Determination method of Blonanserin related substances

Abstract

The invention provides a method for determining a Blonanserin related substance, and belongs to the technical field of pharmaceutical analys.The method comprises the steps that Blonanserin is taken to be prepared into a test solution, then a sodium dihydrogen phosphate buffer solution serves as a mobile phase A, a mixed solution of methyl alcohol and acetonitrile serves as a mobile phase B, high performance liquid chromatography detection is conducted through gradient elution, and the Blonanserin related substance is obtained. The method is used for detecting related substances in blonanserin. According to the present invention, with the specific high performance liquid chromatography condition, the variety and the content of the Blonanserin-related substance can be effectively determined, and the accuracy and the precision of the determination result are good.

Description

Technical Field

[0001] This invention relates to pharmaceutical analysis techniques, and more particularly to a method for determining related substances of bromelain. Background Technology

[0002] Bunanserin is a 5-HT and dopamine antagonist (SDA), a second-generation atypical antipsychotic drug. It has dopamine D2 receptor blocking activity comparable to haloperidol and also has a strong blocking effect on 5-HT2A. Its selectivity for both receptors is stronger than that of other antipsychotic drugs.

[0003] Bunam Serin products include Bunam Serin tablets, but current standards do not include Bunam Serin or its products. Related substances present in Bunam Serin products include process impurities and degradation impurities; process impurities include impurities A, B, C, D, E, F, and G; degradation impurities include impurities I and H. The structural formulas of these impurities are as follows: Impurity A: Impurity B: Impurity C: Impurity D: Impurity E: Impurity F: Impurity G: Impurity H: Impurity I: .

[0004] However, existing methods are not effective in detecting and separating these impurities in Bunan Serin. Therefore, it is necessary to develop a method for determining related substances in Bunan Serin. Summary of the Invention

[0005] To address the above problems, this invention provides a method for determining related substances of Bunansserin.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for determining related substances in Bunam Serin, wherein the method involves preparing a test solution of Bunam Serin, using sodium dihydrogen phosphate buffer as mobile phase A and a mixture of methanol and acetonitrile as mobile phase B, and performing high performance liquid chromatography with gradient elution to detect related substances in Bunam Serin. The elution conditions for the gradient elution are as follows: 0~15min, 47% mobile phase A, 53% mobile phase B; 15~45min, 47%→20% mobile phase A, 53%→80% mobile phase B; 45~55 min, 20% mobile phase A, 80% mobile phase B; 55~56 min, 20%→47% mobile phase A, 80%→53% mobile phase B; 56~65min, 47% mobile phase A, 53% mobile phase B; The relevant substances include impurities A to I, specifically impurities A, B, C, D, E, F, G, H and I.

[0007] Furthermore, the determination method includes the following specific steps: Prepare the test solution by adding methanol to Brønsserin; Prepare reference solutions for each impurity, from A to I. The test solution and impurity reference solution were subjected to high performance liquid chromatography to detect relevant substances in Bunanserin.

[0008] Furthermore, the method also includes using high-performance liquid chromatography to detect the chromatogram obtained, and calculating the content of each relevant substance by area normalization.

[0009] Furthermore, the sodium dihydrogen phosphate buffer is prepared by dissolving sodium dihydrogen phosphate and sodium heptanesulfonate in purified water, and then adjusting the pH to 2.3-2.7 with phosphoric acid.

[0010] Furthermore, the sodium dihydrogen phosphate buffer solution contains 3.85~3.95 g / 1000 mL of sodium dihydrogen phosphate and 4.9~5.1 g / 1000 mL of sodium heptane sulfonate.

[0011] Furthermore, the volume ratio of methanol to acetonitrile in the methanol-acetonitrile mixture is 30:34~36.

[0012] Furthermore, the chromatographic column uses octadecylsilane-bonded silica gel as the packing material.

[0013] Furthermore, the high-performance liquid chromatography (HPLC) detection is performed at a flow rate of 0.9–1.1 mL / min, a column temperature of 28–32 °C, and a detection wavelength of 234–238 nm.

[0014] Furthermore, the content of bromelain in the test solution was 1.2~1.8 mg / mL.

[0015] Furthermore, the reference solutions for each impurity are prepared by dissolving impurities A through I in methanol.

[0016] The beneficial effects of the method for determining related substances of Bunanserin according to the present invention are as follows: This invention can effectively determine the types and contents of related substances in Bunansserin under specific high-performance liquid chromatography conditions, and the determination results have good accuracy and precision. Since there are many kinds of process impurities and degradation impurities in bunamserin, effective detection and control of these impurities can better ensure the safety of bunamserin medication. This invention constructs a method for determining bunamserin-related substances, which can more comprehensively detect bunamserin-related substances and solves the problem that the existing technology cannot detect multiple impurities in bunamserin. This invention uses specific high-performance liquid chromatography (HPLC) detection, which can effectively separate impurities A to I from Bunam Serin, and has high sensitivity and good accuracy. It can more accurately determine the types and contents of impurities A to I in Bunam Serin, and realize the detection and control of multiple impurities in Bunam Serin. The present invention provides a method for determining related substances of bromelain, which is simple to operate and yields easy chromatographic results. The present invention provides a method for determining related substances of bromelain, which, by adjusting process parameters such as chromatographic conditions, fully takes into account the requirement of complete separation of nine impurities (A to I) from bromelain, and successfully establishes a method for calculating the content of each impurity, thereby enabling better control of the quality of bromelain. The present invention provides a method for determining the related substances of bromelain, which can quickly, easily, accurately and reliably determine the quality of bromelain, providing a new reference standard for the quality control of bromelain. Attached Figure Description

[0017] Figure 1 The chromatogram of the Brønsserin mixed solution in Example 1; Figure 2 The chromatogram of related substances in the blank solution of Example 1; Figure 3 The chromatogram of related substances in the positioning solution of impurity A reference standard in Example 1; Figure 4 The chromatogram of related substances in the positioning solution of impurity B reference standard in Example 1; Figure 5 The chromatogram of related substances in the positioning solution of impurity C reference standard in Example 1; Figure 6 The chromatogram of related substances in the positioning solution of impurity D reference standard in Example 1; Figure 7 The chromatogram of related substances in the positioning solution of impurity E reference standard in Example 1; Figure 8 The chromatogram of related substances in the positioning solution of impurity F reference standard in Example 1; Figure 9 The chromatogram of related substances in the positioning solution of impurity G reference standard in Example 1; Figure 10 The chromatogram of related substances in the positioning solution of impurity H reference standard in Example 1; Figure 11 The chromatogram of related substances in the positioning solution of impurity I reference standard in Example 1; Figure 12 The chromatogram of the relevant substances in the system suitability solution in Example 2; Figure 13 This is a standard curve of total other unknown impurities in Bunansserin in Example 2; Figure 14 This is the standard curve diagram of impurity A in Example 2; Figure 15 This is the standard curve of impurity B in Example 2; Figure 16 This is the standard curve diagram of impurity C in Example 2; Figure 17 This is the standard curve diagram of impurity D in Example 2; Figure 18 This is a standard curve diagram of impurity E in Example 2; Figure 19 This is the standard curve of impurity F in Example 2; Figure 20 This is the standard curve of impurity G in Example 2; Figure 21 This is the standard curve of impurity H in Example 2; Figure 22 This is the standard curve diagram of impurity I in Example 2; Figure 23 The first result of the robustness test in Example 2 was obtained using a 041577 column, a flow rate of 1.0 mL / min, a column temperature of 30°C, a pH of 2.5, and a wavelength of 236 nm. Figure 24 The second result of the robustness test in Example 2 was obtained using a 040699 column, a flow rate of 1.0 mL / min, a column temperature of 30°C, a pH of 2.5, and a wavelength of 236 nm. Figure 25 The third result of the robustness test in Example 2 was obtained using a 040696 column, a flow rate of 1.0 mL / min, a column temperature of 30°C, a pH of 2.5, and a wavelength of 236 nm. Figure 26 The fourth result of the robustness test in Example 2 was obtained using a 041577 column, a flow rate of 0.9 mL / min, a column temperature of 30°C, a pH of 2.5, and a wavelength of 236 nm; the chromatogram of related substances was obtained at a flow rate of 1.1 mL / min. Figure 27The fifth result of the robustness test in Example 2 was obtained using a 041577 column, a flow rate of 1.1 mL / min, a column temperature of 30°C, a pH of 2.5, and a wavelength of 236 nm. Figure 28 The robustness test result six in Example 2 was obtained using a 041577 column, a flow rate of 1.0 mL / min, a column temperature of 28°C, a pH of 2.5, and a wavelength of 236 nm. Figure 29 The robustness test result seven in Example 2 was obtained using a 041577 column, a flow rate of 1.0 mL / min, a column temperature of 32°C, a pH of 2.5, and a wavelength of 236 nm. Figure 30 The robustness test result eight in Example 2 was obtained using a 041577 column, a flow rate of 1.0 mL / min, a column temperature of 30°C, a pH of 2.3, and a wavelength of 236 nm. Figure 31 The robustness test result nine in Example 2 was obtained using a 041577 column, a flow rate of 1.0 mL / min, a column temperature of 30°C, a pH of 2.7, and a wavelength of 236 nm. Figure 32 The robustness test result in Example 2 was obtained using a 041577 column, a flow rate of 1.0 mL / min, a column temperature of 30°C, a pH of 2.5, and a wavelength of 234 nm. Figure 33 The robustness test result eleven in Example 2 was obtained using a 041577 column, a flow rate of 1.0 mL / min, a column temperature of 30°C, a pH of 2.5, and a wavelength of 238 nm. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0019] Experimental materials: Brenaseline reference standard, 20 mg / vial, 98.85%, batch number T006-211001, sourced from Renhe Lijian Pharmaceutical (Tianjin) Co., Ltd.; Impurity A reference standard, 15 mg / vial, 99.42%, batch number T006-ZA-210701, sourced from Renhe Lijian Pharmaceutical (Tianjin) Co., Ltd.; Impurity B reference standard, 15 mg / vial, 99.19%, batch number T006-ZB-210701, sourced from Renhe Lijian Pharmaceutical (Tianjin) Co., Ltd.; Impurity C reference standard, 15 mg / vial, 98.86%, batch number T006-ZC-210701, sourced from Renhe Lijian Pharmaceutical (Tianjin) Co., Ltd.; Impurity D reference standard, 15 mg / vial, 96.3%, batch number T006-ZD-210901, sourced from Renhe Lijian Pharmaceutical (Tianjin) Co., Ltd. The following reference standards were found: Impurity E (15 mg / vial, 98.53%), batch number T006-ZE-210701, sourced from Renhe Lijian Pharmaceutical (Tianjin) Co., Ltd.; Impurity F (15 mg / vial, 98.4%), batch number T006-ZE-210901, sourced from Renhe Lijian Pharmaceutical (Tianjin) Co., Ltd.; Impurity G (15 mg / vial, 94.7%), batch number T006-ZG-210901, sourced from Renhe Lijian Pharmaceutical (Tianjin) Co., Ltd.; Impurity H (15 mg / vial, 91.36%), batch number T006-ZH-210701, sourced from Renhe Lijian Pharmaceutical (Tianjin) Co., Ltd.; Impurity I (15 mg / vial, 97.53%), batch number T006-ZI-210701, sourced from Renhe Lijian Pharmaceutical (Tianjin) Co., Ltd. The structural formula of the aforementioned impurities is as follows: Impurity A: Impurity B: Impurity C: Impurity D: Impurity E: Impurity F: Impurity G: Impurity H: Impurity I: ; Among them, impurities A to G are process impurities, while impurities H and I are degradation impurities.

[0020] Example 1: A method for determining related substances of Bunan Serin This embodiment is a method for determining related substances of Bunanserin, specifically including the following steps: S1, Solution Preparation Blank solvent: methanol.

[0021] Test solution: Accurately weigh the bromelain sample, dissolve and dilute it with methanol to prepare a solution containing 1.5 mg bromelain per 1 mL, which is used as the test solution.

[0022] Preparation of the control solution: Accurately measure an appropriate amount of the test solution and dilute it with methanol to prepare a solution containing 1.5 μg of bromelain per 1 mL to obtain the control solution; Preparation of mixed solution: Take appropriate amounts of impurity A to impurity H reference standards and bromelain reference standards, add methanol to dissolve and dilute to prepare a solution containing 1.5 mg of bromelain reference standard and 2.25 μg of each of impurity A to impurity H per 1 mL, thus obtaining the mixed solution; Preparation of impurity I reference solution: Accurately weigh an appropriate amount of impurity I, add methanol to dissolve and dilute to prepare a solution containing 0.1 μg of impurity I per 1 mL, thus obtaining impurity I reference solution; Preparation of reference solutions for each impurity: Accurately weigh appropriate amounts of reference standards for impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, and budesonide, respectively, and dissolve and dilute them with methanol to prepare solutions containing 2.25 μg of each impurity per 1 mL, which are the reference solutions for the corresponding impurities. Preparation of system suitability solution: Weigh approximately 15 mg of this product accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with impurity I reference solution, shake well, and prepare a mixed solution containing approximately 1.5 mg of bromelain and 2.25 μg of impurity I per ml.

[0023] S2, Determination of related substances Accurately measure 10 μL each of blank solution, test solution, control solution, mixed solution, impurity reference solution and system suitability solution, inject them into the high performance liquid chromatograph, record the chromatogram, and calculate the content of related substances by the area normalization method. The results are shown in Table 1.

[0024] The high-performance liquid chromatography (HPLC) conditions are as follows: The chromatographic column was packed with octadecylsilane-bonded silica gel (Venusil XBP C18(L), 5μm, 4.6×150mm or a column with equivalent performance). The mobile phase consisted of sodium dihydrogen phosphate buffer solution as mobile phase A, and a methanol-acetonitrile mixture (methanol and acetonitrile mixture) at a volume ratio of 30:35 as mobile phase B; the detection wavelength was 236 nm; the flow rate was 1.0 mL / min; the column temperature was 30 °C; the injection volume was 10 μL; the elution method was gradient elution; and the elution program was as follows: 0~15min, 47% mobile phase A, 53% mobile phase B; 15~45min, 47%→20% mobile phase A, 53%→80% mobile phase B; 45~55 min, 20% mobile phase A, 80% mobile phase B; 55~56 min, 20%→47% mobile phase A, 80%→53% mobile phase B; 56~65 min, 47% mobile phase A, 53% mobile phase B.

[0025] The sodium dihydrogen phosphate buffer solution used in mobile phase A was prepared by weighing 3.90 g of sodium dihydrogen phosphate and 5.0 g of sodium heptanesulfonate, dissolving them in 1000 mL of purified water, and adjusting the pH value to 2.3-2.7 with phosphoric acid (in this example, the pH value was adjusted to 2.5).

[0026] Table 1. Determination results of Bronasidium and various impurities in Bronasidium samples (area normalization method)

[0027] The chromatogram of the Brønsted serin mixed solution obtained in this invention is as follows: Figure 1 As shown, the chromatogram of related substances in the blank solution is as follows: Figure 2 As shown, the chromatograms of related substances in the solutions for impurities A to I are respectively as follows: Figures 4-11 As shown in Table 1, the determination method of the present invention can not only effectively separate and determine the types of related substances (impurities A to I) in Bunan Selin, but also determine the content of related substances in Bunan Selin.

[0028] In the determination method of related substances in Bunan Serin, the limits for each impurity in Bunan Serin are: impurity I ≤ 0.00625%, other single impurities ≤ 0.10%, and total impurities ≤ 0.2%.

[0029] Example 2: Validation of the determination method This embodiment conducts a methodological investigation of the determination method in Example 1. During the methodological investigation, the chromatographic conditions for high performance liquid chromatography detection are the same as those in Example 1.

[0030] I. Solution Preparation System suitability solution: Accurately weigh an appropriate amount of Bronyasserin sample and impurity I, dissolve and dilute with methanol to prepare a mixed solution containing approximately 1.5 mg of Bronyasserin sample and 2.25 μg of impurity I per 1 mL, which is used as the system suitability solution.

[0031] Reference stock solutions: Accurately weigh appropriate amounts of impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard, impurity E reference standard, impurity F reference standard, impurity G reference standard, impurity H reference standard, and impurity I reference standard, dissolve them in methanol and dilute them to prepare solutions containing approximately 0.225 mg of the corresponding impurity per 1 mL, which are used as reference stock solutions for the corresponding impurities.

[0032] Bronangserine reference stock solution: Accurately weigh an appropriate amount of Bronangserine reference standard, dissolve and dilute it in methanol to prepare a solution containing approximately 1.5 mg of Bronangserine per 1 mL, which is used as the Bronangserine reference stock solution.

[0033] Acid-degrading test solution: Accurately weigh approximately 15 mg of Bronasidium sample and place it in a 10 mL volumetric flask. Add 1 mL of 1 mol / L sulfuric acid aqueous solution and let stand for 24 h. Then, add an appropriate amount of 1 mol / L sodium hydroxide solution to neutralize to a neutral pH (approximately 7.0). Dissolve the solution in methanol (if it does not dissolve, sonicate appropriately) and dilute to the mark. Shake well to obtain the acid-degrading test solution. The acid-degrading blank solution is prepared in the same manner, except that no Bronasidium sample is added.

[0034] Alkali-degraded test solution: Accurately weigh approximately 15 mg of Bronasidium sample and place it in a 10 mL volumetric flask. Add 1 mL of 1 mol / L sodium hydroxide solution and let stand for 24 h. Then, add an appropriate amount of 1 mol / L sulfuric acid solution to neutralize to a pH of approximately 7.0. Dissolve the solution in methanol (if it does not dissolve, sonicate appropriately) and dilute to the mark. Shake well to obtain the alkali-degraded test solution. The alkali-degraded blank solution is prepared in the same manner, except that no Bronasidium sample is added.

[0035] High-temperature destruction test solution: Accurately weigh approximately 15 mg of Bunanserin sample subjected to high temperature (60℃±2℃) for 10 days under the influencing factors test item, place it in a 10 mL volumetric flask, dissolve it in methanol (if it does not dissolve, it can be sonicated appropriately) and dilute to the mark, shake well, and use it as the high-temperature destruction test solution.

[0036] Oxidative Degradation Test Solution: Accurately weigh approximately 15 mg of Bronasidium sample and place it in a 10 mL volumetric flask. Add 1 mL of 30% hydrogen peroxide solution and let stand for 6 hours. Then, dissolve the sample in methanol (if it does not dissolve, sonicate appropriately) and dilute to the mark. Shake well to obtain the oxidative degradation test solution. The oxidative blank solution is prepared in the same way, except that no Bronasidium sample is added.

[0037] Light-damage test solution: Accurately weigh approximately 15 mg of Bunanserin sample exposed to light (4500 lux ± 500 lux) for 10 days under the influence factor test item, place it in a 10 mL volumetric flask, dissolve it in methanol (if it does not dissolve, it can be sonicated appropriately) and dilute to the mark, shake well, and use it as the light-damage test solution.

[0038] High humidity destruction test solution: Accurately weigh approximately 15 mg of Bunanserin sample subjected to high humidity (75%RH±5%RH) for 10 days under the influence factor test item, place it in a 10 mL volumetric flask, dissolve it in methanol (if it does not dissolve, it can be sonicated appropriately) and dilute to the mark, shake well, and use it as the high humidity destruction test solution.

[0039] II. Exclusivity 1) Full wavelength scanning Accurately measure blank solution, oxidation blank solution, acid-base blank solution, test solution, acid-degraded test solution, base-degraded test solution, oxidation-degraded test solution, high-temperature-degraded test solution, light-degraded test solution, and high-humidity-degraded test solution. Detect them according to the method in Example 1. Record the chromatogram using a DAD detector. Calculate the material balance for the Bunansserin sample. See Tables 2-3 for specific results.

[0040] Table 2 Material conservation data for the samples

[0041] Table 3. Experimental data of DAD on damaged samples

[0042] The results showed that, through different specific experiments, the blank solvent did not interfere with the detection under various destructive conditions; the degradation products generated under various destructive conditions could achieve baseline separation from the main peak of Bronang Serin and did not affect the related substances test, proving that the chromatographic method has high specificity and can be used for the determination of related substances in Bronang Serin. Simultaneously, this invention also performed material balance calculations on Bronang Serin samples. The calculations (with the total peak area of ​​the undestructed sample as 100%) showed that the sum of the main peak area and the area of ​​all impurity peaks under each destructive condition was above 95%, indicating that the material balance was essentially maintained. The specificity test (DAD) results showed that the purity of the main peak under each destructive condition was greater than 0.999, and the threshold value was greater than 0.999. The experiments demonstrated that this chromatographic system can achieve good separation between the impurity peaks generated under various destructive conditions and the main component peak; furthermore, the solvent peak did not interfere with the determination under these conditions. Therefore, this method demonstrates high specificity.

[0043] III. System Applicability Accurately measure the blank solution (i.e., methanol), system suitability solution, stock solutions of each reference standard corresponding to impurities A to I, the test solution, and the mixed solution prepared in Example 1. Perform the analysis according to the method in Example 1, record the chromatograms, and the results are shown in Tables 4 and 5. The chromatograms of related substances in the system suitability solution are shown below. Figure 12 .

[0044] Table 4. Peak elution of the mixed solution

[0045] Table 5. Peak elution of solutions suitable for system application

[0046] The results showed that the resolution between the main peak of Bronsenserin and each known impurity peak in the mixed solution met the requirements. In the system suitability solution, the resolution between the Bronsenserin peak and impurity I peak was 4.837, greater than 1.5, which meets the specified requirements.

[0047] IV. Detection Limit The reference solution of bromelain and the reference solutions of each impurity prepared in Example 1 were diluted with methanol to prepare a standard solution of appropriate concentration. The detection limit concentration was calculated with a signal / noise ratio of 3:1. The results are shown in Table 6.

[0048] Table 6 Detection Limit Results

[0049] The results show that the detection capability of Bunanserin and other impurities is good, and this method has good detection capability.

[0050] V. Limit of Quantification The reference solution of bromelain and the reference solutions of each impurity prepared in Example 1 were diluted with methanol to prepare a standard solution of appropriate concentration. The limit of quantitation was calculated with a signal / noise ratio of 10:1. The results were measured six times and are shown in Table 7.

[0051] Table 7 Results of Limit of Quantification

[0052] The results showed that the RSD of the detection results of Brønsserin and each impurity in six parallel determinations of the limit of quantitation solution was less than 20%, which met the requirements.

[0053] VI. Linearity and Range Take appropriate amounts of each impurity stock solution and Brønsserin reference standard stock solution to establish a linearity study from the limit of quantitation to 500% based on their limits. Prepare a series of linear solutions of impurity reference standards and Brønsserin reference standard (specific concentrations are shown in Table 8). Accurately measure 10 µL of each of the above linear solutions and inject them into the liquid chromatograph. Detect according to the chromatographic conditions of Example 1, record the chromatograms, and perform linear regression with the injection concentration (μg / mL) as the X-axis and the peak area as the Y-axis. The results are shown in Table 8. The standard curves for other unknown impurities in Brønsserin are shown in Table 8. Figure 13 The standard curves for impurities A through I are shown below. Figures 14-22 .

[0054] Table 8 Linearity and Range

[0055] The results showed that bromelain and its impurities exhibited good linearity within the limit of quantitation (LOQ) to 500%. When the peak areas of each impurity were within the linear range, the content of each impurity in the sample could also be calculated using the standard curve method. In addition to the previously mentioned requirement that impurities C and E have a resolution of less than 1.5 and need to be detected separately, this invention can also mix the impurities into two mixed series of linear solutions and plot a standard curve. It should be noted that the two mixed series solutions are used to distinguish impurity C from impurity E; other impurities can be mixed with either impurity C or impurity E. Furthermore, other unknown impurities in bromelain were detected using a linear solution of bromelain reference standard.

[0056] VII. Instrument Precision Accurately measure the mixed solution prepared in Example 1, and perform the determination according to the method in Example 1. Perform the determination 6 times consecutively, record the chromatogram, and the results are shown in Table 9.

[0057] Table 9 Results of Instrument Precision Test

[0058] The results showed that Bunanserin and its impurities exhibited good instrument precision.

[0059] 8. Repetitiveness The mixed solution prepared according to the method in Example 1 was prepared in 6 parallel portions. The samples were accurately measured and determined according to the method in Example 1. The chromatograms were recorded, and the results are shown in Table 10.

[0060] Table 10 Repeatability Test Results

[0061] The results show that the analytical method of the present invention has good repeatability.

[0062] 9. Intermediate Precision Following the repeatability test procedure, the precision of the results obtained in the same laboratory under conditions of changing personnel, date and changing the high performance liquid chromatograph (without changing the chromatographic conditions) was evaluated. Groups 1-6 and 7-12 were the second group. The results are shown in Table 11.

[0063] Table 11 Results of intermediate precision test

[0064] The results show that the analytical method of the present invention has good intermediate precision.

[0065] 10. Solution stability The mixed solution prepared in Example 1 was tested at 0, 3, 5, 8, 10, 13, 15, 20, 22 and 24 h according to the method in Example 1 to examine the stability of the solution. The results are shown in Table 12.

[0066] Table 12 Results of solution stability test

[0067] As shown in Table 12, the mixed solution exhibits good stability within 24 hours.

[0068] XI. Durability Blank solution, system suitability solution, and mixed solution prepared in Example 1 were used. Different chromatographic columns were used, and the flow rate, column temperature, pH of the sodium dihydrogen phosphate buffer solution used in mobile phase A, and detection wavelength were adjusted. Except for the changed chromatographic conditions, other process parameters were tested according to the method in Example 1. The results are shown in Tables 13-22. The results of the robustness column investigation are shown in... Figures 23-25 The results of the durability flow rate test are shown in [link to relevant documentation]. Figure 23 and Figures 26-27 The results of the durability column temperature test are shown in [link to test]. Figure 23 and Figures 28-29 The results of the durability pH test are shown in [link to relevant documentation]. Figure 23 and Figures 30-31 The results of the durability wavelength test are shown in [link to relevant documentation]. Figure 23 and Figures 32-33 .

[0069] Table 13. Durability test results (different chromatographic columns)

[0070] Table 14. Results of the robustness test system suitability (different chromatographic columns)

[0071] Table 15 Durability test results (at different flow rates)

[0072] Table 16. Results of the durability test system's suitability (at different flow rates)

[0073] Table 17 Durability Test Results (at different column temperatures)

[0074] Table 18. Results of the durability test system's suitability (at different column temperatures)

[0075] Table 19. Durability test results (pH values ​​of buffer salt solutions used in different mobile phases A)

[0076] Table 20. Results of the durability test system's suitability (pH values ​​of the buffer salt solutions used for different mobile phases A)

[0077] Table 21 Durability test results (different wavelengths)

[0078] Table 22. Results of the durability test system suitability (different wavelengths)

[0079] The results showed that changing different chromatographic columns, adjusting flow rates, column temperatures, the pH of the sodium dihydrogen phosphate buffer solution used in mobile phase A, and the detection wavelength did not interfere with the determination of related substances in the blank solution. Under different chromatographic columns, flow rates of 0.9–1.1 mL / min, column temperatures of 28–32 °C, pH values ​​of the sodium dihydrogen phosphate buffer solution used in mobile phase A of 2.3–2.7, and detection wavelengths of 234–238 nm, the separation rates of bromelain and impurity I in the system suitability solution met the requirements. The RSDs of all impurities and bromelain in the mixed sample solution were all 20%, indicating that the method of the present invention has good robustness. Furthermore, it also shows that during the determination using the method of the present invention, flow rates of 0.9–1.1 mL / min, column temperatures of 28–32 °C, pH values ​​of the sodium dihydrogen phosphate buffer solution used in mobile phase A of 2.3–2.7, and detection wavelengths of 234–238 nm can also be used as chromatographic conditions, without limiting the specific chromatographic column.

[0080] 12. Accuracy Bronsen studied and controlled impurity I as a specific impurity, while other impurities (impurities A, B, C, D, E, F, G, H) were controlled as single impurities. Therefore, this invention conducted an accuracy test on the known impurity I.

[0081] Accurately weigh appropriate amounts of bromelain reference standard and impurity I reference standard, and prepare three portions each of low (50%), medium (100%), and high (150%) concentration solutions as accuracy test solutions. Perform the test according to the method in Example 1, calculate the recovery rate, and the results are shown in Table 23.

[0082] Table 23 Accuracy Test Results for Impurity I

[0083] The results showed that the average recovery rate of impurity I was 101.7% and the RSD value was 4.5%, indicating that the method has high accuracy.

[0084] Examples 3-6: A method for determining related substances of Bunan Serin Examples 3-6 are methods for determining related substances of bromelain. Their procedures are basically the same as in Example 1, differing only in some process parameters, as detailed in Table 24. Table 24 Summary of process parameters in Examples 3-6

[0085] The contents of other parts of Examples 3 to 6 are the same as those of Example 1, and will not be repeated here.

[0086] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for determining related substances of Bunan Serin, characterized in that, The determination method is to prepare a test solution of Bunam Serin, and then use sodium dihydrogen phosphate buffer as mobile phase A and a mixture of methanol and acetonitrile as mobile phase B, and perform high performance liquid chromatography detection by gradient elution to detect related substances in Bunam Serin. The elution conditions for the gradient elution are as follows: 0~15min, 47% mobile phase A, 53% mobile phase B; 15~45min, 47%→20% mobile phase A, 53%→80% mobile phase B; 45~55 min, 20% mobile phase A, 80% mobile phase B; 55~56 min, 20%→47% mobile phase A, 80%→53% mobile phase B; 56~65min, 47% mobile phase A, 53% mobile phase B; The relevant substances include impurities A to I, specifically impurities A, B, C, D, E, F, G, H and I.

2. The method for determining related substances of Bunansserin according to claim 1, characterized in that, The determination method includes the following specific steps: Prepare the test solution by adding methanol to Brønsserin; Prepare reference solutions for each impurity, from A to I. The test solution and impurity reference solution were subjected to high performance liquid chromatography to detect relevant substances in Bunanserin.

3. The method for determining related substances of Bunan Serin according to claim 2, characterized in that, The method also includes using high-performance liquid chromatography to detect the chromatogram and calculating the content of each relevant substance by area normalization.

4. The method for determining the related substances of Bunansserin according to any one of claims 1-3, characterized in that, Sodium dihydrogen phosphate buffer is prepared by dissolving sodium dihydrogen phosphate and sodium heptanesulfonate in purified water, and then adjusting the pH to 2.3-2.7 with phosphoric acid.

5. The method for determining related substances of Bunansserin according to claim 4, characterized in that, The sodium dihydrogen phosphate buffer solution contains 3.85~3.95 g / 1000 mL of sodium dihydrogen phosphate and 4.9~5.1 g / 1000 mL of sodium heptane sulfonate.

6. The method for determining the related substances of Bunansserin according to any one of claims 1-3 and 5, characterized in that, The volume ratio of methanol to acetonitrile in the methanol-acetonitrile mixture is 30:34~36.

7. The method for determining the related substances of Bunansserin according to any one of claims 1-3 and 5, characterized in that, The chromatographic column was packed with octadecylsilane-bonded silica gel.

8. The method for determining the related substances of Bunansserin according to any one of claims 1-3 and 5, characterized in that, The high-performance liquid chromatography (HPLC) detection was performed at a flow rate of 0.9–1.1 mL / min, a column temperature of 28–32 °C, and a detection wavelength of 234–238 nm.

9. The method for determining the related substances of Bunansserin according to any one of claims 1-3 and 5, characterized in that, The content of bromelain in the test solution was 1.2~1.8 mg / mL.

10. The method for determining the related substances of Bunansserin according to claim 2, 3 or 5, characterized in that, The reference solutions for each impurity were prepared by dissolving impurity A through impurity I in methanol.

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