CN101696959B - A kind of method for the content determination of atosiban acetate and its preparation and the determination of related substances - Google Patents

Abstract

The invention relates to an acetic acid atosiban, and a method for detecting content of preparation of acetic acid atosiban and relevant substances. The detection method is an efficient liquid-phase chromatography method, which comprises the following chromatography conditions that a chromatographic column takes octadecylsilane chemically bonded silica (C18) as filling agent; 0.05mol/L phosphate buffer solution is taken as mobile phase A and acetonitrile as mobile phase B to carry out gradient elution; a detection wavelength ranges from 205 to 225nm; the flow speed ranges between 0.8 and 1.2ml/min and the concentration of a prepared sample ensures that each milliliter of the sample contains 0.1 to 15mg of polypeptide; and the sample size is controlled between 5 and 200mu l. The efficient liquid-phase chromatography method can accurately detect a sample and the content of impurities of the sample at the same time and realizes complete sample separation during analysis and ideal reproducibility of analysis result, thereby providing a simple and reliable method for quality control and analysis during production process.

Description

The assay of a kind of acetic acid Atosiban and preparation thereof and the method for determination of related substances

Technical field:

The present invention relates to the method detection method of a kind of active peptides raw material medicine and preparation, especially relate to the assay of acetic acid Atosiban and preparation thereof and the method for determination of related substances.

Background technology:

Atosiban (atosiban), its chemical name is: 1-(3-mercaptan lactic acid)-2-(O-ethyl-D tyrosine)-4-L-threonine-8-L-ornithine-oxytocins.

Its structural formula is:

Atosiban is a kind of oxytocins analog, is the ring type polypeptide of synthetic, is the oxytocins competitive antagonist of acceptor on intrauterine and decidua, the fetal membrane, uses its acetate treatment premature labor clinically.Acetic acid Atosiban parenteral solution (AtosibanAcetate Injection) is developed by Huiling Co.,Ltd (Ferring AB); On March 23rd, 2000, in Austria's listing, commodity were by name: first

The related substance and the content detecting method of acetic acid Atosiban and acetic acid Atosiban parenteral solution are not appeared in the newspapers as yet; The difficulty of its assay method is in its building-up process, may produce disappearance (not exclusively) peptide, the peptide that ruptures, removes kinds of processes impurity such as acid amides polypeptide, diastereoisomeric polypeptide and oligomer; After carrying out purifying through chromatography; The impurity that can't remove is more similar with the chromatographic behavior of main ingredient, is difficult for separating fully.

Summary of the invention:

The purpose of this invention is to provide and a kind ofly can measure acetic acid Atosiban [1-(3-mercaptan lactic acid)-2-(O-ethyl-D tyrosine)-4-threonine-8-ornithine-oxytocins acetate] and the content of acetic acid Atosiban preparation and the detection method of related substance.

Acetic acid Atosiban content assaying method of the present invention is following:

Adopt reversed-phased high performace liquid chromatographic to detect the content of acetic acid Atosiban in 1-(3-mercaptan lactic acid)-2-(O-ethyl-D tyrosine)-4-threonine-8-ornithine-oxytocins acetate and the preparation thereof, its step comprises:

1) sample thief is an amount of, is mixed with the solution that contains 0.75mg among every 1ml with mobile phase A, as need testing solution;

2) it is an amount of to take by weighing reference substance in addition, processes the solution that contains 0.75mg among every 1ml with mobile phase A dissolving and dilution, as reference substance solution;

3) measure each 20 μ l of need testing solution and reference substance solution, inject liquid chromatograph respectively, the record chromatogram with calculated by peak area, obtains the content of acetic acid Atosiban in the sample by external standard method.

Chromatographic condition:

Use octadecylsilane chemically bonded silica to be filling agent; Phosphate buffer (get potassium dihydrogen phosphate 6.8g, add water to 1000ml, with phosphorus acid for adjusting pH value to 2.3, add 120 μ l triethylamines again, promptly get) with 0.05mol/L is a mobile phase A, and acetonitrile is a Mobile phase B, carries out gradient elution; The detection wavelength is 215nm; Flow velocity is 1.0ml/min.Theoretical cam curve is calculated by the Atosiban peak should be not less than 5000.The gradient elution program is following:

Wherein reference substance is the pure article of acetic acid Atosiban, can buy from the reagent shop.

Its related substances assay method of the present invention, step is following:

1) sample thief is an amount of, adds moving phase and is mixed with the solution that contains 1.5mg among every 1ml approximately, as need testing solution;

2) measure need testing solution 1ml, put in the 100ml measuring bottle, add mobile phase A and be diluted to scale, shake up, as contrast solution;

3) get contrast solution 20 μ l and inject liquid chromatograph; Regulate detection sensitivity; Make major component chromatographic peak peak height be about 20% of full scale, precision is measured need testing solution and each 20 μ l of contrast solution again, injects liquid chromatograph respectively; 2 times of record chromatogram to major component peak retention time calculate its related substances.

Chromatographic condition:

Use octadecylsilane chemically bonded silica to be filling agent; Phosphate buffer (get potassium dihydrogen phosphate 6.8g, add water 1000ml, with phosphorus acid for adjusting pH value to 2.3, add 120 μ l triethylamines again, promptly get) with 0.05mol/L is a mobile phase A, and acetonitrile is a Mobile phase B, carries out gradient elution; The detection wavelength is 220nm; Flow velocity is 1.0ml/min.Theoretical cam curve is calculated by the Atosiban peak should be not less than 5000.The gradient elution program is following:

Wherein said related substance is meant the impurity component that has in the acetic acid Atosiban building-up process, like solvent, and intermedium, raw material, amino acid fragments etc., impurity component are measured the related substance purpose and are to limit its content for being constrained to branch.

The characteristics of assay method of the present invention are: separate between each composition in the analyte thoroughly, workable, the analytical approach specificity is strong, accuracy and highly sensitive, adaptability is by force for guaranteeing drug safety, effective, the controlled foundation that provides.

Description of drawings:

Fig. 1 is 0.1% trifluoroacetic acid: acetonitrile is the chromatogram of moving phase

Fig. 2 is the 0.05mol/L phosphate buffer: acetonitrile is the chromatogram of moving phase

Fig. 3 is specificity experiment chromatogram.

Fig. 4 is the detectability chromatogram.

Fig. 5 is the chromatogram of sample determination of related substances.

The chromatogram that Fig. 6 measures for sample size.

Embodiment:

Following examples are used to explain the present invention, but not as limitation of the present invention.

Embodiment 1

Acetic acid Atosiban content assaying method

Experimental apparatus and chromatographic condition:

Waters 515-717-2996 high performance liquid chromatograph, Agilent C18 chromatographic column (5 μ m, 150 * 4.6mm); Phosphate buffer with 0.05mol/L (is got potassium dihydrogen phosphate 6.8g, is added water 1000ml, with phosphorus acid for adjusting pH value to 2.0～3.0; Add 120 μ l triethylamines again, promptly get) be mobile phase A, acetonitrile is a Mobile phase B; Carry out gradient elution, the detection wavelength is 205～225nm; Flow velocity is 0.8～1.2ml/min; Sample size is 5～200 μ l.。

Reagent:

Acetonitrile (HPLC level) is from U.S. world company, and ultrapure water is the self-control of Millipore ultrapure water machine.

The selection of chromatographic condition

1.1 moving phase

We have compared 0.1% trifluoroacetic acid (TFA) through experiment: acetonitrile (ACN) and 0.05mol/L phosphate buffer (PBS): ACN, and as moving phase, baseline fluctuation is big with 0.1% trifluoroacetic acid aqueous solution, and impurity peaks can't separate interference measurement fully with main peak; And be moving phase with 0.05mol/L, baseline is steady, and impurity peaks can separate with main peak fully, and the symmetry of main peak good (seeing Fig. 1 and Fig. 2).

1.2 detection wavelength

Carry out continuous sweep with ultraviolet spectrophotometer at 200～400nm, sample has absorption maximum at the 215nm place, and related substance has stronger absorption at the 220nm place mostly.Consider the baseline noise simultaneously to interference that detects and the sensitivity that takes into account mensuration, the detection wavelength of selection determination of related substances is decided to be 220nm, and the detection wavelength of assay is 215nm.

1.3 sample concentration

The concentration of these article preparation is 7.5mg/ml; Accurate for convenient experimental operation, having compared concentration respectively is the chromatogram of 0.75mg/ml and 1.5mg/ml test sample, and experimental result shows; Concentration is that the peak height and the area of sample impurity peaks of 1.5mg/ml is all more remarkable; And do not cause the chromatographic column overload, the concentration that therefore defines the related substance working sample is 1.5mg/ml, and the concentration of assay sample is 0.75mg/ml.

The checking of testing conditions

2.1 specificity

Sample thief is an amount of, adds mobile phase A dissolving and dilution and processes the solution that concentration is 1.5mg/ml, and every 1ml adds about 5 of 10% sodium hydroxide solution, and room temperature was placed 25 minutes, got 20 μ l injecting chromatographs, record chromatogram (see figure 3).The result shows that under the testing conditions, other components and main peak all can reach baseline separation in the sample after the destruction once more, and specificity is strong.

2.2 detectability

Sample thief is an amount of, add mobile phase A dissolving and dilution after, get 20 μ l and inject liquid chromatograph, the record chromatogram is three times of baseline noise until the main peak peak height, records limit of identification and is about 33.4ng.

2.3 the recovery

Sample thief is an amount of, is mixed with 0.6mg/ml, a 0.75mg/ml and 0.9mg/ml3 concentration respectively, and each 3 increment of each concentration are got 20 μ l injecting chromatographs, calculate recovery rate.The average recovery rate of this method is 100.0%, and RSD% is 0.1%.

Sample determination

3.1 related substance

These article of getting are an amount of, add moving phase and are mixed with the solution that contains 1.5mg among every 1ml approximately, as need testing solution; Precision is measured need testing solution 1ml, puts in the 100ml measuring bottle, adds mobile phase A and is diluted to scale, shakes up, as contrast solution.Get contrast solution 20 μ l and inject liquid chromatograph; Regulate detection sensitivity, make major component chromatographic peak peak height be about 20% of full scale, precision is measured need testing solution and each 20 μ l of contrast solution again; Inject liquid chromatograph respectively, 2 times of (see figure 5)s of record chromatogram to major component peak retention time.

3.2 assay

These article of getting are an amount of, are mixed with the solution that contains 0.75mg among every 1ml with mobile phase A, as need testing solution; It is an amount of that precision takes by weighing reference substance in addition, processes the solution that contains 0.75mg among every 1ml with mobile phase A dissolving and dilution, as reference substance solution.Precision is measured need testing solution and each 20 μ l of reference substance solution, injects liquid chromatograph respectively, and the record chromatogram with calculated by peak area, promptly gets (see figure 6) by external standard method.

Claims (2)

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；2) In addition, accurately weigh an appropriate amount of the reference substance, dissolve and dilute it with mobile phase A to make a solution containing 0.75 mg per 1 ml, as the reference substance solution;3)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，得到样品中醋酸阿托西班的含量；3) Precisely measure 20 μ l of the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, calculate the peak area by the external standard method, and 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：1.0ml/min，Flow rate: 1.0ml/min,检测波长：UV 215nm，Detection wavelength: UV 215nm,其中，对照品为醋酸阿托西班纯品，Wherein, the reference substance is atosiban acetate pure product,含量测定的方法为：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 for content determination is: measure according to two appendices V D high performance liquid chromatography of Chinese Pharmacopoeia edition in 2005;色谱条件与系统适用性试验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 The theoretical plate number 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 as follows: 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 the product.2.一种醋酸阿托西班有关物质的检测方法，其特征在于：2. 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)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；2) Precisely 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 control 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：1.0ml/min，Flow rate: 1.0ml/min,检测波长：UV220nm，Detection wavelength: UV220nm,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The method for the detection of related substances is: according to the 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 The theoretical plate number 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 as follows: 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 the product.

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