CN117368352A - Method for separating oseltamivir intermediate and two chiral isomers thereof by HPLC (high Performance liquid chromatography) - Google Patents
Method for separating oseltamivir intermediate and two chiral isomers thereof by HPLC (high Performance liquid chromatography) Download PDFInfo
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- CN117368352A CN117368352A CN202311330331.7A CN202311330331A CN117368352A CN 117368352 A CN117368352 A CN 117368352A CN 202311330331 A CN202311330331 A CN 202311330331A CN 117368352 A CN117368352 A CN 117368352A
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- VSZGPKBBMSAYNT-RRFJBIMHSA-N oseltamivir Chemical compound CCOC(=O)C1=C[C@@H](OC(CC)CC)[C@H](NC(C)=O)[C@@H](N)C1 VSZGPKBBMSAYNT-RRFJBIMHSA-N 0.000 title claims abstract description 65
- 229960003752 oseltamivir Drugs 0.000 title claims abstract description 60
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000004458 analytical method Methods 0.000 claims abstract description 32
- 239000007788 liquid Substances 0.000 claims abstract description 28
- 239000003495 polar organic solvent Substances 0.000 claims abstract description 24
- 230000014759 maintenance of location Effects 0.000 claims abstract description 15
- 238000001514 detection method Methods 0.000 claims abstract description 13
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 5
- 150000004676 glycans Chemical class 0.000 claims abstract description 4
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 4
- 239000005017 polysaccharide Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 42
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical group CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000010829 isocratic elution Methods 0.000 claims description 3
- 241000023813 Isia Species 0.000 claims description 2
- 239000012535 impurity Substances 0.000 abstract description 10
- 239000000543 intermediate Substances 0.000 description 42
- SPWVRYZQLGQKGK-UHFFFAOYSA-N dichloromethane;hexane Chemical compound ClCCl.CCCCCC SPWVRYZQLGQKGK-UHFFFAOYSA-N 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 8
- OAYLNYINCPYISS-UHFFFAOYSA-N ethyl acetate;hexane Chemical compound CCCCCC.CCOC(C)=O OAYLNYINCPYISS-UHFFFAOYSA-N 0.000 description 7
- 238000004090 dissolution Methods 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 206010022000 influenza Diseases 0.000 description 6
- JXOHGGNKMLTUBP-JKUQZMGJSA-N shikimic acid Natural products O[C@@H]1CC(C(O)=O)=C[C@H](O)[C@@H]1O JXOHGGNKMLTUBP-JKUQZMGJSA-N 0.000 description 6
- 229940079593 drug Drugs 0.000 description 5
- JXOHGGNKMLTUBP-HSUXUTPPSA-N shikimic acid Chemical compound O[C@@H]1CC(C(O)=O)=C[C@@H](O)[C@H]1O JXOHGGNKMLTUBP-HSUXUTPPSA-N 0.000 description 5
- 229960002194 oseltamivir phosphate Drugs 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- AAWZDTNXLSGCEK-LNVDRNJUSA-N (3r,5r)-1,3,4,5-tetrahydroxycyclohexane-1-carboxylic acid Chemical compound O[C@@H]1CC(O)(C(O)=O)C[C@@H](O)C1O AAWZDTNXLSGCEK-LNVDRNJUSA-N 0.000 description 2
- AAWZDTNXLSGCEK-UHFFFAOYSA-N Cordycepinsaeure Natural products OC1CC(O)(C(O)=O)CC(O)C1O AAWZDTNXLSGCEK-UHFFFAOYSA-N 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- AAWZDTNXLSGCEK-ZHQZDSKASA-N Quinic acid Natural products O[C@H]1CC(O)(C(O)=O)C[C@H](O)C1O AAWZDTNXLSGCEK-ZHQZDSKASA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 208000028399 Critical Illness Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- 208000002979 Influenza in Birds Diseases 0.000 description 1
- 201000007100 Pharyngitis Diseases 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 206010064097 avian influenza Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- -1 ethyl amino ethyl propoxy cyclohexene carboxylate Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
- PGZUMBJQJWIWGJ-ONAKXNSWSA-N oseltamivir phosphate Chemical compound OP(O)(O)=O.CCOC(=O)C1=C[C@@H](OC(CC)CC)[C@H](NC(C)=O)[C@@H](N)C1 PGZUMBJQJWIWGJ-ONAKXNSWSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 125000001757 shikimic acid group Chemical group 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229940061367 tamiflu Drugs 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention provides a method for separating oseltamivir intermediate and two chiral isomers thereof by an HPLC method, which comprises the following steps: s1, preparing an analysis solution which contains oseltamivir intermediate and isomers 1 and 2 thereof; s2, injecting the analysis solution into a liquid chromatograph, performing chromatographic analysis, and recording a chromatogram; oseltamivir intermediate main peak retention time is 9.1 min, isomer 1 retention time is 16.8 min, isomer 2 retention time is 20.1 min; the chromatographic column of the liquid chromatograph is a polysaccharide derivative chiral column, and the column temperature is 20-40 ℃; the mobile phase comprises a weak polar organic solvent and a strong polar organic solvent which are eluted at equal proportions, and the flow rate of the mobile phase is 0.5-0.8 ml/min; the detector of the liquid chromatograph is an ultraviolet detector, and the detection wavelength is 254nm. The method can simply, conveniently, rapidly and accurately analyze the oseltamivir intermediate and the isomer 1 and isomer 2 impurities, can completely separate, and has good specificity.
Description
Technical Field
The invention belongs to the technical field of chromatographic analysis, and particularly relates to a method for separating oseltamivir intermediate and two chiral isomers thereof by an HPLC method.
Background
Influenza is an acute respiratory infection caused by influenza virus. It is mainly spread by air, enters the human body through the respiratory tract, and can spread by contact between people, such as handshake, hug, etc., even by contaminated objects. Influenza is prevalent in large numbers in the autumn and winter of the temperate zone each year, and severe complications associated with viruses (e.g., pneumonia or heart-lung failure) lead to higher mortality in critically ill patients.
Tamiflu, which is commercially available as an anti-influenza drug produced exclusively by pharmacy, is commonly known as oseltamivir phosphate, which is considered to be the most effective and highest specific influenza therapeutic drug at present, and its chemical name is ethyl amino ethyl propoxy cyclohexene carboxylate. Duffy was approved for sale in 1999 by the united states and in china for 7 months 2004. Duffy is a national strategic reserve of medicine, and its demand in the united states is 20-30 tons per year. Although the cost of the drug using the daphne is not low from the perspective of pharmaceutical economy, the daphne is a very effective drug for treating influenza, has good safety, drug resistance and bioavailability, can greatly reduce the occurrence of complications (mainly trachea and bronchitis, pneumonia, pharyngitis and the like) and the use of antibiotics, and is one of the most commonly used drugs for treating influenza at present and one of the most effective drugs for resisting avian influenza and A viruses.
Oseltamivir phosphate (II-1) is quite similar in structure to shikimic acid (II-2) and quinic acid (II-3), except for the functional groups at the 3,4,5 positions and chiral centers. Since the 3,4 and 5 positions of shikimic acid and quinic acid are active hydroxyl groups, oseltamivir phosphate can be synthesized through simple conversion, and the difficulty is that three hydroxyl groups are secondary hydroxyl groups, the properties are quite similar, and unique strategies and control are needed to accurately introduce various functional groups at the 3,4 and 5 positions.
There is a simple method for synthesizing an epoxy intermediate by using shikimic acid as a starting material, but since shikimic acid is mostly extracted from plants and contains isomer impurities, oseltamivir phosphate is a single substance and not a mixture of various isomers, it is necessary to control the starting material to be a single substance. Because the shikimic acid has larger polarity, the isomer impurities are difficult to remove, and after the shikimic acid is synthesized to obtain the oseltamivir intermediate, the steric hindrance is increased, and the impurities are easier to separate and purify. Therefore, it is particularly important to develop a process that can isolate oseltamivir intermediates and their isomeric impurities.
Disclosure of Invention
In order to solve the problems in the background art, the invention provides a method for separating oseltamivir intermediate and two chiral isomers thereof by an HPLC method, which can simply, conveniently, rapidly and accurately analyze oseltamivir intermediate and isomer 1 and isomer 2 impurities, can completely separate, and has good specificity.
In order to achieve the above object, the present invention provides the following technical solutions:
a method for separating oseltamivir intermediate and two chiral isomers thereof by an HPLC method, comprising the steps of:
s1, preparing an analysis solution, wherein the analysis solution comprises oseltamivir intermediate, isomer 1 and isomer 2 thereof, and the mass of the oseltamivir intermediate, the isomer 1 and the isomer 2 is equal;
s2, injecting the analysis solution obtained in the step S1 into a liquid chromatograph, performing chromatographic analysis, and recording a chromatogram; oseltamivir intermediate main peak retention time is 9.1 min, isomer 1 retention time is 16.8 min, isomer 2 retention time is 20.1 min;
wherein the chromatographic column of the liquid chromatograph is a polysaccharide derivative chiral column, and the column temperature of the chromatographic column is 20-40 ℃;
the mobile phase comprises a weak polar organic solvent and a strong polar organic solvent, the flow rate of the mobile phase is 0.5-0.8 ml/min, and the weak polar organic solvent and the strong polar organic solvent are eluted at equal scale;
the detector of the liquid chromatograph is an ultraviolet detector, and the detection wavelength is 254nm.
Preferably, the weak polar organic solvent is n-hexane, and the strong polar organic solvent is dichloromethane or ethyl acetate.
Preferably, the isocratic elution ratio of the weak polar organic solvent to the strong polar organic solvent is 85-90:15-10.
Preferably, the concentration of oseltamivir intermediate in the assay solution is 1-10mg/ml.
Preferably, in S2, the sample injection amount of the analysis solution is 5 to 30 μl.
Preferably, the chromatographic column isIA 5um 4.6mm x 150mm column.
Preferably, the specific operation steps of S1 are as follows: taking an equal amount of oseltamivir intermediate and isomers 1 and 2 thereof; and dissolving the mobile phase, fixing the volume, and uniformly mixing to obtain the analysis solution.
Preferably, the oseltamivir intermediate and the structural formulas of the isomer 1 and the isomer 2 thereof are formula 1, formula 2 and formula 3 respectively, as follows:
the application has the following beneficial effects:
the method can simply, conveniently, rapidly and accurately analyze the oseltamivir intermediate and the impurities of two isomers thereof. The main peak oseltamivir intermediate and the isomer 1 impurity and the isomer 2 impurity can be completely separated, and the specificity is good. The detection limit is 0.01 mug, and the sensitivity is high. And the sample is continuously injected for 6 times, the RSD is 0.13%, and the precision is good.
Drawings
Fig. 1-3: HPLC graphs of oseltamivir intermediate, isomer 1 and isomer 2 in example 1 of the present application;
fig. 4-6: 1H NMR chart of oseltamivir intermediate, isomer 1 and isomer 2 in example 1 of this application.
Detailed Description
The present application is described in further detail below with reference to examples.
The raw materials of the examples and comparative examples herein are commercially available in general unless otherwise specified.
The invention provides a method for separating oseltamivir intermediate and two chiral isomers thereof by an HPLC method, which comprises the following steps:
s1, preparing an analysis solution, wherein the analysis solution contains oseltamivir intermediate, isomer 1 and isomer 2, and the mass of the oseltamivir intermediate, the isomer 1 and the isomer 2 is equal.
The specific operation steps of S1 are as follows: taking an equal amount of oseltamivir intermediate and isomers 1 and 2 thereof; dissolving with mobile phase, fixing volume, and mixing to obtain analytical solution with oseltamivir intermediate concentration of 1-10mg/ml.
The mobile phase comprises a weak polar organic solvent and a strong polar organic solvent, wherein the weak polar organic solvent is normal hexane, and the strong polar organic solvent is dichloromethane or ethyl acetate, preferably ethyl acetate. The isocratic elution ratio of the weak polar organic solvent to the strong polar organic solvent is 85-90:15-10; the flow rate of the mobile phase is 0.5-0.8 ml/min.
Oseltamivir intermediates and isomers 1 and 2 have the structural formulae of formula 1, formula 2 and formula 3, respectively, as follows:
s2, injecting the analysis solution obtained in the step S1 into a liquid chromatograph, carrying out chromatographic analysis with the sample injection amount of 5-30ml, and recording a chromatogram; oseltamivir intermediate main peak retention time was 9.1 min, isomer 1 retention time was 16.8 min, and isomer 2 retention time was 20.1 min.
Wherein the chromatographic column of the liquid chromatograph is a polysaccharide derivative chiral column, in particularIA 5um 4.6mm x 150mm column. The column temperature of the chromatographic column is 20-40 ℃. The detector of the liquid chromatograph is an ultraviolet detector, and the detection wavelength is 254nm.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
(1) Instrument and conditions
High performance liquid chromatograph: siemens flying UltiMate 3000 high performance liquid chromatograph system and workstation; chromatographic column and cellophaneIA 5um 4.6mm*150mm。
Mobile phase, n-hexane-ethyl acetate (88:12), flow rate: 0.7mL/min, detection wavelength of 254nm, column temperature: 35 ℃, sample volume: 20. Mu.L, isocratic for 30 min.
(2) Experimental procedure
And (3) taking 60mg of oseltamivir intermediate, and each of the isomer 1 and the isomer 2, adding n-hexane-dichloromethane (88:12) for dissolution, and fixing the volume to 20ml, and uniformly mixing to obtain a solution containing 3mg/ml of oseltamivir intermediate as an analysis solution.
Taking analysis solution, performing high performance liquid chromatography under the above conditions, and recording chromatogram. The results are shown in FIG. 1, with a main peak retention time of 9.1 minutes, isomer 1 retention time of 16.8 minutes, and isomer 2 retention time of 20.1 minutes. It can be seen that under this condition the main peak of oseltamivir intermediate and the two isomeric impurities can be completely separated.
Example 2
(1) Instrument and conditions
High performance liquid chromatograph: siemens flying UltiMate 3000 high performance liquid chromatograph system and workstation; chromatographic column and cellophaneIA 5um 4.6mm*150mm。
Mobile phase, n-hexane-dichloromethane (85:15), flow rate, 0.8mL/min, detection wavelength of 254nm, column temperature, 35 ℃, sample volume, 20. Mu.L, isocratic for 30 min.
(2) Experimental procedure
Taking 40mg of oseltamivir intermediate, and isomer 1 and isomer 2 thereof, respectively, adding n-hexane-dichloromethane (85:15) for dissolution and constant volume to 20ml, and uniformly mixing to obtain a solution containing 2mg/ml of oseltamivir intermediate as an analysis solution.
Taking analysis solution, performing high performance liquid chromatography under the above conditions, and recording chromatogram. Under this condition the chromatogram is identical to that of figure 1.
Example 3
(1) Instrument and conditions
High performance liquid chromatograph: siemens flying UltiMate 3000 high performance liquid chromatograph system and workstation; chromatographic column and cellophaneIA 5um 4.6mm*150mm。
Mobile phase, n-hexane-ethyl acetate (85:15), flow rate: 0.6mL/min, detection wavelength of 254nm, column temperature: 30deg.C, sample volume: 20 μL, isocratic for 30 min.
(2) Experimental procedure
Taking 20mg of oseltamivir intermediate, and each of isomer 1 and isomer 2, adding n-hexane-ethyl acetate (85:15) to dissolve and fix the volume to 20ml, and uniformly mixing to obtain a solution containing 1mg/ml of oseltamivir intermediate as an analysis solution.
Taking analysis solution, performing high performance liquid chromatography under the above conditions, and recording chromatogram. Under this condition the chromatogram is identical to that of figure 1.
Example 4
(1) Instrument and conditions
High performance liquid chromatograph: siemens flying UltiMate 3000 high performance liquid chromatograph system and workstation; chromatographic column and cellophaneIA 5um 4.6mm*150mm；
Mobile phase, n-hexane-ethyl acetate (90:10), flow rate: 0.8mL/min, detection wavelength of 254nm, column temperature: 40 ℃, sample volume: 15. Mu.L, isocratic for 30 min.
(2) Experimental procedure
And (3) taking 60mg of oseltamivir intermediate, and each of the isomer 1 and the isomer 2, adding n-hexane-ethyl acetate (90:10) for dissolution, and fixing the volume to 20ml, and uniformly mixing to obtain a solution containing 3mg/ml of oseltamivir intermediate as an analysis solution.
Taking analysis solution, performing high performance liquid chromatography under the above conditions, and recording chromatogram. Under this condition the chromatogram is identical to that of figure 1.
Example 5
(1) Instrument and conditions
High performance liquid chromatograph: siemens flying UltiMate 3000 high performance liquid chromatograph system and workstation; chromatographic column and cellophaneIA 5um 4.6mm*150mm；
Mobile phase, n-hexane-dichloromethane (86:14), flow rate, 0.7mL/min, detection wavelength of 254nm, column temperature, 40 ℃, sample volume, 10. Mu.L, isocratic for 30 min.
(2) Experimental procedure
And (3) taking 140mg of oseltamivir intermediate, and each of the isomer 1 and the isomer 2, adding n-hexane-dichloromethane (86:14) for dissolution, and fixing the volume to 20ml, and uniformly mixing to obtain a solution containing 7mg/ml of oseltamivir intermediate as an analysis solution.
Taking analysis solution, performing high performance liquid chromatography under the above conditions, and recording chromatogram. Under this condition the chromatogram is identical to that of figure 1.
Example 6
(1) Instrument and conditions
High performance liquid chromatograph: siemens flying UltiMate 3000 high performance liquid chromatograph system and workstation; chromatographic column and cellophaneIA 5um 4.6mm*150mm；
Mobile phase, n-hexane-dichloromethane (87:13), flow rate, 0.5mL/min, detection wavelength of 254nm, column temperature, 30 ℃, sample injection volume, 25. Mu.L, isocratic for 30 min.
(2) Experimental procedure
Taking 100mg of oseltamivir intermediate, and 100mg of each of isomers 1 and 2, adding n-hexane-dichloromethane (87:13), dissolving and fixing the volume to 20ml, and uniformly mixing to obtain a solution containing 5mg/ml of oseltamivir intermediate as an analysis solution.
Taking analysis solution, performing high performance liquid chromatography under the above conditions, and recording chromatogram. Under this condition the chromatogram is identical to that of figure 1.
Example 7
(1) Instrument and conditions
High performance liquid chromatograph: siemens flying UltiMate 3000 high performance liquid chromatograph system and workstation; chromatographic column and cellophaneIA 5um 4.6mm*150mm；
Mobile phase, n-hexane-ethyl acetate (87:13), flow rate: 0.6mL/min, detection wavelength of 254nm, column temperature: 40 ℃, sample volume: 20. Mu.L, isocratic for 30 min.
(2) Experimental procedure
And (3) taking 60mg of oseltamivir intermediate, and each of the isomer 1 and the isomer 2, adding n-hexane-dichloromethane (87:13) for dissolution, and fixing the volume to 20ml, and uniformly mixing to obtain a solution containing 3mg/ml of oseltamivir intermediate as an analysis solution.
Taking analysis solution, performing high performance liquid chromatography under the above conditions, and recording chromatogram. Under this condition the chromatogram is identical to that of figure 1.
Example 8
(1) Instrument and conditions
High performance liquid chromatograph: siemens flying UltiMate 3000 high performance liquid chromatograph system and workstation; chromatographic column and cellophaneIA 5um 4.6mm*150mm；
Mobile phase, n-hexane-ethyl acetate (86:14), flow rate: 0.8mL/min, detection wavelength of 254nm, column temperature: 25 ℃, sample volume: 25. Mu.L, isocratic for 30 min.
(2) Experimental procedure
And taking 20mg of oseltamivir intermediate, and each of the isomer 1 and the isomer 2, adding n-hexane-dichloromethane (86:14) for dissolution, and fixing the volume to 20ml, and uniformly mixing to obtain a solution containing 1mg/ml of oseltamivir intermediate as an analysis solution.
Taking analysis solution, performing high performance liquid chromatography under the above conditions, and recording chromatogram. Under this condition the chromatogram is identical to that of figure 1.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (8)
1. A method for separating oseltamivir intermediate and two chiral isomers thereof by an HPLC method, comprising the steps of:
s1, preparing an analysis solution, wherein the analysis solution comprises oseltamivir intermediate, isomer 1 and isomer 2 thereof, and the mass of the oseltamivir intermediate, the isomer 1 and the isomer 2 is equal;
s2, injecting the analysis solution obtained in the step S1 into a liquid chromatograph, performing chromatographic analysis, and recording a chromatogram; oseltamivir intermediate main peak retention time is 9.1 min, isomer 1 retention time is 16.8 min, isomer 2 retention time is 20.1 min;
wherein the chromatographic column of the liquid chromatograph is a polysaccharide derivative chiral column, and the column temperature of the chromatographic column is 20-40 ℃;
the mobile phase comprises a weak polar organic solvent and a strong polar organic solvent, the flow rate of the mobile phase is 0.5-0.8 ml/min, and the weak polar organic solvent and the strong polar organic solvent are eluted at equal scale;
the detector of the liquid chromatograph is an ultraviolet detector, and the detection wavelength is 254nm.
2. The method for separating oseltamivir intermediate and its two chiral isomers by HPLC according to claim 1, wherein said weak polar organic solvent is n-hexane and said strong polar organic solvent is dichloromethane or ethyl acetate.
3. The method for separating oseltamivir intermediate and its two chiral isomers by HPLC according to claim 2, wherein the isocratic elution ratio of the weak polar organic solvent and the strong polar organic solvent is 85-90:15-10.
4. The method for separating oseltamivir intermediate and its two chiral isomers by HPLC according to claim 1, wherein the concentration of oseltamivir intermediate in the analysis solution is 1-10mg/ml.
5. The method for separating oseltamivir intermediate and its two chiral isomers by HPLC according to claim 1, wherein in S2, the sample injection amount of the analysis solution is 5 to 30 μl.
6. The method for separating oseltamivir intermediate and its two chiral isomers by HPLC according to claim 1, wherein said chromatographic column isIA 5um 4.6mm x 150mm column.
7. The method for separating oseltamivir intermediate and two chiral isomers thereof by HPLC according to any one of claims 1 to 6, wherein the specific operation steps of S1 are as follows: taking an equal amount of oseltamivir intermediate and isomers 1 and 2 thereof; and dissolving the mobile phase, fixing the volume, and uniformly mixing to obtain the analysis solution.
8. The method for separating oseltamivir intermediate and its two chiral isomers by HPLC according to any one of claims 1 to 6, wherein the oseltamivir intermediate and its isomers 1 and 2 have the structural formulae of formula 1, formula 2 and formula 3, respectively, as follows:
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311330331.7A CN117368352A (en) | 2023-10-16 | 2023-10-16 | Method for separating oseltamivir intermediate and two chiral isomers thereof by HPLC (high Performance liquid chromatography) |
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