Method for determining atosiban acetate polymer by liquid chromatography-mass spectrometry

Abstract

The invention relates to a method for determining an atosiban acetate polymer. The method is carried out by adopting high performance liquid chromatography-liquid mass spectrometry chromatography, and comprises the following steps: (1) preparing a polymer-containing test solution of atosiban acetate; (2) injecting the solution obtained in the step (1) into a high performance liquid chromatography and liquid chromatograph-mass spectrometer, and recording a chromatogram; wherein the chromatographic conditions of the liquid chromatography are as follows: the chromatographic column is TSK-GEL G2000SWXL, 7.8 mm * 300 mm * 5 [mu] m, and the mobile phase is prepared from trifluoroacetic acid-water-acetonitrile (0.05: 70: 30); the liquid and mass conditions are as follows: the chromatographic column is ACQUITY UPLC BEH C18 Column, 100 mm * 2.1 mm, 1.7 [mu] m; a mobile phase A is a 0.1% formic acid solution, and a mobile phase B is acetonitrile; an ESI ion source is adopted, and the detection mode is positive ions. The method can be used for measuring the content of high-molecular polymers and identifying the molecular weight of multiple polymers, so that the total amount of the polymers can be measured and controlled more accurately and effectively, and the detection method is simple and convenient to operate and high in specificity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of chemical analysis, in particular, to a method for determining atosiban acetate polymers. BACKGROUND

[0002] Atosiban acetate is a synthetic cyclic polypeptide, which is an oxytocin analogue, and its chemical name is 1-(3-mercapto propionic acid)-2-(O-ethyl-D-tyrosine)-4-L-threonine-8-L-ornithine oxytocin. Clinical results show that after binding to the oxytocin receptor, the product can reduce the contraction frequency and tension of the uterus and inhibit uterine contraction.

[0003] Since the product is a polypeptide drug, and the amino acid contains a thio group, it is easy to form a disulfide bond during synthesis, and multiple disulfide bonds are easy to form a ring, so it is easy to polymerize with each other, which may produce monomers, dimers and trimers. Polymers are one of the key contents of quality control research of this type of drugs. Most of the existing detection methods are for determining the total amount of polymers, which are not strong in specificity and are not accurate in measurement. Therefore, the method of liquid chromatography-mass spectrometry is used to improve the accuracy of measurement data.

[0004] For dimers and trimers in complex mixtures, liquid chromatography-mass spectrometry (LC-MS) technology is needed to make more accurate qualitative and quantitative analysis. Mass spectrometry is a powerful tool for analyzing different polymerization degree compounds in complex mixtures. Through techniques such as electrospray ionization or atmospheric pressure chemical ionization, characteristic ion peaks in the mass spectrum are used to identify and quantify monomers, dimers and trimers.

[0005] Compared with other patents, such as the method for determining the total amount of atosiban acetate polymers disclosed in CN110658296A, which uses liquid phase method to detect the total amount of polymers, the present application uses liquid chromatography-mass spectrometry method, which can not only detect the total amount of polymers, but also identify the molecular weight of dimers and trimers respectively, improve the specificity of the method, and thus more accurately determine the total amount of polymers.

[0006] Compared with the conventional liquid chromatography-mass spectrometry method, due to low ionization efficiency, background interference, and easy dissociation of polymers, the sensitivity of the polymer detection method cannot meet the requirements of actual detection and is not stable, therefore, it is urgent to provide a method for accurately qualitatively / quantitatively detecting atosiban acetate polymers. SUMMARY

[0007] The present application provides a method for determining atosiban acetate polymers, which can accurately detect the polymers contained in atosiban acetate and improve the operability of the detection method.

[0008] In order to solve the above technical problems, the present application provides the following technical solutions:

[0009] A method for determining atosiban acetate polymers, the method comprising the following steps:

[0010] (1) Preparation of test sample: Take the atosiban acetate sample to be tested, dissolve in water to prepare the atosiban acetate test sample;

[0011] (2) Preparation of self-control solution: accurately take the test sample solution, dilute with water to prepare the control solution; preferably, the dilution factor is 100 times;

[0012] (3) Test sample enrichment:

[0013] The molecular exclusion chromatography method is used to separate and control the polymer peaks before the main peak of atosiban acetate, that is, the larger the molecular weight, the shorter the retention time in the chromatographic column.

[0014] The atosiban acetate test sample prepared in step (1) is injected into the liquid chromatograph, and the components close to the main peak are enriched as the polymer enrichment solution containing dimers;

[0015] The other components except the components close to the main peak are enriched as the polymer enrichment solution containing atosiban acetate dimers and trimers; the main peak components are enriched as the polymer enrichment solution containing atosiban acetate monomers.

[0016] The chromatographic conditions are as follows:

[0017] Spherical hydrophilic modified silica gel is used as the filler, trifluoroacetic acid-water-acetonitrile in a volume ratio of 0.05:70:30 is used as the mobile phase, isocratic elution is performed, the running time is not less than 2 times the retention time of the main peak atosiban acetate, the flow rate is 0.4 ml / min to 0.6 ml / min, the detection wavelength is 220 nm, the column temperature is 20℃ to 30℃, and the injection volume is 20 μl;

[0018] (4) Polymer LC-MS analysis: The solution enriched in step (3) is dried, redissolved with ultrapure water, dried again, redissolved with 0.1% formic acid water, centrifuged to take the supernatant, and subjected to LC-MS analysis; the control solution prepared in step (2) is injected;

[0019] Chromatographic conditions:

[0020] Chromatographic column: chromatographic column with octadecylsilane bonded silica gel as the filler;

[0021] Mobile phase A is 0.1% formic acid solution, and mobile phase B is acetonitrile,

[0022] Flow rate 0.3 ml / min, detection wavelength 220 nm,

[0023] Column temperature 40℃,

[0024] Injection volume 1 μl,

[0025] The elution gradient is as follows:

[0026]

[0027]

[0028] Mass spectrometry conditions:

[0029] Electrospray ion source: ESI,

[0030] Detection mode: positive ion,

[0031] Capillary voltage: 3.0KV,

[0032] Cones hole voltage: 120V,

[0033] Source temperature: 120℃.

[0034] Desolvation zone temperature: 450℃,

[0035] Collision gas: argon,

[0036] Pressure: 1.0 x 10 -4 mbar,

[0037] Gas curtain flow rate: 50L / h,

[0038] Desolvation gas flow rate is 800L / h.

[0039] Further, (1) the concentration of atosiban acetate in the test sample is not less than 0.99 ng / ml.

[0040] Further, (3) the chromatographic column is TSK-GEL G2000SWXL, 7.8mm x 300mm x 5μm,

[0041] Further, (3) the flow rate is 0.5 ml per minute, and the column temperature is 25℃.

[0042] Further, (3) the enrichment is repeatedly injecting atosiban acetate test sample or preliminary enrichment solution.

[0043] Further, (3) the enrichment is injecting atosiban acetate test sample or preliminary enrichment solution not less than 3 times.

[0044] Further, (4) the chromatographic column is ACQUITY UPLC BEH C18 Column, 100mm x 2.1mm, 1.7μm.

[0045] Further, the method uses self-control method for calculation.

[0046] Further, the calculation formula is as follows: In the formula: ΣA 杂质峰 - peak area of impurity peak detected in the test solution; A 对照主峰 - peak area of main peak of the self-control solution.

[0047] The impurity peak is a chromatographic peak with an elution time less than the retention time of the main peak, i.e., a dimer and a trimer peak.

[0048] Specifically, since the molecular weight of the trimer is greater than that of the dimer and the monomer in turn, according to the exclusion method, the trimer, the dimer and the monomer are eluted in descending order of molecular weight.

[0049] Further, the theoretical Mass of atosiban acetate is 993.4412, the theoretical Mass of atosiban acetate dimer is 1986.8796, and the theoretical Mass of atosiban acetate trimer is 2980.3236.

[0050] The beneficial effects of the present application are:

[0051] The present application adopts a liquid quality combined method to determine the polymer, which can not only determine the total amount of the polymer, but also identify the molecular weight of the dimer and the trimer respectively, improve the specificity of the method, and thus more accurately determine the total amount of the polymer. At the same time, through the enrichment sample pretreatment strategy, the sensitivity of the polymer detection is significantly improved, the detection limit is reduced to the nanogram level, and the content of multiple levels of polymer can be quantitatively determined. Through analysis of the liquid quality detection result, it can be known that atosiban acetate mainly contains dimer and trimer, and the molecular weight is basically consistent with the calculated molecular weight. Compared with the prior art, the method can more accurately determine the content of the polymer, and the quantitative limits of atosiban, dimer and trimer are 0.99 ng, 1.33 ng and 1.30 ng respectively. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 Polymer enrichment graph;

[0053] Figure 2 Polymer enrichment graph containing dimer;

[0054] Figure 3 Polymer enrichment graph containing trimer;

[0055] Figure 4 Atosiban dimer-containing collection liquid BPI, TIC graph;

[0056] Figure 5 For Figure 4 Local enlarged view of selected area;

[0057] Figure 6Atosiban containing dimer sample prototype peak MS chart (upper chart after correction, lower chart before correction);

[0058] Figure 7 Atosiban containing dimer sample main peak 1 MS chart (upper chart after correction, lower chart before correction);

[0059] Figure 8 Atosiban containing dimer sample main peak 2 MS chart (upper chart after correction, lower chart before correction);

[0060] Figure 9 Atosiban containing trimer sample shoulder peak MS chart (upper chart after correction, lower chart before correction);

[0061] Figure 10 Atosiban containing trimer sample shoulder peak MS chart (upper chart after correction, lower chart before correction);

[0062] Figure 11 For Figure 10 Selected area local magnification;

[0063] Figure 12 Atosiban containing trimer sample shoulder peak MS chart (upper chart after correction, lower chart before correction);

[0064] Figure 13 Atosiban containing trimer sample shoulder peak MS chart (upper chart after correction, lower chart before correction);

[0065] Figure 14 Atosiban containing trimer sample shoulder peak MS chart (upper chart after correction, lower chart before correction);

[0066] Figure 15 Atosiban containing trimer sample shoulder peak MS chart (upper chart after correction, lower chart before correction); DETAILED DESCRIPTION

[0067] The present application is further explained in conjunction with the following examples, which do not limit the application in any way.

[0068] Example 1

[0069] Since the polymer production route of atosiban acetate is mainly through degradation under strong light irradiation, in this example, the sample of atosiban acetate placed under strong light irradiation is detected for polymers using a polymer analysis method, and a certain amount of polymers is enriched for LC-MS analysis. Through LC-MS analysis, the reliability of detection of dimer peaks and trimer peaks is increased.

[0070] (1) Preparation of test sample: take an appropriate amount of the product and place it under light conditions (light intensity 5000lx±500lx), and after 12 days of sufficient light irradiation, take it out and add water to prepare a test sample containing 0.75mg of atosiban acetate per 1ml;

[0071] (2) Self-control solution: precisely take 1.0 ml of the test solution, add water to a 100 ml volumetric flask, dissolve and dilute to the mark.

[0072] (3) Polymer enrichment preparation: according to the molecular exclusion method, each high molecular compound is sequentially eluted from large to small molecular weight, and the test solution is precisely taken according to the test preparation method of step (1) and injected into the liquid chromatograph. The enrichment chart is shown in Figure 1 , wherein the retention time is 13 min to 15 min, which is a polymer, and the retention time is about 17 min, which is the main peak of atosiban acetate.

[0073] Since the molecular weight of atosiban acetate dimer is 1986.8796, and the molecular weight of atosiban acetate trimer is 2980.3236, the trimer elutes earlier than the dimer, therefore, the components enriched for 15 min to 17 min are injected at least 3 times, and the components enriched for 15 min to 17 min are injected at least 3 times. as a polymer enrichment solution containing dimer, see Figure 2 ;

[0074] The components enriched for 13 min to 15 min are injected at least 3 times, and the components enriched for 13 min to 15 min are injected at least 3 times. as a polymer enrichment solution containing trimer, see Figure 3 , and the main peak of 17 min is further enriched as a monomer solution.

[0075] The chromatographic conditions of this high performance liquid chromatography are as follows:

[0076] The chromatographic column is TSK-GEL G2000SWXL, 7.8 mm x 300 mm x 5 μm, with trifluoroacetic acid-water-acetonitrile (volume ratio 0.05:70:30) as the mobile phase, isocratic elution, running to 2 times the retention time of the main peak, flow rate is 0.5 ml per minute, detection wavelength is 220 nm, column temperature is 25℃.

[0077] (4) Polymer LC-MS analysis: the polymer-containing solution enriched in step (3) is subjected to sample treatment, dried, redissolved with an appropriate amount of water, desalted, and then dried. 0.1% formic acid is used for redissolution, low-speed centrifugation for 10 min at 10000 rpm, and the supernatant is injected under the LC-MS conditions. The self-control solution prepared in step (2) is injected.

[0078] The chromatographic conditions of LC-MS are as follows:

[0079] Liquid chromatography conditions: chromatographic column: ACQUITY UPLC BEH C18 Column, 100 mm x 2.1 mm, 1.7 μm, mobile phase A: 0.1% formic acid solution, mobile phase B: acetonitrile, flow rate: 0.3 ml / min, detection wavelength: 220 nm, column temperature: 40℃, injection volume: 1 μl, gradient according to the following table;

[0080]

[0081]

[0082] Mass spectrometry conditions: Electrospray ion source: ESI, detection mode: positive ion, capillary voltage: 3.0KV, cone hole voltage: 120V, source temperature: 120℃, desolvation zone temperature: 450℃, collision gas: argon, pressure: 1.0x10 -4 mbar, curtain gas flow rate: 50L / h, desolvation gas flow rate: 800L / h.

[0083] The detection results show that in the sample solution containing the dimer, the theoretical molecular weight of atosiban acetate is 993.4412, the measured m / z of atosiban monovalent peak is 994.4489, the calculated measured molecular weight is 993.4410, and the ppm of the theoretical value is 0.1; the main peak 1 MS diagram, mainly atosiban dimer bivalent isotope peak, the theoretical molecular weight of the dimer is 1986.8824, the measured m / z of the dimer bivalent is 994.4476, the calculated measured molecular weight is 1986.8796, and the ppm of the theoretical value is 1.4; the main peak 2 MS diagram, mainly atosiban dimer bivalent isotope peak, the measured bivalent peak m / z is 994.4480, the calculated measured molecular weight is 1986.8804, and the ppm of the theoretical value is 1.0; the main peak 2 shoulder peak MS diagram, mainly atosiban trimer trivalent isotope peak, the theoretical molecular weight of the trimer is 2980.3236, the measured m / z of the trimer trivalent peak is 994.4504, the calculated measured molecular weight is 2980.3278, and the ppm of the theoretical value is 1.4. Therefore, the sample mainly contains atosiban dimer, but a small amount of atosiban and trimer exists. The results are shown in Figures 4 to 9 .

[0084] In the sample solution without dimer, the theoretical molecular weight of atosiban acetate is 993.4412, the measured m / z of atosiban monovalent peak is 994.4474, the calculated measured molecular weight is 993.4396, the ppm with the theoretical value is 1.6; the main peak MS diagram, in which the main isotope peak of atosiban trimer trivalent, the theoretical molecular weight of trimer is 2980.3236, the measured m / z of trimer trivalent peak is 994.4486, the calculated measured molecular weight is 2980.3224, the ppm with the theoretical value is 0.4; the chromatographic small peak with the retention time of 5.54 min, the trivalent m / z of the main component is 993.7794, the calculated measured molecular weight is 2978.3148, which is speculated to be the product of atosiban trimer after losing 2 H, the theoretical molecular weight of the product is 2978.3079, the ppm with the measured value is 2.3; the chromatographic small peak with the retention time of 5.54 min, the trivalent m / z of the main component is 993.7792, the calculated measured molecular weight is 2978.3142, which is also speculated to be the product of atosiban trimer after losing 2 H, the ppm with the theoretical value is 2.1. Therefore, the main components of the sample are atosiban and its trimer, in addition, there are a small amount of unknown components, which are speculated to be the products of atosiban trimer after losing 2 H. The results are shown in Figures 10 to 15 .

[0085] Self-control method is used for calculation. The calculation formula is as follows: In the formula: ΣA 杂质峰 - the peak area of the impurity detected in the test solution; A 对照主峰 - the peak area of the main peak of the self-control solution.

[0086] The contents of dimer and trimer are quantitatively detected respectively, in this embodiment, the dimer (%) = the detected dimer impurity peak area / control peak area*1%*100=0.05%, the trimer (%) = the detected trimer impurity peak area / control peak area*1%*100=0.11%. The total content of dimer and trimer is less than the limit of 0.5%.

[0087] Example 2: Selection of mobile phase

[0088] 1. Type of mobile phase: the type of mobile phase is selected, and the three types of mobile phase, phosphoric acid-water-acetonitrile (0.1:70:30), trifluoroacetic acid-water-acetonitrile (0.05:70:30), and perchloric acid-water-acetonitrile (0.05:70:30) are compared. After comparative experiment, under trifluoroacetic acid-water-acetonitrile (0.05:70:30), the main peak type is symmetrical, and the separation degree from adjacent impurities is better.

[0089] 2. Mobile phase ratio: The mobile phase in the chromatographic condition is trifluoroacetic acid-water-acetonitrile (0.05:70:30), different ratios of the mobile phase are tried, trifluoroacetic acid-water-acetonitrile (0.05:60:40), trifluoroacetic acid-water-acetonitrile (0.05:50:50) and trifluoroacetic acid-water-acetonitrile (0.05:70:30), after comparison, under the condition of trifluoroacetic acid-water-acetonitrile (0.05:70:30), the separation degree of the main peak and the polymer is better, about 4.2.

[0090] Screening of column temperature in Example 3

[0091] The column temperature in the chromatographic condition is 25℃, the column temperature is set to 25℃, 30℃, 35℃ and 40℃ respectively, by comparison, the column temperature has no great influence on the polymer, therefore 25℃ is preferred

[0092] Specificity test in Example 4

[0093] Chromatographic conditions are as follows:

[0094] Liquid phase conditions: Chromatographic column: ACQUITY UPLC BEH C18 Column, 100mm x 2.1mm, 1.7μm, mobile phase A is 0.1% formic acid solution, mobile phase B is acetonitrile, flow rate is 0.3ml / min, detection wavelength is 220nm, column temperature is 40℃, sample amount is 1μl, gradient is carried out according to the following table;

[0095] Time (min) A(％) B(％) 0 90 10 1 90 10 4 60 40 8 15 85 8.1 15 85 10 90 10 12 90 10

[0096] Mass spectrometry conditions: Electrospray ion source: ESI, detection mode: positive ion, capillary voltage: 3.0KV, cone hole voltage: 120V, source temperature: 120℃, desolvation zone temperature: 450℃, collision gas: argon, pressure: 1.0x10 -4 mbar, curtain gas flow rate: 50L / h, desolvation gas flow rate is 800L / h.

[0097] Blank solvent: water.

[0098] Test sample solution: Take the atosiban acetate sample to be tested, dissolve in water to prepare the atosiban acetate test sample.

[0099] Polymer enrichment solution: Enrich the dimer and trimer solutions respectively according to the method of Example 1.

[0100] Mixed solution: Take appropriate amount of atosiban acetate and each polymer, dissolve and dilute in water to obtain a solution containing about 0.75mg of atosiban acetate, 75μg of dimer and 75μg of trimer per 1ml.

[0101] Take 1μl of each of the above solutions, inject into the liquid chromatograph, record the chromatogram and investigate the specificity.

[0102]

[0103] Conclusion: 1. The blank solvent does not interfere with the detection of atosiban acetate and polymers. 2. In the mixed solution, the separation degree between each polymer and between atosiban acetate and the adjacent polymer is good.

[0104] Example 5 Quantitative limit test

[0105] After identifying the molecular weight, the quantitative limit test of the liquid phase method was carried out to verify the feasibility of the method.

[0106] The chromatographic conditions are as follows:

[0107] Liquid phase conditions: chromatographic column: ACQUITY UPLC BEH C18 Column, 100mm x 2.1mm, 1.7μm, mobile phase A is 0.1% formic acid solution, mobile phase B is acetonitrile, flow rate 0.3ml / min, detection wavelength 220nm, column temperature 40℃, injection volume 1μl, gradient according to the following table;

[0108] Time (min) A(％) B(％) 0 90 10 1 90 10 4 60 40 8 15 85 8.1 15 85 10 90 10 12 90 10

[0109] Mass spectrometry conditions: electrospray ion source: ESI, detection mode: positive ion, capillary voltage: 3.0KV, cone hole voltage: 120V, source temperature: 120℃, desolvation zone temperature: 450℃, collision gas: argon, pressure: 1.0x10 -4 mbar, curtain gas flow rate: 50L / h, desolvation gas flow rate: 800L / h.

[0110] After diluting the enriched solution of the polymer and the atosiban acetate monomer solution, 1μl was injected into the liquid chromatograph, and the chromatogram was recorded until the signal-to-noise ratio was about 10, as the quantitative limit solution, and 6 consecutive injections were made to investigate the quantitative limit injection precision. The data are as follows:

[0111]

[0112] The test sample concentration provided in this example is 2mg / ml, and the quantitative limits of atosiban, dimer and trimer are 0.99ng, 1.33ng and 1.30ng respectively, which are about 0.002%, 0.003% and 0.003% of the test sample concentration respectively, all of which are lower than the reporting limit 0.05%, and the sensitivity is high, indicating that the chromatographic conditions provided by the present application have high sensitivity.

[0113] Example 5 Accuracy test

[0114] The chromatographic conditions are as follows:

[0115] Liquid phase condition: chromatographic column: ACQUITY UPLC BEH C18 Column, 100mm x 2.1mm, 1.7um, mobile phase A is 0.1% formic acid solution, mobile phase B is acetonitrile, flow rate 0.3ml / min, detection wavelength 220nm, column temperature 40 DEG C, sample amount 1ul, gradient is carried out according to the following table:

[0116] Time (min) A(％) B(％) 0 90 10 1 90 10 4 60 40 8 15 85 8.1 15 85 10 90 10 12 90 10

[0117] Mass spectrum condition: electrospray ion source: ESI, detection mode: positive ion, capillary voltage: 3.0KV, cone hole voltage: 120V, source temperature: 120 DEG C, desolvation zone temperature: 450 DEG C, collision gas: argon, pressure: 1.0x10 -4 Mbar, curtain gas flow rate: 50L / h, desolvation gas flow rate is 800L / h.

[0118] The atosiban acetate reference solution and each recovery solution (80%, 100%, 120%) are prepared, 1ul of each is injected, chromatograms are recorded, and the recovery is calculated.

[0119]

[0120]

[0121] Conclusion: from the table, the recovery of the method is good, and the RSD of the recovery of the dimer and trimer is 0.14% and 0.45% respectively.

[0122] Therefore, the detection method of the atosiban acetate polymer of the present application can not only effectively detect the content of the polymer in atosiban acetate injection, but also can exclude whether other substances except the polymer are contained in the detected substance through liquid chromatography-mass spectrometry. The present application reduces the risk of the polymer exceeding the control limit, and can more accurately and conveniently detect the content of the high molecular polymer in atosiban acetate.

[0123] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method of determining atosiban acetate polymers characterized in that, The method comprises the following steps: (1) Preparation of the sample: the sample of atosiban acetate to be tested is dissolved in water to prepare the sample of atosiban acetate; (2) Preparation of the self-control solution: the sample solution is precisely measured and diluted with water to prepare the control solution; Preferably, the dilution factor is 100 times. (3) Enrichment of the sample: The polymer peak before the main peak of atosiban acetate is separated and controlled by using the molecular exclusion chromatography, The sample of atosiban acetate prepared in step (1) is injected into the liquid chromatograph to enrich the components close to the main peak as the polymer enrichment solution containing dimers; The other components except the components close to the main peak are enriched as the polymer enrichment solution containing atosiban acetate dimers and trimers; and the main peak components are enriched as the polymer enrichment solution containing atosiban acetate monomers. The chromatographic conditions are as follows: The spherical hydrophilic modified silica gel is used as the filler, the trifluoroacetic acid-water-acetonitrile with a volume ratio of 0.05:70:30 is used as the mobile phase, the isocratic elution is performed, the running time is not less than 2 times of the retention time of the main peak atosiban acetate, the flow rate is 0.4-0.6 ml / min, the detection wavelength is 220 nm, the column temperature is 20-30℃, and the injection amount is 20 μl; (4) LC-MS analysis of the polymer: the solutions enriched in step (3) are dried, redissolved with ultrapure water, dried again, redissolved with 0.1% formic acid solution, centrifuged to take the supernatant, and subjected to LC-MS analysis; the self-control solution prepared in step (2) is injected; The chromatographic conditions are as follows: The chromatographic column is a chromatographic column with octadecylsilane-bonded silica gel as the filler; The mobile phase A is 0.1% formic acid solution, and the mobile phase B is acetonitrile, The flow rate is 0.3 ml / min, The detection wavelength is 220 nm, The column temperature is 40℃, The injection amount is 1 μl, The elution gradient is as follows: The mass spectrometry conditions are as follows: The electrospray ion source is ESI, The detection mode is positive ion, The capillary voltage is 3.0 KV, The cone hole voltage is 120 V, The source temperature is 120℃, The desolvation zone temperature is 450℃, The collision gas is argon, Pressure: 1.0 x 10 -4 mbar, The gas curtain gas flow rate is 50 L / h, The desolvation gas flow rate is 800 L / h.

2. The method of claim 1, wherein, (1) The concentration of atosiban acetate in the sample is not less than 0.99 ng / ml.

3. The method of claim 1, wherein, (3) The chromatographic column is TSK-GEL G2000SWXL, 7.8 mm x 300 mm x 5 μm, 4. The method of claim 1, wherein, (3) The flow rate is 0.5 ml / min, and the column temperature is 25℃.

5. The method of claim 1, wherein, (3) The enrichment is repeatedly injecting the sample of atosiban acetate or the preliminary enrichment solution.

6. The method of claim 5, wherein, The repeated injection is not less than 3 times.

7. The method of claim 1, wherein, (4) The chromatographic column is ACQUITY UPLC BEHC18 Column, 100 mm x 2.1 mm, 1.7 μm.

8. The method of claim 1, wherein, The method uses the self-control method for calculation.

9. The detection method according to claim 8, characterized in that, The calculation formula is as follows: wherein: ∑A 杂质峰 - area of the impurity peak detected in the test solution; A 对照主峰 - area of the main peak of the self-control solution; said impurity peak is a chromatographic peak with an elution time less than the retention time of the main peak.

10. The method of claim 1, wherein, The theoretical mass of atosiban acetate is 993.4412, the theoretical mass of atosiban acetate dimer is 1986.8796, and the theoretical mass of atosiban acetate trimer is 2980.3236.

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