Analysis method for detecting related substances in ornithine aspartate

Abstract

The invention discloses an analysis method for detecting ornithine aspartate related substances. The analysis method comprises the following steps: carrying out high performance liquid chromatography detection on a sample to be detected; determining the content of malic acid, maleic acid and fumaric acid in the to-be-detected sample based on the detection result; wherein a mobile phase of the high performance liquid chromatography detection is a monopotassium phosphate solution; the pH (Potential of Hydrogen) value of the mobile phase is 1.5 to 3.5. The method is high in detection result sensitivity, high in accuracy, low in detection limit, high in separation degree, safe, effective, good in stability, easy, convenient and rapid to operate and suitable for large-scale application.

Description

Technical Field

[0001] The present application relates to the field of medicine, and in particular to a method for detecting related substances in ornithine aspartate and a quality control method for ornithine aspartate drugs. Background Art

[0002] Ornithine aspartate is an amino acid drug, mainly used to improve liver function and lower blood ammonia. The chemical structure of ornithine aspartate is as follows:

[0003]

[0004] Ornithine aspartate injection can be used to treat or prevent elevated blood ammonia caused by acute or chronic liver diseases (such as various types of hepatitis, cirrhosis, fatty liver, and post-hepatitis syndrome) and to treat hepatic encephalopathy, such as latent or acute hepatic encephalopathy associated with or secondary to impaired liver detoxification function (such as cirrhosis). It is particularly suitable for treating the early stages of hepatic coma or the state of confusion in hepatic coma.

[0005] Most publicly available information on methods for detecting related substances in ornithine aspartate is based on high-performance liquid chromatography (HPLC), and amino columns are often used for related substance inspections (e.g., "Improved HPLC Method for Determination of Related Substances in Ornithine aspartate API," "High-Performance Liquid Chromatography for Determination of S-3-Aminopiperidin-2-one, Arginine, and Glutamic Acid in Ornithine aspartate Raw Materials," "Inspection of Related Substances in Ornithine aspartate Injection," "Study on the Effect of Temperature on Related Substances in Ornithine aspartate Injection," CN104807924A, CN108593815A, CN108254463B, CN114965746B, etc.). In this type of method, amino columns are used to separate and determine the relevant substances. However, experiments have consistently found that amino columns suffer from poor durability and reproducibility. The specific conditions and procedures must be highly consistent each time the mobile phase is prepared. Otherwise, the retention times of the peaks in the spectra obtained at different times and by different analysts will vary significantly. This can even result in suboptimal resolution between the malic, maleic, and fumaric acid peaks and the main peak, and the order of peak elution for malic, maleic, and fumaric acids can also change. Therefore, addressing this issue is urgent.

[0006] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for detecting related substances in ornithine aspartate with better analysis accuracy of related substances. Summary of the Invention

[0007] The present application aims to, at least to some extent, address at least one of the technical problems existing in the prior art. To this end, the present application provides a method for detecting related substances of ornithine aspartate, specifically malic acid, maleic acid, and fumaric acid. This analytical method can effectively detect the content of malic acid, maleic acid, and fumaric acid in ornithine aspartate, and has the advantages of high sensitivity, high accuracy, low detection limit, high resolution, safety, effectiveness, good stability, simple and rapid operation, and is suitable for a wide range of applications.

[0008] This application is based on the following findings of the inventors:

[0009] "Improved HPLC method for determining related substances in ornithine aspartate raw materials", "High performance liquid chromatography method for determining the contents of S-3-aminopiperidin-2-one, arginine and glutamic acid in ornithine aspartate raw materials", "Inspection of related substances in ornithine aspartate injection", "Study on the influence of temperature on related substances in ornithine aspartate injection", CN104807924A, CN108593815A, CN108254463B, CN114965746B, etc. all use amino columns, but in the experiments, it was found that the use of amino columns generally has the disadvantages of poor durability and reproducibility.

[0010] Based on this, in one aspect of the present application, a method for detecting related substances in ornithine aspartate is proposed. According to an embodiment of the present application, the method comprises:

[0011] The sample to be tested is subjected to high performance liquid chromatography detection;

[0012] Based on the test results, determining the content of malic acid, maleic acid, and fumaric acid in the test sample;

[0013] Wherein, the mobile phase of the high performance liquid chromatography detection is potassium dihydrogen phosphate solution;

[0014] The pH value of the mobile phase is 1.5 to 3.5.

[0015] The method of the present application uses a potassium dihydrogen phosphate solution as the mobile phase, and the mobile phase has a pH of 1.5 to 3.5, which can effectively separate and detect related substances in ornithine aspartate. The content of related substances in ornithine aspartate can also be calculated based on the resulting liquid chromatogram. Therefore, the method of the present application has high sensitivity, strong accuracy, low detection limit, high resolution, safety, effectiveness, good stability, simple and rapid operation, and is suitable for large-scale application.

[0016] In this article, "ornithine aspartate" or "ornithine aspartate drug" refers to L-aspartic acid and L-ornithine; "ornithine aspartate-related substances" refers to malic acid, maleic acid, and fumaric acid.

[0017] According to an embodiment of the present application, the above method further includes at least one of the following technical features:

[0018] According to an embodiment of the present application, the concentration of the potassium dihydrogen phosphate solution in the mobile phase is 0.005-0.035 M, for example, 0.005 M, 0.015 M, 0.02 M, 0.025 M, 0.03 M, 0.035 M, and a range value between any two of them as endpoint values.

[0019] According to an embodiment of the present application, the concentration of the potassium dihydrogen phosphate solution in the mobile phase is 0.01 to 0.03M.

[0020] According to an embodiment of the present application, the elution method in the high performance liquid chromatography detection is isocratic elution.

[0021] According to an embodiment of the present application, the detection wavelength used in the high performance liquid chromatography detection is 190nm~220nm, for example, 190nm, 195nm, 200nm, 205nm, 210nm, 215nm, 220nm and any two point values ​​therein as the range value between the endpoint values.

[0022] According to an embodiment of the present application, the column temperature used in the high performance liquid chromatography detection is 20°C to 40°C, for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C and any two point values ​​therein as the range value between the endpoint values.

[0023] According to an embodiment of the present application, the chromatographic column used in the high performance liquid chromatography detection is YMC-TriartC18.

[0024] According to an embodiment of the present application, the flow rate used in the high performance liquid chromatography detection is 0.70 mL / min to 0.90 mL / min.

[0025] According to an embodiment of the present application, the sample to be tested is selected from ornithine aspartate injection.

[0026] In another aspect of this application, a method for controlling the quality of an ornithine aspartate drug is proposed. According to an embodiment of this application, the method comprises: testing an ornithine aspartate drug for ornithine-related substances using the aforementioned method; and determining, based on the test results, whether the ornithine aspartate drug meets quality standards. Thus, the method of this application can accurately determine whether an ornithine aspartate-related substance sample meets requirements, thereby effectively ensuring drug safety and controlling product quality.

[0027] According to an embodiment of the present application, the ornithine aspartate drug to be tested is ornithine aspartate injection.

[0028] According to an embodiment of the present application, the ornithine aspartate drug to be tested is diluted in advance before the test.

[0029] In an optional embodiment of the present application, the dilution factor of the dilution treatment is 10 to 100 times, for example, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times.

[0030] In an optional embodiment of the present application, when the content of malic acid ≤ 0.15%, maleic acid ≤ 0.10%, and fumaric acid ≤ 0.10% in the ornithine aspartate injection sample, the ornithine aspartate injection sample is of qualified quality;

[0031] When the malic acid content in the ornithine aspartate injection sample is greater than 0.15%, or the maleic acid content is greater than 0.10%, or the fumaric acid content is greater than 0.10%, the quality of the ornithine aspartate injection sample is unqualified.

[0032] In another aspect of the present application, a method for quality control of ornithine aspartate pharmaceuticals is provided. According to an embodiment of the present application, the method comprises:

[0033] Diluting the ornithine aspartate injection sample to obtain a test solution;

[0034] The test solution is subjected to high performance liquid chromatography to determine the content of related substances in ornithine aspartate in the test solution;

[0035] When the malic acid content in the ornithine aspartate injection sample is ≤0.15%, the maleic acid content is ≤0.10%, and the fumaric acid content is ≤0.10%, the ornithine aspartate injection sample is of qualified quality;

[0036] When the malic acid content in the ornithine aspartate injection sample is greater than 0.15%, or the maleic acid content is greater than 0.10%, or the fumaric acid content is greater than 0.10%, the ornithine aspartate injection sample is considered unqualified;

[0037] Wherein, the mobile phase of the high performance liquid chromatography detection is potassium dihydrogen phosphate solution;

[0038] The pH value of the mobile phase is 1.5 to 3.5;

[0039] The concentration of potassium dihydrogen phosphate in the mobile phase is 0.01 to 0.03 M;

[0040] The elution mode in the high performance liquid chromatography detection is isocratic elution;

[0041] The detection wavelength used in the high performance liquid chromatography detection is 190nm to 220nm;

[0042] The column temperature used in the high performance liquid chromatography detection is 20°C to 40°C;

[0043] The chromatographic column used in the HPLC detection was YMC-Triart C18;

[0044] The flow rate used in the high performance liquid chromatography detection is 0.70 mL / min to 0.90 mL / min.

[0045] Therefore, the method of the present application can accurately determine whether the relevant substances in ornithine aspartate meet the requirements, thereby effectively ensuring drug safety and controlling product quality.

[0046] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0048] Figure 1 This is the HPLC chromatogram in Example 1 of the present application;

[0049] Figure 2 This is the HPLC chromatogram with a column temperature of 32° C. in Example 2 of the present application;

[0050] Figure 3 This is the HPLC chromatogram with a column temperature of 28° C. in Example 2 of the present application;

[0051] Figure 4 This is the HPLC chromatogram of Example 2 of the present application with the mobile phase being 0.01M KH2PO4;

[0052] Figure 5This is the HPLC chromatogram of Example 2 of the present application with the mobile phase being 0.03M KH2PO4;

[0053] Figure 6 This is the HPLC chromatogram of the mobile phase pH 2.4 in Example 2 of the present application;

[0054] Figure 7 This is the HPLC chromatogram of the mobile phase pH 2.6 in Example 2 of the present application;

[0055] Figure 8 This is the HPLC chromatogram at a flow rate of 0.7 ml / min in Example 2 of the present application;

[0056] Figure 9 This is the HPLC chromatogram at a flow rate of 0.9 ml / min in Example 2 of the present application;

[0057] Figure 10 This is the HPLC chromatogram with a detection wavelength of 204 nm in Example 2 of the present application;

[0058] Figure 11 This is the HPLC chromatogram with a detection wavelength of 206 nm in Example 2 of the present application. DETAILED DESCRIPTION

[0059] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

[0060] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of this application, unless otherwise specified, "plurality" means two or more.

[0061] In this document, the terms "include" or "comprising" are open expressions, that is, including the contents specified in this application, but not excluding other contents.

[0062] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0063] Below in conjunction with embodiment, the scheme of the application will be explained. Those skilled in the art will appreciate that the following examples are merely for illustration of the application and should not be considered as limiting the scope of the application. Where specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents used or instruments not indicated by the manufacturer are conventional products that can be obtained commercially.

[0064] Example 1: Detection of ornithine-aspartate related substances

[0065] The experimental steps are as follows:

[0066] Preparation of mobile phase: Dissolve 2.7 g of potassium dihydrogen phosphate in 1000 ml of water and adjust the pH to 2.5 with phosphoric acid.

[0067] Preparation of sample solution: Accurately measure 1 ml of the test sample (ornithine aspartate injection) and place it in a 50 ml volumetric flask. Dilute to the mark with water and shake well.

[0068] Detection: Take 20 μl of sample solution and inject it into liquid chromatograph. Detection is performed using Agilent 1260 high performance liquid chromatograph. The chromatographic conditions are as follows:

[0069] Column: YMC-Triart C18, 4.6 mm × 250 mm, 5 μm;

[0070] Column temperature: 30°C;

[0071] Mobile phase: 0.02 M potassium dihydrogen phosphate solution;

[0072] Flow rate: 0.8 ml / min;

[0073] Injection volume: 20 μl;

[0074] Detection wavelength: 205nm.

[0075] Record the chromatogram, see Figure 1 .

[0076] The chromatographic results showed that malic acid, maleic acid, and fumaric acid peaked in sequence. The separation degree of maleic acid and malic acid in the spiked test solution was 14.3, and the separation degree of fumaric acid and maleic acid was 11.3.

[0077] Example 2: Screening by Detection Method

[0078] 1. Adjustment of column temperature

[0079] Preparation of mobile phase: Dissolve 2.7 g of potassium dihydrogen phosphate in 1000 ml of water and adjust the pH to 2.5 with phosphoric acid.

[0080] Preparation of sample solution: Accurately measure 1 ml of the test sample (ornithine aspartate injection) and place it in a 50 ml volumetric flask. Dilute to the mark with water and shake well.

[0081] Detection: Take 20 μl of sample solution and inject it into liquid chromatograph. Detection is performed using Agilent 1260 high performance liquid chromatograph. The chromatographic conditions are as follows:

[0082] Column: YMC-Triart C18, 4.6 mm × 250 mm, 5 μm;

[0083] Column temperature: 32°C and 28°C;

[0084] Mobile phase: 0.02 M potassium dihydrogen phosphate solution;

[0085] Flow rate: 0.8 ml / min;

[0086] Injection volume: 20 μl;

[0087] Detection wavelength: 205nm.

[0088] Record the chromatograms. The column temperatures are 32°C and 28°C. Figure 2 、 Figure 3 .

[0089] It can be seen from the spectrum that when the column temperature is 28°C, the separation degree of maleic acid and malic acid in the spiked test solution is 14.1, and the separation degree of fumaric acid and maleic acid is 10.3; when the main temperature is 32°C, the separation degree of maleic acid and malic acid in the spiked test solution is 13.4, and the separation degree of fumaric acid and maleic acid is 9.8.

[0090] (2) Adjustment of mobile phase composition

[0091] Preparation of mobile phase: see Example 1;

[0092] Preparation of sample solution: Accurately measure 1 ml of the test sample (ornithine aspartate injection) and place it in a 50 ml volumetric flask. Dilute to the mark with water and shake well.

[0093] Detection: Take 20 μl of sample solution and inject it into liquid chromatograph. Detection is performed using Agilent 1260 high performance liquid chromatograph. The chromatographic conditions are as follows:

[0094] Column: YMC-Triart C18, 4.6 mm × 250 mm, 5 μm;

[0095] Column temperature: 30°C;

[0096] Mobile phase: 0.01 M potassium dihydrogen phosphate, 0.03 M potassium dihydrogen phosphate;

[0097] Flow rate: 0.8 ml / min;

[0098] Injection volume: 20 μl;

[0099] Detection wavelength: 205nm.

[0100] Record the chromatogram, such as Figure 4 (0.01M potassium dihydrogen phosphate), Figure 5 (0.03M potassium dihydrogen phosphate).

[0101] It can be seen from the spectrum that at 0.01M KH2PO4, the separation degree of maleic acid and malic acid in the spiked test solution is 14.9, and the separation degree of fumaric acid and maleic acid is 12.6; at 0.03M KH2PO4, the separation degree of maleic acid and malic acid in the spiked test solution is 14.1, and the separation degree of maleic acid and fumaric acid is 11.3.

[0102] (3) Adjustment of mobile phase pH

[0103] Preparation of mobile phase: Dissolve 2.7 g of potassium dihydrogen phosphate in 1000 ml of water and adjust the pH to 2.4 or 2.6 with phosphoric acid.

[0104] Preparation of sample solution: Accurately measure 1 ml of the test sample (ornithine aspartate injection) and place it in a 50 ml volumetric flask. Dilute to the mark with water and shake well.

[0105] Detection: Take 20 μl of sample solution and inject it into liquid chromatograph. Detection is performed using Agilent 1260 high performance liquid chromatograph. The chromatographic conditions are as follows:

[0106] Column: YMC-Triart C18, 4.6 mm × 250 mm, 5 μm;

[0107] Column temperature: 30°C;

[0108] Mobile phase: 0.02 M potassium dihydrogen phosphate, adjusted to pH 2.4 or 2.6 with phosphoric acid;

[0109] Flow rate: 0.8 ml / min;

[0110] Injection volume: 20 μl;

[0111] Detection wavelength: 205nm.

[0112] Record the chromatogram, such as Figure 6 (pH 2.4), Figure 7 (pH 2.6).

[0113] It can be seen from the spectrum that when the pH value is 2.4, the separation degree of maleic acid and malic acid in the spiked test solution is 14.3, and the separation degree of fumaric acid and maleic acid is 11.3; when the pH value is 2.6, the separation degree of maleic acid and malic acid in the spiked test solution is 13.6, and the separation degree of fumaric acid and maleic acid is 8.6.

[0114] (3) Flow rate screening

[0115] Preparation of mobile phase: Dissolve 2.7 g of potassium dihydrogen phosphate in 1000 ml of water and adjust the pH to 2.5 with phosphoric acid.

[0116] Preparation of sample solution: Accurately measure 1 ml of the test sample (ornithine aspartate injection) and place it in a 50 ml volumetric flask. Dilute to the mark with water and shake well.

[0117] Detection: Take 20 μl of sample solution and inject it into liquid chromatograph. Detection is performed using Agilent 1260 high performance liquid chromatograph. The chromatographic conditions are as follows:

[0118] Column: YMC-Triart C18, 4.6 mm × 250 mm, 5 μm;

[0119] Column temperature: 30°C;

[0120] Mobile phase: 0.02 M potassium dihydrogen phosphate solution;

[0121] Flow rate: 0.7ml / min, 0.9ml / min;

[0122] Injection volume: 5 μl;

[0123] Detection wavelength: 254nm.

[0124] Record the chromatograms at flow rates of 0.7 ml / min and 0.9 ml / min. Figure 8 、 Figure 9 .

[0125] It can be seen from the spectrum that at 0.7 ml / min, the separation degree of maleic acid and malic acid in the spiked test solution is 13.9, and the separation degree of fumaric acid and maleic acid is 10.5; at a flow rate of 0.9 ml / min, the separation degree of maleic acid and malic acid in the spiked test solution is 13.9, and the separation degree of fumaric acid and maleic acid is 10.5; at a flow rate of 0.9 ml / min, the separation degree of maleic acid and malic acid in the spiked test solution is 13.5, and the separation degree of fumaric acid and maleic acid is 10.1.

[0126] (4) Wavelength screening

[0127] Preparation of mobile phase: Dissolve 2.7 g of potassium dihydrogen phosphate in 1000 ml of water and adjust the pH to 2.5 with phosphoric acid.

[0128] Preparation of sample solution: Accurately measure 1 ml of the test sample (ornithine aspartate injection) and place it in a 50 ml volumetric flask. Dilute to the mark with water and shake well.

[0129] Detection: Take 20 μl of sample solution and inject it into liquid chromatograph. Detection is performed using Agilent 1260 high performance liquid chromatograph. The chromatographic conditions are as follows:

[0130] Column: YMC-Triart C18, 4.6 mm × 250 mm, 5 μm;

[0131] Column temperature: 30°C;

[0132] Mobile phase: 0.02 M potassium dihydrogen phosphate solution;

[0133] Flow rate: 0.8 ml / min;

[0134] Injection volume: 20 μl;

[0135] Detection wavelength: 204nm, 206nm.

[0136] Record the chromatogram, and detect the spectra at wavelengths of 204 nm and 206 nm respectively. Figure 10 、 Figure 11 .

[0137] From the spectrum, it can be seen that there is no significant difference in the separation between impurities and the main peak at wavelengths of 204nm, 206nm and 205nm, and there is no significant difference in the detection amount of impurities, indicating that the wavelength durability of this method is good.

[0138] Example 3: Methodological Investigation

[0139] The detection method of Example 1 was methodologically validated using conventional methods in the art to verify system adaptability, specificity, detection limit, quantification limit, linear range, accuracy, repeatability, precision, stability, and durability. The specific test results are as follows:

[0140]

[0141]

[0142]

[0143] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0144] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A method for detecting related substances in ornithine aspartate, characterized in that: include: The sample to be tested is subjected to high performance liquid chromatography detection; Based on the test results, determining the content of the relevant substance in the sample to be tested; Wherein, the mobile phase for the high performance liquid chromatography detection is potassium dihydrogen phosphate solution; The pH value of the mobile phase is 1.5-3.

5.

2. The method according to claim 1, characterized in that The concentration of potassium dihydrogen phosphate in the mobile phase is 0.005-0.035 M; And / or, the elution method in the high performance liquid chromatography detection is isocratic elution; And / or, the detection wavelength used in the high performance liquid chromatography detection is 190nm to 220nm; And / or, the column temperature used in the high performance liquid chromatography detection is 20°C to 40°C; And / or, the chromatographic column used in the high performance liquid chromatography detection is YMC-Triart C18; And / or, the flow rate used in the high performance liquid chromatography detection is 0.70 mL / min to 0.90 mL / min.

3. The method according to claim 1, characterized in that The concentration of potassium dihydrogen phosphate in the mobile phase is 0.01-0.03M.

4. The method according to claim 1, characterized in that: The sample to be tested is selected from ornithine aspartate injection.

5. A quality control method for ornithine aspartate medicine, characterized in that: include: The method according to any one of claims 1 to 4 is used to detect the related substances in the ornithine aspartate drug to be tested; Based on the test results, determine whether the quality of the ornithine aspartate drug to be tested is qualified.

6. The method according to claim 5, characterized in that The malic acid ≤0.15%, the maleic acid ≤0.10%, and the fumaric acid ≤0.10% in the tested ornithine aspartate drug are indicative of qualified quality of the tested ornithine aspartate drug.

7. The method according to claim 5, characterized in that The ornithine aspartate drug to be tested is ornithine aspartate injection.

8. The method according to claim 5, characterized in that Before the detection, the ornithine aspartate drug to be detected is diluted in advance.

9. A method for quality control of ornithine aspartate medicine, characterized in that: include: diluting the ornithine aspartate injection sample to obtain a test solution; The liquid to be tested is subjected to high performance liquid chromatography to determine the content of the relevant substance in the liquid to be tested; When the malic acid content in the ornithine aspartate injection sample is ≤0.15%, the maleic acid content is ≤0.10%, and the fumaric acid content is ≤0.10%, the ornithine aspartate injection sample is of qualified quality; When the malic acid in the ornithine aspartate injection sample is greater than 0.15%, or the maleic acid is greater than 0.10%, or the fumaric acid is greater than 0.10%, the quality of the ornithine aspartate injection sample is unqualified; Wherein, the mobile phase for the high performance liquid chromatography detection is potassium dihydrogen phosphate solution; The pH value of the mobile phase is 1.5 to 3.5; The concentration of potassium dihydrogen phosphate in the mobile phase is 0.01 to 0.03 M; The elution method in the high performance liquid chromatography detection is isocratic elution; The detection wavelength used in the high performance liquid chromatography detection is 190nm to 220nm; The column temperature used in the high performance liquid chromatography detection is 20°C to 40°C; The chromatographic column used in the HPLC detection is YMC-Triart C18; The flow rate used in the high performance liquid chromatography detection is 0.70 mL / min to 0.90 mL / min.

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