CN116609474A - Isolation assay of crizotinib intermediate Z 2 HPLC method for related substances - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 68
- 239000002146 L01XE16 - Crizotinib Substances 0.000 title claims abstract description 50
- KTEIFNKAUNYNJU-GFCCVEGCSA-N crizotinib Chemical compound O([C@H](C)C=1C(=C(F)C=CC=1Cl)Cl)C(C(=NC=1)N)=CC=1C(=C1)C=NN1C1CCNCC1 KTEIFNKAUNYNJU-GFCCVEGCSA-N 0.000 title claims abstract description 50
- 229960005061 crizotinib Drugs 0.000 title claims abstract description 50
- 239000000126 substance Substances 0.000 title claims abstract description 36
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 21
- 238000003556 assay Methods 0.000 title description 2
- 238000002955 isolation Methods 0.000 title description 2
- 239000012535 impurity Substances 0.000 claims abstract description 281
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 48
- 238000001514 detection method Methods 0.000 claims abstract description 40
- 238000000926 separation method Methods 0.000 claims abstract description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 21
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 11
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 9
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 8
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 69
- 239000003085 diluting agent Substances 0.000 claims description 39
- 230000014759 maintenance of location Effects 0.000 claims description 17
- 238000012937 correction Methods 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 12
- 239000012085 test solution Substances 0.000 claims description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 238000004364 calculation method Methods 0.000 claims description 5
- 238000010812 external standard method Methods 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 239000000945 filler Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 239000003054 catalyst Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 238000005259 measurement Methods 0.000 abstract description 9
- 238000004458 analytical method Methods 0.000 abstract description 4
- 238000003908 quality control method Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 230000005526 G1 to G0 transition Effects 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 31
- 239000011550 stock solution Substances 0.000 description 22
- 238000007865 diluting Methods 0.000 description 21
- 239000012488 sample solution Substances 0.000 description 20
- 239000000047 product Substances 0.000 description 16
- 238000012360 testing method Methods 0.000 description 14
- 238000005303 weighing Methods 0.000 description 14
- 239000012467 final product Substances 0.000 description 8
- IVDFJHOHABJVEH-UHFFFAOYSA-N pinacol Chemical compound CC(C)(O)C(C)(C)O IVDFJHOHABJVEH-UHFFFAOYSA-N 0.000 description 8
- 239000011259 mixed solution Substances 0.000 description 7
- 238000004090 dissolution Methods 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 239000012452 mother liquor Substances 0.000 description 5
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 239000012490 blank solution Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 3
- 102100033793 ALK tyrosine kinase receptor Human genes 0.000 description 2
- 101710168331 ALK tyrosine kinase receptor Proteins 0.000 description 2
- PCLIMKBDDGJMGD-UHFFFAOYSA-N N-bromosuccinimide Chemical compound BrN1C(=O)CCC1=O PCLIMKBDDGJMGD-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical group O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012803 optimization experiment Methods 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012795 verification Methods 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
- 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
-
- 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
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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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
- 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
- G01N2030/8872—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 impurities
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- General Health & Medical Sciences (AREA)
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- Spectroscopy & Molecular Physics (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention belongs to the technical field of chemical analysis, and particularly relates to a method for measuring a crizotinib intermediate Z 2 HPLC methods of the related substances. The invention takes octadecylsilane chemically bonded silica as a stationary phase, takes a mobile phase A, a mobile phase B and a mobile phase C as mobile phases, wherein the mobile phase A is potassium dihydrogen phosphate solution, the mobile phase B is acetonitrile, and the mobile phase C is methanol; sequentially eluting the impurities SM by gradient 2e SM impurity 1 Impurity Z 2c Crizotinib intermediate Z 2 SM impurity 2 Impurity Z 2b And impurity Z 1 And (5) separating. Then a detector with the wavelength of 220nm is adopted for detection, thereby realizing the crizotinib intermediate Z 2 And the qualitative and quantitative detection of the related substances. The self-built high performance liquid chromatography has high sensitivity, high accuracy and good durability, and can realize the crizotinib intermediate Z within 60 minutes 2 And the separation and measurement of related substances thereof are of great significance to the quality control of crizotinib.
Description
Technical Field
The invention belongs to the field of chemical industryThe technical field of academic analysis, in particular to a method for measuring a crizotinib intermediate Z 2 HPLC methods of the related substances.
Background
Non-small cell lung cancer is the most predominant type of lung cancer, and has high mortality. Crizotinib is one of the targeted drugs for treating non-small cell lung cancer, and is mainly used for treating Anaplastic Lymphoma Kinase (ALK) -positive locally advanced and metastatic non-small cell lung cancer. The market of crizotinib marks the important progress of accurate medical treatment in the field of tumor treatment.
Crizotinib intermediate Z 2 Is an important intermediate in the preparation process of crizotinib, and the structural formula of the intermediate is shown as formula I.
Found by researches, the crizotinib intermediate Z 2 May have the impurity SM 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b And impurity Z 1 . The existence of the impurities is very likely to interfere the purity of crizotinib, thereby affecting the targeting effect of the medicine and reducing the medicine effect. Thus, there is a need for an intermediate Z to crizotinib 2 The impurities contained are effectively controlled to improve the yield of the subsequent steps and the purity of the final product. However, at present, a definite analysis method and literature data can be used for preparing the intermediate Z of crizotinib 2 And impurity SM 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b And impurity Z 1 Effective separation and measurement were performed.
The invention patent with publication number of CN111007158A discloses a method for separating and measuring related substances in the preparation process of crizotinib, wherein acetonitrile and 0.1% trifluoroacetic acid aqueous solution are used as mobile phases, a diode array or ultraviolet is used as a detector, and the detection wavelength is 254nm. The patent uses reverse phase high performance liquid chromatography for detection. Although the invention achieves the intermediate Z of crizotinib 2 However, this method cannot achieve gramAzotinib intermediate Z 2 Medium impurity SM 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b And impurity Z 1 Is used for separation and measurement.
Disclosure of Invention
Accordingly, one of the objects of the present invention is to provide a method for separating crizotinib intermediate Z by high performance liquid chromatography 2 And related substances thereof, which can simultaneously realize the intermediate Z of crizotinib 2 And 6 kinds of impurities are separated, and the method has high accuracy, high sensitivity and good reproducibility.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
separation of crizotinib intermediate Z based on high performance liquid chromatography 2 And related substances thereof, said crizotinib intermediate Z 2 The structural formula of the catalyst is shown as formula I, and the related substance is impurity SM 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b And impurity Z 1 Any one or more of the following; the impurity SM 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b And impurity Z 1 The structural formula of (a) is shown as a formula II-a formula VII in sequence; the chromatographic column of the high performance liquid chromatography adopts octadecylsilane chemically bonded silica as a filler, and takes a mobile phase A, a mobile phase B and a mobile phase C as mobile phases, wherein the mobile phase A is a potassium dihydrogen phosphate solution, the mobile phase B is acetonitrile, and the mobile phase C is methanol; sequentially eluting the impurities SM by gradient 2e SM impurity 1 Impurity Z 2c Crizotinib intermediate Z 2 SM impurity 2 Impurity Z 2b And impurity Z 1 Separating;
the components can be characterized according to the speed of separation.
Further, the gradient elution procedure was:
at 0min, the volume ratio of the mobile phase A to the mobile phase B to the mobile phase C is 63-67:29-31:5, a step of;
at 5 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5, a step of;
at 45 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 35:60:5, a step of;
at 50 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 35:60:5, a step of;
at 50.1 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5, a step of;
at 60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5.
preferably, the gradient elution procedure is:
at 0 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5, a step of;
at 5 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5, a step of;
at 45 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 35:60:5, a step of;
at 50 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 35:60:5, a step of;
at 50.1 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5, a step of;
at 60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5.
further, in the mobile phase A, the concentration of the potassium dihydrogen phosphate solution is 0.008mol/L to 0.012mol/L, preferably 0.01mol/L; the pH of the mobile phase A is adjusted to 3.0-3.2, preferably 3.1, with phosphoric acid.
Further, the flow rate of the mobile phase is 0.9ml/min to 1.1ml/min, preferably 1.0ml/min; the column temperature of the chromatographic column is 32-37 ℃, preferably 35 ℃.
Further, the sample volume was 10. Mu.l.
Preferably, the specification of the chromatographic column is 4.6mm×250mm×5 μm.
Further, the separation time is preferably 60 minutes.
Further, a ghost peak trapping column is added to the high performance liquid chromatography.
The second purpose of the invention is to provide a qualitative identification Bie Ke tinib intermediate Z 2 And related substances thereof, which are useful for the detection of crizotinib intermediate Z 2 Whether or not to contain the impurity SM 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b And impurity Z 1 Has important significance for controlling the quality of crizotinib medicines.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
qualitative authentication Bie Ke Tinib intermediate Z 2 And related substances, and processes for preparing the intermediate Z of crizotinib by the method 2 SM impurity 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b And impurity Z 1 Separating; and then detected by a detector having a wavelength of 220 nm.
Further, the retention time of the seven components is sequentially from short to long: impurity SM 2e SM impurity 1 Impurity Z 2c Crizotinib intermediate Z 2 SM impurity 2 Impurity Z 2b And impurity Z 1 。
The components can be characterized according to the sequence of the retention time.
As a preferable technical scheme, the mobile phase A is potassium dihydrogen phosphate solution with the concentration of 0.01mol/L, the mobile phase B is acetonitrile, and the mobile phase C is methanol; the pH value of the mobile phase A is 3.1; the specification of the chromatographic column is 4.6mm multiplied by 250mm multiplied by 5 mu m; the flow rate of the mobile phase is 1.0ml/min; the column temperature of the chromatographic column is 35 ℃; the detection wavelength is 220nm; performing linear gradient elution according to a gradient elution program to obtain a chromatogram; the gradient elution procedure was as follows:
at 0 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5, a step of;
at 5 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5, a step of;
at 45 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 35:60:5, a step of;
at 50 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 35:60:5, a step of;
at 50.1 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5, a step of;
at 60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5.
further, when the retention time was 9.5.+ -. 0.5min, it was determined as impurity SM 2e The method comprises the steps of carrying out a first treatment on the surface of the When the retention time was 11.9.+ -. 0.5min, it was determined as impurity SM 1 The method comprises the steps of carrying out a first treatment on the surface of the When the retention time was 21.8.+ -. 0.5min, it was determined as impurity Z 2c The method comprises the steps of carrying out a first treatment on the surface of the When the retention time is 35.5+/-0.5 min, the crizotinib intermediate Z is judged 2 The method comprises the steps of carrying out a first treatment on the surface of the When the retention time was 40.2.+ -. 0.5min, it was determined as impurity SM 2 The method comprises the steps of carrying out a first treatment on the surface of the When the retention time was 41.3.+ -. 0.5min, it was determined as impurity Z 2b The method comprises the steps of carrying out a first treatment on the surface of the When the retention time was 44.5.+ -. 0.5min, it was determined as impurity Z 1 。
The invention further aims to provide a method for quantitatively determining the crizotinib intermediate Z 2 And related substances, which can achieve a crizotinib intermediate Z in less than 60 minutes or in more than 60 minutes 2 And the content measurement of related substances.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
quantitative determination of crizotinib intermediate Z 2 And a method for producing a substance related thereto, which is the impurity SM 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b And impurity Z 1 Any one or more of the following; the method comprises the following steps:
(1) Separating: the crizotinib intermediate Z is prepared by the method of the purpose 2 And related substances thereof are separated;
(2) Detection of: intermediate Z of crizotinib by using method described in second aim 2 And related substances thereof are detected;
(3) And (3) content calculation: calculating the crizotinib intermediate Z according to the chromatogram measured in the step (2) and a main component self-comparison method with a correction factor and/or an external standard method 2 And the content of impurities.
Further, impurity SM 2e The quantitative limit concentration of (C) is 0.2575 mug/ml, and the impurity SM 1 The quantitative limit concentration of (C) is 0.2998 mug/ml, and the impurity Z 2c The quantitative limit concentration of (C) is 0.2869 mug/ml, and the impurity SM 2 The quantitative limit concentration of (C) is 0.2956 mug/ml, and the impurity Z 2b The quantitative limit concentration of (C) is 0.2918 mug/ml, and the impurity Z 1 The quantitative limit concentration of (C) is 0.2940 mug/ml, and the crizotinib intermediate Z 2 The quantitative limit concentration of (C) was 0.3003. Mu.g/ml.
Further, impurity SM 2e The detection limit concentration of (C) is 0.1287 mug/ml, and the impurity SM 1 The detection limit concentration of (C) is 0.1499 mug/ml, and the impurity Z 2c The detection limit concentration of (C) is 0.1435 mug/ml, and the impurity SM 2 The detection limit concentration of (C) is 0.1478 mug/ml, and the impurity Z 2b The detection limit concentration of (C) is 0.1459 mug/ml, and the impurity Z 1 The detection limit concentration of (2) is 0.1470 mug/ml, and the crizotinib intermediate Z 2 The detection limit concentration of (C) was 0.1502. Mu.g/ml.
Further, impurity SM 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b Impurity Z 1 The correction factors of (1) are 1.76, 1.10, 1.79, 1.87, 2.18 and 1.22, respectively.
Further, preparing a standard substance to obtain a standard sample chromatogram; and (3) testing the chromatograms obtained by the samples, and calculating the peak areas according to a main component self-comparison method with correction factors and/or an external standard method.
The standard substance is the known component content, and the component content of the test sample is unknown.
The obtained standard sample chromatograms can be stored in a database and used as quality control map materials in an intelligent pharmaceutical factory, and the quality control map materials are used for helping a robot to judge whether products produced by the robot are qualified or not, and quantitatively improved analysis suggestions are provided for unqualified products.
Preferably, the impurity content is calculated by a principal component self-correlation method with a correction factor.
Further, prior to separation, a test solution and a control solution were prepared using acetonitrile as a diluent.
The impurity SM was calculated according to the peak area measured by the above method using the principal component self-correlation method with correction factor 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b And impurity Z 1 Specifically comprising the following steps:
step 1, weighing a proper amount of the product, precisely weighing, adding acetonitrile for dissolving and diluting to prepare a solution with the concentration of about 1mg/ml, and taking the solution as a sample solution;
step 2, precisely measuring a proper amount of a sample solution, and adding acetonitrile to quantitatively dilute the sample solution to obtain a solution with the concentration of about 1.5 mug/ml as a control solution;
step 3, taking the sample solution in the step 1 and the control solution in the step 2, injecting the sample solution into a high performance liquid chromatograph, and recording a chromatogram; then, the impurity SM is calculated from the measured peak area by using the principal component self-correlation method with correction factors 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b And impurity Z 1 Is contained in the composition.
The impurity content calculation formula is as follows:
total impurity (%) =each known impurity (%) +total unknown impurity (%)
Wherein Ar represents the peak area of a single impurity peak in the chromatogram of the sample solution, asum represents the sum of the peak areas of unknown impurities in the chromatogram of the sample solution, as Control Representing the main peak area in the chromatogram of the control solutionRRF represents each impurity correction factor.
The invention has the beneficial effects that:
1. the self-built high performance liquid chromatography method is used for the crizotinib intermediate Z 2 And related substances thereof, realizing the intermediate Z of crizotinib 2 Medium impurity SM 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b And impurity Z 1 The method has the advantages of simplicity, rapidness, high accuracy and the like, and is not reported in the literature.
2. The high performance liquid chromatography provided by the invention can separate 6 impurities, the separation degree among the 6 impurities is more than 1.5, other unknown impurities in the sample do not interfere with the detection of known impurities, and the requirements of related substances are met.
3. The high performance liquid chromatography provided by the invention adopts three-phase elution, so that the selectivity of flowing relative to different impurities is increased, and a plurality of impurities can be better separated; meanwhile, the invention carries out specific selection on the PH value of the mobile phase, thereby obtaining the separation degree meeting the requirement.
4. Impurity Z 2 Further impurity SM is generated in water 2e Thereby influencing the impurity SM 2e The method selects pure acetonitrile as a diluent and is matched with the HPLC method constructed by the method to accurately realize the impurity SM 2e And the recovery rate meets the requirement.
Drawings
FIG. 1 is a chromatogram of a blank solution;
FIG. 2 is a chromatogram of a specificity-mixture solution;
FIG. 3 is a proprietary-SM 1 Locating a chromatogram of the solution;
FIG. 4 is a specific-SM 2 Locating a chromatogram of the solution;
FIG. 5 is a specific-SM 2e Locating a chromatogram of the solution;
FIG. 6 is a diagram of specificity-Z 2b Locating a chromatogram of the solution;
FIG. 7 is a diagram of specificity-Z 2c Chromatography of positioning solutionsA figure;
FIG. 8 is a diagram of specificity-Z 1 Locating a chromatogram of the solution;
FIG. 9 is a chromatogram of a system adaptation-self control solution;
FIG. 10 is a chromatogram of a quantitative limiting solution;
FIG. 11 is a chromatogram of a detection limit solution;
FIG. 12 is a chromatogram of a control solution;
FIG. 13 is a chromatogram of an unlabeled test solution;
FIG. 14 is a chromatogram of accuracy-addition of a labeled test solution-80%;
FIG. 15 is a chromatogram of accuracy-labeled test solution-100%;
FIG. 16 is a chromatogram of accuracy-labeled test solution-120%;
FIG. 17 is a chromatogram of durability-mixed solution-flow rate 0.9 ml/min;
FIG. 18 is a chromatogram of durability-mixed solution-flow rate 1.1ml/min;
FIG. 19 is a chromatogram of durability-mixed solution-column temperature 32 ℃;
FIG. 20 is a chromatogram of durability-mixed solution-column temperature 37 ℃;
FIG. 21 is a chromatogram of durability-mixed solution-mobile phase ratio (A: B: C=63:31:5);
fig. 22 is a chromatogram of durability-mixed solution-mobile phase ratio (a: B: c=66:29:5);
FIG. 23 is a chromatogram of durability-mixed solution-mobile phase pH 3.0;
FIG. 24 is a chromatogram of durability-mixed solution-mobile phase pH 3.2;
FIG. 25 is a chromatogram of a durability-mixed solution-potassium dihydrogen phosphate solution at a concentration of 0.008 mol/L;
FIG. 26 is a chromatogram of a durability-mixed solution-potassium dihydrogen phosphate solution at a concentration of 0.012mol/L;
FIG. 27 is an HPLC chart of mixed solution 1 in example 7;
FIG. 28 is an HPLC chart of mixed solution 2 in example 7;
FIG. 29 is an HPLC chart of mixed solution 3 in example 7.
Detailed Description
The technical scheme of the present invention will be further clearly and completely described in connection with specific embodiments. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. Therefore, all other embodiments obtained by those skilled in the art without undue burden are within the scope of the invention based on the embodiments of the present invention.
In the embodiment of the invention, the measurement is carried out according to high performance liquid chromatography (the rule 0512 of the fourth edition of Chinese pharmacopoeia 2020).
In the embodiment of the invention, the quantitative limit calculation method comprises the following steps:
in the embodiment of the invention, the detection limit calculation method comprises the following steps:
in the embodiment of the invention, crizotinib intermediate Z 2 Related substances of (a) are impurities SM 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b And impurity Z 1 。
In the embodiment of the invention, impurity Z 1a N-bromosuccinimide of formula C 4 H 4 BrNO 2 The molecular weight is 177.98, and the structural formula is shown in formula VIII; impurity Z 1b Is succinimide with a chemical formula of C 4 H 5 NO 2 The molecular weight is 99.09, and the structural formula is shown as formula IX; impurity Z 2a Has the chemical formula of C 16 H 28 NP, molecular weight 265.37, structural formula is shown in formula X; N-acetyl-L-cysteine has a chemical formula of C 5 H 9 NO 3 S, molecular weight is 163.19, structureFormula XI; pinacol has the formula C 6 H 14 O 2 The molecular weight is 118.18, and the structural formula is shown in formula XII.
Example 1
The invention establishes the separation detection of the crizotinib intermediate Z 2 The HPLC method of the related substances comprises the following specific steps:
1. preparing a solution to be tested
Test solution: the product is taken out in proper amount, precisely weighed, dissolved and diluted by acetonitrile to prepare a solution with the concentration of about 1 mg/ml.
Control solution: accurately measuring a proper amount of the sample solution, and adding acetonitrile to quantitatively dilute the sample solution into a solution with the concentration of about 1.5 mug/ml.
System applicability solution: taking Z 2 System applicability reference (intermediate Z) 2 SM impurity 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b Impurity Z 1 ) Proper amount of the mixture is precisely weighed, and acetonitrile is added for dissolution and dilution to prepare a solution with the concentration of about 1 mg/ml.
2. Chromatographic conditions
Adopting octadecylsilane chemically bonded silica as a filler, adopting 0.01mol/L potassium dihydrogen phosphate solution (pH value is regulated to 3.1 by phosphoric acid) as a mobile phase A, acetonitrile as a mobile phase B, and methanol as a mobile phase C, and performing linear gradient elution; the flow rate is 1.0ml/min; column temperature is 35 ℃; the detection wavelength is 220nm; the sample volume was 10. Mu.l. The chromatographic conditions are specifically shown in table 1.
TABLE 1 chromatographic conditions
3. Measurement
(1) System applicability requirements: in the system applicability solution chromatogram, according to the impurity SM 2e SM impurity 1 Impurity Z 2c Intermediate Z 2 SM impurity 2 Impurity Z 2b Impurity Z 1 And (5) sequentially outputting peaks, wherein the separation degree of each component meets the requirement.
(2) Limit: the chromatogram of the sample solution has impurity peaks, except blank solvent peaks, and impurity SM 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b Impurity Z 1 The peak areas calculated after correction (multiplied by correction factors 1.76, 1.10, 1.79, 1.87, 2.18, 1.22 respectively) are not greater than the main peak area of the control solution (0.15%), the peak areas of other individual impurities are not greater than the main peak area of the control solution (0.15%), and the sum of the peak areas of the impurities is not greater than 6.66 times (1.0%) of the main peak area of the control solution calculated according to the corrected peak areas. Any peak in the chromatogram of the test solution, which is smaller than the main peak area of the control solution by 0.33 times, is negligible (0.05%).
(3) The measuring method comprises the following steps: precisely measuring the sample solution and the control solution, respectively injecting into a liquid chromatograph, and recording the chromatograms.
EXAMPLE 2 specificity
(1) Preparing a solution to be tested
The specificity verification comprises a blank solvent interference experiment and separation of impurities possibly existing by a chromatographic system, wherein the blank solution is diluent acetonitrile. Meanwhile, the following solutions to be tested are prepared by adopting a diluent:
impurity N-acetyl-L-cysteine stock solution: accurately weighing 24.30mg of impurity N-acetyl-L-cysteine, placing into a 25ml measuring flask, adding diluent to dissolve, diluting to scale, and shaking to obtain the final product.
Impurity SM 2e Stock solution: accurate weighing of impurity SM 2e 10.48mg, placing into a 10ml measuring flask, adding diluent, dissolving, diluting to scale, and shaking.
Impurity SM 2e Positioning solution: accurate weighing of impurity SM 2e 1.5ml of stock solution is put into a 100ml measuring flask, diluted to scale by adding diluent, and uniformly shaken to obtain the product.
Impurity Z 1 Stock solution: precisely weigh impurity Z 1 25.03mg, put 25ml measuring flaskAdding diluent to dissolve and dilute to scale, and shaking to obtain the final product.
Impurity Z 1 Positioning solution: precise measuring impurity Z 1 1.5ml of stock solution is put into a 100ml measuring flask, diluted to scale by adding diluent, and uniformly shaken to obtain the product.
Impurity SM 1 Stock solution: accurate weighing of impurity SM 1 25.03mg, putting into a 25ml measuring flask, adding a diluent for dissolution, diluting to a scale, and shaking uniformly to obtain the product.
Impurity SM 1 Positioning solution: precision measuring impurity SM 1 1.5ml of stock solution is put into a 100ml measuring flask, diluted to scale by adding diluent, and uniformly shaken to obtain the product.
Impurity SM 2 Stock solution: accurate weighing of impurity SM 2 24.66mg, putting into a 25ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking uniformly to obtain the final product.
Impurity SM 2 Positioning solution: precision measuring impurity SM 2 1.5ml of stock solution is put into a 100ml measuring flask, diluted to scale by adding diluent, and uniformly shaken to obtain the product.
Impurity Z 2a Stock solution: precisely weigh impurity Z 2a 24.06mg, put into a 25ml measuring flask, add diluent to dissolve and dilute to scale, shake well, get the final product.
Impurity Z 2b Stock solution: precisely weigh impurity Z 2b 25.73mg, putting into a 25ml measuring flask, adding a diluent for dissolution, diluting to a scale, and shaking uniformly to obtain the product.
Impurity Z 2b Positioning solution: precise measuring impurity Z 2b 1.5ml of stock solution is put into a 100ml measuring flask, diluted to scale by adding diluent, and uniformly shaken to obtain the product.
Impurity Z 2c Stock solution: precisely weigh impurity Z 2c 24.70mg, putting into a 25ml measuring flask, adding a diluent for dissolution, diluting to a scale, and shaking uniformly to obtain the product.
Impurity Z 2c Positioning solution: precise measuring impurity Z 2c 1.5ml of stock solution is put into a 100ml measuring flask, diluted to scale by adding diluent, and uniformly shaken to obtain the product.
Impurity Z 1a Stock solution: precisely weigh impurity Z 1a 25.42mg, putDissolving in diluent in 25ml measuring flask, diluting to scale, and shaking.
Impurity Z 1b Stock solution: precise measuring impurity Z 1b 25.64mg, putting into a 25ml measuring flask, adding a diluent for dissolution, diluting to a scale, and shaking uniformly to obtain the product.
Impurity pinacol stock solution: accurately weighing 24.98mg of pinacol impurity, placing into a 25ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking to obtain the final product.
Other impurity mixed solution positioning solution: precise measuring impurity Z 2a Impurity Z 1a Impurity Z 1b And (3) placing 1.5ml of each of pinacol and N-acetyl-L-cysteine stock solution into a same 100ml volumetric flask, adding a diluent to dissolve and dilute to a scale, and shaking uniformly to obtain the compound.
Z 2 Control stock solution: precisely weigh Z 2 Placing reference substance 25.18mg into 25ml measuring flask, adding diluent, dissolving, diluting to scale, and shaking.
Test solution: precisely weighing intermediate Z 2 25.69mg, putting into a 25ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking uniformly to obtain the final product.
Self-control solution: precisely transferring 1.5ml of sample solution, placing into a 100ml measuring flask, diluting to scale with diluent, shaking, precisely measuring 1.0ml, placing into a 10ml measuring flask, dissolving with diluent, diluting to scale, and shaking.
Mixing solution: precisely weighing intermediate Z 2 25.30mg, put into a 25ml measuring flask, and precisely measure 2.5ml of other impurity mixed solution positioning solution and impurity SM 2e 、Z 2b 、SM 1 、SM 2 、Z 1 、Z 2c 2.5ml of each positioning solution is placed in the bottle, the diluent is added for dissolution and dilution to the scale, and the positioning solution is uniformly shaken to obtain the product.
(2) Detection of
And (3) precisely measuring 10 mu l of each sample such as the blank solvent, each positioning solution, other impurity mixed positioning solution, mixed solution and the like in the step (1), performing HPLC detection according to chromatographic conditions shown in Table 1 in the example 1, and recording a chromatogram.
Results: as shown in table 2 and fig. 1-9, the integration results of fig. 2 are shown in table 3. The blank solution does not interfere with the measurement of the sample, the separation degree of the main peak and the adjacent impurities is more than 2.0, the separation degree of other known impurities is more than 1.5, and the unknown impurities in the sample solution do not interfere with the detection of 6 known impurities. The result shows that the main peak and the impurity peak are well separated, and the method has good specificity.
TABLE 2 determination results of specificity test
TABLE 3 integral results Table corresponding to FIG. 2
Example 3 quantitative limit
Linear stock: taking SM under "specialization" item 2e 、SM 1 、Z 2c 、SM 2 、Z 2b 、Z 1 、Z 2 And (3) placing 1.5ml of the reference stock solution into the same 100ml measuring flask, diluting to a scale with a diluent, and shaking uniformly to obtain the product.
Quantitative limiting solution: precisely transferring 1.0ml of linear stock solution, placing into a 50ml measuring flask, diluting to scale with diluent, and shaking.
Taking the quantitative limiting solution to continuously sample for 6 times, calculating the ratio (signal to noise ratio) of the peak height of the main peak to the noise, and recording a chromatogram. The detection results are shown in Table 4 and FIG. 10, impurity SM 2e The quantitative limit concentration of (2) is 0.2575 mug/ml, the concentration in the sample is 0.026%, the signal to noise ratio average value is 16.9, and the peak area RSD is 8.9%; impurity SM 1 The quantitative limit concentration of (2) is 0.2998 mug/ml, the concentration in the sample is 0.030%, the signal to noise ratio average value is 37.6, and the peak area RSD is 3.5%; impurity Z 2c The quantitative limit concentration of (2) is 0.2869 mug/ml, the concentration is 0.029% in the sample, the signal to noise ratio average value is 27.0, and the peak area RSD is 9.96%; impurity SM 2 The quantitative limit concentration of (2) was 0.2956. Mu.g/ml, and the concentration was 0 in the sample.030%, signal to noise ratio mean 23.5, peak area RSD 8.7%; impurity Z 2b The quantitative limit concentration of (2) is 0.2918 mug/ml, the concentration is 0.029% in the sample, the signal to noise ratio average value is 20.9, and the peak area RSD is 3.4%; impurity Z 1 The quantitative limit concentration of (2) is 0.2940 mug/ml, the concentration in the sample is 0.029%, the signal to noise ratio average value is 34.3, and the peak area RSD is 3.4%; z is Z 2 The quantitative limit concentration of (2) is 0.3003 mug/ml, the concentration of the compound is 0.030% in the sample, the signal to noise ratio average value is 45.0, and the peak area RSD is 9.2%; the signal-to-noise ratio of the quantitative concentration-limiting chromatographic peaks of each component is more than or equal to 10, the peak area RSD is less than 10%, and the requirements of the determination of related substances are met, so that the impurities can be accurately quantified at the level.
TABLE 4 quantitative limit test results Table
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Example 4 detection Limit
Detection limit solution: precisely measuring 5.0ml of quantitative limiting solution, placing into a 10ml measuring flask, diluting to scale with diluent, and shaking to obtain the final product.
And taking the detection limit solution to continuously sample for 3 times, and calculating the ratio (signal to noise ratio) of the peak height of the main peak to noise. The chromatograms were recorded and the test results are shown in table 5 and fig. 11. Impurity SM 2e The detection limit concentration of (2) is 0.1287 mug/ml, the concentration in the sample is 0.013%, and the signal to noise ratio average value is 6.3; impurity SM 1 The detection limit concentration of (2) is 0.1499 mug/ml, the concentration in the sample is 0.015%, and the signal to noise ratio average value is 13.7; impurity Z 2c The detection limit concentration of (2) is 0.1435 mug/ml, the concentration in the sample is 0.014%, and the signal-to-noise ratio average value is 9.8; impurity SM 2 The detection limit concentration of (2) is 0.1478 mug/ml, the concentration of the sample is 0.015%, and the signal-to-noise ratio average value is 9.2; impurity Z 2b The detection limit concentration of (2) was 0.1459. Mu.g/ml, and the concentration present in the sample was 0.015, the signal-to-noise ratio average value is 8.1; impurity Z 1 The detection limit concentration of (2) is 0.1470 mug/ml, the concentration in the sample is 0.015%, and the signal-to-noise ratio average value is 13.3; z is Z 2 The detection limit concentration of (2) was 0.1502. Mu.g/ml, the concentration of (3) was 0.015% in the sample, and the signal-to-noise ratio was 17.8. The signal to noise ratio of the detection limit concentration chromatographic peak of each component is more than 3, so that the requirements of the determination of related substances are met, and the impurities can be effectively detected at the level.
TABLE 5 detection limit measurement results Table
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Example 5 accuracy
Reference mother liquor: precision measurement of impurity SM in example 2 2e 、SM 1 、Z 2c 、SM 2 、Z 2b 、Z 1 1.5ml of each stock solution is placed in a 100ml measuring flask, diluted to the scale with a diluent and uniformly shaken to obtain the medicine.
Control solution: precisely measuring 2.5ml of reference mother liquor, placing into a 25ml measuring flask, diluting to scale with diluent, and shaking.
Unlabeled test solution: precisely weighing 25.43mg of the sample, placing into a 25ml measuring flask, diluting to scale with diluent, and shaking.
Labeling test sample solution 1#: precisely weighing 25mg of sample, placing into 25ml measuring flask, adding 2.0ml of reference mother liquor, diluting to scale with diluent, and shaking; the sample amounts were 24.89mg, 25.07mg and 25.79mg, respectively.
Labeling test sample solution 2#: precisely weighing 25mg of sample, placing into 25ml measuring flask, adding 2.5ml of reference mother liquor, diluting to scale with diluent, and shaking; the sample amounts were 25.34mg, 25.72mg and 25.49mg, respectively.
Adding a standard test sample solution 3#: precisely weighing 25mg of sample, placing into 25ml measuring flask, adding 3.0ml of reference mother liquor, diluting to scale with diluent, and shaking; the sample amounts were 25.23mg, 25.13mg and 25.41mg, respectively.
Self-control solution: precisely measuring 1.5ml of each marked sample solution and each unmarked sample solution, respectively placing into a 100ml measuring flask, diluting to scale with a diluent, and shaking; and precisely measuring 1.0ml of the solution, respectively placing the solution into 10ml measuring bottles, diluting the solution to a scale with a diluent, and shaking the solution uniformly to obtain the corresponding self-control solution.
And respectively precisely measuring 10 mu l of the blank solvent, the reference substance solution, the untagged sample solution, the self-reference solution and the tagged sample solution, injecting into a liquid chromatograph, and recording a chromatogram. The impurity content and recovery rate were calculated by external standard method and correction factor-added self-comparison method, and the test results are shown in Table 6 and FIGS. 1, 9 and 12-16. 9 parts of standard adding solution (3 concentrations) are measured by an external standard method and a self-comparison method for adding correction factors, and the recovery rate of each impurity is between 90% and 108%, so that the method meets the requirements.
TABLE 6 accuracy test results Table
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EXAMPLE 6 durability
The mixed solution in example 2 was tested under normal chromatography conditions and under the following conditions (initial ratio of mobile phase.+ -. 1, flow rate.+ -. 0.1ml/min, column temperature.+ -. 2 ℃ C. And potassium dihydrogen phosphate solution concentration.+ -. 0.02%) and the degree of separation between peaks was recorded. The results are shown in Table 7 and FIGS. 17 to 26, and SM when the flow rate was 0.9ml/min 2 And Z is 2b The degree of separation was 1.40; under other conditions, the separation degree of all known impurities is greater than 1.5, the separation degree of the main component and the adjacent impurities is greater than 1.5, the impurities can be separated, the detection requirement is met, and the method has good durability.
TABLE 7 chromatographic conditions durability test results Table (separation degree)
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Example 7 establishment of the detection method
In the early research, the invention explores the influence of the two-phase and three-phase flow on the impurity separation condition, discovers that the three-phase mobile phase separation effect is better, and carries out optimization experiments on the mobile phase and gradient elution conditions on the basis. The comparative experimental conditions for specific chromatographic condition selection are as follows:
(1) The screening conditions for the two-phase and three-phase mobile phases are shown in table 8.
TABLE 8
Results: in the double phase, only the impurity SM is added 1 SM impurity 2 Impurity Z 1 And impurity Z 2 As a result, Z is as shown in FIG. 27 1 And Z 2 And completely overlap. The three-phase results are shown in FIG. 28, Z 1 And Z 2 Good separation, indicating that the methanol is opposite to Z 1 And Z 2 More selective and thus optimize the process on a three-phase basis.
(2) A gradient elution method was established using a three-phase mobile phase, as shown in table 9.
TABLE 9
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Results: as shown in FIG. 29 and Table 10, 6 known impurities can be substantially separated, but SM 1 The separation degree of the peak and the adjacent unknown impurity peak is low; subsequent studies have found that this method is poorly durable.
Table 10. Integral result table corresponding to fig. 29
(3) The three-phase mobile phase is adopted for gradient elution, and the detection method is further optimized, and the scheme is shown in table 11.
TABLE 11
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Results: as shown in FIG. 2, the separation degree between 6 known impurity peaks and adjacent impurity peaks is larger than 1.5, and the separation degree between the main peak and the adjacent impurity peaks meets the requirement, so that the method has feasibility.
Claims (10)
1. Separation of crizotinib intermediate Z based on high performance liquid chromatography 2 And related substances, characterized in that the crizotinib intermediate Z 2 The structural formula of the catalyst is shown as formula I, and the related substance is impurity SM 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b And impurity Z 1 Any one or more of the following; the impurity SM 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurities (e.g. impurities)Z 2b And impurity Z 1 The structural formula of (a) is shown as a formula II-a formula VII in sequence; the chromatographic column of the high performance liquid chromatography adopts octadecylsilane chemically bonded silica as a filler, and takes a mobile phase A, a mobile phase B and a mobile phase C as mobile phases, wherein the mobile phase A is a potassium dihydrogen phosphate solution, the mobile phase B is acetonitrile, and the mobile phase C is methanol; sequentially eluting the impurities SM by gradient 2e SM impurity 1 Impurity Z 2c Crizotinib intermediate Z 2 SM impurity 2 Impurity Z 2b And impurity Z 1 Separating;
2. the method of claim 1, wherein the gradient elution procedure is:
at 0min, the volume ratio of the mobile phase A to the mobile phase B to the mobile phase C is 63-67:29-31:5, a step of;
at 5 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5, a step of;
at 45 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 35:60:5, a step of;
at 50 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 35:60:5, a step of;
at 50.1 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5, a step of;
at 60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5.
3. the method according to claim 1, wherein the concentration of the potassium dihydrogen phosphate solution in the mobile phase a is 0.008mol/L to 0.012mol/L; and regulating the pH value of the mobile phase A to 3.0-3.2 by adopting phosphoric acid.
4. The method of claim 1, wherein the mobile phase has a flow rate of 0.9ml/min to 1.1ml/min; the column temperature of the chromatographic column is 32-37 ℃.
5. Qualitative authentication Bie Ke Tinib intermediate Z 2 A process for the preparation of crizotinib intermediate Z by the process according to any one of claims 1 to 4 2 SM impurity 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b And impurity Z 1 Separating; and then detected by a detector having a wavelength of 220 nm.
6. The method of claim 5, wherein the retention time of the seven components is, in order from short to long: impurity SM 2e SM impurity 1 Impurity Z 2c Crizotinib intermediate Z 2 SM impurity 2 Impurity Z 2b And impurity Z 1 。
7. The method according to claim 5, wherein mobile phase A is a potassium dihydrogen phosphate solution with a concentration of 0.01mol/L, mobile phase B is acetonitrile, and mobile phase C is methanol; the pH value of the mobile phase A is 3.1; the specification of the chromatographic column is 4.6mm multiplied by 250mm multiplied by 5 mu m; the flow rate of the mobile phase is 1.0ml/min; the column temperature of the chromatographic column is 35 ℃; the detection wavelength is 220nm;
performing linear gradient elution according to a gradient elution program to obtain a chromatogram; the gradient elution procedure was as follows:
at 0 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5, a step of;
at 5 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5, a step of;
at 45 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 35:60:5, a step of;
at 50 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 35:60:5, a step of;
at 50.1 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5, a step of;
at 60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is 65:30:5.
8. the method according to claim 6, wherein the impurity SM is determined when the retention time is 9.5.+ -. 0.5min 2e The method comprises the steps of carrying out a first treatment on the surface of the When the retention time was 11.9.+ -. 0.5min, it was determined as impurity SM 1 The method comprises the steps of carrying out a first treatment on the surface of the When the retention time was 21.8.+ -. 0.5min, it was determined as impurity Z 2c The method comprises the steps of carrying out a first treatment on the surface of the When the retention time is 35.5+/-0.5 min, the crizotinib intermediate Z is judged 2 The method comprises the steps of carrying out a first treatment on the surface of the When the retention time was 40.2.+ -. 0.5min, it was determined as impurity SM 2 The method comprises the steps of carrying out a first treatment on the surface of the When the retention time was 41.3.+ -. 0.5min, it was determined as impurity Z 2b The method comprises the steps of carrying out a first treatment on the surface of the When the retention time was 44.5.+ -. 0.5min, it was determined as impurity Z 1 。
9. Quantitative determination of crizotinib intermediate Z 2 A method for producing a substance of interest, characterized in that the substance of interest is an impurity SM 2e SM impurity 1 Impurity Z 2c SM impurity 2 Impurity Z 2b And impurity Z 1 Any one or more of the following; the method comprises the following steps:
(1) Separating: crizotinib intermediate Z by the method of any one of claims 1-4 2 And related substances thereof are separated;
(2) And (3) detection: crizotinib intermediate Z by the method of any one of claims 5-8 2 And related substances thereof are detected;
(3) And (3) content calculation: calculating the crizotinib intermediate Z according to the chromatogram measured in the step (2) and a main component self-comparison method with a correction factor and/or an external standard method 2 And the content of impurities.
10. The method of claim 9, wherein prior to separation, the test solution and the control solution are prepared using acetonitrile as a diluent.
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