CN115494169B - Separation and detection method for Boc-His (Trt) -Aib-OH and isomer thereof - Google Patents
Separation and detection method for Boc-His (Trt) -Aib-OH and isomer thereof Download PDFInfo
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- CN115494169B CN115494169B CN202210919212.4A CN202210919212A CN115494169B CN 115494169 B CN115494169 B CN 115494169B CN 202210919212 A CN202210919212 A CN 202210919212A CN 115494169 B CN115494169 B CN 115494169B
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- 238000000926 separation method Methods 0.000 title claims abstract description 43
- POZCMPSLNOUKQN-NDEPHWFRSA-N 2-methyl-2-[[(2S)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-3-(1-tritylimidazol-4-yl)propanoyl]amino]propanoic acid Chemical compound CC(C)(C)OC(=O)N[C@@H](CC1=CN(C=N1)C(C1=CC=CC=C1)(C1=CC=CC=C1)C1=CC=CC=C1)C(=O)NC(C)(C)C(O)=O POZCMPSLNOUKQN-NDEPHWFRSA-N 0.000 title claims abstract description 38
- 238000001514 detection method Methods 0.000 title claims abstract description 36
- 239000000203 mixture Substances 0.000 claims abstract description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 45
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 36
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 36
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 24
- 229960000583 acetic acid Drugs 0.000 claims description 12
- 239000012362 glacial acetic acid Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 9
- 238000012856 packing Methods 0.000 claims description 7
- 239000002245 particle Substances 0.000 claims description 7
- 238000010606 normalization Methods 0.000 claims description 6
- 238000004007 reversed phase HPLC Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims 4
- KMYQCBMXXMOPSD-UHFFFAOYSA-N 3-(2,2,2-trifluoroethylsulfanyl)aniline Chemical compound NC1=CC=CC(SCC(F)(F)F)=C1 KMYQCBMXXMOPSD-UHFFFAOYSA-N 0.000 abstract description 16
- 230000000694 effects Effects 0.000 abstract description 9
- 239000012450 pharmaceutical intermediate Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 24
- 239000000523 sample Substances 0.000 description 17
- 238000012360 testing method Methods 0.000 description 14
- 230000000052 comparative effect Effects 0.000 description 13
- 239000011550 stock solution Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 8
- 239000007788 liquid Substances 0.000 description 6
- 239000013558 reference substance Substances 0.000 description 6
- 239000012488 sample solution Substances 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- LOUPRKONTZGTKE-LHHVKLHASA-N quinidine Chemical class C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@H]2[C@@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-LHHVKLHASA-N 0.000 description 3
- 239000000945 filler Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
Classifications
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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
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- 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
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a separation detection method of Boc-His (Trt) -Aib-OH and isomers thereof, and relates to the technical field of detection of pharmaceutical intermediates. The separation is carried out by adopting reversed-phase chromatographic conditions, and the separation effect of Boc-His (Trt) -Aib-OH and isomers thereof [ Boc-D-His (Trt) -Aib-OH ] can be obviously improved by optimizing the chromatographic conditions, particularly optimizing the composition of a mobile phase, and the separation degree between the peaks of the main components and the peaks of the isomers is more than 1.5, so that the isomers can be accurately quantified.
Description
Technical Field
The invention relates to the technical field of detection of pharmaceutical intermediates, in particular to a separation detection method of Boc-His (Trt) -Aib-OH and isomers thereof.
Background
Boc-His (Trt) -Aib-OH is the presently more commonly used amino acid derivative, which exists as isomer Boc-D-His (Trt) -Aib-OH. Because the properties of the enantiomers are very similar, and the isomers are difficult to separate under the common reversed phase chromatographic condition, the separation detection of most of the inter-substance isomers mainly adopts a normal phase chromatographic mode.
However, under the existing normal phase chromatographic condition, the configuration of the strain (Boc-His (Trt) -Aib-OH) is easy to turn over, so that the measured isomer value is inaccurate, the chromatographic peak tailing is serious, and the baseline separation between isomers is difficult to achieve, so that the accurate quantitative detection of the isomers in the strain is difficult. At present, related reports for effectively solving the isomer separation of the variety are not found in the literature.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a separation and detection method of Boc-His (Trt) -Aib-OH and isomers thereof, and aims to solve the problems of poor peak type and difficult separation and quantification of Boc-His (Trt) -Aib-OH and isomers thereof.
The invention is realized in the following way:
in a first aspect, the invention provides a method for separating and detecting Boc-His (Trt) -Aib-OH and isomers thereof, comprising:
dissolving a sample to be tested by using a mobile phase to obtain a solution to be tested;
detecting the solution to be detected by using a reversed-phase high performance liquid chromatography, wherein a mobile phase is a mixture formed by acetonitrile, methanol, glacial acetic acid and triethylamine, and the volume ratio of the acetonitrile to the methanol to the glacial acetic acid to the triethylamine is 260-300:100-140:0.5-2:0.05-0.2;
the content of Boc-His (Trt) -Aib-OH and its isomer was calculated according to the area normalization method.
In an alternative embodiment, the volume ratio of acetonitrile, methanol, glacial acetic acid, and triethylamine in the mobile phase is 270-290:110-130:1.0-1.5:0.1-0.2.
In an alternative embodiment, the concentration of the test sample in the test solution is 1-3mg/mL.
In an alternative embodiment, a chromatographic column having an inner diameter of 4-5mm, a length of 150mm and a packing particle size of 3-6 μm is used.
In an alternative embodiment, a chromatography column model CHIRALPAK QD-AX is used, the column having an inner diameter of 4.6mm, a length of 150mm and a packing particle size of 5 μm.
In an alternative embodiment, the packing of the chromatographic column is selected from any one of the quinidine derivative-containing bonded silica gels.
In an alternative embodiment, chromatographic conditions: the column temperature is 23-27 ℃, the flow rate is 0.4-0.6mL/min, and the detection wavelength is 252-256nm.
In an alternative embodiment, the sample volume is 5-15 μl; preferably 9-11. Mu.L.
In an alternative embodiment, the test is run isocratically for 20-40 minutes.
In an alternative embodiment, the isomer content is calculated according to the following formula:
wherein Ai represents an isomer peak area;
a represents the main component peak area.
The invention has the following beneficial effects: according to the invention, reversed-phase chromatographic conditions are adopted for separation, and the chromatographic conditions are optimized, especially the composition of a mobile phase is optimized, so that the separation effect of Boc-His (Trt) -Aib-OH and isomers thereof [ Boc-D-His (Trt) -Aib-OH ] can be remarkably improved, the separation degree between a main component peak and an isomer peak is greater than 1.5, and the peak type is better, so that the isomers can be accurately quantified.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a liquid chromatogram of example 1 of the present invention;
FIG. 2 is a liquid chromatogram of comparative example 1 of the present invention;
FIG. 3 is a liquid chromatogram of comparative example 2 of the present invention;
FIG. 4 is a liquid chromatogram of comparative example 3 of the present invention;
FIG. 5 is a liquid chromatogram of comparative example 4 of the present invention;
FIG. 6 is a liquid chromatogram of comparative example 5 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention provides a separation and detection method of Boc-His (Trt) -Aib-OH and isomers thereof, which comprises the following steps:
s1, solution to be measured
The mobile phase is utilized to dissolve the sample to be measured to obtain a solution to be measured, and the concentration of the sample to be measured in the solution to be measured is 1-3mg/mL, such as 1.0mg/mL, 1.5mg/mL, 2.0mg/mL, 2.5mg/mL, 3.0mg/mL and the like.
In practical application, the sample to be tested is dissolved by the mobile phase, and the solution to be tested meeting the concentration requirement is prepared for detection. In order to verify the separation effect of the separation detection method provided by the embodiment of the invention, the inventor adopts the following method to prepare a solution to be detected:
dissolving a proper amount of a test sample in a mobile phase and preparing a test sample solution containing about 1-3mg of the test sample solution in each 1 mL; taking a proper amount of Boc-D-His (Trt) -Aib-OH reference substance, dissolving the reference substance by using a mobile phase and preparing an isomer stock solution containing about 0.05mg of the reference substance per 1 mL; a suitable amount of Boc-His (Trt) -Aib-OH control was taken and a suitable amount of isomer stock solution was added to prepare a system-suitable solution containing about 2mg Boc-His (Trt) -Aib-OH and 0.003mg Boc-D-His (Trt) -Aib-OH per 1 mL.
Specifically, boc-His (Trt) -Aib-OH and Boc-D-His (Trt) -Aib-OH are commercially available products.
S2, chromatographic detection
The solution to be detected is detected by using a reversed-phase high performance liquid chromatography, the mobile phase adopts a mixture formed by acetonitrile, methanol, glacial acetic acid and triethylamine, and the volume ratio of the acetonitrile to the methanol to the glacial acetic acid to the triethylamine is 260-300:100-140:0.5-2:0.05-0.2. The selection of the mobile phase is very important for the separation effect of Boc-His (Trt) -Aib-OH and isomers thereof, and the mobile phase provided by the embodiment of the invention can obviously improve the separation effect of the Boc-His (Trt) -Aib-OH and isomers thereof.
Specifically, the volume ratio of acetonitrile, methanol, glacial acetic acid and triethylamine may be 260:100:0.5:0.05, 270:110:0.8:0.07, 280:120:1.0:0.10, 290:130:1.5:0.12, 300:140:2.0:0.15, etc., or may be any value between the above adjacent ratio values.
In a preferred embodiment, the volume ratio of acetonitrile, methanol, glacial acetic acid and triethylamine in the mobile phase is 270-290:110-130:1.0-1.5:0.1-0.2, such as may be 280:120:1.0:0.10. The separation effect of Boc-His (Trt) -Aib-OH and isomers thereof can be further improved by optimizing the use amounts of the four raw materials in the mobile phase.
In some embodiments, a chromatographic column having an inner diameter of 4-5mm, a length of 150mm and a packing particle size of 3-6 μm is used. Preferably, the model of the chromatographic column is CHIRALPAK QD-AX, the inner diameter of the chromatographic column is 4.6mm, the length of the chromatographic column is 150mm, and the particle size of the filling material is 5 mu m; the packing of the chromatographic column is selected from any one of the quinidine derivative-containing bonded silica gel. The type and parameters of the chromatographic column are in the above range, the chromatographic column is convenient to realize, and the separation effect is also good.
Specifically, the inner diameter of the column may be 4.0mm, 4.2mm, 4.4mm, 4.6mm, 4.8mm, 5.0mm, etc., and the filler particle diameter may be 3 μm, 3.5 μm, 4.0 μm, 4.5 μm, 5.0 μm, 5.5 μm, 6.0 μm, etc.
The inventors have further optimized the chromatographic conditions: the column temperature is 23-27 ℃, the flow rate is 0.4-0.6mL/min, and the detection wavelength is 252-256nm; the sample injection volume is 5-15 mu L; preferably 9-11. Mu.L; the test is performed for 20-40min at equal temperature. The separation effect of Boc-His (Trt) -Aib-OH and isomers thereof can be further improved by controlling the chromatographic conditions in the above range, and the separation degree between a main peak and an isomer peak in a system applicability solution is not lower than 1.5.
Specifically, the column temperature may be 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, etc.; the flow rate can be 0.4mL/min, 0.5mL/min, 0.6mL/min, etc.; the detection wavelength may be 252nm, 253nm, 254nm, 255nm, 256nm, etc.; the sample volume may be 5. Mu.L, 6. Mu.L, 7. Mu.L, 8. Mu.L, 9. Mu.L, 10. Mu.L, 11. Mu.L, 12. Mu.L, 13. Mu.L, 14. Mu.L, 15. Mu.L, etc.; the test is performed at an equal degree for 20min, 25min, 30min, 35min, 40min, etc.
S3, calculating the content
The content of Boc-His (Trt) -Aib-OH and the isomer thereof is calculated according to an area normalization method, and the isomer content can be calculated according to the following formula:
wherein Ai represents an isomer peak area; a represents the main component peak area.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The embodiment provides a separation and detection method of Boc-His (Trt) -Aib-OH and isomers thereof, which comprises the following steps:
(1) Preparation of solution to be detected
A suitable amount of the test sample was dissolved in the mobile phase and a test sample solution containing about 2mg per 1mL was prepared.
A suitable amount of Boc-D-His (Trt) -Aib-OH control was taken, dissolved in the mobile phase and made into a stock solution of 0.05mg of isomer per 1 mL.
A suitable amount of Boc-His (Trt) -Aib-OH control was taken, added with a suitable amount of isomer stock solution, and diluted with mobile phase to prepare a system-suitable solution containing approximately 2mg Boc-His (Trt) -Aib-OH and 0.003mg Boc-D-His (Trt) -Aib-OH per 1 mL.
(2) Chromatographic detection
Detecting the solution to be detected by using a reversed-phase high performance liquid chromatography, wherein the chromatographic conditions are as follows:
chromatographic column (CHIRALPAK QD-AX 150X 4.6mm,5 μm); the filler of the chromatographic column is quinidine derivative bonded silica gel;
acetonitrile: methanol: glacial acetic acid: triethylamine (560:240:2:0.2) as mobile phase;
the column temperature is 24 ℃, the flow rate is 0.6mL/min, the detection wavelength is 254nm, the sample injection volume is 10 mu L, and the operation is performed at an isocratic for 30min.
(3) Content calculation
The isomer content is calculated according to an area normalization method, and the content calculation formula is as follows:
wherein Ai represents an isomer peak area;
a represents the main component peak area.
As shown in FIG. 1, the separation degree between the main peak and the isomer is 3.42, and the peak symmetry is good.
Comparative example 1
The comparative example provides a separation and detection method of Boc-His (Trt) -Aib-OH and isomers thereof, which comprises the following steps:
(1) Preparation of solution to be detected
Taking a proper amount of test sample, and using n-hexane: ethanol (90:10) was dissolved and made into a test solution containing approximately 2mg per 1 mL.
Taking a proper amount of Boc-D-His (Trt) -Aib-OH reference substance, and using n-hexane: ethanol (90:10) was dissolved and made up to 0.05mg of isomer stock per 1 mL.
Taking a proper amount of Boc-His (Trt) -Aib-OH reference substance, adding a proper amount of isomer stock solution, and using n-hexane: ethanol (90:10) was diluted to make a system-suitable solution containing approximately 2mg Boc-His (Trt) -Aib-OH and 0.003mg Boc-D-His (Trt) -Aib-OH per 1 mL.
(2) Chromatographic detection
The chromatographic conditions are as follows: chromatographic column (CHIRALCEL OX-3.6X250 mm,3 μm) with n-hexane: ethanol: trifluoroacetic acid (90:10:0.1) as mobile phase; the column temperature is 35 ℃; the flow rate is 0.8mL/min; the detection wavelength is 210nm; sample volume 5. Mu.l; the operation is performed at an isocratic for 50min.
(3) Content calculation
The isomer content is calculated according to an area normalization method, and the content calculation formula is as follows:
wherein Ai represents an isomer peak area;
a represents the main component peak area.
The results show that: as shown in FIG. 2, under the normal phase chromatographic condition, the main peak and the isomer are not separated at all, and the peak type is poor, so that the method is to be optimized.
Comparative example 2
The comparative example provides a separation and detection method of Boc-His (Trt) -Aib-OH and isomers thereof, which comprises the following steps:
(1) Preparation of solution to be detected
A suitable amount of the test sample was taken, dissolved in acetonitrile and made into a test sample solution containing about 2mg per 1 mL.
A suitable amount of Boc-D-His (Trt) -Aib-OH control was taken, dissolved in acetonitrile and made into a stock solution of 0.05mg of isomer per 1 mL.
A suitable amount of Boc-His (Trt) -Aib-OH reference substance was taken, added with a suitable amount of isomer stock solution, and diluted with acetonitrile to prepare a system-suitable solution containing about 2mg Boc-His (Trt) -Aib-OH and 0.003mg Boc-D-His (Trt) -Aib-OH per 1 mL.
(2) Chromatographic detection
Chromatographic conditions: chromatographic column (Kenetex C18.6X250 mm,5 μm) with 0.1% trifluoroacetic acid solution as mobile phase A and acetonitrile as mobile phase B; the column temperature is 35 ℃; the flow rate is 0.8mL/min; the detection wavelength is 254nm; sample volume 10. Mu.l; mobile phase a: mobile phase B (70:30) was run isocratically for 20min.
The results show that: under this condition, as shown in fig. 3, the main peak is not separated from the isomer, and the process is to be optimized.
Comparative example 3
The comparative example provides a separation and detection method of Boc-His (Trt) -Aib-OH and isomers thereof, which comprises the following steps:
(1) Preparation of solution to be detected
A suitable amount of the test sample was dissolved in the mobile phase and a test sample solution containing about 2mg per 1mL was prepared.
A suitable amount of Boc-D-His (Trt) -Aib-OH control was taken, dissolved in the mobile phase and made into a stock solution of 0.05mg of isomer per 1 mL.
A suitable amount of Boc-His (Trt) -Aib-OH control was taken, added with a suitable amount of isomer stock solution, and diluted with mobile phase to prepare a system-suitable solution containing approximately 2mg Boc-His (Trt) -Aib-OH and 0.003mg Boc-D-His (Trt) -Aib-OH per 1 mL.
(2) Chromatographic detection
The chromatographic conditions are as follows: chromatographic column (CHIRALPAK QD-AX 150X 4.6mm,5 μm) with methanol: glacial acetic acid: triethylamine (100:2:0.2) as mobile phase; column temperature is 24 ℃; the flow rate is 0.6mL/min; the detection wavelength is 254nm; sample volume 10. Mu.l; the isocratic run was for 27min.
(3) Content calculation
The isomer content is calculated according to an area normalization method, and the content calculation formula is as follows:
wherein Ai represents an isomer peak area;
a represents the main component peak area.
The results show that: under this condition, the main peak is not completely separated from the isomer and the peak is more trailing, as shown in fig. 4, the method is to be optimized.
Comparative example 4
The only difference from example 1 is that: the Boc-His (Trt) -Aib-OH and Boc-D-His (Trt) -Aib-OH concentrations in the system applicability solution were 2mg/mL.
The flow rate was 0.7mL/min in chromatographic conditions.
The results show that: the main peak was reduced from the isomer separation as shown in FIG. 5, compared to example 1.
Comparative example 5
The only difference from example 1 is that: the flow rate was 0.3mL/min.
The results show that: the main peak and isomer appeared later in time with a broader peak pattern as shown in figure 6.
In summary, the invention provides a separation and detection method for Boc-His (Trt) -Aib-OH and isomers thereof, which adopts reversed phase chromatographic conditions for separation, and can remarkably improve the separation effect of Boc-His (Trt) -Aib-OH and isomers thereof [ Boc-D-His (Trt) -Aib-OH ] by optimizing chromatographic conditions, particularly optimizing the composition of a mobile phase. Specifically, the method has the following advantages:
(1) The normal phase chromatographic condition is avoided, the use of normal phase low-toxicity and volatile solvents is reduced, and a good environment is created for the laboratory environment and the physical health of the laboratory staff;
(2) The content of the detected isomer can be separated in a short time, so that the cost of time, materials and the like is saved;
(3) The separation degree between the main component peak and the isomer peak is more than 1.5, and the peak type is good, so that the isomer can be accurately quantified;
(4) The method provides effective data basis for controlling isomers in the process;
(5) The isomer is accurately detected and controlled, the quality of subsequent products after the intermediate is put into subsequent reaction can be effectively controlled, the yield is improved, and the income is increased.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A method for the separation and detection of Boc-His (Trt) -Aib-OH and its isomers, comprising:
dissolving a sample to be tested by using a mobile phase to obtain a solution to be tested;
detecting the solution to be detected by using a reversed-phase high performance liquid chromatography, wherein the mobile phase adopts a mixture formed by acetonitrile, methanol, glacial acetic acid and triethylamine, and the volume ratio of the acetonitrile to the methanol to the glacial acetic acid to the triethylamine is 260-300:100-140:0.5-2:0.05-0.2;
calculating the content of Boc-His (Trt) -Aib-OH and isomers thereof according to an area normalization method;
the model of the chromatographic column is CHIRALPAK QD-AX, the column temperature is 23-27 ℃, the flow rate is 0.4-0.6mL/min, and the detection wavelength is 252-256nm.
2. The separation detection method according to claim 1, wherein the volume ratio of acetonitrile, methanol, glacial acetic acid and triethylamine in the mobile phase is 270-290:110-130:1.0-1.5:0.1-0.2.
3. The separation detection method according to claim 1 or 2, wherein the concentration of the sample to be detected in the solution to be detected is 1-3mg/mL.
4. The separation and detection method according to claim 1, wherein the column has an inner diameter of 4-5mm, a length of 150mm, and a packing particle diameter of 3-6 μm.
5. The separation detection method according to claim 4, wherein the column has an inner diameter of 4.6mm, a length of 150mm, and a packing particle diameter of 5. Mu.m.
6. The separation assay of claim 1, wherein the sample volume is 5-15 μl.
7. The separation assay of claim 6, wherein the sample volume is 9-11 μl.
8. The separation assay of claim 1, wherein the assay is run isocratically for 20-40 minutes.
9. The separation detection method according to claim 1, wherein the isomer content is calculated according to the following formula:
wherein Ai represents an isomer peak area;
a represents the main component peak area.
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