Detection method of briracetam related substances

Abstract

The invention relates to a method for detecting a briracetam related substance, and the related substance is one or more of (S)-2-((R)-2-oxo-4-propyl pyrrolidine-1-yl) butyric acid, (S)-2-((R)-2, 5-dioxo-3-propyl pyrrolidine-1-yl) butyramide, and (S)-N-((S)-1-amino-1-oxobutane-2-((R)-2-oxo-4-propyl pyrrolidine-1-yl) butyramide, and the briracetam related substance is one or more of the following substances. High performance liquid chromatography is adopted for detection, and chromatographic conditions are as follows: octadecylsilane chemically bonded silica is taken as a filler of a chromatographic column; a mobile phase is composed of a mobile phase A and a mobile phase B, the mobile phase A is a 0.1% phosphoric acid aqueous solution, the mobile phase B is acetonitrile, and gradient elution is carried out. The method disclosed by the invention can be used for simply, quickly and accurately separating the briracetam from the related substances in the briracetam tablet and accurately determining the content of the related substances, the result is stable and reliable, the separation efficiency is high, and the method has good specificity and reproducibility.

Description

Technical Field

[0001] The present application relates to the technical field of drug analysis, and in particular to a method for detecting brivaracetam-related substances. Background Art

[0002] Brivaracetam is a 2-pyrrolidone derivative with the chemical name (2S)-2-[(4R)-2-oxo-4-propyl-pyrrolidin-1-yl]butanamide and the molecular formula is C 11 H 20 N 2 O 2 , CAS registration number 357336-20-0, chemical structure is as follows:

[0003]

[0004] Brivaracetam is a third-generation anti-epileptic drug developed by UCB of Belgium. It is a 4-carbon-attached n-propyl analogue of the pyrrolidine of the second-generation anti-epileptic drug levetiracetam. Brivaracetam is a selective and high-affinity ligand for the brain salience vesicle protein 2A (SV2A), and has a superior affinity to levetiracetam in inhibiting synaptic transmission and vesicle release. Brivaracetam has high lipophilicity, rapid brain penetration, and a short latency of action, which is of great significance for the clinical emergency treatment of status epilepticus or epileptic cluster seizures. Brivaracetam was approved for marketing by the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) on January 14, 2016 and February 18, 2016, respectively, under the trade name Briviact, for the treatment of partial-onset epilepsy patients aged 16 years and above, and as an adjuvant treatment with or without secondary generalized seizures.

[0005] In the process of synthesizing this compound, impurities such as by-products produced in subsequent reactions due to incomplete reaction of key intermediates and starting materials, reaction by-products caused by low purity of starting materials, degradation by-products produced due to degradation of the drug itself, and process by-products in the synthesis process are all related substances of the drug and require strict quality control.

[0006] Invention patent 202010632316.8 provides a method for detecting the impurity (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butyric acid (impurity D) of brivaracetam hydrolysis. The method uses a high performance liquid chromatograph, octadecylsilane bonded silica gel as a filler, phosphoric acid aqueous solution and acetonitrile as mobile phases for gradient elution, and the detection wavelength is 200-220nm. However, the determination method described in the patent cannot effectively separate and determine brivaracetam and its other two related substances: (S)-2-((R)-2,5-dioxo-3-propylpyrrolidin-1-yl)butyramide (impurity E) and (S)-N-((S)-1-amino-1-oxobutane-2-yl)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butyramide (impurity F).

[0007] Since there is no documented detection method for simultaneously detecting and separating the above-mentioned related substances, but the control of impurities plays a vital role in the entire life cycle of drugs, it is urgent to establish an easy-to-operate detection method for brivaracetam-related substances in order to better promote the research and development of the drug and ensure product quality control. Summary of the invention

[0008] In order to achieve the above-mentioned purpose of the invention, the present application provides a method for detecting brivaracetam-related substances. The detection method adopts high performance liquid chromatography and can simply, quickly and accurately analyze and detect brivaracetam and its related substances by selecting specific chromatographic conditions, thereby ensuring the quality controllable of brivaracetam and its preparations.

[0009] The first aspect of the present application provides a method for detecting related substances in brivaracetam, which uses high-performance liquid chromatography to detect one or more brivaracetam-related substances in a brivaracetam sample.

[0010] In an exemplary embodiment, the related substance is:

[0011] Impurity D: (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butyric acid, the chemical structure is as follows:

[0012]

[0013] Impurity E: (S)-2-((R)-2,5-dioxo-3-propylpyrrolidin-1-yl)butanamide, the chemical structure is as follows:

[0014]

[0015] Impurity F: (S)-N-((S)-1-amino-1-oxobutane-2-yl)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide, the chemical structure is as follows:

[0016]

[0017] In an exemplary embodiment, when the high-performance liquid chromatograph is used for detection, the chromatographic column is filled with octadecylsilane bonded silica gel.

[0018] In an exemplary embodiment, when the high performance liquid chromatograph is used for detection, the chromatographic column model is Welch LP-C18 250 mm×4.6 mm, 5 μm.

[0019] In an exemplary embodiment, when detecting by high performance liquid chromatography, the mobile phase consists of mobile phase A and mobile phase B; wherein the mobile phase A is aqueous phosphoric acid solution, and the mobile phase B is acetonitrile.

[0020] In an exemplary embodiment, the mobile phase A is a phosphoric acid aqueous solution with a volume fraction of 0.1%.

[0021] In an exemplary embodiment, when the high-performance liquid chromatograph is used for detection, the mobile phase elution program is a gradient elution program, and the gradient elution program is as follows:

[0022] Time / min Mobile phase A (%, volume percentage) Mobile phase B (%, volume percentage) 0 90 10 60 75 25 65 75 25 70 90 10 75 90 10

[0023] In an exemplary embodiment, when the high-performance liquid chromatograph is used for detection, the chromatographic conditions further include:

[0024] The detection wavelength is 210nm;

[0025] The flow rate of the mobile phase is 0.9 to 1.1 mL / min, preferably 1.0 mL / min;

[0026] The column temperature is 27-33°C, preferably 30°C;

[0027] The injection volume is 10-30 μL, preferably 10 μL;

[0028] The detector is a UV detector.

[0029] In an exemplary embodiment, the detection method comprises the following steps:

[0030] 1) Prepare solution

[0031] a. Prepare the reference solutions of impurities D, E, and F respectively;

[0032] b. Prepare a reference substance mixture solution of brivaracetam and impurities D, E, and F;

[0033] c. Prepare the test solution of brivaracetam sample;

[0034] d. Prepare a control solution of the brivaracetam test solution;

[0035] 2) Detection

[0036] The positioning solutions of impurities D, E, and F in step a, the impurity mixed solution in step b, the test solution in step c, and the control solution in step d are respectively injected into a high performance liquid chromatograph for detection, and the chromatogram is recorded;

[0037] 3) Analysis

[0038] Determine the retention times of impurities D, E, and F based on the chromatograms of each impurity location solution;

[0039] According to the peak area of ​​each impurity in the chromatograms of the test solution and the control solution, calculate the content of impurities D, E, and F in the test sample.

[0040] In an exemplary embodiment, the preparation method of the localization solution of impurities D, E, and F in step a is: weigh the impurity D, E, and F reference substances respectively, dissolve them in acetonitrile, and obtain the localization solution of each impurity; optionally, the concentration of the localization solution of each impurity can be 0.01 mg / ml.

[0041] In an exemplary embodiment, the preparation method of the mixture solution in step b is: weigh brivaracetam, impurity D, E, and F reference substances, dissolve them in the same volumetric flask with acetonitrile to make up the volume, and obtain an impurity mixture solution; optionally, in the mixture solution, the concentration of brivaracetam can be 5 mg / ml, and the concentration of each impurity can be 0.01 mg / ml.

[0042] In an exemplary embodiment, the preparation method of the brivaracetam test solution in step c is: weighing the brivaracetam test sample, dissolving it with acetonitrile to a fixed volume, filtering, and taking the filtrate to obtain a test solution; optionally, the concentration of the brivaracetam test sample is 5 mg / ml.

[0043] In an exemplary embodiment, the brivaracetam test sample is a brivaracetam bulk drug or a brivaracetam preparation, and the brivaracetam preparation includes but is not limited to brivaracetam tablets (powder after crushing), brivaracetam injection or brivaracetam oral solution.

[0044] In an exemplary embodiment, the preparation method of the control solution in step d is: take 100 μL of the test solution in step c and place it in a 100 mL volumetric flask, add acetonitrile to make up to volume and shake well to obtain a control solution.

[0045] In an exemplary embodiment, in step 3), the separation degree between the chromatographic peaks of brivaracetam and impurities D, E, and F in the chromatogram of the reference substance mixture solution should be greater than 1.5.

[0046] In an exemplary embodiment, in step 3), the content of impurities D, E, and F in the sample is calculated as follows:

[0047]

[0048]

[0049] Compared with the prior art, the detection method described in this application has the following advantages:

[0050] The detection method provided in the present application adopts high performance liquid chromatography, which can simultaneously effectively separate brivaracetam from its impurities (impurity D) (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butyric acid, (impurity E) (S)-2-((R)-2,5-dioxo-3-propylpyrrolidin-1-yl)butyramide, and (impurity F) (S)-N-((S)-1-amino-1-oxobutane-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butyramide, and can be used to detect impurities D, E, and F in brivaracetam, and accurately determine the content of impurities D, E, and F in brivaracetam. The operation is simple, the measurement results are stable and reliable, and the specificity is strong. The detection method provided in the present application can effectively monitor the synthesis process of brivaracetam, be used for the quality detection of brivaracetam, ensure its quality, and have broad application prospects.

[0051] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0053] Figure 1 This is the HPLC spectrum of the impurity D localization solution of the initial elution procedure of Example 1;

[0054] Figure 2 This is the HPLC spectrum of the solution for locating impurity E in the initial elution procedure of Example 1;

[0055] Figure 3 This is the HPLC spectrum of the impurity F location solution of the initial elution procedure of Example 1;

[0056] Figure 4 This is the HPLC spectrum of the impurity mixed solution of the initial elution procedure of Example 1;

[0057] Figure 5 This is the HPLC spectrum of the impurity mixed solution of elution procedure 1 of Example 1;

[0058] Figure 6 This is the HPLC spectrum of the impurity mixed solution of elution procedure 2 of Example 1;

[0059] Figure 7 This is the HPLC spectrum of the impurity mixed solution of elution procedure 3 of Example 1;

[0060] Figure 8 This is the HPLC spectrum of the impurity mixed solution of elution procedure 4 of Example 1;

[0061] Fig. 9 This is the HPLC spectrum of the impurity mixed solution of elution procedure 5 of Example 1;

[0062] Fig.10 This is the HPLC spectrum of the impurity mixed solution of elution procedure 6 of Example 1;

[0063] Fig.11 This is the HPLC spectrum of the impurity mixed solution of elution procedure 7 in Example 1;

[0064] Fig.12 HPLC spectrum of the blank solution in Example 2;

[0065] Fig.13 HPLC spectrum of the blank auxiliary material solution in Example 2;

[0066] Fig.14 This is the HPLC spectrum of the solution for locating impurity D in Example 2;

[0067] Fig.15 This is the HPLC spectrum of the solution for locating impurity E in Example 2;

[0068] Fig.16 This is the HPLC spectrum of the solution for locating impurity F in Example 2;

[0069] Fig.17 is the HPLC spectrum of the system suitability solution in Example 2;

[0070] Fig.18 is the HPLC spectrum of the impurity mixed solution in Example 2;

[0071] Fig.19 is the linear graph of brivaracetam in Example 5;

[0072] Fig. 20is the linear graph of impurity D in Example 5;

[0073] Fig.21 is the linear graph of impurity E in Example 5;

[0074] Fig. 22 is the linear graph of impurity F in Example 5;

[0075] Fig.23 It is the HPLC spectrum of the blank solution in Comparative Example 1;

[0076] Fig.24 This is the HPLC spectrum of the solution for locating impurity D in Comparative Example 1;

[0077] Fig.25 This is the HPLC spectrum of the solution for locating impurity E in Comparative Example 1;

[0078] Fig.26 This is the HPLC spectrum of the solution for locating impurity F in Comparative Example 1;

[0079] Fig. 27 The HPLC spectrum of the impurity mixed solution in Comparative Example 1. DETAILED DESCRIPTION

[0080] In order to make the purpose, technical solution and advantages of the present application more clear, the embodiments of the present invention will be described in detail below. It should be noted that the embodiments and features in the embodiments of the present application can be combined with each other arbitrarily without conflict.

[0081] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0082] Example 1 Investigation of elution procedure

[0083] 1.1. Main technical parameters of HPLC:

[0084] Chromatographic column: Welch LP-C18 250 mm × 4.6 mm, 5 μm;

[0085] Detection wavelength: UV-210nm;

[0086] Flow rate: 1 mL / min;

[0087] Column temperature: 30°C;

[0088] Injection volume: 10 μL;

[0089] Mobile phase: Mobile phase A is 0.1% phosphoric acid aqueous solution; Mobile phase B is acetonitrile;

[0090] 1.2. Solution preparation:

[0091] Impurity D Location Solution D 2 : Weigh the brivaracetam impurity D reference substance, dissolve it in acetonitrile and prepare a solution with a concentration of 0.10942 mg / mL to obtain;

[0092] Impurity E Location Solution E 2 : Weigh the brivaracetam impurity E reference substance, dissolve it in acetonitrile and prepare a solution with a concentration of 0.11542 mg / mL to obtain;

[0093] Impurity F Location Solution F 2 : Weigh the brivaracetam impurity F reference substance, dissolve it in acetonitrile and prepare a solution with a concentration of 5 mg / mL to obtain;

[0094] Impurity mixed solution II: Weigh 100.28 mg of brivaracetam API and place it in a 20 mL volumetric flask ①, and transfer 1 mL of brivaracetam impurity D and E localization solutions to the volumetric flask ① respectively; take 1 mL of brivaracetam impurity F localization solution and place it in a 50 mL volumetric flask ②, add acetonitrile to the mark, shake well, then take 1 mL of the solution in the volumetric flask ② and place it in a volumetric flask ①, add acetonitrile to dilute to the mark, shake well, and obtain. The concentration of brivaracetam is 5.014 mg / mL, the concentration of impurity D is 5.471 μg / mL, the concentration of impurity E is 5.771 μg / mL, and the concentration of impurity F is 5 μg / mL.

[0095] 1.3 Elution procedure

[0096] (1) Initial elution procedure:

[0097] Time / min Mobile phase A (%, volume percentage) Mobile phase B (%, volume percentage) 0 90 10 10 10 90 15 10 90 25 90 10 30 90 10

[0098] (2) Elution procedure 1:

[0099] Time / min Mobile phase A (%, volume percentage) Mobile phase B (%, volume percentage) 0 80 20 70 30 70 75 30 70 80 80 20 85 80 20

[0100] (3) Elution procedure 2:

[0101] Time / min Mobile phase A (%, volume percentage) Mobile phase B (%, volume percentage) 0 80 20 60 50 50 65 50 50 70 80 20 75 80 20

[0102] (4) Elution procedure 3:

[0103] Elution method: isocratic elution

[0104] Mobile phase: 0.1% phosphoric acid aqueous solution: acetonitrile = 75:25 (volume ratio);

[0105] Running time: 60 minutes

[0106] (5) Elution procedure 4:

[0107] Elution method: isocratic elution

[0108] Mobile phase: 0.1% phosphoric acid aqueous solution: acetonitrile = 80:20 (volume ratio);

[0109] Running time: 60 minutes

[0110] (6) Elution procedure 5:

[0111] Time / min Mobile phase A (%, volume percentage) Mobile phase B (%, volume percentage) 0 80 20 60 70 30 65 70 30 70 80 20 75 80 20

[0112] (7) Elution procedure 6:

[0113] Time / min Mobile phase A (%, volume percentage) Mobile phase B (%, volume percentage) 0 90 10 60 80 20 65 80 20 70 90 10 75 90 10

[0114] (8) Elution procedure 7:

[0115] Time / min Mobile phase A (%, volume percentage) Mobile phase B (%, volume percentage) 0 90 10 60 75 25 65 75 25 70 90 10 75 90 10

[0116] 1.4. Sampling analysis

[0117] Remove impurities D, E, and F respectively and position solution D 2 、E 2 、F 2 The impurity mixed solution II was injected into a high performance liquid chromatograph and tested and analyzed under the above chromatographic conditions and elution program conditions, respectively, to obtain the following Figure 1-11 HPLC chromatogram shown.

[0118] Conclusion: (1) Through analysis Figures 1 to 4 From the HPLC chromatogram shown, it can be seen that under the initial elution program conditions, impurities E and F cannot be effectively separated from brivaracetam, and the brivaracetam isomer peak next to the brivaracetam chromatographic peak cannot be effectively separated from the main peak.

[0119] (2) Through analysis Figures 5 to 10 From the HPLC chromatogram shown, it can be seen that under the conditions of elution procedures 1 to 5, although impurities D, E, and F can be effectively separated from brivaracetam (the separation degrees are all greater than 1.5), the brivaracetam isomer peak next to the brivaracetam chromatographic peak cannot be effectively separated from the main peak of brivaracetam; under the conditions of elution procedure 6, impurity D and brivaracetam isomer peaks were not detected.

[0120] (3) Through analysis Fig.11 From the HPLC chromatogram shown, it can be seen that under the conditions of elution procedure 7, the brivaracetam isomer peak at the relative retention time of 1.05 can be effectively separated from the brivaracetam main peak, and brivaracetam, impurity D, impurity E, and impurity F can all be effectively separated, and the separation degrees are all greater than 1.5.

[0121] In summary, elution procedure 7 as an elution procedure in high performance liquid chromatography can effectively separate brivaracetam, impurity D, impurity E and impurity F.

[0122] Example 2 Specificity Test

[0123] 2.1. Main technical parameters of HPLC:

[0124] Chromatographic column: Welch LP-C18 250 mm × 4.6 mm, 5 μm;

[0125] Detection wavelength: UV-210nm;

[0126] Flow rate: 1.0 mL / min;

[0127] Column temperature: 30°C;

[0128] Injection volume: 10 μL;

[0129] Mobile phase: Mobile phase A is 0.1% phosphoric acid aqueous solution; Mobile phase B is acetonitrile;

[0130] Perform gradient elution according to the following elution program:

[0131] Time / min Mobile phase A (%, volume percentage) Mobile phase B (%, volume percentage) 0 90 10 60 75 25 65 75 25 70 90 10 75 90 10

[0132] 2.2 Solution preparation

[0133] Blank solution: acetonitrile;

[0134] Blank excipient solution: weigh 575 mg of blank excipient solution into a 25 ml volumetric flask, add an appropriate amount of acetonitrile and dissolve it by ultrasonication, then dilute it to the scale with acetonitrile, shake well, filter, and obtain the filtrate.

[0135] 2.2.1 Preparation of impurity stock solution:

[0136] Impurity D stock solution 1 : Weigh 10.48 mg of impurity D reference substance, place in a 50 ml volumetric flask, add acetonitrile to dissolve, dilute to scale, shake well, and obtain;

[0137] Impurity E stock solution 1 : Weigh 10.64 mg of impurity E reference substance, place in a 50 ml volumetric flask, add acetonitrile to dissolve, dilute to scale, shake well, and obtain;

[0138] Impurity F stock solution 1 : Take 100 mg of impurity F reference substance, dissolve it in a 50 ml volumetric flask with about 30 ml of acetonitrile, add acetonitrile to dilute to the scale, shake well, and you have it.

[0139] 2.2.2 System suitability solution:

[0140] Weigh 99.35 mg of brivaracetam reference substance, place it in a 20 mL volumetric flask, fully dissolve it with acetonitrile, and add impurity D stock solution d 1、Impurity E stock solution e 1 1mL each, impurity F stock solution f 1 100 μL, add acetonitrile and dilute to the scale.

[0141] 2.2.3 Impurity location solution:

[0142] Impurity D Location Solution D 3 : Weigh 13.03 mg of impurity D reference substance into a 50 mL volumetric flask, add acetonitrile to dissolve and dilute to scale, accurately measure 1 mL of the solution into a 25 mL volumetric flask, add acetonitrile to dilute to scale, and obtain;

[0143] Impurity E Location Solution E 3 : Weigh 12.64 mg of impurity E reference substance into a 50 mL volumetric flask, add acetonitrile to dissolve and dilute to scale, accurately measure 1 mL of the solution into a 25 mL volumetric flask, add acetonitrile to dilute to scale, and obtain;

[0144] Impurity F Location Solution F 3 :Accurately measure 1mL of impurity F stock solution f 1 Place in a 10mL volumetric flask, add acetonitrile to dilute to the mark, then accurately measure 1mL of the solution and place in a 25mL volumetric flask, add acetonitrile to dilute to the mark.

[0145] 2.2.4 Impurity mixed solution III:

[0146] Take appropriate amounts of brivaracetam reference substance and impurity D, E, and F reference substances, respectively, add acetonitrile to dissolve and dilute to prepare a mixed solution containing about 5 mg / mL of brivaracetam and about 0.01 mg / mL of impurities D, E, and F;

[0147] 2.2.5 Test solution a 1 :

[0148] Take 10 brivaracetam tablets, crush and mix them, weigh about 700 mg of the powder and put it into a 25 mL volumetric flask, add an appropriate amount of acetonitrile and dissolve it by ultrasonic, then dilute it to the scale with acetonitrile, shake well, filter, and take the filtrate to obtain the product.

[0149] 2.2.6 Control solution b 1 :

[0150] Accurately measure 100 μL of test solution a 1 Place in a 100mL volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain.

[0151] 2.2.7 Sensitivity solution:

[0152] Accurately measure 5 mL of control solution b 1Place in a 10mL volumetric flask, dilute to scale with acetonitrile, shake well, and obtain.

[0153] 2.3. Sample injection analysis

[0154] Take 10 μL of the blank solution, blank excipient solution, system suitability solution, and test solution a respectively. 1 , control solution b 1 , sensitivity solution, impurity D, E, F positioning solution, and impurity mixed solution III are injected into the high performance liquid chromatograph, wherein the sensitivity solution is used to test the instrument response capability, and the system suitability solution and impurity mixed solution III are used to detect the separation degree of each substance.

[0155] The chromatograms of blank solution, blank excipient solution, system suitability solution, impurity D, E, F positioning solution, and impurity mixed solution III are as follows: Figures 12 to 18 As shown; Table 1 below gives the specificity test results of the system suitability solution.

[0156] Table 1. Specificity test results

[0157] Impurity Name Retention time Separation Impurity E 42.420 4.9 Brivaracetam 46.517 2.4 Brivaracetam isomers 48.563 6.7 Impurity F 54.523 11.5 Impurity D 64.350 NA

[0158] Conclusion: Through the analysis of Figures 12 to 18 From the HPLC chromatogram shown in the figure and the specificity experimental results shown in Table 1, it can be seen that the separation between the peaks of impurities E, F, D and brivaracetam is greater than 1.5, and the blank solvent and blank excipient solution have no interference with the main peak and impurities, indicating that the detection method has good specificity and system applicability.

[0159] Example 3 Precision Test

[0160] Chromatographic conditions are the same as in Example 2

[0161] Impurity D stock solution 3 : Weigh 13.03 mg of impurity D reference substance, place in a 50 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale to obtain;

[0162] Impurity E stock solution 3 : Weigh 12.64 mg of impurity E reference substance into a 50 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale to obtain;

[0163] Impurity F stock solution 3 : Weigh 100 mg (whole bottle) of impurity F reference substance and place it in a 50 mL volumetric flask, dissolve it in acetonitrile and dilute to the scale, accurately transfer 1 mL of the solution and place it in a 10 mL volumetric flask, dilute it to the scale with acetonitrile, and you have it.

[0164] Test solution a 2Brivaracetam tablets were crushed and mixed, and about 700 mg of the powder was weighed and placed in a 25 mL volumetric flask. Acetonitrile was added and ultrasonically dissolved. The impurity D, E, and F stock solutions were accurately pipetted. 3 、e 3 、f 3 1 mL of each, add acetonitrile to dilute to the mark, shake well, filter, and take the filtrate as the test solution a 2 .

[0165] Control solution b 2 :Precisely pipette 1mL of test solution a 2 Place it in a 100ml volumetric flask, add acetonitrile to make up the volume, then transfer 1mL to a 10mL volumetric flask, dilute to make up the volume with acetonitrile, shake well, and you have it.

[0166] (1) Repeatability test

[0167] Prepare 6 portions of the above test solution a in parallel 2 and the corresponding control solution b 2 , respectively take 10 μL of sample for analysis, record the peak area in the chromatogram, and use the following formula to calculate the content of each related substance in the 6 test solutions:

[0168]

[0169]

[0170] (2) Intermediate precision test

[0171] Two different analysts prepared six samples of the above test solution at different times. 2 and the corresponding control solution b 2 , and perform detection and analysis on different instruments. Take 10 μL of each solution for injection analysis, record the peak area of ​​each peak in the chromatogram, and use the above formula to calculate the content of each related substance in the 6 sample solutions.

[0172] The results of the repeatability test and intermediate precision test are shown in Table 2 below.

[0173] Table 2. Repeatability and intermediate precision test results

[0174]

[0175] Conclusion: According to the results in Table 2, the RSDs (n=12) of the impurities D, E, F and total impurities in the 12 spiked test solutions were 16.9%, 2.3%, 2.5% and 3.1% (≤20%), respectively, which met the acceptance criteria, indicating that this detection method has good precision.

[0176] Example 4 Stability Test

[0177] Chromatographic conditions are the same as in Example 2

[0178] The test solution and control solution were prepared in the same manner as the test solution a in Example 2. 1 and control solution b 1 .

[0179] The test solution and the control solution were placed at room temperature for 48 hours, and samples were injected at 5 random time points within 0 to 48 hours to examine the stability of the solution.

[0180] Take 10 μL of each solution for sample analysis and record the peak area of ​​each peak in the chromatogram.

[0181] Table 3. Control solution stability results

[0182] Time Brivaracetam peak area in control solution 0h 0.815 9h 0.828 17h 0.835 26h 0.843 48h 0.820 average value% 0.828 RSD 1.4％

[0183] Table 4. Stability results of test solution

[0184] Time (h) Impurity D Impurity E Impurity F Other unknown impurities Total Miscellaneous 0h Not detected Not detected 0.03％ Not detected 0.03％ 9h Not detected Not detected 0.04％ Not detected 0.04％ 17h Not detected Not detected 0.03％ Not detected 0.03％ 26h Not detected Not detected 0.03％ Not detected 0.03％ 48h Not detected Not detected 0.03％ Not detected 0.03％ Maximum Absolute Difference NA NA 0.01％ NA 0.01％

[0185] Conclusion: The results in the above table show that the test solution was placed at room temperature for 48 hours, and the impurity content did not change significantly over time, and the absolute difference in impurity content was no more than 0.01%. The control solution was placed at room temperature for 48 hours, and the RSD value of the brivaracetam peak area was 1.4%, which was less than 5.0%. This shows that the brivaracetam test solution and the control solution were stable within 48 hours at room temperature.

[0186] Example 5 Linearity Test

[0187] The chromatographic conditions were the same as in Example 2.

[0188] The preparation of impurity D, E, F stock solutions is the same as in Example 3.

[0189] Brivaracetam stock solution: Weigh 12.58 mg of brivaracetam reference substance into a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well, and obtain;

[0190] 25% linear test: Accurately measure 1 ml of brivaracetam stock solution, 1 ml of impurity D and E stock solutions, and 2.5 ml of impurity F stock solution into the same 100 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain;

[0191] 50% linearity test: accurately measure 2 ml of brivaracetam stock solution, 2 ml of impurity D and E stock solutions, and 5 ml of impurity F stock solution into the same 100 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain;

[0192] 100% linearity test: accurately measure 4 ml of brivaracetam stock solution, 4 ml of impurity D and E stock solutions, and 10 ml of impurity F stock solution into the same 100 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain;

[0193] 150% linearity test: accurately measure 6 ml of brivaracetam stock solution, 6 ml of impurity D and E stock solutions, and 15 ml of impurity F stock solution into the same 100 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain;

[0194] 200% linearity test: accurately measure 8 ml of brivaracetam stock solution, 8 ml of impurity D and E stock solutions, and 20 ml of impurity F stock solution into the same 100 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain;

[0195] Take 10 μL of each solution for sample analysis and record the peak area in the chromatogram to obtain the data results and Figures 19 to 22 .

[0196] Table 5. Brivaracetam linearity test results

[0197]

[0198] Table 6. Impurity D linearity test results

[0199]

[0200] Table 7. Impurity E linearity test results

[0201]

[0202] Table 8. Impurity F linearity test results

[0203]

[0204] Conclusion: By analyzing the data in Tables 5 to 8 and Figures 19 to 22 From the linear equation shown, it can be seen that brivaracetam has a good linear relationship with the peak area in the concentration range of 0.252 μg / ml to 10.064 μg / ml, and the linear equation is y=0.171x+0.004, r=1.000; impurity D has a good linear relationship with the peak area in the concentration range of 0.261 μg / ml to 20.848 μg / ml, and the linear equation is y=0.153x-0.014, r=1.000, and the correction factor (f=k 主 / k 杂, the same below) is 1.1, no correction is required; impurity E has a good linear relationship with the peak area in the concentration range of 0.253μg / ml to 20.224μg / ml, the linear equation is y=0.222x-0.009, r=1.000, and the correction factor relative to the main component of brivaracetam is 0.8; impurity F has a good linear relationship with the peak area in the concentration range of 0.500μg / ml to 20.000μg / ml, the linear equation is y=0.129x-0.012, r=1.000, and the correction factor relative to the main component of brivaracetam is 1.3.

[0205] Example 6 Limit of Quantitation and Limit of Detection Test

[0206] Chromatographic conditions are the same as in Example 2

[0207] Take the solution under Example 5, for example, 25% linear solution, and dilute it step by step. When the signal-to-noise ratio S / N is about 10, the concentration of the solution is the quantitative limit concentration, and when the signal-to-noise ratio S / N is about 3, the concentration of the solution is the detection limit concentration.

[0208] Take 10 μL of each solution for sample analysis and record the chromatogram.

[0209] Table 9. Quantitation limit results

[0210]

[0211] Table 10. Detection limit results

[0212]

[0213] Conclusion: The quantitative limit of impurity D is 0.2606μg / ml, 6 injections were made continuously, the peak area RSD (n=6) was 8.5% (<20%), and the S / N was greater than 10; the detection limit was 0.1303μg / ml, S / N=5. The quantitative limit of impurity E was 0.2528μg / ml, 6 injections were made continuously, the peak area RSD (n=6) was 7.8% (<20%), and the S / N was greater than 10; the detection limit was 0.1264μg / ml, S / N=5. The quantitative limit of impurity F was 0.5000μg / ml, 6 injections were made continuously, the peak area RSD (n=6) was 10.7% (<20%), and the S / N was greater than 10; the detection limit was 0.1250μg / ml, S / N=3.

[0214] Example 7 Durability Test

[0215] Chromatographic conditions are the same as in Example 2

[0216] 10 μL of the system suitability solution under the specificity test item of Example 2 was injected for analysis, and the chromatographic conditions were adjusted according to the following Table 11 to obtain the data results in Table 11:

[0217] Table 11. Durability test results

[0218]

[0219] Conclusion: As shown in the table above, the retention time of brivaracetam and impurities did not change significantly after fine-tuning the flow rate, column temperature and replacing the chromatographic column, the separation between the peaks was greater than 1.5, and the sensitivity solution S / N was greater than 10. It can be seen that this method has good durability for detecting brivaracetam-related substances.

[0220] Example 8 Determination of related substances in brivaracetam samples by HPLC

[0221] The chromatographic conditions were the same as in Example 2.

[0222] Randomly measure the content of impurities, such as impurity F, in the test sample.

[0223] 8.1 Solution preparation

[0224] Three batches of brivaracetam samples shown in Table 12 were taken to prepare test solution, control solution and sensitivity solution in the same manner as in Example 2.

[0225] 8.2. Sample injection analysis

[0226] Take one injection of each of the above solutions for analysis, and after testing the instrument response capability with the sensitivity solution, the data results shown in Table 12 below are obtained:

[0227] Table 12

[0228]

[0229] Conclusion: The content of single impurity F in the test sample does not exceed the limit (0.2%), and the three batches of samples in the instruction manual meet the quality standard requirements

[0230] Comparative Example 1

[0231] 1) Main technical parameters of HPLC:

[0232] Chromatographic column: Inertsil ODS-3250×4.6mm, 5μm;

[0233] Detection wavelength: UV-210nm

[0234] Flow rate: 1.0ml / min

[0235] Column temperature: 30°C

[0236] Injection volume: 10 μl

[0237] Mobile phase: It consists of mobile phase A and mobile phase B. Mobile phase A is a 0.1% by volume phosphoric acid aqueous solution, and mobile phase B is acetonitrile.

[0238] Gradient elution program:

[0239] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 30 40 60 35 40 60 36 70 30 50 70 30

[0240] 2) Solution preparation

[0241] Blank solution: acetonitrile;

[0242] Impurity D stock solution 1 : Weigh 10.48 mg of impurity D reference substance, place in a 50 ml volumetric flask, add acetonitrile to dissolve, dilute to scale, shake well, and obtain;

[0243] Impurity E stock solution 1 : Weigh 10.64 mg of impurity E reference substance, place in a 50 ml volumetric flask, add acetonitrile to dissolve, dilute to scale, shake well, and obtain;

[0244] Impurity F stock solution 1 : Take 100 mg of impurity F reference substance (whole bottle), dissolve it in a 50 ml volumetric flask with about 30 ml of acetonitrile, dilute to the mark with acetonitrile, shake well, and obtain;

[0245] Impurity D Location Solution D 1 :Accurately measure 500μl of impurity D stock solution d 1 Place in a 20ml volumetric flask, add acetonitrile to dilute to scale, shake well, and you have it.

[0246] Impurity E Location Solution E 1 :Accurately measure 500μl of impurity E stock solution e 1 Place in a 20ml volumetric flask, add acetonitrile to dilute to scale, shake well, and you have it.

[0247] Impurity F Location Solution F 1 :Accurately measure 1 ml of impurity F stock solution f 1 Place in a 20ml volumetric flask, add acetonitrile to dilute to the mark, shake well (solution ①); accurately measure 1ml of solution ① and place in a 20ml volumetric flask, add acetonitrile to dilute to the mark, shake well, and you have it;

[0248] Impurity mixed solution I: Accurately weigh 20.62 mg of brivaracetam reference substance and place it in a 20 ml volumetric flask, then accurately measure 500 μl of impurity D and E stock solutions and 1 ml of solution ① and place them in the volumetric flask, add acetonitrile solvent and dilute to the scale, shake well, and obtain;

[0249] The concentrations of the substances are as follows:

[0250] Brivaracetam: 20.62 mg / 20 ml = 1.031 mg / ml;

[0251] Impurity D: 10.48 mg / 50 / 20 ml*0.5*1000=5.24 μg / ml;

[0252] Impurity E: 10.64 mg / 50 / 20 ml*0.5*1000=5.32 μg / ml;

[0253] Impurity F: 100mg / 50 / 20 / 20ml*1*1000=5μg / ml;

[0254] 3) Sampling analysis

[0255] Take the blank solution, impurity D, E, F location solutions, and impurity mixed solution I and inject one injection each. Figures 23 to 27 HPLC profile shown.

[0256] Conclusion: If Figures 23 to 27 The HPLC chromatogram shown shows that when the chromatographic conditions in Comparative Example 1 were used to detect and separate the related substances in brivaracetam, impurities E and impurity F could not be effectively separated.

Claims

1. A method for detecting brivaracetam-related substances, wherein the brivaracetam-related substances are one or more of the following: Impurity D: (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butyric acid, the chemical structure is as follows: Impurity E: (S)-2-((R)-2,5-dioxo-3-propylpyrrolidin-1-yl)butanamide, the chemical structure is as follows: Impurity F: (S)-N-((S)-1-amino-1-oxobutane-2-yl)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide, the chemical structure is as follows: The detection is carried out by high-performance liquid chromatography, which comprises the following steps: 1) Prepare solution a. Prepare the reference solutions of impurities D, E, and F respectively; b. Prepare a reference substance mixture solution of brivaracetam and impurities D, E, and F; c. Prepare brivaracetam test solution; d. Prepare a control solution of the brivaracetam test solution; 2) Detection The positioning solutions of impurities D, E, and F in step a, the impurity mixed solution in step b, the test solution in step c, and the control solution in step d are respectively injected into a high performance liquid chromatograph for detection, and the chromatogram is recorded; Wherein, in step 2), the chromatographic conditions of the high performance liquid chromatograph include: Chromatographic column: Octadecylsilane bonded silica gel is used as the filling column. Mobile phase: It consists of mobile phase A and mobile phase B. The mobile phase A is a phosphoric acid aqueous solution, and the mobile phase B is acetonitrile, with gradient elution. 2 . The detection method according to claim 1 , wherein the mobile phase A is a 0.1% by volume phosphoric acid aqueous solution.

3. The detection method according to claim 1, wherein: The gradient elution conditions are as follows:

4. The detection method according to any one of claims 1 to 3, wherein other chromatographic conditions further include: The detection wavelength is 210nm; The column model is Welch LP-C18 250 mm × 4.6 mm, 5 μm; The flow rate is 0.9-1.1 mL / min, preferably 1.0 mL / min; The column temperature is 27-33°C, preferably 30°C; The injection volume was 10 μL; The detector is a UV detector.

5. According to the detection method of claim 4, the preparation method of the localization solution of impurities D, E, and F in step a is: weigh the impurity D, E, and F reference substances respectively, dissolve them in acetonitrile to obtain the localization solution of each impurity; optionally, the concentration of the localization solution of each impurity can be 0.01 mg / ml.

6. The detection method according to claim 4, wherein the preparation method of the mixture solution in step b is: weighing brivaracetam, impurity D, E, and F reference substances, dissolving them in the same volumetric flask with acetonitrile to make up the volume, and obtaining an impurity mixed solution; optionally, in the mixture solution, the concentration of brivaracetam can be 5 mg / ml, and the concentration of each impurity can be 0.01 mg / ml.

7. The detection method according to claim 4, wherein the preparation method of the brivaracetam test solution in step c is: weighing the brivaracetam test sample, dissolving it in acetonitrile to a fixed volume, filtering, and taking the filtrate to obtain a test solution; optionally, the concentration of the brivaracetam test sample is 5 mg / ml; The brivaracetam test sample is selected from brivaracetam bulk drug or brivaracetam preparation, and the brivaracetam preparation includes but is not limited to brivaracetam tablets, brivaracetam injection or brivaracetam oral solution; preferably, the brivaracetam test sample is powder after pulverizing brivaracetam tablets.

8. The detection method according to claim 4, wherein the preparation method of the control solution in step d is: take 100 μL of the test solution in step c and place it in a 100 mL volumetric flask, add acetonitrile to the volume and shake well to obtain a control solution.

9. The detection method according to claim 4, further comprising: 3) Analysis Determine the retention times of impurities D, E, and F based on the chromatograms of each impurity location solution; According to the chromatograms of the test solution and the control solution, calculate the content of impurities D, E, and F in the test sample.

10. The detection method according to claim 9, in step 3), the content calculation formula of impurities D, E and F in the test sample is:

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