CN101696959B - Acetic acid atosiban, and method for detecting content of preparation of acetic acid atosiban and relevant substances - Google Patents
Acetic acid atosiban, and method for detecting content of preparation of acetic acid atosiban and relevant substances Download PDFInfo
- Publication number
- CN101696959B CN101696959B CN200910008881A CN200910008881A CN101696959B CN 101696959 B CN101696959 B CN 101696959B CN 200910008881 A CN200910008881 A CN 200910008881A CN 200910008881 A CN200910008881 A CN 200910008881A CN 101696959 B CN101696959 B CN 101696959B
- Authority
- CN
- China
- Prior art keywords
- atosiban
- acetic acid
- solution
- mobile phase
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
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
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)
1. acetic acid Atosiban content assaying method is characterized in that its step comprises:
1) it is an amount of to get acetic acid Atosiban raw material or formulation samples, 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 that other precision takes by weighing reference substance, processes the solution that contains 0.75mg among every 1ml with mobile phase A dissolving and dilution, as reference substance solution;
3) 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, obtains the content of acetic acid Atosiban in the sample by external standard method;
Chromatographic condition is:
Chromatographic column: with octadecylsilane chemically bonded silica C18 is filling agent,
Moving phase: the phosphate buffer with 0.05mol/L is a mobile phase A, and acetonitrile is a Mobile phase B,
Type of elution: gradient elution,
Flow velocity: 1.0ml/min,
Detect wavelength: UV 215nm,
Wherein, reference substance is the pure article of acetic acid Atosiban,
The method of assay is: according to two appendix V of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring;
The chromatographic condition and the system suitability test theory number of plates are calculated by the Atosiban peak should be not less than 5000, and the gradient elution program is following:
Wherein, the compound method of phosphate buffer is: 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.
2. the detection method of an acetic acid Atosiban related substance is characterized in that:
1) it is an amount of to get acetic acid Atosiban raw material or formulation samples, adds moving phase and is mixed with the solution that contains 1.5mg among every 1ml approximately, as need testing solution;
2) 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;
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 is:
Chromatographic column: with octadecylsilane chemically bonded silica C18 is filling agent,
Moving phase: the phosphate buffer with 0.05mol/L is a mobile phase A, and acetonitrile is a Mobile phase B,
Type of elution: gradient elution,
Flow velocity: 1.0ml/min,
Detect wavelength: UV220nm,
Related substance detect method be: according to two appendix V of Chinese Pharmacopoeia version in 2005 D high effective liquid chromatography for measuring
The chromatographic condition and the system suitability test theory number of plates are calculated by the Atosiban peak should be not less than 5000, and the gradient elution program is following:
Wherein, the compound method of phosphate buffer is: 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910008881A CN101696959B (en) | 2009-02-11 | 2009-02-11 | Acetic acid atosiban, and method for detecting content of preparation of acetic acid atosiban and relevant substances |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910008881A CN101696959B (en) | 2009-02-11 | 2009-02-11 | Acetic acid atosiban, and method for detecting content of preparation of acetic acid atosiban and relevant substances |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101696959A CN101696959A (en) | 2010-04-21 |
| CN101696959B true CN101696959B (en) | 2012-10-17 |
Family
ID=42142074
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910008881A Active CN101696959B (en) | 2009-02-11 | 2009-02-11 | Acetic acid atosiban, and method for detecting content of preparation of acetic acid atosiban and relevant substances |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101696959B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421092B (en) * | 2013-09-05 | 2015-05-13 | 杭州阿德莱诺泰制药技术有限公司 | Atosiban purification method |
| CN105503828A (en) * | 2015-12-24 | 2016-04-20 | 北京康立生医药技术开发有限公司 | Preparation method of fumarate of pyrrole derivatives |
| CN106018584A (en) * | 2016-05-12 | 2016-10-12 | 华润双鹤利民药业（济南）有限公司 | Detection method for dezocine related substances |
| CN106932347B (en) * | 2017-05-17 | 2019-04-12 | 浙江遂昌利民科技有限公司 | A kind of mezlocillin and its quality index detection method |
| CN107312072A (en) * | 2017-06-20 | 2017-11-03 | 浙江湃肽生物有限公司 | A kind of method of purifies and separates Atosiban |
| CN109142580A (en) * | 2018-09-12 | 2019-01-04 | 南京康舟医药科技有限公司 | Measure method of the atosiban acetate in relation to substance |
| CN110218242A (en) * | 2019-06-26 | 2019-09-10 | 海南中和药业股份有限公司 | A kind of preparation method of atosiban acetate impurity |
| CN110658297B (en) * | 2019-10-30 | 2021-12-10 | 海南通用三洋药业有限公司 | Method for detecting high-molecular polymer in terlipressin for injection |
| CN110658296A (en) * | 2019-10-30 | 2020-01-07 | 海南通用三洋药业有限公司 | Method for detecting high-molecular polymer in atosiban acetate injection |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101314613A (en) * | 2008-05-08 | 2008-12-03 | 吉尔生化（上海）有限公司 | Solid phase synthesis method for atosiban |
| CN101357937A (en) * | 2007-07-31 | 2009-02-04 | 崔颀 | Method for synthesizing atosiban acetate from solid phase polypeptide |
-
2009
- 2009-02-11 CN CN200910008881A patent/CN101696959B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101357937A (en) * | 2007-07-31 | 2009-02-04 | 崔颀 | Method for synthesizing atosiban acetate from solid phase polypeptide |
| CN101314613A (en) * | 2008-05-08 | 2008-12-03 | 吉尔生化（上海）有限公司 | Solid phase synthesis method for atosiban |
Non-Patent Citations (2)
| Title |
|---|
| Alan R. Oyler，et al.Hydrophilic interaction chromatography on amino-silica phases complements reversed-phase high-performance liquid chromatography and capillary electrophoresis for peptide analysis.《Journal of Chromatography A》.1996,第724卷第378－383页. * |
| 李萍等.纳升电喷雾串联质谱在阿托西班一级结构确证中的应用.《分析测试学报》.2008,第27卷第16-18页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101696959A (en) | 2010-04-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101696959B (en) | Acetic acid atosiban, and method for detecting content of preparation of acetic acid atosiban and relevant substances | |
| Kang et al. | Modern methods for analysis of antiepileptic drugs in the biological fluids for pharmacokinetics, bioequivalence and therapeutic drug monitoring | |
| CN104237421A (en) | Related substance detection method for trelagliptin succinate and preparation thereof | |
| CN104655751A (en) | Method for detecting residual organic solvents in dapoxetine | |
| CN110146621B (en) | Method for determining content of polymer in cephalosporin antibiotic medicine | |
| CN102375033B (en) | High performance liquid chromatographic analysis method of bendamustine hydrochloride and its related substances | |
| CN103076409A (en) | Analysis and detection method for oxiracetam and impurities thereof | |
| CN107515255A (en) | Utilize high performance liquid chromatograph measure Dapagliflozin and its method about material | |
| WO2011064574A1 (en) | Hplc method for detecting lenalidomide | |
| CN103926335B (en) | The high-efficient liquid phase chromatogram process measuring method of related substance in a kind of Dapoxetine hydrochloride | |
| CN101661019B (en) | Method for separating and measuring Palonosetron hydrochloride and optical isomers thereof | |
| CN101609069A (en) | A kind of method of measuring Entecavir and Pharmaceutical composition related substance thereof with the HPLC method | |
| CN103487545A (en) | Liquid chromatography detection method for carbachol content and impurity content | |
| CN100387988C (en) | Content detecting method for Ibuprofen, chlorphenamine maleate and Pseudoephedrine Hydrochloride compound preparation | |
| CN104897832A (en) | High performance liquid chromatography method for detecting free amino acid content in transfer factor solution and preparation thereof | |
| CN102579961B (en) | Detection method for aristolochic acid A in swelling-reducing and pain-alleviating tincture | |
| CN104007185A (en) | HPLC determination method for detecting impurities in zanamivir and zanamivir-containing preparation | |
| CN101936959B (en) | High performance liquid chromatography (HPLC) for fast separating and analyzing zafirlukast and isomers thereof | |
| CN104678006B (en) | A kind of Sunitinib malate Related substance method | |
| Youssef et al. | Development and validation of RP-HPLC method for the estimation and separation of valsartan, losartan and irbesartan in bulk and pharmaceutical formulation | |
| Agrawal | Validated stability indicating method for determination of indapamide in pharmaceutical formulation | |
| CN102109499A (en) | Method for simultaneously detecting acetone and ethyl acetate residues in drug by gas chromatography | |
| CN1786706B (en) | Cyclovirobuxine D raw medicine and method for determining its content in preparation by chromatography | |
| CN102109501B (en) | Method for detecting related substances in quinapril hydrochloride and hydrochlorothiazide composition | |
| CN101776658A (en) | Method for measuring content of vitamin D2 in vitamin D2 calcium levulinate injection solution |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: 570216 Haikou Free Trade Zone, Nanhai Road, Hainan, Haikou, China, 168 Co-patentee after: Hainan Zhonghe Polypeptide Research Co., Ltd. Patentee after: Hainan Zhonghe Pharmaceutical Co., Ltd. Address before: 570216 Hainan Zhonghe Pharmaceutical Co., Ltd., Haikou, Nanhai Road, No. 168, Hainan, China Co-patentee before: Hainan Zhonghe Polypeptide Research Co., Ltd. Patentee before: Hainan Zhonghe Pharmaceutical Co., Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20170425 Address after: 570216 Haikou Free Trade Zone, Nanhai Road, Hainan, Haikou, China, 168 Patentee after: Hainan Zhonghe Pharmaceutical Co., Ltd. Address before: 570216 Haikou Free Trade Zone, Nanhai Road, Hainan, Haikou, China, 168 Co-patentee before: Hainan Zhonghe Polypeptide Research Co., Ltd. Patentee before: Hainan Zhonghe Pharmaceutical Co., Ltd. |