CN107515269A - A kind of ultra performance liquid chromatography analysis method of Exenatide - Google Patents
A kind of ultra performance liquid chromatography analysis method of Exenatide Download PDFInfo
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- CN107515269A CN107515269A CN201710695491.XA CN201710695491A CN107515269A CN 107515269 A CN107515269 A CN 107515269A CN 201710695491 A CN201710695491 A CN 201710695491A CN 107515269 A CN107515269 A CN 107515269A
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- exenatide
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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
- G01N30/89—Inverse chromatography
Abstract
The invention discloses a kind of ultra performance liquid chromatography analysis method of Exenatide, the sample solution containing Exenatide is passed through in chromatographic column, using reverse phase liquid chromatography external standard percentage method quantitative analysis, the liquid phase chromatogram condition is：Chromatographic column：UPLC；Mobile phase：Volume proportion is 80：20~20：80 mobile phase A and the mixed solution of Mobile phase B, the mobile phase A are 0.1M/L tetraethyl ammonium perchlorate (TEAP) aqueous solution, adjust PH to 2.05 with perchloric acid, the Mobile phase B is 100% acetonitrile；Gradient elution；Detection wavelength：210~220nm；Flow velocity：0.18~0.23ml/min；Column temperature：40~60 DEG C；The μ L of sampling volume 1~5.The present invention have the characteristics that it is accurate quickly, theoretical cam curve it is high, can effectively measure Exenatide content；Sample introduction concentration is lower, can reduce the consumption of expensive Exenatide, reduce cost；Meanwhile appearance time is faster, mobile phase consumption is less.
Description
Technical field
The invention belongs to Pharmaceutical Analysis technical field, in particular to a kind of ultra performance liquid chromatography of Exenatide
Analysis method.
Background technology
Exenatide (Exenatide) is a kind of human glucagon-like-peptide-1 (GLP-1) analog, can treat II types
Diabetic.It is the 1st GLP-1 analog for being ratified listing by food and medicine Surveillance Authority of the U.S. (FDA) in 2005,
In 2009 in the granted listing of China, and the 1st GLP-1 analog in Discussion on Chinese Listed.
In order to ensure the follow-up research and development of Exenatide and the quality of production, it is necessary to enter to the quality of bulk drug and its drug bearing microsphere
Row control.Therefore, research obtains a kind of detection method of Exenatide assay, and this seems outstanding for medicine manufacture
To be urgent.
A kind of Exenatide and its impurity ultra high efficiency liquid is disclosed in Patent No. 201310101282.X patent of invention
Phase chromatographic detection method, this method press the flowing of different proportion preparation with buffer solution and organic solvent respectively in column chromatography
Phase A and Mobile phase B are used as mobile phase, sample of the gradient elution containing Exenatide and each impurity, wherein flowing by different proportion mixing
Dynamic phase A is ammonium sulfate or metabisulfite solution containing 10-25 volume % acetonitriles and methanol, and Mobile phase B is to contain 50-80 volumes %
The ammonium sulfate or metabisulfite solution of acetonitrile and methanol；Wherein flow rate of mobile phase is 0.4-0.6ml/min, sample concentration 1mg/
ML, sampling volume are 5 μ L；And find out from the embodiment of the invention, the detection time of Exenatide is more than 10mins.
A kind of high effective liquid chromatography for measuring Ai Saina is disclosed in the patent of invention of Patent No. 201410205748.5
The method of peptide and its impurity, this method pass through high effective liquid chromatography for measuring Exenatide and its impurity, gradient elution；Wherein,
Chromatogram flow phase aqueous phase uses 1.2~1.8mmol/L alkyl sulfonic acid sodium solution, and adjusts pH to 4.0~4.8 with phosphoric acid；Using
Mobile phase A be acetonitrile-abovementioned alkyl sodium sulfonate solution=10：90, Mobile phase B is acetonitrile-alkyl sulfonic acid sodium solution=52：
48；Flow rate of mobile phase is 0.8~1.2ml/min, and the concentration of Exenatide sample is 0.4~1.6mg/ml, and sampling volume is 40 μ
L。
One kind liquid chromatography for separating and determining Ai Sai is disclosed in the patent application of Application No. 200910247706.7
The method of that peptide and wherein miscellaneous peptide, in the chromatographic column used in this method, stationary phase is alkyl linked silica gel, mobile phase be acetonitrile-
Phosphate solution, wherein, the phosphate solution using phosphate solution or containing appropriate acetonitrile as mobile phase A, phosphate solution
Concentration is 0.01~0.1mol/L；Acetonitrile using acetonitrile or containing appropriate phosphate solution carries out ladder as Mobile phase B, using acetonitrile
Degree elution, and the concentration gradient scope that meter Mobile phase B accounts for whole mobile phase by volume is 10~90%；Exenatide sample
Concentration is 0.1~5mg/ml, and the flow velocity of mobile phase is 0.5~1.5ml/min, and sampling volume is 2~100 μ L.
In the above prior art, using efficient liquid phase chromatographic analysis；And ultra high efficiency liquid phase compares efficient liquid phase,
Sample introduction concentration is lower, can reduce the consumption of expensive Exenatide, reduce cost；Meanwhile appearance time is faster, stream
It is dynamic mutually to consume less.In view of this, it is necessary to the content analysis method of Exenatide of the prior art is improved, with solution
Certainly above mentioned problem.
The content of the invention
The present invention is good, again present invention aims at a kind of stability is provided for defect present in above prior art
Renaturation is good, simple to operate, theoretical cam curve is high, accurate quick, the ultra performance liquid chromatography analysis method for Exenatide.
The technical proposal for solving the technical problem of the invention is：A kind of ultra performance liquid chromatography analysis of Exenatide
Method, the sample solution containing Exenatide is passed through in chromatographic column, quantitatively divided using reverse phase liquid chromatography external standard percentage method
Analysis, the liquid phase chromatogram condition are：
Chromatographic column：UPLC；
Mobile phase：Volume proportion is 80：20~20：80 mobile phase A and the mixed solution of Mobile phase B, the mobile phase A
For 0.1M/L tetraethyl ammonium perchlorate (TEAP) aqueous solution, PH to 2.05 is adjusted with perchloric acid, the Mobile phase B is 100% second
Nitrile；
Gradient elution；
Detection wavelength：210~220nm；
Flow velocity：0.18~0.23ml/min；
Column temperature：40~60 DEG C；
The μ L of sampling volume 1~5.
The sample introduction concentration of the sample solution of the Exenatide is 0.004~0.2mg/mL.
The sample solution is made up of Exenatide bulk drug with deionized water.
The Detection wavelength is 215nm, flow velocity 0.21ml/min, 50 DEG C of column temperature, the μ L of sampling volume 1.4.
The detailed process of the gradient elution is that elution time and mobile phase A volume ratio order are transported for 0~15min72%
OK, 15~15.1min is from 32% → 20%, and 15.1~16.7min is again from 20% → 30%, and 16.7~16.8min is again from 30%
→ 72%, then 72% operation to 18.4min.
The beneficial effects of the invention are as follows：Compared with traditional HPLC methods, the ultra high efficiency of Exenatide provided by the invention
That liquid phase analysis method has the characteristics that is accurate quick, theoretical cam curve is high, can effectively measure Exenatide content and (compare Fig. 1
With Fig. 2 it can be found that).Sample introduction concentration is lower, can reduce the consumption of expensive Exenatide, reduce cost；Meanwhile
Faster, mobile phase consumption is less for appearance time.
Brief description of the drawings
Fig. 1 is the ultra performance liquid chromatography analysis result spectrogram of the Exenatide sample in embodiment 1.
Fig. 2 is the efficient liquid phase chromatographic analysis result spectrogram of currently used Exenatide sample in comparative example 1.
Embodiment
With reference to the accompanying drawings and detailed description, the present invention is furture elucidated, it should be understood that following embodiments are only
For illustrating the present invention rather than limitation the scope of the present invention.
Embodiment 1
Exenatide assay.
Ultra performance liquid chromatography condition：
Chromatographic column：AcquityUPLC (BEH, C18), specification 2.1mm × 50mm, 1.7 μm.
Proportion of mobile phase is：Volume proportion is 20：80~80：20 mobile phase A and the mixed solution of Mobile phase B, gradient
Elution, the mobile phase A are 0.1M/L tetraethyl ammonium perchlorate (TEAP) aqueous solution, and PH to 2.05 is adjusted with perchloric acid, described
Mobile phase B is 100% acetonitrile.The detailed process of the gradient elution is, elution time and mobile phase A volume ratio order for 0~
15min 72% is run, 15~15.1min from 32% → 20%, 15.1~16.7min again from 20% → 30%, 16.7~
16.8min from 30% → 72%, then 72% is run to 18.4min again.
Detection wavelength is 215nm；Flow velocity is 0.21ml/min；50 DEG C of column temperature；The μ L of sampling volume 1.4.
Solution is prepared：
The preparation of mobile phase A：Tetraethyl ammonium perchlorate 2.297g is weighed, is dissolved with 100ml ultra-pure waters, then adjusted with perchloric acid
PH to 2.05 is saved, is then filtered stand-by.
Exenatide raw material about 10mg is taken, it is accurately weighed, it is placed in 100ml volumetric flasks, adds appropriate diluent to dissolve and dilute
Release to scale, shake up, as need testing solution.
According to Fig. 1 as can be seen that Exenatide, peak shape is good, peak purity is good.
Embodiment 2
The system precision test of Exenatide assay
Ultra performance liquid chromatography condition：
Chromatographic column：AcquityUPLC (BEH, C18), specification 2.1mm × 50mm, 1.7 μm.
Proportion of mobile phase is：Volume proportion is 20：80~80：20 mobile phase A and the mixed solution of Mobile phase B, gradient
Elution, the mobile phase A are 0.1M/L tetraethyl ammonium perchlorate (TEAP) aqueous solution, and PH to 2.05 is adjusted with perchloric acid, described
Mobile phase B is 100% acetonitrile.The detailed process of the gradient elution is, elution time and mobile phase A volume ratio order for 0~
15min 72% is run, 15~15.1min from 32% → 20%, 15.1~16.7min again from 20% → 30%, 16.7~
16.8min from 30% → 72%, then 72% is run to 18.4min again.
Detection wavelength is 215nm；Flow velocity is 0.21ml/min；50 DEG C of column temperature；The μ L of sampling volume 1.4.
Solution is prepared：
Precision weighs 10.000mg Exenatide bulk drugs, puts in 100mL volumetric flasks, adds ultra-pure water to shake up, make to scale
It is 0.10012mg/mL Exenatide solution into concentration, as need testing solution.
Table 1：System Precision test result
According to table 1 as can be seen that the peak area RSD% for repeating same sample 6 times is 0.7%, meet RSD% in pharmacopeia
<2% regulation, illustrate that system precision is good, experiment is with a high credibility.
Embodiment 3
The replica test of Exenatide assay
Ultra performance liquid chromatography condition：
Chromatographic column：AcquityUPLC (BEH, C18), specification 2.1mm × 50mm, 1.7 μm.
Proportion of mobile phase is：Volume proportion is 20：80~80：20 mobile phase A and the mixed solution of Mobile phase B, gradient
Elution, the mobile phase A are 0.1M/L tetraethyl ammonium perchlorate (TEAP) aqueous solution, and PH to 2.05, the stream are adjusted with phosphoric acid
Dynamic phase B is 100% acetonitrile.The detailed process of the gradient elution is, elution time and mobile phase A volume ratio order for 0~
15min 72% is run, 15~15.1min from 32% → 20%, 15.1~16.7min again from 20% → 30%, 16.7~
16.8min from 30% → 72%, then 72% is run to 18.4min again.
Detection wavelength is 215nm；Flow velocity is 0.21ml/min；50 DEG C of column temperature；The μ L of sampling volume 1.4.
Solution is prepared：
Precision weighs 6 parts of Exenatide bulk drug sample respectively：(10.023mg, 10.056mg, 10.087mg,
10.095mg, 10.074mg, 10.019mg), and scale is diluted to ultra-pure water, shake up, obtain the Exenatide of various concentrations
Solution (0.10023mg/mL, 0.10056mg/mL, 0.10087mg/mL, 0.10095mg/mL, 0.10074mg/mL,
0.10019mg/mL), as need testing solution.
Table 2：Replica test result
According to table 2 as can be seen that the average content of Exenatide is 99.1% in sample, it is 0.2% to calculate RSD, is had very
Good reappearance.
Comparative example 1
High-efficient liquid phase chromatogram condition：
Chromatographic column：ACE, C18,300A, specification 150*4.6mm, 300A, 3um.
Proportion of mobile phase is：Volume proportion is 20：80~80：20 mobile phase A and the mixed solution of Mobile phase B, gradient
Elution, the mobile phase A are 0.1M/L tetraethyl ammonium perchlorate (TEAP) aqueous solution, and PH to 2.05 is adjusted with perchloric acid, described
Mobile phase B is 100% acetonitrile；
Detection wavelength is 215nm；Flow velocity is 1ml/min；50 DEG C of column temperature；The μ L of sampling volume 20.
Solution is prepared：
The preparation of mobile phase A：Tetraethyl ammonium perchlorate 2.297g is weighed, is dissolved with 100ml ultra-pure waters, then adjusted with perchloric acid
PH to 2.05 is saved, is then filtered stand-by.
Exenatide raw material about 10mg is taken, it is accurately weighed, it is placed in 100ml volumetric flasks, adds appropriate diluent to dissolve and dilute
Release to scale, shake up, as need testing solution.
Compare Fig. 1 and Fig. 2 it can be found that the method for the present invention is faster than high performance liquid chromatography appearance；Pass through experiment condition
It can be found that the method flow velocity of the present invention is low, appearance time section, therefore mobile phase can be saved.
Embodiment of above is merely to illustrate the present invention, and not limitation of the present invention, about the common of technical field
Technical staff, without departing from the spirit and scope of the present invention, it can also make a variety of changes and modification, thus it is all
Equivalent technical scheme falls within scope of the invention, and scope of patent protection of the invention should be defined by the claims.
Claims (5)
- A kind of 1. ultra performance liquid chromatography analysis method of Exenatide, it is characterised in that：Sample containing Exenatide is molten Liquid is passed through in chromatographic column, and using reverse phase liquid chromatography external standard percentage method quantitative analysis, the liquid phase chromatogram condition is：Chromatographic column：UPLC；Mobile phase：Volume proportion is 80：20~20：80 mobile phase A and the mixed solution of Mobile phase B, the mobile phase A are 0.1M/L tetraethyl ammonium perchlorate (TEAP) aqueous solution, PH to 2.05 is adjusted with perchloric acid, the Mobile phase B is 100% acetonitrile；Gradient elution；Detection wavelength：210~220nm；Flow velocity：0.18~0.23ml/min；Column temperature：40~60 DEG C；The μ L of sampling volume 1~5.
- A kind of 2. ultra performance liquid chromatography analysis method of Exenatide according to claim 1, it is characterised in that：It is described The sample introduction concentration of the sample solution of Exenatide is 0.004~0.2mg/mL.
- A kind of 3. ultra performance liquid chromatography analysis method of Exenatide according to claim 1, it is characterised in that：It is described Sample solution is made up of Exenatide bulk drug with deionized water.
- A kind of 4. ultra performance liquid chromatography analysis method of Exenatide according to claim 1, it is characterised in that：It is described Detection wavelength is 215nm, flow velocity 0.21ml/min, 50 DEG C of column temperature, the μ L of sampling volume 1.4.
- A kind of 5. ultra performance liquid chromatography analysis method of Exenatide according to claim 1, it is characterised in that：It is described The detailed process of gradient elution is that elution time and mobile phase A volume ratio order are run for 0~15min 72%, 15~ 15.1min from 32% → 20%, 15.1~16.7min again from 20% → 30%, 16.7~16.8min again from 30% → 72%, Then 72% run to 18.4min.
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| CN114280179A (en) * | 2021-12-22 | 2022-04-05 | 北京美福润医药科技股份有限公司 | Pretreatment of exenatide and method for detecting isomer in His amino acid eluate obtained by pretreatment |
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| CN114280179B (en) * | 2021-12-22 | 2024-03-15 | 北京美福润医药科技股份有限公司 | Pretreatment of exenatide and detection method of isomer in His amino acid eluent obtained by pretreatment |
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Application publication date: 20171226 |