Method for detecting impurities in brivaracetam injection

Abstract

The application provides a method for detecting impurities of brivaracetam injection by using a non-salt mobile phase system combined with reversed-phase liquid chromatography, the detection method is detected by using an HPLC method, and the mobile phase system is a mixed solution of a phosphoric acid solution and acetonitrile, wherein in the mixed solution, the volume percentage of acetonitrile is 30%-45%, and the concentration of the phosphoric acid solution is greater than or equal to 0.05%; the chromatographic column is a chiral column containing tris(4-chloro-3-methylphenyl aminomethyl ester). By using the above detection method, the related substances and isomers of brivaracetam can be separated and determined in the same analysis method, and the blank excipients do not interfere with the detection of the above components.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis, specifically relating to a method for detecting impurities in bricetramine injection, particularly bricetramine-related substances and its isomers. Background Technology

[0002] Bricetane, a novel antiepileptic drug, has a structure similar to levetiracetam and is used to treat partial-onset epilepsy in patients aged 16 years and older, as adjunctive therapy with or without secondary generalized seizures. In January 2016, bricetane injection, developed by UCB and marketed under the brand name Briviact, was first approved for marketing in the European Union, and in February 2016, it was approved for marketing in the United States. The structural formula of bricetane is as follows:

[0003]

[0004] Formula V: (2S)-2-[(4R)-2-oxo-4-propyltetrahydro-1-pyrrolidinyl]butyramide (Bricetan)

[0005] As shown in the above structural formula, briracetam has two chiral centers. Under conditions such as high temperature or light exposure, the chiral centers may flip, thus introducing three isomer impurities. Furthermore, when briracetam is produced as an injectable solution, its amide group is easily hydrolyzed to a carboxylic acid, generating related impurities.

[0006] CN112834674A discloses a method for detecting briracetam isomers using high-performance liquid chromatography (HPLC), but it is not applicable to aqueous solutions of injectable solutions and cannot simultaneously detect related substances. CN110824093A discloses a method for detecting briracetam and its related substances using HPLC, but the structural and polarity differences between stereoisomers are small and difficult to separate, thus preventing isomer separation. CN114076799A discloses a method for detecting briracetam isomers using HPLC, employing a potassium phosphate system to separate isomers in injectable solutions, but it does not simultaneously detect briracetam related substances.

[0007] Therefore, it is necessary to establish a new detection and analysis method that can control and detect bricertan isomer impurities and related substances together, ensuring product quality and safety are controllable. Summary of the Invention

[0008] To address the deficiencies in existing technologies, this invention provides a method for simultaneously separating and detecting impurities, including related substances and isomers, in briracetam injection using a non-salt mobile phase system combined with a chiral column containing specific functional groups. Specifically, the detection method employs HPLC, and the mobile phase system is a mixture of phosphoric acid solution and acetonitrile, wherein the volume percentage of acetonitrile in the mixture is 30%-45%, and the concentration of phosphoric acid solution is ≥0.05%; the chromatographic column is a chiral column containing tris(4-chloro-3-methylphenylaminomethyl ester).

[0009] The impurities described in this invention include chiral isomers and related substances, including at least three isomers (diastereomer A, enantiomer B, and diastereomer C) and related substance D.

[0010] Preferably, the resolution between the components of the bricesteran-related substances and isomers in the detection method is >1.5, more preferably >2.

[0011] In some embodiments of the present invention, the flow rate of the mobile phase in the detection method is 0.1-2.0 ml / min, preferably 0.5-1.0 ml / min; preferably, the elution mode of the mobile phase is isocratic elution.

[0012] In some embodiments of the present invention, the chromatographic column temperature of the detection method is 10-60℃, preferably 25±5℃.

[0013] In some embodiments of the present invention, the detector wavelength of the detection method is 205-215nm, preferably 210nm.

[0014] In some embodiments of the present invention, the stationary phase of the chiral chromatographic column of the detection method is selected from amylose-tris(4-chloro-3-methylphenylaminomethyl ester) or amylose-tris(4-chloro-3-methylphenylaminomethyl ester), including but not limited to commercially available models such as Chiral NX(2)-RH, CHI RALCEL OX-H, and Lux ​​Cellouse-4.

[0015] In some embodiments of the present invention, the injection volume of the detection method is 1-20 μl, preferably 5-10 μl;

[0016] In some embodiments of the present invention, the analysis time of the detection method is ≥30 min, preferably 40 ± 5 min;

[0017] This invention also provides a method for detecting impurities in bricetan injection, which employs HPLC. The mobile phase system is a mixture of 1% phosphoric acid solution and acetonitrile, wherein the volume percentage of acetonitrile in the mixture is 30%-45%. The stationary phase of the chromatographic column is amylose-tris(4-chloro-3-methylphenylaminomethyl ester), the flow rate of the mobile phase is 0.5-1.0 ml / min, the elution mode of the mobile phase is isocratic elution, the column temperature is 25±5℃, and the detector wavelength is 210 nm.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention, through the optimization of the mobile phase system, specifically limiting the use of common inorganic salts such as phosphates as the mobile phase, combined with a chiral chromatographic column with specific functional groups, enables the separation of briracetam impurities, particularly at least three isomers and key related substances, in the same analytical method (resolution > 1.5), while ensuring that blank excipients do not interfere with the detection of the above components. While ensuring the robustness, sensitivity, and accuracy of the method, it simplifies the analytical conditions, is easy to operate, and is very suitable for industrial operation. Attached Figure Description

[0020] Figure 1 The chromatogram for the system suitability of Example 1 is shown, where peaks 1, 2, 3, 4, and 5 correspond to formulas IV, II, II I, I, and V (impurities D, B, C, A, and bricetan), respectively.

[0021] Figure 2 The image shows the blank excipient of the sample bricetan injection, with the blank excipient eluting before 7.5 min.

[0022] Figure 3 HPLC chromatogram of the detection method in Comparative Example 1

[0023] Figure 4 Blank excipient diagram for the detection method of Comparative Example 1;

[0024] Figure 5 This is the HPLC chromatogram of the detection method in Example 1;

[0025] Figure 6 This is the HPLC chromatogram of the detection method in Example 2;

[0026] Figure 7 This is the HPLC chromatogram of the detection method in Example 3;

[0027] Figure 8 The image shows the HPLC chromatogram of the detection method in Example 4. Detailed Implementation

[0028] The present invention is further illustrated below by way of embodiments, but these embodiments are not intended to limit the invention to the scope of the embodiments described. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0029] Unless otherwise specified, all raw materials and equipment used in the specific embodiments of this invention are commercially available. Among them, bricilactan injection, used as the test sample, was purchased under the trade name Briviact and manufactured by UCB Pharma SA.

[0030] Except as specifically described in the following embodiments, the detection method of the present invention includes the following steps:

[0031] 1) Prepare the test solution: Take the test sample to obtain the solution; 2) Prepare the control solution: Measure an appropriate amount of the test solution and dilute it with a solvent to obtain the control solution; 3) Prepare the system suitability solution: Take the mixed reference standards of bricetramine and impurities A, B, C, and D respectively, and dissolve and dilute them with a solvent to prepare the system suitability solution; 4) Inject the test solution described in step 1), the control solution in step 2), and the system suitability solution in step 3) respectively, perform high performance liquid chromatography analysis, record the chromatogram, determine the retention time of bricetramine and impurities A, B, C, and D, and calculate the content of bricetramine-related substances and isomer impurities in the bricetramine injection in the test solution according to the main component self-comparison method.

[0032] The relevant substances and their bricetam isomer structural formulas are as follows:

[0033]

[0034] Formula I: (S)-2-((S)-2-oxo-4-propylpyrrolidine-1-yl)butyramide (bricecetam diastereomer - impurity A)

[0035]

[0036] Formula II: (R)-2-((S)-2-oxo-4-propylpyrrolidine-1-yl)butyramide. (Briceran enantiomer - impurity B)

[0037]

[0038] Formula III: (R)-2-((R)-2-oxo-4-propylpyrrolidine-1-yl)butyramide (bricecetan diastereomer - impurity C)

[0039]

[0040] Formula IV: (S)-2-((R)-2-oxo-4-propylpyrrolidine-1-yl)butyric acid. (Briracetam related substances - impurity D).

[0041] In this invention, the phosphoric acid solution refers to an aqueous solution of phosphoric acid.

[0042] Comparative Example 1:

[0043] Instrument: Dionex U3000

[0044] Column: Amylose-tris(3-chloro-5-methylphenylcarbamate) DAICEL CHI RALPAK-IG-34.6×250mm, 5μm

[0045] Mobile phase: 20 mmol / L potassium dihydrogen phosphate (adjusted to pH 7.5 with 1 mmol / L sodium hydroxide solution) - acetonitrile (80:20)

[0046] Flow rate: 1.0 ml / min

[0047] Column temperature: 25℃

[0048] Detector: UV

[0049] Detection wavelength: 210nm

[0050] Injection volume: 5 μl

[0051] Take an appropriate amount of sample and dilute it with the mobile phase to prepare a solution containing approximately 5 mg of bricetram per ml, which is used as the test solution; accurately measure an appropriate amount of the test solution and dilute it with the mobile phase to prepare a solution containing approximately 50 μg of bricetram per ml, which is used as the control solution.

[0052] Take appropriate amounts of impurity A, impurity B, impurity C, impurity D and bricetan reference standard, dissolve and dilute them with solvent to prepare a solution containing 20 μg each of impurities A, B, C, and D and 10 mg of bricetan per 1 ml, which is used as the system suitability solution.

[0053] Example 1:

[0054] Instrument: Dionex U3000

[0055] Column: Chral NX(2)-RH 4.6×250mm, 5μm

[0056] Mobile phase: 0.1% phosphoric acid-acetonitrile (65:35)

[0057] Flow rate: 1.0 ml / min

[0058] Column temperature: 25℃

[0059] Detector: UV

[0060] Detection wavelength: 210nm

[0061] Injection volume: 5 μl

[0062] Take an appropriate amount of this product as the test solution; accurately measure an appropriate amount of the test solution and dilute it with the mobile phase to prepare a solution containing about 50 μg of brucetam per ml as the control solution.

[0063] Take appropriate amounts of impurity A, impurity B, impurity C, impurity D, and bricetam reference standard, dissolve and dilute them in solvent to prepare a solution containing 250 μg of impurity A, 20 μg each of impurities B, C, and D, and 10 mg of bricetam per 1 ml. This solution is used as a system suitability solution.

[0064] Example 2:

[0065] Column: Chral NX(2)-RH 4.6×250mm, 5μm

[0066] Mobile phase: 0.1% phosphoric acid-acetonitrile (70:30)

[0067] Other testing conditions are the same as in Example 1.

[0068] Example 3:

[0069] Column: Chral NX(2)-RH 4.6×250mm, 5μm

[0070] Mobile phase: 0.1% phosphoric acid-acetonitrile (60:40)

[0071] Other testing conditions are the same as in Example 1.

[0072] Example 4:

[0073] Column: Chral NX(2)-RH 4.6×250mm, 5μm

[0074] Mobile phase: 0.1% phosphoric acid-acetonitrile (55:45)

[0075] Other testing conditions are the same as in Example 1.

[0076] The test data results are as follows:

[0077] Table 1. Resolution data for different embodiments

[0078]

[0079]

[0080] The results of Examples 1-4 show that the detection method of the present invention, under chromatographic conditions where the volume percentage of acetonitrile in the mobile phase is 30%-45%, meets the requirements for the detection and separation of briracetam and its isomers (A, B, C) and related substance D in briracetam injection, without interference from excipient peaks, and exhibits good method tolerance, strong operability, high sensitivity, and good accuracy.

Claims

1. A method for detecting impurities in bricetam injection, wherein the detection method employs HPLC, and the mobile phase system is a mixed solution of phosphoric acid and acetonitrile, wherein... The mixed solution contains 30%-45% acetonitrile by volume and ≥0.05% phosphoric acid solution; the chromatographic column is a chiral column containing tris(4-chloro-3-methylphenylaminomethyl ester).

2. The detection method according to claim 1, characterized in that, The impurities include diastereomer A, enantiomer B, diastereomer C) and related substances D; preferably, the separation degree between the components of the related substances and isomers of bricetan is >1.

5.

3. The detection method according to claim 2, characterized in that, The flow rate of the mobile phase in the detection method is 0.1-2.0 ml / min, preferably 0.5-1.0 ml / min; preferably, the elution mode of the mobile phase is isocratic elution.

4. The detection method according to claim 3, characterized in that, The column temperature of the detection method is 10-60℃, preferably 25±5℃.

5. The detection method according to claim 4, characterized in that, The detector wavelength of the detection method is 205-215nm, preferably 210nm.

6. The detection method according to claim 5, characterized in that, The chromatographic column used in the detection method is selected from Chiral NX(2)-RH, CHIRALCEL OX-H, and Lux ​​Cellulose-4.

7. A method for detecting impurities in bricetan injection, comprising HPLC detection, wherein the mobile phase system is a mixed solution of 1% phosphoric acid and acetonitrile, wherein... In the mixed solution, the volume percentage of acetonitrile is 30%-45%, the stationary phase of the chromatographic column is amylose-tris(4-chloro-3-methylphenylaminomethyl ester), the flow rate of the mobile phase is 0.5-1.0 ml / min, the elution mode of the mobile phase is isocratic elution, the column temperature is 25±5℃, the detector wavelength is 210 nm, and the resolution between the components of bricetam related substances and isomers in the detection method is >1.

5.

8. A method for detecting and separating bricetam from its chiral isomers and related substances, characterized in that, The detection method employs high performance liquid chromatography (HPLC), with the following chromatographic conditions: column: Chiral NX(2)-RH 4.6×250mm; mobile phase: a mixture of phosphoric acid solution and acetonitrile, wherein the concentration of phosphoric acid solution is 0.1% and the volume percentage of acetonitrile in the mixture is 30%-45%; elution method: isocratic elution; mobile phase flow rate: 1mL / min; column temperature: 25℃; and the resolution between the components of bricetam related substances and isomers in the detection method is >2.

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