Carbetocin injection isomer detection method

Abstract

The invention relates to a carbetocin injection isomer detection method, and belongs to the technical field of polypeptide drug impurity analysis. The carbetocin injection isomer detection method comprises the following steps: detecting Ile position isomer impurities in 8-site Leu in a carbetocin raw material by adopting a high performance liquid chromatography; the adopted chromatographic conditions are as follows: octadecyl silane bonded silica gel is taken as a filling agent, acetonitrile-0. 02 mol / L ammonium acetate-0.03 mol / L monopotassium phosphate is taken as a mobile phase A, methanol-acetonitrile is taken as a mobile phase B, and gradient elution is performed; the detection wavelength is 210 to 230 nm; the column temperature is 40-70 DEG C; the flow velocity is 0.5 to 1.2 mL / min; and the sample injection volume is 10-40 [mu] L. The carbetocin injection isomer detection method is high in accuracy and good in sensitivity, repeatability and precision meet requirements, position isomer impurities can be accurately detected, and the product quality can be better controlled.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide drug impurity analysis, and particularly relates to a method for detecting isomers in carbetocin injection. Background Art

[0002] Carbetocin injection was developed by Ferring Pharmaceuticals AG of Switzerland. It is a synthetic long-acting oxytocin nonapeptide agonist analogue, and was first marketed in Canada in June 1997. It can be used for preventing uterine atony and postpartum hemorrhage after cesarean section under selective epidural or spinal anesthesia, and has the advantages of rapid onset, long action time, clinical effectiveness, and low side effects.

[0003] The molecular formula of carbetocin is (C 45 H 69 N 11 O 12 S), its chemical name is desamino-2-oxo-methyltyrosine-1-k-oxytocin, with a molecular weight of 988.16, and its structural formula is as follows:

[0004] <{

[0005] The molecular formula of the isomer impurity at the Ile position in Leu at position 8 of carbetocin is (C 45 H 69 N 11 O 12 S), with a molecular weight of 988.16, and its structural formula is as follows:

[0006]

[0007] This isomer impurity is a possible impurity in the raw material drug. Its existence affects the quality of the raw material drug and the preparation. However, there is no method for detecting this isomer impurity in the existing technology. Therefore, it is necessary to develop a method for detecting this isomer impurity. Summary of the Invention

[0008] The object of the present invention is to provide a method for detecting isomers in carbetocin injection, which is a method for detecting the isomer impurity at the Ile position in Leu at position 8 in the carbetocin raw material. Using the detection method of the present invention can better control the product quality.

[0009] To solve the above technical problems, the technical solution of the present invention is specifically as follows:

[0010] A method for detecting isomers in carbetocin injection, comprising the following steps:

[0011] Determine the Ile positional isomer impurity in the 8 - Leu of carbetocin raw material in carbetocin injection by high - performance liquid chromatography;

[0012] The chromatographic conditions are as follows:

[0013] Using octadecylsilane chemically bonded silica gel as the filler, acetonitrile - 0.02mol / L ammonium acetate - 0.03mol / L potassium dihydrogen phosphate as mobile phase A, and methanol - acetonitrile as mobile phase B, with gradient elution; the detection wavelength is 210 - 230nm; the column temperature is 40 - 70°C; the flow rate is 0.5 - 1.2mL / min; the injection volume is 10 - 40μL.

[0014] In the above technical solution, the volume ratio of acetonitrile, ammonium acetate and potassium dihydrogen phosphate in mobile phase A is 70:20:10 - 40:30:30, and the pH value is 4.5 - 7.0.

[0015] In the above technical solution, the volume ratio of acetonitrile, ammonium acetate and potassium dihydrogen phosphate in mobile phase A is 60:25:15, and the pH value is 6.1.

[0016] In the above technical solution, the volume ratio of methanol to acetonitrile in mobile phase B is 15:85 - 90:10.

[0017] In the above technical solution, the volume ratio of methanol to acetonitrile in mobile phase B is 65:35.

[0018] In the above technical solution, the detection wavelength is 220nm; the column temperature is 45°C; the flow rate is 0.8mL / min; the injection volume is 10μL.

[0019] In the above technical solution, the chromatographic column used is selected from any one of the following chromatographic columns:

[0020] Thermo Hyersil BDS C18, 150mm×4.6mm, 3.5μm;

[0021] Agilent ZOBAX C18, 150mm×4.6mm, 3.5μm;

[0022] Waters Xbridge C18, 150mm×4.6mm, 3.5μm.

[0023] The beneficial effects of the present invention are:

[0024] The present invention is a method for detecting isomers of carbetocin injection, with high accuracy, good sensitivity, and repeatability and precision meeting the requirements, enabling accurate detection of positional isomer impurities and better controlling the product quality. Description of the Drawings

[0025] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0026] Figure 1 It is the linear graph of the carbetocin position isomer in Example 1.

[0027] Figure 2 It is the linear graph of carbetocin in Example 1.

[0028] Figure 3 It is the linear graph of the carbetocin position isomer in Example 2.

[0029] Figure 4 It is the linear graph of carbetocin in Example 2.

[0030] Figure 5 It is the linear graph of the carbetocin position isomer in Example 3.

[0031] Figure 6 It is the linear graph of carbetocin in Example 3.

[0032] Figure 7 It is the linear graph of the carbetocin position isomer in Example 4.

[0033] Figure 8 It is the linear graph of carbetocin in Example 4. Specific Embodiments

[0034] The inventive concept of the present invention is: The object of the present invention is to provide a detection method for detecting the Ile position isomer impurity in the 8 - Leu of carbetocin raw material, so as to better control the product quality.

[0035] A method for detecting carbetocin injection isomers of the present invention specifically includes the following steps:

[0036] Using high - performance liquid chromatography to detect the Ile position isomer impurity in the 8 - Leu of carbetocin raw material in carbetocin injection;

[0037] The chromatographic conditions are: using octadecylsilane - bonded silica gel as the filler (chromatographic column: 150mm×4.6mm, 3.5μm or a chromatographic column with equivalent efficiency); using acetonitrile - 0.02mol / L ammonium acetate - 0.03mol / L potassium dihydrogen phosphate (volume ratio 70:20:10 - 40:30:30, pH value 4.5 - 7.0) as mobile phase A, and methanol - acetonitrile (volume ratio 15:85 - 90:10) as mobile phase B for gradient elution; detection wavelength: 210 - 230nm; column temperature: 40 - 70°C; flow rate 0.5 - 1.2mL / min; injection volume: 10 - 40μL.

[0038] Test solution: Accurately measure an appropriate amount of this product (carbetocin injection), and dilute it with mobile phase A to prepare a solution containing about 20-100 μg of carbetocin per 1 mL.

[0039] Reference solution: Take an appropriate amount of carbetocin reference substance, dissolve and dilute it with mobile phase A to prepare a solution containing about 0.2-1 μg of carbetocin per 1 mL.

[0040] System suitability: Take appropriate amounts of carbetocin reference substance and carbetocin position isomer reference substance, dissolve and dilute them with mobile phase A to prepare a mixed solution containing about 20-100 μg of carbetocin and 0.2-1 μg of carbetocin position isomer per 1 mL.

[0041] System suitability requirements: In the system suitability chromatogram, the resolution between the carbetocin peak and the carbetocin position isomer peak should meet the requirements, and the number of theoretical plates calculated based on the carbetocin isomer peak should be greater than 2000.

[0042] Assay method:

[0043] Accurately measure the above solutions respectively, inject them into a high performance liquid chromatograph, record the chromatogram, and calculate by the external standard method based on the peak area.

[0044] The present invention will be described in more detail below with reference to the accompanying drawings and embodiments.

[0045] Example 1

[0046] A method for detecting isomers of carbetocin injection, wherein the isomer is the Ile position isomer impurity in Leu at position 8 in the carbetocin raw material;

[0047] (1) Experimental conditions, reagents and test articles:

[0048] Instrument: Dionex U3000 high performance liquid chromatograph, DAD detector;

[0049] Balance: XSE 205DU electronic balance, Mettler Toledo (Shanghai) Instruments Co., Ltd.;

[0050] Reagents: Acetonitrile and methanol are both chromatographically pure, ammonium acetate and potassium dihydrogen phosphate are both analytically pure;

[0051] Reference substances:

[0052] Carbetocin (purchased from TRC), carbetocin position isomer (purchased from SINCO);

[0053] (2) Experimental method:

[0054] Preparation of test solution: This product (carbetocin injection, prepared by oneself according to the following composition);

[0055] The composition of carbetocin injection is as follows:

[0056] Carbetocin 0.1 mg, methionine 1 mg, succinic acid 1.19 mg, mannitol 47 mg. The amount of sodium hydroxide is added according to the control of the solution pH value to 5.5, and water for injection is added to 1 mL.

[0057] Carbetocin reference solution: Take an appropriate amount of carbetocin reference substance, weigh it accurately, dissolve it with mobile phase A and quantitatively dilute it to prepare a solution containing about 1 μg per 1 mL.

[0058] Carbetocin position isomer reference solution: Take an appropriate amount of carbetocin position isomer reference substance, weigh it accurately, dissolve it with mobile phase A and quantitatively dilute it to prepare a solution containing about 1 μg per 1 mL.

[0059] System suitability: Take appropriate amounts of carbetocin reference substance and carbetocin position isomer reference substance, dissolve them with mobile phase A and dilute to prepare a mixed solution containing about 100 μg of carbetocin and 1 μg of carbetocin position isomer per 1 mL.

[0060] Assay method:

[0061] Use Thermo Hyersil BDS C18 (150 mm×4.6 mm, 3.5 μm) as the chromatographic column; use acetonitrile - 0.02 mol / L ammonium acetate - 0.03 mol / L potassium dihydrogen phosphate (60:25:15, pH value 6.1) as mobile phase A, and methanol - acetonitrile (65:35) as mobile phase B for gradient elution; detection wavelength: 220 nm; column temperature: 45 °C; flow rate: 0.8 mL / min; injection volume: 10 μL.

[0062]

[0063]

[0064] System suitability requirements: In the system suitability chromatogram, the resolution between the carbetocin peak and the carbetocin position isomer peak should meet the requirements, and the number of theoretical plates calculated based on the carbetocin isomer peak should be greater than 8000.

[0065] (3) Methodology investigation and validation:

[0066] 1) Specificity:

[0067] Solvent: Acetonitrile - 0.02 mol / L ammonium acetate - 0.03 mol / L potassium dihydrogen phosphate (volume ratio 60:25:15, pH value 6.1) (mobile phase A).

[0068] Blank excipient solution: Prepare a blank excipient solution without carbetocin according to the prescription ratio and preparation process of carbetocin injection, and you will get it.

[0069] Test solution: This product (carbetocin injection).

[0070] Carbetocin reference solution: Take an appropriate amount of carbetocin reference substance, weigh it accurately, dissolve it with a solvent and quantitatively dilute it to prepare a solution containing about 1 μg per 1 mL.

[0071] Carbetocin position isomer reference solution: Take an appropriate amount of carbetocin position isomer reference substance, weigh it accurately, dissolve it with a solvent and quantitatively dilute it to prepare a solution containing about 1 μg per 1 mL.

[0072] System suitability solution: Take appropriate amounts of carbetocin reference substance and carbetocin position isomer reference substance, dissolve them with a solvent and dilute them to prepare a mixed solution containing about 100 μg of carbetocin and 1 μg of carbetocin position isomer per 1 mL.

[0073] Take 10 μL of each of the above solutions and inject them into a high performance liquid chromatograph respectively, and record the chromatogram.

[0074] Experimental results: The solvent and the blank excipient have no interference, and the resolution of the system suitability meets the requirements.

[0075] 2) Quantification limit:

[0076] Take appropriate amounts of carbetocin and carbetocin position isomer reference substances, dissolve them with a solvent respectively and dilute them step by step until the signal-to-noise ratio is about 10. The quantification limit results are shown in Table 1.

[0077] Table 1 Quantification limit results

[0078]

[0079] Take the test solution at the quantification limit concentration and inject it continuously for 6 times. The test results are used as the results for investigating the repeatability of the quantification limit. The statistical results of the retention time and peak area for the repeatability determination of the quantification limit are shown in Table 2-3 below.

[0080] Table 2 Repeatability of the quantification limit of carbetocin

[0081]

[0082] Table 3 Repeatability of the quantification limit of carbetocin position isomer

[0083]

[0084] It can be seen from Tables 1-3 that the conclusion is:

[0085] The quantitation limit of carbetocin is 0.39 ng, which is equivalent to 0.04% of the concentration of the test solution. The RSD of the retention time of carbetocin is 0.2%, less than 2%, and the RSD of the peak area is 2.3%, less than 15%, meeting the requirements. The quantitation limit of the positional isomer of carbetocin is 0.40 ng, which is equivalent to 0.04% of the concentration of the test solution. The RSD of the retention time of the positional isomer of carbetocin is 0.2%, less than 2%, and the RSD of the peak area is 5.4%, less than 15%, meeting the requirements. Thus, it can be seen that the method of the present invention has good sensitivity and can meet the detection requirements.

[0086] 3) Detection limit:

[0087] Take appropriate amounts of the reference substance solutions of carbetocin and the positional isomer of carbetocin, dissolve them separately with the solvent and dilute step by step until the signal-to-noise ratio is about 3. The results are shown in Table 4.

[0088] Table 4 Results of detection limit

[0089]

[0090] The experimental results in Table 4 show that: the detection limit of the impurity of the positional isomer of carbetocin is 0.13 ng, which is approximately equivalent to 0.01% of the concentration of the test solution, and the detection limit of carbetocin is 0.13 ng, which is approximately equivalent to 0.01% of the concentration of the test solution. Thus, it can be seen that the method of the present invention can ensure the effective detection of the impurity of the positional isomer of carbetocin.

[0091] 4) Linearity:

[0092] Take appropriate amounts of the reference substance solutions of carbetocin and the positional isomer of carbetocin, dissolve them separately with the solvent and dilute step by step to prepare reference substance solutions at the quantitation limit, 50% limit to 150% limit, and determine the linearity. The results are shown in Tables 5 and 6, and Figure 1 and 2 :

[0093] Table 5 Results of linearity determination of the positional isomer of carbetocin

[0094]

[0095]

[0096] Table 6 Results of linearity determination of carbetocin

[0097]

[0098] The experimental results show that: for the cabergoline position isomers, in the concentration range of 0.0410 μg / mL to 1.5614 μg / mL, the correlation coefficient r is 0.9998, indicating a good linear relationship; for cabergoline, in the concentration range of 0.0388 μg / mL to 1.5320 μg / mL, the correlation coefficient r is 1.0000, indicating a good linear relationship.

[0099] 5) Repeatability:

[0100] Referring to the detection method for the position isomers of this product (cabergoline injection), prepare 1 portion of the system suitability solution, 6 portions of the test sample spiked solution, and 2 portions of the reference solution, inject them into the high performance liquid chromatograph respectively, record the chromatogram, and the results are shown in Table 7.

[0101] System suitability solution: Take appropriate amounts of cabergoline reference substance and cabergoline position isomer reference substance, dissolve and dilute with the solvent to prepare a mixed solution containing about 100 μg of cabergoline and 1 μg of cabergoline position isomer per 1 mL.

[0102] Test sample spiked solution: Take an appropriate amount of cabergoline position isomer reference substance, dissolve and dilute with this product to prepare a solution containing about 1 μg of the position isomer per 1 mL.

[0103] Cabergoline reference solution: Take an appropriate amount of cabergoline reference substance, accurately weigh it, dissolve and quantitatively dilute with the solvent to prepare a solution containing about 1 μg per 1 mL.

[0104] Table 7 Repeatability results

[0105]

[0106] From the results in Table 7, the conclusion can be drawn that: the repeatability mean is 0.99%, and the RSD is 2.2%, meeting the standard requirements. Therefore, it shows that the method of this product has good repeatability.

[0107] 6) Intermediate precision:

[0108] By different people at different times, referring to the detection method for the cabergoline position isomers of this product, see 5) Repeatability to prepare 1 portion of the system suitability solution, 6 portions of the test sample spiked solution, and 2 portions of the reference solution, inject them into the high performance liquid chromatograph respectively, record the chromatogram, and the results are shown in Table 8.

[0109] Table 8 Precision results

[0110]

[0111] As can be seen from the results in Table 8, the conclusion is that the mean intermediate precision is 1.03% and the RSD is 3.1%, meeting the standard requirements. The mean of 12 data from the repeatability test and the intermediate precision test is 1.01% and the RSD is 3.1%, indicating that this method has good precision.

[0112] 7) Accuracy:

[0113] Stock solution of carbetocin position isomer reference substance: Take an appropriate amount of carbetocin position isomer reference substance, weigh accurately, dissolve and dilute with solvent to prepare a solution containing about 100 μg per 1 mL.

[0114] Carbetocin position isomer reference substance solution: Take an appropriate amount of carbetocin position isomer reference substance, weigh accurately, dissolve and dilute with solvent to prepare a solution containing about 1 μg per 1 mL.

[0115] Test solution: This product (carbetocin injection).

[0116] High test solution: Accurately measure an appropriate amount of the reference substance solution, and dilute it with this product (carbetocin injection) to prepare a solution containing about 1.5 μg of carbetocin position isomer per 1 mL.

[0117] Medium test solution: Accurately measure an appropriate amount of the reference substance solution, and dilute it with this product (carbetocin injection) to prepare a solution containing about 1.0 μg of carbetocin position isomer per 1 mL.

[0118] Low test solution: Accurately measure an appropriate amount of the reference substance solution, and dilute it with this product (carbetocin injection) to prepare a solution containing about 0.5 μg of carbetocin position isomer per 1 mL.

[0119] Test solution (quantitation limit concentration): Accurately measure an appropriate amount of the reference substance solution, and dilute it with this product (carbetocin injection) to prepare a solution containing about 0.041 μg of carbetocin position isomer per 1 mL.

[0120] Accurately measure the above solutions respectively and inject them into the high performance liquid chromatograph, record the chromatogram, and the results are shown in Table 9.

[0121] Table 9 Results of accuracy

[0122]

[0123]

[0124] The experimental results in Table 9 show that the recoveries of carbetocin position isomers are 98.34%, 99.63%, 99.98%, and 99.72% in sequence, the RSD value is 1.0%, the recoveries are all between 75% and 120%, and the RSD is within 8%, indicating that the method of the present invention has good accuracy.

[0125] Conclusion: The method of the present invention has high accuracy, good sensitivity, and the specificity, linearity, repeatability, and precision all meet the requirements, and can well control the isomer impurities in carbetocin injection. Example 1 is the best example, with simple operation method, stable baseline, high sensitivity and good repeatability.

[0126] Example 2

[0127] A method for detecting isomers in carbetocin injection, wherein the isomers are the isomer impurities at the Ile position in Leu at the 8th position in the carbetocin raw material;

[0128] (1) Experimental conditions, reagents and test drugs:

[0129] Instrument: Agilent 1260 high performance liquid chromatograph, DAD detector;

[0130] Balance: XSE 205DU electronic balance, Mettler Toledo (Shanghai) Instruments Co., Ltd.;

[0131] Reagents: Acetonitrile and methanol are both chromatographically pure, and ammonium acetate and potassium dihydrogen phosphate are both analytically pure;

[0132] Reference substances: Carbetocin (purchased from TRC), carbetocin position isomer (purchased from SINCO);

[0133] (2) Experimental method:

[0134] Solvent: Acetonitrile - 0.02 mol / L ammonium acetate - 0.03 mol / L potassium dihydrogen phosphate (70:20:10, pH value is 5.5), that is, mobile phase A.

[0135] System suitability solution: Take appropriate amounts of carbetocin reference substance and carbetocin position isomer reference substance, dissolve and dilute with mobile phase A to prepare a mixed solution containing about 20 μg of carbetocin and 0.2 μg of carbetocin position isomer per 1 mL.

[0136] Preparation of test solution: Precisely measure an appropriate amount of this product (carbetocin injection), dissolve and dilute with mobile phase A to prepare a solution containing about 20 μg of carbetocin per 1 mL.

[0137] Carbetocin reference substance solution: Take an appropriate amount of carbetocin reference substance, precisely weigh it, dissolve and quantitatively dilute with mobile phase A to prepare a solution containing about 0.2 μg per 1 mL.

[0138] Carbetocin position isomer reference substance solution: Take an appropriate amount of carbetocin position isomer reference substance, precisely weigh it, dissolve and quantitatively dilute with mobile phase A to prepare a solution containing about 0.2 μg per 1 mL.

[0139] Determination method:

[0140] Use Agilent ZOBAX C18 (150 mm × 4.6 mm, 3.5 μm) as the chromatographic column; use acetonitrile - 0.02 mol / L ammonium acetate - 0.03 mol / L potassium dihydrogen phosphate (70:20:10, pH value is 5.5) as mobile phase A, and methanol - acetonitrile (20:80) as mobile phase B for gradient elution; detection wavelength: 210 nm; column temperature: 40 °C; flow rate 1.2 mL / min; injection volume: 40 μL.

[0141]

[0142] System suitability requirements: In the system suitability chromatogram, the resolution between the carbetocin peak and the carbetocin positional isomer peak should meet the requirements, and the theoretical plate number calculated based on the carbetocin positional isomer peak should be greater than 8000.

[0143] (3) Methodology investigation and verification:

[0144] 1) Specificity:

[0145] Solvent: Acetonitrile - 0.02 mol / L ammonium acetate - 0.03 mol / L potassium dihydrogen phosphate (70:20:10, pH value is 5.5), that is, mobile phase A.

[0146] System suitability solution: Take appropriate amounts of carbetocin reference substance and carbetocin positional isomer reference substance, dissolve and dilute with the solvent to prepare a mixed solution containing about 20 μg of carbetocin and 0.2 μg of carbetocin isomer per 1 mL.

[0147] Blank excipient solution: Prepare a blank excipient solution without carbetocin according to the prescription ratio and preparation process of carbetocin injection, and that's it.

[0148] Test solution: Accurately measure an appropriate amount of this product (carbetocin injection), dissolve with the solvent and quantitatively dilute to prepare a solution containing about 20 μg of carbetocin per 1 mL.

[0149] Carbetocin reference substance solution: Take an appropriate amount of carbetocin reference substance, accurately weigh it, dissolve with the solvent and quantitatively dilute to prepare a solution containing about 0.2 μg per 1 mL.

[0150] Carbetocin positional isomer reference substance solution: Take an appropriate amount of carbetocin positional isomer reference substance, accurately weigh it, dissolve with the solvent and quantitatively dilute to prepare a solution containing about 0.2 μg per 1 mL.

[0151] Take 40 μL of each of the above solutions and inject them into the high - performance liquid chromatograph respectively, and record the chromatogram.

[0152] Experimental results: The solvent and blank excipients showed no interference, and the resolution of the system suitability met the requirements.

[0153] 2) Quantitation limit:

[0154] Weigh appropriate amounts of carbetocin and carbetocin position isomer reference substances, dissolve them separately in the solvent and dilute step by step until the signal-to-noise ratio is approximately 10. The quantitation limit results are shown in Table 10.

[0155] Table 10 Quantitation limit results

[0156]

[0157] Take the test sample at the quantitation limit concentration and inject it continuously for 6 times. The test results are used as the results for investigating the repeatability of the quantitation limit. The statistical results of the retention time and peak area for the repeatability determination of the quantitation limit are shown in Tables 11 - 12.

[0158] Table 11 Repeatability of the quantitation limit of carbetocin

[0159]

[0160] Table 12 Repeatability of the quantitation limit of carbetocin position isomer

[0161]

[0162] From the results in Tables 10 - 12, the conclusion is as follows: The quantitation limit of carbetocin is 0.14 ng, which is equivalent to 0.02% of the concentration of the test sample solution. The RSD of the retention time of carbetocin is 0.3%, less than 2%, and the RSD of the peak area is 1.6%, less than 15%, meeting the requirements. The quantitation limit of carbetocin position isomer is 0.15 ng, which is equivalent to 0.02% of the concentration of the test sample solution. The RSD of the retention time of carbetocin position isomer is 0.1%, less than 2%, and the RSD of the peak area is 2.5%, less than 15%, meeting the requirements. The method of the present invention has good sensitivity and can meet the detection requirements.

[0163] 3) Detection limit:

[0164] Weigh appropriate amounts of carbetocin and carbetocin position isomer reference substance solutions, dissolve them separately in the solvent and dilute step by step until the signal-to-noise ratio is approximately 3. The results are shown in Table 13.

[0165] Table 13 Detection limit results

[0166]

[0167] The experimental results in Table 13 show that the detection limit of carbetocin position isomers is 0.07 ng, and the detection limit of carbetocin is 0.08 ng, both of which are approximately equivalent to 0.01% of the test sample. This indicates that the method of the present invention can ensure the effective detection of carbetocin position isomer impurities.

[0168] 4) Linearity:

[0169] Take appropriate amounts of carbetocin and carbetocin position isomer reference substance solutions, dissolve them separately with the solvent and dilute step by step to prepare reference substance solutions at the limit of quantitation, 50% limit to 150% limit, and determine the linearity. The results are shown in Tables 14 - 15 and Figure 3 and 4 :

[0170] Table 14 Results of linearity determination of carbetocin position isomers

[0171]

[0172] Table 15 Results of linearity determination of carbetocin

[0173]

[0174] The experimental results show that for carbetocin position isomers, in the concentration range of 0.0038 μg / mL - 0.3200 μg / mL, the correlation coefficient r is 0.9999, indicating a good linear relationship; for carbetocin, in the concentration range of 0.0039 μg / mL - 0.3247 μg / mL, the correlation coefficient r is 0.9999, indicating a good linear relationship.

[0175] 5) Repeatability:

[0176] According to the method for detecting position isomers of this product (carbetocin injection), prepare 1 portion of system suitability solution, 6 portions of test sample spiked solution, and 2 portions of reference substance solution, inject them into the high - performance liquid chromatograph respectively, and record the chromatograms. The results are shown in Table 16.

[0177] System suitability solution: Take appropriate amounts of carbetocin reference substance and carbetocin position isomer reference substance, dissolve them with the solvent and dilute to prepare a mixed solution containing approximately 20 μg of carbetocin and 0.2 μg of carbetocin position isomers per 1 mL.

[0178] Carbetocin reference substance solution: Take an appropriate amount of carbetocin reference substance, accurately weigh it, dissolve it with the solvent and quantitatively dilute it to prepare a solution containing approximately 0.2 μg per 1 mL.

[0179] Test sample spiked solution: Take appropriate amounts of this product (carbetocin injection) and carbetocin position isomer reference substance, dissolve them with the solvent and dilute to prepare a mixed solution containing approximately 20 μg of carbetocin and 0.2 μg of position isomers per 1 mL.

[0180] Table 16 Repeatability Results

[0181]

[0182] From the results in Table 16, the conclusion is that the repeatability mean is 0.99% and the RSD is 1.6%, meeting the standard requirements. This indicates that the method of the present invention has good repeatability.

[0183] 6) Intermediate precision:

[0184] By different persons at different times, referring to the method for detecting the positional isomers of this product (carbetocin injection), prepare 1 portion of the system suitability solution, 6 portions of the test sample spiked solution, and 2 portions of the reference solution according to the repeatability in 5), inject them into the liquid chromatograph respectively, and record the chromatograms. The results are shown in Table 17.

[0185] Table 17 Precision Results

[0186]

[0187] From the results in Table 17, the conclusion is that the intermediate precision mean is 1.00% and the RSD is 1.7%, meeting the standard requirements. The mean of the 12 data from the repeatability test and the intermediate precision test is 0.99% and the RSD is 1.7%, indicating that this method has good precision.

[0188] 7) Accuracy:

[0189] Stock solution of carbetocin positional isomer reference substance: Take an appropriate amount of carbetocin positional isomer reference substance, weigh it accurately, dissolve and dilute it with a solvent to prepare a solution containing about 100 μg per 1 mL.

[0190] Carbetocin positional isomer reference solution: Take an appropriate amount of carbetocin positional isomer reference substance, weigh it accurately, dissolve and dilute it with a solvent to prepare a solution containing about 0.2 μg per 1 mL.

[0191] Test sample solution: Accurately measure an appropriate amount of this product (carbetocin injection), and dilute it with a solvent to prepare a solution containing about 20 μg of carbetocin per 1 mL.

[0192] High-concentration test sample: Accurately measure appropriate amounts of this product (carbetocin injection) and the stock solution of carbetocin positional isomer reference substance, and dilute them with a solvent to prepare a solution containing about 20 μg of carbetocin and 0.3 μg of carbetocin positional isomer per 1 mL.

[0193] In the test sample: Accurately measure an appropriate amount of this product (carbetocin injection) and the mother liquor of the carbetocin position isomer reference substance, and dilute with a solvent to prepare a solution containing approximately 20 μg of carbetocin and 0.2 μg of carbetocin position isomer per 1 mL.

[0194] Low test sample: Accurately measure an appropriate amount of this product (carbetocin injection) and the mother liquor of the carbetocin position isomer reference substance, and dilute with a solvent to prepare a solution containing approximately 20 μg of carbetocin and 0.1 μg of carbetocin position isomer per 1 mL.

[0195] Test sample (quantitation limit concentration): Accurately measure an appropriate amount of this product (carbetocin injection) and the mother liquor of the carbetocin position isomer reference substance, and dilute with a solvent to prepare a solution containing approximately 20 μg of carbetocin and 0.0035 μg of carbetocin position isomer per 1 mL.

[0196] Accurately measure the above solutions respectively and inject them into a high performance liquid chromatograph, record the chromatogram, and the results are shown in Table 18.

[0197] Table 18 Results of accuracy

[0198]

[0199] The experimental results in Table 18 show that the recovery rates of carbetocin position isomers are 106.67%, 96.39%, 99.67%, and 99.29% in sequence, the RSD value is 5.1%, the recovery rates are all between 75% and 120%, and the RSD is within 8%, indicating that the method of the present invention has good accuracy.

[0200] Conclusion: The method of the present invention has high accuracy, good sensitivity, and the specificity, linearity, repeatability, and precision all meet the requirements, and can well control the isomer impurities in this product (carbetocin injection).

[0201] Example 3

[0202] A method for detecting isomers in carbetocin injection, wherein the isomer is the Ile position isomer impurity in Leu at position 8 in the carbetocin raw material;

[0203] (1) Experimental conditions and reagents and test articles:

[0204] Instrument: Agilent 1260 high performance liquid chromatograph;

[0205] Balance: XSE 205DU electronic balance, Mettler-Toledo (Shanghai) Instruments Co., Ltd.;

[0206] Reagents: Acetonitrile and methanol are both chromatographically pure, and ammonium acetate and potassium dihydrogen phosphate are both analytically pure;

[0207] Reference Substances: Carbetocin (purchased from TRC), Carbetocin Positional Isomer (purchased from SINCO);

[0208] (2) Experimental Method:

[0209] Solvent: Acetonitrile - 0.02 mol / L Ammonium Acetate - 0.03 mol / L Potassium Dihydrogen Phosphate (50:30:20, pH 7.0), i.e., Mobile Phase A.

[0210] System Suitability Solution: Take appropriate amounts of carbetocin reference substance and carbetocin positional isomer reference substance, dissolve and dilute with Mobile Phase A to prepare a mixed solution containing approximately 50 μg of carbetocin and 0.5 μg of carbetocin positional isomer per 1 mL.

[0211] Preparation of Test Solution: Accurately measure an appropriate amount of this product (carbetocin injection), dissolve and dilute with Mobile Phase A to prepare a solution containing approximately 50 μg of carbetocin per 1 mL.

[0212] Carbetocin Reference Solution: Take an appropriate amount of carbetocin reference substance, accurately weigh it, dissolve and quantitatively dilute with Mobile Phase A to prepare a solution containing approximately 0.5 μg per 1 mL.

[0213] Carbetocin Positional Isomer Reference Solution: Take an appropriate amount of carbetocin positional isomer reference substance, accurately weigh it, dissolve and quantitatively dilute with Mobile Phase A to prepare a solution containing approximately 0.5 μg per 1 mL.

[0214] Determination Method:

[0215] Use Waters Xbridge C18 (150 mm × 4.6 mm, 3.5 μm) as the chromatographic column; use acetonitrile - 0.02 mol / L ammonium acetate - 0.03 mol / L potassium dihydrogen phosphate (50:30:20, pH 7.0) as Mobile Phase A, and methanol - acetonitrile (85:15) as Mobile Phase B for gradient elution; detection wavelength: 230 nm; column temperature: 70 °C; flow rate: 0.6 mL / min; injection volume: 20 μL.

[0216]

[0217] System Suitability Requirements: In the system suitability chromatogram, the resolution between the carbetocin peak and the carbetocin positional isomer peak should meet the requirements, and the number of theoretical plates calculated based on the carbetocin positional isomer peak should be greater than 8000.

[0218] (3) Methodology Investigation and Validation:

[0219] 1) Specificity:

[0220] Solvent: Acetonitrile - 0.02 mol / L ammonium acetate - 0.03 mol / L potassium dihydrogen phosphate (50:30:20, pH 7.0) (Mobile phase A).

[0221] System suitability solution: Take appropriate amounts of carbetocin reference substance and carbetocin positional isomer reference substance, dissolve and dilute with the solvent to prepare a mixed solution containing approximately 50 μg of carbetocin and 0.5 μg of carbetocin positional isomer per 1 mL.

[0222] Blank excipient solution: Prepare the blank excipient solution without carbetocin according to the prescription ratio and preparation process of carbetocin injection, and you will get it.

[0223] Test solution: Accurately measure an appropriate amount of this product (carbetocin injection), dissolve with the solvent and quantitatively dilute to prepare a solution containing approximately 50 μg of carbetocin per 1 mL.

[0224] Carbetocin reference solution: Take an appropriate amount of carbetocin reference substance, accurately weigh it, dissolve with the solvent and quantitatively dilute to prepare a solution containing approximately 0.5 μg per 1 mL.

[0225] Carbetocin positional isomer reference solution: Take an appropriate amount of carbetocin positional isomer reference substance, accurately weigh it, dissolve with the solvent and quantitatively dilute to prepare a solution containing approximately 0.5 μg per 1 mL.

[0226] Inject 20 μL of each of the above solutions into the high performance liquid chromatograph respectively, and record the chromatogram.

[0227] Experimental results: The solvent and the blank excipient have no interference, and the resolution of the system suitability meets the requirements.

[0228] 2) Quantification limit:

[0229] Take appropriate amounts of carbetocin and carbetocin positional isomer reference substances, dissolve with the solvent and dilute step by step until the signal-to-noise ratio is approximately 10. The quantification limit results are shown in Table 19.

[0230] Table 19 Quantification limit results

[0231]

[0232] Take the test solution at the quantification limit concentration and inject it continuously for 6 times. The test results are used as the results for investigating the repeatability of the quantification limit. The statistical results of the retention time and peak area for the repeatability determination of the quantification limit are shown in Tables 20 - 21.

[0233] Table 20 Repeatability of the quantification limit of carbetocin

[0234]

[0235] Table 21 Repeatability of the Quantitation Limit of the Cabergoline Position Isomers

[0236]

[0237] From the results in Tables 19 - 21, the conclusion is as follows: The quantitation limit of cabergoline is 0.63 ng, which is equivalent to 0.06% of the concentration of the test solution. The RSD of the retention time of cabergoline is 0.1%, less than 2%, and the RSD of the peak area is 1.0%, less than 15%, meeting the requirements. The quantitation limit of the cabergoline position isomers is 0.62 ng, which is equivalent to 0.06% of the concentration of the test solution. The RSD of the retention time of the cabergoline position isomers is 0.1%, less than 2%, and the RSD of the peak area is 1.6%, less than 15%, meeting the requirements. The method of the present invention has good sensitivity and can meet the detection requirements.

[0238] 3) Detection Limit:

[0239] Take appropriate amounts of the reference substance solutions of cabergoline and its position isomers, dissolve them separately with the solvent and dilute step by step until the signal - to - noise ratio is about 3. The results are shown in Table 22.

[0240] Table 22 Results of the Detection Limit

[0241]

[0242] The experimental results in Table 22 show that the detection limits of both the cabergoline position isomers and cabergoline are 0.31 ng, both approximately equivalent to 0.03% of the test substance. This indicates that the method of the present invention can ensure the effective detection of the cabergoline position isomer impurities.

[0243] 4) Linearity:

[0244] Take appropriate amounts of the reference substance solutions of cabergoline and its position isomers, dissolve them separately with the solvent and dilute step by step to prepare reference substance solutions at the quantitation limit, 50% limit - 150% limit, and determine the linearity. The results are shown in Tables 23 - 24 and Figure 5 and 6 :

[0245] Table 23 Results of the Linearity Determination of the Cabergoline Position Isomers

[0246]

[0247] Table 24 Results of the Linearity Determination of Cabergoline

[0248]

[0249]

[0250] The experimental results show that: for the carboprost isomers, in the concentration range of 0.0309 μg / mL to 0.7728 μg / mL, the correlation coefficient r is 0.9999, indicating a good linear relationship; for carboprost, in the concentration range of 0.0313 μg / mL to 0.7836 μg / mL, the correlation coefficient r is 0.9998, indicating a good linear relationship.

[0251] 5) Repeatability:

[0252] According to the detection method for the position isomers of this product (carboprost injection), prepare 1 portion of the system suitability solution, 6 portions of the test sample spiked solution, and 2 portions of the reference solution, and inject them into the high performance liquid chromatograph respectively. Record the chromatograms, and the results are shown in Table 25.

[0253] System suitability solution: Take appropriate amounts of carboprost reference substance and carboprost position isomer reference substance, dissolve and dilute with solvent to prepare a mixed solution containing about 50 μg of carboprost and 0.5 μg of carboprost position isomer per 1 mL.

[0254] Carboprost reference solution: Take an appropriate amount of carboprost reference substance, accurately weigh it, dissolve and quantitatively dilute with solvent to prepare a solution containing about 0.5 μg per 1 mL.

[0255] Test sample spiked solution: Take appropriate amounts of this product (carboprost injection) and carboprost position isomer reference substance, dissolve and dilute with solvent to prepare a mixed solution containing about 50 μg of carboprost and 0.5 μg of carboprost position isomer per 1 mL.

[0256] Table 25 Repeatability results

[0257]

[0258] Conclusion: The repeatability mean value is 0.97%, and the RSD is 1.3%, meeting the standard requirements. The method of the present invention has good repeatability.

[0259] 6) Intermediate precision:

[0260] By different persons at different times, according to the detection method for the position isomers of this product (carboprost injection), refer to the preparation of 1 portion of the system suitability solution, 6 portions of the test sample spiked solution, and 2 portions of the reference solution in 5) Repeatability, and inject them into the liquid chromatograph respectively. Record the chromatograms, and the results are shown in Table 26.

[0261] Table 26 Precision results

[0262]

[0263] As can be seen from the results in Table 26, the conclusion is that the mean intermediate precision is 0.99% and the RSD is 2.4%, meeting the standard requirements. The mean of 12 data from the repeatability test and the intermediate precision test is 0.98% and the RSD is 2.4%, indicating that the method of the present invention has good precision.

[0264] 7) Accuracy:

[0265] Stock solution of carbetocin position isomer reference substance: Take an appropriate amount of carbetocin position isomer reference substance, weigh accurately, dissolve and dilute with solvent to prepare a solution containing about 80 μg per 1 mL.

[0266] Position isomer reference substance solution: Take an appropriate amount of carbetocin position isomer reference substance, weigh accurately, dissolve and dilute with solvent to prepare a solution containing about 0.5 μg per 1 mL.

[0267] Test solution: Accurately measure an appropriate amount of this product (carbetocin injection), and dilute with solvent to prepare a solution containing about 50 μg of carbetocin per 1 mL.

[0268] High-concentration test solution: Accurately measure an appropriate amount of this product (carbetocin injection) and the stock solution of carbetocin position isomer reference substance, and dilute with solvent to prepare a solution containing about 50 μg of carbetocin and 0.75 μg of carbetocin position isomer per 1 mL.

[0269] Medium-concentration test solution: Accurately measure an appropriate amount of this product (carbetocin injection) and the stock solution of carbetocin position isomer reference substance, and dilute with solvent to prepare a solution containing about 50 μg of carbetocin and 0.5 μg of carbetocin position isomer per 1 mL.

[0270] Low-concentration test solution: Accurately measure an appropriate amount of this product (carbetocin injection) and the stock solution of carbetocin position isomer reference substance, and dilute with solvent to prepare a solution containing about 50 μg of carbetocin and 0.25 μg of carbetocin position isomer per 1 mL.

[0271] Test solution (quantitation limit concentration): Accurately measure an appropriate amount of this product (carbetocin injection) and the stock solution of carbetocin position isomer reference substance, and dilute with solvent to prepare a solution containing about 50 μg of carbetocin and 0.03 μg of carbetocin position isomer per 1 mL.

[0272] Accurately measure the above solutions respectively and inject them into a high-performance liquid chromatograph, record the chromatogram, and the results are shown in Table 27 below.

[0273] Table 27 Accuracy results

[0274]

[0275]

[0276] The experimental results in Table 27 show that the recovery rates of the carbetocin position isomers are 92.62%, 98.84%, 97.98%, and 96.84% in sequence, the RSD value is 2.9%, and the recovery rates are all between 75% and 120%, and the RSD is within 8%. This indicates that the method of the present invention has good accuracy.

[0277] Conclusion: The method of the present invention has high accuracy and good sensitivity, and its specificity, linearity, reproducibility, and precision all meet the requirements, and it can well control the content of the carbetocin position isomers in this product (carbetocin injection).

[0278] Example 4

[0279] A method for detecting the isomers of carbetocin injection, wherein the isomers are the Ile position isomer impurities in Leu at position 8 in the carbetocin raw material;

[0280] (1) Experimental conditions and reagents and test articles:

[0281] Instrument: Agilent 1260 high performance liquid chromatograph, DAD detector;

[0282] Balance: XSE 205DU electronic balance, Mettler-Toledo (Shanghai) Instrument Co., Ltd.;

[0283] Reagents: Acetonitrile and methanol are both chromatographically pure, and ammonium acetate and potassium dihydrogen phosphate are both analytically pure;

[0284] Reference substances: Carbetocin (purchased from TRC), carbetocin position isomers (purchased from SINCO);

[0285] (2) Experimental method:

[0286] Solvent: Acetonitrile - 0.02 mol / L ammonium acetate - 0.03 mol / L potassium dihydrogen phosphate (40:30:30, pH value is 4.5), that is, mobile phase A.

[0287] Preparation of test solution: Precisely measure an appropriate amount of this product (carbetocin injection), dissolve and dilute it with mobile phase A to prepare a solution containing about 60 μg of carbetocin per 1 mL.

[0288] Carbetocin reference substance solution: Take an appropriate amount of carbetocin reference substance, accurately weigh it, dissolve it with mobile phase A and quantitatively dilute it to prepare a solution containing about 0.6 μg per 1 mL.

[0289] Carbetocin position isomer reference substance solution: Take an appropriate amount of carbetocin position isomer reference substance, accurately weigh it, dissolve it with mobile phase A and quantitatively dilute it to prepare a solution containing about 0.6 μg per 1 mL.

[0290] System suitability solution: Take appropriate amounts of carbetocin reference substance and carbetocin positional isomer reference substance, dissolve and dilute with mobile phase A to prepare a mixed solution containing about 60 μg of carbetocin and 0.6 μg of carbetocin positional isomer per 1 mL.

[0291] Determination method:

[0292] Use Waters Xbridge C18 (150 mm × 4.6 mm, 3.5 μm) as the chromatographic column; use acetonitrile - 0.02 mol / L ammonium acetate - 0.03 mol / L potassium dihydrogen phosphate (40:30:30, pH 4.5) as mobile phase A, and methanol - acetonitrile (90:10) as mobile phase B for gradient elution; detection wavelength: 225 nm; column temperature: 60 °C; flow rate: 1.0 mL / min; injection volume: 40 μL.

[0293]

[0294] System suitability requirements: In the system suitability chromatogram, the resolution between the carbetocin peak and the carbetocin positional isomer peak should meet the requirements, and the number of theoretical plates calculated based on the carbetocin positional isomer peak should be greater than 8000.

[0295] (3) Methodology investigation and validation:

[0296] 1) Specificity:

[0297] Solvent: Acetonitrile - 0.02 mol / L ammonium acetate - 0.03 mol / L potassium dihydrogen phosphate (40:30:30, pH 4.5) (mobile phase A).

[0298] System suitability: Take appropriate amounts of carbetocin reference substance and carbetocin positional isomer reference substance, dissolve and dilute with the solvent to prepare a mixed solution containing about 60 μg of carbetocin and 0.6 μg of carbetocin positional isomer per 1 mL.

[0299] Blank excipient solution: Prepare a blank excipient solution without carbetocin according to the prescription ratio and preparation process of carbetocin injection.

[0300] Test solution: Precision measure an appropriate amount of this product (carbetocin injection), dissolve with the solvent and quantitatively dilute to prepare a solution containing about 60 μg of carbetocin per 1 mL.

[0301] Carbetocin reference substance solution: Take an appropriate amount of carbetocin reference substance, accurately weigh, dissolve with the solvent and quantitatively dilute to prepare a solution containing about 0.6 μg per 1 mL.

[0302] Carbetocin positional isomer reference substance solution: Take an appropriate amount of carbetocin positional isomer reference substance, weigh it accurately, dissolve it with a solvent and quantitatively dilute it to prepare a solution containing about 0.6 μg per 1 mL.

[0303] Take 40 μL of each of the above solutions and inject them into a high performance liquid chromatograph respectively, and record the chromatogram.

[0304] Experimental results: The solvent and blank excipients have no interference, and the system suitability resolution meets the requirements.

[0305] 2) Limit of quantitation:

[0306] Take appropriate amounts of carbetocin and carbetocin positional isomer reference substances, dissolve them with a solvent respectively and dilute them step by step until the signal-to-noise ratio is about 10. The limit of quantitation results are shown in Table 28.

[0307] Table 28 Limit of quantitation results

[0308]

[0309] Take the test sample at the limit of quantitation concentration and inject it continuously for 6 times. The test results are used as the results for investigating the repeatability of the limit of quantitation. The statistical results of the retention time and peak area for the repeatability determination of the limit of quantitation are shown in Tables 29 - 30.

[0310] Table 29 Repeatability of the limit of quantitation of carbetocin

[0311]

[0312] Table 30 Repeatability of the limit of quantitation of carbetocin positional isomer

[0313]

[0314] From the results in Tables 28 - 30, the conclusion can be drawn that: the limit of quantitation of carbetocin is 1.03 ng, which is equivalent to 0.04% of the concentration of the test sample solution. The RSD of the retention time of carbetocin is 0.2%, less than 2%, and the RSD of the peak area is 0.4%, less than 15%, meeting the requirements. The limit of quantitation of carbetocin positional isomer is 1.05 ng, which is equivalent to 0.04% of the concentration of the test sample solution. The RSD of the retention time of carbetocin positional isomer is 0.3%, less than 2%, and the RSD of the peak area is 2.2%, less than 15%, meeting the requirements. It shows that the method of the present invention has good sensitivity and can meet the detection requirements.

[0315] 3) Limit of detection:

[0316] Take appropriate amounts of carbetocin and carbetocin positional isomer reference substance solutions, dissolve them with a solvent respectively and dilute them step by step until the signal-to-noise ratio is about 3. The results are shown in Table 31.

[0317] Table 31 Detection Limit Results

[0318]

[0319] The experimental results show that: the detection limits of carbetocin position isomers and carbetocin are both 0.52 ng, which are both approximately equivalent to 0.02% of the concentration of the test solution. This indicates that the method of the present invention can ensure the effective detection of carbetocin position isomers.

[0320] 4) Linearity:

[0321] Take appropriate amounts of carbetocin and carbetocin position isomer reference substance solutions, dissolve them separately with a solvent and dilute step by step to prepare reference substance solutions at the limit of quantitation, 50% limit to 150% limit, and determine the linearity. The results are shown in Tables 32 - 33 and Figure 7 and 8 :

[0322] Table 32 Results of Linear Determination of Position Isomers

[0323]

[0324]

[0325] Table 33 Results of Linear Determination of Carbetocin

[0326]

[0327] The experimental results show that: for carbetocin position isomers, in the concentration range of 0.0263 μg / mL to 0.9004 μg / mL, the correlation coefficient r is 1.0000, indicating a good linear relationship; for carbetocin, in the concentration range of 0.0269 μg / mL to 0.9213 μg / mL, the correlation coefficient r is 0.9999, indicating a good linear relationship.

[0328] 5) Repeatability:

[0329] According to the method for detecting position isomers of this product (carbetocin injection), prepare 1 portion of system suitability solution, 6 portions of test sample spiked solution, and 2 portions of reference substance solution, inject them into the high - performance liquid chromatograph respectively, and record the chromatograms. The results are shown in Table 34.

[0330] System suitability: Take appropriate amounts of carbetocin reference substance and carbetocin position isomer reference substance, dissolve them with a solvent and dilute to prepare a mixed solution containing approximately 60 μg of carbetocin and 0.6 μg of carbetocin position isomer per 1 mL.

[0331] Carbetocin reference substance solution: Take an appropriate amount of carbetocin reference substance, accurately weigh it, dissolve it with a solvent and quantitatively dilute it to prepare a solution containing approximately 0.6 μg per 1 mL.

[0332] Test sample spiked solution: Take appropriate amounts of this product (carbetocin injection) and the reference substance of carbetocin positional isomer, dissolve and dilute with a solvent to prepare a mixed solution containing about 60 μg of carbetocin and 0.6 μg of carbetocin positional isomer per 1 mL.

[0333] Table 34 Repeatability results

[0334]

[0335] From the results in Table 34, the conclusion is: the repeatability mean is 0.97%, the RSD is 1.9%, meeting the standard requirements. It shows that the method of the present invention has good repeatability.

[0336] 6) Intermediate precision:

[0337] By different persons at different times, according to the detection method of the positional isomer of this product (carbetocin injection), prepare 1 portion of system suitability solution, 6 portions of test sample spiked solution, and 2 portions of reference substance solution with reference to 5) Repeatability, inject them into the high performance liquid chromatograph respectively, record the chromatograms, and the results are shown in Table 35.

[0338] Table 35 Precision results

[0339]

[0340] From the results in Table 35, the conclusion is: the intermediate precision mean is 0.98%, the RSD is 2.5%, meeting the standard requirements. The mean of 12 data from the repeatability test and the intermediate precision test is 0.98%, the RSD is 2.2%, indicating that the method of the present invention has good precision.

[0341] 7) Accuracy:

[0342] Stock solution of reference substance of carbetocin positional isomer: Take appropriate amount of the reference substance of carbetocin positional isomer, weigh accurately, dissolve and dilute with a solvent to prepare a solution containing about 60 μg per 1 mL.

[0343] Reference substance solution of carbetocin positional isomer: Take appropriate amount of the reference substance of carbetocin positional isomer, weigh accurately, dissolve and dilute with a solvent to prepare a solution containing about 0.6 μg per 1 mL.

[0344] Test sample solution: Accurately measure an appropriate amount of this product (carbetocin injection), dilute with a solvent to prepare a solution containing about 60 μg of carbetocin per 1 mL.

[0345] High concentration of test sample: Accurately measure appropriate amounts of this product (carbetocin injection) and the stock solution of reference substance of carbetocin positional isomer, dilute with a solvent to prepare a solution containing about 60 μg of carbetocin and 0.9 μg of carbetocin positional isomer per 1 mL.

[0346] In the test sample: accurately measure appropriate amounts of this product (carbetocin injection) and the mother liquor of the carbetocin positional isomer reference substance, and dilute with a solvent to prepare a solution containing approximately 60 μg of carbetocin and 0.6 μg of carbetocin positional isomer per 1 mL.

[0347] Low test sample: accurately measure appropriate amounts of this product (carbetocin injection) and the mother liquor of the carbetocin positional isomer reference substance, and dilute with a solvent to prepare a solution containing approximately 60 μg of carbetocin and 0.3 μg of carbetocin positional isomer per 1 mL.

[0348] Test sample (quantification limit concentration): accurately measure appropriate amounts of this product (carbetocin injection) and the mother liquor of the carbetocin positional isomer reference substance, and dilute with a solvent to prepare a solution containing approximately 60 μg of carbetocin and 0.025 μg of carbetocin positional isomer per 1 mL.

[0349] Accurately measure the above solutions and inject them into a high performance liquid chromatograph, record the chromatogram, and the results are shown in Table 36.

[0350] Table 36 Results of accuracy

[0351]

[0352]

[0353] The experimental results in Table 36 show that the recovery rates of carbetocin positional isomers are 94.81%, 97.89%, 98.30%, and 98.14% in sequence, the RSD value is 2.0%, the recovery rates are all between 75% and 120%, and the RSD is within 8%, indicating that the method of the present invention has good accuracy.

[0354] The conclusion is that the method of the present invention has high accuracy, good sensitivity, and the specificity, linearity, repeatability, and precision all meet the requirements, and can well control the content of carbetocin positional isomer in this product (carbetocin injection).

[0355] Obviously, the above embodiments are only examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for detecting the isomers of carbetocin injection, characterized in that, Comprising the following steps: Using high performance liquid chromatography to detect the Ile positional isomer impurity in Leu at position 8 in the carbetocin raw material in the carbetocin injection; The chromatographic conditions used are as follows: Using octadecylsilyl silica gel as the filler, using acetonitrile - 0.02 mol / L ammonium acetate - 0.03 mol / L potassium dihydrogen phosphate as mobile phase A, using methanol - acetonitrile as mobile phase B, gradient elution; the detection wavelength is 210 - 230 nm; the column temperature is 40 - 70 °C; the flow rate is 0.5 - 1.2 mL / min; the injection volume is 10 - 40 μL.

2. The detection method of the cabergoline injection isomers according to claim 1, characterized in that, The volume ratio of acetonitrile, ammonium acetate and potassium dihydrogen phosphate in mobile phase A is 70:20:10 - 40:30:30, and the pH value is 4.5 - 7.

0.

3. The method for detecting the isomers of carbetocin injection according to claim 2, wherein The volume ratio of acetonitrile, ammonium acetate and potassium dihydrogen phosphate in mobile phase A is 60:25:15, and the pH value is 6.

1.

4. The detection method of carbetocin injection isomers according to claim 1, characterized in that, The volume ratio of methanol to acetonitrile in mobile phase B is 15:85 - 90:

10.

5. The method for detecting the isomers of carbetocin injection according to claim 4, characterized in that, The volume ratio of methanol to acetonitrile in mobile phase B is 65:

35.

6. The method for detecting the isomers of carbetocin injection according to claim 1, wherein The detection wavelength is 220 nm; the column temperature is 45 °C; the flow rate is 0.8 mL / min; the injection volume is 10 μL.

7. The detection method of carbetocin injection isomers according to any one of claims 1-6, characterized in that, The chromatographic column used is selected from any one of the following chromatographic columns: Thermo Hyersil BDS C18, 150 mm × 4.6 mm, 3.5 μm; Agilent ZOBAX C18, 150 mm × 4.6 mm, 3.5 μm; Waters Xbridge C18, 150 mm × 4.6 mm, 3.5 μm.

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