CN116338067A - Method for separating and measuring enantiomer in L-alanine isopropyl ester hydrochloride by high performance liquid chromatography - Google Patents
Method for separating and measuring enantiomer in L-alanine isopropyl ester hydrochloride by high performance liquid chromatography Download PDFInfo
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- CN116338067A CN116338067A CN202111558471.0A CN202111558471A CN116338067A CN 116338067 A CN116338067 A CN 116338067A CN 202111558471 A CN202111558471 A CN 202111558471A CN 116338067 A CN116338067 A CN 116338067A
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- 238000000034 method Methods 0.000 title claims abstract description 29
- YAQKNCSWDMGPOY-JEDNCBNOSA-N propan-2-yl (2s)-2-aminopropanoate;hydrochloride Chemical compound Cl.CC(C)OC(=O)[C@H](C)N YAQKNCSWDMGPOY-JEDNCBNOSA-N 0.000 title claims abstract description 23
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 9
- 238000001514 detection method Methods 0.000 claims abstract description 14
- QKGYJVXSKCDGOK-UHFFFAOYSA-N hexane;propan-2-ol Chemical compound CC(C)O.CCCCCC QKGYJVXSKCDGOK-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000000926 separation method Methods 0.000 claims abstract description 7
- 239000007983 Tris buffer Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 44
- 239000002904 solvent Substances 0.000 claims description 26
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 239000000047 product Substances 0.000 claims description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
- 239000012085 test solution Substances 0.000 claims description 9
- 238000003556 assay Methods 0.000 claims description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 239000000523 sample Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 3
- PASDCCFISLVPSO-UHFFFAOYSA-N benzoyl chloride Chemical compound ClC(=O)C1=CC=CC=C1 PASDCCFISLVPSO-UHFFFAOYSA-N 0.000 claims description 3
- 239000012295 chemical reaction liquid Substances 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 3
- 238000001212 derivatisation Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- HDEDKYAIXDAMKA-UHFFFAOYSA-N CC1=CC=C(Cl)C=C1OC(N)=O Chemical compound CC1=CC=C(Cl)C=C1OC(N)=O HDEDKYAIXDAMKA-UHFFFAOYSA-N 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims 4
- 238000010790 dilution Methods 0.000 claims 1
- 239000012895 dilution Substances 0.000 claims 1
- 238000004090 dissolution Methods 0.000 claims 1
- 239000000945 filler Substances 0.000 claims 1
- 239000012467 final product Substances 0.000 claims 1
- 238000007689 inspection Methods 0.000 claims 1
- 239000000463 material Substances 0.000 claims 1
- 238000012856 packing Methods 0.000 claims 1
- 238000005191 phase separation Methods 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 5
- 230000005526 G1 to G0 transition Effects 0.000 abstract description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 abstract description 3
- 239000000741 silica gel Substances 0.000 abstract description 3
- 229910002027 silica gel Inorganic materials 0.000 abstract description 3
- -1 5-chloro-2-methyl phenyl Chemical group 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract description 2
- 239000011259 mixed solution Substances 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 8
- 239000013558 reference substance Substances 0.000 description 7
- 239000011550 stock solution Substances 0.000 description 6
- 238000011084 recovery Methods 0.000 description 5
- 239000007858 starting material Substances 0.000 description 5
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000012088 reference solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
-
- 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 relates to the field of medicine analysis, in particular to a high performance liquid chromatography detection method for determining enantiomer content in L-alanine isopropyl ester hydrochloride. The method adopts high performance liquid chromatography, the chromatographic column adopts stationary phase silica gel surface coated chiral chromatographic column of amylose-tris (5-chloro-2-methyl phenyl carbamate), the mobile phase adopts mixed solution with the ratio of n-hexane-isopropanol of 70:30, the flow rate is 1.0ml/min, the detection wavelength is 220nm, and the column temperature is 30 ℃. The method can effectively separate the L-alanine isopropyl ester hydrochloride and the enantiomer thereof, and the separation degree meets the requirements. Can strictly and accurately control the quality of the L-alanine isopropyl ester hydrochloride.
Description
Technical Field
The invention relates to the field of medicine analysis, in particular to a high performance liquid chromatography detection method for determining enantiomer content in L-alanine isopropyl ester hydrochloride.
Background
L-alanine isopropyl ester hydrochloride: the chemical structural formula is as follows:
the molecular formula is: c (C) 6 H 14 ClNO 2 The molecular weight is: 167.63
The L-alanine isopropyl ester hydrochloride is an important starting material in the synthesis process of the propionofovir fumarate, has a chiral center, and the enantiomer of the L-alanine isopropyl ester hydrochloride is one of chiral impurity sources of the propionofovir fumarate, so that the content of the enantiomer in the L-alanine isopropyl ester hydrochloride needs to be strictly controlled.
The method of separating most isomers of a drug by high performance liquid chromatography is known, but factors such as mobile phase, solvent, column temperature, chromatographic column and the like have great influence on detection results or are not suitable for target detection objects. At present, the enantiomer of the L-alanine isopropyl ester hydrochloride has no legal standards such as pharmacopoeia and the like. Therefore, it is necessary to develop a method for detecting the enantiomer in L-alanine isopropyl ester hydrochloride, which is capable of effectively separating L-alanine isopropyl ester hydrochloride and its enantiomer to ensure the validity of the detection. The quality of the L-alanine isopropyl ester hydrochloride can be strictly and accurately controlled.
Disclosure of Invention
The invention solves the technical problem of providing a method for separating and measuring enantiomer in L-alanine isopropyl ester hydrochloride by high performance liquid chromatography, which can effectively separate the L-alanine isopropyl ester hydrochloride and enantiomer thereof, realize the quality control of the enantiomer of the L-alanine isopropyl ester hydrochloride, and ensure the drug quality of the propionofovir fumarate bulk drug and the safety of clinical use.
The invention provides a method for separating and measuring L-alanine isopropyl ester hydrochloride enantiomer by high performance liquid chromatography, which is characterized in that the method adopts chromatographic conditions as follows: the chromatographic column adopts stationary phase silica gel surface coated chiral chromatographic column of amylose-tris (5-chloro-2-methyl phenyl carbamate), the mobile phase adopts mixed solution of n-hexane-isopropanol in the ratio of 70:30, the flow rate is 0.8-1.2 ml/min, the detection wavelength is 215-225 nm, and the column temperature is 25-35 ℃.
System applicability solution: the derivative and enantiomer (LB-SM 5-c) were dissolved in n-hexane-isopropanol (10:90) and diluted to give solutions containing about 1mg each per 1 ml.
Test solution: the derivative (LB-SM 5-b) was taken in an appropriate amount, precisely weighed, dissolved in n-hexane-isopropanol (10:90) and quantitatively diluted to prepare a solution containing about 2mg per 1 ml.
Derivatization of samples: about 5.0g of the product is taken and placed in a reaction bottle, about 48ml of dichloromethane is added to dissolve the product, about 10ml of triethylamine is dripped, then stirring reaction is carried out for 1h, about 3.8ml of benzoyl chloride is dripped when the temperature of the reaction liquid is reduced to below 5 ℃, the reaction is continued to be stirred for 30min at the temperature below 5 ℃, after the reaction is finished, the temperature is slowly raised to room temperature and stirred for 15-20 h, about 50ml of water is added to continue stirring, the solution of the dichloromethane layer at the lower layer is collected, and the solution is washed by about 50ml of saturated sodium bicarbonate solution and about 50ml of water in sequence. And (3) adding 5g of anhydrous magnesium sulfate after washing, drying for 30min, concentrating the filtrate, and purifying by adopting a column chromatography method to obtain the magnesium sulfate.
The chromatographic column is a chiral chromatographic column with amylose-tris (5-chloro-2-methylphenyl carbamate) coated on the surface of stationary phase silica gel, and is preferably CHIRALPAK AY-H (250X 4.6mm,5 μm).
The ratio of the mobile phase to the n-hexane-isopropanol is 60:40-80:20, preferably 70:30.
The detector is an ultraviolet detector, and the detection wavelength is 215-225 nm, preferably 220nm.
The column temperature is 25 to 35 ℃, preferably 30 ℃.
The flow rate is 0.8 to 1.2ml/min, preferably 1.0ml/min.
The ratio of n-hexane-isopropanol in the solvent of the formulated sample is 5:95 to 15:85, preferably 10:90.
In the system applicability solution, the peak sequence is enantiomer (LB-SM 5-c) peak and derivative peak, and the separation degree of the two peaks is not less than 3.
The invention provides a separation and determination method of an enantiomer of L-alanine isopropyl ester hydrochloride, wherein chromatographic conditions of the method can effectively separate the L-alanine isopropyl ester hydrochloride and the enantiomer thereof, the specificity is strong, the accuracy and the durability are good, and the product quality of the L-alanine isopropyl ester hydrochloride can be strictly controlled.
The accompanying drawings:
fig. 1: results for blank solvent;
fig. 2: results of the system applicability solution;
fig. 3: results of the test solution;
fig. 4: results of the control solution;
fig. 5: a linear relationship graph.
Detailed Description
Example 1
Relevant chromatographic conditions in the present invention
Instrument: SHIMADZU LC-20AT high performance liquid chromatograph, SPD-20Avp ultraviolet detector;
chromatographic column: CHIRALPAK AY-H20X14.6mm, 5 μm;
mobile phase: n-hexane-isopropanol (70:30);
detection wavelength: 220nm;
column temperature: 30 DEG C
Flow rate: 1.0mL/min
Blank solvent: n-hexane-isopropyl alcohol (10:90)
System applicability solution: the derivative and enantiomer (LB-SM 5-c) are dissolved in a solvent and diluted to prepare solutions containing about 1mg of each 1 ml.
Test solution: the derivative is taken to be a proper amount, precisely weighed, dissolved by a solvent and quantitatively diluted to prepare a solution containing about 2mg of the derivative per 1 ml.
Derivatization of samples: about 5.0g of the product is taken and placed in a reaction bottle, about 48ml of dichloromethane is added to dissolve the product, about 10ml of triethylamine is dripped, then stirring reaction is carried out for 1h, about 3.8ml of benzoyl chloride is dripped when the temperature of the reaction liquid is reduced to below 5 ℃, the reaction is continued to be stirred for 30min at the temperature below 5 ℃, after the reaction is finished, the temperature is slowly raised to room temperature and stirred for 15-20 h, about 50ml of water is added to continue stirring, the solution of the dichloromethane layer at the lower layer is collected, and the solution is washed by about 50ml of saturated sodium bicarbonate solution and about 50ml of water in sequence. And (3) adding 5g of anhydrous magnesium sulfate after washing, drying for 30min, concentrating the filtrate, and purifying by adopting a column chromatography method to obtain the magnesium sulfate.
And (3) measuring: taking 5 μl of the above solution, respectively injecting into high performance liquid chromatograph, and recording chromatogram. The patterns are shown in figures 1-4. The blank solvent does not interfere with the determination of the enantiomer, the enantiomer and the derivative of the product sequentially show peaks in the system applicability solution, the separation degree of the two peaks is far greater than 2, and the baseline separation can be achieved. The theoretical plate numbers and the separation degrees of the two peaks are specifically shown in the following table:
example 2
Special property test (quantitative limit and detection limit)
The chromatographic conditions and assay methods of example 1 were used.
Detection limit and quantitative limit: and (3) taking a proper amount of enantiomer reference substances, respectively adding solvents to dissolve and dilute the enantiomer reference substances to prepare solutions with certain concentrations, and determining according to a method. The determination is generally made by a signal-to-noise ratio method, the quantitative limit is determined by using a signal-to-noise ratio of 10:1, and the detection limit is determined by using a signal-to-noise ratio of 3:1. Meanwhile, the sample injection precision test under the quantitative concentration limit item needs to be examined, and the results are shown in the following table:
example 3
Linearity and range
The chromatographic conditions and assay methods of example 1 were used.
Linear test solution: and (3) taking a proper amount of the derivative and an enantiomer reference substance, respectively dissolving in a solvent, diluting to prepare a series of solutions with a concentration, and determining according to a law.
Results: the enantiomer is in the concentration range of 0.203ug/ml to 20.31 mug/ml, the linear equation is y=11344x+2578.7, the correlation coefficient r is 0.9987, and the linear correlation is good. The results are shown in the following table:
example 4
Accuracy test
The chromatographic conditions and assay methods of example 1 were used.
Control stock solution: about 20mg of enantiomer reference substance is taken, precisely weighed, placed in a 10ml measuring flask, dissolved by adding a solvent and diluted to a scale, shaken uniformly, precisely measured 2ml, placed in a 100ml measuring flask, diluted to the scale by using the solvent, and shaken uniformly.
Control solution: precisely measuring 5ml of reference stock solution, placing into a 20ml measuring flask, diluting to scale with solvent, and shaking.
Blank solution: about 20mg of the derivative of the product is taken, precisely weighed, placed in a 20ml measuring flask, and dissolved and diluted to a scale by adding a proper amount of solvent.
Test solution: about 20mg of the derivative of the product is precisely weighed, placed in a 20ml measuring flask, dissolved by adding a proper amount of solvent, respectively precisely measured by a proper amount of reference stock solution, diluted by the solvent to prepare a solution containing enantiomer with quantitative limit concentration, 50%, 100% and 150%, and uniformly shaken, wherein 3 parts of each concentration is prepared in parallel.
And (3) measuring: precisely measuring 5 μl of the above solution, respectively injecting into high performance liquid chromatograph, and recording chromatogram. The recovery rate was calculated according to the external standard method.
Results: the recovery rate of enantiomer at each concentration is 94.24% -103.27%, the average recovery rate is 99.5%, the relative standard deviation is 3.0%, and the recovery rate is good. The results are shown in the following table:
enantiomer recovery test results
Example 5
Solution stability test
The chromatographic conditions and assay methods of example 1 were used.
And (3) placing the mixture at room temperature for different times, respectively retrieving 5 μl of the reference substance solution and the test substance solution containing 100% of enantiomer under the yield term, injecting the mixture into a liquid chromatograph, recording the chromatogram, and inspecting the stability of the solution.
Results: the enantiomer control solution and the test solution were stable for 12 h. The results are shown in the following table.
Solution stability investigation results
Example 6
Durability test
The chromatographic conditions and assay methods of example 1 were used.
Control stock solution: about 20mg of enantiomer reference substance is taken, precisely weighed, placed in a 10ml measuring flask, dissolved by adding a solvent and diluted to a scale, shaken uniformly, precisely measured 2ml, placed in a 100ml measuring flask, diluted to the scale by using the solvent, and shaken uniformly.
Control solution: precisely measuring 5ml of reference stock solution, placing into a 20ml measuring flask, diluting to scale with solvent, and shaking.
Test solution: about 40mg of the derivative of the product is taken, precisely weighed, placed in a 20ml measuring flask, dissolved by adding a proper amount of solvent, precisely measured with 5ml of reference stock solution, diluted to a scale by the solvent and shaken uniformly.
And (3) measuring: precisely measuring 5 μl of reference solution and 5 μl of sample solution, respectively injecting into liquid chromatograph, recording chromatogram, and examining enantiomer content under different conditions such as temperature, flow rate, wavelength, mobile phase ratio, etc.
Results: as is evident from the test, the measurement results of the enantiomer did not change significantly when the column temperature, the flow rate, the detection wavelength, etc. changed slightly, indicating that the method was excellent in durability. The specific results are shown in the following table:
enantiomer durability test results
Example 7
Determination of samples
The chromatographic conditions and assay methods of example 1 were used.
Control solution: about 20mg of enantiomer reference substance is taken, precisely weighed, placed in a 10ml measuring flask, dissolved by adding a solvent and diluted to a scale, shaken uniformly, precisely measured 2ml of the solution, placed in a 100ml measuring flask, diluted to the scale by using the solvent and shaken uniformly. Precisely weighing 5ml, placing in a 20ml measuring flask, diluting to scale with solvent, and shaking.
Test solution: about 40mg of the derivative of the product is taken, precisely weighed, placed in a 20ml measuring flask, dissolved by adding a solvent, diluted to a scale and shaken uniformly.
And (3) measuring: precisely measuring 5 μl of the reference solution and the sample solution, respectively injecting into a liquid chromatograph, and recording the chromatograms.
Results: the results of enantiomer assays in three batches of samples are shown in the following table,
enantiomer determination results
| Lot number | 180301 | 2018080601 | 2019031601 |
| Enantiomer (%) | 0.003 | 0.005 | 0.002 |
Conclusion: the series of verification results show that the method can well control the enantiomer in the L-alanine isopropyl ester hydrochloride.
Claims (11)
1. A method for separating and measuring isopropyl L-alaninate hydrochloride and enantiomer thereof by adopting high performance liquid chromatography, which is characterized by comprising the following conditions: fillers (e.g., CHIRALPAK AY-H250X 4.6mm,5 μm),
1) Adopting a normal phase separation mode;
2) Column packing material: the surface is coated with amylose-tris (5-chloro-2-methylphenyl carbamate);
3) Mobile phase: n-hexane/isopropanol combination solution;
4) Column temperature: 25-35 ℃;
5) Flow rate: 0.8 ml/min-1.2 ml/min;
6) Wavelength of inspection: 215 nm-225 nm;
7) Sample injection volume: 5 μl;
8) Solvent: n-hexane/isopropanol combination solution;
9) Preparing a system applicability solution: taking a proper amount of the derivative and enantiomer (LB-SM 5-c), adding n-hexane-isopropanol (10:90) for dissolution and dilution to prepare solutions containing about 1mg of each 1 ml;
10 Test solution preparation: taking a proper amount of the derivative, precisely weighing, dissolving with n-hexane-isopropanol (10:90), and quantitatively diluting to prepare a solution containing about 2mg of the derivative per 1 ml;
11 Derivatization of the sample: about 5.0g of the product is taken and placed in a reaction bottle, about 48ml of dichloromethane is added to dissolve the product, about 10ml of triethylamine is dripped, then stirring reaction is carried out for 1h, about 3.8ml of benzoyl chloride is dripped when the temperature of the reaction liquid is reduced to below 5 ℃, the reaction is continued to be stirred for 30min at the temperature below 5 ℃, after the reaction is finished, the temperature is slowly raised to room temperature and stirred for 15-20 h, about 50ml of water is added to continue stirring, the solution of the dichloromethane layer at the lower layer is collected, and the solution is washed by about 50ml of saturated sodium bicarbonate solution and about 50ml of water in sequence. Adding anhydrous magnesium sulfate about 5g after washing, drying for 30min, concentrating the filtrate, and purifying by column chromatography to obtain the final product;
12 Assay: the solution was injected into a high performance liquid chromatograph, and the chromatogram was recorded and analyzed.
2. The method of claim 1, wherein the chromatographic column is CHIRALPAK AY-H (250 x 4.6mm,5 μm).
3. The method according to claim 1, characterized in that the ratio of mobile phase n-hexane-isopropanol is 60:40 to 80:20, preferably 70:30.
4. The process according to claim 1, wherein the column temperature is 25-35 ℃, preferably 30 ℃.
5. The method according to claim 1, characterized in that the flow rate is 0.8-1.2 ml/min, preferably 1.0ml/min.
6. The method according to claim 1, wherein the detector is an ultraviolet detector with a detection wavelength of 215nm to 225nm, preferably 220nm.
7. The method according to claim 1, characterized in that the sample volume is 5-10 μl, preferably 5 μl.
8. The method according to claim 1, characterized in that the solvent n-hexane-isopropanol ratio of the formulated sample is 5:95 to 15:85, preferably 10:90.
9. The method of claim 1, wherein the system applicability solution is formulated as a solution containing 0.5mg/mL to 1.0mg/mL of the derivative and enantiomer.
10. The method of claim 1, wherein the peak sequence in the system applicability solution is enantiomer (LB-SM 5-c) peak and derivative peak, and the separation degree of the two peaks is not less than 3.
11. The method of claim 1, wherein the sample solution is formulated as a solution containing 2mg/ml of the derivative.
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