and zero with the reaction reagent (aluminum chloride methanol solution) as the blank solvent.

[0061] (3) Immediately after adding the reaction reagent to the sample solution, place it in a thermostatic heating and stirring device for reaction. The water bath temperature is 50 °C and the reaction time is 3 minutes. Immediately conduct the detection after the reaction is completed, calculate the ratio and use the formula to obtain the actual ratio of the main components in the bulk drug.

[0062] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0063] Example 1

[0064] Under the same detection conditions, compare the differences in the ultraviolet-visible spectra of 3’-methylhesperidin and 3’,5-dimethylhesperidin before and after treatment with aluminum chloride methanol solution. Specifically, it includes the following steps:

[0065] (1) Weigh 4.00 mg of methylhesperidin bulk drug, add 4 ml of purified water to prepare a 1 mg / ml bulk drug solution; weigh 0.61 mg of 3’-methylhesperidin, add 610 μl of purified water to prepare a 1 mg / ml 3’-methylhesperidin solution; weigh 1.57 mg of 3’,5-dimethylhesperidin, add 1.57 ml of purified water to prepare a 1 mg / ml 3’,5-dimethylhesperidin solution.

[0066] (2) Turn on the ultraviolet-visible spectrophotometer, enter the spectral scanning function, and adjust the parameter settings: the scanning range is 600~190 nm; the breadth mode is Abs; the scanning speed is fast; the scanning interval is 0.2 nm. Use purified water as the blank control, add it to the cuvette for baseline adjustment.

[0067] (3) The raw drug solution, 3'-methylhesperidin solution, and 3',5-dimethylhesperidin solution were each diluted to 0.03 mg / mL and then tested. The test results are as Figures 1 to 3 .

[0068] (4) Weighed 14.8 mg of anhydrous aluminum chloride and dissolved it in 30 mL of methanol. Took 120 μL each of the 3'-methylhesperidin solution and 3',5-dimethylhesperidin solution, mixed them evenly with 3880 μL of the aluminum chloride methanol solution, placed them in a thermostatic heating and stirring device with heat collection for reaction. The water bath temperature was 40 °C and the reaction time was 10 minutes. Immediately tested after the reaction was completed. The blank control was a mixture of 120 μL of water and 3880 μL of the aluminum chloride methanol solution. The test results are as Figures 4 to 5 .

[0069] The test results showed that before the reaction, the maximum absorption wavelengths of the raw drug of hesperidin, 3'-methylhesperidin, and 3',5-dimethylhesperidin were the same. According to the current standard of the raw drug [WS-10001(HD-1386)-2003], it was impossible to distinguish the actual contents of 3'-methylhesperidin and 3',5-dimethylhesperidin and the ratio between them by using ultraviolet spectrophotometry. The results after the reaction showed that the aluminum chloride methanol solution could cause a red shift in the maximum absorption wavelength of 3'-methylhesperidin. It should be due to the complexation reaction with aluminum ions, the energy required for π-π* transition decreased, so the energy difference became smaller and a red shift occurred; while the maximum absorption wavelength of 3',5-dimethylhesperidin did not change, which should be because the methyl substitution in the A ring of its parent nucleus structure affected the benzoyl group and could not produce a band II migration.

[0070] Example 2

[0071] Under the conditions of the same substrate and the same reaction time, the reaction temperature of the complexation reaction was optimized. The specific steps were as follows:

[0072] (1) Turned on the ultraviolet-visible spectrophotometer, entered the spectral scanning function, and adjusted the parameter settings: the scanning range was 600 - 190 nm; the bandwidth mode was Abs; the scanning speed was fast; the scanning interval was 0.2 nm. Using a mixture of 120 μL of water and 3880 μL of the aluminum chloride methanol solution as the blank control, added it to the cuvette for baseline adjustment.

[0073] (2) Prepared four test solution samples as follows:

[0074] Test sample I: 120 μL of 3'-methylhesperidin solution was mixed evenly with 3880 μL of the aluminum chloride methanol solution

[0075] Test sample II: 120 μL of 3'-methylhesperidin solution was mixed evenly with 3880 μL of the aluminum chloride methanol solution

[0076] Test sample III: Mix 120 μL of 3′,5-dimethylhesperidin solution evenly with 3880 μL of aluminum chloride methanol solution.

[0077] Test sample IV: Mix 120 μL of 3′,5-dimethylhesperidin solution evenly with 3880 μL of methanol solution.

[0078] (3) Place test samples I, III, and IV in a thermostatic heating and stirring device with a heating coil for reaction. The water bath temperature is 50 °C, and the reaction time is 10 minutes, while test sample II is not heated and is left at room temperature for 10 minutes. Then, immediately perform the detection. The detection results are as Figures 6 to 9 .

[0079] Combining the detection results of this example with those of Example 1 shows that water bath heating can promote the complexation reaction. Under the condition of a 50 °C water bath, the aluminum chloride methanol solution reacts with 3′-methylhesperidin and does not react with 3′,5-dimethylhesperidin.

[0080] Example 3

[0081] Optimize the reaction time of the complexation reaction by the method of controlling variables. The specific steps are as follows:

[0082] (1) Turn on the ultraviolet-visible spectrophotometer, enter the spectral scanning function, and adjust the parameter settings: the scanning range is 450–200 nm; the breadth mode is Abs; the scanning speed is fast; the scanning interval is 0.2 nm.

[0083] (2) Weigh 36.7 mg of anhydrous aluminum chloride, dissolve it in 80 mL of methanol to prepare an aluminum chloride methanol solution; weigh 2.65 mg of 3′-methylhesperidin, dissolve it in 2.65 mL of purified water to prepare a 3′-methylhesperidin solution. Use a mixture of 120 μL of water and 3880 μL of aluminum chloride methanol solution as a blank control, add it to a cuvette for baseline adjustment.

[0084] (3) Take 120 μL of 3′-methylhesperidin solution and mix it evenly with 3880 μL of aluminum chloride methanol solution, immediately place it in a thermostatic heating and stirring device with a heating coil for reaction. The water bath temperature is 50 °C, and perform the detection at the 1st, 3rd, 5th, 7.5th, and 10th minutes respectively. The detection results are as Figures 10 to 14 .

[0085] The detection results show that the optimal reaction time is 3 minutes.

[0086] Example 4

[0087] A method for rapidly detecting the proportion of the main components of methylhesperidin raw material drug. The specific steps are as follows:

[0088] (1) Turn on the ultraviolet-visible spectrophotometer. After the instrument self-checks, enter the spectral scanning function and adjust the parameter settings as follows: the scanning range is 450 - 200 nm; the photometric mode is Abs; the scanning speed is fast; the scanning interval is 0.2 nm.

[0089] (2) Weigh 58.6 mg of anhydrous aluminum chloride and dissolve it in 117.2 mL of methanol to prepare an aluminum chloride methanol solution; weigh 2.85 mg of 3’,5-dimethylhesperidin and dissolve it in 2.85 mL of purified water to prepare a 3’,5-dimethylhesperidin solution; weigh 2.65 mg of 3’-methylhesperidin and dissolve it in 2.65 mL of purified water to prepare a 3’-methylhesperidin solution.

[0090] (3) Take the 3’,5-dimethylhesperidin solution and 3’-methylhesperidin solution to prepare test sample solutions with different ratios. The specific liquid addition is as follows in the table:

[0091]

[0092] (4) After detecting the test sample solutions by an analytical liquid chromatograph, automatically integrate to obtain the peak areas, and calculate the ratio of the peak area of 3’,5-dimethylhesperidin to the peak area of 3’-methylhesperidin.

[0093] (5) Take 1200 μL of each ratio of the test sample solutions in (3) and mix them evenly with 2800 μL of the aluminum chloride methanol solution in parallel for 3 times. Place them in a thermostatic heating and stirring device with heat collection for reaction. The water bath temperature is 50 °C and the reaction time is 3 minutes. Immediately conduct detection after the reaction. The detection results are as follows in the table:

[0094]

[0095]

[0096] Taking the ratio of peak areas as x and the average value of absorbance ratios as y, perform linear regression analysis to obtain y = 0.540x + 0.461. The results are as Figure 15 .

[0097] (6) Weigh 1.56 mg of methyl hesperidin raw material, add 1.56 ml of purified water to prepare a sample solution. Take 200 μl of the sample solution and dilute it 10 times. Detect it with an analytical liquid chromatograph. The detection results show that the peak area of 3’,5-dimethyl hesperidin is 8290161, and the peak area of 3’-methyl hesperidin is 17458696. Calculate the peak area ratio to be 0.4748. Take 1200 μl of the diluted sample solution and mix it evenly with 2800 μl of aluminum chloride methanol solution, and immediately place it in a thermostatic heating and stirring device with heat collection for reaction. The water bath temperature is 50 °C, and the reaction time is 3 minutes. Immediately detect it after the reaction is completed. The detection results show that the absorbance at the maximum absorption wavelength of 3’,5-dimethyl hesperidin is 0.5083, and the absorbance at the maximum absorption wavelength of 3’-methyl hesperidin is 0.7125. Calculate the absorbance ratio to be 0.7134, and substitute it into the regression equation to calculate the x value to be 0.4674.

[0098] The above-disclosed are only the preferred embodiments of the present invention. Of course, the scope of the rights of the present invention cannot be limited thereby. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope covered by the present invention.

Claims

1. A method for rapid detection of hesperidin modified derivatives, characterized in that, Including reacting a sample with a reaction reagent, followed by ultraviolet-visible light detection to determine the proportion of each hesperidin modified derivative in the sample; the reaction reagent includes an aluminum salt reagent.

2. The method for rapid detection of hesperidin modified derivatives according to claim 1, wherein The reaction reagent includes a methanol solution of aluminum chloride.

3. The method for rapidly detecting the hesperidin modified derivative according to claim 1, characterized in that, The concentration of the reaction reagent is 0.5 - 1.5 mg / ml.

4. The method for rapid detection of hesperidin modified derivatives according to claim 1, characterized in that, The reaction temperature of the reaction is 40 - 60 °C, and the reaction time is 3 - 10 minutes.

5. The method for rapid detection of hesperidin modified derivatives according to claim 1, characterized in that, The hesperidin modified derivatives include one or more of 3'-methyl hesperidin and 3',5-dimethyl hesperidin.

6. The method for rapidly detecting the hesperidin modified derivative according to claim 1, wherein Including the following steps: A. Dissolve the sample to prepare a sample solution for standby. B. Add anhydrous aluminum chloride to methanol to prepare the reaction reagent for standby. C. Set the detection parameters for the ultraviolet-visible light detection and zero with the reaction reagent as the blank solvent. D. After adding the reaction reagent to the sample solution, carry out the reaction. After the reaction is completed, carry out the ultraviolet-visible light detection to obtain the absorbance, substitute it into the standard linear regression formula to determine the proportion of the hesperidin modified derivative in the sample.

7. The method for rapid detection of hesperidin modified derivatives according to claim 6, characterized in that, The standard linear regression formula is obtained by carrying out the ultraviolet-visible light detection on the standard curve solution under the same conditions; the standard curve solution includes a standard reference substance solution and a standard test solution. The preparation of the standard curve solution includes: a. Weigh the 3'-methyl hesperidin reference substance and dissolve it to prepare a standard reference substance solution A. b. Weigh the 3',5-dimethyl hesperidin reference substance and dissolve it to prepare a standard reference substance solution B. c. Configure the standard test solution by mixing the standard reference substance solution A and the standard reference substance solution B at a volume ratio of 0.5 - 1.

5.

8. The method for rapid detection of hesperidin modified derivatives according to claim 6, characterized in that, The standard linear regression formula includes: S1. Obtain the absorbance of the standard curve solution through the ultraviolet-visible light detection. S2. Obtain the peak area of the standard curve solution through liquid chromatography detection. S3. Carry out linear regression analysis on the absorbance obtained in S1 and the peak area obtained in S2 to obtain the standard linear regression formula.

9. The method for rapidly detecting the hesperidin modified derivative according to claim 7, wherein In step D, the volume ratio of the sample solution to the reaction reagent is 1:30; the ultraviolet-visible light detection includes one or more of ultraviolet-visible light spectral scanning and dual-wavelength detection; the concentrations of the sample solution, the standard reference substance solution A, and the standard reference substance solution B are the same.

10. The method for rapidly detecting the hesperidin modified derivative according to claim 9, wherein The ultraviolet-visible light detection is ultraviolet-visible light spectral scanning, and the parameter settings include: the scanning range is 600 - 190 nm, the photometric mode is Abs, the scanning speed is fast, and the scanning interval is 0.2 nm; the concentrations of the sample solution, the standard reference substance solution A, and the standard reference substance solution B are all 1 mg / ml; the reaction is carried out in a thermostatic heating and stirring device with heat collection.

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