Method for detecting related substances of briracetam oral liquid

Abstract

The invention discloses a method for detecting related substances of a briracetam oral liquid, and belongs to the technical field of related substance detection. According to the method for detecting the related substances of the briracetam oral liquid, the related substances are detected by adopting a high performance liquid chromatography. According to the detection method, the separation degree between the main peak and each impurity meets the requirement, the detection amount of each impurity meets the requirement, the system is good in applicability, high in specificity and high in sensitivity, the problems that part of impurities cannot be detected and the durability and the detection accuracy are poor in the detection of the related substances of the briracetam oral solution in the prior art are solved, and the detection method is suitable for large-scale popularization and application. The method can effectively monitor the impurity condition in the Briracetam oral solution and ensure the product quality of the oral solution.

Description

Technical Field

[0001] The present application relates to a method for detecting related substances in brivaracetam oral solution, and belongs to the technical field of related substance detection. Background Art

[0002] Brivaracetam oral solution, marketed as Briviact, is an anti-epileptic drug used primarily to treat partial-onset seizures (with or without secondarily generalized tonic-clonic seizures) in adults and adolescents 16 years of age and older. It contains the active ingredient brivaracetam, which is a new anti-epileptic drug from the acetamide class.

[0003] The detection of related substances in brivaracetam oral solution is mainly carried out by high performance liquid chromatography. However, the existing detection of related substances in brivaracetam oral solution has the problems of failure to detect some impurities, poor durability and detection accuracy, and cannot effectively monitor the impurities in brivaracetam oral solution and ensure the product quality of the oral solution. Summary of the invention

[0004] According to one aspect of the present application, a method for detecting related substances of brivaracetam oral solution is provided. The separation degree between the main peak and each impurity of the detection method meets the requirements, the detection amount of each impurity meets the requirements, the system has good applicability, strong specificity and high sensitivity, and solves the problems of failure to detect some impurities, poor durability and detection accuracy in the detection of related substances of brivaracetam oral solution in the prior art, and can effectively monitor the impurity situation in brivaracetam oral solution and ensure the product quality of the oral solution.

[0005] The method for detecting related substances in the brivaracetam oral solution described in the present application uses high performance liquid chromatography to detect the related substances;

[0006] Wherein, mobile phase A is phosphoric acid aqueous solution;

[0007] Mobile phase B was acetonitrile;

[0008] The related substances are p-hydroxybenzoic acid, p-hydroxybenzoic acid 1-glyceride, p-hydroxybenzoic acid 2-glyceride, impurity E, and impurity E isomers;

[0009] The impurity E is (2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butyric acid;

[0010] The impurity E isomer is (S)-2-[(S)-2-oxo-4-propyltetrahydro-1-pyrrolidinyl]butyric acid.

[0011] The structure of impurity E described in this application is as follows:

[0012]

[0013] The structure of the impurity E isomer described in this application is as follows:

[0014]

[0015] Optionally, the concentration of the phosphoric acid aqueous solution is 0.085-0.115 wt %; preferably, the concentration of the phosphoric acid aqueous solution is 0.095-0.105 wt %.

[0016] Optionally, the concentration of the aqueous phosphoric acid solution is independently selected from any value of 0.085wt%, 0.09wt%, 0.095wt%, 0.1wt%, 0.105wt%, 0.1wt%, 0.115wt% or a range between any two of the above values.

[0017] Optionally, the column length of the chromatographic column is 150-250 mm; preferably, the column length of the chromatographic column is 250 mm.

[0018] Optionally, the inner diameter of the chromatographic column is 3.0-4.6 mm; preferably, the inner diameter of the chromatographic column is 4.6 mm.

[0019] Optionally, the particle size of the chromatographic column is 2.5-5 μm; preferably, the particle size of the chromatographic column is 5 μm.

[0020] Optionally, the filler of the chromatographic column is octadecylsilane bonded silica gel.

[0021] Optionally, the chromatographic column is selected from any one of Agilent Zorbax Rx-C18, Venusil MP C18, HuaXue-Bio C18, HamiltonPRP-C18, and Waters XBridge C18.

[0022] Optionally, the elution gradient is as follows:

[0023] 0min, mobile phase A 90vol%, mobile phase B 10vol%;

[0024] 18 min, mobile phase A 90 vol%, mobile phase B 10 vol%;

[0025] 30 min, mobile phase A 35 vol%, mobile phase B 65 vol%;

[0026] 40 min, mobile phase A 35 vol%, mobile phase B 65 vol%;

[0027] 40.1 min, mobile phase A 90 vol%, mobile phase B 10 vol%;

[0028] 50 min, mobile phase A 90 vol%, mobile phase B 10 vol%.

[0029] Optionally, the detection wavelength is 200-220 nm; preferably, the detection wavelength is 210 nm.

[0030] Optionally, the column temperature is 35-45°C.

[0031] Optionally, the injection volume is 5-15 μl.

[0032] Specifically, the injection volume was 10 μl.

[0033] Optionally, before detection, the brivaracetam test sample is prepared into a test solution with a brivaracetam concentration of 1 mg / ml.

[0034] Optionally, the flow rate of the mobile phase is 0.8-1.2 ml / min; preferably, the flow rate of the mobile phase is 0.9-1.1 ml / min.

[0035] The beneficial effects of this application include:

[0036] 1. The detection method of related substances in brivaracetam oral solution of the present application has the following requirements: the separation degree between the main peak and each impurity meets the requirements; the detection amount of each impurity meets the requirements; and the system has good applicability.

[0037] 2. The method of this application can effectively detect related substances in brivaracetam oral solution with strong specificity and high sensitivity.

[0038] 3. The detection method of related substances of brivaracetam oral solution of the present application has strong durability and high detection accuracy.

[0039] 4. The detection method of related substances of brivaracetam oral solution of the present application solves the problems in the prior art that some impurities in brivaracetam oral solution cannot be detected, and the durability and poor detection accuracy can effectively monitor the impurities in brivaracetam oral solution and ensure the product quality of the oral solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 HPLC spectrum of the mixed solution of Screening Example 1;

[0041] Figure 2 HPLC spectrum of the mixed solution of Screening Example 2;

[0042] Figure 3 The HPLC spectrum of the mixed solution of Screening Example 3 is shown below;

[0043] Figure 4 HPLC spectrum of the system solution of Screening Example 4;

[0044] Figure 5 HPLC spectrum of the blank solution of Screening Example 4;

[0045] Figure 6 This is the HPLC spectrum of the test sample of Screening Example 5;

[0046] Figure 7 HPLC spectrum of the system solution of Screening Example 6;

[0047] Figure 8 This is the HPLC spectrum of the test sample in Screening Example 6. DETAILED DESCRIPTION

[0048] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0049] Unless otherwise specified, the raw materials in the examples of this application were purchased through commercial channels.

[0050] The test products are as follows:

[0051] name Dosage (mg / ml) Brivaracetam 10mg / ml glycerin 152mg / ml Methylparaben 1mg / ml Sodium Citrate 2.94mg / ml Anhydrous citric acid 0.895mg / ml Sodium carboxymethylcellulose 5mg / ml Sucralose 40mg / ml Non-crystallized sorbitol solution 168mg / ml (as sorbitol) Raspberry flavor 5.6mg / ml Purified water Add to 1ml

[0052] The impurity calculation formula is as follows:

[0053]

[0054] Note: A 单杂 : The peak area of ​​a single impurity in the chromatogram of the test solution;

[0055] A 对照 : Main peak area in the chromatogram of the reference solution;

[0056] A 总峰 : Total peak area in the chromatogram of the test solution;

[0057] A 主峰 : Main peak area in the chromatogram of the test solution

[0058] Impurity A: (R)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide; the structure is as follows:

[0059]

[0060] Impurity B: (S)-2-((S)-2-oxo-4-propylpyrrolidin-1-yl)butanamide; the structure is as follows:

[0061]

[0062] Impurity C: (R)-2-((S)-2-oxo-4-propylpyrrolidin-1-yl)butanamide; the structure is as follows:

[0063]

[0064] Impurity D: (S)-N-((S)-1-amino-1-oxobutan-2-yl)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide, the structure is as follows:

[0065]

[0066] Screening Example 1

[0067] The chromatographic conditions were as follows:

[0068]

[0069]

[0070] Figure 1 For the HPLC spectrogram of the mixed solution of Screening Example 1, Figure 1 It can be seen that under this method, brivaracetam + impurity C, impurity A + B, methylparaben, impurity D, and impurity E appear in sequence, the separation degree of impurity A + B and methylparaben is less than 1.5, and the distance between impurity D and methylparaben is relatively close. The method has poor durability and sample detection accuracy.

[0071] Screening Example 2

[0072] The chromatographic conditions are the same as those in Comparative Example 1, except that the elution gradient is different, and the elution gradient is as follows:

[0073]

[0074] Figure 2 This is the HPLC spectrum of the mixed solution of Screening Example 2.

[0075] Figure 2 The peak table is as follows:

[0076] Detector A 205nm

[0077] Peak Compound Name RT RRT area area% high Theoretical plates Tailing Factor Separation 1 Brivaracetam + Impurity C 10.516 1.00 13358861 69.00 1604550 33885 1.288 2 Impurity A + Impurity B 10.939 1.04 40635 0.21 8335 10273 -- 1.281 3 Methylparaben 11.144 1.06 5896446 30.46 719165 39115 1.109 0.623 4 Impurity D 11.787 1.12 33205 0.17 5106 65874 1.150 3.145 5 Impurity E 13.829 1.32 31702 0.16 4818 88939 1.120 11.062 total 19360849 100.00 2341974

[0078] from Figure 2 As can be seen from the test results of the peak table, the separation degree between methylparaben and impurity D is increased, and the separation degree between impurity A+B and methylparaben is less than 1.5.

[0079] Screening Example 3

[0080] The chromatographic conditions were the same as those in Comparative Example 1, except that the chromatographic column was Ultimate XB-C8 4.6*250*5μm, and the elution gradient was as follows:

[0081]

[0082] Figure 3 This is the HPLC spectrum of the mixed solution of Screening Example 3.

[0083] Figure 3 The peak table is as follows:

[0084] Detector A205nm

[0085] Peak RT area area% high Theoretical plates Tailing Factor Separation 1 15.234 13980004 69.21 1068866 34387 0.908 2 16.014 6156302 30.48 385878 27946 0.824 2.191 3 18.686 34020 0.17 2721 65261 0.739 7.907 4 23.585 30484 0.15 2121 64502 0.751 14.754 total 20200810 100.00 1459586

[0086] from Figure 3 From the test results of the peak table, it can be seen that after replacing the chromatographic column (increasing the column length and increasing the pore size) on the basis of adjusting the gradient, impurities A+B are included with the brivaracetam peak or the methylparaben peak, and the separation degree of the brivaracetam peak and the methylparaben peak meets the requirements, and all chromatographic peak fronts using this chromatographic column.

[0087] Screening Example 4

[0088] The chromatographic conditions were the same as those in Comparative Example 1, except that the chromatographic column was Agilent ZORBAX SB-C8 4.6*150*5 μm, and the elution gradient was as follows:

[0089]

[0090]

[0091] Figure 4 This is the HPLC spectrum of the system solution of Screening Example 4.

[0092] Figure 4 The system solution test peak table is as follows:

[0093] Detector A205nm

[0094] Peak Compound Name RT RRT area area% high Theoretical plates Tailing Factor Separation 1 Brivaracetam + Impurity C 10.733 1.00 13694935 99.06 1683274 37018 1.297 2 Impurity A+B 11.148 1.04 59021 0.43 9313 59539 1.197 2.043 3 Impurity D 11.990 1.12 36392 0.26 5605 69560 1.210 4.621 4 Impurity E 14.041 1.31 34208 0.25 5206 93262 1.173 11.215 total 13824556 100.00 1703398

[0095] Figure 5 This is the HPLC spectra of the blank solution of Screening Example 4.

[0096] from Figure 4 and Figure 5 It can be seen that after replacing (increasing the pore size) the chromatographic column on the basis of adjusting the gradient, the separation degree between the brivaracetam peak and the methylparaben peak meets the requirements, the peak shape is good, the impurity A+B peak is included in the methylparaben peak, and the air-assisted interference impurity D detection.

[0097] Screening Example 5

[0098] The chromatographic conditions were the same as those of Comparative Example 1, except that the chromatographic column was Waters XBridge C18 (250 mm×4.6 mm, 5 μm), and the elution gradient was as follows:

[0099]

[0100] Figure 6 This is the HPLC spectrum of the test sample in Screening Example 5.

[0101] Figure 6 The peak table is as follows:

[0102] Detector A205nm

[0103] Peak RT RRT area area% high Theoretical plates Tailing Factor Separation 1 5.332 0.36 78001 0.37 7454 7555 0.732 2 12.202 0.82 7034 0.03 527 20024 0.847 23.277 3 14.800 1.00 20867445 98.89 1385219 39777 0.726 8.098 4 15.293 1.03 143343 0.68 14167 45934 1.692 5 16.353 1.10 5606 0.03 432 35616 0.999 3.354 total 21101429 100.00 1407800

[0104] from Figure 6 It can be seen that after changing the chromatographic column based on the adjustment of the gradient, the brivaracetam peak is included with the methylparaben peak, and the air-assisted interference detection.

[0105] Screening Example 6

[0106] The chromatographic conditions were as follows:

[0107]

[0108] Figure 7 This is the HPLC spectrum of the system solution of Screening Example 6.

[0109] Figure 7 The peak table of the system solution is as follows:

[0110] Detector A210nm

[0111] Peak Compound Name RT RRT area area% high Theoretical plates Tailing Factor Separation 1 2-Glyceryl p-hydroxybenzoate 10.449 0.33 9317 0.06 433 5615 0.985 2 Parabens 12.168 0.39 269897 1.65 14415 9757 1.065 3.273 3 1-Glyceryl p-hydroxybenzoate 15.243 0.49 273016 1.67 10060 7285 0.997 5.095 4 Brivaracetam + Impurity B 31.260 1.00 15743493 96.07 2042411 382978 1.004 34.954 5 Impurity D 31.908 1.02 43426 0.26 7271 551181 0.968 3.469 6 Impurity E 33.023 1.06 48608 0.30 6572 466805 1.003 6.102 total 16387758 100.00 2081163

[0112] Figure 8 This is the HPLC spectrum of the test sample in Screening Example 6.

[0113] Figure 8 The peak table of the test product is as follows:

[0114] Detector A210nm

[0115] Peak RT RRT area area% high Theoretical plates Tailing Factor Separation 1 12.263 0.40 5998 0.12 386 14321 1.048 2 15.422 0.50 3581 0.07 186 14817 0.994 6.893 3 30.690 1.00 5058653 99.81 750115 422546 1.034 43.896 total 5068233 100.00 750688

[0116] from Figure 7 and Figure 8 From the test results, it can be seen that the chromatographic peaks of the system suitability solution have good peak shapes, and the air-assisted method does not interfere with the detection of related substances, so it is determined as the final detection method.

[0117] Methodological validation:

[0118] 1. System adaptability:

[0119] (1) Related substances 1

[0120] Solution preparation:

[0121] Solvent: Accurately measure 100 ml of acetonitrile, add it to 900 ml of water, and mix well.

[0122] Impurity stock solution: Accurately weigh 1 mg of impurities E and E isomer, place them in different 10 ml volumetric bottles, add solvent to dissolve them and dilute to the scale, shake well, and use them as impurity stock solution.

[0123] System suitability solution: Accurately weigh about 20 mg of brivaracetam reference substance, place in a 10 ml volumetric flask, add an appropriate amount of solvent, shake thoroughly to dissolve it completely, add 1 ml each of impurities E and E isomer, dilute to the scale with solvent, and shake well.

[0124] Test solution: Accurately measure 1 ml of the product, place it in a 10 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0125] Blank auxiliary material: Accurately measure 1 ml of blank auxiliary material, place it in a 10 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0126] 0.2% control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the scale with solvent, and shake well; accurately measure 2 ml of the above solution, place it in a 10 ml volumetric flask, add solvent to dilute it to the scale, and shake well as the control solution.

[0127] Sensitivity solution: Accurately measure 5 ml of the control solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0128] 10 μl of each of the above solutions was measured and injected into the liquid chromatograph.

[0129] The system suitability results are as follows:

[0130]

[0131] The test results of the test solution are as follows:

[0132]

[0133] From the test results, it can be seen that the blank excipient does not interfere with the test sample detection; the separation between the main peak and each impurity in the system applicability solution meets the requirements, and other chromatographic parameters meet the requirements; the signal-to-noise ratio of the sensitivity solution is greater than 10; the detection amounts of impurity E and impurity E isomers in the related substances in the test sample solution meet the requirements; the method has good system applicability.

[0134] (2) Related substances 2

[0135] Solution preparation:

[0136] Solvent: Accurately measure 100 ml of acetonitrile, add it to 900 ml of water, and mix well.

[0137] Impurity stock solution: Accurately weigh 1 mg each of the impurities p-hydroxybenzoic acid, p-hydroxybenzoic acid 1-glyceride, and p-hydroxybenzoic acid 2-glyceride, and place them in different 50ml volumetric flasks, add solvent to dissolve them and dilute to the scale, shake well, and use them as impurity stock solution.

[0138] Impurity localization solution: Accurately measure 1 ml of each of the above impurity stock solutions, place them in different 5 ml volumetric bottles, add solvent to dilute to the scale, and shake well.

[0139] System suitability solution: Accurately weigh about 100 mg of methylparaben reference substance, place in a 10 ml volumetric flask, add an appropriate amount of solvent and shake thoroughly to dissolve it completely, add 1 ml each of the impurity p-hydroxybenzoic acid, p-hydroxybenzoic acid 1-glyceride and p-hydroxybenzoic acid 2-glyceride stock solutions, add solvent to dilute to the scale, and shake well.

[0140] Test solution: Use a pipette to accurately measure 1 ml of the product, place it in a 10 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0141] Blank auxiliary material: Use a pipette to accurately measure 1 ml of blank auxiliary material, place it in a 10 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0142] 10 μl of each of the above solutions was measured and injected into the liquid chromatograph.

[0143] The system suitability results are as follows:

[0144]

[0145] System Suitability - Test Solution, test results are as follows:

[0146]

[0147] From the above results, it can be seen that the separation between methylparaben and various impurities in the system suitability solution meets the requirements, and other chromatographic parameters meet the requirements. The system applicability of this method is good.

[0148] 2. Exclusivity

[0149] Solution preparation:

[0150] Undegraded: Accurately measure 1 ml of this product, place it in a 10 ml volumetric flask, add solvent to dilute to the scale, shake well, and use it as the test solution; Blank excipient: Accurately measure 1 ml of blank excipient, place it in a 10 ml volumetric flask, add solvent to dilute to the scale, shake well.

[0151] Light degradation for 24 hours: Measure 20 ml of the product and put it into a volumetric bottle. After placing it under 5000 Lux light conditions for 24 hours, accurately measure 1 ml of the product and put it into a 10 ml volumetric bottle. Add solvent to dilute to the scale and shake well. Prepare the light blank auxiliary material solution in the same way.

[0152] High temperature degradation for 24 hours: Measure 20 ml of this product, put it into a volumetric bottle, put it in an 80℃ oven, put it in an oven for 24 hours, take it out, cool it to room temperature, accurately measure 1 ml of this product, put it into a 10 ml volumetric bottle, add dissolution and dilute to the scale, shake well, and you have it. Prepare high temperature blank auxiliary material solution in the same way.

[0153] Oxidative degradation for 16.5 h: accurately measure 1 ml of the product and place it in a 10 ml volumetric flask, add 2 ml of 30% hydrogen peroxide solution, shake thoroughly, leave for 16.5 h, add solvent to dilute to the scale, shake well, and obtain; prepare oxidative degradation blank auxiliary material solution in the same way.

[0154] Acid degradation for 4 hours: Measure 1 ml of this product, place it in a 10 ml volumetric flask, add 1 ml of 5 mol / L HCI solution, leave it at room temperature for 4 hours, add 1 ml of 5 mol / L sodium hydroxide solution to neutralize, dilute to the scale, and shake well. Prepare the acid degradation blank auxiliary material solution in the same way.

[0155] Alkali degradation for 1 hour: Take 1 ml of this product and place it in a 10 ml volumetric flask, add 1 ml of 1 mol / L sodium hydroxide solution; leave it at room temperature for 1 hour, add 1 ml of 1 mol / L HCl to neutralize the solution until it is neutral, dilute to the scale, and shake well. Prepare the blank excipient solution for alkaline degradation in the same way.

[0156] Accurately measure 10 μl of each of the above solutions and inject them into the liquid chromatograph.

[0157] The test results of relevant substance 1 are as follows:

[0158]

[0159] peak

[0160] The test results of relevant substance 2 are as follows:

[0161]

[0162]

[0163] The test results show that: under each degradation condition, the blank excipients do not interfere with the impurity detection; under each degradation condition, the separation between the main peak and the adjacent impurities is greater than 1.5, and the purity index of the minimum peak of the main component is ≥0; under each degradation condition, the material conservation is within the range of 90%-110%. In summary, this method detects related substances 1 and related substances 2 of this product, and the specificity meets the requirements.

[0164] 3. Durability

[0165] The parameters of the chromatographic conditions are as follows:

[0166] Chromatographic parameters Specified value Changing parameters Column temperature 40℃ ±5℃ Flow rate 1.0ml / min ±0.1ml / min Phosphoric acid addition amount 0.1% Phosphoric acid ±0.005％ Chromatographic columns WatersXBridgeC18; Different batches of columns instrument Shimadzu 20-AT Agilent

[0167] Solution preparation:

[0168] Diluent: acetonitrile-water (10:90) vol%

[0169] Impurity stock solution: Accurately weigh 1 mg each of impurities A, B, C, D, E, p-hydroxybenzoic acid, p-hydroxybenzoic acid 1-glyceride, and p-hydroxybenzoic acid 2-glyceride, and place them in different 25 ml volumetric flasks, add solvent to dissolve them and dilute to the scale, shake well, and use them as impurity stock solution.

[0170] System suitability solution: Accurately weigh about 20 mg of brivaracetam reference substance, place in a 10 ml volumetric flask, add an appropriate amount of solvent, and shake thoroughly to completely dissolve;

[0171] Add 1 ml of impurity B, D, and E stock solutions, dilute to the mark with solvent, and shake well; or add 1 ml of impurity B, p-hydroxybenzoic acid, p-hydroxybenzoic acid 1-glyceride, and p-hydroxybenzoic acid 2-glyceride, dilute to the mark with solvent, and shake well;

[0172] Test solution: Accurately measure 1 ml of the product, place it in a 10 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0173] Blank auxiliary material: Accurately measure 1 ml of blank auxiliary material, place it in a 10 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0174] 0.2% control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the scale with solvent, and shake well; Accurately measure 2 ml of the above solution, place it in a 10 ml volumetric flask, add solvent to dilute it to the scale, and shake well as the control solution.

[0175] Blank: solvent.

[0176] For substance 1, the system suitability solution separation test results are as follows:

[0177]

[0178]

[0179] For substance 2, the results of the system suitability solution separation test are as follows:

[0180] For substance 1, the system suitability solution retention time / min results are as follows:

[0181]

[0182] For substance 2, the system suitability solution retention time / min results are as follows:

[0183] For substance 1, the test results of theoretical plate number of system suitability solution are as follows:

[0184] For substance 2, the results of the theoretical plate number test of the system suitability solution are as follows:

[0185]

[0186]

[0187] For substance 1, the system suitability solution tailing factor test results are as follows:

[0188]

[0189] For substance 2, the system suitability solution tailing factor test results are as follows:

[0190] Related substances 1. Test results of related substances in the test solution are as follows:

[0191] Related substance 2, the test results of related substances in the test solution are as follows:

[0192]

[0193] From the above results, it can be seen that when there are slight changes in the flow rate (0.9ml / min~1.1ml / min), column temperature (35℃~45℃), and the amount of phosphoric acid added to the mobile phase (0.095%~0.105%), as well as when changing to different batches of chromatographic columns from the same brand or changing to different instruments (Agilent 1260), the test results of the test samples do not change significantly compared with the standard conditions, indicating that this method has good durability.

[0194] 4. Sensitivity

[0195] Brivaracetam stock solution: Accurately weigh 2 mg of brivaracetam reference substance, place in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well; prepare impurity E and impurity E isomer stock solutions in the same way.

[0196] Methylparaben mother solution: Accurately weigh 2 mg of methylparaben reference substance, place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well; prepare p-hydroxybenzoic acid, p-hydroxybenzoic acid 1-glyceride, and p-hydroxybenzoic acid 2-glyceride mother solutions in the same way.

[0197] Methylparaben stock solution: Accurately measure 1 ml of methylparaben mother solution, place it in a 10 ml volumetric flask, add solvent to dilute to the scale and shake well; prepare p-hydroxybenzoic acid, p-hydroxybenzoic acid 1-glyceride, and p-hydroxybenzoic acid 2-glyceride stock solutions in the same way.

[0198] Take an appropriate amount of each stock solution, quantitatively dilute it to make a solution with a signal-to-noise ratio of about 10 to 30 as the quantitative limit solution; dilute it to make a solution with a signal-to-noise ratio of about 3 to 8 as the detection limit solution.

[0199] Accurately measure 10 μl of each of the above solutions and inject them into the liquid chromatograph.

[0200] The test results of relevant substance 1 are as follows:

[0201]

[0202] From the test results, it can be seen that the quantitative limit concentration percentages of brivaracetam and each known impurity are all less than the reporting limit concentration (0.05%), which meets the requirements. This method has good sensitivity and the test sample concentration of 1 mg / ml is suitable.

[0203] The test results of relevant substance 2 are as follows:

[0204]

[0205] From the test results, we can see that the quantitative limit concentration percentages of methylparaben and each impurity are no more than the reporting limit concentration (0.05%), which meets the requirements. This method has good sensitivity and the test sample concentration of 0.1mgml (methylparaben) is suitable.

[0206] 5. Accuracy

[0207] Related substance 1 accuracy:

[0208] Solution preparation:

[0209] Blank auxiliary material: Use a volumetric pipette to accurately measure 1 ml of blank auxiliary material, place it in a 10 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0210] Impurity E stock solution: Accurately weigh 1 mg of impurity E reference substance, place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0211] Impurity E reference solution: Accurately measure 1 ml of the impurity E stock solution, place it in a 10 ml volumetric flask, add solvent to dilute to the mark, and shake well. Prepare 2 portions in parallel.

[0212] Test solution: Use a volumetric pipette to accurately measure 1 ml of sample solution, place it in a 10 ml volumetric flask, add solvent to dilute to the mark, and shake well. Prepare 2 portions in parallel.

[0213] (a) Low concentration recovery solution: Use a volumetric pipette to accurately measure 2 ml of the sample solution and place it in a 20 ml volumetric flask. Add 1 ml of the impurity E reference solution and dilute to the mark with solvent. Shake to obtain the solution. Prepare 3 copies in parallel.

[0214] (a) Medium concentration recovery solution: Use a volumetric pipette to accurately measure 1 ml of the sample solution and place it in a 10 ml volumetric flask. Add 1 ml of the impurity E stock solution and dilute to the mark with solvent. Shake well to obtain the solution. Prepare 3 portions in parallel.

[0215] (a) High concentration recovery solution: Use a volumetric pipette to accurately measure 1 ml of the sample solution and place it in a 10 ml volumetric flask. Add 1.5 ml of the impurity E stock solution and dilute to the mark with solvent. Shake well to obtain the solution. Prepare three portions in parallel.

[0216] Impurity E isomer stock solution: Accurately weigh 1 mg of the impurity E isomer reference substance, place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0217] Impurity E isomer reference solution: Accurately measure 1 ml of the impurity E isomer stock solution, place it in a 10 ml volumetric flask, add solvent to dilute to the mark, and shake well. Prepare 2 portions in parallel.

[0218] Test solution: Use a volumetric pipette to accurately measure 1 ml of the sample solution, place it in a 10 ml volumetric flask, add solvent to dilute to the mark, and shake well. Prepare 2 portions in parallel.

[0219] (b) Low concentration recovery solution: Use a volumetric pipette to accurately measure 2 ml of the sample solution and place it in a 20 ml volumetric flask. Add 1 ml of the impurity E isomer reference solution and dilute to the mark with solvent. Shake well to obtain the solution. Prepare 3 copies in parallel.

[0220] (b) Medium concentration recovery solution: Use a volumetric pipette to accurately measure 1 ml of the sample solution and place it in a 10 ml volumetric flask. Add 1 ml of the impurity E isomer stock solution and dilute to the mark with solvent. Shake well to obtain the solution. Prepare 3 portions in parallel.

[0221] (b) High concentration recovery solution: Use a volumetric pipette to accurately measure 1 ml of the sample solution and place it in a 10 ml volumetric flask. Add 1.5 ml of the impurity E isomer stock solution and dilute to the mark with solvent. Shake well to obtain the solution. Prepare 3 copies in parallel.

[0222] Accurately measure 10 μl of each of the above solutions and inject them into the liquid chromatograph respectively.

[0223] The recovery results of impurity E are as follows:

[0224]

[0225] The recovery results of impurity E isomer are as follows:

[0226]

[0227] From the above results, it can be seen that the recoveries of the known impurities within the reporting limit, 100%, and 150% concentration range are all within the range of 95-105%, which meets the requirements; the RSD values ​​are all less than 10.0%, which meets the requirements.

[0228] Related substance 2 accuracy:

[0229] Solution preparation:

[0230] Stock solution of 2-p-hydroxybenzoic acid glyceride: accurately weigh 2 mg of 2-p-hydroxybenzoic acid glyceride reference substance, place it in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well; prepare the stock solutions of p-hydroxybenzoic acid and 1-p-hydroxybenzoic acid glyceride in the same way as above.

[0231] Accuracy reference solution: Accurately measure 1 ml of each of the impurity p-hydroxybenzoic acid 2-glyceride, p-hydroxyformic acid, and p-hydroxybenzoic acid 1-glyceride stock solutions, place in a 10 ml volumetric flask, add solvent to dilute to the mark, and shake well. Prepare 2 portions in parallel.

[0232] Test solution: Use a volumetric pipette to accurately measure 1 ml of sample solution, place it in a 10 ml volumetric flask, add solvent to dilute to the mark, and shake well. Prepare 2 portions in parallel.

[0233] Report limit mother solution: accurately measure 1 ml of each of p-hydroxybenzoic acid 2-glyceride, p-hydroxybenzoic acid, and p-hydroxybenzoic acid 1-glyceride stock solutions, place in the same 100 ml volumetric flask, add solvent to dilute to the mark, and shake well. Prepare 2 portions in parallel.

[0234] (b) Low concentration recovery solution: Use a volumetric pipette to accurately measure 1 ml of the sample solution, place it in a 10 ml volumetric flask, add 2.5 ml of the reporting limit stock solution, add solvent to dilute to the scale, shake well, and prepare 3 copies in parallel.

[0235] (b) Medium concentration recovery solution: Use a volumetric pipette to accurately measure 1 ml of the sample solution, place it in a 10 ml volumetric flask, add 1 ml each of the impurity p-hydroxybenzoic acid 2-glyceride, p-hydroxybenzoic acid, and p-hydroxybenzoic acid 1-glyceride stock solutions, add solvent to dilute to the scale, shake well, and prepare 3 copies in parallel.

[0236] (b) High concentration recovery solution: Use a volumetric pipette to accurately measure 1 ml of the sample solution and place it in a 10 ml volumetric flask. Add 1.5 ml each of the impurity 2-hydroxybenzoic acid glyceride, p-hydroxybenzoic acid, and 1-hydroxybenzoic acid glyceride stock solutions. Add solvent to dilute to the scale and shake well. Prepare 3 copies in parallel.

[0237] Accurately measure 10 μl of each of the above solutions and inject them into the liquid chromatograph respectively.

[0238] The recovery results of 2-hydroxybenzoic acid glyceride are as follows:

[0239]

[0240] The recovery results of p-hydroxybenzoic acid are as follows:

[0241]

[0242]

[0243] The recovery results of 1-hydroxybenzoic acid glyceride are as follows:

[0244]

[0245] From the above results, it can be seen that the recoveries of the known impurities within the reporting limit, 100%, and 150% concentration ranges are all within the range of 95-105%, which meets the requirements; the RSD values ​​are all less than 10.0%, which meets the requirements.

[0246] The relevant substances of the test samples were detected by the chromatographic conditions finally screened out and the chromatographic conditions in Screening Example 1, and the results are as follows. Among them, the method of Screening Example 6 is calculated by the area normalization method with methylparaben as the main peak, and the remaining impurities are calculated by the self-control method with brivaracetam as the main peak. The calculation method of the method of Screening Example 1: All impurities are calculated by the self-control method with brivaracetam as the main peak.

[0247]

[0248] According to the test spectra and results before and after optimization, the impurities detected by the optimized method are more complete and accurate.

[0249] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A method for detecting related substances in brivaracetam oral solution, characterized in that: The relevant substances were tested by high performance liquid chromatography; Wherein, mobile phase A is phosphoric acid aqueous solution; Mobile phase B was acetonitrile; The related substances are p-hydroxybenzoic acid, p-hydroxybenzoic acid 1-glyceride, p-hydroxybenzoic acid 2-glyceride, impurity E, and impurity E isomers; The impurity E is (2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butyric acid; The impurity E isomer is (S)-2-[(S)-2-oxo-4-propyltetrahydro-1-pyrrolidinyl]butyric acid.

2. The method for detecting related substances in brivaracetam oral solution according to claim 1, characterized in that: The concentration of the phosphoric acid aqueous solution is 0.085-0.115 wt %.

3. The method for detecting related substances in brivaracetam oral solution according to claim 1, characterized in that: The column length of the chromatographic column is 150-250 mm; Preferably, the inner diameter of the chromatographic column is 3.0-4.6 mm; Preferably, the particle size of the chromatographic column is 2.5-5 μm.

4. The method for detecting related substances in brivaracetam oral solution according to claim 1, characterized in that: The filler of the chromatographic column is octadecylsilane bonded silica gel.

5. The method for detecting related substances in brivaracetam oral solution according to claim 1, characterized in that: The elution gradient is as follows: 0min, mobile phase A 90vol%, mobile phase B 10vol%; 18 min, mobile phase A 90 vol%, mobile phase B 10 vol%; 30 min, mobile phase A 35 vol%, mobile phase B 65 vol%; 40 min, mobile phase A 35 vol%, mobile phase B 65 vol%; 40.1min, mobile phase A 90vol%, mobile phase B 10vol%; 50min, mobile phase A 90vol%, mobile phase B 10vol%.

6. The method for detecting related substances in brivaracetam oral solution according to claim 1, characterized in that: The detection wavelength is 200-220nm.

7. The method for detecting related substances in brivaracetam oral solution according to claim 1, characterized in that: The column temperature was 35-45°C.

8. The method for detecting related substances in brivaracetam oral solution according to claim 1, characterized in that: The injection volume is 5-15μl.

9. The method for detecting related substances in brivaracetam oral solution according to claim 1, characterized in that: Before testing, the brivaracetam test sample was prepared into a test solution with a brivaracetam concentration of 1 mg / ml.

10. The method for detecting related substances in brivaracetam oral solution according to claim 1, characterized in that: The flow rate of the mobile phase was 0.8-1.2 ml / min.

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