CN108344832B - Detection method for completely separating main component from impurities thereof - Google Patents
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- 239000012535 impurity Substances 0.000 title claims abstract description 30
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 18
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims abstract description 5
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 3
- 238000004007 reversed phase HPLC Methods 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 78
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 239000008213 purified water Substances 0.000 claims description 18
- 239000000523 sample Substances 0.000 claims description 15
- CKFVSMPWXAASIQ-MRXNPFEDSA-N 2-[(2r)-2-hydroxy-3-[4-(3-oxomorpholin-4-yl)anilino]propyl]isoindole-1,3-dione Chemical group C([C@@H](O)CN1C(C2=CC=CC=C2C1=O)=O)NC(C=C1)=CC=C1N1CCOCC1=O CKFVSMPWXAASIQ-MRXNPFEDSA-N 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 6
- -1 3-ethoxypropyl Chemical group 0.000 claims description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 239000007858 starting material Substances 0.000 claims description 4
- MHCRLDZZHOVFEE-UHFFFAOYSA-N 4-(4-aminophenyl)morpholin-3-one Chemical compound C1=CC(N)=CC=C1N1C(=O)COCC1 MHCRLDZZHOVFEE-UHFFFAOYSA-N 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 239000006227 byproduct Substances 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- XKJCHHZQLQNZHY-UHFFFAOYSA-N phthalimide Chemical compound C1=CC=C2C(=O)NC(=O)C2=C1 XKJCHHZQLQNZHY-UHFFFAOYSA-N 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- 239000000243 solution Substances 0.000 abstract description 33
- 239000012071 phase Substances 0.000 abstract description 13
- 239000012074 organic phase Substances 0.000 abstract description 4
- 239000000337 buffer salt Substances 0.000 abstract description 3
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 abstract description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000002156 mixing Methods 0.000 description 9
- 239000012490 blank solution Substances 0.000 description 8
- 238000005303 weighing Methods 0.000 description 8
- 239000012452 mother liquor Substances 0.000 description 7
- 239000000543 intermediate Substances 0.000 description 6
- 239000012085 test solution Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- 238000007865 diluting Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 150000002466 imines Chemical class 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000004807 localization Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 159000000000 sodium salts Chemical class 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Inorganic materials [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- KGFYHTZWPPHNLQ-AWEZNQCLSA-N rivaroxaban Chemical compound S1C(Cl)=CC=C1C(=O)NC[C@@H]1OC(=O)N(C=2C=CC(=CC=2)N2C(COCC2)=O)C1 KGFYHTZWPPHNLQ-AWEZNQCLSA-N 0.000 description 1
- 229960001148 rivaroxaban Drugs 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 235000011121 sodium hydroxide Nutrition 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/89—Inverse chromatography
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a detection method for completely separating main components and impurities thereof, which is characterized by comprising the following steps: the reversed-phase high performance liquid chromatography is adopted, a phenyl liquid chromatography column is adopted, a diode array detector is adopted, a gradient elution program is adopted, and a certain proportion of buffer salt solution monosodium salt-organic phase is taken as a mobile phase. The invention has the advantages that: the method has the advantages of strong specificity, high accuracy and simple and convenient operation.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a detection method for completely separating a main component from impurities of the main component.
Background
The 2- [ (2R) -2-hydroxy-3- [ [4- (3-oxo-4-morpholinyl) phenyl ] amino ] propyl ] -1H-isoindole-1, 3(2H) -dione (main component) is produced by reacting 4- (4-aminophenyl) morpholin-3-one with (S) - (+) -N- (2, 3-ethoxypropyl) phthalimide. These two starting materials have impurities remaining and by-products are formed during the synthesis of the main component, and these impurities remain in the detection process. The existing detection method for completely separating 2- [ (2R) -2-hydroxy-3- [ [4- (3-oxo-4-morpholinyl) phenyl ] amino ] propyl ] -1H-isoindole-1, 3(2H) -diketone from impurities thereof has the disadvantages of low specificity, low accuracy and complex operation. Therefore, a new technical solution should be provided to solve the above problems.
Disclosure of Invention
The invention aims to provide a detection method for completely separating main components and impurities thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
a high performance liquid chromatography detection method for completely separating rivaroxaban intermediates and impurities thereof adopts reversed-phase high performance liquid chromatography, phenyl liquid chromatography columns, diode array detectors and gradient elution procedures, and takes a certain proportion of buffer salt solution monosodium salt-organic phase as a mobile phase.
The further technical scheme is as follows:
the column is selected from ZORBAX-SB, 250mm 4.6mm 5 μm.
The organic phase is one of methanol, acetonitrile, propanol and isopropanol.
Preferably, the organic phase is methanol.
The buffer salt solution is one of phosphate, formate, acetate, citrate and perchlorate.
Preferably, the buffered salt solution is phosphate and the buffered salt solution has a pH of 6.8.
The sodium salt is one of sodium hydroxide, sodium acetate and sodium sulfate.
Preferably, the sodium salt is sodium hydroxide.
A method for detecting 2- [ (2R) -2-hydroxy-3- [ [4- (3-oxo-4-morpholinyl) phenyl ] amino ] propyl ] -1H-isoindole-1, 3(2H) -diketone and completely separating impurities thereof comprises the following steps,
1) taking a proper amount of a sample of 2- [ (2R) -2-hydroxy-3- [ [4- (3-oxo-4-morpholinyl) phenyl ] amino ] propyl ] -1H-isoindole-1, 3(2H) -dione, dissolving the sample with methanol respectively to prepare a sample solution containing 0.1-1.5 mg of 2- [ (2R) -2-hydroxy-3- [ [4- (3-oxo-4-morpholinyl) phenyl ] amino ] propyl ] -1H-isoindole-1, 3(2H) -dione per lmL;
2) setting the flow rate of a mobile phase to be 0.5-1.5 mL/min, the detection wavelength to be 200-240 nm, the temperature of a chromatographic column incubator to be 20-40 ℃, and the sample injection amount to be 5-30 mu L;
3) mobile phase a is ph6.8 phosphate buffer, methanol is 95:5, the mobile phase B is methanol, and the gradient elution procedure is as follows:
further, the flow rate of the detection wavelength of 220nm in the step 2) is 1.0ml/min, the temperature of a chromatographic column incubator is 30 ℃, and the sample injection amount is 10 mu L
The invention has the advantages that: the method has the advantages of strong specificity, high accuracy and simple and convenient operation.
Drawings
FIG. 1: specificity-blank solution chromatogram
FIG. 2: chromatogram of solution for specific 4-nitrone localization
FIG. 3: specific-imine positioning solution chromatogram
FIG. 4: solution chromatogram for specificity-principal component localization
FIG. 5: specific-two-substituted positioning solution chromatogram
FIG. 6: system applicability solution chromatogram
FIG. 7: quantitative limit test chromatogram
FIG. 8: detection limit test chromatogram
FIG. 9: sample introduction precision test chromatogram
FIG. 10: chromatogram for repeatability test
FIG. 11: chromatogram of intermediate precision test
FIG. 12: durability-unchanged chromatogram
FIG. 13: durability-pH-6.6 chromatogram
FIG. 14: durability-pH-7.0 chromatogram
FIG. 15: durability-wavelength-218 nm chromatogram
FIG. 16: durability-wavelength-222 nm chromatogram
FIG. 17: durability-flow-0.8 ml/min chromatogram
FIG. 18: durability-flow-1.2 ml/min chromatogram
FIG. 19: durability-column temperature-28 ℃ chromatogram
FIG. 20: durability-column temperature-32 ℃ chromatogram
Detailed Description
2- [ (2R) -2-hydroxy-3- [ [4- (3-oxo-4-morpholinyl) phenyl ] amino ] propyl ] -1H-isoindole-1, 3(2H) -dione (hereinafter referred to as main component) is produced by reacting 4- (4-aminophenyl) morpholin-3-one with (S) - (+) -N- (2, 3-ethoxypropyl) phthalimide. These two starting materials have impurities remaining and by-products are formed during the synthesis of the main component, and these impurities remain in the detection process.
The method considers 4 kinds of impurities, and the names and structural formulas of the impurities to be controlled are shown in table 1:
TABLE 1 impurity Structure and Source
The method for verifying the purity of the main components to be verified comprises the following steps:
purity detection conditions to be verified:
a chromatographic column: ZoRBAX-SB 250mm 4.6mm 5um agilent
Sample introduction amount: 10ul, flow rate: 1.0ml/min, column temperature: 30 ℃, wavelength: 220nm mobile phase composition:
mobile phase A: ph6.8 phosphate buffer (1L of water with 1.36g of monopotassium phosphate +0.188g of sodium hydroxide): methanol 95:5, mobile phase B: methanol
| t/min | A％ | B％ |
| 0.0 | 95 | 5 |
| 20.0 | 90 | 10 |
| 37.0 | 65 | 35 |
| 42.0 | 65 | 35 |
| 43.0 | 95 | 5 |
| 55.0 | 95 | 5 |
Methodological validation of chromatographic methods
Specificity test:
solution/sample concentration for localization of principal component (500 ug/ml):
precisely weighing 25mg of the product into a 50ml volumetric flask, adding 10ml of methanol and a proper amount of purified water for dissolving, then using the purified water to fix the volume to a scale, and uniformly mixing.
4-aminoketone mother liquor:
precisely weighing 25mg to 50ml volumetric flask, adding 10ml methanol and a proper amount of purified water to dissolve, then using the purified water to fix the volume to the scale, and mixing uniformly.
4-nitrone mother liquor:
precisely weighing 25mg to 50ml volumetric flask, adding 10ml methanol and a proper amount of purified water to dissolve, then using the purified water to fix the volume to the scale, and mixing uniformly.
Imine mother liquor:
precisely weighing 25mg to 50ml volumetric flask, adding 10ml methanol and a proper amount of purified water to dissolve, then using the purified water to fix the volume to the scale, and mixing uniformly.
A disubstituted mother liquor:
accurately weighing 100mg to 100ml volumetric flask, adding 20ml methanol and proper amount of purified water to dissolve, then using purified water to fix the volume to the scale, and mixing uniformly.
Blank solution (20% aqueous methanol):
adding 10ml of methanol into a 50ml volumetric flask, diluting with purified water to a constant volume to a scale, and uniformly mixing. Specific test solutions:
precisely transferring appropriate amount of each impurity mother liquor and main component mother liquor into the same volumetric flask to prepare mixed solution of each impurity and main component of 50ug/ml, and mixing. Used for investigating specificity and separation degree.
Positioning solution of each impurity and main component:
precisely transferring each impurity and appropriate amount of main component mother liquor, and respectively preparing into 50ug/ml positioning solution.
And respectively injecting the blank solution, the special test solution, the main component positioning solution, the 4-aminoketone positioning solution, the 4-nitrone positioning solution, the imine positioning solution and the disubstituted positioning solution into a liquid chromatograph, and recording the chromatogram.
TABLE 2 results of the specificity test
And (4) conclusion: 1. the separation degree between peaks meets the preset requirement; 2. the theoretical plate number meets the preset requirement; 3. the peak purity of each peak is less than the purity threshold.
And (3) sample injection precision test:
accurately weighing 49.05mg to 100ml of the product in a volumetric flask, adding 20ml of methanol and a proper amount of purified water to dissolve, and diluting with the purified water to a constant volume to be calibrated. Preparing blank solution by the same method.
And respectively injecting the blank solution and the sample injection repeatability test solution into a liquid chromatograph, and recording the chromatogram.
TABLE 3 sample introduction precision test results of impurities and main components
And (4) conclusion: 4-aminoketone, 4-nitrone, imine, main component and disubstituted RSD are 2.06%, 2.38%, 0.52%, 0.12% and 3.10%; the maximum single impurity RSD is 0.36%, but the relative retention time of unknown impurities of 0.34 is gradually increased, the RSD is 57.93%, and the RSD of the main component meets the requirement. The solution needs to be prepared in situ.
Quantitative limit test:
and (3) calculating the dilution volume required when the signal-to-noise ratio is directly diluted to 10:1 according to the signal-to-noise ratio of each peak of the system applicability solution, and diluting to obtain the quantitative limit test solution.
The ratio of the signal intensity of each spectral peak to the baseline noise was about 10: 1.
TABLE 4 quantitative limit test results of each impurity and main component
Detection limit test:
and diluting the quantitative limit test solution by 3 times to obtain a detection limit test solution.
The ratio of the signal intensity of each spectral peak to the baseline noise was about 3: 1.
TABLE 5 test results of detection limits of impurities and main components
Main component linear test:
linear solution 6 (150%): accurately weighing 37.5mg of the product into a 50ml volumetric flask, adding 10ml of methanol and a proper amount of purified water for dissolving, then using the purified water to fix the volume to a scale, and uniformly mixing.
Linear solution 5 (100%): precisely weighing 25mg of the product into a 50ml volumetric flask, adding 10ml of methanol and a proper amount of purified water for dissolving, then using the purified water to fix the volume to a scale, and uniformly mixing.
Linear solution 4 (70%): the linear solution 5 (100%) 7ml is removed precisely to a 10ml volumetric flask and is made to volume with 20% aqueous methanol solution to the mark.
Linear solution 3 (50%): the linear solution 5 (100%) was removed precisely 5ml to 10ml volumetric flask and made up to the mark with 20% aqueous methanol solution.
Linear solution 2 (20%): 2ml of the linear solution 5 (100%) is precisely transferred to a 10ml volumetric flask and is made to volume with 20% aqueous methanol solution.
Linear solution 1 (2%): precisely transferring 1ml of the linear solution 2 (20%) into a 10ml volumetric flask, and fixing the volume to the scale with 20% methanol aqueous solution.
Injecting 6 parts of linear solution into a liquid chromatograph, recording chromatogram, performing linear regression analysis with the actual concentration of the main component as abscissa and the peak area of the main component as ordinate, and obtaining linear correlation coefficient (R) of the main component2) Should be greater than 0.999.
TABLE 6 Main component Linear test results
And (4) conclusion: the main component presents a good linear relation between 0.01mg/ml and 0.69mg/ml, the linear equation is that y is 56407x +296.58, and the correlation coefficient R2Is 0.9997.
And (3) repeatability test:
6 parts of sample solution are prepared respectively and are prepared at present. Preparing blank solution by the same method.
The main component RSD is less than or equal to 2.0 percent.
TABLE 7 repeatability test results for each impurity and main component
And (4) conclusion: the RSD of the principal component is satisfactory.
Intermediate precision test:
6 parts of sample solution is prepared by using different manufacturers and instruments at different time and using chromatographic columns with the same type and different batch numbers by different personnel respectively, and the preparation is carried out at present. Preparing blank solution by the same method.
The repeatability of the main component is compared with 12 groups of data with intermediate precision, and the RSD of the purity detection result is less than or equal to 2.0 percent.
TABLE 8 intermediate precision test results of main component and impurities
And (4) conclusion: the RSD of the 12 sets of data for reproducibility and intermediate precision of the principal component was 0.036%. Meets the requirements.
And (3) durability test:
the purity of the main component of the product was measured by changing the flow rate (0.9ml/min, 1.1ml/min), wavelength (218nm, 222nm), column temperature (28 ℃, 32 ℃), pH of the mobile phase (6.6, 7.0), and comparing with the results of measurements without changing the conditions.
Preparing a sample solution, and preparing the sample solution at present. Preparing blank solution by the same method.
Comparing the detection result after changing the conditions with the detection result for changing, the RSD of the purity of the main component is less than or equal to 2.0 percent.
TABLE 9 durability test results
And (4) conclusion: the method has good durability to flow velocity, column temperature, detection wavelength and pH value of mobile phase, and has no interference among impurities, and the separation degree is more than 1.5.
Verified main component purity detection method
The detection method comprises the following steps:
a chromatographic column: ZoRBAX-SB 250mm 4.6mm 5um agilent
Sample introduction amount: 10ul, flow rate: 1.0ml/min, column temperature: 30 ℃, wavelength: 220nm
Mobile phase composition:
mobile phase A: ph6.8 phosphate buffer: methanol 95:5, mobile phase B: methanol
| t/min | A％ | B％ |
| 0.0 | 95 | 5 |
| 20.0 | 90 | 10 |
| 37.0 | 65 | 35 |
| 42.0 | 65 | 35 |
| 43.0 | 95 | 5 |
| 55.0 | 95 | 5 |
Main component purity detection result
Claims (2)
1. A detection method for completely separating a main component and impurities thereof, wherein the main component is 2- [ (2R) -2-hydroxy-3- [ [4- (3-oxo-4-morpholinyl) phenyl ] amino ] propyl ] -1H-isoindole-1, 3(2H) -dione, starting materials for generating the main component are 4- (4-aminophenyl) morpholin-3-one and (S) - (+) -N- (2, 3-ethoxypropyl) phthalimide, and the impurities are impurities contained in starting materials for generating the main component and byproducts generated in a synthesis process, and the detection method is characterized in that: adopts a reversed phase high performance liquid chromatography and a phenyl liquid chromatography column, adopts a diode array detector, adopts a gradient elution program, comprises the following steps,
1) taking a proper amount of a 2- [ (2R) -2-hydroxy-3- [ [4- (3-oxo-4-morpholinyl) phenyl ] amino ] propyl ] -1H-isoindole-1, 3(2H) -dione sample, dissolving the sample with methanol and purified water, and preparing 1mL of sample solution containing 0.1-1.5 mg;
2) setting the flow rate of a mobile phase to be 0.5-1.5 mL/min, the detection wavelength to be 200-240 nm, the temperature of a chromatographic column incubator to be 20-40 ℃, and the sample injection amount to be 5-30 mu L;
3) mobile phase a was ph6.8 phosphate buffer: methanol in a ratio of 95:5, flow ofThe mobile phase B is methanol, and the gradient elution procedure is as follows:
。
2. the method according to claim 1, wherein the method comprises the steps of: the detection wavelength in the step 2) is 220nm, the flow rate is 1.0ml/min, the temperature of a chromatographic column incubator is 30 ℃, and the sample injection amount is 10 mu L.
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