CN116735759A - Method for detecting ulipam intermediate and enantiomer impurities thereof by high performance liquid chromatography - Google Patents
Method for detecting ulipam intermediate and enantiomer impurities thereof by high performance liquid chromatography Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 49
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 96
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 90
- 238000001514 detection method Methods 0.000 claims abstract description 27
- 239000000523 sample Substances 0.000 claims abstract description 25
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims abstract description 21
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims abstract description 21
- 235000019838 diammonium phosphate Nutrition 0.000 claims abstract description 21
- 239000012488 sample solution Substances 0.000 claims abstract description 7
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 4
- 239000000945 filler Substances 0.000 claims abstract description 3
- 150000004676 glycans Chemical class 0.000 claims abstract description 3
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 3
- 239000005017 polysaccharide Substances 0.000 claims abstract description 3
- 239000005696 Diammonium phosphate Substances 0.000 claims description 16
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 2
- AUALQMFGWLZREY-UHFFFAOYSA-N acetonitrile;methanol Chemical compound OC.CC#N AUALQMFGWLZREY-UHFFFAOYSA-N 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- 239000012085 test solution Substances 0.000 claims description 2
- 238000000926 separation method Methods 0.000 abstract description 30
- 238000002347 injection Methods 0.000 abstract description 20
- 239000007924 injection Substances 0.000 abstract description 20
- 238000003908 quality control method Methods 0.000 abstract description 2
- 239000011248 coating agent Substances 0.000 abstract 1
- 238000000576 coating method Methods 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 97
- 230000014759 maintenance of location Effects 0.000 description 18
- 238000010829 isocratic elution Methods 0.000 description 15
- 239000007853 buffer solution Substances 0.000 description 11
- 238000012360 testing method Methods 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 8
- 238000010998 test method Methods 0.000 description 7
- OTLNPYWUJOZPPA-UHFFFAOYSA-N 4-nitrobenzoic acid Chemical compound OC(=O)C1=CC=C([N+]([O-])=O)C=C1 OTLNPYWUJOZPPA-UHFFFAOYSA-N 0.000 description 5
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 5
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 5
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 5
- 239000001099 ammonium carbonate Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- HZNVUJQVZSTENZ-UHFFFAOYSA-N 2,3-dichloro-5,6-dicyano-1,4-benzoquinone Chemical compound ClC1=C(Cl)C(=O)C(C#N)=C(C#N)C1=O HZNVUJQVZSTENZ-UHFFFAOYSA-N 0.000 description 4
- 229920002160 Celluloid Polymers 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- OTLNPYWUJOZPPA-UHFFFAOYSA-M 4-nitrobenzoate Chemical compound [O-]C(=O)C1=CC=C([N+]([O-])=O)C=C1 OTLNPYWUJOZPPA-UHFFFAOYSA-M 0.000 description 1
- 229940127597 CGRP antagonist Drugs 0.000 description 1
- 208000019695 Migraine disease Diseases 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- ANRVCYAFMSFYQR-UHFFFAOYSA-N butan-1-amine;ethanol Chemical compound CCO.CCCCN ANRVCYAFMSFYQR-UHFFFAOYSA-N 0.000 description 1
- 239000003735 calcitonin gene related peptide receptor antagonist Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 206010027599 migraine Diseases 0.000 description 1
- 229940126701 oral medication Drugs 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 238000011165 process development Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- General Health & Medical Sciences (AREA)
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- Treatment Of Liquids With Adsorbents In General (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention relates to a method for detecting a Ubbelopam intermediate and enantiomer impurities thereof by a high performance liquid chromatography, wherein a chromatographic column CHIRALCEL OJ-RH, 4.6X106 mm and 5 mu m adopt polysaccharide derivative coating as a filler; the diammonium hydrogen phosphate buffer solution with the concentration of 10-30 mMol/L is adopted as a mobile phase A, acetonitrile and methanol (30-40:70-60) are adopted as a mobile phase B, and the ratio is 40-45:60-55; the detection wavelength is 205 nm-215 nm; the column temperature is 20-25 ℃; the flow rate of the mobile phase is 0.3-0.7 mL/min; the method for detecting the ulipam intermediate and the enantiomer impurities thereof by using the high performance liquid chromatography provided by the invention can realize that the separation degree R of the main component of the ulipam intermediate and the enantiomer impurities thereof is more than or equal to 1.6, the content RSD of the enantiomer impurities of a sample solution is less than or equal to 1.2% after continuous sample injection for 6 times, the sample injection repeatability is good, and the method durability is good, so that the method can be used for quality control of the ulipam intermediate.
Description
Technical Field
The invention belongs to the field of medicine analysis, and particularly relates to a method for detecting a ubenimpam intermediate and enantiomer impurities thereof by using a high performance liquid chromatography.
Background
Ubregeepan (Ubregeepant) is an oral drug developed by Merck in the United states for the treatment of migraine, whose mechanism of action is a calcitonin gene-related peptide receptor antagonist, which was marketed in the United states in 2019. The structural formula is as follows:
。
the Ubbelopam intermediate exists in the form of p-nitrobenzoate, has obvious basic groups, has 3 chiral centers in the compound, has poor enantiomer separation and has certain challenges in process development. There is no literature report on the analysis method of the Ubbelopam intermediate (I) and the enantiomer impurity (II) and diastereomer impurity. The structural formula of the Ubbelopam intermediate (I) and the enantiomer impurity (II) thereof is as follows:
。
disclosure of Invention
The invention provides a method for detecting a ubenimpam intermediate (I) and enantiomer impurities (II) thereof by using a high performance liquid chromatography method, which comprises the following steps:
,
1) Chromatographic conditions: a chromatographic column CHIRALCEL OJ-RH, which adopts polysaccharide derivative coated silica gel as a filler; adopting a diammonium hydrogen phosphate buffer solution as a mobile phase A and acetonitrile-methanol as a mobile phase B; the detection wavelength is 205 nm-215 nm; the column temperature is 20-25 ℃; the flow rate of the mobile phase is 0.3-0.7 mL/min;
2) Preparing a test solution: dissolving the sample with 70% acetonitrile water;
3) And (3) measuring: precisely measuring the sample solution, injecting into a liquid chromatograph, and recording the chromatogram.
The concentration of diammonium phosphate in the mobile phase A in the method is 10-30 mMol/L.
The ratio of acetonitrile to methanol in the mobile phase B is 30-40:70-60.
The ratio of acetonitrile to methanol in the mobile phase B in the method is 30:70 or 40:60.
The ratio of the mobile phase A to the mobile phase B is 60-55:40-45.
The ratio of the mobile phase A to the mobile phase B is 60:40 or 55:45.
The flow rate of the mobile phase is 0.4-0.6 mL/min.
The detection wavelength of the method is 210 nm.
The column temperature of the method is 22-25 ℃.
The chromatographic column used in the method is CHIRALCEL OJ-RH, 4.6X106 mm and 5 μm.
The separation degree is used for evaluating the separation degree between a substance to be detected and a substance to be separated, is a key index for measuring the separation efficiency of a chromatographic system, and is expressed by R, wherein R is equal to the ratio of the difference between the retention time of adjacent chromatographic peaks to the peak-to-peak width average value of two chromatographic peaks. The larger R indicates the better separation of the adjacent two components. Generally, when R < 1.0, the two peaks overlap partially, and when r=1.0, the degree of separation can reach 98%, and when r=1.5, the degree of separation can reach 99.7%.
The method for detecting the ulipam intermediate (I) and the enantiomer impurity (II) thereof by using the high performance liquid chromatography can realize that the separation degree R of the ulipam intermediate (I) and the enantiomer impurity (II) thereof is more than or equal to 1.6, the content RSD of the enantiomer impurity of a sample solution is less than or equal to 1.2 percent after continuous sample injection for 6 times, the sample injection repeatability is good, the method durability is good, and the method can be used for quality control of the ulipam intermediate (I).
Drawings
FIG. 1, example 2 method proprietary experiments-blank solution.
FIG. 2, example 2 method specialization experiment-para-nitrobenzoic acid positioning.
FIG. 3, example 2 method proprietary experiments-System applicability solution.
FIG. 4, example 2 method specific experiments-test sample solutions.
Detailed Description
Example 1: screening of chromatographic conditions
The chromatographic conditions are determined by screening through chromatographic columns, mobile phases, wavelengths, flow rates, etc.
Example 1-a
Chromatographic column: macroxylonite OD-H4.6X1250 mm,5 μm;
detection wavelength: 210 nm; column temperature: 25. the temperature is lower than the temperature; sample injection amount: 10. mu L; flow rate: 0.5 mL/min;
mobile phase a: n-hexane;
mobile phase B:0.4% n-butylamine ethanol; gradient elution was performed according to the following conditions;
conclusion: by adopting an alkaline system and a large xylol OD-H chromatographic column, p-nitrobenzoic acid has a late peak, a poor base line and interference.
Example 1-b
Attempts to use reverse systems;
chromatographic column: the macrocelluloid ID 4.6X1250 mm,5 μm;
detection wavelength: 210 nm; column temperature: 25. the temperature is lower than the temperature; sample injection amount: 5. mu L; flow rate: 0.5 mL/min;
mobile phase a is acetonitrile;
mobile phase B was 20 mMol/L potassium hexafluorophosphate pH 2.0;
isocratic elution was performed with mobile phase a: mobile phase b=30:70, 0 min, 60 min;
conclusion: the ulipam intermediate (I), the enantiomeric impurity (II) and p-nitrobenzoic acid were not completely separated.
Example 1-c
Attempts to exchange column macrocelluloid OD-RH on the basis of example 1-b
Chromatographic column: macroxylonite OD-RH 4.6X105 mm,5 μm;
detection wavelength: 210 nm; column temperature: 25. the temperature is lower than the temperature; sample injection amount: 5. mu L; flow rate: 0.5 mL/min;
mobile phase a was acetonitrile: methanol=70:30;
mobile phase B was 20 mMol/L potassium hexafluorophosphate pH 2.0;
mobile phase a: mobile phase b=30:70; isocratic elution is carried out according to 0 min and 40 min;
conclusion: the chromatographic column was adjusted on the basis of example 1-b, and the peak of the ubenimpam intermediate (I) and the enantiomeric impurity (II) was earlier and completely coincident.
Example 1-d
Attempts were made to replace column macrocelluloid AY-RH on the basis of example 1-c;
chromatographic column: large xylonite AY-RH 4.6X105 mm,5 μm;
detection wavelength: 210 nm; column temperature: 25. the temperature is lower than the temperature; sample injection amount: 5. mu L; flow rate: 0.5 mL/min;
mobile phase a was acetonitrile: methanol=40:60;
mobile phase B is 20 mMol/L ammonium bicarbonate;
isocratic elution was performed for 0 min, 30 min, with mobile phase a: mobile phase b=40:60;
conclusion: the large xylonite AY-RH, the Ubbelopam intermediate (I) and the enantiomer impurity (II) are not completely separated, the peak type is bad and the response is low.
Example 1-e
Attempts were made to replace column macrocelluloid AS-RH on the basis of example 1-d;
chromatographic column: large xylonite AS-RH 4.6X105 mm,5 μm;
detection wavelength: 210 nm; column temperature: 25. the temperature is lower than the temperature; sample injection amount: 5. mu L; flow rate: 0.5 mL/min;
mobile phase a was acetonitrile: methanol=40:60;
mobile phase B is 20 mMol/L ammonium bicarbonate;
isocratic elution was performed for 0 min, 30 min, with mobile phase a: mobile phase b=40:60;
conclusion: the chromatographic column was adjusted on the basis of example 1-d, the ubenimpam intermediate (I) and the enantiomeric impurity (II) were completely coincident and the peak pattern was poor.
Example 1-f
Attempts were made to replace the chromatographic column CHIRALCEL OJ-RH on the basis of example 1-e;
chromatographic column: CHIRALCEL OJ-RH 4.6X105 mm,5 μm;
detection wavelength: 210 nm; column temperature: 25. the temperature is lower than the temperature; sample injection amount: 10. mu L; flow rate: 0.5 mL/min;
mobile phase a was acetonitrile: methanol=70:30;
mobile phase B was 20 mMol/L potassium hexafluorophosphate pH 2.0;
isocratic elution was performed with mobile phase a: mobile phase b=30:70, 0 min, 60 min;
conclusion: the separation of the ubenimpam intermediate (I) and the enantiomeric impurity (II) was improved but not completely separated by using the organic phase acetonitrile: methanol=70:30.
EXAMPLE 1-g
Attempts were made to change mobile phase a to acetonitrile: methanol=60:40 on the basis of example 1-f;
chromatographic column: CHIRALCEL OJ-RH 150X 4.6 mm,5 μm;
detection wavelength: 210 nm; column temperature: 25. the temperature is lower than the temperature; sample injection amount: 5. mu L; flow rate: 0.5 mL/min;
mobile phase a was acetonitrile: methanol=60:40;
mobile phase B was 20 mMol/L potassium hexafluorophosphate pH 2.0;
isocratic elution was performed with mobile phase a: mobile phase b=30:70, 0 min, 60 min;
conclusion: the separation of the ubenimpam intermediate (I) and the enantiomer impurity (II) is not obviously improved by adopting acetonitrile to methanol=60:40 as a mobile phase A.
Example 1-h
Attempts were made to change mobile phase B to 20 mMol/L ammonium bicarbonate based on example 1-g;
chromatographic column: CHIRALCEL OJ-RH 4.6X105 mm,5 μm;
detection wavelength: 210 nm; column temperature: 25. the temperature is lower than the temperature; sample injection amount: 5. mu L; flow rate: 0.5 mL/min;
mobile phase a was acetonitrile: methanol=60:40;
mobile phase B is 20 mMol/L ammonium bicarbonate;
isocratic elution was performed for 0 min, 60 min, with mobile phase a: mobile phase b=40:60;
conclusion: 20 mMol/L ammonium bicarbonate is adopted, and the separation degree of the Ubbelopam intermediate (I) and the enantiomer impurity (II) is 0.88 and is not completely separated.
Example 1-i
Attempts were made to change mobile phase B to 20 mMol/L diammonium phosphate based on example 1-h;
chromatographic column: CHIRALCEL OJ-RH 150X 4.6 mm,5 μm;
detection wavelength: 210 nm; column temperature: 25. the temperature is lower than the temperature; sample injection amount: 5. mu L; flow rate: 0.5 mL/min;
mobile phase a was acetonitrile: methanol=40:60;
mobile phase B was 20 mMol/L diammonium phosphate;
isocratic elution was performed for 0 min, 40 min with mobile phase a: mobile phase b=45:55;
conclusion: the 20 mMol/L diammonium hydrogen phosphate is adopted, the separation degree of the Ubbelopam intermediate (I) and the enantiomer impurity (II) is 1.89, the full separation is realized, the peak type is good, and no interference exists in the blank.
Example 2: method specificity
Preparation of mobile phase:
mobile phase a:20 Preparation of mMol/L diammonium phosphate buffer solution: the diammonium phosphate 2.64 and g are weighed, and water is added to 1000 mL for dissolution.
Mobile phase B: acetonitrile: methanol=40:60: acetonitrile 400 mL and methanol 600 mL are sequentially measured and placed in a 1L beaker, stirred and mixed uniformly and ultrasonically for standby.
Solution preparation:
the sample of the Ubbelopam intermediate (I), the paranitrobenzoic acid sample and the system applicability solution are respectively dissolved by 70% acetonitrile, and the concentration is 5.0 mg/mL.
The chromatographic conditions were as follows:
chromatographic column: CHIRALCEL, IJ-RH, 150X4.6 mm,5 μm;
detection wavelength: 210 nm; column temperature: 25. the temperature is lower than the temperature; sample injection amount: 5. mu L; flow rate: 0.5 mL/min;
mobile phase A was 20 mMol/L diammonium phosphate buffer solution
Mobile phase B was acetonitrile: methanol=40:60
Isocratic elution was performed for 0 min, 40 min with mobile phase a: mobile phase b=55:45;
results: retention time of the ubenimpam intermediate (I): 15.152 min; enantiomeric impurity (II) retention time: 13.577 min; p-nitrobenzoic acid retention time: 3.5 And (5) min.
The method can realize the complete separation of the Ubbelopam intermediate (I), the enantiomer impurity (II) and the p-nitrobenzoic acid, and has good method specificity.
Example 3: repeatability experiments
Taking 6 parts of sample solution, continuously injecting sample, recording a chromatogram, and obtaining the following results:
(1) Enantiomeric impurity (II) retention time: 13.589 min; peak area: 1.3263; enantiomer content: 0.10%; retention time of the ubenimpam intermediate (I): 15.118 min;
(2) Enantiomeric impurity (II) retention time: 13.582 min; peak area: 1.3208; enantiomer content: 0.10%; retention time of the ubenimpam intermediate (I): 15.111 min;
(3) Enantiomeric impurity (II) retention time: 13.581 min; peak area: 1.3160; enantiomer content: 0.10%; retention time of the ubenimpam intermediate (I): 15.109 min;
(4) Enantiomeric impurity (II) retention time: 13.572 min; peak area: 1.3406; enantiomer content: 0.10%; retention time of the ubenimpam intermediate (I): 15.103 min;
(5) Enantiomeric impurity (II) retention time: 13.576 min; peak area: 1.3403; enantiomer content: 0.10%; retention time of the ubenimpam intermediate (I): 15.112 min;
(6) Enantiomeric impurity (II) retention time: 13.568 min; peak area: 1.3218; enantiomer content: 0.10%; retention time of the ubenimpam intermediate (I): 15.101 min;
the repeated experiment results show that: 6 parts of sample solution are continuously sampled, the retention time of the Ubbelopam intermediate (I) and the retention time of the enantiomer impurity (II) are respectively 0.04%, 0.06% and 1.11%, the content RSD is far lower than 2.0%, and the experimental method has good repeatability.
Example 4: durability test
The degree of tolerance of the results, which is not affected, was measured by adjusting the flow rate, mobile phase concentration, mobile phase ratio, column temperature, etc. of the chromatographic conditions.
Example 4-a, different flow durability experiments
The chromatographic conditions were as follows:
chromatographic column: CHIRALCEL, IJ-RH, 150X4.6 mm,5 μm;
detection wavelength: 210 nm; column temperature: 25. the temperature is lower than the temperature; sample injection amount: 5. mu L; flow rate: 0.3 to 0.7 per minute mL per minute;
mobile phase A was 20 mMol/L diammonium phosphate buffer solution
Mobile phase B was acetonitrile: methanol=40:60
Isocratic elution was performed for 0 min, 40 min with mobile phase a: mobile phase b=55:45;
the results were as follows:
(1) The flow rate is 0.3 mL/min, and the separation degree is 1.98;
(2) The flow rate is 0.5 mL/min, and the separation degree is 1.75;
(3) The flow rate is 0.7 mL/min, and the separation degree is 1.65;
the test results for different flow rates of durability showed that: the test method changes between the flow rate of 0.3 mL/min and the flow rate of 0.7 mL/min, and has little influence on the measurement result, thus indicating that the method has good durability.
EXAMPLE 4-b buffer salt concentration durability experiments in different mobile phases
The chromatographic conditions were as follows:
chromatographic column: CHIRALCEL, IJ-RH, 150X4.6 mm,5 μm;
detection wavelength: 210 nm; column temperature: 25. the temperature is lower than the temperature; sample injection amount: 5. mu L; flow rate: 0.5 mL/min;
mobile phase A is 10-30 mMol/L diammonium hydrogen phosphate buffer solution
Mobile phase B was acetonitrile: methanol=40:60
Isocratic elution was performed for 0 min, 40 min with mobile phase a: mobile phase b=55:45;
the results were as follows:
(1) The concentration of the diammonium phosphate buffer solution is 10 mMol/L, and the separation degree is 1.79;
(2) The concentration of the diammonium phosphate buffer solution is 20 mMol/L, and the separation degree is 1.75;
(3) The concentration of the diammonium phosphate buffer solution is 30 mMol/L, and the separation degree is 1.78;
the test results of different mobile phase concentrations of durability show that: the mobile phase diammonium phosphate buffer solution concentration of the test method is changed between 10 mMol/L and 30 mMol/L, and the test method has little influence on the test result, so that the method is good in durability.
EXAMPLE 4-c, durability experiments with different Mobile phases starting ratios
The chromatographic conditions were as follows:
chromatographic column: CHIRALCEL, IJ-RH, 150X4.6 mm,5 μm;
detection wavelength: 210 nm; column temperature: 25. the temperature is lower than the temperature; sample injection amount: 5. mu L; flow rate: 0.5 mL/min;
mobile phase A is 20 mMol/L diammonium phosphate buffer solution;
mobile phase B was acetonitrile: methanol=40:60;
adopting mobile phase A and mobile phase B=60-55:40-45, and performing isocratic elution according to 0 min and 40 min;
the results were as follows:
(1) Mobile phase a: mobile phase b=60:40, separation degree 2.12;
(2) Mobile phase a: mobile phase b=55:45, separation degree 1.75;
the test results for durability at different initial ratios of mobile phases show that: the initial ratio of mobile phase A to mobile phase B in the test method is changed between 60:40 and 55:45, and the influence on the measurement result is small, so that the method has good durability.
Example 4-d, different column temperature durability experiments
The chromatographic conditions were as follows:
chromatographic column: CHIRALCEL, IJ-RH, 150X4.6 mm,5 μm;
detection wavelength: 210 nm; column temperature: 20-25 ℃; sample injection amount: 5. mu L; flow rate: 0.5 mL/min;
mobile phase A was 20 mMol/L diammonium phosphate buffer solution
Mobile phase B was acetonitrile: methanol=40:60;
isocratic elution was performed for 0 min, 40 min with mobile phase a: mobile phase b=55:45;
the results were as follows:
(1) The column temperature is 20 ℃, and the separation degree is 1.76;
(2) The column temperature was 25℃and the degree of separation was 1.75.
The test results of different column temperatures of durability show that: the test method changes between the column temperature of 20 ℃ and 25 ℃ and has little influence on the measurement result, thus showing that the method has good durability.
Example 4-e, durability experiments at different detection wavelengths
The chromatographic conditions were as follows:
chromatographic column: CHIRALCEL, IJ-RH, 150X4.6 mm,5 μm;
detection wavelength: 205 nm-215 nm; column temperature: 25 ℃; sample injection amount: 5. mu L; flow rate: 0.5 mL/min;
mobile phase A was 20 mMol/L diammonium phosphate buffer solution
Mobile phase B was acetonitrile: methanol=40:60
Isocratic elution was performed for 0 min, 40 min with mobile phase a: mobile phase b=55:45;
the results were as follows:
(1) The detection wavelength is 205 nm, and the separation degree is 1.75;
(2) The detection wavelength is 210 nm, and the separation degree is 1.75;
(3) The detection wavelength is 215 nm, and the separation degree is 1.75;
the test results of different detection wavelengths of durability show that: the test method changes between the detection wavelengths 205, nm and 215, nm, and has little influence on the measurement result, thus indicating that the method has good durability.
Example 4-f, durability test of acetonitrile to methanol ratio in mobile phase B
The chromatographic conditions were as follows:
chromatographic column: CHIRALCEL, IJ-RH, 150X4.6 mm,5 μm;
detection wavelength: 210 nm; column temperature: 25 ℃; sample injection amount: 5. mu L; flow rate: 0.5 mL/min;
mobile phase A is 20 mMol/L diammonium phosphate buffer solution;
the mobile phase B is acetonitrile and methanol=30-40:70-60;
isocratic elution at 0 min, 40 min, with mobile phase a: mobile phase b=55:45
The results were as follows:
(1) Acetonitrile: methanol=30:70 in mobile phase B, separation degree 2.16;
(2) Acetonitrile: methanol=40:60 in mobile phase B, separation degree 1.75;
the test results of different acetonitrile to methanol ratios for durability showed that: the test method is changed between acetonitrile: methanol=30:70 and acetonitrile: methanol=40:60, and has little influence on the measurement result, thus indicating that the method has good durability.
Claims (10)
1. A method for detecting a ubenimpam intermediate (I) and enantiomer impurities (II) thereof by using a high performance liquid chromatography method is characterized in that:
，
1) Chromatographic conditions: a chromatographic column CHIRALCEL OJ-RH, which adopts polysaccharide derivative coated silica gel as a filler; adopting a diammonium hydrogen phosphate buffer solution as a mobile phase A and acetonitrile-methanol as a mobile phase B; the detection wavelength is 205-215 nm; the column temperature is 20-25 ℃; the flow rate of the mobile phase is 0.3-0.7 mL/min;
2) Preparing a test solution: dissolving the sample with 70% acetonitrile water;
3) And (3) measuring: precisely measuring the sample solution, injecting into a liquid chromatograph, and recording the chromatogram.
2. The method for detecting the ubenimpam intermediate (I) and the enantiomer impurities (II) thereof by using the high performance liquid chromatography according to claim 1, wherein the method comprises the following steps of: the concentration of diammonium phosphate in the mobile phase A is 10-30 mMol/L.
3. The method for detecting the ubenimpam intermediate (I) and the enantiomer impurities (II) thereof by using the high performance liquid chromatography according to claim 1, wherein the method comprises the following steps of: the ratio of acetonitrile to methanol in the mobile phase B is 30-40:70-60.
4. A method for detecting the umbegiline intermediate (I) and its enantiomeric impurity (II) by high performance liquid chromatography according to claim 3, characterized in that: the ratio of acetonitrile to methanol in the mobile phase B is 30:70 or 40:60.
5. The method for detecting the ubenimpam intermediate (I) and the enantiomer impurities (II) thereof by using the high performance liquid chromatography according to claim 1, wherein the method comprises the following steps of: the ratio of the mobile phase A to the mobile phase B is 60-55:40-45.
6. The method for detecting the ubenimpam intermediate (I) and the enantiomer impurities (II) thereof by using the high performance liquid chromatography according to claim 5, wherein the method comprises the following steps of: the ratio of the mobile phase A to the mobile phase B is 60:40 or 55:45.
7. The method for detecting the ubenimpam intermediate (I) and the enantiomer impurities (II) thereof by using the high performance liquid chromatography according to claim 1, wherein the method comprises the following steps of: the flow rate of the mobile phase is 0.4-0.6 mL/min.
8. The method for detecting the ubenimpam intermediate (I) and the enantiomer impurities (II) thereof by using the high performance liquid chromatography according to claim 1, wherein the method comprises the following steps of: the detection wavelength is 210 nm.
9. The method for detecting the ubenimpam intermediate (I) and the enantiomer impurities (II) thereof by using the high performance liquid chromatography according to claim 1, wherein the method comprises the following steps of: the column temperature is 22-25 ℃.
10. The method for detecting the ubenimpam intermediate (I) and the enantiomer impurities (II) thereof by using the high performance liquid chromatography according to claim 1, wherein the method comprises the following steps of: the column was CHIRALCEL OJ-RH, 4.6X106 mm,5 μm.
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