Method for determining gallic acid in berberine tannate

Abstract

The invention provides a method for determining gallic acid in berberine tannate, and relates to the field of medicine component detection. The method comprises the following steps: S1, preparation of a reference substance solution: taking a gallic acid reference substance, adding a solvent to dissolve the gallic acid reference substance, and quantitatively diluting the gallic acid reference substance to prepare a solution containing 0.02-0.04 mg of the gallic acid reference substance per 1mL; s2, preparation of a test solution: taking berberine tannate, adding a solvent to dissolve the berberine tannate, and quantitatively diluting the berberine tannate to prepare a solution containing 0.5-0.7 mg of berberine tannate per 1mL; and S3, determining the content by using a liquid chromatography. The invention develops the method for determining the gallic acid in the berberine tannate, the good separation of the tannic acid, the berberine and the gallic acid in the berberine tannate is realized, the detection time is short, the separation degree is high, and a good foundation is provided for the control of the safety and the stability of the berberine tannate.

Description

Technical Field

[0001] The invention relates to the technical field of drug component detection, and in particular to a method for determining gallic acid in berberine tannate. Background Art

[0002] Berberine tannate, also known as coptis chinensis, is an important alkaloid that can be extracted from plants such as coptis chinensis, phellodendron amurense, and trifoliate needles. It has antibacterial, anti-inflammatory, antiviral and antioxidant effects and is often used to treat intestinal infections, sores, inflammation and other diseases. Berberine tannate, a drug for gastrointestinal diseases, decomposes into berberine and tannic acid in the intestines. The former has antibacterial effects and has inhibitory effects on a variety of gram-positive and gram-negative bacteria in vitro, including hemolytic streptococci, Staphylococcus aureus, Vibrio cholerae, meningococci, Shigella, Salmonella typhi, and Corynebacterium diphtheriae; the latter has an astringent effect.

[0003] However, gallic acid is present in berberine tannate. Gallic acid, chemical name is 3,4,5-trihydroxybenzoic acid; the structural formula is shown in Formula I below. Gallic acid has multiple biological activities and application values, but its safety and toxicity also need to be paid attention to. Excessive intake of gallic acid may irritate the gastrointestinal tract, causing abdominal pain, diarrhea and other discomfort symptoms, and even nausea and vomiting in severe cases. In addition, long-term and excessive intake of gallic acid may also increase the risk of cancer and cause damage to the nervous system.

[0004]

[0005] The gallic acid in tannic acid berberine is introduced from the starting material tannic acid on the one hand, and on the other hand, in the process of synthesizing tannic acid berberine from tannic acid and berberine hydrochloride, gallic acid is produced by the degradation of tannic acid. Tannic acid is a compound composed of five to twelve-0-galloyl-β-D-glucose, and the structural formula containing ten gallic acids is shown in the following formula II.

[0006]

[0007] Tannic acid is unstable under strong acid and high temperature conditions, and one of its degradation products is gallic acid. Therefore, the content of gallic acid in tannic acid berberine can be detected to monitor the stability of tannic acid in the reaction.

[0008] There are methods for determining the content of berberine tannate in the prior art, such as "Determination of the content of berberine tannate by high performance liquid chromatography" (Lu Ying, Zhang Zheng, Deng Yueting); or methods for determining gallic acid in other substances are recorded, such as Chinese patent CN 117491529 A discloses a method for determining the content of gallic acid in Terminalia chebula, but does not record the method and research for determining gallic acid in berberine tannate.

[0009] Since berberine tannate contains two main components, tannic acid and berberine, the final degradation product of tannic acid in the production process and stable storage is gallic acid. However, the prior art does not record the method and research for determining gallic acid in berberine tannate, which has a great impact on the safety and effectiveness of drugs after listing.

[0010] Based on this, developing a method to determine gallic acid in berberine tannate to improve the safety and stability of the drug is the research focus of researchers in this field. Summary of the invention

[0011] In view of the above problems, the present invention provides a method for determining gallic acid in berberine tannate. Through the specific chromatographic conditions of the present invention, the gallic acid content in berberine tannate can be directly, quickly and accurately measured.

[0012] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0013] In one aspect, the present invention provides a method for determining gallic acid in berberine tannate, comprising the following steps:

[0014] S1: Preparation of reference solution:

[0015] Take the gallic acid reference substance, dissolve it in a solvent and quantitatively dilute it to make a solution containing 0.02-0.04 mg per 1 mL;

[0016] S2: Preparation of test solution:

[0017] Take berberine tannate, dissolve it in a solvent and quantitatively dilute it to make a solution containing 0.5-0.7 mg per 1 mL;

[0018] S3: Liquid chromatography was used for content determination. The chromatographic conditions were as follows:

[0019] Chromatographic column: Octadecyl bonded silica gel as filler;

[0020] Mobile phase: 0.05%-0.15% phosphoric acid solution is mobile phase A, and 95%-100% methanol solution is mobile phase B;

[0021] Column temperature: 28℃-32℃;

[0022] Detection wavelength: 265-280nm;

[0023] The elution gradient is:

[0024] 0-8min, the volume fraction of mobile phase A is 75%-85%, and the volume fraction of mobile phase B is 15%-25%;

[0025] 8-25min, the volume fraction of mobile phase A changes from 75%-85% to 20%-30%, and the volume fraction of mobile phase B changes from 15%-25% to 70%-80%;

[0026] 25-27min, the volume fraction of mobile phase A is 20%-30%, and the volume fraction of mobile phase B is 70%-80%;

[0027] 27-30min, the volume fraction of mobile phase A increases from 20%-30% to 75%-85%, and the volume fraction of mobile phase B increases from 70%-80% to 15%-25%;

[0028] 30-40min, the volume fraction of mobile phase A is 75%-85%, and the volume fraction of mobile phase B is 15%-25%.

[0029] Preferably, in S1, the solvent is a mixed solution of a phosphoric acid solution with a volume fraction of 0.05%-0.15% and a methanol solution with a volume fraction of 95%-100%.

[0030] Further preferably, in S1, the solvent is a mixed solution of a phosphoric acid solution with a volume fraction of 0.1% and a methanol solution with a volume fraction of 100%.

[0031] Preferably, in S1, the volume ratio of the phosphoric acid solution to the methanol solution is 75-85:15-25; further preferably, in S1, the volume ratio of the phosphoric acid solution to the methanol solution is 80:20.

[0032] Preferably, in S2, the solvent is a mixed solution of a phosphoric acid solution with a volume fraction of 0.05%-0.15% and a methanol solution with a volume fraction of 95%-100%.

[0033] Further preferably, in S2, the solvent is a mixed solution of a phosphoric acid solution with a volume fraction of 0.1% and a methanol solution with a volume fraction of 100%.

[0034] Preferably, in S2, the volume ratio of the phosphoric acid solution to the methanol solution is 75-85:15-25; further preferably, in S2, the volume ratio of the phosphoric acid solution to the methanol solution is 80:20.

[0035] Preferably, in S3, the chromatographic column is Welch Ultimate XB-C18, 4.6 mm×250 mm, 5 μm.

[0036] Preferably, in S3, the mobile phase is: a phosphoric acid solution with a volume fraction of 0.05%-0.1% as mobile phase A, and a methanol solution with a volume fraction of 98%-100% as mobile phase B.

[0037] Further preferably, in S3, the mobile phase is: a phosphoric acid solution with a volume fraction of 0.1% as mobile phase A, and a methanol solution with a volume fraction of 100% as mobile phase B.

[0038] Preferably, in S3, the column temperature is 30°C.

[0039] Preferably, in S3, the detection wavelength is 271 nm.

[0040] Preferably, in S3, the elution gradient is:

[0041] 0-8min, the volume fraction of mobile phase A is 75%-80%, and the volume fraction of mobile phase B is 20%-25%;

[0042] 8-25min, the volume fraction of mobile phase A changes from 75%-80% to 20%-25%, and the volume fraction of mobile phase B changes from 20%-25% to 75%-80%;

[0043] 25-27min, the volume fraction of mobile phase A is 20%-25%, and the volume fraction of mobile phase B is 75%-80%;

[0044] 27-30min, the volume fraction of mobile phase A increased from 20%-25% to 75%-80%, and the volume fraction of mobile phase B decreased from 75%-80% to 20%-25%;

[0045] 30-40min, the volume fraction of mobile phase A is 75%-80%, and the volume fraction of mobile phase B is 20%-25%.

[0046] Further preferably, in S3, the elution gradient is:

[0047] 0-8min, the volume fraction of mobile phase A was 80%, and the volume fraction of mobile phase B was 20%;

[0048] 8-25min, the volume fraction of mobile phase A increased from 80% to 25%, and the volume fraction of mobile phase B increased from 20% to 75%;

[0049] 25-27min, the volume fraction of mobile phase A is 25%, and the volume fraction of mobile phase B is 75%;

[0050] 27-30min, the volume fraction of mobile phase A increased from 25% to 80%, and the volume fraction of mobile phase B increased from 75% to 20%;

[0051] 30-40min, the volume fraction of mobile phase A is 80%, and the volume fraction of mobile phase B is 20%.

[0052] Preferably, in S3, the flow rate of the chromatographic condition is 1-1.5 mL / min; further preferably, in S3, the flow rate of the chromatographic condition is 1 mL / min.

[0053] Preferably, in S3, the injection volume of the chromatographic condition is 15-25 μL; further preferably, in S3, the injection volume of the chromatographic condition is 20 μL.

[0054] Preferably, in S3, the injection tailing factor in the above-mentioned condition changes is between 0.80-1.20. Further preferably, the conditions are formulated such that the number of theoretical plates calculated based on the gallic acid peak is not less than 3000, and the tailing factors are between 0.95-1.05.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] The present invention develops a method for determining gallic acid in berberine tannate. Through the specific mobile phase and elution gradient of the present invention, good separation of tannic acid, berberine and gallic acid in berberine tannate is achieved, the detection time is short, and the separation degree is high, which provides a good basis for controlling the safety and stability of berberine tannate. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The chromatogram of the test solution of Example 1.

[0058] Figure 2 This is the chromatogram of tannic acid reference solution.

[0059] Figure 3 This is the chromatogram of berberine hydrochloride reference solution.

[0060] Figure 4 The chromatogram of the gallic acid reference substance solution of Comparative Example 1 is shown in FIG.

[0061] Figure 5 The chromatogram of the test solution of Comparative Example 3 is shown in FIG.

[0062] Figure 6 This is the chromatogram of the test solution of Comparative Example 5.

[0063] Figure 7 This is the chromatogram of the test solution of Comparative Example 6. DETAILED DESCRIPTION

[0064] In order to make the technical means, creative features, purpose and effect of the present invention easy to understand, the present invention is further explained below in conjunction with specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without making creative work all belong to the protection scope of the present invention. It is worth noting that the raw materials used in the present invention are all common commercial products, and their sources are not specifically limited. The technology and scientific terms used in the embodiments have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0065] raw material:

[0066] Table 1. Sample information table

[0067] name batch number source Berberine Tannate BTH-07-1401 Boji Pharmaceutical Technology Co., Ltd.

[0068] Table 2. Reagent information

[0069] Reagent name Manufacturer batch number level Phosphoric acid Guangzhou Chemical Reagent Factory 2022050221 AR Methanol Sigma WXBF1598V HPLC Acetonitrile Sigma WXBF07940 HPLC water Boji Pharmaceutical Technology Co., Ltd. N / A Ultrapure water

[0070] Table 3. Reference material information table

[0071]

[0072] Example 1

[0073] A method for determining gallic acid in berberine tannate, specifically comprising the following steps:

[0074] S1: Preparation of reference solution:

[0075] Take an appropriate amount of gallic acid reference substance, weigh it accurately, dissolve it in a solvent and quantitatively dilute it to make a solution containing about 0.03 mg per 1 mL. The solvent is a mixed solution of 0.1% by volume phosphoric acid solution and 100% by volume methanol solution, with a volume ratio of 80:20.

[0076] S2: Preparation of test solution:

[0077] Take an appropriate amount of berberine tannate, weigh it accurately, dissolve it in a solvent and dilute it quantitatively to make a solution containing about 0.6 mg per 1 mL. The solvent is a mixed solution of 0.1% by volume phosphoric acid solution and 100% by volume methanol solution, and the volume ratio of the two is 80:20.

[0078] S3: Liquid chromatography was used for content determination. The chromatographic conditions were as follows:

[0079] Chromatographic column: Welch Ultimate XB-C18, 4.6 mm × 250 mm, 5 μm;

[0080] Mobile phase: 0.1% by volume phosphoric acid solution was used as mobile phase A, and 100% by volume methanol solution was used as mobile phase B;

[0081] Column temperature: 30°C;

[0082] Detection wavelength: 271nm;

[0083] Flow rate: 1.0 mL / min;

[0084] Injection volume 20 μL;

[0085] The elution gradient is:

[0086] Table 4. Elution gradient

[0087] .

[0089] Test Example 1

[0090] According to the detection method of Example 1, the system applicability, specificity, detection limit, linear relationship, repeatability, stability and filter membrane adsorption were investigated.

[0091] 1. System applicability

[0092] 1) In the spectrum of the test solution of Example 1 (such as Figure 1 As shown), the spectrum of the reference tannic acid solution (as shown Figure 2 As shown in the figure), the spectrum of the reference substance berberine hydrochloride (as shown in the figure) Figure 3 As shown in the figure, the separation between the gallic acid peak and the adjacent impurity peaks in the spectrum is greater than 1.5, the separation is good, and there is no peak interference in the determination.

[0093] 2) Precision experiment

[0094] The reference solution was accurately aspirated and injected 5 times, 10 μL each time, and the retention time and peak area were examined. The results showed that the RSD of the peak area of ​​gallic acid was 0.04% and the RSD of the retention time was 0.05%, indicating that the method had good precision.

[0095] 3) Recovery rate

[0096] 80% accuracy solution: Take about 60 mg of this product, weigh accurately, put it in a 100 mL volumetric flask, accurately add 1 mL of the above accuracy stock solution ①, dissolve it with solvent and dilute to the scale, shake well. Prepare 3 copies in parallel.

[0097] 100% accuracy solution: Take about 60 mg of this product, weigh accurately, place in a 100 mL volumetric flask, accurately add 2.5 mL of the above accuracy stock solution ①, dissolve with solvent and dilute to scale, shake well. Prepare 3 copies in parallel.

[0098] 120% accuracy solution: Take about 60 mg of this product, weigh accurately, put it in a 100 mL volumetric flask, accurately add 4 mL of the above accuracy stock solution ①, dissolve it with solvent and dilute to the scale, shake well. Prepare 3 copies in parallel.

[0099] It fully complies with the standard requirements of "Content of the component to be determined in the sample and the limit of recovery rate" under "9101 Validation Guidelines for Analytical Methods" in Part IV of the "Chinese Pharmacopoeia" (2020 Edition), that is, when the content of the component to be determined is 1 mg / g, the limit of recovery rate is 90%-108%, indicating a good recovery rate.

[0100]

[0101] Table 5. Results

[0102]

[0103] 2. Exclusivity

[0104] Tannic acid solution: Take an appropriate amount of tannic acid, accurately measure it, dissolve it in a solvent and dilute it to make a solution containing about 1 mg per 1 mL;

[0105] Berberine hydrochloride solution: Take an appropriate amount of berberine hydrochloride solution, accurately measure, add solvent to dissolve and dilute to make a solution containing about 1 mg per 1 mL;

[0106] The preparation methods of blank solution, berberine tannate solution and gallic acid reference solution are the same as above.

[0107] Blank solvent, tannic acid solution, berberine hydrochloride solution, and berberine tannate solution were injected respectively. The blank solvent and berberine hydrochloride did not emit peaks at the retention time of gallic acid. The separation between gallic acid and adjacent peaks in tannic acid solution and berberine tannate was greater than 1.5, and the separation was good, indicating good specificity.

[0108] 3. Detection limit

[0109] The gallic acid reference solution was taken and continuously diluted with solvent. The gallic acid concentration was 15.44 ng / mL and the signal-to-noise ratio (S / N) was 19.1, which was defined as the quantitative limit solution. The RSD value of the peak area after 6 repeated injections was 2.4%, which was equivalent to 0.003% of the berberine tannate concentration, proving that the detection ability was good.

[0110] 4. Linear Relationship

[0111] Take 5 mg of gallic acid reference substance, dilute it with solvent to concentrations of 0.3 μg / mL, 3 μg / mL, 15 μg / mL, 30 μg / mL, and 45 μg / mL, and inject them for determination respectively.

[0112] In the concentration range of 0.018 μg / mL-44.758 μg / mL (limit concentration LOQ-150%), the linear regression equation of gallic acid concentration and its peak area is y=71184.71653x-1374.29787, the correlation coefficient (r) is 0.99999, greater than 0.999, the Y-axis intercept should be 0.06% of the 100% theoretical concentration response value, less than 2.0%, and the RSD value of the response factor of the gallic acid peak in each linear solution is 1.6%, less than 2.0%. In the above-mentioned proposed range, the linear relationship between gallic acid concentration and peak area is good.

[0113] 5. Repeatability

[0114] For the same batch of samples, 6 test solutions were prepared in parallel and the contents were determined. The RSD of the gallic acid content results of the 6 test solutions was 0.15%, which was in line with the regulations, demonstrating good repeatability.

[0115] 6. Stability

[0116] When the reference solution was placed at room temperature for 26 hours, the RD value of the peak area of ​​gallic acid at each time point was compared with that at 0 hour, and was 0.1%, which proved that the reference solution had good stability when placed at room temperature for 26 hours.

[0117] When the test solution was placed at room temperature for 21 hours, the RD value of gallic acid content at each time point was between 0.0% and 0.1% compared with that at 0 hour, which proved that the test solution had good stability when placed at room temperature for 21 hours.

[0118] 7. Filter membrane adsorption

[0119] By filtering with a filter membrane, 1 mL, 3 mL, and 5 mL of the test solution were discarded, respectively. The adsorption rates of the filtrates were between 0% and 0.2%, all less than 1.0%, indicating that the selected filter membrane had basically no adsorption.

[0120] Comparative Example 1

[0121] Compared with Example 1, only the mobile phase was changed to:

[0122] Phosphoric acid solution with a volume fraction of 0.1% was used as mobile phase A, and acetonitrile was used as mobile phase B;

[0123] The rest is the same as Example 1.

[0124] Experimental results: Figure 4 As shown, the elution time of the gallic acid peak in the gallic acid reference solution is consistent with that in the blank solution, which affects the determination of gallic acid results. Replacing the mobile phase with acetonitrile cannot meet the determination requirements.

[0125] Comparative Example 2

[0126] Compared with Example 1, only the mobile phase was changed to:

[0127] The mobile phase A was a formic acid solution with a volume fraction of 0.1%, and the mobile phase B was a methanol solution with a volume fraction of 100%;

[0128] The rest is the same as Example 1.

[0129] Experimental results: Gallic acid peak was not detected in the gallic acid reference solution and berberine tannate solution within 40 minutes. When mobile phase A was replaced with formic acid, the detection time was too long and the efficiency was too low.

[0130] Comparative Example 3

[0131] Compared with Example 1, only the mobile phase was changed to:

[0132] 20 mM potassium dihydrogen phosphate solution was used as mobile phase A, and 100% methanol solution was used as mobile phase B;

[0133] The rest is the same as Example 1.

[0134] Experimental results: Figure 5 As shown, compared with Example 1, the peak elution time of gallic acid and tannic acid in the berberine tannate solution is consistent, which interferes with the determination of the main peak of gallic acid.

[0135] Comparative Example 4

[0136] Compared with Example 1, only the solvent in S2 was changed to purified water and the mobile phase composition was changed to a different proportion;

[0137] The rest is the same as Example 1.

[0138] Solvent: 0.1% phosphoric acid solution (precisely measure 1 mL of phosphoric acid and dilute to 1000 mL with water) - methanol (80:20, V / V)

[0139] After selecting different ratios of 0.1% phosphoric acid solution and methanol and mixing them evenly (see Table 6 below), the berberine tannate API was dissolved and sampled separately. The specific results are as follows: the extraction in purified water was incomplete, and the ratio of 0.1% phosphoric acid solution: methanol (60:40) and increasing the ratio of 0.1% phosphoric acid solution by volume was close to the theoretical value. The preferred ratio of 0.1% phosphoric acid solution: methanol (80:20) was selected as the solvent.

[0140] Table 6.

[0141]

[0142] Comparative Example 5

[0143] A method for determining gallic acid in berberine tannate comprises the following steps:

[0144] S1: Preparation of reference solution:

[0145] Take an appropriate amount of gallic acid reference substance, weigh it accurately, dissolve it in solvent and quantitatively dilute it to make a solution containing about 0.03 mg per 1 mL.

[0146] S2: Preparation of test solution:

[0147] Take an appropriate amount of berberine tannate, weigh it accurately, add a solvent to dissolve it and quantitatively dilute it to make a solution containing about 0.6 mg per 1 mL.

[0148] S3: Liquid chromatography was used for content determination. The chromatographic conditions were as follows:

[0149] Chromatographic column: Welch Ultimate XB-C18, 4.6 mm × 250 mm, 5 μm;

[0150] Mobile phase: 0.05% by volume phosphoric acid solution was used as mobile phase A, and 100% by volume methanol solution was used as mobile phase B;

[0151] Column temperature: 30°C;

[0152] Detection wavelength: 271nm;

[0153] Flow rate: 1.0 mL / min;

[0154] Injection volume 20 μL;

[0155] The elution gradient was isocratic elution: the volume fraction of mobile phase A was 93%, and the volume fraction of mobile phase B was 7%.

[0156] Its chromatogram is as follows Figure 6 shown.

[0157] Experimental results: There is only one peak in the spectrum of the test solution, which contains tannic acid, berberine hydrochloride and gallic acid. Gallic acid cannot be determined alone, and the method is not suitable.

[0158] Comparative Example 6

[0159] Compared with Example 1, only the elution gradient was changed to:

[0160] Table 7. Elution gradient

[0161] T / min A％ B％ 0 90 10 40 20 80 45 20 80 50 90 10 60 90 10

[0162] The rest is the same as Example 1.

[0163] Its chromatogram is as follows Figure 7 shown.

[0164] Experimental conclusion: The test solution contains gallic acid, tannic acid, and berberine hydrochloride, all of which have peaks and are separated from gallic acid. However, there are no relevant impurity peaks in the spectrum after 40 minutes, resulting in too long analysis time. In order to improve analysis efficiency and reduce instrument analysis time, this method is not applicable.

[0165] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for determining gallic acid in berberine tannate, characterized in that: The following steps are involved: S1: Preparation of reference solution: Take the gallic acid reference substance, dissolve it in a solvent and quantitatively dilute it to make a solution containing 0.02-0.04 mg per 1 mL; S2: Preparation of test solution: Take berberine tannate, dissolve it in a solvent and quantitatively dilute it to make a solution containing 0.5-0.7 mg per 1 mL; S3: Liquid chromatography was used for content determination. The chromatographic conditions were as follows: Chromatographic column: Octadecyl bonded silica gel as filler; Mobile phase: 0.05%-0.15% phosphoric acid solution is mobile phase A, and 95%-100% methanol solution is mobile phase B; Column temperature: 28℃-32℃; Detection wavelength: 265-280nm; The elution gradient is: 0-8min, the volume fraction of mobile phase A is 75%-85%, and the volume fraction of mobile phase B is 15%-25%; 8-25min, the volume fraction of mobile phase A changes from 75%-85% to 20%-30%, and the volume fraction of mobile phase B changes from 15%-25% to 70%-80%; 25-27min, the volume fraction of mobile phase A is 20%-30%, and the volume fraction of mobile phase B is 70%-80%; 27-30min, the volume fraction of mobile phase A increased from 20%-30% to 75%-85%, and the volume fraction of mobile phase B decreased from 70%-80% to 15%-25%; 30-40min, the volume fraction of mobile phase A is 75%-85%, and the volume fraction of mobile phase B is 15%-25%.

2. The method according to claim 1, characterized in that In S1, the solvent is a mixed solution of a phosphoric acid solution with a volume fraction of 0.05%-0.15% and a methanol solution with a volume fraction of 95%-100%, and the volume ratio of the phosphoric acid solution to the methanol solution is 75-85:15-25.

3. The method according to claim 1, characterized in that In S2, the solvent is a mixed solution of a phosphoric acid solution with a volume fraction of 0.05%-0.15% and a methanol solution with a volume fraction of 95%-100%, and the volume ratio of the phosphoric acid solution to the methanol solution is 75-85:15-25.

4. The method according to claim 1, characterized in that: In S3, the chromatographic column is Welch Ultimate XB-C18, 4.6 mm×250 mm, 5 μm.

5. The method according to claim 1, characterized in that In S3, the mobile phases are: a phosphoric acid solution with a volume fraction of 0.05%-0.1% as mobile phase A, and a methanol solution with a volume fraction of 98%-100% as mobile phase B.

6. The method according to claim 1, characterized in that In S3, the column temperature is 30°C.

7. The method according to claim 1, characterized in that In S3, the detection wavelength is 271 nm.

8. The method according to claim 1, characterized in that In S3, the elution gradient is: 0-8min, the volume fraction of mobile phase A is 75%-80%, and the volume fraction of mobile phase B is 20%-25%; 8-25min, the volume fraction of mobile phase A changes from 75%-80% to 20%-25%, and the volume fraction of mobile phase B changes from 20%-25% to 75%-80%; 25-27min, the volume fraction of mobile phase A is 20%-25%, and the volume fraction of mobile phase B is 75%-80%; 27-30min, the volume fraction of mobile phase A increased from 20%-25% to 75%-80%, and the volume fraction of mobile phase B decreased from 75%-80% to 20%-25%; 30-40min, the volume fraction of mobile phase A is 75%-80%, and the volume fraction of mobile phase B is 20%-25%.

9. The method according to claim 8, characterized in that In S3, the elution gradient is: 0-8min, the volume fraction of mobile phase A was 80%, and the volume fraction of mobile phase B was 20%; 8-25min, the volume fraction of mobile phase A increased from 80% to 25%, and the volume fraction of mobile phase B increased from 20% to 75%; 25-27min, the volume fraction of mobile phase A is 25%, and the volume fraction of mobile phase B is 75%; 27-30min, the volume fraction of mobile phase A increased from 25% to 80%, and the volume fraction of mobile phase B increased from 75% to 20%; 30-40min, the volume fraction of mobile phase A is 80%, and the volume fraction of mobile phase B is 20%.

10. The method according to claim 1, characterized in that In S3, the flow rate of the chromatographic condition is 1-1.5 mL / min; the injection volume of the chromatographic condition is 15-25 μL.

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