CN107305198B - Method for separating and measuring apremilast and related substances by high performance liquid chromatography - Google Patents

Method for separating and measuring apremilast and related substances by high performance liquid chromatography Download PDF

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CN107305198B
CN107305198B CN201610242289.7A CN201610242289A CN107305198B CN 107305198 B CN107305198 B CN 107305198B CN 201610242289 A CN201610242289 A CN 201610242289A CN 107305198 B CN107305198 B CN 107305198B
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张吉
周春燕
谭辉
颜波
周维
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Chongqing Huabangshengkai Pharm Co ltd
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Abstract

The invention belongs to the technical field of analytical chemistry, relates to separation and detection of apremilast and related substances, and particularly relates to a method for separating and determining apremilast and related substances by adopting a high performance liquid chromatography. The method takes octadecylsilane chemically bonded silica as a stationary phase, takes an aqueous solution of acetonitrile and/or methanol as a mobile phase, and the mobile phase also contains phosphoric acid with the volume percentage concentration of 0.02-0.15%. The method is applicable to the five related substances possibly generated in the process of synthesizing the apremilast at present, can be used for simultaneously separating and detecting one or more related substances, has stable baseline, and can more accurately and more effectively detect and separate the five related substances, thereby more accurately ensuring the quality control of the apremilast raw material and the preparation thereof and finally determining the safety and effectiveness of the product.

Description

Method for separating and measuring apremilast and related substances by high performance liquid chromatography
Technical Field
The invention belongs to the technical field of analytical chemistry, relates to separation and detection of apremilast and related substances, and particularly relates to a method for separating and determining apremilast and related substances by adopting a high performance liquid chromatography.
Background
Apremilast is a phosphodiesterase inhibitor, the first small molecule drug approved by FDA in 2014 at 21/3 for the treatment of psoriasis-induced arthritis (PsA), Celgene, formerly known as research. Apremilast molecular formula is C22H24N2O7S, chemical name is N- [2- [ (1S) -1- (3-ethoxy-4-methoxyphenyl 1) -2- (methylsulfonyl) ethyl]-2, 3-dihydro-1, 3-dione-1H-isoindol-4-yl]Acetamide, having the chemical formula:
Figure GDA0002916986490000011
in the apremilast synthesis process, some related impurity substances such as by-products or intermediates generated in the production process or degradation products and the like are generated, and these impurities may affect the purity and quality of the medicine due to incomplete removal, and at present, 5 kinds of impurity substances are known, which are respectively: related substances 1 (process impurities/degradation products) shown as a formula I, related substances 2 (process impurities/degradation products) shown as a formula II, related substances 3 (process impurities) shown as a formula III, related substances 4 (process impurities) shown as a formula IV and related substances 5 (process impurities/degradation products) shown as a formula V, wherein the structural formulas of the 5 related substances are as follows:
Figure GDA0002916986490000021
for the by-products and intermediates introduced in the Apremilast synthesis process, the quality control needs to be carried out in Apremilast bulk drugs, and the quality control needs to be carried out on Apremilast related degradation impurities in Apremilast bulk drugs and preparations, so that the realization of the separation and the determination of Apremilast and related substances has important significance for the production and the storage of Apremilast raw materials and preparations.
In the prior art, the invention patent with the application number of 201410765717.5 discloses a method for separating and measuring related substances of Apremilast by using a liquid chromatography, which uses a chromatographic column with octadecylsilane chemically bonded silica as a filler and uses 0.1 percent trifluoroacetic acid and acetonitrile as mobile phases to quantitatively measure the content of the Apremilast and the related substances thereof; however, the method disclosed in the invention patent with the application number of 201410765717.5 cannot completely separate apremilast and related impurities, the system baseline drift is severe, and the method cannot be applied to other related impurities, and cannot completely separate and detect five impurities described in the application at the same time, which means that the method disclosed in the prior art cannot be applied to the detection of the five impurities at the same time.
Disclosure of Invention
In view of the above, the inventor of the present application searches a quality control method for apremilast-related substances through a large number of experiments, and strictly performs method verification, so that the method is scientific and rigorous, and the requirements of research, development and production are met. Therefore, the present invention is directed to a method for separating apremilast and related substances. The invention also aims to provide a method for separating and measuring apremilast and related substances by using the high performance liquid chromatography.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the method for separating apremilast and related substances uses octadecylsilane chemically bonded silica as a stationary phase and uses an aqueous solution of acetonitrile and/or methanol as a mobile phase.
Further, in the method, the mobile phase also contains phosphoric acid with a volume percentage concentration of 0.02-0.15%, preferably 0.02-0.1%, and more preferably 0.05%.
Further, in the method, the related substances are one or more of formula I, formula II, formula III, formula IV and formula V; the structural formulas of the formula I, the formula II, the formula III, the formula IV and the formula V are shown as follows:
Figure GDA0002916986490000031
the method for separating apremilast and related substances is suitable for separating the 5 impurities, can be used for separating one impurity independently and also can be used for separating the 5 impurities simultaneously, and compared with the prior art, the detection method has the advantages that the baseline is stable, the apremilast and the 5 related substances can be separated more accurately and more effectively, and the method has remarkable progress.
The invention also discloses a method for separating and measuring apremilast and related substances by using the high performance liquid chromatography, wherein a chromatographic column adopted in the analysis process of the high performance liquid chromatography uses octadecylsilane chemically bonded silica as a filling agent, and a solution A and an organic solvent B are mixed according to a certain proportion as a mobile phase for elution; the solution A is water, and the organic solvent B is acetonitrile and/or methanol.
Further, in the method, the solution A is a phosphoric acid aqueous solution with the volume percentage concentration of 0.02-0.15%.
Further, the method preferably comprises a phosphoric acid aqueous solution having a concentration of 0.02 to 0.1% by volume, and more preferably a phosphoric acid aqueous solution having a concentration of 0.05% by volume.
Further, in the method, it is preferable that the organic solvent B is acetonitrile.
Furthermore, in the method, the organic solvent B also contains phosphoric acid with a volume percentage concentration of 0.02-0.15%, preferably a volume percentage concentration of 0.02-0.1%, and more preferably a volume percentage concentration of 0.05%.
Further, the related substances are one or more of compounds shown in formula I, formula II, formula III, formula IV and formula V.
Further, the method is characterized in that the high performance liquid chromatography is reversed phase liquid chromatography, the adopted chromatographic column takes octadecylsilane chemically bonded silica as a filler, the particle size of the octadecylsilane chemically bonded silica is 3-5 μm, preferably 5 μm, and the column length of the chromatographic column is 150-250mm, preferably 250 mm. When the chromatographic column is used for separation, the applicable column temperature range of the chromatographic column is 30-40 ℃, and the preferred column temperature is 40 ℃.
Further, the method, when the elution is carried out by using the mobile phase, the flow rate of the mobile phase is 0.5-1.5ml/min, and preferably 1.0 ml/min.
Further, in the method, an ultraviolet detector is adopted for detection after elution, and both a DAD detector and a VWD detector are suitable. Preferably, in the analysis process of the high performance liquid chromatography, an ultraviolet detector is adopted for detection after the mobile phase is eluted, and the detection wavelength is 230 +/-5 nm.
Further, the method comprises the step of mixing the solution A and the organic solvent B according to a certain proportion to form a mobile phase for gradient elution, wherein the gradient proportion is as follows:
0min, wherein the volume ratio of the solution A to the organic solvent B is 80: 20;
for 10min, the volume ratio of the solution A to the organic solvent B is 80: 20;
35min, wherein the volume ratio of the solution A to the organic solvent B is 25: 75;
45min, wherein the volume ratio of the solution A to the organic solvent B is 25: 75;
46min, wherein the volume ratio of the solution A to the organic solvent B is 80: 20;
and (3) 52min, wherein the volume ratio of the solution A to the organic solvent B is 80: 20.
Further, in the method, in the analysis process of the high performance liquid chromatography, a reference substance or a sample to be detected needs to be dissolved firstly, and in the invention, acetonitrile and 0.01M KH are adopted2PO4The mixed solution is a reference substance or a sample to be detected which is dissolved or diluted by a solvent, the pH of the mixed solution is 3.5 +/-0.2, and the pH is preferably adjusted by phosphoric acid.
Further, it is preferable that acetonitrile and 0.01M KH are contained in the mixture2PO4Is 50: 50.
the method of the invention is realized according to the following method in a specific embodiment:
taking proper amount of reference substances of apremilast, related substances 1, related substances 2, related substances 3, related substances 4 and related substances 5, and adding acetonitrile: 0.01M KH2PO4(pH 3.5 with phosphoric acid) 50: 50, dissolving to prepare a system suitability solution containing 300 mu g of Apremilast and 3 mu g of related substances 1, 2,3, 4 and 5 respectively per 1 ml.
Taking an apremilast control, adding acetonitrile: 0.01M KH2PO4 (pH 3.5 with phosphoric acid) 50: 50, and prepared into a control solution containing 1.5. mu.g of the total amount per 1 ml.
Taking a proper amount of a sample to be detected (apremilast or a medicinal composition containing apremilast), adding acetonitrile: 0.01M KH2PO4(pH 3.5 with phosphoric acid) 50: dissolving 50% to obtain a sample solution containing Apremilast 300 μ g per 1ml, respectively adding the system applicability solution, the sample solution and the reference solution into a liquid chromatograph, and performing chromatography under the conditions of the present invention (the filler of the chromatographic column is octadecylsilane chemically bonded silica, the column length is 250mm, the detection wavelength is 230nm, and 0.05% phosphoric acid solution and 0.05% acetonitrile phosphate are used as the detection wavelengthGradient elution is carried out on the mobile phase, the proportion of the gradient elution is shown in table 1, the flow rate of the mobile phase is 1.0ml/min, the temperature of a chromatographic column incubator is 40 ℃, and the injection volume is 20 mul), the content of each known impurity in the sample is calculated according to a self-contrast method with a correction factor, the content of other single impurities in the sample is calculated according to the self-contrast method, and the total impurities are the sum of each known impurity and other single impurities.
TABLE 1 volume of gradient elution solution A and organic solvent B
Time (min) 0 10 35 45 46 52
Solution A (%) 80 80 25 25 80 80
Organic solvent B (%) 20 20 75 75 20 20
The invention has the beneficial effects that:
the method for separating apremilast and related substances has stable baseline, and can detect and separate the 5 related substances more accurately and effectively, while the baseline of the method with the application number of 201410765717.5 has serious drift and can not completely separate and detect the 5 related substances.
The method for separating and measuring apremilast and related substances by using the high performance liquid chromatography is applicable to the 5 related substances, can be used for simultaneously separating and detecting one or more related substances, and improves the separation and detection efficiency compared with the prior art. Compared with the prior art, the detection result is more accurate, so that the quality of the apremilast raw material and the preparation thereof can be more accurately controlled, and the safety and effectiveness of the product can be finally determined.
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FIG. 1 is a HPLC chart of a system suitability solution in example 1 and example 2;
FIG. 2 is an HPLC chart of the substances related to Apremilast measured in example 1;
FIG. 3 is an HPLC chart of substances related to the determination of Apremilast tablets in example 2;
FIG. 4 is a HPLC chart of a system suitability solution in comparative example 1;
FIG. 5 is a HPLC chart of a solution of the substance 2 of interest in comparative example 1;
FIG. 6 is a HPLC chart of the determination of apremilast acid degradation solution in example 3 using the method of the present invention;
FIG. 7 is an HPLC chart of the apremilast acid degradation solution measured by the method of application No. 201410765717.5 in comparative example 2;
FIG. 8 is a chromatogram of a pool HPLC of the RRT0.97 peak in the acid-degraded solution determined by the method of the invention in example 3;
FIG. 9 is a mass spectrum of [ M + H ] with a retention time of 25.303min peak in comparative example 2;
FIG. 10 is a mass spectrum of [ M-H ] of the peak having a retention time of 25.303min in comparative example 2.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings, which are used for better illustrating the contents of the present invention, but not for limiting the contents of the present invention to the embodiments. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
In the following examples, the apparatus and chromatographic conditions used in the process of the invention are as follows:
(1) high performance liquid chromatograph: LC-2010 (Shimadzu);
a detector: UV;
a chromatographic workstation: LCsolution.
(2) A chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the particle size of silica particles is 5 μm, the length of a chromatographic column is 250mm, and the diameter is 4.6 mm.
(3) Mass spectrometry: API 3000;
a workstation: analyst 1.4.2;
an ion source: ESI.
(4) Mobile phase
Solution A: 0.05% phosphoric acid solution;
an organic solvent B: 0.05% acetonitrile phosphate.
(5) Detection conditions
Mobile phase: mixing the solution A and the organic solvent B according to the volume ratio shown in the following table to be used as a mobile phase for gradient elution;
flow rate of mobile phase: 1.0 ml/min;
temperature of the column oven: 40 ℃;
detector detection wavelength after elution: 230 nm;
sample introduction amount: 20 μ l.
Time (min) 0 10 35 45 46 52
Solution A (%) 80 80 25 25 80 80
Organic solvent B (%) 20 20 75 75 20 20
Example 1 determination of substances related to Apremilast bulk drug
Taking proper amount of reference substances of apremilast, related substances 1, related substances 2, related substances 3, related substances 4 and related substances 5, and adding acetonitrile: 0.01M KH2PO4(pH 3.5 with phosphoric acid) 50: 50 (volume ratio) and preparing a system suitability solution containing 300 mug of apremilast and 3 mug of related substance 1, related substance 2, related substance 3, related substance 4 and related substance 5 respectively in each 1 ml.
Taking a proper amount of apremilast raw material medicine, adding acetonitrile: 0.01M KH2PO4(pH 3.5 with phosphoric acid) 50: 50 (volume ratio) and preparing a test solution containing 300 mu g of apremilast per 1 ml.
Precisely measuring a proper amount of the test solution, namely acetonitrile: 0.01M KH2PO4(pH 3.5 with phosphoric acid) 50: 50 (by volume) were dissolved and prepared as a control solution containing 1.5. mu.g per 1 ml.
Respectively injecting the system applicability solution, the control solution and the sample solution into a liquid chromatograph, measuring according to the chromatographic conditions, and recording the chromatogram, wherein the results are shown in figure 1 and figure 2. And calculating the content of each known impurity in the sample according to a main component self-comparison method with a correction factor, and calculating the content of other single impurities in the sample according to the self-comparison method, wherein the total impurity is the sum of each known impurity and other single impurities.
The calculation formula is as follows:
Figure GDA0002916986490000081
Figure GDA0002916986490000082
Figure GDA0002916986490000083
in the formula: a. thex-peak area of each impurity peak, mAu;
Ar-0.5% of the peak area of the main peak of the control solution, mAu;
fx-a known impurity correction factor;
∑Ax-sum of peak areas of individual impurities, mAu.
EXAMPLE 2 determination of related substances in Apremilast formulations (Apremilast tablets)
Taking proper amount of reference substances of apremilast, related substances 1, related substances 2, related substances 3, related substances 4 and related substances 5, and adding acetonitrile: 0.01M KH2PO4(pH 3.5 with phosphoric acid) 50: 50 (volume ratio) and preparing a system suitability solution containing 300 mug of apremilast and 3 mug of related substance 1, related substance 2, related substance 3, related substance 4 and related substance 5 respectively in each 1 ml.
Taking apremilast tablets, grinding, taking a proper amount of acetonitrile: 0.01M KH2PO4(pH 3.5 with phosphoric acid) 50: a suitable amount of 50 (by volume) was sonicated for 30 minutes (shaking at any time) to prepare a test solution containing 300. mu.g of apremilast per 1 ml.
Precisely measuring a proper amount of the test solution, namely acetonitrile: 0.01M KH2PO4(pH 3.5 with phosphoric acid) 50: 50 (by volume) were dissolved and prepared as a control solution containing 1.5. mu.g per 1 ml.
Respectively injecting the system applicability solution, the control solution and the sample solution into a liquid chromatograph, measuring according to the chromatographic conditions, and recording the chromatogram, wherein the results are shown in fig. 1 and fig. 3. And calculating the content of each known impurity in the sample according to a main component self-comparison method with a correction factor, and calculating the content of other single impurities in the sample according to the self-comparison method, wherein the total impurity is the sum of each known impurity and other single impurities.
The calculation formula is as follows:
Figure GDA0002916986490000091
Figure GDA0002916986490000092
Figure GDA0002916986490000093
in the formula: a. thex-peak area of each impurity peak, mAu;
Ar-0.5% of the peak area of the main peak of the control solution, mAu;
fx-a known impurity correction factor;
∑Ax-sum of peak areas of individual impurities, mAu.
Comparative example 1
The assay was performed according to the method disclosed in the prior art (application No. 201410765717.5) under the following chromatographic conditions:
a chromatographic column: octadecylsilane bonded silica (Hypersil BDS C18) had a column length of 150mm and a diameter of 4.6mm (acid-resistant column), and the particle diameter of the octadecylsilane bonded silica was 5 μm.
The mobile phase is the mixture of the A phase and the B phase.
Phase A: 0.1mol/L trifluoroacetic acid solution; phase B: and (3) acetonitrile. The mixture was mixed in the volume ratio shown in the following table as a mobile phase to perform gradient elution.
Time (min) 0 30 35 40 50
Solution A (%) 90 40 40 90 90
Organic solvent B (%) 10 60 60 10 10
Detection wavelength: 220 nm.
Sample introduction amount: 20 μ l.
Temperature of the column oven: at 25 ℃.
Detection time: 2 times retention time of the main peak.
Appropriate amount of reference substances of apremilast, related substance 1, related substance 2, related substance 3, related substance 4 and related substance 5 are dissolved in acetonitrile to prepare a system-applicable solution containing 300. mu.g of apremilast and 3. mu.g of related substance 1, related substance 2, related substance 3, related substance 4 and related substance 5 per 1ml, the solution is injected into a liquid chromatograph, the measurement is carried out according to the chromatographic condition with the application number of 201410765717.5, and the chromatogram is recorded, and the result is shown in figure 4.
In fig. 4, the number of detected known impurity peaks was 4, and 1 known impurity-related substance 2 was not detected, as compared with fig. 1 in example 1.
And taking a proper amount of related substance 2 reference substances, adding acetonitrile: 0.01M KH2PO4(pH 3.5 with phosphoric acid) 50: 50 (volume ratio), and preparing a solution containing 20. mu.g of the substance 2 per 1ml, injecting the solution into a liquid chromatograph, measuring under the chromatographic conditions of application No. 201410765717.5, and recording the chromatogram, wherein the result is shown in FIG. 5.
As is clear from FIG. 5, even if the concentration of the substance 2 in the sample solution was increased, the substance 2 could not be detected under the chromatographic conditions of application No. 201410765717.5.
It can be seen that the method of the present invention can simultaneously detect the related substance 1, the related substance 2, the related substance 3, the related substance 4 and the related substance 5, but cannot detect the related substance 2 by the method of application No. 201410765717.5.
Example 3 detection of related substances after degradation of Apremilast acid
Taking a proper amount of apremilast, placing the apremilast in a measuring flask, adding 2.0ml of 1mol/L hydrochloric acid solution, placing the apremilast in a water bath at 80 ℃ for 1h, taking out the apremilast, cooling, adding 2.0ml of 1mol/L sodium hydroxide solution for neutralization, and adding acetonitrile: 0.01M KH2PO4(pH 3.5 with phosphoric acid) 50: 50 (volume ratio) mixed solution 40ml, ultrasonic treatment for 10 minutes (shaking at any time), taking out, cooling, adding acetonitrile: 0.01M KH2PO4(pH 3.5 with phosphoric acid) 50: the 50 (volume ratio) mixed solution is fixed to the scale, shaken up and filtered, the subsequent filtrate is taken as the test solution, the test solution is taken and injected into a high performance liquid chromatograph, the analysis is carried out according to the chromatographic conditions of the invention, the chromatogram is recorded, and the result is shown in figure 6.
Comparative example 2
The detection of the relevant substances after the degradation of apremilast acid was carried out according to the method disclosed in the prior art (application No. 201410765717.5), with the chromatographic conditions:
a chromatographic column: octadecylsilane bonded silica (Hypersil BDS C18) had a column length of 150mm and a diameter of 4.6mm (acid-resistant column), and the particle diameter of the octadecylsilane bonded silica was 5 μm.
Mobile phase a phase: 0.1mol/L trifluoroacetic acid solution; phase B: and (3) acetonitrile. The A phase and the B phase are mixed according to the volume ratio shown in the following table to be used as mobile phases for gradient elution.
Time (min) 0 30 35 40 50
Solution A (%) 90 40 40 90 90
Organic solvent B (%) 10 60 60 10 10
Detection wavelength: 220 nm;
sample introduction amount: 20 mu l of the mixture;
temperature of the column oven: 25 ℃;
detection time: 2 times retention time of the main peak.
The sample solution prepared in example 3 was injected into a high performance liquid chromatograph, detection was performed under the chromatographic conditions of this example, and a chromatogram was recorded, with the results shown in fig. 7.
And (2) injecting 20 mu l of the test sample solution prepared in the example 3 into a liquid chromatograph, detecting according to the chromatographic conditions of the invention, collecting a chromatographic peak solution of the maximum degradation impurity RRT0.97, injecting 20 mu l of the collected solution into the liquid chromatograph, detecting according to the chromatographic conditions of the invention, recording the chromatogram, and collecting the chromatographic peak solution with the retention time of 25.303min, and analyzing by adopting the positive ion and negative ion modes of API3000 MS and ESI ion source, wherein the results are shown in figures 9 and 10.
As is clear from fig. 6 and 7, the number of impurity peaks detected in fig. 6 is larger, and the substance 2 related to acid degradation impurities is not detected in fig. 7.
As can be seen from FIG. 8, RRT0.97 actually shows an acid degradation peak, and application No. 201410765717.5 failed to detect this degradation impurity.
As can be seen from FIGS. 9 and 10, the molecular weight of the peak with a retention time of 25.303min was 418.46, which is consistent with the molecular weight of the substance 2.
From the results of example 3 and comparative example 2, it is understood that the related substance 2 can be effectively detected by detecting the related substance after the apremilast acid degradation by using the chromatographic conditions of the present invention, and the method of application No. 201410765717.5 has poor impurity separation capability, cannot effectively detect the related substance 2, and has more accurate detection result.
In summary, compared with the prior art, the method for separating and determining apremilast and related substances by high performance liquid chromatography of the invention comprises the following steps: firstly, the method is applicable to 5 related substances, and can be used for simultaneously separating and detecting one or more related substances, so that compared with the prior art, the separation and detection efficiency is improved; secondly, the chromatographic condition of the invention is utilized to detect the apremilast acid degradation impurities, the related substance 2 can be separated from other degradation impurities more accurately and effectively, the method with the application number of 201410765717.5 can not detect the related substance 2, the quality of the apremilast raw material and the preparation thereof can be more accurately ensured to be controllable, and the safety and the effectiveness of the product can be finally determined. Thus, the chromatographic conditions of the present invention bring beneficial effects with significant advancement.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Claims (3)

1. A method for separating and measuring apremilast and related substances by high performance liquid chromatography is characterized in that a chromatographic column adopted in the analysis process of the high performance liquid chromatography takes octadecylsilane chemically bonded silica as a filling agent, and a solution A and an organic solvent B are mixed according to a certain proportion as a mobile phase for gradient elution; the organic solvent B is acetonitrile and/or methanol; the solution A is a phosphoric acid aqueous solution with the volume percentage concentration of 0.02-0.15%; the organic solvent B also contains phosphoric acid with the volume percentage concentration of 0.02-0.15%; the related substances are compounds shown in a formula I, a formula II, a formula III, a formula IV and a formula V, and the specific structural formula is as follows:
Figure FDA0002938271590000011
the gradient elution ratio is as follows:
0min, wherein the volume ratio of the solution A to the organic solvent B is 80: 20;
for 10min, the volume ratio of the solution A to the organic solvent B is 80: 20;
35min, wherein the volume ratio of the solution A to the organic solvent B is 25: 75;
45min, wherein the volume ratio of the solution A to the organic solvent B is 25: 75;
46min, wherein the volume ratio of the solution A to the organic solvent B is 80: 20;
and (3) 52min, wherein the volume ratio of the solution A to the organic solvent B is 80: 20.
2. The method according to claim 1, wherein during the HPLC analysis, the mobile phase is eluted and then detected by an ultraviolet detector, and the detection wavelength is 230 ± 5 nm.
3. The method of claim 1, wherein the HPLC analysis is performed using acetonitrile and 0.01M KH2PO4The mixed solution is a reference substance or a sample to be detected which is dissolved or diluted by a solvent, and the pH value of the mixed solution is 3.5 +/-0.2.
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