A method for analyzing carbetocin-related substances

Abstract

The invention discloses an analysis method for carbetocin-related substances. The invention aims at the problem that the existing technology and method cannot simultaneously detect 29 related substances in carbetocin, which brings difficulties to the quality control of carbetocin raw material medicine products, and establishes a method for simultaneously detecting 29 related substances in carbetocin. The method can separate carbetocin-related substances (including carbetocin and 29 impurities) in a high-performance liquid chromatogram; and through system suitability, destruction test, quantitative limit and detection limit, linearity and range, accuracy, precision (repeatability and intermediate precision), solution stability, and durability verification experiments, it is shown that the method can achieve the purpose of accurate quantitative analysis, which is conducive to stricter quality control of carbetocin.

Description

Technical Field

[0001] The invention belongs to the technical field of medical analysis, and particularly relates to an analysis method for carbetocin-related substances. Background Art

[0002] Carbetocin is a synthetic, long-acting oxytocin with agonist properties. It can be administered intravenously in a single dose immediately after cesarean section under epidural or spinal anesthesia to prevent uterine hypotonia and postpartum hemorrhage. Its clinical and pharmacological properties are similar to those of naturally produced oxytocin. It can bind to the oxytocin receptors of uterine smooth muscle, causing rhythmic contractions of the uterus, increasing its frequency and uterine tension on the basis of the original contractions. In the non-pregnant state, the uterine oxytocin receptor content is very low, which increases during pregnancy and reaches a peak during delivery. Therefore, carbetocin has no effect on the non-pregnant uterus, and only has an effective uterine contraction effect on the pregnant uterus and the uterus that has just given birth.

[0003] In the process of carbetocin production, preparation and synthesis, impurity peptides such as missing peptides, differential peptides, oxidized peptides, degraded peptides, dimers, etc. may be produced. Common impurities are shown in the following table:

[0004]

[0005]

[0006]

[0007] Impurities contained in drugs will affect the quality of carbetocin. In order to strictly control the quality of carbetocin and ensure the safety and effectiveness of clinical medication, it is necessary to establish an effective detection method for carbetocin-related substances. At present, the existing carbetocin impurity detection method cannot effectively separate and detect the 29 known impurities in carbetocin, and cannot perform strict quality control. Therefore, a detection method applicable to all 29 impurities in carbetocin is urgently needed. Summary of the invention

[0008] The object of the present invention is to provide a high performance liquid chromatography method for simultaneously detecting the 29 related substances of the above-mentioned carbetocin. In view of the problem that the existing technology and methods cannot simultaneously detect the 29 related substances of the above-mentioned carbetocin, which brings difficulties to the quality control of the carbetocin raw material, the inventor has established a method for simultaneously detecting the 29 related substances in carbetocin through a large number of experimental studies, which can be used for the quality research and quality control of the carbetocin raw material product.

[0009] The present invention adopts high performance liquid chromatography to detect carbetocin related substances, and the specific chromatographic conditions are as follows:

[0010] Octadecylsilane bonded silica gel is used as filler, and the column specifications are Waters C18, 3.0mm×150mm, 3.5μm; mobile phase A is ammonium acetate buffer (weigh 0.3g of ammonium acetate, add 1000ml of water to dissolve, add 380ml of acetonitrile, then add 8ml of PIC B-8Low UV reagent, and dilute to 2000ml with water); the mobile phase B is acetonitrile-mobile phase A (1:1); gradient mobile phase elution is adopted, and the mobile phase gradient is set as follows based on the mobile phase volume ratio of 100%:

[0011]

[0012] In the above-mentioned related substance detection method, taking the volume ratio of the mobile phase as 100%, the preferred mobile phase gradient is set as follows:

[0013]

[0014] The column temperature of the chromatographic column in the above-mentioned related substance detection method is 45°C-75°C, preferably 60°C.

[0015] The chromatographic column flow rate of the above-mentioned related substance detection method is 0.5-1.2 ml / min, preferably 0.8 ml / min.

[0016] The injection volume of the above-mentioned related substance detection method is 10 μl.

[0017] In the wavelength selection test of the present invention, carbetocin reference solution and impurity reference solution were taken and detected by PDA detector. Carbetocin and each impurity have maximum absorption near 220nm wavelength, so 220nm was selected as the detection wavelength.

[0018] The present invention determines the related substances of carbetocin by an area normalization method, and the scheme of the present invention can achieve baseline separation of 29 related substances of carbetocin and the main peak of carbetocin, and can accurately determine the content of the 29 related substances of carbetocin. The method is simple, fast, accurate, efficient, and has good reproducibility, and establishes a good foundation for the formulation of the quality standard of carbetocin. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram showing the separation of carbetocin and the impurity [Des-N-α-Butyryl1] carbetocin in Example 1.

[0020] Figure 2 This is a diagram showing the separation of carbetocin and the impurity [Des-Tyr(Me)2] carbetocin in Example 1.

[0021] Figure 3This is a diagram showing the separation of carbetocin and the impurity [Des-Ile3] carbetocin in Example 1.

[0022] Figure 4 This is a diagram showing the separation of carbetocin and the impurity [Des-Asn5] carbetocin in Example 1.

[0023] Figure 5 This is a diagram showing the separation of carbetocin and the impurity [Des-N-α-Butyryl1, Des-Cys6] carbetocin in Example 1.

[0024] Figure 6 This is a diagram showing the separation of carbetocin and the impurity [Des-Pro7] carbetocin in Example 1.

[0025] Figure 7 This is a diagram showing the separation of carbetocin and the impurity [Des-Leu8] carbetocin in Example 1.

[0026] Figure 8 This is a diagram showing the separation of carbetocin and the impurity [Des-Gly9] carbetocin in Example 1.

[0027] Fig. 9 This is a diagram showing the separation of carbetocin and the impurity [D-Tyr(Me)2] carbetocin in Example 1.

[0028] Fig.10 This is a diagram showing the separation of carbetocin and the impurity [D-allo-Ile3] carbetocin in Example 1.

[0029] Fig.11 This is a diagram showing the separation of carbetocin and the impurity [D-Gln4] carbetocin in Example 1.

[0030] Fig.12 This is a diagram showing the separation of carbetocin and the impurity [D-Leu8] carbetocin in Example 1.

[0031] Fig.13 This is a diagram showing the separation of carbetocin and the impurity [Leu3] carbetocin in Example 1.

[0032] Fig.14 This is a diagram showing the separation of carbetocin and the impurity [Ile8] carbetocin in Example 1.

[0033] Fig.15 This is a diagram showing the separation of carbetocin and the impurity [Leu3, Ile8] carbetocin in Example 1.

[0034] Fig.16This is a diagram showing the separation of carbetocin and the impurity [Tyr2] carbetocin in Example 1.

[0035] Fig.17 This is a diagram showing the separation of carbetocin and the impurity [Ava7] carbetocin in Example 1.

[0036] Fig.18 This is a diagram showing the separation of carbetocin and the impurity [Pro7ˋ-Pro7] carbetocin in Example 1.

[0037] Fig.19 This is a diagram showing the separation of carbetocin and the impurity [Gly9ˋ-Gly9] carbetocin in Example 1.

[0038] Fig. 20 This is a diagram of the separation of carbetocin and 10 impurities in Example 1 (from left to right: [Gly9-OH] carbetocin, [Asp5] carbetocin, [β-Asp5] carbetocin, [Glu4] carbetocin, carbetocin sulfoxide I, carbetocin sulfoxide II, [D-Cys6] carbetocin, [Des-Gln4] carbetocin, carbetocin, [D-Asn5] carbetocin, and carbetocin dimer). DETAILED DESCRIPTION

[0039] Example 1

[0040] Preparation of mixed solution: Accurately weigh carbetocin, [Gly 9 -OH]carbetocin, [Asp 5 ]Carbetocin, [β-Asp 5 ]Carbetocin, [Glu 4 ]Carbetocin, Carbetocin sulfoxide I, Carbetocin sulfoxide II, [D-Cys 6 ]Carbetocin, [Des-Gln 4 ]Carbetocin, [D-Asn 5 ]Add appropriate amount of carbetocin and carbetocin dimer reference substance to water, dissolve and dilute to make a solution containing about 1 mg of the product and 5 μg of impurities per 1 ml, shake well, and the solution is ready.

[0041] Impurity separation solution: accurately weigh carbetocin, [Des-N-α-Butyryl 1 ]Carbetocin, [Des-Tyr(Me) 2 ]Carbetocin, [Des-Ile 3 ]Carbetocin, [Des-Asn 5 ]Carbetocin, [Des-N-α-Butyryl 1,Des-Cys 6 ]Carbetocin, [Des-Pro 7 ]Carbetocin, [Des-Leu 8 ]Carbetocin, [Des-Gly 9 ]Carbetocin, [D-Tyr(Me) 2 ]Carbetocin, [D-allo-Ile 3 ]Carbetocin, [D-Gln 4 ]Carbetocin, [D-Leu 8 ]Carbetocin, [Leu 3 ]Carbetocin, [Ile 8 ]Carbetocin, [Leu 3 ,Ile 8 ]Carbetocin, [Tyr 2 ]Carbetocin, [Ava 7 ]Carbetocin, [Pro 7` -Pro 7 ]Carbetocin, [Gly 9` -Gly 9 ]Add appropriate amount of carbetocin reference substance to water for dissolution and dilute to make a solution containing about 1 mg of this product and 5 μg of impurities per 1 ml.

[0042] The relevant substances were determined according to the following HPLC conditions. 10 μl of each of the above solutions was accurately measured and injected into the liquid chromatograph to record the chromatogram. Figure 1-Figure 20 .

[0043] Chromatographic conditions: Octadecylsilane bonded silica gel was used as filler (Waters C18, 3.0mm×150mm, 3.5μm or chromatographic column with equivalent performance); ammonium acetate buffer (weigh 0.3g of ammonium acetate, add 1000ml of water to dissolve, add 380ml of acetonitrile, then add 8ml of PIC B-8Low UV reagent, and dilute to 2000ml with water) as mobile phase A, acetonitrile-mobile phase A (1:1) as mobile phase B; flow rate of 0.8ml per minute; column temperature of 60℃; detection wavelength of 220nm; injection volume of 10μl; gradient elution by HPLC, the mobile phase gradient is as follows:

[0044]

[0045] Test results

[0046]

[0047]

[0048] The method of the present invention can make the separation degree of 29 carbetocin related substances and the main peak of carbetocin greater than 1.5, and can effectively separate them.

[0049] Example 2

[0050] In the following examples, the substances measured are: [Gly 9 -OH]carbetocin, [Asp 5 ]Carbetocin, [β-Asp 5 ]Carbetocin, [Glu 4 ]Carbetocin, Carbetocin sulfoxide I, Carbetocin sulfoxide II, [D-Cys 6 ]Carbetocin, [Des-Gln 4 ]Carbetocin, [D-Asn 5 ]Carbetocin, carbetocin dimer.

[0051] 1. System suitability

[0052] Preparation of injection precision solution: Take an appropriate amount of this product, weigh it accurately, dissolve it in water and dilute it to make a solution containing 1 mg per 1 ml.

[0053] Accurately measure 10 μl of each injection precision solution (measure 6 times continuously), inject it into the liquid chromatograph, and record the chromatogram.

[0054] System suitability test results

[0055] frequency 1 2 3 4 5 6 RSD% Retention time (min) 21.981 21.982 21.998 21.982 21.976 21.984 0.03 Peak area 11769614 11687280 11754502 11749773 11735187 11734528 0.24

[0056] Conclusion: The injection precision solution was injected 6 times in parallel, the retention time RSD was 0.03%, lower than 1.0%, and the peak area RSD was 0.24%, lower than 2.0%. The injection precision was good.

[0057] 2. Destruction test

[0058] Undamaged: Accurately weigh 10 mg of this product, place in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain.

[0059] Acid destruction (powder): accurately weigh 10 mg of this product, place it in a 10 ml volumetric flask, add 1 ml of 1 mol / L hydrochloric acid solution to dissolve it, leave it for 3.5 hours, add 1 ml of 1 mol / L sodium hydroxide solution, dilute it to the scale with water, shake well, and the product is ready. Perform a blank test at the same time.

[0060] Acid destruction (solution): accurately weigh 10 mg of this product, place it in a 10 ml volumetric flask, add 1 ml of water to dissolve it, add 1 ml of 1 mol / L hydrochloric acid solution, leave it for 3.5 hours, add 1 ml of 1 mol / L sodium hydroxide solution, dilute it to the scale with water, shake well, and the solution is ready. Perform a blank test at the same time.

[0061] Alkali destruction (powder): accurately weigh 10 mg of this product, place it in a 10 ml volumetric bottle, add 1 ml of 0.1 mol / L sodium hydroxide solution to dissolve it, let it stand for 5 minutes, add 1 ml of 0.1 mol / L hydrochloric acid solution, add water to dilute to the scale, shake well, and the product is obtained. Perform a blank test at the same time.

[0062] Alkali destruction (solution): accurately weigh 10 mg of this product, place it in a 10 ml volumetric flask, add 1 ml of water to dissolve it, add 1 ml of 0.1 mol / L sodium hydroxide solution, let it stand for 5 minutes, add 1 ml of 0.1 mol / L hydrochloric acid solution, dilute to the scale with water, shake well, and the solution is ready. Perform a blank test at the same time.

[0063] Light destruction (powder): Accurately weigh 10 mg of this product and place it in a 10 ml volumetric bottle. Place it in a light box with an illumination of 4500 lx ± 500 lx for 2.5 days. Add water to dissolve and dilute to the scale. Shake well to obtain.

[0064] Light destruction (solution): Accurately weigh 10 mg of this product, place it in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and place it in a light box under an illumination of 4500lx±500lx for 2.5 days.

[0065] Oxidative destruction (powder): Weigh 10 mg of this product accurately, place it in a 10 ml volumetric bottle, add 1 ml of 30% hydrogen peroxide to dissolve it, leave it for 3 hours, add water to dilute to the scale, shake well, and the solution is ready. Perform a blank test at the same time.

[0066] Oxidative destruction (solution): Weigh 10 mg of this product accurately, place it in a 10 ml volumetric bottle, add 1 ml of 30% hydrogen peroxide to dissolve it, add water to dissolve and dilute to the scale, leave it for 3 hours, shake well, and the solution is ready. Perform a blank test at the same time.

[0067] High temperature destruction (powder): Accurately weigh 10 mg of this product and place it in a 20 ml volumetric flask. Place it in a 100°C oven for 9 hours, then add water to dissolve and dilute to the scale. Shake well to obtain the product.

[0068] High temperature destruction (solution): accurately weigh 10 mg of this product, place it in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, place it in a 100°C oven for 9 hours, shake well, and the product is ready.

[0069] Accurately measure 10 μl of each of the above solutions, inject into liquid chromatograph, and record the chromatogram.

[0070] Forced degradation test results-1

[0071]

[0072]

[0073] Conclusion: (1) Under each forced degradation condition, each solvent did not interfere with the detection of the main component and each impurity. No impurities were detected under the main component peak. The separation degree between the main peak and the preceding and following impurities was greater than 1.5. The method had good specificity. (2) The mass conservation under each degradation condition was within the range of 95% to 105%, which can be judged as mass conservation. (3) Under oxidative conditions, the main degradation products of this product were carbetocin sulfoxide I and carbetocin sulfoxide II; under alkaline conditions, the main degradation product was [D-Asn5] carbetocin; under acidic conditions, the main degradation products were [Gly9-OH] carbetocin, [Asp5] carbetocin, and [Glu4] carbetocin; under high temperature and light conditions, the related substances were relatively stable.

[0074] 3. Limit of Quantitation and Limit of Detection

[0075] Limit of quantitation: Accurately weigh an appropriate amount of impurity reference substance, dilute it gradually, and take the concentration when the signal-to-noise ratio is 10:1 or the amount injected into the instrument as the limit of quantitation.

[0076] Accurately measure 10 μl of the quantitative limit solution (measured 6 times continuously), inject it into the liquid chromatograph, and record the chromatogram.

[0077] Quantitation limit result -1

[0078] Components Concentration (μg / ml) Injection mass (ng) Equivalent to test sample concentration (%) <![CDATA[[Gly 9 -OH]Carbetocin]]> 0.099 0.986 0.010 <![CDATA[[Asp 5 ]Carbetocin]]> 0.094 0.942 0.009 <![CDATA[[β-Asp 5 ]Carbetocin]]> 0.089 0.895 0.009 <![CDATA[[Glu 4 ]Carbetocin]]> 0.094 0.941 0.009 Carbetocin sulfoxide I 0.081 0.807 0.008 Carbetocin sulfoxide II 0.051 0.506 0.005 <![CDATA[[D-Cys 6 ]Carbetocin]]> 0.094 0.943 0.009 <![CDATA[[Des-Gln 4 ]Carbetocin]]> 0.074 0.741 0.007 Carbetocin 0.197 1.965 0.020 <![CDATA[[D-Asn 5 ]Carbetocin]]> 0.092 0.922 0.009 Carbetocin dimer 0.259 2.588 0.026

[0079] Quantitation limit result -2

[0080]

[0081]

[0082] Detection limit: Take the quantitation limit solution under the quantitation limit item and dilute it step by step until the signal-to-noise ratio (S / N) is about 3, and the concentration corresponding to it is the detection limit concentration.

[0083] Accurately measure 10 μl of the detection limit solution (measured 3 times continuously), inject it into the liquid chromatograph, and record the chromatogram.

[0084] Detection limit results

[0085]

[0086]

[0087] Conclusion: This method has high sensitivity.

[0088] 4. Linearity and range

[0089] Preparation of 200% linear stock solution: accurately weigh appropriate amounts of carbetocin, [Gly9-OH] carbetocin, [Asp5] carbetocin, [Glu4] carbetocin, [β-Asp5] carbetocin, [D-Cys6] carbetocin, [D-Asn5] carbetocin, carbetocin sulfoxide I, carbetocin sulfoxide II, and carbetocin dimer reference substance, dissolve in water ([Des-Gln4] carbetocin is dissolved in 40% acetic acid solution) and dilute to make a solution containing 10 μg per 1 ml.

[0090] Linear solution preparation: Prepare according to the table below

[0091]

[0092] Accurately measure 10 μl of each of the above solutions, inject into a liquid chromatograph, and record the chromatogram. Perform linear regression analysis in the concentration range of LOQ to 150%, with concentration as the abscissa (C) and peak area as the ordinate (A).

[0093] Linearity test results-1

[0094]

[0095]

[0096] Conclusion: The linear relationship between carbetocin and its impurities was good under this method.

[0097] 5. Accuracy

[0098] Preparation of 200% accuracy stock solution: accurately weigh [Gly 9 -OH]carbetocin, [Asp 5 ]Carbetocin, [Glu 4 ]Carbetocin, [β-Asp 5 ]Carbetocin, [D-Cys 6 ]Carbetocin, [D-Asn 5 ]Carbetocin, Carbetocin sulfoxide I, Carbetocin sulfoxide II, [Des-Gln 4 ] carbetocin, carbetocin dimer, add water to dissolve ([Des-Gln 4 Carbetocin is dissolved in 40% acetic acid and diluted to prepare a solution containing about 10 μg per 1 ml.

[0099] Preparation of 80% accuracy solution: accurately weigh about 10 mg of this product, place it in a 10 ml volumetric bottle, add 4 ml of 200% accuracy stock solution to dissolve it, add water to dilute to the scale, shake well, and you have it. (Prepare 3 copies in parallel)

[0100] Preparation of 100% accuracy solution: accurately weigh about 10 mg of this product, place it in a 10 ml volumetric bottle, add 5 ml of 200% accuracy stock solution to dissolve it, add water to dilute to the scale, shake well, and you have it. (Prepare 3 copies in parallel)

[0101] Preparation of 120% accuracy solution: accurately weigh about 10 mg of this product, place it in a 10 ml volumetric bottle, add 6 ml of 200% accuracy stock solution to dissolve it, add water to dilute to the scale, shake well, and you have it. (Prepare 3 copies in parallel)

[0102] Preparation of test solution: Accurately weigh about 10 mg of the product, place in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain.

[0103] Reference solution: accurately weigh [Gly 9 -OH]carbetocin, [Asp 5 ]Carbetocin, [Glu 4 ]Carbetocin, [β-Asp 5 ]Carbetocin, [D-Cys 6 ]Carbetocin, [D-Asn 5 ]Carbetocin, Carbetocin sulfoxide I, Carbetocin sulfoxide II, [Des-Gln 4 ] carbetocin, carbetocin dimer, add water to dissolve ([Des-Gln 4 ] Carbetocin was dissolved in 40% acetic acid and quantitatively diluted to make a solution containing about 5 μg per 1 ml. (Prepare 2 portions in parallel)

[0104] Accurately measure 10 μl of the accuracy solution, test solution and reference solution, inject them into the liquid chromatograph and record the chromatogram.

[0105] [Gly 9 -OH]Carbetocin Accuracy

[0106]

[0107] [Asp 5 ]Carbetocin Accuracy

[0108]

[0109]

[0110] [β-Asp 5]Carbetocin Accuracy

[0111]

[0112] [Glu 4 ]Carbetocin Accuracy

[0113]

[0114] Carbetocin sulfoxide I accuracy

[0116]

[0117] Carbetocin sulfoxide Ⅱ accuracy

[0118]

[0119] [D-Cys 6 ]Carbetocin Accuracy

[0120]

[0121] [Des-Gln 4 ]Carbetocin Accuracy

[0122]

[0123] [D-Asn 5 ]Carbetocin Accuracy

[0124]

[0125] Carbetocin dimer accuracy

[0126]

[0127] Conclusion: The accuracy of each impurity was calculated using external standards, and the results were all in the range of 90% to 108%, with RSDs lower than 3.0%, indicating that the method had good accuracy.

[0128] 6. Precision (repeatability and intermediate precision)

[0129] Preparation of test solution: Take an appropriate amount of the product, weigh accurately, dissolve in water and dilute to make a solution containing about 1 mg per 1 ml. (Prepare 6 portions in parallel)

[0130] Repeatability and intermediate precision were measured by different people at different times using different instruments.

[0131]

[0132] Conclusion: Six test solutions were prepared in parallel, and the range of each impurity detected was less than 0.05%. According to repeatability statistics, the range of each impurity detected in 12 test solutions was less than 0.1%, and the method had good precision.

[0133] 7. Solution stability

[0134] At room temperature, within a period of time from the time of solution preparation, accurately measure 10 μl of the above solution, inject it into the liquid chromatograph in sequence, and record the chromatogram.

[0135] Test solution stability results

[0136]

[0137] Conclusion: The test solution was verified for 22.5 hours, and the detected amount of each impurity had no significant change, and the range was less than 0.05%. Therefore, the test solution was stable within 22.5 hours at room temperature.

[0138] 8. Durability

[0139] Robustness to fine-tune chromatographic conditions

[0140]

[0141]

[0142] Durability Results - Separation

[0143]

[0144] Durability Results - Test Solution

[0145]

[0146]

[0147] Note: Difference %, is the absolute value of the difference between the standard condition and each durability condition.

[0148] Conclusion: Under different flow rates, different initial ratios, different column temperatures, different amounts of ammonium acetate added, and different chromatographic columns, the system suitability of [Gly 9 -OH]carbetocin, [Asp 5 ]Carbetocin, [Glu 4 ]Carbetocin, [β-Asp 5 ]Carbetocin, [D-Cys 6 ]Carbetocin, [D-Asn 5 ]Carbetocin, Carbetocin sulfoxide I, Carbetocin sulfoxide II, [Des-Gln 4The separation degrees of carbetocin, carbetocin dimer and carbetocin were all greater than 1.5, and the system applicability was good under all conditions; the difference in the detected amount of impurities in the test samples under all conditions was less than 0.05%, and the difference in total impurities was less than 0.1%, and the method had good durability.

Claims

1. A method for analyzing carbetocin-related substances, characterized in that: The detection was performed by high performance liquid chromatography, and the specific chromatographic conditions were as follows: the chromatographic column was filled with octadecylsilane bonded silica gel; an ultraviolet detector was used, and a mixture of mobile phase A and mobile phase B was used as the mobile phase, and gradient elution was performed, and the gradient elution was:

2. The analysis method according to claim 1, characterized in that: Mobile phase A is ammonium acetate and water in a mass volume ratio of 0.3g:1000ml, mixed and dissolved, then 380ml of acetonitrile is added, and then 8ml of PIC B-8LowUV reagent is added, and water is added to dilute to 2000ml; the mobile phase B is a mixture of mobile phase A and acetonitrile in a volume ratio of 1:1; gradient mobile phase is used for elution, and the mobile phase gradient is set as follows based on the mobile phase volume ratio of 100%:

3. The analysis method according to claim 1, characterized in that: The specifications of the chromatographic column are: Waters C18, 3.0mm×150mm, 3.5μm.

4. The analysis method according to claim 1, characterized in that: The column temperature was 60°C.

5. The analysis method according to claim 1, characterized in that: The flow rate is 0.8 ml / min.

6. The analysis method according to claim 1, characterized in that The injection volume was 10 μl.

7. The analysis method according to claim 1, characterized in that The detection wavelength is 220 nm.

8. The analysis method according to any one of claims 1 to 7, characterized in that The carbetocin related substances are shown in the following table:

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