Method for detecting related substances in atosiban acetate and preparation thereof

Abstract

The invention discloses a method for detecting related substances in atosiban acetate and a preparation thereof, and belongs to the technical field of chemical analysis. According to the method, a high performance liquid chromatography method is adopted, a C18 chromatographic column is used as a stationary phase, an ultraviolet detector is used as a detector, a buffer salt solution-organic phase is used as a mobile phase, and a gradient elution procedure is adopted to detect the content of related substances in atosiban acetate and the preparation thereof. The related substances comprise Di-Gly9-atosiban, delta-Ava7-atosiban, Des-Gly9-atosiban, Gly9-ol-atosiban, Asp5-atosiban, Gly9-OH-atosiban, a dimer (Gly-Orn), and Des-Gly-Aminoperperidine8-atosiban, and the related substances are used for preparing the medicine for preventing and treating ovarian cancer. The medicine for preventing and treating ovarian cancer can be used for preventing and treating ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer and the like. The method has the advantages of strong specificity, good repeatability and high accuracy, and can effectively control the quality of atosiban acetate and preparation products thereof.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and particularly relates to a method for detecting related substances in atosiban acetate and a preparation thereof. Background Art

[0002] During the peptide synthesis process, some impurities similar to the target peptide chain structure will be generated, such as missing peptides, inserted peptides, staggered peptides and differential peptides. Peptide-related impurities can be introduced by starting materials, side reactions in the production process or degradation during long-term storage. Since the structure and properties of some peptide-related impurities are similar to the main components, a single-principle analysis method may not be able to completely separate them. Usually, a variety of detection methods based on different principles are used to check related substances. In addition to the common high-performance liquid chromatography, ion exchange chromatography, capillary electrophoresis, etc. can also be considered for related substance inspection methods. Molecular exclusion chromatography, polyacrylamide gel electrophoresis, etc. can be used for polymer inspection.

[0003] Atosiban acetate is a cyclic polypeptide of disulfide bond type composed of 9 amino acids, which exists in the form of acetate in medicine, and its general name is atosiban acetate. Atosiban acetate is a synthetic polypeptide, and due to the large number of reaction steps, feeding quantity, etc. in the synthesis process, there are many types of synthetic impurities. In addition, there are also many degradation impurities produced during production and transportation. Some impurities are very similar to the structure of the main component atosiban, and these impurities are easy to produce and difficult to separate during production and storage and transportation, which seriously affects the safety and effectiveness of the medicine. If it is not well controlled, it is possible to produce serious toxic and side effects. Patent document CN101696959A discloses a method for the determination of the content of atosiban acetate and its preparation and the determination of related substances, but the separation effect of impurity B and the main peak in the method is poor; Patent document CN105949283A discloses atosiban acetate impurities, preparation and detection methods, but it cannot separate impurities with polarity close to the main component. Patent document CN117907506A discloses an ultra-high performance liquid chromatography detection method for impurities in atosiban acetate, but it has high requirements for instruments and equipment, a complex elution system, and poor separation between impurities. In addition, the detection method for related substances in the registration standard for imported drugs was used for detection. Due to the high temperature of the chromatographic column and the poor stability of the system, the peak shape was tailed, and the separation degree between impurity B and the main peak was poor.

[0004] Therefore, developing a safe, reliable, and effective analytical method for simultaneously detecting and separating impurities in atosiban acetate raw materials and preparations can not only ensure product quality but also have significant clinical value. Summary of the invention

[0005] The object of the present invention is to provide a method for detecting related substances in atosiban acetate and its preparations, so as to solve the problem that process impurities generated during the synthesis of atosiban acetate and degradation impurities generated during the storage of its preparations are difficult to measure and control.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A method for detecting related substances in atosiban acetate and its preparations, using a high performance liquid chromatography method, a C18 chromatographic column as a stationary phase, an ultraviolet detector as a detector, a buffered salt solution-organic phase as a mobile phase, and a gradient elution program to detect the content of related substances in atosiban acetate and its preparations;

[0008] The related substances include: Di-Gly 9 -Atosiban, δ-Ava 7 -Atosiban, Des-Gly 9 -Atosiban, Gly 9 -ol-Atosiban, Asp 5 -Atosiban, Gly 9 -OH-Atosiban, Dimer (Gly-Orn), Des-Gly-Aminopiperidone 8 -Atosiban.

[0009] Furthermore, the mobile phase is hexafluorophosphate solution-acetonitrile.

[0010] Furthermore, the detection method comprises the following steps:

[0011] 1) Setting chromatographic conditions;

[0012] 2) Prepare the system adaptability solution with diluent for methodological verification; then take an appropriate amount of atosiban acetate raw material or preparation sample and dissolve it with diluent to obtain the test solution; take the test solution and dilute it 100 times with diluent to obtain the control solution;

[0013] 3) Inject the test solution and the control solution into the liquid chromatograph for detection, record the chromatogram, and calculate the percentage of the peak area of ​​the relevant substance in the chromatogram of the test solution relative to the main peak area of ​​the control solution according to the self-control method.

[0014] Furthermore, the diluent is a trifluoroacetic acid aqueous solution with a pH of 3-3.5, and the trifluoroacetic acid aqueous solution is prepared by the following steps: adding 10% trifluoroacetic acid to 2000 mL of deionized water to adjust the pH to 3-3.5.

[0015] Preferably, the diluent is a trifluoroacetic acid aqueous solution with a pH of 3.2.

[0016] Furthermore, the chromatographic conditions of the high performance liquid chromatography are:

[0017] Chromatographic column: C18 column;

[0018] Flow rate: 0.8-1.2mL / min;

[0019] Injection volume: 10-50 μL;

[0020] Column temperature: 30-50°C;

[0021] Detection wavelength: 200-240nm;

[0022] Mobile phase A: 0.01-0.5 mol / L hexafluorophosphate solution, 1% phosphoric acid or 0.1 mol / L potassium dihydrogen phosphate, pH adjusted to 2.0-6.0;

[0023] Mobile phase B: acetonitrile;

[0024] Taking the total volume of the mobile phase as 100%, the gradient elution program is:

[0025]

[0026] Preferably, the chromatographic conditions of the high performance liquid chromatography are:

[0027] Chromatographic column: HiQ sil C18HS 4.6mm×250mm 5μm column;

[0028] Flow rate: 1.0 mL / min;

[0029] Injection volume: 25 μL;

[0030] Column temperature: 37°C;

[0031] Detection wavelength: 220nm;

[0032] Mobile phase A: 0.05 mol / L hexafluorophosphate solution. According to the acidity and alkalinity of the hexafluorophosphate solution, 1% phosphoric acid or 0.1 mol / L potassium dihydrogen phosphate was selected to adjust the pH to 4.5.

[0033] Mobile phase B: acetonitrile;

[0034] Taking the total volume of the mobile phase as 100%, the gradient elution program is as follows:

[0035]

[0036] Furthermore, the hexafluorophosphate solution includes potassium hexafluorophosphate solution, sodium hexafluorophosphate solution, and ammonium hexafluorophosphate solution.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. The present invention provides a method for detecting related substances in atosiban acetate and its preparations, which can effectively detect degradation impurities generated during the synthesis and storage of atosiban acetate: Di-Gly 9 -Atosiban, δ-Ava 7 -Atosiban, Des-Gly 9 -Atosiban, Gly 9 -ol-Atosiban, Asp 5 -Atosiban, Gly 9 -OH-Atosiban, Dimer (Gly-Orn), Des-Gly-Aminopiperidone 8 -Atosiban. This method has strong specificity, good repeatability and high accuracy, and can effectively control the quality of atosiban acetate and its preparations.

[0039] 2. The present invention adopts a special impurity separation method based on the structural characteristics of atosiban acetate impurities, and effectively detects impurities with similar polarity to the main component through self-comparison. The method system is simple and convenient to prepare, especially for impurity B (δ-Ava 7 -atosiban) with a resolution of more than 1.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present invention will be further described below in conjunction with the accompanying drawings.

[0041] Figure 1 It is the chromatogram of Comparative Example 1;

[0042] Figure 2 It is the chromatogram of Comparative Example 2;

[0043] Figure 3 It is the chromatogram of Comparative Example 3;

[0044] Figure 4 is the chromatogram under the condition that the concentration of mobile phase A in Example 1 is 0.05 mol / L;

[0045] Figure 5 is the chromatogram under the condition of mobile phase A pH 4.5 in Example 2;

[0046] Figure 6 is the chromatogram under the condition of column temperature of 37° C. in Example 3;

[0047] Figure 7 is a chromatogram using gradient elution program 5 in Example 4;

[0048] Figure 8This is the chromatogram in Example 5. DETAILED DESCRIPTION

[0049] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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.

[0050] Comparative Example 1

[0051] A method for detecting related substances in atosiban acetate and its preparations, using a high performance liquid chromatography method, comprises the following steps:

[0052] 1) Set the chromatographic conditions:

[0053] Instrument: Thermo Fisher U3000 HPLC

[0054] Chromatographic column: Inertsil ODS-34.6×250mm 5μm

[0055] Flow rate: 1.0mL / min

[0056] Injection volume: 25 μL

[0057] Column temperature: 45°C

[0058] Detection wavelength: 220nm

[0059] Mobile phase A: trifluoroacetic acid aqueous solution at pH 3.2

[0060] Mobile phase B: acetonitrile: methanol = 60:40 (v / v)

[0061] Taking the total volume of mobile phase as 100%, the gradient elution program is:

[0062]

[0063] 2) Use mobile phase A as diluent and prepare a diluent containing approximately total impurities (impurity B: δ-Ava 7 -Atosiban, Impurity C: Des-Gly 9 -Atosiban, Impurity D: Gly 9 -ol-atosiban) 3 μg and atosiban 0.6 mg system suitability solution were used for method validation;

[0064] 3) Accurately measure the system adaptability solution and inject it into the liquid chromatograph for detection, and record the chromatogram. The chromatogram is as follows: Figure 1 As shown;

[0065] Experimental results: Impurity B could not be completely separated from the main peak, while impurities C and D could be completely separated from the main peak.

[0066] Comparative Example 2

[0067] A method for detecting related substances in atosiban acetate and its preparations, using a high performance liquid chromatography method, comprises the following steps:

[0068] 1) Set the chromatographic conditions:

[0069] Instrument: Thermo Fisher U3000 HPLC

[0070] Chromatographic column: ZORBAX SB-C18 4.6×250mm 5μm

[0071] Flow rate: 1.0mL / min

[0072] Injection volume: 20 μL

[0073] Column temperature: 30°C

[0074] Detection wavelength: 220nm

[0075] Mobile phase A: 0.05 mol / L potassium dihydrogen phosphate, adjusted to pH = 2.3 with 1% phosphoric acid, then add 120 μL triethylamine

[0076] Mobile phase B: acetonitrile

[0077] Taking the total volume of mobile phase as 100%, the gradient elution program is:

[0078]

[0079] 2) Use mobile phase A: mobile phase B = 4:1 (v / v) as diluent to prepare a diluent containing about total impurities (impurity B: δ-Ava 7 -Atosiban, Impurity C: Des-Gly 9 -Atosiban, Impurity D: Gly 9 -ol-atosiban) 3 μg and atosiban 0.6 mg system suitability solution were used for method validation;

[0080] 3) Accurately measure the system adaptability solution and inject it into the liquid chromatograph for detection, and record the chromatogram. The chromatogram is as follows: Figure 2 As shown;

[0081] Experimental results: Impurity B could not be completely separated from the main peak, impurities C and D co-eluted but could be completely separated from the main peak.

[0082] Comparative Example 3

[0083] A method for detecting related substances in atosiban acetate and its preparations, using a high performance liquid chromatography method, comprises the following steps:

[0084] 1) Set the chromatographic conditions:

[0085] Instrument: High performance liquid chromatography waters

[0086] Chromatographic column: Inertsil ODS-24.6×250mm 5μm

[0087] Flow rate: 1.2mL / min

[0088] Injection volume: 25 μL

[0089] Column temperature: 60°C

[0090] Detection wavelength: 220nm

[0091] Mobile phase A: pH = 3.2 trifluoroacetic acid aqueous solution: acetonitrile: methanol = 75:15:10

[0092] Mobile phase B: acetonitrile: methanol = 60:40 (v / v)

[0093] Taking the total volume of mobile phase as 100%, the gradient elution program is:

[0094]

[0095] 2) Using a trifluoroacetic acid aqueous solution with a pH of 3.2 as a diluent, prepare a solution containing about 1 mL of impurity B: δ-Ava 7 - System suitability solutions of atosiban 3 μg and atosiban 0.6 mg for method validation;

[0096] 3) Accurately measure the system adaptability solution and inject it into the liquid chromatograph for detection, and record the chromatogram. The chromatogram is as follows: Figure 3 As shown;

[0097] Experimental results: The separation effect of impurity B and the main peak is poor, and the main peak has serious tailing.

[0098] Example 1

[0099] In this embodiment, the concentration gradient of the buffer salt concentration of mobile phase A is set, and the concentrations of mobile phase A are (0.01 mol / L, 0.05 mol / L, 0.08 mol / L, 0.1 mol / L) potassium hexafluorophosphate solution, and according to the acidity and alkalinity of the hexafluorophosphate solution, 1% phosphoric acid or 0.1 mol / L potassium dihydrogen phosphate is selected to adjust the pH to 3.0; and the method verification is performed;

[0100] A method for detecting related substances in atosiban acetate and its preparations, using a high performance liquid chromatography method, comprises the following steps:

[0101] 1) Set the chromatographic conditions:

[0102] Instrument: Thermo Fisher U3000 HPLC

[0103] Chromatographic column: Inertsil ODS-24.6×250mm 5μm

[0104] Flow rate: 1.0mL / min

[0105] Injection volume: 25 μL

[0106] Column temperature: 40°C

[0107] Detection wavelength: 220nm

[0108] Mobile phase B: acetonitrile

[0109] Taking the total volume of mobile phase as 100%, the gradient elution program is:

[0110]

[0111] 2) Using a trifluoroacetic acid aqueous solution with a pH of 3.2 as the diluent, prepare a solution containing approximately 1 mL of total impurities (impurity A: Di-Gly 9 -Atosiban, Impurity B: δ-Ava 7 -Atosiban, Impurity C: Des-Gly 9 -Atosiban, Impurity D: Gly 9 -ol-Atosiban, Impurity E: Asp 5 -Atosiban, Impurity F: Gly 9 -OH-Atosiban, Impurity G: Dimer (Gly-Orn), Impurity H: Des-Gly-Aminopiperidone 8 -Atosiban) 3 μg and atosiban 0.6 mg system suitability solution for method validation;

[0112] 3) Accurately measure the system adaptability solution and inject it into the liquid chromatograph for detection, and record the chromatogram. The chromatogram of the mobile phase A with a concentration of 0.05 mol / L potassium hexafluorophosphate solution adjusted to pH=3.0 with 1% phosphoric acid is as follows: Figure 4 As shown;

[0113] Experimental results: When the buffer concentration of mobile phase A is 0.05 mol / L, δ-Ava 7 -Atosiban peak has a relatively high separation from the main peak and a high number of theoretical plates.

[0114] Example 2

[0115] In this embodiment, the pH value of mobile phase A buffer salt is set to a concentration gradient, mobile phase A: 0.05 mol / L potassium hexafluorophosphate solution, according to the acidity and alkalinity of the hexafluorophosphate solution, 1% phosphoric acid or 0.1 mol / L potassium dihydrogen phosphate is selected to adjust the pH to 2.0, 2.4, 3.0, 3.6, 4.0, 4.5, 5.0, 6.0 respectively; method verification is performed;

[0116] A method for detecting related substances in atosiban acetate and its preparations, using a high performance liquid chromatography method, comprises the following steps:

[0117] 1) Set the chromatographic conditions:

[0118] Instrument: Thermo Fisher U3000 HPLC

[0119] Chromatographic column: Inertsil ODS-24.6×250mm 5μm

[0120] Flow rate: 1.0mL / min

[0121] Injection volume: 25 μL

[0122] Column temperature: 40°C

[0123] Detection wavelength: 220nm

[0124] Mobile phase B: acetonitrile

[0125] Taking the total volume of mobile phase as 100%, the gradient elution program is:

[0126]

[0127] 2) Using a trifluoroacetic acid aqueous solution with a pH of 3.2 as the diluent, prepare a solution containing approximately 1 mL of total impurities (impurity A: Di-Gly 9 -Atosiban, Impurity B: δ-Ava 7 -Atosiban, Impurity C: Des-Gly 9 -Atosiban, Impurity D: Gly 9 -ol-Atosiban, Impurity E: Asp 5 -Atosiban, Impurity F: Gly 9 -OH-Atosiban, Impurity G: Dimer (Gly-Orn), Impurity H: Des-Gly-Aminopiperidone 8 -Atosiban) 3 μg and atosiban 0.6 mg system suitability solution for method validation;

[0128] 3) Accurately measure the system adaptability solution and inject it into the liquid chromatograph for detection, and record the chromatogram. The chromatogram of the mobile phase A with a concentration of 0.05 mol / L potassium hexafluorophosphate solution adjusted to pH = 4.5 with 1% phosphoric acid is as follows: Figure 5 As shown;

[0129] Experimental results: The pH value of mobile phase A was in the range of 4.0-6.0, and δ-Ava 7 - Separation of Atosiban peak from the main peak and Des-Gly 9 -The separation of atosiban remained basically unchanged. Combined with the separation between other impurity peaks, when the pH value of mobile phase A was 4.5, the separation between the main peaks and impurity peaks and between the impurity peaks was relatively good.

[0130] Example 3

[0131] In this embodiment, the column temperature is set to a concentration gradient, and the column temperature is set to 30°C, 35°C, 37°C, 40°C, 45°C, and 50°C respectively; a methodological verification is performed; a method for detecting related substances in atosiban acetate and its preparations, using a high performance liquid chromatography method, comprising the following steps:

[0132] 1) Set the chromatographic conditions:

[0133] Instrument: Thermo Fisher U3000 HPLC

[0134] Chromatographic column: Inertsil ODS-24.6×250mm 5μm

[0135] Flow rate: 1.0mL / min

[0136] Injection volume: 25 μL

[0137] Detection wavelength: 220nm

[0138] Mobile phase A: 0.05 mol / L potassium hexafluorophosphate solution. According to the acidity and alkalinity of the hexafluorophosphate solution, 1% phosphoric acid or 0.1 mol / L potassium dihydrogen phosphate was selected to adjust the pH to 4.5;

[0139] Mobile phase B: acetonitrile

[0140] Taking the total volume of mobile phase as 100%, the gradient elution program is:

[0141]

[0142]

[0143] 2) Using a trifluoroacetic acid aqueous solution with a pH of 3.2 as the diluent, prepare a solution containing approximately 1 mL of total impurities (impurity A: Di-Gly 9-Atosiban, Impurity B: δ-Ava 7 -Atosiban, Impurity C: Des-Gly 9 -Atosiban, Impurity D: Gly 9 -ol-Atosiban, Impurity E: Asp 5 -Atosiban, Impurity F: Gly 9 -OH-Atosiban, Impurity G: Dimer (Gly-Orn), Impurity H: Des-Gly-Aminopiperidone 8 -Atosiban) 3 μg and atosiban 0.6 mg system suitability solution for method validation;

[0144] 3) Accurately measure the system adaptability solution and inject it into the liquid chromatograph for detection. Record the chromatogram. The column temperature is 37°C. Figure 6 As shown;

[0145] Experimental results: When the column temperature is 37℃, δ-Ava 7 -Atosiban peak has the best separation from the main peak and is closely related to Des-Gly 9 - The resolution of atosiban is relatively good.

[0146] Example 4

[0147] This embodiment sets 5 groups of different gradient elution program concentrations.

[0148] Taking the total volume of mobile phase as 100%, gradient elution program 1:

[0149]

[0150] Taking the total volume of mobile phase as 100%, gradient elution program 2:

[0151]

[0152]

[0153] Taking the total volume of mobile phase as 100%, gradient elution program 3:

[0154]

[0155] Taking the total volume of mobile phase as 100%, gradient elution program 4:

[0156]

[0157] Taking the total volume of mobile phase as 100%, gradient elution program 5:

[0158]

[0159] Conduct methodological validation;

[0160] A method for detecting related substances in atosiban acetate and its preparations, using a high performance liquid chromatography method, comprises the following steps:

[0161] 1) Set the chromatographic conditions:

[0162] Instrument: Thermo Fisher U3000 HPLC

[0163] Chromatographic column: Inertsil ODS-24.6×250mm 5μm

[0164] Flow rate: 1.0mL / min

[0165] Injection volume: 25 μL

[0166] Column temperature: 37°C

[0167] Detection wavelength: 220nm

[0168] Mobile phase A: 0.05 mol / L potassium hexafluorophosphate solution. According to the acidity and alkalinity of the hexafluorophosphate solution, 1% phosphoric acid or 0.1 mol / L potassium dihydrogen phosphate was selected to adjust the pH to 4.5;

[0169] Mobile phase B: acetonitrile

[0170] 2) Using a trifluoroacetic acid aqueous solution with a pH of 3.2 as the diluent, prepare a solution containing approximately 1 mL of total impurities (impurity A: Di-Gly 9 -Atosiban, Impurity B: δ-Ava 7 -Atosiban, Impurity C: Des-Gly 9 -Atosiban, Impurity D: Gly 9 -ol-Atosiban, Impurity E: Asp 5 -Atosiban, Impurity F: Gly 9 -OH-Atosiban, Impurity G: Dimer (Gly-Orn), Impurity H: Des-Gly-Aminopiperidone 8 -Atosiban) 3 μg and atosiban 0.6 mg system suitability solution for method validation;

[0171] 3) Accurately measure the system adaptability solution and inject it into the liquid chromatograph for detection, and record the chromatogram. The chromatogram of gradient elution program 5 is as follows: Figure 7 As shown;

[0172] Experimental results: When gradient elution program 5 was used, δ-Ava 7 -Atosiban peak has the best separation from the main peak and is closely related to Des-Gly9 - The resolution of atosiban is relatively good.

[0173] Example 5

[0174] In this example, two different sets of C18 chromatographic columns (HiQ sil C18 HS 4.6 mm × 250 mm 5 μm, Inertsil ODS-2 4.6 × 250 mm 5 μm) were used for method validation;

[0175] A method for detecting related substances in atosiban acetate and its preparations, using a high performance liquid chromatography method, comprises the following steps:

[0176] 1) Set the chromatographic conditions:

[0177] Instrument: Thermo Fisher U3000 HPLC

[0178] Flow rate: 1.0mL / min

[0179] Injection volume: 25 μL

[0180] Column temperature: 37°C

[0181] Detection wavelength: 220nm

[0182] Mobile phase A: 0.05 mol / L potassium hexafluorophosphate solution. According to the acidity and alkalinity of the hexafluorophosphate solution, 1% phosphoric acid or 0.1 mol / L potassium dihydrogen phosphate was selected to adjust the pH to 4.5;

[0183] Mobile phase B: acetonitrile

[0184] Taking the total volume of mobile phase as 100%, the gradient elution program is:

[0185]

[0186] 2) Using a trifluoroacetic acid aqueous solution with a pH of 3.2 as the diluent, prepare a solution containing approximately 1 mL of total impurities (impurity A: Di-Gly 9 -Atosiban, Impurity B: δ-Ava 7 -Atosiban, Impurity C: Des-Gly 9 -Atosiban, Impurity D: Gly 9 -ol-Atosiban, Impurity E: Asp 5 -Atosiban, Impurity F: Gly 9 -OH-Atosiban, Impurity G: Dimer (Gly-Orn), Impurity H: Des-Gly-Aminopiperidone 8 -Atosiban) 3 μg and atosiban 0.6 mg systemic solution;

[0187] 3) Accurately measure the system adaptability solution and inject it into the liquid chromatograph for detection, and record the chromatogram. The chromatogram of the HiQ silC18 HS 4.6mm×250mm 5μm column is as follows: Figure 8 As shown;

[0188] Experimental results: When using a chromatographic column (HiQ sil C18 HS 4.6mm×250mm 5μm), δ-Ava 7 -Atosiban peak has the best separation from the main peak and is closely related to Des-Gly 9 - Atosiban resolution greater than 1.5.

[0189] The separation results of the test are shown in Table 1:

[0190] Table 1

[0191]

[0192] Through the above-mentioned methodology verification, under the HiQ sil C18 HS 4.6mm×250mm 5μm chromatographic column used in this embodiment and the high performance liquid chromatography conditions provided, take an appropriate amount of atosiban acetate or preparation sample, dissolve it with a diluent to obtain a test solution; take the test solution, dilute it 100 times with the diluent (precisely measure 1mL of the test solution into a 100mL volumetric flask, dilute to the scale with the diluent, and shake well) to obtain a control solution; and perform high performance liquid chromatography detection on it, which can effectively detect 8 impurities in the sample; this method detects the structural characteristics of atosiban acetate impurities, and has high detection accuracy and good applicability.

[0193] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0194] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting related substances in atosiban acetate and its preparations, characterized in that: The content of related substances in atosiban acetate and its preparations was detected by high performance liquid chromatography, using buffered saline solution-organic phase as mobile phase and gradient elution procedure; The related substances include: Di-Gly 9 -Atosiban, δ-Ava 7 -Atosiban, Des-Gly 9 -Atosiban, Gly 9 -ol-Atosiban, Asp 5 -Atosiban, Gly 9 -OH-Atosiban, Dimer (Gly-Orn), Des-Gly-Aminopiperidone 8 -Atosiban.

2. The method for detecting related substances in atosiban acetate and its preparations according to claim 1, characterized in that: The detection method comprises the following steps: 1) Setting chromatographic conditions; 2) Take an appropriate amount of atosiban acetate raw material or preparation sample, dissolve it with diluent to obtain a test solution; take the test solution, dilute it 100 times with diluent to obtain a control solution; 3) Inject the test solution and the control solution into the liquid chromatograph for detection, record the chromatogram, and calculate the percentage of the peak area of ​​the relevant substance in the chromatogram of the test solution relative to the main peak area of ​​the control solution according to the self-control method.

3. The method for detecting related substances in atosiban acetate and its preparations according to claim 2, characterized in that: The chromatographic conditions are: Chromatographic column: C18 column; Flow rate: 0.8-1.2mL / min; Injection volume: 10-50 μL; Column temperature: 30-50°C; Detection wavelength: 200-240nm; Mobile phase A: 0.01-0.5 mol / L hexafluorophosphate solution, 1% phosphoric acid or 0.1 mol / L potassium dihydrogen phosphate, pH adjusted to 2.0-6.0; Mobile phase B: acetonitrile; Taking the total volume of the mobile phase as 100%, the gradient elution program is:

4. The method for detecting related substances in atosiban acetate and its preparations according to claim 3, characterized in that: The chromatographic column is a HiQ silC18HS 4.6mm×250mm 5μm chromatographic column.

5. The method for detecting related substances in atosiban acetate and its preparations according to claim 3, characterized in that: The mobile phase A is 0.05 mol / L hexafluorophosphate solution, 1% phosphoric acid or 0.1 mol / L dipotassium hydrogen phosphate adjusted to pH 4.5; And / or, the hexafluorophosphate solution includes potassium hexafluorophosphate solution, sodium hexafluorophosphate solution, and ammonium hexafluorophosphate solution.

6. The method for detecting related substances in atosiban acetate and its preparations according to claim 3, characterized in that: The column temperature was 37°C.

7. The method for detecting related substances in atosiban acetate and its preparations according to claim 3, characterized in that: Taking the total volume of the mobile phase as 100%, the gradient elution program is:

8. The method for detecting related substances in atosiban acetate and its preparations according to claim 2, characterized in that: The diluent is a trifluoroacetic acid aqueous solution with a pH of 3-3.5.

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