Method for detecting related substances of brexpiprazole tablets

Abstract

The invention discloses a method for detecting related substances in a brexpiprazole tablet, and belongs to the field of medicine detection.The amount of the related substances in the brexpiprazole tablet is detected through high performance liquid chromatography, and chromatographic detection conditions of the high performance liquid chromatography are defined as follows: octadecylsilane chemically bonded silica is used as a filler; according to the present invention, by using a chromatographic column as a chromatographic column, using a 0.01 mol / L potassium dihydrogen phosphate-0. 05mol / L sodium perchlorate buffer solution as a mobile phase A and using an acetonitrile solution as a mobile phase B, gradient elution is performed, the amount of the related substances in the brexpiprazole tablet can be accurately detected, and the detection method has characteristics of high sensitivity and good separation degree.

Description

Technical Field

[0001] The invention relates to a method for detecting chemical substances, in particular to a method for detecting related substances of brepazine tablets, and belongs to the technical field of drug detection. Background Art

[0002] The indications of brepirazole tablets are to be used as an adjunct to antidepressants in the treatment of major depressive disorder (MDD) in adults and for the treatment of schizophrenia in patients aged 13 years and above. Brapirazole tablets are mainly prepared from the raw material brepirazole and excipients through tableting process. In the process of preparing and producing brepirazole tablets, related substances may be produced that affect the quality of the finished product. The finished product needs to be tested to control the quality of brepirazole tablets and ensure the production of high-quality products.

[0003] Currently, there is no good method to detect the amount of various impurities in the related substances in brepirazoline tablets, which may have a potential impact on the quality control of brepirazoline tablets. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for detecting related substances in brepirazole tablets, using high performance liquid chromatography to detect the amount of related substances in brepirazole tablets. The method of the present invention can accurately detect the amount of related substances in brepirazole tablets with high sensitivity and good separation.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A method for detecting related substances in brepirazoline tablets, using high performance liquid chromatography to detect the amount of related substances in brepirazoline tablets, wherein the related substances include impurity A, impurity C, impurity D, and impurity E;

[0007] The chemical name of impurity A is 7-hydroxy-2-quinolone;

[0008] The chemical name of impurity C is 4-(benzo[b]thiophen-4-yl)-1-(4-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)butyl)piperazine-1-oxide;

[0009] The chemical name of impurity D is 4-(4-(benzothiophen-4-yl)piperazin-1-yl)butan-1-ol;

[0010] The chemical name of impurity E is 8-(benzothiophen-4-yl)-5,8-diazaspiro[4.5]dec-5-ammonium bromide;

[0011] The chromatographic detection conditions include: using octadecylsilane bonded silica gel as a filler; using 0.01 mol / L potassium dihydrogen phosphate-0.05 mol / L sodium perchlorate buffer as a mobile phase A, and using acetonitrile solution as a mobile phase B, and performing gradient elution.

[0012] Further, the gradient elution program was: 0 min, 85% A, 15% B; 50 min, 30% A, 70% B; 50.1 min, 85% A, 15% B; 60 min, 85% A, 15% B.

[0013] Furthermore, the chromatographic detection conditions also include: a detection wavelength of 254 nm and a column temperature of 35°C.

[0014] Furthermore, the chromatographic detection conditions also include: a flow rate of 1.0 ml per minute and an injection volume of 10 μl.

[0015] Further, mobile phase A was prepared by taking potassium dihydrogen phosphate and sodium perchlorate, adding water to dissolve them, and adjusting the pH to 2.5 with phosphoric acid to prepare a 0.01 mol / L potassium dihydrogen phosphate-0.05 mol / L sodium perchlorate buffer.

[0016] Furthermore, the detection method comprises the following steps:

[0017] S1. Preparation of solvent: Mix water, acetonitrile, methanol and acetic acid to obtain a solvent;

[0018] S2. Preparation of the test solution: Take brepazole tablets, add a solvent and sonicate to dissolve brepazole, dilute with solvent to prepare a solution containing 0.5 mg of brepazole per 1 ml, centrifuge, and take the supernatant to obtain the test solution;

[0019] S3. Preparation of control solution: accurately measure the test solution and quantitatively dilute it with a solvent to prepare a solution containing 2.5 μg of brepazine per 1 ml as a control solution;

[0020] S4. Preparation of mixed reference solution: Take impurity A, impurity C, impurity D, impurity E reference substances, add solvent and dilute to prepare a solution containing 0.1 mg per 1 ml as impurity reference stock solution; take brepirazole and impurity reference stock solution respectively, dissolve in solvent and dilute to prepare mixed reference solution containing 0.5 mg of brepirazole and 5 μg of each impurity per 1 ml;

[0021] S5. The control solution, the mixed reference solution and the test solution are respectively detected by high performance liquid chromatography to obtain the chromatogram data of the control solution, the mixed reference solution and the test solution, and the amount of each impurity in the test sample is calculated based on the chromatogram data of the mixed reference solution and the test sample.

[0022] Furthermore, the peak areas of impurity A, impurity D, impurity E, and impurity C shall not be greater than the main peak area of ​​the control solution.

[0023] Further, in step S1, the volume ratio of water-acetonitrile-methanol-acetic acid is 50:40:10:1.

[0024] Furthermore, in step S5, the chromatogram data is calculated using the following calculation method:

[0025] Self-control + correction factor method: single impurity C = (A 样 ×f / A 自对 )×C 自对 ;

[0026] A 样 : The peak area of ​​each impurity in the chromatogram of the test solution;

[0027] f: correction factor for each impurity;

[0028] A 自对 : The main component peak area in the chromatogram of the test sample’s own reference solution;

[0029] C 自对 : The percentage concentration of the test sample itself as the control solution is 0.5%.

[0030] Further, the correction factor f includes the correction factors corresponding to the following impurities: the correction factor of impurity A is 1.2, the correction factor of impurity C is 1.3, the correction factor of impurity D is 1.1, and the correction factor of impurity E is 1.1.

[0031] Due to the adoption of the above technical solution, the technical progress achieved by the present invention is:

[0032] The method of the present invention can accurately detect the related substances of brepirazole tablets with high sensitivity and good separation. By limiting the detection conditions of the high performance liquid chromatograph, the related substances in the brepirazole tablets can be better separated, the amount of each impurity can be accurately detected, the corresponding chromatogram data can be obtained, the baseline is separated, and the separation is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0034] Figure 1 It is a test chromatogram of the test solution of the present invention;

[0035] Figure 2 It is a blank solution detection chromatogram of the present invention;

[0036] Figure 3 It is a chromatogram of the sensitivity solution detection of the present invention;

[0037] Figure 4 It is the chromatogram of the location detection of impurity A of the present invention;

[0038] Figure 5 It is the chromatogram of the location detection of impurity C of the present invention;

[0039] Figure 6 This is the chromatogram of the location detection of impurity D of the present invention;

[0040] Figure 7 This is the chromatogram of the location detection of impurity E of the present invention;

[0041] Figure 8 The present invention is a sample injection precision experiment reference solution detection chromatogram Figure 1 ;

[0042] Fig. 9 The present invention is a sample injection precision experiment reference solution detection chromatogram Figure 2 ;

[0043] Fig.10 The present invention is a sample injection precision experiment reference solution detection chromatogram Figure 3 ;

[0044] Fig.11 The present invention is a sample injection precision experiment reference solution detection chromatogram Figure 4 ;

[0045] Fig.12 The present invention is a sample injection precision experiment reference solution detection chromatogram Figure 5 ;

[0046] Fig.13 The present invention is a sample injection precision experiment reference solution detection chromatogram Figure 6 ;

[0047] In the accompanying drawings, the ordinate is mAU, and the abscissa is Time. DETAILED DESCRIPTION

[0048] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention and the related drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Below is a combination of the embodiments and Figure 1-13 The present invention is described in further detail:

[0050] A method for detecting related substances in brepirazoline tablets, using a high performance liquid chromatograph to detect related substances in brepirazoline tablets, wherein the related substances include impurity A, impurity C, impurity D, and impurity E;

[0051] The chemical name of impurity A is 7-hydroxy-2-quinolone, and its chemical structure is as follows:

[0052]

[0053] The chemical name of impurity C is 4-(benzo[b]thiophen-4-yl)-1-(4-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)butyl)piperazine-1-oxide, and the chemical structure is as follows:

[0054]

[0055] The chemical name of impurity D is 4-(4-(benzothiophene-4-yl)piperazine-1-yl)butan-1-ol, and the chemical structure is as follows:

[0056]

[0057] The chemical name of impurity E is 8-(benzothiophene-4-yl)-5,8-diazaspiro[4.5]decane-5-ammonium bromide, and the chemical structure is as follows:

[0058]

[0059] Chromatographic detection conditions: octadecylsilane bonded silica gel is used as filler (YMC-Pack ODS-AQ, 4.6×150mm, 3μm or chromatographic column with equivalent performance); 0.01mol / L potassium dihydrogen phosphate-0.05mol / L sodium perchlorate buffer is used as mobile phase A, acetonitrile solution is used as mobile phase B, gradient elution is performed, the detection wavelength is 254nm, the column temperature is 35°C, the flow rate is 1.0ml per minute, and the injection volume is 10μl.

[0060] Gradient elution program: 0 min, 85% A, 15% B; 50 min, 30% A, 70% B; 50.1 min, 85% A, 15% B; 60 min, 85% A, 15% B.

[0061] Preparation of 0.01 mol / L potassium dihydrogen phosphate-0.05 mol / L sodium perchlorate buffer: Take 2.72 g of potassium dihydrogen phosphate and 14.05 g of sodium perchlorate, add 2000 ml of water to dissolve, and adjust the pH to 2.5 with phosphoric acid.

[0062] The detection method includes the following steps:

[0063] S1. Preparation of solvent: water, acetonitrile, methanol and acetic acid are mixed uniformly to obtain a solvent, wherein the volume ratio of water-acetonitrile-methanol-acetic acid is 50:40:10:1;

[0064] S2. Preparation of the test solution: Take brepazole tablets, add a solvent and sonicate to dissolve brepazole, dilute with solvent to prepare a solution containing 0.5 mg of brepazole per 1 ml, centrifuge, and take the supernatant to obtain the test solution;

[0065] S3. Preparation of control solution: accurately measure the test solution and quantitatively dilute it with a solvent to prepare a solution containing 2.5 μg of brepazine per 1 ml as a control solution;

[0066] S4. Preparation of mixed reference solution: Take impurity A, impurity C, impurity D, and impurity E reference substances, add solvent and dilute to prepare a solution containing 0.1 mg per 1 ml as the impurity reference stock solution, take brepirazole and the impurity reference stock solution, respectively, dissolve and dilute with solvent to prepare a mixed reference solution containing 0.5 mg of brepirazole and 5 μg of each impurity per 1 ml;

[0067] S5. The control solution, the mixed reference solution and the test solution are respectively detected by high performance liquid chromatography to obtain the chromatogram data of the control solution, the mixed reference solution and the test solution, and the amount of each impurity in the test sample is calculated based on the chromatogram data of the mixed reference solution and the test sample.

[0068] The calculation formula used for chromatogram data calculation is:

[0069] Self-control + correction factor method: single impurity C (%) = (A 样 ×f / A 自对 )×C 自对

[0070] A 样 : The peak area of ​​each impurity in the chromatogram of the test solution;

[0071] f: correction factor for each impurity;

[0072] A 自对 : The main component peak area in the chromatogram of the test sample’s own reference solution;

[0073] C 自对 : The percentage concentration of the test sample's own control solution is 0.5%;

[0074] Total impurities Ctotal (%) = ΣC (%)

[0075] C (%): calculated value of each individual impurity content.

[0076] The correction factors corresponding to each impurity are: the correction factor of impurity A is 1.2, the correction factor of impurity C is 1.3, the correction factor of impurity D is 1.1, and the correction factor of impurity E is 1.1.

[0077] Limit: If there are impurity peaks in the test solution, the peak areas of impurities A, D, E and C shall not be greater than the main peak area of ​​the control solution (0.5%), the peak area of ​​other individual impurities shall not be greater than the main peak area of ​​the control solution (0.5%), and the sum of the peak areas of all impurities shall not be greater than 4 times the main peak area of ​​the control solution (2.0%).

[0078] Example 1

[0079] The specific test process of this embodiment 1 is as follows:

[0080] 1. Instruments, equipment, reagents and samples

[0081] 1.1 Instruments and Equipment

[0082] Table 1: Instrument information

[0083] Instruments and Equipment Device Model Equipment manufacturers High performance liquid chromatography U3000 Thermo Analytical Balance ME104E / 02, XS3DU Mettler Toledo Ultrasound KH7200B Kunshan Hechuang Ultrasonic Instrument Co., Ltd. Centrifuge TG16MW Hunan Hexi Instrument Equipment Co., Ltd.

[0084] 1.2 Reagents and consumables

[0085] Table 2: Reagent and consumables information

[0086] name level supplier Purified water First-class water self made Methanol solution HPLC Adamas-beta Acetonitrile solution HPLC Adamas-beta Acetic acid solution AR GENERAL-REAGENT

[0087] 1.3 Test and reference articles

[0088] Table 3: Test and control sample information

[0089]

[0090]

[0091] Note: N / A means “not applicable”.

[0092] 2. Testing conditions

[0093] Table 4: Instrument detection parameter information

[0094]

[0095] 3. Solution preparation

[0096] 3.1 Preparation of solvent: Put 500 ml of water, 400 ml of acetonitrile, 100 ml of methanol and 10 ml of acetic acid into a 2L conical flask, mix well, seal and set aside.

[0097] 3.2 Preparation of test solution: Take 10 brepazine tablets (specification 0.5 mg), add solvent and ultrasonically dissolve the brepazine tablets, dilute to 10 ml, and make a solution containing 0.5 mg of brepazine per 1 ml, centrifuge, take the supernatant, seal and set aside.

[0098] 3.3 Preparation of control solution: Accurately measure 1 ml of the test solution and quantitatively dilute it to 200 ml with solvent to prepare a solution containing 2.5 μg of brexpiprazole per 1 ml.

[0099] 3.4 Preparation of mixed reference solution: accurately weigh 100 mg of each impurity A, impurity C, impurity D and impurity E reference substance, dissolve in solvent, dilute to volume in a 1L conical flask, and prepare a solution containing 0.1 mg per 1 ml as the impurity reference stock solution; respectively take 10 mg of brepirazole and 1 ml of the impurity reference stock solution, continue to dissolve in solvent and dilute to 20 ml, to prepare a mixed reference solution containing 0.5 mg of brepirazole and 5 μg of each impurity per 1 ml.

[0100] 3.5 Preparation of mobile phase A (0.01 mol / L potassium dihydrogen phosphate-0.05 mol / L sodium perchlorate buffer): Take 2.72 g of potassium dihydrogen phosphate and 14.05 g of sodium perchlorate, add 2000 ml of water to dissolve, and adjust the pH to 2.5 with phosphoric acid.

[0101] 4. Test results and calculations

[0102] The control solution, mixed reference solution and test solution are respectively detected by high performance liquid chromatography to obtain chromatogram data of the control solution, mixed reference solution and test solution; the amount of each impurity in the test sample is calculated through the chromatogram data of the mixed reference sample and the test sample.

[0103] The calculation formula used for chromatogram data calculation is:

[0104] Self-control + correction factor method: single impurity C (%) = (A 样 ×f / A 自对 )×C 自对

[0105] A 样 : The peak area of ​​each impurity in the chromatogram of the test solution;

[0106] f: correction factor for each impurity;

[0107] A 自对 : The main component peak area in the chromatogram of the test sample’s own reference solution;

[0108] C 自对 : The percentage concentration of the test sample's own control solution is 0.5%;

[0109] Total impurities Ctotal (%) = ΣC (%)

[0110] C (%): calculated value of each individual impurity content.

[0111] The correction factors corresponding to each impurity are: the correction factor of impurity A is 1.2, the correction factor of impurity C is 1.3, the correction factor of impurity D is 1.1, and the correction factor of impurity E is 1.1.

[0112] Limit: If there are impurity peaks in the test solution, the peak areas of impurities A, D, E and C shall not be greater than the main peak area of ​​the control solution (0.5%), the peak area of ​​other individual impurities shall not be greater than the main peak area of ​​the control solution (0.5%), and the sum of the peak areas of all impurities shall not be greater than 4 times the main peak area of ​​the control solution (2.0%).

[0113] Results: In the test solution of breprazol tablets, the amounts of impurities detected were impurity A not detected, impurity C 0.02%, impurity D not detected, impurity E not detected, the largest single impurity was 0.04%, and the total impurity was 0.17%. Figure 1 Therefore, it can be seen that the relevant substances are controlled within the qualified range and the quality of brexpiprazole tablets is well controlled.

[0114] 5. Method Validation

[0115] 5.1 Linearity and range

[0116] (1) Solution preparation

[0117] Reference substance stock solution: Take 1.25 mg of brexpiprazole reference substance and each impurity reference substance, weigh accurately, place in a 50 ml volumetric flask, add solvent to dissolve and dilute to scale, shake well. This is equivalent to 10% of the concentration of the test solution.

[0118] Take the reference substance stock solution and dilute it quantitatively with solvent according to the table below.

[0119] Table 5 Dilution methods of various concentration solutions

[0120] Concentration ratio Accurately pipette 10% stock solution (ml) Volume of measuring flask (ml) 1.2％ 3 25 1.0％ 2 20 0.5％ 1 20 0.2％ 2 100 0.1％ 1 100 0.05％ 1 200

[0121] (2) Acceptance criteria and measurement results

[0122] Determination method: Take the above-mentioned solutions of each concentration, conduct determination, record the peak area (A) of each finished product, and perform linear regression analysis with the concentration of each solution as the horizontal axis (x) and the corresponding peak area as the vertical axis (y). The relevant acceptance criteria and determination results are shown in the table below.

[0123] Table 6 Impurity A linearity and range test acceptance criteria and results

[0124]

[0125]

[0126] Table 7 Impurity C linearity and range test acceptance criteria and results

[0127]

[0128] Table 8 Impurity D linearity and range test acceptance criteria and results

[0129]

[0130] Table 9 Impurity E linearity and range test acceptance criteria and results

[0131]

[0132] Table 10 Acceptance criteria and results of linearity and range tests for brexpiprazole

[0133]

[0134]

[0135] Note: Intercept deviation = linear equation y-axis intercept / peak area corresponding to percentage limit concentration × 100%

[0136] Conclusion: The concentration of each impurity is in the range of 0.05%-1.2%, with good linearity (r>0.99) and an intercept of no more than 15%, indicating that the single-point external standard method can be used to determine the content and meets the acceptance criteria.

[0137] 5.2 Correction Factor

[0138] The linear equation slope (K) measured under the linearity and range items is taken to calculate the correction factor for each impurity. The calculation formula is as follows: f = Kbreprazol / Kimpurity.

[0139] Table 11 Correction factor calculation results and acceptance criteria

[0140]

[0141] Note: RD=|f1-f2| / (f1+f2)×100%

[0142] Results: The calibration factor was measured twice in parallel using the standard curve method, and RD met the acceptance criteria. The average calibration factor of impurities D and E was in the range of 0.9-1.1, calculated as 1.0; the average calibration factor of impurities A and C was in the range of 0.2-2.0, and the peak area was multiplied by the calibration factor before the self-control method was used to determine its content; the calibration factor of each impurity was calculated as the average value.

[0143] 5.3 Investigation of injection precision and stability of control solution

[0144] (1) Solution preparation

[0145] Select the 1% concentration reference solution under Linearity and Range.

[0146] (2) Acceptance criteria and measurement results

[0147] Precision determination method: Take the above solution, measure it 6 times continuously, record the peak area (A) of each component, and calculate the peak area RSD value. The relevant acceptance criteria and determination results are shown in the table below.

[0148] Solution stability determination method: The sampling intervals for the stability of the reference solution are shown in the table below. The relevant acceptance criteria and determination results are shown in the table below.

[0149] Table 12 Acceptance criteria and experimental results for injection precision of reference solution

[0150] Element Jmd1 Jmd2 Jmd3 Jmd4 Jmd5 Jmd6 RSD≤2.5% Impurity A 40393 40376 40489 39259 39870 39863 1.18 Impurity C 37549 37104 37709 36177 36694 36584 1.61 Impurity D 44533 43953 44115 42707 43412 43193 1.54 Impurity E 35794 35309 35494 34232 34992 34739 1.60 Brepiraazole 46147 45676 46148 44682 45347 45151 1.28

[0151] Conclusion: The RSD of the 1% concentration solution of the reference substance and each impurity reference substance did not exceed the limit, indicating that the method of the present invention has good sampling precision for the determination of related substances in brepazine tablets.

[0152] Table 13 Acceptance criteria (change value ≤ 5%) and experimental results of stability study of reference substance solution

[0153]

[0154] Note: Change value = peak area at each time point / peak area at 0h - 1

[0155] Conclusion: The peak area change values ​​of the reference substance and each impurity reference substance 1% concentration solution within 99 hours did not exceed the acceptance standard, indicating that the 1% reference substance solution had good stability when placed in the greenhouse for 99 hours.

[0156] 5.4 Limit of quantitation

[0157] (1) Solution preparation

[0158] Reference solution (0.05%): Same as standard curve.

[0159] (2) Acceptance criteria and measurement results

[0160] Determination method: Take the above solution and measure it continuously for 6 times, record the peak area (A) and signal-to-noise ratio (S / N) of each component, and calculate the peak area RSD value. The relevant acceptance criteria and determination results are shown in the table below.

[0161] Table 14 Acceptance criteria and experimental results for the quantitative limit determination of each component

[0162] LOQ C (μg / ml) Sensitivity ≤ 0.05% Peak area mean RSD≤15% S / N≥10 Impurity A 0.1251 0.050％ 1976 3.2％ 29.0-45.8 Impurity C 0.1236 0.049％ 1763 4.3％ 24.6-37.7 Impurity D 0.1186 0.047％ 2150 2.4％ 28.3-44.1 Impurity E 0.0956 0.038％ 1619 3.8％ 17.3-28.7 Brepiraazole 0.1183 0.047％ 2217 3.1％ 31.4-48.7

[0163] Conclusion: The sensitivity of brepazine tablets and the quantitative limit of each impurity is not lower than the impurity reporting limit, the signal-to-noise ratio is not lower than 10, and the peak area RSD does not exceed 15%, indicating that the method of the present invention can accurately quantify each impurity above the reporting limit and meets the acceptance criteria.

[0164] 5.5 Detection limit

[0165] (1) Solution preparation

[0166] Take the solution below the quantitative limit and dilute it quantitatively with solvent, which is equivalent to 0.015% of the concentration (sensitivity) of the test solution.

[0167] (2) Acceptance criteria and measurement results

[0168] Determination method: Take the above solution, measure once, and record the signal-to-noise ratio (S / N) of each finished product peak. The relevant acceptance criteria and determination results are shown in the table below.

[0169] Table 15 Acceptance criteria and experimental results for detection limits of each component

[0170] LOD C (μg / ml) Sensitivity ≤ 0.015% S / N≥3 Impurity A 0.0375 0.015％ 13.8 Impurity C 0.0371 0.015％ 11.5 Impurity D 0.0356 0.014％ 14.2 Impurity E 0.0287 0.011％ 8.4 Brepiraazole 0.0355 0.014％ 13.1

[0171] Conclusion: After diluting brepazole tablets and the quantitative concentration solutions of each impurity by 3.3 times, the signal-to-noise ratio is not less than 3, indicating that the detection sensitivity of the method of the present invention is good and meets the acceptance criteria.

[0172] 5.6 Recovery rate

[0173] (1) Solution preparation

[0174] Reference solution (5%): Take 1.25 mg of each impurity reference substance, weigh accurately, place in a 100 ml volumetric flask, add solvent to dissolve and dilute to scale, shake well. This is equivalent to 5% of the concentration of the test solution.

[0175] Reference solution (1%): Accurately measure 4 ml of reference solution (5%), place in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0176] Background solution: Grind 964 mg of brepirazole tablets (0.5 mg) into fine powder, weigh accurately, place in a 20 ml volumetric flask, add solvent and ultrasonically dissolve brepirazole and dilute to scale, shake well, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant. Prepare 6 copies in parallel. Take each solution and quantitatively dilute 200 times as 0.5% control solution.

[0177] 0.1% (reporting limit) recovery solution: Take 964 mg of brepirazole tablets, grind them into fine powder, weigh accurately, put them into a 20 ml volumetric flask, accurately add 2 ml of reference solution (1%), add solvent and ultrasonically dissolve brepirazole and dilute to scale, shake well, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant. Prepare 3 copies in parallel. Take each solution, quantitatively dilute 200 times, and use it as 0.5% reference solution.

[0178] 0.5% (limit) recovery solution: Take 964 mg of brepirazole tablets, grind them into fine powder, weigh accurately, put them into a 20 ml volumetric bottle, accurately add 2 ml of control solution (0.5%), add solvent and ultrasonic to dissolve brepirazole and dilute to scale, shake well, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant. Prepare 6 copies in parallel. Take each solution, quantitatively dilute 200 times, and use it as 0.5% control solution.

[0179] 0.75% (150% limit) recovery solution: Take 964 mg of brepirazole tablets, grind them into fine powder, weigh accurately, put them into a 20 ml volumetric bottle, accurately add 3 ml of control solution (5%), add solvent and ultrasonically dissolve brepirazole and dilute to scale, shake well, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant. Prepare 3 copies in parallel. Take each solution, quantitatively dilute 200 times, and use it as 0.5% control solution.

[0180] (2) Acceptance criteria and measurement results

[0181] Take 3 concentration gradient data (limit concentration, take the first three data for calculation), calculate the recovery rate, and the relevant acceptance criteria and measurement results are shown in the table below.

[0182] Calculation formula: Recovery rate (%) = (detected amount - original amount) / added amount × 100%

[0183] Table 16 Impurity recovery test acceptance criteria and results (0.5 mg specification)

[0184]

[0185] Conclusion: The recoveries and RSDs of the three gradient concentrations of brepazole tablets all met the acceptance criteria, indicating that the detection accuracy of the method of the present invention meets the requirements.

[0186] 5.7 Repeatability

[0187] (1) Repeatability without spike addition

[0188] Take 6 sets of data under the recovery rate item to calculate the mean and deviation of the detected amount.

[0189] The relevant acceptance criteria and test results are shown in the table below.

[0190] Table 17 Repeatability (without spike) acceptance criteria

[0191]

[0192] Table 18 Repeatability (no spike) test results and acceptance criteria (0.5 mg specification)

[0193]

[0194]

[0195] Conclusion: Without adding spikes, the repeatability of the detected amounts of each impurity meets the acceptance criteria, indicating that the detection repeatability of the method of the present invention is good.

[0196] (2) Repeatability of spike addition

[0197] Take 6 sets of data at the lower limit concentration of the recovery rate item to calculate the recovery rate and its RSD value.

[0198] The relevant acceptance criteria and test results are shown in the table below.

[0199] Table 19 Repeatability (spiking) test results and acceptance criteria (unit: %) (0.5 mg specification)

[0200] Impurities 1 2 3 4 5 6 Mean RSD≤10% Impurity A 103.2 103.1 101.8 103.0 104.0 103.7 103.1 0.75 Impurity C 99.8 101.2 100.6 101.4 102.5 103.2 101.5 1.23 Impurity D 95.9 96.4 95.9 97.7 98.1 99.3 97.2 1.41 Impurity E 93.9 94.4 94.2 95.5 94.9 96.3 94.9 0.97

[0201] Conclusion: The repeatability of each impurity measured by the spiked method meets the acceptance criteria, indicating that the repeatability of the detection method of the present invention is good.

[0202] 5.8 Intermediate precision test

[0203] Determination method: Determined by different experimenters at different times using different instruments according to the method under Repeatability (spiking) (n=6).

[0204] Take 6 data points measured under the repeatability (spiked) item and 6 data points measured under the intermediate precision item, combine them to calculate the RSD, and the relevant acceptance criteria (same as recovery rate) and measurement results are shown in the table below.

[0205] Table 20 Intermediate precision test results and acceptance criteria (unit: %) (0.5 mg specification)

[0206]

[0207]

[0208] Conclusion: The repeatability and intermediate precision of the detected impurities measured by the spiked method were in line with the acceptance criteria, indicating that the repeatability and intermediate precision of the detection method of the present invention were good.

[0209] 5.9 Conclusion

[0210] Based on the above verification, it can be seen that: the injection precision and the stability of the control solution meet the requirements, indicating that the injection of the method of the present invention is precise and accurate, and the detection operation process is stable, and the result obtained is highly accurate; the quantitative limit test meets the requirements, indicating that the method of the present invention can accurately determine the amount of related substances therein, and has sensitive quantitative detection capabilities; the detection limit test meets the requirements, indicating that the detection limit of the method of the present invention is reasonably set, and the detection method has high sensitivity; the recovery test meets the acceptance standard, indicating that the detection accuracy of the method of the present invention meets the requirements; the repeatability test meets the requirements, indicating that the method of the present invention is repeatable and feasible; the intermediate precision test meets the requirements, that is, the precision obtained under the conditions between the repeatability conditions and the reproducibility conditions is high, indicating that the measurement precision of the method of the present invention is good. All of the above verification items meet the requirements, and the method of the present invention can be used to detect the amount of impurities A, impurity C, impurity D and impurity E in brepazole tablets.

[0211] Comparative Example 1

[0212] Take 1 ml of the 5.7 test sample spiked solution in Example 1 and use a high performance liquid chromatograph for determination and analysis. The chromatographic detection conditions are as follows:

[0213] Chromatographic column: octadecylsilane bonded silica gel;

[0214] Mobile phase A: 0.1% ammonium dihydrogen phosphate solution;

[0215] Mobile phase B: acetonitrile solution;

[0216] Column temperature: 35 °C;

[0217] Flow rate: 1.0ml / min;

[0218] Injection volume: 10 μl;

[0219] Detection wavelength: 254nm;

[0220] Gradient elution program: 0 min, 85% A, 15% B; 50 min, 30% A, 70% B; 50.1 min, 85% A, 15% B; 60 min, 85% A, 15% B.

[0221] Conclusion: Comparative Example 1 is a comparative test of Example 1. The method of Comparative Example 1 is to use a high performance liquid chromatograph for detection and analysis, which is the same as the detection method used in Example 1, except that the mobile phase A in the detection conditions is different. Result: The chromatograms of various impurities were not detected, and the chromatographic detection conditions used in the method of Comparative Example 1 are not applicable.

[0222] According to the comparison between the detection results of Comparative Example 1 and Example 1, it can be seen that: in the same detection method, different mobile phase A combinations play a vital role in the detection process, and different mobile phase solution combinations can dissolve and separate different substances, thereby detecting different substances. In contrast, the mobile phase A combination used in Example 1 can well dissolve and separate the impurities in the breprazol tablets, thereby detecting the corresponding chromatograms of each impurity, with baseline separation, accurate and clear, and the amount of each impurity can be obtained by calculation.

[0223] Comparative Example 2

[0224] Take 1 ml of the 5.7 test sample spiked solution in Example 1 and use a high performance liquid chromatograph for determination and analysis. The chromatographic detection conditions are as follows:

[0225] Chromatographic column: octadecylsilane bonded silica gel;

[0226] Mobile phase A: 0.01 mol / L potassium dihydrogen phosphate-0.05 mol / L sodium perchlorate buffer;

[0227] Mobile phase B: acetonitrile solution;

[0228] Column temperature: 35°C;

[0229] Flow rate: 1.0ml / min;

[0230] Injection volume: 10 μl;

[0231] Detection wavelength: 254nm;

[0232] Gradient elution program: 0 min, 2% A, 98% B; 40 min, 20% A, 80% B; 60 min, 55% A, 45% B; 65 min, 60% A, 40% B; 75 min, 30% A, 70% B; 85 min, 10% A, 90% B.

[0233] Conclusion: Comparative Example 2 is a comparative test of Example 1. The detection methods used in Comparative Example 2 and Example 1 are the same, except that the gradient elution procedures in the detection conditions are different. According to the comparison of the detection results of Comparative Example 2 with those of Example 1, it can be seen that in the same detection method, the substances detected by changing the gradient elution procedure are significantly different. In contrast, the gradient elution procedure used in Example 1 can better elute and separate the impurities in the breprazol tablets, accurately detect the substances of each impurity, obtain the corresponding chromatogram, baseline separation, and good separation degree.

[0234] From the above comparative examples 1-2, it can be seen that:

[0235] First, in the same detection method, different mobile phase solution combinations can be used to dissolve and separate different substances, thereby detecting different substances. The mobile phase A combination used in the present invention can well dissolve and separate the impurities in the breprazol tablets, thereby detecting the corresponding chromatogram of each impurity, with baseline separation, accurate and clear, and the amount of each impurity can be obtained by calculation.

[0236] Second, in the same detection method, different compounds can be detected by changing the gradient elution program. The gradient elution program used in the present invention can better elute and separate the impurities in the breprazol tablets, accurately detect the substances of each impurity, obtain the corresponding chromatogram, baseline separation, and good separation degree.

[0237] In summary, this example confirms that the use of high performance liquid chromatography and by limiting the chromatographic detection conditions can accurately detect the amounts of impurities A, C, D and E in breprazol tablets, and the detection method has high sensitivity and good separation.

[0238] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention, and all of them belong to the protection scope of the present invention.

Claims

1. A method for detecting related substances in breprazol tablets, characterized in that: High performance liquid chromatography was used to detect the amount of related substances in breprazol tablets, and the related substances included impurity A, impurity C, impurity D, and impurity E; The chemical name of impurity A is 7-hydroxy-2-quinolone; The chemical name of impurity C is 4-(benzo[b]thiophen-4-yl)-1-(4-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)butyl)piperazine-1-oxide; The chemical name of impurity D is 4-(4-(benzothiophen-4-yl)piperazin-1-yl)butan-1-ol; The chemical name of impurity E is 8-(benzothiophen-4-yl)-5,8-diazaspiro[4.5]dec-5-ammonium bromide; The chromatographic detection conditions include: using octadecylsilane bonded silica gel as a filler; using 0.01 mol / L potassium dihydrogen phosphate-0.05 mol / L sodium perchlorate buffer as a mobile phase A, and using acetonitrile solution as a mobile phase B, and performing gradient elution.

2. The method for detecting related substances in breprazol tablets according to claim 1, characterized in that: Gradient elution program: 0 min, 85% A, 15% B; 50 min, 30% A, 70% B; 50.1min, 85%A, 15%B; 60min, 85%A, 15%B.

3. The method for detecting related substances in breprazol tablets according to claim 1, characterized in that: The chromatographic detection conditions also include: detection wavelength of 254 nm and column temperature of 35°C.

4. The method for detecting related substances in breprazol tablets according to claim 1, characterized in that: The chromatographic detection conditions also include: a flow rate of 1.0 ml per minute and an injection volume of 10 μl.

5. The method for detecting related substances in breprazol tablets according to claim 1, characterized in that: Preparation of mobile phase A: Take potassium dihydrogen phosphate and sodium perchlorate, add water to dissolve them, adjust the pH to 2.5 with phosphoric acid, and prepare 0.01 mol / L potassium dihydrogen phosphate-0.05 mol / L sodium perchlorate buffer.

6. The method for detecting related substances in breprazol tablets according to claim 1, characterized in that: The detection method includes the following steps: S1. Preparation of solvent: Mix water, acetonitrile, methanol and acetic acid to obtain a solvent; S2. Preparation of the test solution: Take brepazole tablets, add a solvent and sonicate to dissolve brepazole, dilute with solvent to prepare a solution containing 0.5 mg of brepazole per 1 ml, centrifuge, and take the supernatant to obtain the test solution; S3. Preparation of control solution: accurately measure the test solution and quantitatively dilute it with a solvent to prepare a solution containing 2.5 μg of brepazine per 1 ml as a control solution; S4. Preparation of mixed reference solution: Take impurity A, impurity C, impurity D, impurity E reference substances, add solvent and dilute to prepare a solution containing 0.1 mg per 1 ml as impurity reference stock solution; take brepirazole and impurity reference stock solution respectively, dissolve in solvent and dilute to prepare mixed reference solution containing 0.5 mg of brepirazole and 5 μg of each impurity per 1 ml; S5. The control solution, the mixed reference solution and the test solution are respectively detected by high performance liquid chromatography to obtain the chromatogram data of the control solution, the mixed reference solution and the test solution, and the amount of each impurity in the test sample is calculated based on the chromatogram data of the mixed reference solution and the test sample.

7. The method for detecting related substances in breprazol tablets according to claim 6, characterized in that: The peak areas of impurity A, impurity D, impurity E and impurity C shall not be greater than the main peak area of ​​the control solution.

8. The method for detecting related substances in breprazol tablets according to claim 6, characterized in that: In step S1, the volume ratio of water-acetonitrile-methanol-acetic acid is 50:40:10:

1.

9. The method for detecting related substances in breprazol tablets according to claim 6, characterized in that: In step S5, the chromatogram data is calculated using the following calculation method: Self-control + correction factor method: single impurity C = (A 样 ×f / A 自对 )×C 自对 ; A 样 : The peak area of ​​each impurity in the chromatogram of the test solution; f: correction factor for each impurity; A 自对 : The main component peak area in the chromatogram of the test sample’s own reference solution; C 自对 : The percentage concentration of the test sample itself as the control solution is 0.5%.

10. A method for detecting related substances in breprazol tablets according to claim 9, characterized in that: The f correction factor includes the correction factors corresponding to the following impurities: the correction factor of impurity A is 1.2, the correction factor of impurity C is 1.3, the correction factor of impurity D is 1.1, and the correction factor of impurity E is 1.1.

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