CN101696959A - Acetic acid atosiban, and method for detecting content of preparation of acetic acid atosiban and relevant substances - Google Patents

Abstract

Translated fromChinese

本发明涉及一种醋酸阿托西班及其制剂的含量测定和有关物质测定的方法，检测方法为高效液相色谱法，色谱条件为：色谱柱：以十八烷基硅烷键合硅胶(C18)为填充剂；以0.05mol/L的磷酸盐缓冲液为流动相A，乙腈为流动相B，进行梯度洗脱；检测波长为205～225nm；流速为0.8～1.2ml/min，配制的样品浓度为每1ml中约含多肽0.1～15mg，进样量：5-200μl，采用本发明的高效液相色谱分析法，可以同时准确测定样品及其杂质的含量，分析过程中样品分离彻底，分析结果重现性好，为生产进程中的质量控制分析提供了简单可靠的方法。

The invention relates to a method for determining the content of atosiban acetate and its preparations and related substances. The detection method is high performance liquid chromatography. ) as filler; 0.05mol/L phosphate buffer as mobile phase A, acetonitrile as mobile phase B, gradient elution; detection wavelength is 205 ~ 225nm; flow rate is 0.8 ~ 1.2ml/min, the prepared sample The concentration is about 0.1-15mg of polypeptide per 1ml, and the injection volume: 5-200μl. Using the high-performance liquid chromatography analysis method of the present invention, the content of the sample and its impurities can be accurately measured at the same time. During the analysis process, the sample is completely separated and analyzed The results are reproducible and provide a simple and reliable method for quality control analysis in the production process.

Description

Translated fromChinese

一种醋酸阿托西班及其制剂的含量测定和有关物质测定的方法A kind of method for the content determination of atosiban acetate and its preparation and the determination of related substances

技术领域：Technical field:

本发明涉及一种活性多肽原料药和制剂的方法检测方法，尤其是涉及醋酸阿托西班及其制剂的含量测定和有关物质测定的方法。The invention relates to a method for detecting active polypeptide raw materials and preparations, in particular to a method for determining the content of atosiban acetate and its preparations and related substances.

背景技术：Background technique:

阿托西班(atosiban)，其化学名称为：1-(3-硫醇丙醇酸)-2-(O-乙基-D酪氨酸)-4-L-苏氨酸-8-L-鸟氨酸-催产素。Atosiban (atosiban), its chemical name is: 1-(3-mercaptopropanol acid)-2-(O-ethyl-D-tyrosine)-4-L-threonine-8-L - Ornithine - Oxytocin.

其结构式为：Its structural formula is:

阿托西班是一种催产素类似物，为人工合成的环状多肽，是子宫内及蜕膜、胎膜上受体的催产素竞争性拮抗剂，在临床上使用其醋酸盐治疗早产。醋酸阿托西班注射液(AtosibanAcetate Injection)由辉凌公司(Ferring AB)研制，2000年3月23日首次在奥地利上市，商品名为：

Atosiban is an oxytocin analog, a synthetic cyclic polypeptide, and a competitive antagonist of oxytocin on receptors in the uterus, decidua, and fetal membrane. Its acetate is used clinically to treat premature labor . Atosiban Acetate Injection (AtosibanAcetate Injection) was developed by Ferring AB and was first launched in Austria on March 23, 2000. The trade name is:

醋酸阿托西班和醋酸阿托西班注射液的有关物质和含量的检测方法尚未见报道，其测定方法的难度在于在其合成过程中可能会产生缺失(不完全)肽、断裂肽、去酰胺多肽、非对映异构的多肽和低聚物等多种工艺杂质，通过色谱法进行纯化后，无法去除的杂质与主药的色谱行为比较相似，不易完全分离。The detection method of related substance and content of atosiban acetate and atosiban acetate injection has not been reported yet, and the difficulty of its determination method is that missing (incomplete) peptide, fragmentation peptide, de- Amide peptides, diastereoisomeric peptides and oligomers and other process impurities, after purification by chromatography, the impurities that cannot be removed are similar to the chromatographic behavior of the main drug, and it is not easy to completely separate them.

发明内容：Invention content:

本发明的目的是提供一种能测定醋酸阿托西班[1-(3-硫醇丙醇酸)-2-(O-乙基-D酪氨酸)-4-苏氨酸-8-鸟氨酸-催产素醋酸盐]及醋酸阿托西班制剂的含量和有关物质的检测方法。The object of the present invention is to provide a method capable of determining atosiban acetate [1-(3-thiol propanoic acid)-2-(O-ethyl-D tyrosine)-4-threonine-8- Ornithine-Oxytocin Acetate] and Atosiban Acetate Preparations and the detection methods of related substances.

本发明的醋酸阿托西班含量测定方法如下：Atosiban acetate content assay method of the present invention is as follows:

采用反相高效液相色谱法检测1-(3-硫醇丙醇酸)-2-(O-乙基-D酪氨酸)-4-苏氨酸-8-鸟氨酸-催产素醋酸盐及其制剂中醋酸阿托西班的含量，其步骤包括：Determination of 1-(3-thiolpropanoic acid)-2-(O-ethyl-D-tyrosine)-4-threonine-8-ornithine-oxytocin ester by RP-HPLC Content of atosiban acetate in acid salt and preparation thereof, its steps comprise:

1)取样品适量，用流动相A配制成每1ml中含0.75mg的溶液，作为供试品溶液；1) get sample appropriate amount, be mixed with the solution that contains 0.75mg in every 1ml with mobile phase A, as need testing solution;

2)另称取对照品适量，用流动相A溶解并稀释制成每1ml中含0.75mg的溶液，作为对照品溶液；2) Another appropriate amount of reference substance was weighed, dissolved and diluted with mobile phase A to make a solution containing 0.75mg per 1ml, as the reference substance solution;

3)量取供试品溶液与对照品溶液各20μl，分别注入液相色谱仪，记录色谱图，按外标法以峰面积计算，得到样品中醋酸阿托西班的含量。3) Take 20 μl each of the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, calculate the peak area according to the external standard method, and obtain the content of atosiban acetate in the sample.

色谱条件：Chromatographic conditions:

用十八烷基硅烷键合硅胶为填充剂；以0.05mol/L的磷酸盐缓冲液(取磷酸二氢钾6.8g，加水至1000ml，用磷酸调节pH值至2.3，再加入120μl三乙胺，即得)为流动相A，乙腈为流动相B，进行梯度洗脱；检测波长为215nm；流速为1.0ml/min。理论塔板数按阿托西班峰计算应不低于5000。梯度洗脱程序如下：Use octadecylsilane bonded silica gel as filler; use 0.05mol/L phosphate buffer (take 6.8g potassium dihydrogen phosphate, add water to 1000ml, adjust pH value to 2.3 with phosphoric acid, then add 120μl triethylamine , to get) as mobile phase A, acetonitrile as mobile phase B, gradient elution; detection wavelength is 215nm; flow rate is 1.0ml/min. The number of theoretical plates should not be less than 5000 based on the calculation of the atosiban peak. The gradient elution procedure is as follows:

其中对照品为醋酸阿托西班纯品，可从试剂商店购买。The reference substance is pure atosiban acetate, which can be purchased from a reagent store.

本发明的有关物质含量测定方法，步骤如下：Related substance content determination method of the present invention, step is as follows:

1)取样品适量，加流动相配制成每1ml中约含1.5mg的溶液，作为供试品溶液；1) Take an appropriate amount of sample, add mobile phase to be mixed with a solution containing about 1.5 mg in every 1 ml, as the test solution;

2)量取供试品溶液1ml，置100ml量瓶中，加流动相A稀释至刻度，摇匀，作为对照溶液；2) Measure 1ml of the test solution, put it in a 100ml measuring bottle, add mobile phase A to dilute to the mark, shake well, and use it as a contrast solution;

3)取对照溶液20μl注入液相色谱仪，调节检测灵敏度，使主成分色谱峰峰高约为满量程的20％，再精密量取供试品溶液和对照溶液各20μl，分别注入液相色谱仪，记录色谱图至主成分峰保留时间的2倍，计算有关物质含量。3) Take 20 μl of the control solution and inject it into the liquid chromatograph, adjust the detection sensitivity so that the peak height of the main component chromatographic peak is about 20% of the full scale, then accurately measure 20 μl each of the test solution and the control solution, and inject them into the liquid chromatograph respectively Instrument, record the chromatogram to twice the retention time of the main component peak, and calculate the content of related substances.

色谱条件：Chromatographic conditions:

用十八烷基硅烷键合硅胶为填充剂；以0.05mol/L的磷酸盐缓冲液(取磷酸二氢钾6.8g，加水1000ml，用磷酸调节pH值至2.3，再加入120μl三乙胺，即得)为流动相A，乙腈为流动相B，进行梯度洗脱；检测波长为220nm；流速为1.0ml/min。理论塔板数按阿托西班峰计算应不低于5000。梯度洗脱程序如下：Use octadecylsilane-bonded silica gel as a filler; use 0.05mol/L phosphate buffer (take 6.8g of potassium dihydrogen phosphate, add 1000ml of water, adjust the pH value to 2.3 with phosphoric acid, then add 120μl of triethylamine, Obtained) as mobile phase A, acetonitrile as mobile phase B, gradient elution is carried out; detection wavelength is 220nm; flow rate is 1.0ml/min. The number of theoretical plates should not be less than 5000 based on the calculation of the atosiban peak. The gradient elution procedure is as follows:

其中所述有关物质是指醋酸阿托西班合成过程中带有的杂质成分，如溶剂，中间体，原料，氨基酸片段等，杂质成分为限制成分，测定有关物质目的在于限制其含量。The related substances refer to the impurity components in the synthesis process of atosiban acetate, such as solvents, intermediates, raw materials, amino acid fragments, etc. The impurity components are restricted components, and the purpose of determining the related substances is to limit their content.

本发明测定方法的特点是：被分析物中各成分间分离彻底，可操作性强，分析方法专属性强、准确性和灵敏度高、适应性强为保证药品安全、有效、可控提供依据。The characteristics of the determination method of the present invention are: the components in the analyte are completely separated, the operability is strong, the analysis method is specific, the accuracy and sensitivity are high, and the adaptability is strong, which provides a basis for ensuring the safety, effectiveness and controllability of medicines.

附图说明：Description of drawings:

图1为0.1％三氟乙酸∶乙腈为流动相的色谱图Fig. 1 is 0.1% trifluoroacetic acid: acetonitrile is the chromatogram of mobile phase

图2为0.05mol/L磷酸盐缓冲液∶乙腈为流动相的色谱图Fig. 2 is 0.05mol/L phosphate buffer: acetonitrile is the chromatogram of mobile phase

图3为专属性实验色谱图。Figure 3 is the specificity experimental chromatogram.

图4为检测限色谱图。Figure 4 is a detection limit chromatogram.

图5为样品有关物质测定的色谱图。Figure 5 is a chromatogram of the determination of related substances in the sample.

图6为样品含量测定的色谱图。Fig. 6 is the chromatogram of sample content determination.

具体实施方式：Detailed ways:

以下实施例用于说明本发明，但不作为对本发明的限制。The following examples are used to illustrate the present invention, but not to limit the present invention.

实施例1Example 1

醋酸阿托西班含量测定方法Determination method of atosiban acetate

实验仪器及色谱条件：Experimental equipment and chromatographic conditions:

Waters 515-717-2996高效液相色谱仪，Agilent C18色谱柱(5μm，150×4.6mm)，以0.05mol/L的磷酸盐缓冲液(取磷酸二氢钾6.8g，加水1000ml，用磷酸调节pH值至2.0～3.0，再加入120μl三乙胺，即得)为流动相A，乙腈为流动相B，进行梯度洗脱，检测波长为205～225nm；流速为0.8～1.2ml/min；进样量为5～200μl。。Waters 515-717-2996 high-performance liquid chromatography, Agilent C18 chromatographic column (5μm, 150×4.6mm), with 0.05mol/L phosphate buffer (take 6.8g of potassium dihydrogen phosphate, add water 1000ml, adjust with phosphoric acid pH value to 2.0 ~ 3.0, then add 120μl triethylamine, to get) as mobile phase A, acetonitrile as mobile phase B, carry out gradient elution, detection wavelength is 205 ~ 225nm; flow rate is 0.8 ~ 1.2ml/min; The sample volume is 5-200 μl. .

试剂：Reagent:

乙腈(HPLC级)来自美国天地公司，超纯水为Millipore超纯水机自制。Acetonitrile (HPLC grade) was from American Tiandi Company, and ultrapure water was made by Millipore ultrapure water machine.

色谱条件的选择Choice of Chromatographic Conditions

1.1流动相1.1 mobile phase

我们通过实验比较了0.1％三氟乙酸(TFA)∶乙腈(ACN)与0.05mol/L磷酸盐缓冲液(PBS)∶ACN，以0.1％三氟乙酸水溶液作为流动相，基线波动大，杂质峰与主峰无法完全分离，干扰测定；而以0.05mol/L为流动相，基线平稳，杂质峰与主峰可完全分离，且主峰的对称性好(见图1和图2)。We have compared 0.1% trifluoroacetic acid (TFA): acetonitrile (ACN) and 0.05mol/L phosphate buffer saline (PBS): ACN by experiment, with 0.1% trifluoroacetic acid aqueous solution as mobile phase, the baseline fluctuates greatly, impurity peak It cannot be completely separated from the main peak, which interferes with the determination; while using 0.05mol/L as the mobile phase, the baseline is stable, the impurity peak can be completely separated from the main peak, and the symmetry of the main peak is good (see Figure 1 and Figure 2).

1.2检测波长1.2 Detection wavelength

用紫外分光光度计在200～400nm进行连续扫描，样品在215nm处有最大吸收，而有关物质大多在220nm处有较强的吸收。同时考虑基线噪音对检测的干扰并兼顾测定的灵敏度，选择有关物质测定的检测波长定为220nm，含量测定的检测波长为215nm。Continuously scan at 200-400nm with an ultraviolet spectrophotometer, the sample has a maximum absorption at 215nm, and most of the related substances have strong absorption at 220nm. At the same time, considering the interference of baseline noise on the detection and taking into account the sensitivity of the determination, the detection wavelength for the determination of related substances is selected as 220nm, and the detection wavelength for content determination is 215nm.

1.3样品浓度1.3 Sample concentration

本品制剂的浓度为7.5mg/ml，为了实验操作方便准确，分别比较了浓度为0.75mg/ml与1.5mg/ml供试品的色谱图，实验结果表明，浓度为1.5mg/ml的样品杂质峰的峰高及面积均较显著，且未造成色谱柱过载，因此确定有关物质测定样品的浓度为1.5mg/ml，含量测定样品的浓度为0.75mg/ml。The concentration of this product preparation is 7.5mg/ml. For the convenience and accuracy of the experimental operation, the chromatograms of the test samples with a concentration of 0.75mg/ml and 1.5mg/ml were compared respectively. The experimental results show that the sample with a concentration of 1.5mg/ml The peak height and area of the impurity peaks are all more significant, and the chromatographic column is not overloaded, so it is determined that the concentration of the related substance determination sample is 1.5mg/ml, and the concentration of the content determination sample is 0.75mg/ml.

检测条件的验证Verification of detection conditions

2.1专属性2.1 Specificity

取样品适量，加流动相A溶解并稀释制成浓度为1.5mg/ml的溶液，每1ml加10％氢氧化钠溶液约5滴，室温放置25分钟，取20μl注入色谱仪，记录色谱图(见图3)。结果表明，再次检测条件下，破坏后的样品中其他组分与主峰均可达到基线分离，专属性强。Take an appropriate amount of sample, add mobile phase A to dissolve and dilute to make a solution with a concentration of 1.5 mg/ml, add about 5 drops of 10% sodium hydroxide solution per 1 ml, leave it at room temperature for 25 minutes, take 20 μl and inject it into the chromatograph, and record the chromatogram ( See Figure 3). The results showed that under the re-testing conditions, other components and the main peak in the destroyed sample could achieve baseline separation with strong specificity.

2.2检测限2.2 Detection limit

取样品适量，加流动相A溶解并稀释后，取20μl注入液相色谱仪，记录色谱图，直至主峰峰高为基线噪音的三倍，测得最低检出量约为33.4ng。Take an appropriate amount of sample, add mobile phase A to dissolve and dilute, take 20 μl and inject it into the liquid chromatograph, record the chromatogram until the peak height of the main peak is three times of the baseline noise, and the minimum detected amount is about 33.4ng.

2.3回收率2.3 Recovery rate

取样品适量，分别配制成0.6mg/ml、0.75mg/ml和0.9mg/ml3个浓度，每个浓度各3份样，取20μl注入色谱仪，计算回收率。该方法的平均回收率为100.0％，RSD％为0.1％。Take an appropriate amount of sample and prepare three concentrations of 0.6mg/ml, 0.75mg/ml and 0.9mg/ml respectively, and take 3 samples for each concentration, inject 20μl into the chromatograph, and calculate the recovery rate. The average recovery of this method was 100.0%, and the RSD% was 0.1%.

样品测定Sample determination

3.1有关物质3.1 Related substances

取本品适量，加流动相配制成每1ml中约含1.5mg的溶液，作为供试品溶液；精密量取供试品溶液1ml，置100ml量瓶中，加流动相A稀释至刻度，摇匀，作为对照溶液。取对照溶液20μl注入液相色谱仪，调节检测灵敏度，使主成分色谱峰峰高约为满量程的20％，再精密量取供试品溶液和对照溶液各20μl，分别注入液相色谱仪，记录色谱图至主成分峰保留时间的2倍(见图5)。Take an appropriate amount of this product, add mobile phase to prepare a solution containing about 1.5 mg per 1 ml, as the test solution; accurately measure 1 ml of the test solution, put it in a 100 ml measuring bottle, add mobile phase A to dilute to the mark, shake Uniform, as a control solution. Get contrast solution 20 μ l and inject liquid chromatograph, adjust detection sensitivity, make the chromatographic peak height of main component be about 20% of full scale, then accurately measure each 20 μ l of need testing solution and contrast solution, inject liquid chromatography respectively, Record the chromatogram to twice the retention time of the main component peak (see Figure 5).

3.2含量测定3.2 Content determination

取本品适量，用流动相A配制成每1ml中含0.75mg的溶液，作为供试品溶液；另精密称取对照品适量，用流动相A溶解并稀释制成每1ml中含0.75mg的溶液，作为对照品溶液。精密量取供试品溶液与对照品溶液各20μl，分别注入液相色谱仪，记录色谱图，按外标法以峰面积计算，即得(见图6)。Take an appropriate amount of this product, and use mobile phase A to prepare a solution containing 0.75 mg per 1 ml, as the test solution; in addition, accurately weigh an appropriate amount of reference substance, dissolve and dilute with mobile phase A to prepare a solution containing 0.75 mg per 1 ml. solution, as a reference solution. Precisely measure 20 μl each of the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the peak area according to the external standard method (see Figure 6).

Claims (4)

Translated fromChinese

1.一种醋酸阿托西班含量测定方法，其特征是，其步骤包括：1. a method for assaying atosiban acetate content, is characterized in that, its step comprises:1)取醋酸阿托西班原料或制剂样品适量，用流动相A配制成每1ml中含0.75mg的溶液，作为供试品溶液；1) get an appropriate amount of atosiban acetate raw material or preparation sample, be mixed with the solution that contains 0.75mg in every 1ml with mobile phase A, as need testing solution;2)另称取对照品适量，用流动相A溶解并稀释制成每1ml中含0.75mg的溶液，作为对照品溶液；2) Another appropriate amount of reference substance was weighed, dissolved and diluted with mobile phase A to make a solution containing 0.75mg per 1ml, as the reference substance solution;3)量取供试品溶液与对照品溶液各20μl，分别注入液相色谱仪，记录色谱图，按外标法以峰面积计算，得到样品中醋酸阿托西班的含量；3) Measure need testing solution and reference substance solution each 20 μ l, inject liquid chromatograph respectively, record chromatogram, calculate with peak area by external standard method, obtain the content of atosiban acetate in the sample;色谱条件为：The chromatographic conditions are:色谱柱：以十八烷基硅烷键合硅胶C18为填充剂，Chromatographic column: Octadecylsilane bonded silica gel C18 as filler,流动相：以0.05mol/L的磷酸盐缓冲液为流动相A，乙腈为流动相B，Mobile phase: 0.05mol/L phosphate buffer as mobile phase A, acetonitrile as mobile phase B,洗脱方式：梯度洗脱，Elution method: gradient elution,流速：0.8～1.2ml/min，Flow rate: 0.8～1.2ml/min,检测波长：UV 205～225nm，Detection wavelength: UV 205~225nm,配制的样品溶液浓度：每1ml中约含醋酸阿托西班0.1～15mgConcentration of the prepared sample solution: about 0.1-15mg of atosiban acetate per 1ml进样量：5～200μl。Injection volume: 5-200μl.2.根据权利要求1所述的方法，其特征在于：2. The method according to claim 1, characterized in that:含量测定的的方法为：照中国药典2005年版二部附录V D高效液相色谱法测定；The method of content determination is: according to Chinese Pharmacopoeia 2005 edition two appendix V D high performance liquid chromatography determination;色谱条件与系统适用性试验用十八烷基硅烷键合硅胶为填充剂；以0.05mol/L的磷酸盐缓冲液为流动相A，乙腈为流动相B，进行梯度洗脱；检测波长为215nm；流速为1.0ml/min，理论塔板数按阿托西班峰计算应不低于5000，梯度洗脱程序如下：Chromatographic conditions and system suitability test use octadecylsilane bonded silica gel as filler; use 0.05mol/L phosphate buffer as mobile phase A, acetonitrile as mobile phase B, and perform gradient elution; detection wavelength is 215nm ; The flow rate is 1.0ml/min, the number of theoretical plates should not be less than 5000 based on the calculation of the atosiban peak, and the gradient elution procedure is as follows:其中，磷酸盐缓冲液的配制方法为：取磷酸二氢钾6.8g，加水1000ml，用磷酸调节pH值至2.3，再加入120μl三乙胺，即得；Among them, the preparation method of the phosphate buffer solution is: take 6.8 g of potassium dihydrogen phosphate, add 1000 ml of water, adjust the pH value to 2.3 with phosphoric acid, and then add 120 μl of triethylamine to obtain it;测定法取醋酸阿托西班原料或制剂样品适量，用流动相A配制成每1ml中含0.75mg的溶液，作为供试品溶液；另精密称取对照品适量，用流动相A溶解并稀释制成每1ml中含0.75mg的溶液，作为对照品溶液，精密量取供试品溶液与对照品溶液各20μl，分别注入液相色谱仪，记录色谱图，按外标法以峰面积计算，即得。Determination method Take an appropriate amount of atosiban acetate raw material or preparation sample, and use mobile phase A to prepare a solution containing 0.75 mg per 1 ml, as the test solution; in addition, accurately weigh an appropriate amount of reference substance, dissolve and dilute with mobile phase A Make the solution that contains 0.75mg in every 1ml, as reference substance solution, accurately measure each 20 μ l of need testing solution and reference substance solution, inject liquid chromatograph respectively, record chromatogram, calculate with peak area by external standard method, Instantly.3.一种醋酸阿托西班有关物质的检测方法，其特征在于：3. A detection method for atosiban acetate related substances, characterized in that:1)取醋酸阿托西班原料或制剂样品适量，加流动相配制成每1ml中约含1.5mg的溶液，作为供试品溶液；1) Get an appropriate amount of atosiban acetate raw material or preparation sample, add mobile phase and be mixed with the solution that contains about 1.5mg in every 1ml, as need testing solution;2)量取供试品溶液1ml，置100ml量瓶中，加流动相A稀释至刻度，摇匀，作为对照溶液；2) Measure 1ml of the test solution, put it in a 100ml measuring bottle, add mobile phase A to dilute to the mark, shake well, and use it as a contrast solution;3)取对照溶液20μl注入液相色谱仪，调节检测灵敏度，使主成分色谱峰峰高约为满量程的20％，再精密量取供试品溶液和对照溶液各20μl，分别注入液相色谱仪，记录色谱图至主成分峰保留时间的2倍，计算有关物质含量；3) Take 20 μl of the control solution and inject it into the liquid chromatograph, adjust the detection sensitivity so that the peak height of the main component chromatographic peak is about 20% of the full scale, then accurately measure 20 μl each of the test solution and the control solution, and inject them into the liquid chromatograph respectively instrument, record the chromatogram to twice the retention time of the main component peak, and calculate the content of related substances;色谱条件为：The chromatographic conditions are:色谱柱：以十八烷基硅烷键合硅胶C18为填充剂，Chromatographic column: Octadecylsilane bonded silica gel C18 as filler,流动相：以0.05mol/L的磷酸盐缓冲液为流动相A，乙腈为流动相B，Mobile phase: 0.05mol/L phosphate buffer as mobile phase A, acetonitrile as mobile phase B,洗脱方式：梯度洗脱，Elution method: gradient elution,流速：0.8～1.2ml/min，Flow rate: 0.8～1.2ml/min,检测波长：UV 205～225nm，Detection wavelength: UV 205 ~ 225nm,配制的样品溶液浓度：每1ml中约含醋酸阿托西班0.1～15mgConcentration of the prepared sample solution: about 0.1-15mg of atosiban acetate per 1ml进样量：5～200μl，Injection volume: 5～200μl,4.根据权利要求3所述的方法，其特征在于：4. The method according to claim 3, characterized in that:有关物质检测的的方法为：照中国药典2005年版二部附录V D高效液相色谱法测定色谱条件与系统适用性试验用十八烷基硅烷键合硅胶为填充剂；以0.05mol/L的磷酸盐缓冲液为流动相A，乙腈为流动相B，进行梯度洗脱；检测波长为220nm；流速为1.0ml/min，理论塔板数按阿托西班峰计算应不低于5000，梯度洗脱程序如下：The method that relevant substance detects is: according to Chinese Pharmacopoeia 2005 edition two appendices V D high performance liquid chromatographic method determination chromatographic condition and system suitability test use octadecylsilane bonded silica gel as filler; With 0.05mol/L Phosphate buffer is mobile phase A, acetonitrile is mobile phase B, and gradient elution is carried out; the detection wavelength is 220nm; the flow rate is 1.0ml/min, and the number of theoretical plates should not be less than 5000 based on the atosiban peak. The elution procedure is as follows:

其中，磷酸盐缓冲液的配制方法为：取磷酸二氢钾6.8g，加水1000ml，用磷酸调节pH值至2.3，再加入120μl三乙胺，即得；Among them, the preparation method of the phosphate buffer solution is: take 6.8 g of potassium dihydrogen phosphate, add 1000 ml of water, adjust the pH value to 2.3 with phosphoric acid, and then add 120 μl of triethylamine to obtain it;测定法取醋酸阿托西班原料或制剂样品适量，加流动相A配制成每1ml中约含1.5mg的溶液，作为供试品溶液；精密量取供试品溶液1ml，置100ml量瓶中，加流动相A稀释至刻度，摇匀，作为对照溶液，取对照溶液20μl注入液相色谱仪，调节检测灵敏度，使主成分色谱峰峰高约为满量程的20％，再精密量取供试品溶液和对照溶液各20μl，分别注入液相色谱仪，记录色谱图至主成分峰保留时间的2倍。Determination method: Take an appropriate amount of atosiban acetate raw material or preparation sample, add mobile phase A to prepare a solution containing about 1.5mg in every 1ml, as the test solution; accurately measure 1ml of the test solution, and put it in a 100ml measuring bottle , add mobile phase A to dilute to the mark, shake well, as a control solution, take 20 μl of the control solution and inject it into the liquid chromatograph, adjust the detection sensitivity so that the peak height of the chromatographic peak of the main component is about 20% of the full scale, and then accurately measure the supply 20 μl each of the test solution and the control solution were injected into the liquid chromatograph, and the chromatogram was recorded to twice the retention time of the main component peak.

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