CN111474262A - Method for detecting crizotinib intermediate - Google Patents

Method for detecting crizotinib intermediate Download PDF

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CN111474262A
CN111474262A CN202010338170.6A CN202010338170A CN111474262A CN 111474262 A CN111474262 A CN 111474262A CN 202010338170 A CN202010338170 A CN 202010338170A CN 111474262 A CN111474262 A CN 111474262A
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detecting
crizotinib intermediate
crizotinib
sample solution
chromatogram
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CN111474262B (en
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蒙发明
陈舒婷
甘鑫钰
李胜辉
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Foshan Yiansai Pharmaceutical Technology Co ltd
Enantiotech Corp Ltd
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Foshan Yiansai Pharmaceutical Technology Co ltd
Enantiotech Corp Ltd
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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
    • G01N30/04—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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/047—Standards external
    • 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 a method for detecting a crizotinib intermediate. The method comprises the following steps: dissolving the crizotinib intermediate racemate in an organic solvent to obtain a control sample solution; dissolving a crizotinib intermediate to be detected in an organic solvent to obtain a sample solution to be detected; taking the control sample solution, detecting by liquid chromatography, and recording a chromatogram map A; taking the sample solution to be tested, detecting by liquid chromatography, and recording a chromatogram map B; calculating according to the chromatogram A and the chromatogram B; wherein the structural formula of the crizotinib intermediate is shown as a formula (1). The detection method is simple to operate, is not influenced by sample batches, has high applicability, and can accurately, quickly and reliably obtain the enantiomer content of the crizotinib intermediate.
Figure DDA0002467529890000011

Description

Method for detecting crizotinib intermediate
Technical Field
The invention relates to the field of analysis, and particularly relates to a method for detecting a crizotinib intermediate.
Background
Crizotinib, the chemical name of which is 3- [ (R) -1- (2, 6-dichloro-3-fluorophenyl) ethoxy ] -5- [1- (piperidin-4-yl) -1H-pyrazol-4-yl ] pyridine-2-amine, has a structural formula shown in formula 1-1, is an oral targeted drug for treating NSC L C, is a drug taking a fusion gene (EM L4-A L K) formed by echinoderm microtubule binding protein 4 and anaplastic lymphoma kinase as a target, and is approved by FDA in 2011 to be a small-molecule targeted drug for treating anaplastic lymphoma kinase (A L K) positive NSC L C late-stage patients.
Figure BDA0002467529870000011
An important intermediate, chemically named (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol, having the CAS number of 877397-65-4, is involved in the process of preparing crizotinib, and the structure of the intermediate is shown as follows:
Figure BDA0002467529870000021
the chiral purity and isomer content of the intermediate directly influence the drug effect of crizotinib. At present, no relevant documents and reports of the enantiomer detection method of the crizotinib intermediate are found.
Disclosure of Invention
Based on the above, the invention aims to provide a simple, accurate, rapid and reliable method for detecting crizotinib intermediate.
The technical scheme is as follows:
a method for detecting a crizotinib intermediate comprises the following steps:
dissolving the crizotinib intermediate racemate in an organic solvent to obtain a control sample solution;
dissolving a crizotinib intermediate to be detected in an organic solvent to obtain a sample solution to be detected;
taking the control sample solution, detecting by liquid chromatography, and recording a chromatogram map A;
taking the sample solution to be tested, detecting by liquid chromatography, and recording a chromatogram map B;
calculating according to the chromatogram A and the chromatogram B;
wherein the structural formula of the crizotinib intermediate is shown as a formula (1):
Figure BDA0002467529870000022
the conditions of the liquid chromatography include: adopting mobile phase isocratic elution; the mobile phase is prepared from (97 +/-2) by volume: (0.075 ± 0.025): (2 ± 0.5): (1 +/-0.5) n-hexane, an amine solvent, isopropanol and ethanol.
In one embodiment, the volume ratio of the n-hexane, the amine solvent, the isopropanol and the ethanol is (97 ± 1): (0.05 ± 0.025): (2 ± 0.25): (1. + -. 0.25).
In one embodiment, the flow velocity of the mobile phase is 0.8m L/min-2 m L/min.
In one embodiment, the amine solvent is selected from at least one of diethylamine and triethylamine.
In one embodiment, the conditions of the liquid chromatography comprise:
the volume ratio is 97:0.05: 2: 1 of n-hexane, diethylamine, isopropanol and ethanol.
In one embodiment, the organic solvent is prepared from n-hexane, isopropanol and tertiary amine according to a volume ratio of (50 +/-0.5): 0.5 +/-0.1).
In one embodiment, the tertiary amine is selected from at least one of diethylamine and triethylamine.
In one embodiment, the concentration of the crizotinib intermediate racemate in the control sample solution is 0.3mg/m L-0.8 mg/m L, and the concentration of the crizotinib intermediate to be detected in the sample solution is 0.3mg/m L-0.8 mg/m L.
In one preferable embodiment, the concentration of the crizotinib intermediate racemate in the control sample solution is 0.4mg/m L-0.6 mg/m L, and the concentration of the crizotinib intermediate in the sample solution to be tested is 0.4mg/m L-0.6 mg/m L.
In one embodiment, the step of calculating is: and comparing the chromatogram A with the chromatogram B, and calculating according to an area normalization method by taking peaks with consistent retention time.
In one embodiment, the conditions of the liquid chromatography comprise: the chromatographic column is selected from a chiralcel AD-H column or a chiralcel AD column;
the detection wavelength was 210nm, and the column temperature was room temperature.
In one preferred embodiment, the column length, the inner diameter and the particle size of the filler particles of the chiralcel AD-H are 250mm, 4.6mm and 5 μm, respectively;
the column length, the inner diameter and the particle size of the filler particles of the chiralcel ad column were 250mm, 4.6mm and 5 μm, respectively.
The invention has the following beneficial effects:
the method for detecting the crizotinib intermediate comprises the steps of preparing a reference sample solution and a test sample solution, adopting liquid chromatography as an analysis means, selecting a specific mobile phase composition, and testing to obtain chromatograms of the reference sample and the test sample; and recording chromatographic peaks with consistent retention time in the chromatograms of the two, and calculating the content of the enantiomer.
The crizotinib intermediate racemate is used as a control sample, so that the influence of other components on a detection result can be effectively removed, and the accuracy and the reproducibility of the detection result are greatly improved; and the solvent with a specific ratio is used as a mobile phase, and the ratio is proper, so that the separation effect of the enantiomers can be improved. The detection method is simple to operate, is not influenced by sample batches, has high applicability, and can accurately, quickly and reliably obtain the enantiomer content of the crizotinib intermediate.
Drawings
FIG. 1 is a chromatogram of test sample 1 of example 1;
FIG. 2 is a chromatogram of test sample 2 in example 2;
FIG. 3 is a chromatogram of test sample 3 in example 3;
FIG. 4 is a linear regression curve of the control sample of example 4;
FIG. 5 is a chromatogram of a working control of crizotinib intermediate racemate in example 5;
FIG. 6 is a chromatogram of test sample 2 in comparative example 1;
fig. 7 is a chromatogram of test sample 2 in comparative example 2.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Crizotinib, the chemical name of which is 3- [ (R) -1- (2, 6-dichloro-3-fluorophenyl) ethoxy ] -5- [1- (piperidin-4-yl) -1H-pyrazol-4-yl ] pyridine-2-amine, has a structural formula shown in formula 1-1, is an oral targeted drug for treating NSC L C, is a drug taking a fusion gene (EM L4-A L K) formed by echinoderm microtubule binding protein 4 and anaplastic lymphoma kinase as a target, and is approved by FDA in 2011 to be a small-molecule targeted drug for treating anaplastic lymphoma kinase (A L K) positive NSC L C late-stage patients.
Figure BDA0002467529870000051
An important intermediate, chemically named (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol, having the CAS number of 877397-65-4, is involved in the process of preparing crizotinib, and the structure of the intermediate is shown as follows:
Figure BDA0002467529870000061
the chiral purity and isomer content of the intermediate directly influence the drug effect of crizotinib. At present, no relevant documents and reports of the enantiomer detection method of the crizotinib intermediate are found. The invention provides a crizotinib intermediate and a method for detecting the content of enantiomers thereof.
The technical scheme is as follows:
a method for detecting contents of crizotinib intermediate and enantiomer thereof comprises the following steps:
dissolving the crizotinib intermediate racemate in an organic solvent to obtain a control sample solution;
dissolving a crizotinib intermediate to be detected in an organic solvent to obtain a sample solution to be detected;
taking the control sample solution, detecting by liquid chromatography, and recording a chromatogram map A;
taking the sample solution to be tested, detecting by liquid chromatography, and recording a chromatogram map B;
calculating according to the chromatogram A and the chromatogram B;
wherein the structural formula of the crizotinib intermediate is shown as a formula (1):
Figure BDA0002467529870000062
the conditions of the liquid chromatography include: adopting mobile phase isocratic elution; the mobile phase is prepared from (97 +/-2) by volume: (0.075 ± 0.025): (2 ± 0.5): (1 +/-0.5) n-hexane, an amine solvent, isopropanol and ethanol.
The method for detecting the crizotinib intermediate comprises the steps of preparing a reference sample solution and a test sample solution, adopting liquid chromatography as an analysis means, selecting a specific mobile phase composition, and testing to obtain chromatograms of the reference sample and the test sample; and recording chromatographic peaks with consistent retention time in the chromatograms of the two, and calculating the content of the enantiomer.
In the invention, the crizotinib intermediate racemate is used as a control sample, so that the influence of other components on the detection result can be effectively removed, and the accuracy and the reproducibility of the test result are greatly improved; and the solvent with a specific ratio is used as a mobile phase, and the ratio is proper, so that the separation effect of enantiomers can be improved. The detection method is simple to operate, is not influenced by sample batches, has high applicability, and can accurately, quickly and reliably obtain the enantiomer content of the crizotinib intermediate.
In the present invention, the conditions of the liquid chromatography include: adopting mobile phase isocratic elution; the mobile phase is prepared from (97 +/-2) by volume: (0.075 ± 0.025): (2 ± 0.5): (1 +/-0.5) of normal hexane, an amine solvent, isopropanol and ethanol.
The specific ratio and the specific solvent are adopted as the eluent, the gradient is proper, the flowing and the migration of a sample in a liquid chromatographic column are facilitated, the separation effect of enantiomers can be improved, and the peak emergence time of the sample can be observed. It is understood that the volume ratio of the n-hexane, the amine solvent, the isopropanol and the ethanol can be set to be, but is not limited to be: 95:0.05:1.5:0.5, 95:0.06:1.5:0.5, 95:0.07:1.5:0.5, 95:0.08:1.5:0.5, 95:0.09:1.5:0.5, 95:0.1:1.5:0.5, 95:0.05:1.6:0.5, 95:0.05:1.7:0.5, 95:0.05:1.8:0.5, 95:0.05:1.9:0.5, 95:0.05: 2.0.5, 95:0.05:2.1:0.5, 95:0.05:2.2:0.5, 95:0.05:2.5, 95:0.05:2.4:0.5, 95:0.05: 1.05: 0.5, 95:0.05: 1.05: 0.5, 0.05:1.5, 95:0.05:1.5, 0.05: 0.5, 0.05:1.5, 95:0.05:1.5, 0.5, 95:0.05:1.5, 0.5, 95: 1.05: 0.1.5, 0.05: 0.1.05: 0.5, 95:0.05: 0.5, 0.1.5, 0.05: 1.05: 0.5, 0.05:1.5, 95: 0.1.5, 0.05: 0.1.5, 0.1.1.5, 95: 0.95: 0.1.1.5, 0.05: 0.5, 0.05: 0.05, 96:0.05:1.6:0.5, 96:0.05:1.7:0.5, 96:0.05:1.8:0.5, 96:0.05:1.9:0.5, 96:0.05:2.0: 5, 96:0.05:2.1:0.5, 96:0.05:2.2:0.5, 96:0.05:2.3:0.5, 96:0.05:2.4:0.5, 96:0.05:2.5:0.5, 96:0.05:1.5:0.6, 96:0.05:1.5:0.7, 96:0.05:1.5:0.8, 96:0.05:1.9, 96:0.05: 1.0.0, 96: 1.05: 1.0.05: 1.0.5: 1.0.0.05: 1.5:0.7, 96: 1.05: 0.05:1.5: 0.05:1.5: 0.5: 1.7, 0.05: 0.5: 1.5: 0.5: 1.7, 0.05: 0.1.05: 0.05: 0.5: 0.1.1.05: 0.05: 0.5: 0.1.05: 0.5: 0.7, 0.1.05: 0.1.1.1.05: 0.05: 0.1.05: 0.1.5, 0.7, 0.1.1.1.1.1.1.1.05: 0.5: 0.05: 0, 97:0.05:2.2:0.5, 97:0.05:2.3:0.5, 97:0.05:2.4:0.5, 97:0.05:2.5:0.5, 97:0.05:1.5:0.6, 97:0.05:1.5:0.7, 97:0.05:1.5:0.8, 97:0.05:1.5:0.9, 97:0.05:1.5:1.0, 97:0.05:1.5:1.1, 97:0.05: 1.2, 97:0.05:1.5:1.3, 97:0.05:1.5:1.4, 97:0.05:1.5:1.5, 98:0.05:1.5:0.5, 98: 0.06.5: 0.5: 1.5, 98:0.07:1.5:0.5, 98:0.08:1.5:0.5, 98:0.09:1.5:0.5, 98:0.1:1.5:0.5, 98:0.05:1.6:0.5, 98:0.05:1.7:0.5, 98:0.05:1.8:0.5, 98:0.05:1.9:0.5, 98:0.05:2.0:0.5, 99:0.05:2.1:0.5, 99:0.05:2.2:0.5, 99:0.05:2.3:0.5, 99:0.05:2.4:0.5, 99:0.05:2.5, 99:0.05: 1.6, 99:0.05: 0.7: 0.5.
Preferably, the volume ratio of the n-hexane to the amine solvent to the isopropanol to the ethanol is (97 +/-1): (0.05 ± 0.025): (2 ± 0.25): (1. + -. 0.25).
In one embodiment, the amine solvent is selected from at least one of diethylamine and triethylamine.
Preferably, the amine solvent is diethylamine.
In one preferred embodiment, the conditions of the liquid chromatograph include: the volume ratio of n-hexane, diethylamine, isopropanol and ethanol is 97:0.05: 2: 1 isocratic elution.
In one embodiment, the flow velocity of the mobile phase is 0.8m L/min-1.2 m L/min, which is beneficial to the flowing and migration of a sample in a liquid chromatographic column and the peak-off time of the sample is observed, the flow velocity is too fast, which causes the two configurations of substances to be detected to be incapable of being effectively separated, thus affecting the accuracy of detection results, and the flow velocity is too slow, which causes the abnormal peak-off type, and it can be understood that the flow velocity of the mobile phase can be set to be, but not limited to, 0.8m L0/min, 0.81m L1/min, 0.82m L2/min, 0.83m L3/min, 0.85m L/min, 0.88m L/min, 0.9m L/min, 0.95m L/min, 1.0m L/min, 1.05m L/min, 1.1m L/min and 1.2m L/min.
Preferably, the flow velocity of the mobile phase is 0.9m L/min to 1.1m L/min.
In one embodiment, the organic solvent for dissolving the crizotinib intermediate racemate and the crizotinib intermediate to be detected is prepared from n-hexane, isopropanol and tertiary amine according to a volume ratio of (50 +/-0.5): 0.5 +/-0.1). The crizotinib intermediate racemate and the crizotinib intermediate to be detected are respectively and fully dissolved in an organic solvent, so that the flow and the migration of a sample in a liquid chromatographic column are facilitated, and the blockage of the liquid chromatographic column is avoided.
Preferably, the organic solvent is prepared from n-hexane: isopropyl alcohol: tertiary amine is added according to the volume ratio of 50:50: 0.5.
In one embodiment, the tertiary amine is selected from at least one of diethylamine or triethylamine.
Preferably, in the present invention, the organic solvent is prepared from n-hexane: isopropyl alcohol: diethylamine according to the volume ratio of 50:50: 0.5.
In one embodiment, the concentration of the crizotinib intermediate racemate in the control sample solution is 0.3mg/m L-0.8 mg/m L, and the concentration of the crizotinib intermediate in the sample solution to be detected is 0.3mg/m L-0.8 mg/m L, so that the flow and the migration of a sample in a liquid chromatographic column are facilitated, and the liquid chromatographic column is prevented from being blocked.
It is understood that, in the present invention, the concentration of crizotinib intermediate racemate in the control sample solution can be set to, but is not limited to, 0.3mg/m L, 0.33mg/m L, 0.35mg/m L0, 0.4mg/m L1, 0.45mg/m L2, 0.48mg/m L3, 0.5mg/m L4, 0.52mg/m L, 0.57mg/m L, 0.6mg/m L, 0.66mg/m L, 0.7mg/m L and 0.8mg/m L. preferably, the concentration of crizotinib intermediate racemate in the control sample solution is 0.4mg/m L-0.6 mg/m L.
It is understood that, in the present invention, the concentration of crizotinib intermediate in the sample solution can be set to be, but not limited to, 0.3mg/m L, 0.33mg/m L, 0.35mg/m L0, 0.4mg/m L1, 0.45mg/m L2, 0.48mg/m L3, 0.5mg/m L4, 0.52mg/m L, 0.57mg/m L, 0.6mg/m L, 0.66mg/m L, 0.7mg/m L, and 0.8mg/m L, preferably, the concentration of crizotinib intermediate in the sample solution is 0.4mg/m L-0.6 mg/m L.
In one embodiment, the step of calculating the percentage of enantiomers is specifically: comparing the chromatograms of the control sample and the test sample, taking peaks with consistent retention time, and calculating the content of the enantiomer according to an area normalization method. The percentage of the content of the enantiomer is calculated by using an area normalization method, so that the experimental error is reduced, and the accuracy and the reproducibility of the test result are greatly improved.
In one embodiment, the conditions of the liquid chromatography comprise: the chromatographic column is selected from a chiralcel AD-H column or a chiralcel AD column;
the detection wavelength was 210nm, and the column temperature was room temperature.
In one preferred embodiment, the column length, the inner diameter and the particle size of the packing particles of the chiralcel AD-H column are 250mm, 4.6mm and 5 μm, respectively;
the column length, the inner diameter and the particle size of the filler particles of the chiralcel AD column were 250mm, 4.6mm and 5 μm, respectively.
In one preferred embodiment, the sample amount of the control sample solution and the sample amount of the test sample solution are both 20 μ L-40 μ L.
The following are specific examples.
Example 1
The embodiment provides a method for detecting a crizotinib intermediate, which comprises the following steps:
(1) dissolving crizotinib intermediate 1 racemate in an organic solvent to obtain a control sample solution A1; dissolving crizotinib intermediate 1 to be detected in an organic solvent to obtain a sample solution B1;
the organic solvent is prepared by mixing n-hexane, isopropanol and diethylamine according to the volume ratio of 50:50:0.5, and the concentrations of the control sample solution A1 and the test sample solution B1 are both 0.5mg/m L.
(2) The control sample solution A1 was injected into a liquid chromatograph, and the chromatogram of the control sample A1 was recorded.
(3) The test sample solution B1 was taken and injected into a liquid chromatograph, the chromatogram of the test sample B1 was recorded, and the percentage of the enantiomer was calculated by the area normalization method, and the results are shown in table 1 and fig. 1.
The chromatographic conditions in this example were:
a chromatographic column: chiralcel AD-H (4.6 x 250mm,5 μm);
the sample injection amount is 20 mu L;
the flow rate is 1.0m L/min;
column temperature: room temperature;
a detector: VWD;
detection wavelength: 210 nm;
mobile phase: the mobile phase A is a mixed solvent of n-hexane and diethylamine, the mobile phase B is a mixed solvent of isopropanol and ethanol,
the volume ratio of n-hexane, diethylamine, isopropanol and ethanol is 97:0.05: 2: 1;
and (3) an elution mode: isocratic elution.
From FIG. 1, peak 1 and peak 2 can be seen, and the results are shown in Table 1.
TABLE 1
Batch number Degree of separation Number of theoretical plate Enantiomers Chiral purity
Sample to be tested B1 1.98 2849 0.09％ 99.91％
Example 2
The embodiment provides a method for detecting a crizotinib intermediate, which comprises the following steps:
(1) the control sample solution was the same as in example 1; dissolving crizotinib intermediate 2 to be detected in an organic solvent to obtain a sample solution B2;
the organic solvent is prepared by mixing n-hexane, isopropanol and diethylamine according to the volume ratio of 50:50:0.5, and the concentrations of the control sample solution A1 and the test sample solution B2 are both 0.5mg/m L.
(2) The test sample solution B2 was taken and injected into a liquid chromatograph, the chromatogram of the test sample B2 was recorded, and the percentage of the enantiomer was calculated by the area normalization method, and the results are shown in table 2 and fig. 2.
The chromatographic conditions in this example were:
a chromatographic column: chiralcel AD-H (4.6 x 250mm,5 μm);
the sample injection amount is 20 mu L;
the flow rate is 1.0m L/min;
column temperature: room temperature;
a detector: VWD;
detection wavelength: 210 nm;
mobile phase: the mobile phase A is a mixed solvent of n-hexane and diethylamine, the mobile phase B is a mixed solvent of isopropanol and ethanol,
the volume ratio of n-hexane, diethylamine, isopropanol and ethanol is 97:0.05: 2: 1;
and (3) an elution mode: isocratic elution.
TABLE 2
Batch number Degree of separation Number of theoretical plate Enantiomers Chiral purity
Sample to be tested B2 - - 0 100％
Example 3
The embodiment provides a method for detecting a crizotinib intermediate, which comprises the following steps:
(1) the control sample solution was the same as in example 1; dissolving crizotinib intermediate 3 to be detected in an organic solvent to obtain a sample solution B3;
the organic solvent is prepared by mixing n-hexane, isopropanol and diethylamine according to the volume ratio of 50:50:0.5, and the concentrations of the control sample solution A1 and the test sample solution B3 are both 0.5mg/m L.
(2) The test sample solution B3 was taken and injected into a liquid chromatograph, the chromatogram of the test sample was recorded, and the percentage of the enantiomer was calculated by the area normalization method, and the results are shown in table 3 and fig. 3.
The chromatographic conditions in this example were:
a chromatographic column: chiralcel AD-H (4.6 x 250mm,5 μm);
the sample injection amount is 20 mu L;
the flow rate is 1.0m L/min;
column temperature: room temperature;
a detector: VWD;
detection wavelength: 210 nm;
mobile phase: the mobile phase A is a mixed solvent of n-hexane and diethylamine, the mobile phase B is a mixed solvent of isopropanol and ethanol,
the volume ratio of n-hexane, diethylamine, isopropanol and ethanol is 97:0.05: 2: 1;
and (3) an elution mode: isocratic elution.
TABLE 3
Batch number Degree of separation Number of theoretical plate Enantiomers Chiral purity
Sample to be tested B3 - - 0 100％
Example 4 Linear test
Precisely weighing about 12.5mg of a control sample, placing the control sample into a 25m L volumetric flask, adding an organic solvent formed by mixing n-hexane, isopropanol and diethylamine according to a volume ratio of 50:50:0.5, dissolving and diluting the organic solvent to a scale, shaking up to be used as a linear test stock solution, precisely weighing 0.02, 0.2, 0.4 and 0.5m L stock solutions into a 100m L volumetric flask, using the organic solvent in the embodiment, diluting to a scale, shaking up to prepare solutions with the concentrations of 0.1ug/m L, 1ug/m L, 2ug/m L and 2.5ug/m L respectively, sampling respectively, recording peak areas, drawing, calculating a linear regression equation, and obtaining a result shown in figure 4.
The chromatographic conditions in this example were:
a chromatographic column: chiralcel AD-H (4.6 x 250mm,5 μm);
the sample injection amount is 20 mu L;
the flow rate is 1.0m L/min;
column temperature: room temperature;
a detector: VWD;
detection wavelength: 210 nm;
mobile phase: the mobile phase A is a mixed solvent of n-hexane and diethylamine, the mobile phase B is a mixed solvent of isopropanol and ethanol,
the volume ratio of n-hexane, diethylamine, isopropanol and ethanol is 97:0.05: 2: 1;
and (3) an elution mode: isocratic elution.
Example 5 System suitability test
The control solutions prepared in examples 1-3 were used as system suitability solutions. The specific method comprises the following steps:
(1) accurately weighing crizotinib intermediate racemate working reference substances, adding an organic solvent formed by mixing n-hexane, isopropanol and diethylamine according to the volume ratio of 50:50:0.5, dissolving and diluting to prepare a solution containing 0.5mg of crizotinib intermediate racemate per 1m L, and using the solution as a system applicability test solution.
(2) And precisely measuring a system applicability test solution, injecting the system applicability test solution into a liquid chromatograph, and recording a chromatogram.
The chromatographic conditions are as follows:
a chromatographic column: chiralcel AD-H (4.6 x 250mm,5 μm);
the sample injection amount is 20 mu L;
the flow rate is 1.0m L/min;
column temperature: room temperature;
a detector: ultraviolet light;
detection wavelength: 210 nm;
mobile phase: the mobile phase A is a mixed solvent of n-hexane and diethylamine, the mobile phase B is a mixed solvent of isopropanol and ethanol,
the volume ratio of n-hexane, diethylamine, isopropanol and ethanol is 97:0.05: 2: 1;
and (3) an elution mode: isocratic elution.
The results of the chromatograms were recorded as shown in table 4 and fig. 5. As can be seen in FIG. 5, two peaks appear at 15.680min and 17.99min with almost no overlap, indicating that the separation was very good. The resolution between the crizotinib intermediate and its enantiomer was greater than 1.5, indicating good system applicability.
TABLE 4
Figure BDA0002467529870000151
Comparative example 1
The comparative example provides a method for detecting a crizotinib intermediate, which comprises the following steps:
(1) the control sample solution A1 and the test sample solution B2 were the same as in example 2, and both had a concentration of 0.5mg/m L.
(2) The control sample solution A1 was injected into a liquid chromatograph, and the chromatogram of the control sample A1 was recorded.
(3) The test sample solution B2 was taken and injected into a liquid chromatograph, and the chromatogram of the test sample B2 was recorded, and the percentage of the enantiomer was calculated by the area normalization method, and the result was shown in fig. 6.
The chromatographic conditions in this comparative example were:
a chromatographic column: chiralcel AD-H (4.6 x 250mm,5 μm);
the sample injection amount is 20 mu L;
the flow rate is 1.0m L/min;
column temperature: room temperature;
a detector: VWD;
detection wavelength: 210 nm;
mobile phase: the mobile phase A is n-hexane, the mobile phase B is a mixed solvent of isopropanol and ethanol,
the volume ratio of n-hexane, isopropanol and ethanol is 97: 5: 1;
and (3) an elution mode: isocratic elution.
As can be seen from fig. 6, the separation was not effective.
Comparative example 2
The comparative example provides a method for detecting a crizotinib intermediate, which comprises the following steps:
(1) the control sample solution A1 and the test sample solution B2 were the same as in example 2, and both had a concentration of 0.5mg/m L.
(2) The control sample solution A1 was injected into a liquid chromatograph, and the chromatogram of the control sample A1 was recorded.
(3) The test sample solution B2 was taken and injected into a liquid chromatograph, and the chromatogram of the test sample B2 was recorded, and the percentage of the enantiomer was calculated by the area normalization method, and the result was shown in fig. 7.
The chromatographic conditions in this comparative example were:
a chromatographic column: chiralcel AD-H (4.6 x 250mm,5 μm);
the sample injection amount is 20 mu L;
the flow rate is 1.0m L/min;
column temperature: room temperature;
a detector: VWD;
detection wavelength: 210 nm;
mobile phase: the mobile phase A is n-hexane, the mobile phase B is isopropanol,
the volume ratio of n-hexane to isopropanol is 90: 10;
and (3) an elution mode: isocratic elution.
As can be seen from fig. 7, the separation was not effective.
Comparative example 3: compared with the embodiment 2, the mobile phase is changed into the volume ratio of the petroleum ether, the diethylamine, the isopropanol and the ethanol is 97:0.05: 2: 1; the rest of the samples were the same as in example 2, and the samples were not separated efficiently.
Comparative example 4: compared with example 2, the mobile phase was changed to a volume ratio of cyclohexane, diethylamine, isopropanol and ethanol of 97:0.05: 2: 1; the remaining conditions were the same as in example 2, and the test sample was not separated efficiently.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. The method for detecting the crizotinib intermediate is characterized by comprising the following steps of:
dissolving the crizotinib intermediate racemate in an organic solvent to obtain a control sample solution;
dissolving a crizotinib intermediate to be detected in an organic solvent to obtain a sample solution to be detected;
taking the control sample solution, detecting by liquid chromatography, and recording a chromatogram map A;
taking the sample solution to be tested, detecting by liquid chromatography, and recording a chromatogram map B;
calculating according to the chromatogram A and the chromatogram B;
wherein the structural formula of the crizotinib intermediate is shown as a formula (1):
Figure FDA0002467529860000011
the conditions of the liquid chromatography include: adopting mobile phase isocratic elution; the mobile phase is prepared from (97 +/-2) by volume: (0.075 ± 0.025): (2 ± 0.5): (1 +/-0.5) of normal hexane, an amine solvent, isopropanol and ethanol.
2. The method for detecting the crizotinib intermediate according to claim 1, wherein the volume ratio of the n-hexane, the amine solvent, the isopropanol and the ethanol is (97 ± 1): (0.05 ± 0.025): (2 ± 0.25): (1. + -. 0.25).
3. The method for detecting the crizotinib intermediate as claimed in claim 1, wherein the flow rate of the mobile phase is 0.8m L/min-1.2 m L/min.
4. The method for detecting a crizotinib intermediate according to any one of claims 1 to 3, wherein the amine solvent is at least one selected from diethylamine and triethylamine.
5. The method for detecting the crizotinib intermediate according to claim 4, wherein the conditions of the liquid chromatography comprise:
the volume ratio is 97:0.05: 2: 1 of n-hexane, diethylamine, isopropanol and ethanol.
6. The method for detecting crizotinib intermediate as claimed in any of claims 1 to 3, wherein the organic solvent is prepared from n-hexane, isopropanol and tertiary amine in a volume ratio of (50 ± 0.5): (50 ± 0.5): (0.5 ± 0.1).
7. The method for detecting the crizotinib intermediate according to claim 6, wherein the tertiary amine is at least one selected from diethylamine and triethylamine.
8. The method for detecting crizotinib intermediate according to any one of claims 1 to 3, wherein the crizotinib intermediate racemate concentration in the control sample solution is 0.3mg/m L-0.8 mg/m L, and the crizotinib intermediate to be detected concentration in the sample solution is 0.3mg/m L-0.8 mg/m L.
9. The method for detecting crizotinib intermediate according to any one of claims 1 to 3, characterized in that the calculating step is: and comparing the chromatogram A with the chromatogram B, and taking peaks with consistent retention time to calculate according to an area normalization method.
10. The method for detecting crizotinib intermediate according to any one of claims 1 to 3, wherein the conditions of the liquid chromatography comprise: the adopted chromatographic column is selected from a chiralcel AD-H column or a chiralcel AD column; the detection wavelength was 210nm, and the column temperature was room temperature.
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