CN116609467A - HPLC detection method for Artemisia principals and intermediates thereof - Google Patents
HPLC detection method for Artemisia principals and intermediates thereof Download PDFInfo
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- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 20
- 238000001514 detection method Methods 0.000 title claims abstract description 17
- 239000000543 intermediate Substances 0.000 title claims description 54
- 235000003826 Artemisia Nutrition 0.000 title claims description 3
- 240000006891 Artemisia vulgaris Species 0.000 title claims description 3
- 235000003261 Artemisia vulgaris Nutrition 0.000 title claims description 3
- 235000009052 artemisia Nutrition 0.000 title claims description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 51
- XDXWLKQMMKQXPV-QYQHSDTDSA-N eltrombopag Chemical compound CC1=NN(C=2C=C(C)C(C)=CC=2)C(=O)\C1=N/NC(C=1O)=CC=CC=1C1=CC=CC(C(O)=O)=C1 XDXWLKQMMKQXPV-QYQHSDTDSA-N 0.000 claims abstract description 48
- 229960001069 eltrombopag Drugs 0.000 claims abstract description 48
- 238000000034 method Methods 0.000 claims abstract description 27
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000010828 elution Methods 0.000 claims abstract description 14
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 8
- 238000005303 weighing Methods 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- 239000012488 sample solution Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 abstract description 9
- 239000007864 aqueous solution Substances 0.000 abstract description 8
- 238000004458 analytical method Methods 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract description 2
- 239000000741 silica gel Substances 0.000 abstract description 2
- 229910002027 silica gel Inorganic materials 0.000 abstract description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 23
- 230000014759 maintenance of location Effects 0.000 description 12
- 238000002347 injection Methods 0.000 description 11
- 239000007924 injection Substances 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 238000002156 mixing Methods 0.000 description 7
- 238000012417 linear regression Methods 0.000 description 6
- 238000000926 separation method Methods 0.000 description 5
- 201000003793 Myelodysplastic syndrome Diseases 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 206010043554 thrombocytopenia Diseases 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- GJBBAPXESBCGRU-UHFFFAOYSA-N 2-(3,4-dimethylphenyl)-5-methyl-4h-pyrazol-3-one Chemical compound O=C1CC(C)=NN1C1=CC=C(C)C(C)=C1 GJBBAPXESBCGRU-UHFFFAOYSA-N 0.000 description 1
- 206010000830 Acute leukaemia Diseases 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- QWXYZCJEXYQNEI-OSZHWHEXSA-N intermediate I Chemical compound COC(=O)[C@@]1(C=O)[C@H]2CC=[N+](C\C2=C\C)CCc2c1[nH]c1ccccc21 QWXYZCJEXYQNEI-OSZHWHEXSA-N 0.000 description 1
- 238000011866 long-term treatment Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
Classifications
-
- 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
- G01N30/02—Column chromatography
-
- 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
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
-
- 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
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
-
- 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
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses an HPLC detection method for eltrombopag and an intermediate thereof, and belongs to the technical field of medicine analysis detection. The analysis method of the present invention includes using an Agilent 1260 liquid chromatograph; filling the chromatographic column with octadecylsilane chemically bonded silica gel; gradient elution is carried out by taking phosphoric acid aqueous solution, methanol and acetonitrile as mobile phases; and (3) selecting proper flow rate and temperature conditions for high performance liquid chromatography analysis. The method provided by the invention is convenient and quick, and can effectively elute and separate the eltrombopag and the intermediate thereof, and the method has economical efficiency and practicability.
Description
Technical Field
The invention belongs to the technical field of medicine analysis and detection, and particularly relates to an HPLC detection method for eltrombopag and an intermediate thereof.
Background
Myelodysplastic syndrome (MDS) is a malignant clonal disease characterized by a low hematopoietic function, myelodysplasia, etc., that is likely to be converted to acute leukemia. The data indicate that the probability of thrombocytopenia is high, whether the patient is at mild or high risk. Clinical trials of some scientific researchers at home and abroad show that the eltrombopag ethanol gel plays a vital role in long-term treatment or short-term treatment of thrombocytopenia, the bleeding condition of a patient is obviously improved, and the medicine can safely and effectively treat various thrombocytopenia diseases and brings new hopes to the patient. The molecular formula of the eltrombopag is C 29 H 36 N 6 O 6 A relative molecular mass of 564.64, obtainable via intermediate 1:2 '-benzyloxy-3' -nitrobiphenyl-3-carboxylic acid and intermediate 2: the preparation is carried out by condensation coupling reaction between two intermediates of 2- (3, 4-dimethyl-phenyl) -5-methyl-2, 4-dihydro-pyrazol-3-one. As shown in the following formula.
Therefore, in order to track and control the quality of medicines, it is necessary to establish an effective detection method capable of simultaneously detecting eltrombopag and two intermediates thereof. Patent CN107870217B discloses a method for detecting related substances of a mugwort-treopap intermediate i, but only two intermediates and related impurities are detected respectively, a finished product of mugwort-treopap and the intermediates thereof are not analyzed synchronously, and the used salt-containing mobile phase may cause buffer salt to permeate into the depth of a chromatographic column bonding phase, damage a silica gel matrix, loss of the chromatographic column bonding phase, loosening of a column bed, reduced column efficiency and higher economic cost.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a method for detecting eltrombopag and two intermediates thereof by using a high-performance liquid chromatography with strong specificity, good accuracy, high economy and convenience and rapidness.
The invention adopts the technical scheme that: an HPLC analysis method of eltrombopag and intermediates thereof, said method employing chromatographic conditions of:
a detector: a diode array detector;
chromatographic column: a C18 chromatographic column;
column temperature: 30 ℃;
wavelength: 230nm;
flow rate: 1mL/min;
elution mode: and (3) carrying out gradient elution by adopting a mobile phase A and a mobile phase B.
The method is used for detecting the eltrombopag and the intermediate thereof,
the structure of eltrombopag is as follows:
the structure of the eltrombopag intermediate is as follows:
further, the concentration of the phosphoric acid aqueous solution of the mobile phase A is 0.05%, and the mobile phase B is acetonitrile;
further, the particle diameter of the chromatographic column is 20um, and the column length is 250mm;
further, the sample injection amount is 5 mu L;
further, the elution procedure was:
| time (min) | Mobile phase a (volume fraction,%) | Mobile phase B (volume fraction,%) |
| 0 | 90 | 10 |
| 6 | 55 | 45 |
| 10 | 50 | 50 |
| 12 | 30 | 70 |
| 20 | 80 | 20 |
Further, the separation degree of the eltrombopag and the intermediate thereof is more than 2.
Further, the theoretical plate numbers of the eltrombopag and the intermediate thereof are all more than or equal to 5097.
The beneficial effects of the invention are as follows: the invention adopts high performance liquid chromatography to effectively analyze and measure the eltrombopag and the intermediate thereof, and the method has good separation degree, accurate result and low economic cost, and can be used for quality control of the intermediate impurities in the eltrombopag production process.
Drawings
FIG. 1 is an HPLC chart under the conditions measured in example 1 of the present invention;
FIG. 2 is an HPLC chart under the conditions measured in comparative example 1 of the present invention;
FIG. 3 is an HPLC chart under the conditions measured in comparative example 2 of the present invention;
FIG. 4 is an HPLC chart under the conditions measured in comparative example 3 of the present invention.
Detailed Description
The invention is further illustrated by the following specific examples. It should be understood that: the examples of the present invention are merely illustrative of the present invention and are not intended to be limiting. Among the reagents used in the examples below, methanol and acetonitrile were of HPLC grade, with the brand being microphone, water being purified water, and a high performance liquid chromatograph was Agilent HPLC 1260. The raw materials used in the invention are all self-made in a laboratory.
Example 1:
preparation of the solution: the method comprises the steps of respectively weighing 25mg of the eltrombopag test sample and two intermediates thereof, precisely weighing the eltrombopag test sample and two intermediates thereof in a 25mL volumetric flask, adding a proper amount of acetonitrile aqueous solution with the volume concentration of 60% to dissolve and dilute the solution to a scale, and uniformly mixing the solution.
Chromatographic determination conditions:
chromatographic column: c18, specification 4.6X105 mm,5 μm;
mobile phase: a:0.05% phosphoric acid in water; b: acetonitrile;
flow rate: 1.0mL/min;
column temperature: 30 ℃;
sample injection amount: 5. Mu.L;
gradient elution procedure:
under the chromatographic conditions of example 1, the detection chromatogram of the solution is shown in FIG. 1. It can be seen that eltrombopag peak shape is good, retention time is 18.623min, tailing factor is 0.99, theoretical plate number is 50434; the retention time of the eltrombopag intermediate 1 is 6.822min, the tailing factor is 1.44, the separation degree is 5.70, and the theoretical plate number is 8400; eltrombopag intermediate 2 retention time was 9.135min, tailing factor was 1.27, degree of separation was 22.47, theoretical plate number was 5097.
Comparative example 1
Preparation of the solution: the method comprises the steps of respectively weighing 25mg of the eltrombopag test sample and the intermediate thereof, precisely weighing the eltrombopag test sample and the intermediate thereof in a 25mL volumetric flask, adding a proper amount of acetonitrile aqueous solution with the volume concentration of 60% to dissolve and dilute to a scale, and uniformly mixing.
Chromatographic determination conditions:
chromatographic column: c18, specification 4.6X105 mm,5 μm;
mobile phase: a: purifying water; b: acetonitrile;
flow rate: 1.0mL/min;
column temperature: 30 ℃;
sample injection amount: 5. Mu.L;
gradient elution procedure:
under the chromatographic conditions of comparative example 1, the detection chromatogram of the solution is shown in FIG. 2. The retention time of eltrombopag is 20.422min, the tailing factor is 1.33, and the theoretical plate number is 28885; intermediate 1 retention time 9.470min, tailing factor 1.43, theoretical plate number 7244; the retention time of eltrombopag intermediate 2 was 11.131min, the tailing factor was 1.71, and the theoretical plate number was 2005. As can be seen from the figure, intermediate 1 and intermediate 2 have a smaller degree of separation, and eltrombopag intermediate 2 has a significant tailing.
Comparative example 2
Preparation of the solution: the method comprises the steps of respectively weighing 25mg of the eltrombopag test sample and the intermediate thereof, precisely weighing the eltrombopag test sample and the intermediate thereof in a 25mL volumetric flask, adding a proper amount of acetonitrile aqueous solution with the volume concentration of 60% to dissolve and dilute to a scale, and uniformly mixing.
Chromatographic determination conditions:
chromatographic column: c18, specification 4.6X105 mm,5 μm;
mobile phase: a:0.05% aqueous triethylamine solution; b: acetonitrile;
flow rate: 1.0mL/min;
column temperature: 30 ℃;
sample injection amount: 5. Mu.L;
gradient elution procedure:
the detection chromatogram of the solution under the chromatographic conditions of comparative example 2 is shown in fig. 3. The retention time of eltrombopag is 17.722min, the tailing factor is 1.91, and the theoretical plate number is 3325; the retention time of the intermediate 1 is 6.673min, the tailing factor is 1.01, the theoretical plate number is 751, and the basic requirement of pharmacopoeia on the plate number is far lower than that of the intermediate; the retention time of the eltrombopag intermediate 2 is 16.092min, the tailing factor is 0.79, and the theoretical plate number is 3734. And as can be seen from the graph, the eltrombopag and the two intermediates thereof have poorer peak shapes and split into two peaks, and the baseline drift is obvious.
Comparative example 3
Preparation of the solution: the method comprises the steps of respectively weighing 25mg of the eltrombopag test sample and the intermediate thereof, precisely weighing the eltrombopag test sample and the intermediate thereof in a 25mL volumetric flask, adding a proper amount of acetonitrile aqueous solution with the volume concentration of 60% to dissolve and dilute to a scale, and uniformly mixing.
Chromatographic determination conditions:
chromatographic column: c18, specification 4.6X105 mm,5 μm;
mobile phase: a:0.05% phosphoric acid in water; b: methanol;
flow rate: 1.0mL/min;
column temperature: 30 ℃;
sample injection amount: 5. Mu.L;
gradient elution procedure:
the detection chromatogram of the solution under the chromatographic conditions of comparative example 3 is shown in fig. 4. The retention time of eltrombopag is 17.803min, the tailing factor is 1.72, and the theoretical plate number is 36267; intermediate 1 retention time 6.547min, tailing factor 1.63, theoretical plate number 4969; the retention time of the eltrombopag intermediate 2 is 9.229min, the tailing factor is 1.75, the theoretical plate number is 1714, and the plate number is lower than the basic requirement of pharmacopoeia. And as can be seen from the figure, the eltrombopag intermediate 2 has obvious tailing.
Example 2: system adaptability test
Preparation of test solution: the method comprises the steps of respectively weighing 25mg of the eltrombopag test sample and the intermediate thereof, precisely weighing the eltrombopag test sample and the intermediate thereof in a 25mL volumetric flask, adding a proper amount of acetonitrile aqueous solution with the volume concentration of 60% to dissolve and dilute to a scale, and uniformly mixing.
Chromatographic determination conditions:
chromatographic column: c18, specification 4.6X105 mm,5 μm;
mobile phase: a:0.05% phosphoric acid in water; b: acetonitrile;
flow rate: 1.0mL/min;
column temperature: 30 ℃;
sample injection amount: 5. Mu.L;
gradient elution procedure:
sample solutions were taken and repeated 6 times, and the test results are shown in table 1:
table 1 results of System adaptability experiments
According to Table 1, it can be seen that the same mixed sample solution is repeated 6 times, and RSD% of eltrombopag, its intermediate 1 and intermediate 2 are respectively 0.938%,1.012% and 0.601%, which all meet the requirements of RSD% < 2.0% in pharmacopoeia, so that the method disclosed by the invention has good system adaptability and high test reliability.
Example 3: repeatability test
Preparation of test solution: respectively weighing 25mg of 6 groups of eltrombopag samples, precisely weighing in a 25mL volumetric flask, dissolving with 60% acetonitrile water solution, diluting to scale, mixing uniformly to obtain mixed solutions with different mass concentrations, and respectively corresponding to sample solutions with the numbers of 1-6 in sequence.
Chromatographic determination conditions:
chromatographic column: c18, specification 4.6X105 mm,5 μm;
mobile phase: a:0.05% phosphoric acid in water; b: acetonitrile;
flow rate: 1.0mL/min;
column temperature: 30 ℃;
sample injection amount: 5. Mu.L;
gradient elution procedure:
sample solutions of No. 1 to No. 6 with different mass concentrations are taken and respectively injected, and the content of each sample solution is measured as shown in Table 2:
TABLE 2 repeatability test results
As can be seen from Table 2, the average content of eltrombopag in the sample is 98.60%, the RSD% is 0.221%, and the RSD% conforming to the pharmacopoeia is not more than 2.0%, which indicates that the method of the invention has good reproducibility.
Example 4: linearity test
Preparation of test solution: respectively weighing 6 parts of eltrombopag and intermediate samples thereof according to the six concentrations of 20%,50%,80%,100%,120% and 150% of the sample volume concentration: 5mg,12.5mg,20mg,25mg,30mg and 37.5mg are precisely weighed into a 25mL volumetric flask, a proper amount of acetonitrile aqueous solution with the volume concentration of 60% is added for dissolution and dilution to a scale, and the mixed solutions with different mass concentrations are obtained after uniform mixing, and the mixed solutions respectively correspond to sample solutions with the numbers of 1 to 6 in sequence.
Chromatographic determination conditions:
chromatographic column: c18, specification 4.6X105 mm,5 μm;
mobile phase: a:0.05% phosphoric acid in water; b: acetonitrile;
flow rate: 1.0mL/min;
column temperature: 30 ℃;
sample injection amount: 5. Mu.L;
gradient elution procedure:
| time (min) | Mobile phase a (volume fraction,%) | Mobile phase B (volume fraction,%) |
| 0 | 90 | 10 |
| 6 | 55 | 45 |
| 10 | 50 | 50 |
| 12 | 30 | 70 |
| 20 | 80 | 20 |
Sample solutions of No. 1 to 6 with different mass concentrations are taken and respectively injected for 6 times, and the average peak area and RSD% of each sample solution are measured as shown in Table 3:
TABLE 3 Linear test results
According to the data in table 3, linear regression was performed by the least square method with the concentrations of eltrombopag, eltrombopag intermediate 1 and eltrombopag intermediate 2 on the abscissa and the peak area on the ordinate, respectively. The eltrombopag is in the range of 0.201-1.516 mg/mL sample injection concentration and returns linearlyThe equation is given by y=12163x-835.48, and the linear regression coefficient r 2 0.9956; the Etrazobopa intermediate 1 has a linear regression equation of y= 22045x-2097.7 and a linear regression coefficient r within the range of 0.197-1.534 mg/mL of sample injection concentration 2 0.9942; the Equ bopa intermediate 2 has a linear regression equation of y=11052x+504.65 and a linear regression coefficient r within the range of 0.212-1.526 mg/mL sample injection concentration 2 0.9929, all three of which meet the pharmacopoeia rule r 2 And more than or equal to 0.99, which indicates that the method has good linearity.
While specific embodiments of the invention have been described above, it will be understood that the invention is not limited to the particular embodiments described above, but is capable of modification and/or variation in form that will occur to those skilled in the art.
Claims (6)
1. An HPLC detection method for the Artemisia principals and intermediates thereof is characterized in that: the chromatographic conditions adopted by the method are as follows:
a detector: a diode array detector;
chromatographic column: a C18 chromatographic column;
column temperature: 30 ℃;
wavelength: 230nm;
flow rate: 1mL/min;
elution mode: and (3) carrying out gradient elution by adopting a mobile phase A and a mobile phase B.
2. The HPLC detection method of claim 1, wherein mobile phase a is 0.05% aqueous phosphoric acid; mobile phase B was acetonitrile.
3. The HPLC detection method according to claim 1, wherein the gradient elution ratio is:
。
4. The HPLC detection method of claim 1, wherein: the method comprises the steps of preparing a sample solution mixed with a certain concentration of eltrombopag and an intermediate thereof.
5. The HPLC detection method of claim 4, wherein: the preparation method of the sample solution comprises the following steps: and respectively taking pure eltrombopag and two intermediates, precisely weighing, adding 60% acetonitrile solution for dissolving and diluting to prepare a mixed solution of 1 mg/mL.
6. The HPLC detection method of claim 1, wherein: the two intermediates of the eltrombopag are respectively: intermediate 1:2 '-benzyloxy-3' -nitrobiphenyl-3-carboxylic acid; intermediate 2:2- (3, 4-dimethyl-phenyl) -5-methyl-2, 4-dihydropyrazol-3-one.
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