A method for the hplc detection of eltrombopag and intermediates thereof

Abstract

The application discloses a HPLC detection method for eltrombopag and intermediates thereof, and belongs to the technical field of drug analysis and detection. The analysis method comprises the following steps: using an Agilent 1260 liquid chromatograph; selecting an octadecylsilane bonded silica gel filling chromatographic column; adopting a phosphoric acid aqueous solution, methanol and acetonitrile as a mobile phase for gradient elution; and selecting appropriate flow rate and temperature conditions for high performance liquid chromatography analysis. The method is convenient and fast, can effectively elute and separate eltrombopag and intermediates, and has economy and practicability.

Description

Technical Field

[0001] This invention belongs to the field of drug analysis and detection technology, specifically relating to an HPLC detection method for eltrombopag and its intermediates. Background Technology

[0002] Myelodysplastic syndrome (MDS) is a malignant clonal disease characterized by hematopoietic insufficiency and abnormal bone marrow proliferation, which may transform into acute leukemia. Data shows that regardless of whether the patient has mild or high-risk MDS, the probability of thrombocytopenia is high. Clinical trials conducted by researchers both domestically and internationally have found that eltrombopag ethanol gel plays a crucial role in both long-term and short-term treatment of thrombocytopenia, significantly improving patients' bleeding conditions. This drug can safely and effectively treat various types of thrombocytopenia, bringing new hope to patients. The molecular formula of eltrombopag is C2. 29 H 36 N6O6, with a relative molecular mass of 564.64, can be prepared by a condensation coupling reaction between two intermediates: intermediate 1: 2'-benzyloxy-3'-nitrobiphenyl-3-carboxylic acid and intermediate 2: 2-(3,4-dimethyl-phenyl)-5-methyl-2,4-dihydropyrazole-3-one. See the figure below.

[0003]

[0004] Therefore, in order to track and control drug quality, it is essential to establish an effective detection method that can simultaneously detect eltrombopag and its two intermediates. Patent CN107870217B discloses a method for detecting related substances of eltrombopag intermediate I, but it only detects the two intermediates and related impurities separately, without simultaneously analyzing the eltrombopag finished product and its intermediates. Furthermore, the salt-containing mobile phase it uses may cause the buffer salt to penetrate deep into the bonded phase of the chromatographic column, damaging the silica matrix, leading to loss of the bonded phase, loosening of the column bed, decreased column efficiency, and high economic costs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a high-performance liquid chromatography (HPLC) method for the detection of eltrombopag and its two intermediates, which is highly specific, accurate, economical, convenient, and fast.

[0006] The technical solution adopted in this invention is: an HPLC analysis method for eltrombopag and its intermediates, wherein the chromatographic conditions used in the method are as follows:

[0007] Detector: Diode array detector;

[0008] Chromatographic column: C18 column;

[0009] Column temperature: 30℃;

[0010] Wavelength: 230nm;

[0011] Flow rate: 1 mL / min;

[0012] Elution method: Gradient elution is performed using mobile phase A and mobile phase B.

[0013] This method is used to detect eltrombopag and its intermediates.

[0014] The structure of eltrombopag is as follows:

[0015]

[0016] The structure of the eltrombopag intermediate is as follows:

[0017]

[0018] Furthermore, the concentration of the mobile phase A (phosphoric acid aqueous solution) is 0.05%, and the mobile phase B is acetonitrile;

[0019] Furthermore, the chromatographic column has a particle size of 20 μm and a column length of 250 mm;

[0020] Furthermore, the injection volume was 5 μL;

[0021] Furthermore, the elution procedure is as follows:

[0022] 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

[0023] Furthermore, the separation degree of eltrombopag and its intermediates is greater than 2.

[0024] Furthermore, the theoretical plate number of eltrombopag and its intermediates is ≥5097.

[0025] The beneficial effects of this invention are as follows: This invention uses high performance liquid chromatography to effectively analyze and determine eltrombopag and its intermediates. The method has good separation, accurate results, and low economic cost, and can be used for quality control of intermediate impurities contained in the production process of eltrombopag. Attached Figure Description

[0026] Figure 1 HPLC chromatograms under the conditions measured in Example 1 of this invention;

[0027] Figure 2 The HPLC chromatogram under the conditions measured in Comparative Example 1 of this invention;

[0028] Figure 3 The HPLC chromatograms under the conditions measured in Comparative Example 2 of this invention;

[0029] Figure 4 The HPLC chromatograms of Comparative Example 3 of this invention are obtained under the conditions measured in this invention. Detailed Implementation

[0030] The present invention will be further illustrated below with specific embodiments. It should be understood that the embodiments of the present invention are merely illustrative and not intended to limit the invention. In the following embodiments, the reagents used include methanol and acetonitrile of HPLC grade, brand name Maclean, and purified water. The high-performance liquid chromatograph used was an Agilent HPLC 1260. All raw materials used in this invention were prepared in the laboratory.

[0031] Example 1:

[0032] Preparation of solution: Weigh 25 mg of eltrombopag test sample and its two intermediates into 25 mL volumetric flasks, add an appropriate amount of 60% acetonitrile aqueous solution to dissolve and dilute to the mark, and mix well.

[0033] Chromatographic determination conditions:

[0034] Chromatographic column: C18, 4.6×250mm, 5μm;

[0035] Mobile phase: A: 0.05% aqueous phosphoric acid solution; B: acetonitrile;

[0036] Flow rate: 1.0 mL / min;

[0037] Column temperature: 30℃;

[0038] Injection volume: 5 μL;

[0039] Gradient elution procedure:

[0040]

[0041] Under the chromatographic conditions of Example 1, the detection chromatogram of the solution is as follows: Figure 1 As shown, eltrobappa showed good peak shape, retention time of 18.623 min, tailing factor of 0.99, and theoretical plate number of 50534; eltrobappa intermediate 1 had a retention time of 6.822 min, tailing factor of 1.44, resolution of 5.70, and theoretical plate number of 8400; eltrobappa intermediate 2 had a retention time of 9.135 min, tailing factor of 1.27, resolution of 22.47, and theoretical plate number of 5097.

[0042] Comparative Example 1

[0043] Preparation of solution: Weigh 25 mg of eltrombopag test sample and its intermediate into 25 mL volumetric flasks, add an appropriate amount of 60% acetonitrile aqueous solution to dissolve and dilute to the mark, and mix well.

[0044] Chromatographic determination conditions:

[0045] Chromatographic column: C18, 4.6×250mm, 5μm;

[0046] Mobile phase: A: purified water; B: acetonitrile;

[0047] Flow rate: 1.0 mL / min;

[0048] Column temperature: 30℃;

[0049] Injection volume: 5 μL;

[0050] Gradient elution procedure:

[0051]

[0052] Under the chromatographic conditions of Comparative Example 1, the detection chromatogram of the solution is as follows: Figure 2 As shown in the figure, the retention time of eltrombopag was 20.422 min, the tailing factor was 1.33, and the theoretical plate number was 28885; the retention time of intermediate 1 was 9.470 min, the tailing factor was 1.43, and the theoretical plate number was 7244; the retention time of eltrombopag intermediate 2 was 11.131 min, the tailing factor was 1.71, and the theoretical plate number was 2005. It can be seen from the figure that the separation between intermediate 1 and intermediate 2 is relatively small, and eltrombopag intermediate 2 exhibits significant tailing.

[0053] Comparative Example 2

[0054] Preparation of solution: Weigh 25 mg of eltrombopag test sample and its intermediate into 25 mL volumetric flasks, add an appropriate amount of 60% acetonitrile aqueous solution to dissolve and dilute to the mark, and mix well.

[0055] Chromatographic determination conditions:

[0056] Chromatographic column: C18, 4.6×250mm, 5μm;

[0057] Mobile phase: A: 0.05% triethylamine aqueous solution; B: acetonitrile;

[0058] Flow rate: 1.0 mL / min;

[0059] Column temperature: 30℃;

[0060] Injection volume: 5 μL;

[0061] Gradient elution procedure:

[0062]

[0063] Under the chromatographic conditions of Comparative Example 2, the detection chromatogram of the solution is as follows: Figure 3As shown in the figure, eltrombopag had a retention time of 17.722 min, a tailing factor of 1.91, and a theoretical plate number of 3325; intermediate 1 had a retention time of 6.673 min, a tailing factor of 1.01, and a theoretical plate number of 751, far below the pharmacopoeia's basic requirement for the number of plates; eltrombopag intermediate 2 had a retention time of 16.092 min, a tailing factor of 0.79, and a theoretical plate number of 3734. Furthermore, the figure shows that eltrombopag and its two intermediates all exhibited poor peak shapes, splitting into two peaks, and significant baseline drift.

[0064] Comparative Example 3

[0065] Preparation of solution: Weigh 25 mg of eltrombopag test sample and its intermediate into 25 mL volumetric flasks, add an appropriate amount of 60% acetonitrile aqueous solution to dissolve and dilute to the mark, and mix well.

[0066] Chromatographic determination conditions:

[0067] Chromatographic column: C18, 4.6×250mm, 5μm;

[0068] Mobile phase: A: 0.05% aqueous phosphoric acid solution; B: methanol;

[0069] Flow rate: 1.0 mL / min;

[0070] Column temperature: 30℃;

[0071] Injection volume: 5 μL;

[0072] Gradient elution procedure:

[0073]

[0074] Under the chromatographic conditions of Comparative Example 3, the detection chromatogram of the solution is as follows: Figure 4 As shown in the figure, eltrombopag intermediate 2 had a retention time of 17.803 min, a tailing factor of 1.72, and a theoretical plate number of 36267; intermediate 1 had a retention time of 6.547 min, a tailing factor of 1.63, and a theoretical plate number of 4969; and eltrombopag intermediate 2 had a retention time of 9.229 min, a tailing factor of 1.75, and a theoretical plate number of 1714, both of which are below the basic requirements of the pharmacopoeia. Furthermore, the figure shows that eltrombopag intermediate 2 exhibits significant tailing.

[0075] Example 2: System Adaptability Test

[0076] Preparation of test solution: Weigh 25 mg of eltrombopag test sample and its intermediate into 25 mL volumetric flasks, add an appropriate amount of 60% acetonitrile aqueous solution to dissolve and dilute to the mark, and mix well.

[0077] Chromatographic determination conditions:

[0078] Chromatographic column: C18, 4.6×250mm, 5μm;

[0079] Mobile phase: A: 0.05% aqueous phosphoric acid solution; B: acetonitrile;

[0080] Flow rate: 1.0 mL / min;

[0081] Column temperature: 30℃;

[0082] Injection volume: 5 μL;

[0083] Gradient elution procedure:

[0084]

[0085] The test solution was injected repeatedly 6 times, and the results are shown in Table 1:

[0086] Table 1 Results of System Adaptability Experiment

[0087]

[0088] As shown in Table 1, after repeated injection of the same mixed sample solution 6 times, the RSD% of eltrombopag and its intermediates 1 and 2 were 0.938%, 1.012%, and 0.601%, respectively, all of which meet the requirements of RSD% < 2.0% in the pharmacopoeia. This indicates that the method of the present invention has good system adaptability and high experimental reliability.

[0089] Example 3: Repeatability Test

[0090] Preparation of test solutions: Weigh 25 mg of each of the 6 groups of eltrombopag test samples into 25 mL volumetric flasks, dissolve and dilute to the mark with 60% acetonitrile aqueous solution, mix well, and obtain mixed solutions of different mass concentrations, which correspond to test solutions numbered 1 to 6 respectively.

[0091] Chromatographic determination conditions:

[0092] Chromatographic column: C18, 4.6×250mm, 5μm;

[0093] Mobile phase: A: 0.05% aqueous phosphoric acid solution; B: acetonitrile;

[0094] Flow rate: 1.0 mL / min;

[0095] Column temperature: 30℃;

[0096] Injection volume: 5 μL;

[0097] Gradient elution procedure:

[0098]

[0099] Different mass concentrations of test sample solutions, numbered 1 to 6, were injected separately, and the contents of each test sample solution were measured as shown in Table 2.

[0100] Table 2 Results of Repeatability Tests

[0101]

[0102] As shown in Table 2, the average content of eltrombopag in the sample was 98.60%, and the RSD% was 0.221%, which meets the pharmacopoeia requirement that the RSD% should not exceed 2.0%, indicating that the method of the present invention has good reproducibility.

[0103] Example 4: Linearity Test

[0104] Preparation of test solutions: Six portions of eltrombopag and its intermediate samples were weighed according to six concentrations of 20%, 50%, 80%, 100%, 120%, and 150% respectively: 5 mg, 12.5 mg, 20 mg, 25 mg, 30 mg, and 37.5 mg. The weights were accurately weighed into 25 mL volumetric flasks, and an appropriate amount of 60% acetonitrile aqueous solution was added to dissolve and dilute to the mark. The mixtures were mixed to obtain mixed solutions of different mass concentrations, which corresponded to test solutions numbered 1 to 6 respectively.

[0105] Chromatographic determination conditions:

[0106] Chromatographic column: C18, 4.6×250mm, 5μm;

[0107] Mobile phase: A: 0.05% aqueous phosphoric acid solution; B: acetonitrile;

[0108] Flow rate: 1.0 mL / min;

[0109] Column temperature: 30℃;

[0110] Injection volume: 5 μL;

[0111] Gradient elution procedure:

[0112] 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

[0113] Six injections were performed on test solutions of different mass concentrations, numbered 1 to 6, and the average peak area and RSD% of each test solution were measured, as shown in Table 3.

[0114] Table 3 Results of the linear experiment

[0115]

[0116] Based on the data in Table 3, linear regression was performed using the least squares method, with the concentrations of eltrombopag, eltrombopag intermediate 1, and eltrombopag intermediate 2 as the x-axis and the peak area as the y-axis, respectively. For eltrombopag in the injection concentration range of 0.201–1.516 mg / mL, the linear regression equation was y = 12163x - 835.48, and the linear regression coefficient r0 was [missing value]. 2 The value was 0.9956; for eltrombopag intermediate 1 within the injection concentration range of 0.197–1.534 mg / mL, the linear regression equation was y = 22045x - 2097.7, and the linear regression coefficient r was 0.9956. 2 The value was 0.9942; for eltrombopag intermediate 2 within the injection concentration range of 0.212–1.526 mg / mL, the linear regression equation was y = 11052x + 504.65, and the linear regression coefficient r was 0.9942. 2 The value was 0.9929, and all three values ​​met the pharmacopoeia requirements. 2 A value ≥0.99 indicates that the method of the present invention has good linearity.

[0117] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above. Any modifications and / or alterations made by those skilled in the art to the present invention will fall within the protection scope of the present invention.

Claims

1. An HPLC detection method for eltrombopag and its intermediates, characterized in that: The chromatographic conditions used in the method are as follows: Detector: Diode array detector; Chromatographic column: C18 column; Column temperature: 30℃; Wavelength: 230nm; Flow rate: 1 mL / min; Elution method: Gradient elution was performed using mobile phase A and mobile phase B. Mobile phase A was a 0.05% aqueous solution of phosphoric acid; mobile phase B was acetonitrile. The two intermediates of eltrombopag tested were: intermediate 1: 2'-benzyloxy-3'-nitrobiphenyl-3-carboxylic acid; intermediate 2: 2-(3,4-dimethyl-phenyl)-5-methyl-2,4-dihydropyrazole-3-one.

2. The HPLC detection method according to claim 1, characterized in that, The gradient elution ratio is: 。 3. The HPLC detection method according to claim 1, characterized in that: The method includes preparing a mixed test solution of eltrombopag and its intermediates at a certain concentration.

4. The HPLC detection method according to claim 3, characterized in that: The test solution is prepared by accurately weighing pure eltrombopag and two intermediates, dissolving and diluting them with 60% acetonitrile solution to prepare a mixed solution of 1 mg / mL.

Images