Method for detecting related substances in terlipressin acetate

Abstract

The invention relates to the field of pharmaceutical analysis, in particular to a method for detecting related substances in terlipressin acetate. The invention provides a method for detecting related substances in terlipressin acetate, which comprises the following steps: weighing a desmopressin acetate sample, and dissolving to prepare a test solution; measuring the test solution, diluting to prepare a contrast solution, respectively injecting the test solution and the contrast solution into a liquid chromatograph, taking octadecylsilane chemically bonded silica as a chromatographic column filler, adopting an ultraviolet detector for detection, selecting a hexafluorophosphate buffer solution as a mobile phase A and acetonitrile as a mobile phase B, carrying out gradient elution, recording a chromatogram, and determining the content of the test solution. And calculating the content of the related substances according to a self-contrast method. According to the method, impurities, such as process impurities and degraded impurities, which are generated in the synthesis process and the storage period of the terlipressin acetate and have similar polarity with main components can be effectively detected, the method is high in specificity, good in repeatability and high in accuracy, and the quality of the terlipressin acetate product can be effectively controlled.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical analysis, and particularly to a method for detecting related substances in terlipressin acetate. Background Art

[0002] Terlipressin acetate is a chemically synthesized polypeptide compound, and its chemical name is N-α-triglycyl-8-lysine-vasopressin. Its structural formula is as follows:

[0003]

[0004] Named L-glycyl-L-glycyl-L-glycyl-L-cysteinyl-L-tyrosyl-L-phenylalanyl-L-glutaminyl-L-asparaginyl-L-cysteinyl-L-prolyl-L-lysyl-L-glycinamide acetate (4→9-disulfide ring).

[0005] Terlipressin is a new type of synthetic long-acting vasopressin preparation. It is a prodrug and is itself inactive. After the action of aminopeptidase in vivo, three glycyl residues at its N-terminus are removed, and then the active lysine vasopressin is slowly "released". Therefore, terlipressin is equivalent to a storage depot that can release lysine vasopressin at a stable rate.

[0006] During the process of polypeptide synthesis, some impurities with structures similar to the target peptide chain will be generated, such as deletion peptides, insertion peptides, knot peptides, and epimers, etc. Peptide-related impurities can be introduced by starting materials, side reactions of the production process, or degradation during long-term storage. Therefore, in the research on the method for detecting related substances in synthetic polypeptide drugs, it is necessary to fully consider the detection efficiency of process impurities and degradation impurities. Since the structures and properties of some peptide-related impurities are similar to those of the main component, a single principle analysis method may not be able to completely separate them, and usually multiple detection methods with different principles are used for the detection of related substances. High performance liquid chromatography is the most commonly used analytical technique for polypeptide-related substances.

[0007] Currently, the publicly disclosed methods for analyzing related substances include: the quality standard of terlipressin acetate for injection of Ferring GmbH (standard number: YBH02332010); patent document CN105301156A discloses a method for analyzing related substances of terlipressin acetate for injection; terlipressin acetate (draft for soliciting opinions of the 2015 edition of the pharmacopoeia). However, the above publicly disclosed analysis methods cannot effectively separate some insertion peptides (such as Di-Gly 1 -terlipressin, etc.) and isomeric impurities (such as D-Cys 9 -terlipressin, etc.) generated during the synthesis of terlipressin acetate.

[0008] Therefore, it is necessary to develop a detection method that can separate these impurities similar to the main component from terlipressin acetate. Summary of the Invention

[0009] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a method for detecting related substances in terlipressin acetate, which can effectively separate and detect process impurities generated during the synthesis of terlipressin acetate and degradation impurities generated during its storage from the main component. This method has high sensitivity, good selectivity, simple operation, and low cost, meeting the requirements for comprehensive quality control of terlipressin acetate.

[0010] To achieve the above purpose and other related purposes, the present invention provides a method for detecting related substances in terlipressin acetate, including the following steps:

[0011] Step 1, weigh a sample of desmopressin acetate, dissolve it, and prepare a test solution.

[0012] Step 2, measure the test solution and dilute it to prepare a control solution.

[0013] Step 3, inject the test solution in Step 1 and the control solution in Step 2 into a liquid chromatograph respectively. Use octadecylsilane-bonded silica gel as the chromatographic column packing material, detect with an ultraviolet detector, select hexafluorophosphate buffer as mobile phase A and acetonitrile as mobile phase B for gradient elution, record the chromatogram, and calculate the percentage of the peak area of related substances in the test solution relative to the peak area of the main peak in the control solution according to the self-control method.

[0014] As described above, compared with the prior art, the present application has the following beneficial effects:

[0015] 1. The present invention provides a method for detecting related substances in terlipressin acetate, which can effectively detect process impurities and degradation impurities generated during the synthesis and storage of terlipressin acetate: process impurities such as deletion peptides, insertion peptides, mislinked peptides, epimeric peptides, and racemization impurities; degradation impurities such as peptide bond cleavage, deamidation, and β-elimination reaction impurities. This method has strong specificity, good repeatability, and high accuracy, and can effectively control the product quality of terlipressin acetate.

[0016] 2. In view of the structural characteristics of the impurities in terlipressin acetate, the present invention adopts a special impurity separation method, and through self-control, effectively detects impurities with polarity similar to the main component, such as insertion peptides (Di-Gly 1 -terlipressin, Di-Gly 12 -terlipressin, etc.), isomeric impurities (D-Cys 9 -terlipressin, D-Lys 11-Terlipressin, etc.). This method system is simple and convenient to formulate and easy to operate. Description of the Drawings

[0017] Figure 1 It is the chromatogram of gradient elution program 1 in Example 1.

[0018] Figure 2 It is the chromatogram of gradient elution program 2 in Example 1.

[0019] Figure 3 It is the chromatogram of gradient elution program 3 in Example 1.

[0020] Figure 4 It is the chromatogram of gradient elution program 4 in Example 1.

[0021] Figure 5 It is the chromatogram with a column temperature of 25°C in Example 2.

[0022] Figure 6 It is the chromatogram with a column temperature of 30°C in Example 2.

[0023] Figure 7 It is the chromatogram with a column temperature of 35°C in Example 2.

[0024] Figure 8 It is the chromatogram with a column temperature of 40°C in Example 2.

[0025] Figure 9 It is the chromatogram with a column temperature of 45°C in Example 2.

[0026] Figure 10 It is the chromatogram with a pH of 2.0 for phase A in Example 3.

[0027] Figure 11 It is the chromatogram with a pH of 2.2 for phase A in Example 3.

[0028] Figure 12 It is the chromatogram with a pH of 2.4 for phase A in Example 3.

[0029] Figure 13 It is the chromatogram with a pH of 2.6 for phase A in Example 3.

[0030] Figure 14 It is the chromatogram with a pH of 2.8 for phase A in Example 3.

[0031] Figure 15 It is the chromatogram with a pH of 3.0 for phase A in Example 3.

[0032] Figure 16 It is the chromatogram with a pH of 3.5 for phase A in Example 3.

[0033] Figure 17It is the chromatogram under the condition that the concentration of mobile phase A is 0.03 mol / L in Example 4.

[0034] Figure 18 It is the chromatogram under the condition that the concentration of mobile phase A is 0.04 mol / L in Example 4.

[0035] Figure 19 It is the chromatogram under the condition that the concentration of mobile phase A is 0.05 mol / L in Example 4.

[0036] Figure 20 It is the chromatogram under the condition that the concentration of mobile phase A is 0.06 mol / L in Example 4.

[0037] Figure 21 It is the chromatogram under the condition that the concentration of mobile phase A is 0.08 mol / L in Example 4.

[0038] Figure 22 It is the chromatogram under the condition that the concentration of mobile phase A is 0.1 mol / L in Example 4.

[0039] Figure 23 It is the representative chromatogram for sample detection in Example 6 Detailed implementation manners

[0040] In order to make the invention purpose, technical solutions and beneficial effects of this application clearer, the following further illustrates this application with reference to examples. It should be understood that the examples are only used to explain this application and are not used to limit the scope of the application. The test methods used in the following examples are all conventional methods unless otherwise specified. Those familiar with this technology can easily understand other advantages and effects of this application from the content disclosed in this description.

[0041] Impurities in drug quality standards refer to impurities introduced by the production process or raw materials in drugs produced according to the legally reviewed and approved process and raw and auxiliary materials, or impurities generated during storage, excluding new impurities generated by changing the production process or raw and auxiliary materials, nor including foreign substances incorporated or contaminated. Drug impurities are usually divided into: organic impurities, inorganic impurities, and residual solvents. Organic impurities can be introduced during the production or storage of drugs, or can be generated by the interaction between drugs and excipients or packaging structures. These impurities may be identified or unidentified, volatile or non-volatile. Among them, organic impurities with chemical structures similar to or related to the active ingredient are usually called related substances. The impurities in terlipressin acetate involved in this article are organic impurities with chemical structures similar to or related to the active ingredient, that is, related substances.

[0042] The inventors of this application have conducted a large number of exploratory researches and found a method for detecting related substances in terlipressin acetate, and completed this application on this basis.

[0043] The present application provides a method for detecting related substances in terlipressin acetate, comprising the following steps:

[0044] Step 1, weighing a desmopressin acetate sample and dissolving it to prepare a test solution;

[0045] Step 2, measuring the test solution and diluting it to prepare a control solution;

[0046] Step 3, injecting the test solution in Step 1 and the control solution in Step 2 into a liquid chromatograph respectively. Using octadecylsilyl silica gel as the chromatographic column packing, detecting with an ultraviolet detector, selecting hexafluorophosphate buffer solution as mobile phase A and acetonitrile as mobile phase B for gradient elution, recording the chromatogram, and calculating the percentage of the peak area of the related substances in the test solution relative to the main peak area of the control solution according to the self-control method.

[0047] Taking the percentage of the peak area of the related substances in the test solution relative to the main peak area of the control solution as the content of the related substances.

[0048] Specifically, the content of the related substances is calculated using the following formula:

[0049]

[0050] Cr——Concentration of the test solution, mg / mL;

[0051] Ar——Peak area of the related substances in the test solution;

[0052] Cs——Concentration of the self-control solution, mg / mL;

[0053] As——Main peak area in the self-control solution;

[0054] In the detection method provided by the present application, Step 1 refers to the preparation of the test solution: weighing a terlipressin acetate sample, dissolving it with a diluent to prepare a test solution, and the diluent can be water. Based on the total volume of the test solution, the concentration of the desmopressin acetate sample is 0.1 - 5 mg / ml; specifically, it can be 0.1 - 0.5 mg / ml, 0.5 - 1 mg / ml, or 1 - 5 mg / ml, etc.

[0055] Among them, optionally, the terlipressin acetate sample contains related substances, and the related substances include process impurities and / or degradation impurities. The process impurities include deletion peptides, insertion peptides, mislinked peptides, epimeric peptides, and / or racemization impurities, and the degradation impurities include peptide bond cleavage impurities, deamidation impurities, and / or β-elimination reaction impurities. More preferably, the related substances are selected from Gly 12 -OH-terlipressin, Glu 7 -Gly12 -OH Terlipressin, D-Lys 11 -Terlipressin, D-Phe 6 -Terlipressin, Lys 11 -OH-Terlipressin, Di-Gly 1 -Gly 2 -Terlipressin, Reduced Terlipressin, Di-Gly 1 -Terlipressin, β-Asp 8 -Terlipressin, Lys 11 (AC)-Terlipressin, D-Pro 10 -Terlipressin, AC-Gly 1 -Terlipressin, Des-Tyr 5 -Terlipressin, Asp 8 -Gly 12 -OH-Terlipressin, Des-Gly 12 -Terlipressin, Reduced Asp 8 -Terlipressin, Des-Phe 6 -Terlipressin, Reduced Glu 7 -Terlipressin, Des-Asn 8 -Terlipressin, Des-Pro 10 -Terlipressin, Di-Gly 12 -Terlipressin, D-Cys 9 -Terlipressin, D-Cys 4 -Terlipressin, D-Asn 8 -Terlipressin, D-Tyr 5 -Terlipressin, Des-Lys 11 -Terlipressin, D-Gln 7 -Terlipressin, Des-Gly 1 -Terlipressin, Des-Gly 1 -Gly 2 -Gly 3 -Terli, Des-Gly 1 -Gly 2 -Terlipressin, Des-Gln 7 -Terlipressin, Di-Gly 1 -Gly 2 -Gly 3 -Terlipressin, Glu 7 -Terlipressin, Asp 8 -Terlipressin, Gly 12 -OEt-Terlipressin, Terlipressin Dimer (Cys 9-Cys 9 )), terlipressin dimer (Cys 4 -Cys 9 ), or any one or more of them.

[0056] In the detection method provided by this application, step 2 refers to the preparation of the control solution: measure the test solution and dilute it with a diluent to prepare the control solution. The diluent can be water. Among them, based on the total volume of the control solution, the concentration of the desmopressin acetate sample is 0.0001 - 0.005 mg / ml; specifically, it can be 0.0001 - 0.001 mg / ml, 0.001 - 0.002 mg / ml, or 0.002 - 0.005 mg / ml, etc. The specific dilution factor of the control solution can be adjusted according to the experimental needs.

[0057] In the detection method provided by this application, step 3 refers to injecting the test solution in step 1 and the control solution in step 2 into a liquid chromatograph respectively. Using octadecylsilane-bonded silica gel as the chromatographic column packing and detecting with an ultraviolet detector, hexafluorophosphate buffer solution is used as mobile phase A and acetonitrile as mobile phase B for gradient elution. Record the chromatogram and calculate the content of related impurities by the self-control method based on the peak area. In some embodiments, the resolution calculation is automatically displayed by the chromatographic workstation report. When the resolution is greater than 1.5, it is considered that the related substances are successfully separated and detected from desmopressin acetate.

[0058] In step 3, the liquid chromatograph is an instrument familiar to those skilled in the art. For example, it can be a high-performance liquid chromatograph U3000, and other suitable chromatographs can also be used in this application.

[0059] In step 3, the mass concentration ratio of the test solution to the control solution is 1000:1 - 100:1; specifically, it can be 1000:1 - 500:1, 500:1 - 200:1, or 200:1 - 100:1, etc.

[0060] In step 3, the chromatographic column is a material familiar to those skilled in the art. For example, it can be Xselect HSS T3 2.5um 4.6×150mm 5μm, and other suitable chromatographic columns can also be used in this application.

[0061] In some embodiments, the gradient elution method is as follows:

[0062] From 0 to 18 minutes, the volume percentage of acetonitrile in the eluent is 15 - 22%; from 18 to 35 minutes, the volume percentage of acetonitrile in the eluent is 15 - 22%; from 35 to 50 minutes, the volume percentage of acetonitrile in the eluent is 32 - 38%; from 50 to 51 minutes, the volume percentage of acetonitrile in the eluent is 15 - 22%; from 51 to 60 minutes, the volume percentage of acetonitrile in the eluent is 15 - 22%.

[0063] In step 3, the detection wavelength of the ultraviolet detector is 200 - 240 nm. The flow rate through the chromatographic column is 0.8 - 1.2 ml / min. The injection volume is 10 - 50 μl. The hexafluorophosphate buffer solution is selected from potassium hexafluorophosphate solution, sodium hexafluorophosphate solution or ammonium hexafluorophosphate solution. The solvent of the above hexafluorophosphate buffer solution is water. The molar concentration of the hexafluorophosphate buffer solution is 0 - 0.5 mol / L, and the pH value is 2 - 6.

[0064] In some embodiments, in step 3, the pH value of the hexafluorophosphate buffer solution is 2.0 - 3.5. The test of Example 3 shows that when the pH value of mobile phase A is 2.6, the separation of the main peak from the front and rear impurities is relatively good, and the number of impurities separated from the main peak is more. Therefore, finally, when the pH value of mobile phase A is 2.6, it is selected as the pH value of mobile phase A in this method.

[0065] In some embodiments, in step 3, the column temperature of the liquid chromatography is 30 - 50 °C; preferably, the column temperature of the liquid chromatography is 40 °C. The test of Example 2 shows that when the column temperature is 40 °C, the separation of the main peak from the front and rear impurities is relatively good, and the number of impurities separated from the main peak is more. In summary, 40 °C is selected as the column temperature in this method.

[0066] In some embodiments, in step 3, the molar concentration of the hexafluorophosphate buffer solution is 0.03 - 0.1 mol / L; preferably, the molar concentration of the hexafluorophosphate buffer solution is 0.1 mol / L. The test of Example 4 shows that when the buffer salt concentration is 0.1 mol / L, the separation effect of the main peak from the front and rear impurities is the best. In summary, 0.1 mol / L is selected as the buffer salt concentration in this method.

[0067] In some embodiments, in step 3, the conditions of the gradient elution are:

[0068]

[0069] The test of Example 1 shows that when the elution gradient is 1 - 3, the separation of the main peak from the impurities and the separation of the impurities from each other are basically unchanged; when the elution gradient is 4, the separation of the main peak from the front and rear impurity peaks is relatively good. In summary, the elution gradient 4, that is, the above conditions, is selected as the elution gradient in this method.

[0070] Under the above optimal conditions, the resolution between the main peak and related substances of the detection method provided by the present invention meets the method requirements and has strong specificity. This application can effectively separate process impurities and degradation impurities from the main component, with strong specificity, good repeatability, and high accuracy, and can effectively control the quality of terlipressin acetate products.

[0071] The following uses specific specific examples to illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0072] Before further describing the specific implementation manners of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific specific implementation manners described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific specific implementation manners and not for limiting the protection scope of the present invention; in the specification and claims of the present invention, unless otherwise clearly indicated in the text, the singular forms "a", "an", and "the" include the plural forms.

[0073] When the embodiments give numerical ranges, it should be understood that unless otherwise specified in the present invention, any value between the two endpoints of each numerical range and any one of the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, equipment, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, equipment, and materials of the prior art similar to or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0074] Example 1

[0075] Four groups of different gradient elution program concentrations are set in this example.

[0076] A method for detecting related substances in terlipressin acetate, using high performance liquid chromatography, includes the following steps:

[0077] 1) Set chromatographic conditions:

[0078] Instrument: Thermo Fisher U3000 high performance liquid chromatograph

[0079] Chromatographic column: Xselect HSS T3 2.5um 4.6×150mm 5μm

[0080] Flow rate: 1.0 mL / min

[0081] Sample volume: 25 μL

[0082] Column temperature: 30 °C

[0083] Detection wavelength: 210 nm

[0084] Mobile phase A: 0.05 mol / L potassium hexafluorophosphate solution (adjusted to pH 2.4 with phosphoric acid)

[0085] Mobile phase B: acetonitrile

[0086] 2) Using water as the diluent, prepare a mixed solution containing about 4 μg of each impurity (a total of 17) and 0.4 mg of terlipressin per 1 mL as the impurity mixed solution.

[0087] Gradient elution program 1:

[0088]

[0089] Gradient elution program 2:

[0090]

[0091] Gradient elution program 3:

[0092]

[0093] Gradient elution program 4:

[0094]

[0095] Impurity information is as follows:

[0096] Table 1 Impurity information

[0097]

[0098]

[0099] 3) Precisely measure 20 μL of the impurity mixed solution and inject it into the liquid chromatograph, and record the chromatogram.

[0100] Experimental results: As Figures 1 to 4 shown, in the chromatogram of the impurity mixed solution, the changes in the program in gradient elution programs 1 to 3 have basically no effect on the separation between the main peak and impurities and between impurities. However, when gradient elution program 4 is used for investigation, the separation between the main peak and the front and rear impurity peaks is relatively better.

[0101] Example 2

[0102] In this example, the column temperature is set gradiently. Column temperature: Set 25 °C, 30 °C, 35 °C, 40 °C, 45 °C respectively.

[0103] A method for detecting related substances in terlipressin acetate, using high performance liquid chromatography, includes the following steps:

[0104] 1) Set the chromatographic conditions:

[0105] Instrument: Thermo Fisher U3000 high performance liquid chromatograph

[0106] Chromatographic column: Xselect HSS T3 2.5um 4.6×150mm 5μm

[0107] Flow rate: 1.0 mL / min

[0108] Injection volume: 25 μL

[0109] Detection wavelength: 210 nm

[0110] Mobile phase A: 0.05 mol / L potassium hexafluorophosphate solution (adjusted to pH 2.4 with phosphoric acid)

[0111] Mobile phase B: acetonitrile

[0112] Gradient elution program:

[0113]

[0114]

[0115] 2) Using water as a diluent, prepare a mixed solution containing about 4 μg of each impurity (a total of 17, see Table 1) and 0.4 mg of terlipressin per 1 mL as the impurity mixed solution.

[0116] 3) Precisely measure 20 μL of the impurity mixed solution and inject it into the liquid chromatograph, and record the chromatogram.

[0117] Experimental results: As Figures 5 to 9 shown, in the chromatogram of the impurity mixed solution, when the column temperature is 40 °C, the separation of the main peak from the front and back impurities is relatively good, and more impurities are separated from the main peak.

[0118] Example 3

[0119] In this example, the pH value of the buffer salt in mobile phase A is set in a gradient. Mobile phase A: 0.05 mol / L potassium hexafluorophosphate solution, adjusted to pH 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.5 with phosphoric acid.

[0120] A method for detecting related substances in atosiban acetate and its preparations, using high performance liquid chromatography, includes the following steps:

[0121] Chromatographic column: Xselect HSS T3 2.5um 4.6×150mm 5μm

[0122] Flow rate: 1.0 mL / min

[0123] Sample injection volume: 25 μL

[0124] Column temperature: 40 °C

[0125] Detection wavelength: 210 nm

[0126] Mobile phase B: Acetonitrile

[0127] Gradient elution program:

[0128]

[0129] 2) Using water as the diluent, prepare a mixed solution containing about 4 μg of each impurity (a total of 17, see Table 1) and 0.4 mg of terlipressin per 1 mL as the impurity mixed solution.

[0130] 3) Precisely measure 20 μL of the impurity mixed solution and inject it into the liquid chromatograph, and record the chromatogram.

[0131] Experimental results: As Figures 10 to 16 shown, in the chromatogram of the impurity mixed solution, when the pH is 2.6, the separation of the main peak from the front and rear impurities is relatively good, and more impurities are separated from the main peak.

[0132] Example 4

[0133] In this example, a concentration gradient is set for the buffer salt concentration of mobile phase A. Mobile phase A: Potassium hexafluorophosphate solutions with concentrations of (0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, 0.08 mol / L, 0.1 mol / L), adjusted to pH = 2.6 with phosphoric acid.

[0134] A method for detecting related substances in terlipressin acetate, using high performance liquid chromatography, includes the following steps:

[0135] 1) Set the chromatographic conditions:

[0136] Instrument: Thermo Fisher U3000 high performance liquid chromatograph

[0137] Chromatographic column: Xselect HSS T3 2.5um 4.6×150mm 5μm

[0138] Flow rate: 1.0 mL / min

[0139] Sample injection volume: 25 μL

[0140] Column temperature: 40 °C

[0141] Detection wavelength: 210 nm

[0142] Mobile phase B: Acetonitrile

[0143] Gradient elution program:

[0144]

[0145] 2) Using water as the diluent, prepare a mixed solution containing about 4 μg of each impurity (a total of 17, see Table 1) and 0.4 mg of terlipressin per 1 mL as the impurity mixed solution.

[0146] 3) Precisely measure 20 μL of the impurity mixed solution and inject it into the liquid chromatograph, and record the chromatogram.

[0147] Experimental results: As Figures 17 to 22 shown, in the chromatogram of the impurity mixed solution, the buffer salt concentration has a significant influence on the separation of the main peak from the impurities before and after. As the buffer salt concentration increases, the separation effect of the main peak from the impurities before and after becomes better. When the buffer salt concentration is 0.1 mol / L, the separation effect of the main peak from the impurities before and after is the best.

[0148] Example 5

[0149] In this example, a specificity test was carried out on the process impurities and degradation impurities that may be generated during the synthesis process and storage period of terlipressin acetate. The impurity information is as follows:

[0150] Table 2 Summary of impurity information

[0151]

[0152]

[0153] A method for detecting related substances in terlipressin acetate, using high performance liquid chromatography, includes the following steps: 1) Set the chromatographic conditions:

[0154] Instrument: Thermo Fisher U3000 high performance liquid chromatograph

[0155] Chromatographic column: Xselect HSS T3 2.5um 4.6×150mm 5μm

[0156] Flow rate: 1.0 mL / min

[0157] Injection volume: 25 μL

[0158] Column temperature: 40 °C

[0159] Detection wavelength: 210 nm

[0160] Mobile phase A: 0.1 mol / L hexafluorophosphate solution, adjusted to pH 2.6 with phosphoric acid;

[0161] Mobile phase B: acetonitrile

[0162] Gradient elution program:

[0163]

[0164]

[0165] 2) Using water as the diluent, prepare a mixed solution containing about 4 μg of each single impurity and 0.4 mg of terlipressin per 1 mL separately as the positioning resolution solution.

[0166] 3) Precisely measure 20 μL of the impurity mixed solution and inject it into the liquid chromatograph, record the chromatogram, and the positioning results are shown in the following table:

[0167] Table 3 Impurity positioning results

[0168]

[0169]

[0170] Experimental results: The chromatogram impurity positioning results show that the resolution between each impurity peak and the main peak is greater than 1.5, meeting the requirements.

[0171] Example 6

[0172] This example uses terlipressin acetate for the detection of related substance content.

[0173] 1) Set the chromatographic conditions:

[0174] Instrument: Thermo Fisher U3000 high performance liquid chromatograph

[0175] Chromatographic column: Xselect HSS T3 2.5um 4.6×150mm 5μm

[0176] Flow rate: 1.0 mL / min

[0177] Injection volume: 25 μL

[0178] Column temperature: 40 °C

[0179] Detection wavelength: 210 nm

[0180] Mobile phase A: 0.1 mol / L hexafluorophosphate solution, adjusted to pH 2.6 with phosphoric acid;

[0181] Mobile phase B: acetonitrile

[0182] Gradient elution program:

[0183]

[0184]

[0185] 2) Use water as the diluent to prepare a solution containing about 0.4 mg of terlipressin acetate per 1 mL as the test solution.

[0186] 3) Take the test sample solution and dilute it 100-fold with the diluent (accurately measure 1 mL of the test sample solution into a 100 mL volumetric flask, dilute to the mark with the diluent, and shake well) to obtain the control solution.

[0187] 4) Accurately measure 20 μL of the test sample solution and the control solution and inject them into the liquid chromatograph, record the chromatogram, and the calculation results are shown in Table 4.

[0188] Table 4 Detection Results of Related Substances in Terlipressin Acetate

[0189]

[0190] Under the high-performance liquid chromatography conditions provided in the above examples, take an appropriate amount of the terlipressin acetate sample, dissolve it with the diluent to obtain the test sample solution; take the test sample solution and dilute it 100-fold with the diluent (accurately measure 1 mL of the test sample solution into a 100 mL volumetric flask, dilute to the mark with the diluent, and shake well) to obtain the control solution; and perform high-performance liquid chromatography detection on it, and 37 impurities in the sample can be effectively detected; this method detects according to the structural characteristics of the impurities in terlipressin acetate, with high detection accuracy and good applicability.

[0191] The following comparative examples are mainly for the comparison of the investigation results of different mobile phase systems.

[0192] Comparative Example 1

[0193] A method for detecting related substances in terlipressin acetate, using high-performance liquid chromatography, includes the following steps:

[0194] 1) Method for using the quality standard of terlipressin acetate in the European Pharmacopoeia:

[0195] Instrument: Thermo Fisher U3000 high-performance liquid chromatograph

[0196] Chromatographic column: Waters Symmetry C18 5μm 3.0*150mm

[0197] Flow rate: 0.6 mL / min

[0198] Injection volume: 100 μL

[0199] Column temperature: 30 °C

[0200] Detection wavelength: 210 nm

[0201] Mobile phase A: (Buffer solution: 3.30 g of ammonium sulfate is dissolved in water, diluted to 5000 ml, and 1.0 ml of sulfuric acid is added and mixed evenly): methanol = 81.5:18.5 (v / v)

[0202] Mobile phase B: Buffer solution: methanol = 70:30 (v / v)

[0203] Gradient elution program:

[0204]

[0205] 2) Using water as the diluent, prepare a mixed solution containing about 4 μg of impurities and 0.4 mg of terlipressin per 1 mL separately, as the positioning resolution solution;

[0206] 3) Precisely measure the positioning resolution solution and inject it into the liquid chromatograph, record the chromatogram, and the experimental results are shown in Table 5

[0207] Table 5: Positioning and resolution results

[0208] Name RT RRT Resolution <![CDATA[Di-Gly 1 -Gly 2 -Gly 3 -Terlipressin]]> Co-eluting with the main peak 1.000 - <![CDATA[Di-Gly 1 -Gly 2 -Terlipressin]]> Co-eluting with the main peak 1.000 - <![CDATA[Di-Gly 1 -Terlipressin]]> Co-eluting with the main peak 1.000 - <![CDATA[Des-Gln 7 -terlipressin]]> Co-eluting with the main peak 1.000 - Reduced terlipressin 24.173 1.320 3.10 <![CDATA[D-Cys 9 -Terlipressin]]> 16.957 0.925 0.71 <![CDATA[D-Pro 10 -terlipressin]]> 21.497 1.170 0.73 <![CDATA[Des-Gly 12 -Terlipressin]]> 21.247 1.156 0.78 <![CDATA[D-Asn 8 -terlipressin]]> Co-eluting with the main peak 1.000 - <![CDATA[D-Lys 11 -terlipressin]]> 21.193 1.148 0.83 <![CDATA[Di-Gly 12 -Terlipressin]]> Co-eluting with the main peak 1.000 -

[0209] Experimental results: There are 11 impurities in the above table. Only the separation degree between the impurity reduced terlipressin and the main peak meets the requirements. 6 impurity peaks co-elute with the main peak, and the separation degree between 4 impurity peaks and the main peak is less than 1.5, not meeting the requirements.

[0210] Comparative Example 2

[0211] A method for detecting related substances in terlipressin acetate, using high performance liquid chromatography, including the following steps:

[0212] 1) Draft for comments of the 2015 Edition Pharmacopoeia:

[0213] Instrument: Thermo Fisher U3000 high performance liquid chromatograph

[0214] Chromatographic column: Welch Ultimate AQ-C18 4.6×250 mm 5 μm

[0215] Flow rate: 1.0 mL / min

[0216] Injection volume: 20 μL

[0217] Column temperature: 40 °C

[0218] Detection wavelength: 210 nm

[0219] Mobile phase A: 0.067 mol / L sodium dihydrogen phosphate solution (adjusted to pH 3.5 with phosphoric acid)

[0220] Mobile phase B: Acetonitrile

[0221] Gradient elution program:

[0222]

[0223] 2) Using water as the diluent, prepare a mixed solution containing about 4 μg of impurities and 0.4 mg of terlipressin per 1 mL separately as the positioning resolution solution;

[0224] 3) Precisely measure the positioning resolution solution and inject it into the liquid chromatograph, record the chromatogram, and the experimental results are shown in Table 6;

[0225] Table 6: Positioning and resolution results

[0226]

[0227]

[0228] Experimental results: There are a total of 17 impurities in the above table. The resolution between 9 impurity peaks and the main peak meets the requirements; 4 impurity peaks co-elute with the main peak; the resolution between 4 impurity peaks and the main peak is less than 1.5, not meeting the requirements.

[0229] The above embodiments are intended to illustrate the embodiments disclosed in the present invention and should not be construed as a limitation of the present invention. In addition, various modifications listed herein and changes in the methods of the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in connection with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, all obvious modifications to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A method for detecting related substances in terlipressin acetate, comprising the following steps: Step 1, weighing a desmopressin acetate sample and dissolving it to prepare a test solution; Step 2, measuring the test solution and diluting it to prepare a control solution; Step 3, injecting the test solution in Step 1 and the control solution in Step 2 into a liquid chromatograph respectively. Using octadecylsilane-bonded silica gel as the chromatographic column packing material, detecting with an ultraviolet detector, selecting hexafluorophosphate buffer as mobile phase A and acetonitrile as mobile phase B, performing gradient elution, recording the chromatogram, and calculating the percentage of the peak area of the related substances in the test solution relative to the main peak area of the control solution according to the self-control method.

2. The detection method according to claim 1, characterized in that, In Step 1, the terlipressin acetate sample contains related substances, and the related substances include process impurities and / or degradation impurities; And / or, based on the total volume of the test solution, the concentration of the desmopressin acetate sample is 0.1 - 5 mg / ml.

3. The detection method according to claim 1 or 2, characterized in that, The related substances are selected from Gly 12 -OH-terlipressin, Glu 7 -Gly 12 -OH terlipressin, D-Lys 11 -terlipressin, D-Phe 6 -terlipressin, Lys 11 -OH-terlipressin, Di-Gly 1 -Gly 2 -terlipressin, reduced terlipressin, Di-Gly 1 -terlipressin, β-Asp 8 -terlipressin, Lys 11 (AC)-terlipressin, D-Pro 10 -terlipressin, AC-Gly 1 -terlipressin, Des-Tyr 5 -terlipressin, Asp 8 -Gly 12 -OH-terlipressin, Des-Gly 12 -terlipressin, reduced Asp 8 -terlipressin, Des-Phe 6 -terlipressin, reduced Glu 7 -terlipressin, Des-Asn 8 -terlipressin, Des-Pro 10 -terlipressin, Di-Gly 12 -terlipressin, D-Cys 9 -terlipressin, D-Cys 4 -terlipressin, D-Asn 8 -terlipressin, D-Tyr 5 -terlipressin, Des-Lys 11 -terlipressin, D-Gln 7 -terlipressin, Des-Gly 1 -terlipressin, Des-Gly 1 -Gly 2 -Gly 3 -terlipressin, Des-Gly 1 -Gly 2 -terlipressin, Des-Gln 7 -terlipressin, Di-Gly 1 -Gly 2 -Gly 3 -terlipressin, Glu 7 -terlipressin, Asp 8 -Terlipressin, Gly 12 -OEt-terlipressin, terlipressin dimer (Cys 9 -Cys 9 ), terlipressin dimer (Cys 4 -Cys 9 ) or any one or more thereof.

4. The detection method according to claim 1, wherein In Step 2, based on the total volume of the control solution, the concentration of the desmopressin acetate sample is 0.0001 - 0.005 mg / ml.

5. The detection method according to claim 1, characterized in that In Step 3, the concentration ratio of the test solution to the control solution is 1000:1 - 100:

1.

6. The detection method according to claim 1, characterized in that In Step 3, the gradient elution method is as follows: From 0 to 18 minutes, the volume percentage of acetonitrile in the eluent is 15 - 22%, from 18 to 35 minutes, the volume percentage of acetonitrile in the eluent is 15 - 22%, from 35 to 50 minutes, the volume percentage of acetonitrile in the eluent is 32 - 38%, from 50 to 51 minutes, the volume percentage of acetonitrile in the eluent is 15 - 22%, from 51 to 60 minutes, the volume percentage of acetonitrile in the eluent is 5 - 22%.

7. The detection method according to claim 1, wherein In Step 3, the detection wavelength of the ultraviolet detector is 200 - 240 nm; And / or, the flow rate through the chromatographic column is 0.8 - 1.2 ml / min; And / or, the injection volume is 10 - 50 μl; And / or, the hexafluorophosphate buffer is selected from potassium hexafluorophosphate solution, sodium hexafluorophosphate solution or ammonium hexafluorophosphate solution; And / or, the concentration of the hexafluorophosphate buffer is 0 - 0.5 mol / L, and the pH value is 2 - 6.

8. The detection method according to claim 7, wherein The pH value of the hexafluorophosphate buffer is 2.0 - 3.5; And / or, the concentration of the hexafluorophosphate buffer is 0.03 - 0.1 mol / L; And / or, the column temperature of the liquid chromatography is 30 - 50 °C.

9. The detection method according to claim 8, wherein, The pH value of the hexafluorophosphate buffer is 2.6; And / or, the column temperature of the liquid chromatography is 40 °C; And / or, the concentration of the hexafluorophosphate buffer is 0.1 mol / L.

10. The detection method according to claim 1, wherein The conditions for the gradient elution are:

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