CN106770777A - HPLC detects method of the desmopressin acetate about material - Google Patents
HPLC detects method of the desmopressin acetate about material Download PDFInfo
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- CN106770777A CN106770777A CN201611242046.XA CN201611242046A CN106770777A CN 106770777 A CN106770777 A CN 106770777A CN 201611242046 A CN201611242046 A CN 201611242046A CN 106770777 A CN106770777 A CN 106770777A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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Abstract
Method of the desmopressin acetate about material is detected the invention provides a kind of HPLC, the method is with acetic acid aqueous solution and acetic acid acetonitrile solution as mobile phase, using Gradient elution, under same chromatographic condition, meet the separating degree requirement of desmopressin acetate and adjacent miscellaneous peak, can accurately judge the relevant levels of substance in desmopressin acetate sample.The method configuration process is simple, low to testing equipment requirement, and detection method is simple, easy to operate, and testing result is true and reliable, and not saliferous in mobile phase, reduction causes damage to chromatographic column, is conducive to popularity.
Description
Technical field
The invention belongs to analytical chemistry field, and in particular to a kind of side of HPLC detection desmopressin acetates about material
Method.
Background technology
Minirin is the analogue of natural Argipressin, is that the chemical constitution of natural hormone is carried out at two
Change and obtain, i.e., 1- cysteines slough amino and replace 8-L- arginine with 8-D- arginine.The peptide sequence knot of minirin
Structure is:Mpa-Tyr-Phe-Gln-Asn-Cys-Pro-D-Arg-Gly-NH2(wherein Mpa and Cys is by disulfide bond cyclization).
Minirin is mainly used in treatment central diabetes insipidus, enuresis nocturna and hemophilia etc., is also used for kidney urine dense
The test of contracting function, also acts as postoperation hemostatic.
At present, the Related substance method on desmopressin acetate, more using phosphate solution and acetonitrile conduct
Mobile phase, by each impurity of gradient elution separation desmopressin acetate.Wherein,《Chinese Pharmacopoeia 2015 editions》Middle acetic acid deammoniation
In pitressin quality standard, using phosphate solution（Take 0.067mol/L dipotassium hydrogen phosphates and 0.067mol/L potassium dihydrogen phosphates
Isometric mixing, regulation pH value to 7.0）It is mobile phase A, with acetonitrile as Mobile phase B, according to the form below gradient elution：
There is mobile phase and prepare complicated in the method, detection time is more long, high to instrument requirements, be unfavorable for the deficiencies such as quick analysis.
The content of the invention
Method of the desmopressin acetate about material is detected it is an object of the invention to provide a kind of HPLC, can be quick
Detect the relevant material situation of desmopressin acetate.
A kind of HPLC that the present invention is provided detects that desmopressin acetate, about the method for material, is comprised the following steps：
（1）Take desmopressin acetate standard items and desmopressin acetate standard liquid is prepared using water, take acetic acid deammoniation pressurization
Each known impurities reference substance of element prepares desmopressin acetate contamination levels solution using water；
（2）Take testing sample and testing sample solution is prepared using water；
（3）System suitability：Take desmopressin acetate appropriate, it is accurately weighed, it is dissolved in water and dilutes and is made in every 1mL
Solution containing desmopressin acetate 0.5mg is used as need testing solution；Precision measures need testing solution 1.0mL and puts 100mL measuring bottles
In, scale is dissolved in water and is diluted to, system suitability solution is configured to, the μ L of system suitability solution sample introduction 20 are taken, acetic acid goes
Ammonia pitressin peak and each impurity peaks separating degree R>1.5；
（4）Test limit is tested：Desmopressin acetate known impurities are taken, accurately weighed, being dissolved in water and diluting is made 1.0 ~ 1.5
The weak solution of μ g/mL, changes sample size successively, works as signal to noise ratio（S/N）The amount of sample is test limit when about 3: 1；
（5）Take step（2）Preparation the μ L of testing sample solution 10 ~ 20 injection liquid chromatograph, flow rate of mobile phase be 0.6 ~
1.0mL/min, Detection wavelength is：210nm；Detector：DAD；Column temperature：33~37℃；
The mobile phase includes A phases and B phases, and A phases are acetic acid-water；B phases：Acetic acidacetonitrile；The body of acetic acid and water in described A phases
Product is than being 0.1:100；Acetic acid and the volume ratio of acetonitrile in B phases are 0.1:100；A phases are 78 with the volume ratio of B phases:22.
Using step（5）Testing conditions determination step（1）The standard liquid or mixed standard solution of configuration, can all reach has
Effect is separated.
Method of the desmopressin acetate about material is detected according to above-mentioned HPLC, it is characterised in that：Step（1）It is described
Desmopressin acetate concentration of standard solution is 0.5 ~ 1.0mg/mL;Mixed standard solution mass concentration is 0.004 ~ 0.01mg/
mL。
Method of the desmopressin acetate about material, step are detected according to above-mentioned HPLC（1）Mixed standard solution is matched somebody with somebody
Method processed is to take desmopressin acetate and its impurity reference substance respectively in right amount, is configured to the standard solution of 0.5mg/mL, point
Do not take the μ L of above-mentioned each standard solution 100 to be placed in 10mL volumetric flasks, dilute with water constant volume obtains the mixing that concentration is 5 μ g/mL
Standard liquid.
Method of the desmopressin acetate about material, step are detected according to above-mentioned HPLC（2）The testing sample is molten
The mass concentration of liquid is 0.3 ~ 0.8mg/mL.
Method of the desmopressin acetate about material, step are detected according to above-mentioned HPLC（1）And step（2）The mark
It is only 0.2 ~ 0.4 μm of filter membrane mistake by hole before quasi- solution, mixed solution and testing sample solution injection liquid chromatograph
Filter.
Method of the desmopressin acetate about material, step are detected according to above-mentioned HPLC（5）Used in chromatogram
Column type number is Diamonsil, column length 250mm, diameter 4.6mm, and 5 μm of granularity, filler is octadecylsilane chemically bonded silica, grain
Footpath 100A.
Method of the desmopressin acetate about material, step are detected according to above-mentioned HPLC（1）Described acetic acid deammoniation
Pitressin impurity includes known impurities and unknown impuritie, and unknown impuritie is accessory substance, intermediate and the drop of generation in building-up process
Solution product.
HPLC of the present invention detects that desmopressin acetate, about the method for material, has the following advantages that：
1st, under same chromatographic condition, the separating degree requirement of desmopressin acetate and adjacent miscellaneous peak can be met, can be accurately judged
Relevant levels of substance in desmopressin acetate sample；
2nd, with acetic acid aqueous solution and acetic acid acetonitrile solution as mobile phase, configuration process is simple, convenient and swift.Be free of in mobile phase
Salt, reduction causes damage to chromatographic column, extends chromatographic column service life；
3rd, using Gradient elution, low to testing equipment requirement without gradient elution, detection method is simple, easy to operate, detection knot
It is really true and reliable, be conducive to popularity.
Brief description of the drawings
The HPLC-UV detection of system suitability solution under the conditions of Fig. 1 embodiment of the present invention 1
The HPLC-UV detection of desmopressin acetate under the conditions of Fig. 2 embodiment of the present invention 1.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Embodiment 1
The relevant material detection of desmopressin acetate.
First, reagent and solution
1st, desmopressin acetate reference substance：With minirin（C46H64N14O12S2）It is calculated as 87.0%
2nd, minirin EY reference substances：100.0%（HPLC methods, area normalization method）
3rd, minirin F reference substances：91.0%（HPLC methods, area normalization method）
4th, minirin D reference substances：88.3%（HPLC methods, area normalization method）
5th, minirin Y reference substances：96.4%（HPLC methods, area normalization method）
6th, the preparation of testing sample solution
Desmopressin acetate 10.02mg is weighed, is placed in 10mL volumetric flasks, plus distilled water dissolves and be settled to scale, shaken up
It is standby
7th, system suitability：Take desmopressin acetate appropriate, it is accurately weighed, it is dissolved in water and dilutes and is made in every 1mL
Solution containing desmopressin acetate 0.5mg is used as need testing solution；Each impurity reference substance is taken respectively appropriate, be configured to 0.5mg/
The need testing solution of mL, precision is measured during need testing solution 0.1mL puts 10mL measuring bottles, is dissolved in water and is diluted to scale, is prepared
Into system suitability solution.
2nd, instrument and equipment
1st, assay balance：Sensibility reciprocal is 0.0001g
2nd, miillpore filter：0.45 μm of aperture
3rd, high performance liquid chromatograph（Equipped with UV-detector）.
3rd, analytical procedure
1st, liquid phase chromatogram condition
Chromatographic column：Diamonsil, column length 250mm, diameter 4.6mm, 5 μm of granularity, filler is octadecylsilane bonded silica
Glue；
Mobile phase：A phases are acetic acid-water；B phases：Acetic acidacetonitrile；Acetic acid and the volume ratio of water are 0.1 in described A phases:100；B
Acetic acid and the volume ratio of acetonitrile in phase are 0.1 ~ 100；A phases are 78 with the volume ratio of B phases:22；
Flow velocity：0.8mL/min；
Detection wavelength：210nm
Sample size：20μL
2nd, liquid chromatogram measuring
Take the μ L of system suitability solution sample introduction 20, the separating degree between desmopressin acetate and each known impurities and other impurities
>1.5.Testing sample solution is taken, 0.45 μm of filter membrane is crossed, precision measures 10 μ L injection liquid chromatographs, records collection of illustrative plates.
Embodiment 2
Test limit is tested.
First, reagent and solution
1st, minirin EY solution： 0.116ng/μL
2nd, minirin F solution： 0.112 ng/μL
3rd, minirin solution D： 0.12 ng/μL
4th, minirin Y solution： 0.108 ng/μL.
2nd, instrument and equipment
1st, assay balance：Sensibility reciprocal is 0.0001g
2nd, miillpore filter：0.45 μm of aperture
3rd, high performance liquid chromatograph（Equipped with UV-detector）.
3rd, analytical procedure
1st, liquid phase chromatogram condition
Chromatographic column：Diamonsil, column length 250mm, diameter 4.6mm, 5 μm of granularity, filler is octadecylsilane bonded silica
Glue；
Mobile phase：A phases are acetic acid-water；B phases：Acetic acidacetonitrile；Acetic acid and the volume ratio of water are 0.1 in described A phases:100；B
Acetic acid and the volume ratio of acetonitrile in phase are 0.1 ~ 100；A phases are 78 with the volume ratio of B phases:22；
Flow velocity：0.8mL/min；
Detection wavelength：210nm
Sample size：20μL~5μL
2nd, testing result
Result shows：Minirin EY detections are limited to 0.58ng（S/N≈3）.Minirin F detections are limited to 0.56ng（S/N
≈3）.Minirin D detections are limited to 0.6ng（S/N≈3）.Minirin Y detections are limited to 0.54ng（S/N≈3）.
Claims (7)
1. a kind of HPLC detects method of the desmopressin acetate about material, it is characterised in that comprise the following steps：
（1）Take desmopressin acetate standard items and desmopressin acetate standard liquid is prepared using water, take acetic acid deammoniation pressurization
Each known impurities reference substance of element prepares desmopressin acetate contamination levels solution using water；
（2）Take testing sample and testing sample solution is prepared using water；
（3）System suitability：Take desmopressin acetate appropriate, it is accurately weighed, it is dissolved in water and dilutes and is made in every 1mL
Solution containing desmopressin acetate 0.5mg is used as need testing solution；Precision measures need testing solution 1.0mL and puts 100mL measuring bottles
In, scale is dissolved in water and is diluted to, system suitability solution is configured to, the μ L of system suitability solution sample introduction 20 are taken, acetic acid goes
Ammonia pitressin peak and each impurity peaks separating degree R>1.5；
（4）Test limit is tested：Take desmopressin acetate known impurities, it is accurately weighed, be dissolved in water and dilute be made 0.1 ~
The weak solution of 0.15 ng/ μ L, changes sample size successively, works as signal to noise ratio（S/N）The amount of sample is test limit when about 3: 1；
（5）Take step（2）Preparation the μ L of testing sample solution 10 ~ 20 injection liquid chromatograph, flow rate of mobile phase be 0.6 ~
1.0mL/min, Detection wavelength is：210nm；Detector：DAD；Column temperature：33~37℃；
The mobile phase includes A phases and B phases, and A phases are acetic acid-water；B phases：Acetic acidacetonitrile；The body of acetic acid and water in described A phases
Product is than being 0.1:100；Acetic acid and the volume ratio of acetonitrile in B phases are 0.1:100；A phases are 78 with the volume ratio of B phases:22；
Using step（5）Testing conditions determination step（1）The standard liquid or mixed standard solution of configuration, can all reach and effectively divide
From；
Described desmopressin acetate structural formula is：
2. HPLC according to claim 1 detects method of the desmopressin acetate about material, it is characterised in that：Step
（1）The desmopressin acetate concentration of standard solution is 0.5 ~ 1.0 mg/mL；Mixed standard solution mass concentration be 0.004 ~
0.01mg/mL。
3. HPLC according to claim 1 detects method of the desmopressin acetate about material, it is characterised in that：Step
（1）Mixed standard solution preparation method is to take desmopressin acetate and its impurity reference substance respectively in right amount, is configured to 0.5mg/
The standard solution of mL, takes the μ L of above-mentioned each standard solution 100 and is placed in 10mL volumetric flasks respectively, and dilute with water constant volume is obtained
Concentration is the mixed standard solution of 5 μ g/mL.
4. HPLC according to claim 1 detects method of the desmopressin acetate about material, it is characterised in that：Step
（2）The mass concentration of the testing sample solution is 0.3 ~ 0.8mg/mL.
5. HPLC according to claim 1 detects method of the desmopressin acetate about material, it is characterised in that：Step
（1）And step（2）It is only by hole before the standard liquid, mixed solution and testing sample solution injection liquid chromatograph
0.2 ~ 0.4 μm of filtering membrane filtration.
6. HPLC according to claim 1 detects method of the desmopressin acetate about material, it is characterised in that：Step
（5）Used in chromatographic column model Diamonsil, column length 250mm, diameter 4.6mm, 5 μm of aperture, filler is octadecane
Base silane bonded silica gel, particle diameter 100A.
7. HPLC according to claim 1 detects method of the desmopressin acetate about material, it is characterised in that：Step
（1）Described desmopressin acetate impurity includes known impurities and unknown impuritie, and unknown impuritie is generation in building-up process
Accessory substance, intermediate and catabolite, it is known that impurity see the table below：
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111812238A (en) * | 2020-07-09 | 2020-10-23 | 苏州旭辉检测有限公司 | Biological sample pretreatment method for vasopressin and analogues thereof |
| CN114409740A (en) * | 2021-12-31 | 2022-04-29 | 江苏诺泰澳赛诺生物制药股份有限公司 | Method for purifying desmopressin impurities |
-
2016
- 2016-12-29 CN CN201611242046.XA patent/CN106770777A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111812238A (en) * | 2020-07-09 | 2020-10-23 | 苏州旭辉检测有限公司 | Biological sample pretreatment method for vasopressin and analogues thereof |
| CN114409740A (en) * | 2021-12-31 | 2022-04-29 | 江苏诺泰澳赛诺生物制药股份有限公司 | Method for purifying desmopressin impurities |
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