CN115469039A

CN115469039A - Butylphthalide and related substance detection method thereof

Info

Publication number: CN115469039A
Application number: CN202211332102.4A
Authority: CN
Inventors: 徐霞; 彭桢利; 李连明; 文涛; 张海
Original assignee: Chengdu Shibeikang Biological Medicine Technology Co ltd
Current assignee: Chengdu Shibeikang Biological Medicine Technology Co ltd
Priority date: 2022-10-28
Filing date: 2022-10-28
Publication date: 2022-12-13
Anticipated expiration: 2042-10-28

Abstract

The invention relates to the field of drug detection and analysis, and particularly relates to a method for detecting butylphthalide and related substances thereof. The detection method adopts high performance liquid chromatography, adopts 5-fluorophenylpropyl bonded silica gel as a filler, adopts an ultraviolet detector, detects the wavelength of 210-280 nm, takes an acid salt solution as a mobile phase A, takes a mixed solution of methanol and acetonitrile as a mobile phase B, and performs gradient elution. The detection method provided by the invention is stable, good in specificity, high in sensitivity and accuracy, and suitable for drug registration quality research of butylphthalide and related substances thereof.

Description

Butylphthalide and related substance detection method thereof

Technical Field

The invention relates to the field of drug detection and analysis, in particular to a method for detecting butylphthalide and related substances thereof.

Background

The butylphthalide is 3-butyl-l (H) -isobenzofuranone, is racemic-3-n-butylphthalide, is also called apigenin, is firstly extracted from celery seeds, is colorless or light yellow clear oily liquid, has specific aroma, and is easy to degrade under the conditions of illumination and high temperature. The structure of butylphthalide is as follows:

。

the common synthesis process of butylphthalide is mainly a route disclosed in patent CN 101962374A:

。

according to the structural property and the main synthesis process of butylphthalide, related substances of the butylphthalide are mainly divided into butylphthalide analogs, degradation products and process impurities, wherein the butylphthalide analogs comprise alkenyl phthalides and alkyl phthalides, for example, the alkenyl phthalides comprise methylene phthalide, ethylene phthalide, propylene phthalide, butylene phthalide and pentene phthalide, and the alkyl phthalides comprise phthalide, methyl phthalide, ethyl phthalide, propylene phthalide and pentyl phthalide; the degradation products comprise hydroxybutylphthalide and o-pentenoic benzoic acid; the process impurities comprise reaction intermediate o-valeryl benzoic acid and products of ring-opening intermediate side reaction, such as butyl phthalate, dibutyl phthalate, 1, 2-dipentyl benzene and the like. Most of the analogs of butylphthalide can be removed by means of process technology, and degradation impurities and process impurities become key points of product quality control, particularly, the main degradation impurity of butylphthalide, namely hydroxy butylphthalide and synthetic intermediate, namely o-valeryl benzoic acid. The prior art does not report an effective detection method for simultaneously detecting the impurities. Therefore, a detection method for effectively detecting butylphthalide and various impurities thereof is urgently needed in the field so as to realize accurate control of product quality.

National drug standard of butylphthalide (WS 1- (X-124) -2005Z) records a high performance liquid chromatography method for detecting related substances and contents, methanol-water (65-35) is used as a mobile phase, and the detection wavelength is 280nm; however, this method cannot effectively detect the degradation impurities. Patent CN110108818A, example 1, discloses a high performance liquid chromatography method for simultaneously detecting various impurities such as hydroxybutylphthalide, butylphthalide and propylphthalide, but is not suitable for detecting process impurities such as 1, 2-dipentacylbenzene. The article name of 'detection of impurities in butylphthalide bulk drug by reversed-phase high performance liquid chromatography' (doi: 10.16155/j.0254-1793.2020.07.16) describes a reversed-phase high performance liquid chromatography which can simultaneously detect impurities such as hydroxybutylphthalide, propylphthalide and the like, but cannot detect impurities such as alkenylphthalide, such as butylphthalide.

Therefore, the method for detecting the relevant substances is developed reliably and simultaneously detecting the impurities such as the degradation impurities, the butylphthalide analogue impurities, the process intermediates and the like, the accuracy of the quality research of the butylphthalide product is improved, the safety and the reliability of the product are improved, and the significance is great.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides a method for detecting butylphthalide and related substances thereof. The method adopts liquid chromatography, and can conveniently and scientifically detect butylphthalide and its degradation impurities, butylphthalide analogue impurities and process impurities.

The invention provides a method for detecting butylphthalide and related substances thereof, which adopts high performance liquid chromatography, takes 5-fluorophenylpropyl bonded silica gel as a filling agent, adopts an ultraviolet detector, detects the wavelength of 210-280 nm, takes a salt solution with acidic pH value as a mobile phase A, takes a mixed solution of methanol and acetonitrile as a mobile phase B, and performs gradient elution.

Further, the above-mentioned related substances include, but are not limited to, one or more of hydroxybutylphthalide, butenylphthalide, o-pentenoic acid, propylphthalide, phthalic acid, monobutyl phthalate, o-valerylbenzoic acid, dibutyl phthalate, and 1, 2-dipentanoylbenzene.

Further, the information of the butylphthalide related to the invention, such as material codes and structures, is as follows:

。

further, the salt of the above salt solution comprises potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium acetate, sodium acetate, or potassium acetate.

Further, the pH of the salt solution is 1.0 to 5.0, preferably 2.0 to 4.0.

Further, the pH adjusting agent of the above salt solution contains phosphoric acid, hydrochloric acid, or acetic acid.

Further, the concentration of the salt solution is 1-50 mmol/L; preferably 1 to 30mmol/L.

Further, the detection wavelength is 230. + -.10 nm, preferably 230. + -.3 nm.

Further, the volume ratio of methanol to acetonitrile in the mobile phase B is 1: (0.8-4).

Further, the above gradient elution comprises the following procedure:

。

preferably, the above gradient elution comprises the following procedure:

。

further, the column temperature of the above-mentioned chromatographic conditions is 1 to 40 ℃, preferably 20 to 40 ℃.

Further, the flow rate of the above-mentioned chromatographic conditions is 0.5 to 1.5ml/min, preferably 1.0ml/min.

Further, the amount of the sample to be used in the above-mentioned chromatographic conditions is 5 to 50. Mu.l, preferably 10 to 20. Mu.l.

Further, the method comprises the following steps:

(1) Precisely weighing appropriate amount of butylphthalide or optional butylphthalide related substances, and diluting with solvent to obtain sample solution or mixed solution;

(2) Injecting the test solution or mixed solution into a liquid chromatograph, and detecting according to chromatographic conditions.

Further, the solvent comprises methanol, ethanol, 30-60% acetonitrile water solution or a mobile phase A and acetonitrile with the volume ratio of 70-40: 30-60 of mixed solution; preferably, the volume ratio of 30-60% acetonitrile water solution or mobile phase A to acetonitrile is 70-40: 30 to 60 portions of mixed solution. Wherein the mobile phase A is as defined above for mobile phase A in the chromatographic conditions; the percent of aqueous acetonitrile is percent by volume.

Furthermore, each 1ml of the test sample contains 0.1-10 mg of butylphthalide; preferably 0.2 to 2mg; more preferably 1mg.

Furthermore, each 1ml of the mixed solution contains 5-20 mu g of hydroxyl butylphthalide and 0.1-10 mu g of other related substances; preferably, each 1ml contains 10 mu g of hydroxyl butylphthalide and 1 mu g of other related substances;

advantageous effects: the detection method of butylphthalide and related substances thereof has good specificity, high sensitivity, good peak type of each peak, and separation degrees between impurity peaks and main peaks and between impurity peaks and impurity peaks larger than 1.5; the method has high accuracy, good repeatability and reproducibility, high precision, meets the quality analysis and detection requirements, and is suitable for the content of the product and the quality research of related substances.

Drawings

FIG. 1: system suitability solution chromatogram of condition 1.

FIG. 2 is a schematic diagram: system suitability solution chromatogram of condition 2.

FIG. 3: system suitability solution chromatogram of condition 3.

Detailed Description

The scheme of the present invention will be explained below with reference to test examples and examples. It will be appreciated by those skilled in the art that the following examples are illustrative only and should not be taken as limiting the scope of the invention. The particular techniques or conditions not specified in the examples are performed according to the techniques or conditions described in the literature in the field or according to the product specifications. The samples, reagents or instruments used are not indicated by the manufacturer, and are all conventional products in the art, which are commercially available.

Test example 1: methodology investigation of butylphthalide related substance detection method

Chinese herbal medicine prescription and its preparing and calculating method

Test solution: taking a proper amount of the product, precisely weighing, adding a solvent to dissolve and quantitatively diluting to prepare a solution containing about 1.0mg of the product in each lml.

Control solution: a proper amount of the test solution is precisely measured and diluted by a solvent to prepare a solution containing about 1.0 mu g of the test solution per lml.

Control solution: taking appropriate amount of reference substances of impurities Z1, Z2, Z3, Z6, M2, M3, M5, M6 and M7, precisely weighing, adding solvent to dissolve, and quantitatively diluting to obtain solution containing 1.0 μ g of each of impurities Z2, Z3, Z6, M2, M3, M5, M6 and M7 and 10 μ g of impurity Z1 in each lml.

System applicability solution: taking appropriate amount of impurities Z1, Z2, Z3, Z6, M2, M3, M5, M6 and M7 reference substances and test samples, precisely weighing, adding solvent to dissolve and quantitatively dilute to obtain solution containing 1.0 μ g of each of impurities Z2, Z3, Z6, M2, M3, M5, M6 and M7, 10 μ g of impurity Z1 and 1.0mg of the product in per lml.

Description of the drawings: the above solution was prepared using a 10mmol/l potassium dihydrogen phosphate solution (pH adjusted to 2.0 with phosphoric acid) -acetonitrile (50. It is understood that the solvent used in the method of the present invention also includes other solvents capable of dissolving the above substances, including but not limited to methanol, ethanol, 30 to 60% acetonitrile in water or a mobile phase A and acetonitrile in a volume ratio of 70 to 40: 30-60, wherein the mobile phase A is a salt solution with an acidic pH value, the salt solution comprises potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium acetate, sodium acetate or potassium acetate, the pH value is 1.0-5.0, and the pH regulator comprises phosphoric acid, hydrochloric acid or acetic acid. However, it is worth noting that the inventors of the present application found through experiments that, when the substance to be detected contains a compound having a carboxylic acid group such as M2 phthalic acid, the solvent preferably has a volume ratio of mobile phase a to acetonitrile of 70 to 40: 30-60, the impurity M2 peak can not be forked or shoulder-peaked, and the M5 peak has stable retention time and can not be greatly drifted.

The determination method comprises the following steps: precisely measuring each solution, respectively injecting into a liquid chromatograph, and recording chromatogram.

The calculation method comprises the following steps: calculated as peak area by external standard method.

Condition screening of spent fuel chromatography

Referring to a detection method of related substances in the national drug standard of butylphthalide, the detection method of related substances of the product is finally determined by optimizing the method, wherein typical chromatographic conditions are as follows:

condition 1: method for preparing related substances in butylphthalide national drug standard

Chromatographic conditions are as follows: by adopting high performance liquid chromatography and an ultraviolet detector, the detection wavelength is 280nm, an SveaTM C18.6 mm multiplied by 250mm 5 mu m is taken as a chromatographic column, methanol-water (63.

Test results and evaluation: the system applicability solution chromatogram of condition 1 is shown in fig. 1 (mixed solution of only Z-series impurities), all of which have low impurity response; the impurities Z1 and Z6 are completely overlapped and cannot be separated; the impurity Z3 has a poor peak shape.

Condition 2: method for example 1 in patent CN110108818A

Chromatographic conditions are as follows: key parameters such as detection wavelength 227nm, and a chromatographic column of Agilent ZORBAX plus phenyl-Hexyl, 4.6mm multiplied by 250mm and 5 mu m; the mobile phase A is 0.1% acetic acid aqueous solution (volume ratio); mobile phase B was acetonitrile-methanol (1, volume ratio); other parameters are shown in patent CN110108818A, example 1.

Test results and evaluation: the system applicability solution chromatogram of the condition 2 is shown in figure 2, and all impurity responses are low; the impurity M2 appears shoulder peaks, and the peak shape is poor; the separation degree of impurities M6 and M7 is 1.2, and the separation degree is poor; the peak emergence of the whole impurity is late, and the peak emergence of the impurity M7 is positioned at the peak inversion position.

Condition 3:

chromatographic conditions are as follows: high performance liquid chromatography and an ultraviolet detector are adopted, the detection wavelength is 230nm, the chromatographic column takes 5-fluorophenylpropyl bonded silica gel as a filler (SHMADZU Shim-pack Velox,4.6 x 250mm,5 mu m), 10mmol/L potassium dihydrogen phosphate solution (phosphoric acid is used for adjusting the pH value to 3.0) is taken as a mobile phase A, methanol: acetonitrile (45: 100, volume ratio) as mobile phase B, gradient elution according to the following table, injection volume of 10. Mu.l, flow rate of 1.0ml/min, column temperature of 35 ℃.

Test results and evaluation: the system applicability solution chromatogram of the condition 3 is shown in figure 3, and the solvent does not interfere with the detection of each impurity in the test sample; the peak type of each peak is good, and the separation degree of each impurity from the main peak and impurities meets the specification.

In conclusion, in order to satisfy detection sensitivity, resolution and peak pattern at the same time, chromatographic condition 3 was prepared as a relevant substance detection condition, and the method was verified.

Test example 2: method for verifying related substances

The solution used in this test and its preparation were the same as those in test example 1, and the chromatographic conditions were those in test example 1, condition 3.

Characteristic for fruit trees

Taking known impurities Z1, Z2, Z3, Z6, M2, M3, M5, M6 and M7, a blank solvent, a system applicability solution and a test solution in proper amount, and injecting samples according to a proposed chromatographic condition for determination. The results are shown in Table 2-1, and the chromatogram is shown in FIG. 3.

The test result shows that: under the condition of the proposed chromatogram, the blank solvent does not interfere the detection of each component to be detected and the main peak, the separation degree of each known impurity and each main component and the adjacent peak thereof is more than 1.5, and in the solution with system applicability, the purity of the main peak and each known impurity meets the specification, which shows that the proposed chromatogram condition has good specificity, the tailing factor of each peak is less than 1.2, and the peak type of each peak is good. Under the condition, the peak sequences of the components are M2, M5, M3, Z1, Z6, Z3-1, butylphthalide main component and Z3-2, Z2, M7 and M6, wherein Z3-1 and Z3-2 are cis-trans isomers of Z3, and the two peaks are combined to calculate Z3 in the following method verification.

Component of a polymer force the degradation test

In order to investigate the possible degradation path of butylphthalide (product for short) and the separation and detection capability of the proposed chromatographic conditions on the degradation products, a forced degradation test is carried out. The sample preparation method comprises the following steps: weighing an appropriate amount of the product (batch No. 000820), strongly destroying the product, dissolving the product with acetonitrile, and diluting the product to prepare a solution containing 1.0mg of butylphthalide per 1ml, thus obtaining the product. The test conditions are shown in Table 2-2.

And (4) measuring the solution according to the established chromatographic conditions, and calculating the content of each impurity according to an area normalization method. The results are shown in tables 2-3.

Under the condition of the chromatographic system, the blank solvent does not interfere with the detection of impurities in each damaged sample, each degraded impurity can be effectively separated from a main peak and each known impurity peak, the purity parameters of the main peaks accord with the regulations, and the material balance of each damaged sample is 95-105%. The method shows that the possibly degraded impurities can be effectively detected under the proposed chromatographic conditions.

\9354

Taking a proper amount of butylphthalide reference substances and known impurity reference substances, precisely weighing, respectively dissolving with a solvent, and gradually diluting to prepare reference substance solutions with certain concentration, wherein the reference substance solution is determined as the quantitative limit of the substance when the signal-to-noise ratio is about 10, and the reference substance solution is determined as the detection limit of the substance when the signal-to-noise ratio is about 3. The results are shown in tables 2-4.

The test result shows that: the sampling concentration planned by the inspection of related substances of the product is 1.0mg/ml, the sampling amount is 10 mu l, and the absolute sampling amount is 10000ng. The absolute sample injection amount is more than 10000 times of the detection limit of the impurities M2, M5, M3, M7, M6, Z1, Z2, Z3 and Z6, which indicates that the proposed sample injection concentration can ensure the effective detection of the impurities in the product.

\9355

Taking appropriate amount of each of MZ2, MZ3, MZ5, MZ6, MZ7 reference substances, precisely weighing, diluting with solvent to make reference substance solution with quantitative limit concentration, 50%, 80%, 100%, 120%, 150%, 200% of limit concentration, precisely weighing 10 μ l of each solution, injecting into liquid chromatograph, and recording chromatogram. The results of the sample injection linear analysis are shown in tables 2-5 with the sample injection concentration as the abscissa and the peak area as the ordinate.

Remarking: and the Z3 linearity adopts the sum of peak areas of a Z3-peak 1 and a Z3-peak 2 and the concentration to carry out linear analysis.

From the test results, it can be seen that when the M2 concentration is between 0.0204. Mu.g/ml and 2.0360. Mu.g/ml, the concentration and the peak area have a good linear relationship; when the concentration of M5 is between 0.0505 mu g/ml and 2.2440 mu g/ml, the concentration and the peak area have good linear relation; when the concentration of M3 is between 0.0453 mug/ml and 2.0120 mug/ml, the concentration and the peak area have good linear relation; when the concentration of M7 is between 0.0462 mu g/ml and 2.0540 mu g/ml, the concentration and the peak area have good linear relation; when the concentration of M6 is between 0.0503 mug/ml and 2.2360 mug/ml, the concentration and the peak area have good linear relation; when the concentration of Z1 is between 0.0847 mu g/ml and 22.4200 mu g/ml (LOQ-200% limit concentration), the concentration and the peak area form a good linear relation; when the concentration of Z2 is between 0.0304 mu g/ml and 2.0260 mu g/ml (LOQ-200% limit concentration), the concentration and the peak area form a good linear relation; when the concentration of Z3 is between 0.0424 mu g/ml and 2.1200 mu g/ml (LOQ-200% limit concentration), the concentration and the peak area form a good linear relation; when the concentration of Z6 is in the range of 0.12000 mu g/ml to 2.4000 mu g/ml (LOQ to 200% limit concentration), the concentration and the peak area have good linear relation.

\935630

And continuously injecting the system applicability solution for 6 times, recording a chromatogram, and calculating the peak area of each impurity and the RSD of the retention time.

Test results show that RSD of each peak area of each impurity and each main component in the system applicability solution is about 0.2 to 0.5 percent, RSD of retention time is about 0.01 to 0.04 percent, and the RSD is less than 2.0 percent, and the precision requirement of liquid chromatography determination is met.

\9357

In order to examine the precision of the measuring method of the related substances of the product, 6 parts of test solution is prepared and detected according to a formulated method. The test results are shown in tables 2 to 6.

The results show that the method has no obvious difference in the determination results of 6 samples for determining the impurities of the product, and has good repeatability.

\9358

In order to examine the degree of closeness between the detection results of the known impurities M2, M5, M3, M7, M6, Z1, Z2, Z3 and Z6 in the product and the true values by the proposed detection method, the accuracy is verified by adopting a sample recovery mode. Taking a proper amount of a test sample with known impurity content, preparing a test sample solution according to a formulated method, adding known impurities with limit concentrations of 50%, 100% and 150% into the test sample solution respectively, then carrying out sample injection measurement, and calculating the recovery rate.

The test results show that: the recovery rate of M2 is 98.04-102.67%, the average recovery rate is 100.9%, and the recovery rate RSD is 1.9%; the recovery rate of M5 is 98.53-100.89%, the average recovery rate is 100.0%, and the recovery rate RSD is 0.9%; the recovery rate of M3 is 98.84-102.56%, the average recovery rate is 100.5%, and the recovery rate RSD is 1.3%; the recovery rate of M7 is 95.25-99.15%, the average recovery rate is 96.8%, and the recovery rate RSD is 1.1%; the recovery rate of M6 is 99.13-102.91%, the average recovery rate is 101.2%, and the recovery rate RSD is 1.3%; the recovery rate of Z1 is 98.45-100.26%, the average recovery rate is 99.4%, and the recovery rate RSD is 0.6%; the recovery rate of Z2 is 98.59-100.40%, the average recovery rate is 99.2%, and the recovery rate RSD is 0.5%; the recovery rate of Z3 is 92.92-97.84%, the average recovery rate is 95.6%, and the recovery rate RSD is 1.0%; the Z6 recovery rate is between 100.69% and 109.10%, the average recovery rate is 103.4%, and the recovery rate RSD is 1.7%; and the recovery rate RSD of all 9 samples containing impurities is less than 2.0 percent, which indicates that the proposed detection method has high accuracy.

\9359

In order to verify the degree of accuracy of the measurement results when the established chromatographic conditions were slightly changed, the durability of the established chromatographic conditions was examined. Each solution was taken and measured under a prescribed condition.

Test results show that when the detection wavelength, the flow rate, the column temperature, the chromatographic column and the mobile phase are slightly changed, such as the detection wavelength is changed by +/-3 nm, the flow rate is changed by +/-0.5 ml/min, the column temperature is changed by +/-5 ℃, different types of chromatographic columns (fillers are unchanged) are changed, and the pH of the mobile phase A is changed by +/-1, under all conditions, the solvent does not interfere with the detection of all impurities in a sample, the measurement results of substances related to butylphthalide in the chromatogram of each sample solution and the system applicability solution are not obviously different, and the separation degree of all the impurities and main peaks and adjacent peaks thereof is more than 1.5, so that the requirements are met.

Example 1:

chromatographic conditions are as follows: the gradient program is shown in Table 3-1 below, and the other conditions are the same as in test example 2.

And (3) determination: and injecting each solution into a chromatographic system, and collecting chromatograms.

Example 2:

chromatographic conditions are as follows: the gradient program is shown in tables 3-2 below, and the other conditions are the same as in test example 2.

Example 3:

chromatographic conditions are as follows: mobile phase a was 50mmol/L potassium dihydrogen phosphate solution (pH adjusted to 5.0 with acetic acid) was mobile phase a, methanol-acetonitrile (1.

Example 4:

chromatographic conditions are as follows: mobile phase a was a 1mmol/L potassium dihydrogen phosphate solution (pH adjusted to 1.0 with hydrochloric acid) was mobile phase a, methanol-acetonitrile (1.

Example 5:

chromatographic conditions are as follows: mobile phase a was 10mmol/L sodium acetate solution (pH adjusted to 3.0 with acetic acid) was mobile phase a, methanol-acetonitrile (45.

And (3) determination: and injecting each solution into each chromatographic system, and collecting chromatograms.

Data statistics and summarization: the chromatograms of test example 2 "under specificity" and examples 1 to 5 above were summarized, and the peak areas, retention times and degrees of separation were counted, and the results showed: in each control solution chromatogram: the separation degree of each impurity, the main medicine and adjacent peaks of the main medicine is over 1.5, the separation degree requirement is met, the purity index of each peak meets the requirement, each method has good specificity, and the quality analysis requirement is met; and the peak areas of all impurities and main peaks are basically consistent, which proves that the accuracy of each method is high.

Claims

1. The method for detecting butylphthalide and related substances thereof adopts high performance liquid chromatography, and is characterized in that 5-fluorophenylpropyl bonded silica gel is adopted as a filling agent, an ultraviolet detector is adopted, the detection wavelength is 210 nm-280 nm, a salt solution with an acidic pH value is adopted as a mobile phase A, a mixed solution of methanol and acetonitrile is adopted as a mobile phase B, and elution is carried out according to gradient.

2. The method of claim 1, wherein the related substances include, but are not limited to, one or more of hydroxybutylphthalide, butenylphthalide, o-pentenoic acid, propylphthalide, phthalic acid, monobutyl phthalate, o-valerylbenzoic acid, dibutyl phthalate, 1, 2-dipentanoylbenzene.

3. The method of claim 1, wherein the salt of the salt solution comprises potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium acetate, sodium acetate, or potassium acetate.

4. The method of claim 1, wherein the salt solution has a concentration of 1 to 50mmol/L.

5. The method of claim 1, wherein the salt solution has a pH of 1.0 to 5.0; the pH adjusting agent of the salt solution comprises phosphoric acid, hydrochloric acid or acetic acid.

6. The method according to claim 1, wherein the detection wavelength is 230 ± 10nm, preferably 230 ± 3nm.

7. The method according to claim 1, wherein the volume ratio of methanol to acetonitrile in the mobile phase B is 1: (0.8-4).

8. The method of claim 1, wherein the gradient elution comprises the following procedure:

。

9. method according to claim 1, characterized in that it comprises the following steps:

10. The method according to claim 9, wherein the solvent comprises methanol, ethanol, 30-60% acetonitrile in water or a mobile phase a to acetonitrile in a volume ratio of 70-40: 30-60 of mixed solution; preferably, the volume ratio of 30-60% acetonitrile water solution or mobile phase A to acetonitrile is 70-40: 30 to 60 portions of mixed solution.