阅读说明：本技术 一种布立西坦及其异构体的检测方法 (Method for detecting brivaracetam and isomers thereof ) 是由 倪晟 夏金强 叶艳蓉 王菲 周亮 赵航 徐兵勇 陈鸿翔 姜维斌 屠芳芳 卜华荣 于 2020-12-29 设计创作，主要内容包括：本发明涉及一种布立西坦及其异构体的检测方法,包括采用高效液相色谱法进行检测的步骤,所述液相色谱条件为：以表面涂敷有直链淀粉-三(3,5-二甲苯基氨基甲酸酯)的硅胶为填充剂；以正己烷、异丙醇和有机碱的混合溶液为流动相,进入检测器进行检测。该方法能有效分离测定布立西坦对映异构体和非对映异构体的含量,操作简便,且灵敏度高、专属性和重复性好,检测中溶剂峰不干扰测定,检测结果准确可靠,对于实现布立西坦及其制剂的质量控制具有及其重要的意义。(The invention relates to a method for detecting brivaracetam and isomers thereof, which comprises the step of detecting by adopting a high performance liquid chromatography, wherein the liquid chromatography conditions are as follows: silica gel coated with amylose-tri (3, 5-xylyl carbamate) on the surface is used as a filling agent; and (3) taking a mixed solution of normal hexane, isopropanol and organic base as a mobile phase, and detecting by using a detector. The method can effectively separate and measure the content of the enantiomer and the diastereomer of the brivaracetam, is simple and convenient to operate, has high sensitivity and good specificity and repeatability, does not interfere with the measurement of a solvent peak in the detection, has accurate and reliable detection results, and has important significance for realizing the quality control of the brivaracetam and the preparation thereof.)

1. A method for detecting brivaracetam and isomers thereof is characterized in that a chromatographic column adopted in the method adopts silica gel coated with amylose-tri (3, 5-xylyl carbamate) on the surface as a filling agent, adopts a mixed solution of normal hexane, isopropanol and organic base as a mobile phase, and the organic base is one of triethylamine and ethanolamine and enters a detector for detection; the specific structural formula of the isomer is as follows:

formula I: isomer impurity 1

Formula II: isomer impurity 2

Formula III: isomer impurity 3.

2. The method for detecting brivaracetam and isomers thereof according to claim 1, wherein the organic base is preferably ethanolamine.

3. The method for detecting brivaracetam and isomers thereof as claimed in claim 1, wherein the mobile phase is a mixed solution of n-hexane, isopropanol and an organic base, wherein the volume ratio of n-hexane to isopropanol is 5: 95-15: 85, and the volume percentage of the organic base in the mixed solution is 0.05% -0.2%.

4. The method for detecting brivaracetam and isomers thereof according to claim 1, wherein the temperature of the chromatographic column is 15-40 ℃.

5. The method for detecting brivaracetam and isomers thereof as claimed in claim 1, wherein the wavelength of the detector is 200nm-220 nm.

6. The assay for brivaracetam and isomers thereof according to any one of claims 1-5, wherein the specific assay comprises the following steps:

1) preparing a test solution: dissolving a test sample in a solvent to obtain a test sample solution;

2) preparing a control solution: measuring a proper amount of a test solution, and diluting with a solvent to obtain a control solution;

3) preparing a system applicability solution: respectively taking a reference substance mixed with brivaracetam and impurity 1 and a reference substance mixed with impurity 2 and impurity 3, and dissolving and diluting the reference substances by using a solvent to prepare a system applicability solution;

4) respectively sampling the test solution in the step 1), the reference solution in the step 2) and the system applicability solution in the step 3), carrying out high performance liquid chromatography analysis, recording a chromatogram, determining the retention time of the brivaracetam and the impurities 1, 2 and 3, and calculating the content of isomer impurities in the test solution according to a main component self-reference method.

7. The method for detecting brivaracetam and isomers thereof according to claim 6, wherein the solvent in the steps 1) and 2) is a mobile phase.

Technical Field

The invention belongs to the field of analytical chemistry, and particularly relates to a method for detecting brivaracetam and isomers thereof.

Background

The molecular formula of Brivaracetam is C11H20N2O2, the English name is Brivaracetam, the CAS number is 357336-20-0, the structural formula is shown as IV, the Brivaracetam is a second generation anti-epileptic drug developed by UCB company of Belgium, and the product name is Briviact. Can be used for treating partial seizure type epilepsies of 16 years old and above with or without secondary systemic seizure as adjuvant therapy.

Formula IV: brivaracetam

The compound has two chiral centers, and under the conditions of high temperature, illumination and the like, the chiral centers can be inverted, so that 3 isomer impurities are introduced. Because the main active ingredient for taking effect is brivaracetam, and the increase of the content of isomer impurities can reduce the drug effect or increase the toxic and side effects, strict quality control is carried out on the isomer impurities according to the CDE evaluation principle and the requirements of the ICH Q3 and other guidelines.

Therefore, there is a need to develop an effective method for rapidly, accurately and sensitively detecting enantiomers and diastereomers in brivaracetam.

Disclosure of Invention

In view of the above, the invention aims to provide a method for detecting brivaracetam and isomers thereof, which can effectively separate and measure the content of brivaracetam and isomers thereof, is simple and convenient to operate, has high sensitivity and good specificity and repeatability, does not interfere with measurement of solvent peaks in detection, and has accurate and reliable detection results.

In order to achieve the purpose, the technical scheme of the invention is as follows:

separating and measuring brivaracetam and enantiomers and diastereomers thereof by an HPLC method, wherein a chromatographic column is adopted in the method, silica gel coated with amylose-tri (3, 5-xylyl carbamate) on the surface is adopted as a filling agent, a mixed solution of n-hexane, isopropanol and an organic base is adopted as a mobile phase, the organic base is one of triethylamine and ethanolamine, and the organic base enters a detector for detection; the specific structural formula of the isomer is as follows:

formula I: isomer impurity 1

Formula II: isomer impurity 2

Formula III: isomer impurity 3.

Further, the mobile phase is a mixed solution of n-hexane, isopropanol and an organic base, wherein the volume ratio of the n-hexane to the isopropanol is 5: 95-15: 85, and the volume percentage of the organic base in the mixed solution is 0.05% -0.2%.

Preferably, the volume ratio of the n-hexane to the isopropanol in the mobile phase is 10:90, and the volume percentage of the organic base in the mixed solution is 0.1%.

Further, the organic base added in the mobile phase is triethylamine and ethanolamine.

Preferably, the organic base is ethanolamine.

Further, the temperature of the chromatographic column is 15-40 ℃.

Preferably, the temperature of the chromatographic column is 20 ℃.

Further, the wavelength of the detector is 200-220 nm.

Preferably, the detector has a wavelength of 205 nm.

Furthermore, the flow rate of the mobile phase for elution is 0.7-1.3 ml/min.

Preferably, the mobile phase is eluted at a flow rate of 1.0 ml/min.

Further, the detection method specifically comprises the following steps:

1) preparing a test solution: dissolving a test sample in a solvent to obtain a test sample solution;

2) preparing a control solution: measuring a proper amount of a test solution, and diluting with a solvent to obtain a control solution;

3) preparing a system applicability solution: respectively taking a reference substance mixed with brivaracetam and impurity 1 and a reference substance mixed with impurity 2 and impurity 3, and dissolving and diluting the reference substances by using a solvent to prepare a system applicability solution;

4) respectively sampling the test solution in the step 1), the reference solution in the step 2) and the system applicability solution in the step 3), carrying out high performance liquid chromatography analysis, recording a chromatogram, determining the retention time of the brivaracetam and the impurities 1, 2 and 3, and calculating the content of isomer impurities in the test solution according to a main component self-reference method.

Further, the solvent in step 1) and step 2) is a mobile phase.

The invention has the beneficial effects that:

the HPLC method provided by the invention can be used for simultaneously separating and measuring the brivaracetam and the isomers thereof, can be used for effectively separating and measuring the brivaracetam and the contents of enantiomers and diastereomers thereof, is simple and convenient to operate, has high sensitivity and good specificity and repeatability, enables the solvent peak in the detection not to interfere with the measurement, is accurate and reliable in detection result, enables the separation degree between brivaracetam isomer impurities and adjacent peaks to be larger than 1.5, and enables other known impurity peaks and brivaracetam main peaks not to influence the detection.

Drawings

FIG. 1 is a high performance liquid chromatogram of a brivaracetam sample.

FIG. 2 is a brivaracetam isomer impurity detection limit spectrum.

Detailed Description

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental methods of the preferred embodiments, which do not indicate specific conditions, are generally performed according to conventional conditions, and the examples are given for better illustration of the present invention, but the present invention is not limited to the examples. 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.

Test 1:

the instrument comprises the following steps: daian U3000

A chromatographic column: chiralpak AD-H, 4.6mm X250 mm, 5 μm or equivalent performance chromatography column

Mobile phase: n-hexane-isopropanol (90:10)

Flow rate: 1.0ml/min

Column temperature: 20 deg.C

A detector: UV (ultraviolet) light

Detection wavelength: 205nm

Sample introduction volume: 20 μ l

Taking a proper amount of the fine powder of the product, adding mobile phase for dissolving and diluting to prepare a solution containing about 2mg of brivaracetam in each 1ml as a test solution; precisely measuring a proper amount of the test solution, and diluting the test solution with a mobile phase to prepare a solution containing about 4 mu g of brivaracetam in each 1ml of the test solution as a control solution. Taking a proper amount of a reference substance mixed by brivaracetam and impurity 1 and a proper amount of a reference substance mixed by impurity 2 and impurity 3, dissolving and diluting by using a mobile phase to prepare a solution containing about 0.5mg of impurity 1, about 0.1mg of impurity 2 and about 0.1mg of impurity 3 in each 1ml of the solution as an impurity stock solution; and (3) taking about 20mg of the brivaracetam reference substance, putting the brivaracetam reference substance into a 10ml measuring flask, adding a proper amount of mobile phase for dissolving, adding 2ml of impurity stock solution, diluting to a scale with the mobile phase, and shaking up to obtain a system applicability solution.

Test 2:

the instrument comprises the following steps: daian U3000

A chromatographic column: chiralpak AD-H, 4.6mm X250 mm, 5 μm or equivalent performance chromatography column

Mobile phase: n-hexane-isopropanol-ethanolamine (90:10:0.01)

Flow rate: 1.0ml/min

Column temperature: 20 deg.C

A detector: UV (ultraviolet) light

Detection wavelength: 205nm

Sample introduction volume: 20 μ l

Taking a proper amount of the fine powder of the product, adding mobile phase for dissolving and diluting to prepare a solution containing about 2mg of brivaracetam in each 1ml as a test solution; precisely measuring a proper amount of the test solution, and diluting the test solution with a mobile phase to prepare a solution containing about 4 mu g of brivaracetam in each 1ml of the test solution as a control solution. Taking a proper amount of a reference substance mixed by brivaracetam and impurity 1 and a proper amount of a reference substance mixed by impurity 2 and impurity 3, dissolving and diluting by using a mobile phase to prepare a solution containing about 0.5mg of impurity 1, about 0.1mg of impurity 2 and about 0.1mg of impurity 3 in each 1ml of the solution as an impurity stock solution; and (3) taking about 20mg of the brivaracetam reference substance, putting the brivaracetam reference substance into a 10ml measuring flask, adding a proper amount of mobile phase for dissolving, adding 2ml of impurity stock solution, diluting to a scale with the mobile phase, and shaking up to obtain a system applicability solution.

Test 3:

the instrument comprises the following steps: daian U3000

A chromatographic column: chiralpak AD-H, 4.6mm X250 mm, 5 μm or equivalent performance chromatography column

Mobile phase: n-hexane-isopropanol-ethanolamine (90:10:0.05)

Flow rate: 1.0ml/min

Column temperature: 20 deg.C

A detector: UV (ultraviolet) light

Detection wavelength: 205nm

Sample introduction volume: 20 μ l

Taking a proper amount of the fine powder of the product, adding mobile phase for dissolving and diluting to prepare a solution containing about 2mg of brivaracetam in each 1ml as a test solution; precisely measuring a proper amount of the test solution, and diluting the test solution with a mobile phase to prepare a solution containing about 4 mu g of brivaracetam in each 1ml of the test solution as a control solution. Taking a proper amount of a reference substance mixed by brivaracetam and impurity 1 and a proper amount of a reference substance mixed by impurity 2 and impurity 3, dissolving and diluting by using a mobile phase to prepare a solution containing about 0.5mg of impurity 1, about 0.1mg of impurity 2 and about 0.1mg of impurity 3 in each 1ml of the solution as an impurity stock solution; and (3) taking about 20mg of the brivaracetam reference substance, putting the brivaracetam reference substance into a 10ml measuring flask, adding a proper amount of mobile phase for dissolving, adding 2ml of impurity stock solution, diluting to a scale with the mobile phase, and shaking up to obtain a system applicability solution.

Test 4:

the instrument comprises the following steps: daian U3000

A chromatographic column: chiralpak AD-H, 4.6mm X250 mm, 5 μm or equivalent performance chromatography column

Mobile phase: n-hexane-isopropanol-ethanolamine (90:10:0.1)

Flow rate: 1.0ml/min

Column temperature: 20 deg.C

A detector: UV (ultraviolet) light

Detection wavelength: 205nm

Sample introduction volume: 20 μ l

Taking a proper amount of the fine powder of the product, adding mobile phase for dissolving and diluting to prepare a solution containing about 2mg of brivaracetam in each 1ml as a test solution; precisely measuring a proper amount of the test solution, and diluting the test solution with a mobile phase to prepare a solution containing about 4 mu g of brivaracetam in each 1ml of the test solution as a control solution. Taking a proper amount of a reference substance mixed by brivaracetam and impurity 1 and a proper amount of a reference substance mixed by impurity 2 and impurity 3, dissolving and diluting by using a mobile phase to prepare a solution containing about 0.5mg of impurity 1, about 0.1mg of impurity 2 and about 0.1mg of impurity 3 in each 1ml of the solution as an impurity stock solution; and (3) taking about 20mg of the brivaracetam reference substance, putting the brivaracetam reference substance into a 10ml measuring flask, adding a proper amount of mobile phase for dissolving, adding 2ml of impurity stock solution, diluting to a scale with the mobile phase, and shaking up to obtain a system applicability solution.

Test 5:

the instrument comprises the following steps: daian U3000

A chromatographic column: chiralpak AD-H, 4.6mm X250 mm, 5 μm or equivalent performance chromatography column

Mobile phase: n-hexane-isopropanol-ethanolamine (90:10:0.2)

Flow rate: 1.0ml/min

Column temperature: 20 deg.C

A detector: UV (ultraviolet) light

Detection wavelength: 205nm

Sample introduction volume: 20 μ l

Taking a proper amount of the fine powder of the product, adding mobile phase for dissolving and diluting to prepare a solution containing about 2mg of brivaracetam in each 1ml as a test solution; precisely measuring a proper amount of the test solution, and diluting the test solution with a mobile phase to prepare a solution containing about 4 mu g of brivaracetam in each 1ml of the test solution as a control solution. Taking a proper amount of a reference substance mixed by brivaracetam and impurity 1 and a proper amount of a reference substance mixed by impurity 2 and impurity 3, dissolving and diluting by using a mobile phase to prepare a solution containing about 0.5mg of impurity 1, about 0.1mg of impurity 2 and about 0.1mg of impurity 3 in each 1ml of the solution as an impurity stock solution; and (3) taking about 20mg of the brivaracetam reference substance, putting the brivaracetam reference substance into a 10ml measuring flask, adding a proper amount of mobile phase for dissolving, adding 2ml of impurity stock solution, diluting to a scale with the mobile phase, and shaking up to obtain a system applicability solution.

Test 6:

the instrument comprises the following steps: daian U3000

A chromatographic column: chiralpak AD-H, 4.6mm X250 mm, 5 μm or equivalent performance chromatography column

Mobile phase: n-hexane-isopropanol-triethylamine (90:10:0.01)

Flow rate: 1.0ml/min

Column temperature: 20 deg.C

A detector: UV (ultraviolet) light

Detection wavelength: 205nm

Sample introduction volume: 20 μ l

Taking a proper amount of the fine powder of the product, adding mobile phase for dissolving and diluting to prepare a solution containing about 2mg of brivaracetam in each 1ml as a test solution; precisely measuring a proper amount of the test solution, and diluting the test solution with a mobile phase to prepare a solution containing about 4 mu g of brivaracetam in each 1ml of the test solution as a control solution. Taking a proper amount of a reference substance mixed by brivaracetam and impurity 1 and a proper amount of a reference substance mixed by impurity 2 and impurity 3, dissolving and diluting by using a mobile phase to prepare a solution containing about 0.5mg of impurity 1, about 0.1mg of impurity 2 and about 0.1mg of impurity 3 in each 1ml of the solution as an impurity stock solution; and (3) taking about 20mg of the brivaracetam reference substance, putting the brivaracetam reference substance into a 10ml measuring flask, adding a proper amount of mobile phase for dissolving, adding 2ml of impurity stock solution, diluting to a scale with the mobile phase, and shaking up to obtain a system applicability solution.

Test 7:

the instrument comprises the following steps: daian U3000

A chromatographic column: chiralpak AD-H, 4.6mm X250 mm, 5 μm or equivalent performance chromatography column

Mobile phase: n-hexane-isopropanol-triethylamine (90:10:0.05)

Flow rate: 1.0ml/min

Column temperature: 20 deg.C

A detector: UV (ultraviolet) light

Detection wavelength: 205nm

Sample introduction volume: 20 μ l

Taking a proper amount of the fine powder of the product, adding mobile phase for dissolving and diluting to prepare a solution containing about 2mg of brivaracetam in each 1ml as a test solution; precisely measuring a proper amount of the test solution, and diluting the test solution with a mobile phase to prepare a solution containing about 4 mu g of brivaracetam in each 1ml of the test solution as a control solution. Taking a proper amount of a reference substance mixed by brivaracetam and impurity 1 and a proper amount of a reference substance mixed by impurity 2 and impurity 3, dissolving and diluting by using a mobile phase to prepare a solution containing about 0.5mg of impurity 1, about 0.1mg of impurity 2 and about 0.1mg of impurity 3 in each 1ml of the solution as an impurity stock solution; and (3) taking about 20mg of the brivaracetam reference substance, putting the brivaracetam reference substance into a 10ml measuring flask, adding a proper amount of mobile phase for dissolving, adding 2ml of impurity stock solution, diluting to a scale with the mobile phase, and shaking up to obtain a system applicability solution.

Test 8:

the instrument comprises the following steps: daian U3000

A chromatographic column: chiralpak AD-H, 4.6mm X250 mm, 5 μm or equivalent performance chromatography column

Mobile phase: n-hexane-isopropanol-triethylamine (90:10:0.1)

Flow rate: 1.0ml/min

Column temperature: 20 deg.C

A detector: UV (ultraviolet) light

Detection wavelength: 205nm

Sample introduction volume: 20 μ l

Taking a proper amount of the fine powder of the product, adding mobile phase for dissolving and diluting to prepare a solution containing about 2mg of brivaracetam in each 1ml as a test solution; precisely measuring a proper amount of the test solution, and diluting the test solution with a mobile phase to prepare a solution containing about 4 mu g of brivaracetam in each 1ml of the test solution as a control solution. Taking a proper amount of a reference substance mixed by brivaracetam and impurity 1 and a proper amount of a reference substance mixed by impurity 2 and impurity 3, dissolving and diluting by using a mobile phase to prepare a solution containing about 0.5mg of impurity 1, about 0.1mg of impurity 2 and about 0.1mg of impurity 3 in each 1ml of the solution as an impurity stock solution; and (3) taking about 20mg of the brivaracetam reference substance, putting the brivaracetam reference substance into a 10ml measuring flask, adding a proper amount of mobile phase for dissolving, adding 2ml of impurity stock solution, diluting to a scale with the mobile phase, and shaking up to obtain a system applicability solution.

Test 9:

the instrument comprises the following steps: daian U3000

A chromatographic column: chiralpak AD-H, 4.6mm X250 mm, 5 μm or equivalent performance chromatography column

Mobile phase: n-hexane-isopropanol-triethylamine (90:10:0.2)

Flow rate: 1.0ml/min

Column temperature: 20 deg.C

A detector: UV (ultraviolet) light

Detection wavelength: 205nm

Sample introduction volume: 20 μ l

Taking a proper amount of the fine powder of the product, adding mobile phase for dissolving and diluting to prepare a solution containing about 2mg of brivaracetam in each 1ml as a test solution; precisely measuring a proper amount of the test solution, and diluting the test solution with a mobile phase to prepare a solution containing about 4 mu g of brivaracetam in each 1ml of the test solution as a control solution. Taking a proper amount of a reference substance mixed by brivaracetam and impurity 1 and a proper amount of a reference substance mixed by impurity 2 and impurity 3, dissolving and diluting by using a mobile phase to prepare a solution containing about 0.5mg of impurity 1, about 0.1mg of impurity 2 and about 0.1mg of impurity 3 in each 1ml of the solution as an impurity stock solution; and (3) taking about 20mg of the brivaracetam reference substance, putting the brivaracetam reference substance into a 10ml measuring flask, adding a proper amount of mobile phase for dissolving, adding 2ml of impurity stock solution, diluting to a scale with the mobile phase, and shaking up to obtain a system applicability solution.

The results of the above 9 tests were compared as follows:

as can be seen from the above table, after ethanolamine and triethylamine are added into the mobile phase, the separation degree between each impurity and the main component is obviously improved, and the trailing condition of the main peak is also obviously improved; when the addition amount of the ethanolamine is more than 0.05 percent or the addition amount of the triethylamine is more than 0.1 percent, the separation degree is more than 1.5, and the tailing factor is not more than 1.5. Wherein, when the addition amount of the ethanolamine is 0.1-0.2%, the separation effect is optimal.

To sum up: the HPLC method provided by the invention can be used for simultaneously separating and measuring brivaracetam and isomers thereof, can be used for effectively separating and measuring the brivaracetam and the contents of enantiomers and diastereomers thereof, is simple and convenient to operate, has high sensitivity (the detection limit concentration of the isomers is 0.07 mu g/ml, which is equivalent to 0.004% of the content of finished products), and is good in specificity and repeatability, the measurement is not interfered by a solvent peak in the detection, the detection result is accurate and reliable, the separation degree between the brivaracetam isomer impurities and adjacent peaks is more than 1.5, and other known impurity peaks and brivaracetam main peaks do not influence the detection.

Finally, the above embodiments are only intended to illustrate the technical solution of the present invention and not to limit the same, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, which should be covered by the claims of the present invention.