CN116482267A - Detection method for 9 related impurities in medicaments for dilating cerebral vessels and protecting nerves - Google Patents
Detection method for 9 related impurities in medicaments for dilating cerebral vessels and protecting nerves Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 79
- 239000003814 drug Substances 0.000 title claims abstract description 70
- 238000001514 detection method Methods 0.000 title claims abstract description 43
- 230000002490 cerebral effect Effects 0.000 title claims abstract description 17
- 210000005036 nerve Anatomy 0.000 title claims abstract description 17
- 230000000916 dilatatory effect Effects 0.000 title claims abstract description 12
- 239000000243 solution Substances 0.000 claims abstract description 101
- 239000002904 solvent Substances 0.000 claims abstract description 53
- 238000000034 method Methods 0.000 claims abstract description 30
- 238000007865 diluting Methods 0.000 claims abstract description 24
- 229940079593 drug Drugs 0.000 claims abstract description 18
- 239000013558 reference substance Substances 0.000 claims abstract description 15
- 230000035945 sensitivity Effects 0.000 claims abstract description 13
- 238000005303 weighing Methods 0.000 claims abstract description 12
- 238000002347 injection Methods 0.000 claims abstract description 10
- 239000007924 injection Substances 0.000 claims abstract description 10
- 238000004090 dissolution Methods 0.000 claims abstract description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 6
- 239000012085 test solution Substances 0.000 claims abstract description 6
- 239000012895 dilution Substances 0.000 claims abstract 3
- 238000010790 dilution Methods 0.000 claims abstract 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 86
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 claims description 16
- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 claims description 16
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 claims description 16
- 235000002906 tartaric acid Nutrition 0.000 claims description 16
- 239000011975 tartaric acid Substances 0.000 claims description 16
- 239000000523 sample Substances 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 8
- 239000012488 sample solution Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 6
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 4
- 239000005695 Ammonium acetate Substances 0.000 claims description 4
- 229940043376 ammonium acetate Drugs 0.000 claims description 4
- 235000019257 ammonium acetate Nutrition 0.000 claims description 4
- 239000012088 reference solution Substances 0.000 claims description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 2
- 239000000463 material Substances 0.000 abstract description 6
- 239000000126 substance Substances 0.000 abstract description 6
- 239000002994 raw material Substances 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- RXPRRQLKFXBCSJ-GIVPXCGWSA-N vincamine Chemical compound C1=CC=C2C(CCN3CCC4)=C5[C@@H]3[C@]4(CC)C[C@](O)(C(=O)OC)N5C2=C1 RXPRRQLKFXBCSJ-GIVPXCGWSA-N 0.000 description 39
- DDNCQMVWWZOMLN-IRLDBZIGSA-N Vinpocetine Chemical compound C1=CC=C2C(CCN3CCC4)=C5[C@@H]3[C@]4(CC)C=C(C(=O)OCC)N5C2=C1 DDNCQMVWWZOMLN-IRLDBZIGSA-N 0.000 description 32
- 229960000744 vinpocetine Drugs 0.000 description 32
- RXPRRQLKFXBCSJ-UHFFFAOYSA-N dl-Vincamin Natural products C1=CC=C2C(CCN3CCC4)=C5C3C4(CC)CC(O)(C(=O)OC)N5C2=C1 RXPRRQLKFXBCSJ-UHFFFAOYSA-N 0.000 description 24
- 229960002726 vincamine Drugs 0.000 description 24
- 239000011550 stock solution Substances 0.000 description 19
- -1 vincamine oxime Chemical class 0.000 description 13
- 239000002253 acid Substances 0.000 description 8
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 229960003048 vinblastine Drugs 0.000 description 6
- 229950006936 apovincamine Drugs 0.000 description 5
- OZDNDGXASTWERN-UHFFFAOYSA-N apovincamine Natural products C1=CC=C2C(CCN3CCC4)=C5C3C4(CC)C=C(C(=O)OC)N5C2=C1 OZDNDGXASTWERN-UHFFFAOYSA-N 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- QGQXAMBOYWULFX-LZWSPWQCSA-N 2-morpholin-4-ylethyl (e)-6-(4,6-dihydroxy-7-methyl-3-oxo-1h-2-benzofuran-5-yl)-4-methylhex-4-enoate Chemical compound OC=1C=2C(=O)OCC=2C(C)=C(O)C=1C\C=C(/C)CCC(=O)OCCN1CCOCC1 QGQXAMBOYWULFX-LZWSPWQCSA-N 0.000 description 4
- OZDNDGXASTWERN-CTNGQTDRSA-N Apovincamine Chemical compound C1=CC=C2C(CCN3CCC4)=C5[C@@H]3[C@]4(CC)C=C(C(=O)OC)N5C2=C1 OZDNDGXASTWERN-CTNGQTDRSA-N 0.000 description 4
- UJAQRLRRCXIJFW-DWLFOUALSA-N dihydrovinpocetine Chemical compound C1=CC=C2C(CCN3CCC4)=C5[C@@H]3[C@]4(CC)C[C@@H](C(=O)OCC)N5C2=C1 UJAQRLRRCXIJFW-DWLFOUALSA-N 0.000 description 4
- ZHFOVMIMMIBXLC-GGAORHGYSA-N ethyl (15S,19S)-15-ethyl-5-methoxy-1,11-diazapentacyclo[9.6.2.02,7.08,18.015,19]nonadeca-2(7),3,5,8(18),16-pentaene-17-carboxylate Chemical compound C1=C(OC)C=C2C(CCN3CCC4)=C5[C@@H]3[C@]4(CC)C=C(C(=O)OCC)N5C2=C1 ZHFOVMIMMIBXLC-GGAORHGYSA-N 0.000 description 4
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 3
- XRMLLVHAALNSGZ-HJNYFJLDSA-N ethylvincaminate Chemical compound C1=CC=C2C(CCN3CCC4)=C5[C@@H]3[C@]4(CC)C[C@@](C(=O)OCC)(O)N5C2=C1 XRMLLVHAALNSGZ-HJNYFJLDSA-N 0.000 description 3
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 206010065559 Cerebral arteriosclerosis Diseases 0.000 description 1
- 206010008111 Cerebral haemorrhage Diseases 0.000 description 1
- 241000207834 Oleaceae Species 0.000 description 1
- 241000863480 Vinca Species 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 206010008118 cerebral infarction Diseases 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000010339 dilation Effects 0.000 description 1
- 229940088679 drug related substance Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 201000005851 intracranial arteriosclerosis Diseases 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of medicine detection, and particularly relates to a detection method for 9 related impurities in a drug for dilating cerebral vessels and protecting nerves. The method comprises the following steps: (1) preparing a system applicability solution: taking impurities A, B, C and D in the medicine and a medicine reference substance or raw materials, precisely weighing, adding a solvent for dissolution and dilution; (2) preparing a test solution: taking the medicinal raw materials, precisely weighing, adding a solvent for dissolution and dilution; (3) preparing a control solution: taking a proper amount of a medicine reference substance, precisely weighing, dissolving with a solvent and diluting; (4) preparing a sensitivity solution: diluting the control solution by 50 times; (5) detection by HPLC method. The invention has the beneficial effects that: under the chromatographic system, 9 impurities in the medicine can be well separated, the risk of missed detection is avoided, the detection accuracy is improved, and the interference of auxiliary materials in injection is avoided, so that related substances in the medicine are more effectively controlled.
Description
Technical Field
The invention belongs to the technical field of medicine detection, and particularly relates to a detection method for simultaneously detecting 9 relevant impurities in a cerebral vessel dilation and nerve protection medicine.
Background
The impurity of the medicine is a substance which has no therapeutic effect on the medicine or is harmful to human body or affects the quality of the medicine, and the impurity detection is an important index for controlling the quality of the medicine. In the aspects of preparation, production, storage or transportation of medicines, clinical application and the like, the quality of the medicines must be ensured, and the effectiveness and the safety of the medicines can be ensured.
The detection method of 9 related impurities in the medicaments for dilating cerebral vessels and protecting nerves mainly refers to a detection method of the medicament vinpocetine, wherein the medicament is a derivative of alkaloid extracted from vinca of oleaceae, and the effective component of the medicament is apovincamine ethyl ester, and has the effect of improving various symptoms induced by cerebral infarction sequela, cerebral hemorrhage sequela, cerebral arteriosclerosis and the like.
The impurities in the vinpocetine raw material mainly originate from reaction raw materials, synthesis intermediates, reaction byproducts, degradation impurities and the like introduced in the synthesis process. The main impurities are as follows: 9 kinds of vincamine acid ethyl ester (impurity A), apovincamine (impurity B), methoxy vinpocetine (impurity C), dihydro vinpocetine (impurity D), vinpocetine, vincamine oxime, vincamine ketone and the like are adopted, and in order to ensure the safety and effectiveness of clinical use of the product, all impurities need to be strictly controlled. At present, impurity control methods carried in domestic and foreign pharmacopoeias such as ChP, EP, USP are used for controlling ethyl vincamine (impurity A), apovincamine (impurity B), methoxy vinpocetine (impurity C) and dihydro vinpocetine (impurity D), and other impurities are not controlled.
The literature "HPLC determination of the content of 4 related substances in vinpocetine injection" (chinese pharmacist, 2014) describes the detection of the content of 4 related substances in vinpocetine by HPLC, in which the mobile phase employed is disclosed as 0.2mol/L ammonium acetate: acetonitrile, volume ratio 40:60 the chromatographic column is Kromasil C 18 150 mm. Times.4.6 mm,5 μm; the flow rate is 1.0mL/min; detection wavelength: 280nm; sample injectionThe amount is as follows: 20. Mu.L.
The drawbacks of the above method are: vinpocetine and vincamine acid have poor retention in the chromatographic system, and have high risk of omission; and the auxiliary material peak in the vinpocetine injection interferes with the inspection of two impurities, so that the detection accuracy still needs to be improved.
Therefore, it is necessary to invent a method capable of effectively improving the defects of the detection method, not only accurately detecting the impurity content in the vinpocetine, but also avoiding the interference of auxiliary materials in the injection so as to improve the accuracy of the detection result.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method capable of quantitatively removing the interference of other impurities and accurately obtaining the content of 9 impurities in the medicaments (vinpocetine, medicament for short) for expanding cerebral vessels and protecting nerves, so that the detection accuracy is greatly improved.
The method has the greatest characteristics that the effective separation of 9 impurities in the medicine is realized, and the error caused by the interference of auxiliary materials is avoided.
The detection method of 9 relevant impurities in the medicaments for dilating cerebral vessels and protecting nerves comprises the following steps:
(1) Preparing a System applicability solution
Adding solvent into impurities A, B, C and D and medicinal reference substance, dissolving and diluting to obtain solution containing medicine, impurity A, impurity B, impurity C and impurity D, and shaking; the medicine is a medicine for expanding cerebral vessels and protecting nerves;
(2) Preparing test solution
Taking the medicine, precisely weighing, adding a solvent for dissolving and diluting to prepare a solution containing the medicine, and shaking uniformly to obtain a sample solution;
(3) Preparing reference substance solution
Precisely measuring the solution of the sample, diluting with solvent to obtain solution containing the drug, and shaking to obtain reference solution;
(4) Preparing a sensitivity solution
Precisely measuring control solution, diluting with solvent to obtain solution containing the drug, and shaking to obtain sensitivity solution;
(5) Detection by HPLC
The conditions for detection were as follows:
chromatographic column: a column of island liquid VP-ODS 250X 4.6mm,5 μm or equivalent performance;
mobile phase a:15.4g/L ammonium acetate solution-acetonitrile, volume ratio 75:25;
mobile phase B: acetonitrile;
solvent: tartaric acid solution-acetonitrile, volume ratio of 75:25;
column temperature: 40 ℃;
detection wavelength: 280nm;
flow rate: 1.0mL/min;
sample injection amount: 20. Mu.L.
Preferably, in the step (1), when preparing the system applicability solution, taking vinpocetine impurity A, impurity B, impurity C, impurity D and a drug reference substance, adding a solvent to dissolve and dilute the mixture to prepare a solution containing 1 mug of the drug, 6 mug of the impurity A, 5 mug of the impurity B, the impurity C and the impurity D, and shaking the solution uniformly to obtain the system applicability solution.
Preferably, in the step (2) of preparing the solution of the test sample, 25mg of the drug is taken, precisely weighed, dissolved and diluted by a solvent to prepare 1mg of solution containing the drug per 1mL, wherein the solvent is a mixed solution of 0.1% tartaric acid solution and acetonitrile, the volume ratio of the 0.1% tartaric acid solution to the acetonitrile is 75:25, and the solution is uniformly shaken to obtain the solution of the test sample.
Preferably, in (3), when preparing the control solution, taking a drug control, precisely weighing, adding a solvent to dissolve and dilute the drug control to prepare a solution containing 10 mug of the drug per 1mL, wherein the solvent is a mixed solution of 0.1% tartaric acid solution and acetonitrile, and the volume ratio of the 0.1% tartaric acid solution to the acetonitrile is 75:25, shaking uniformly to obtain the reference substance solution.
Preferably, in (4), when preparing the sensitivity solution, a proper amount of the control solution is precisely measured, and the control solution is dissolved and diluted with a solvent to prepare a solution containing 0.2 μg of vinpocetine per 1mL, wherein the solvent is a mixed solution of 0.1% tartaric acid solution and acetonitrile, and the volume ratio of 0.1% tartaric acid solution to acetonitrile is 75:25, shaking uniformly to obtain a sensitivity solution.
Preferably, in (5), the solvent is 0.1% tartaric acid solution-acetonitrile, and the volume ratio is 75:25, the column temperature is 40 ℃, and the mobile phase A is acetate solution-acetonitrile; mobile phase B was acetonitrile.
The invention has the beneficial effects that:
(1) The invention adopts a gradient elution method, realizes the effective separation of 9 impurities in the expanded cerebral blood vessel and the protective nerve drugs (mainly comprising vinpocetine), prepares a system applicability solution, is convenient for completing the separation during the detection, and improves the detection accuracy;
(2) The invention increases the control of column temperature, avoids errors caused by the interference of auxiliary materials in the injection, explores a method suitable for solving the technical problem of the invention, and improves the detection accuracy.
Drawings
FIG. 1 is a graph of a drug substance detected by the method of the present invention;
FIG. 2 is a graph of the method of the present invention when detecting an injection;
FIG. 3 is a peak sequence chart of 9 related substances;
FIG. 4 shows a test sample examination chart of the fine tuning chromatographic conditions.
Detailed Description
The present invention will now be further described in connection with specific embodiments in order to enable those skilled in the art to better understand the invention.
Example 1
The method for detecting the content of 9 impurities in the medicaments for dilating cerebral vessels and protecting nerves comprises the following steps:
(1) Preparing a System applicability solution
Taking a proper amount of each of vinpocetine impurity A, impurity B, impurity C, impurity D and a vinpocetine reference substance, adding a solvent for dissolving and diluting to prepare a solution containing 1 mug of vinpocetine, 6 mug of impurity A, 5 mug of impurity B, impurity C and impurity D in each 1mL, and shaking uniformly to obtain a system applicability solution;
(2) Preparing test solution
Taking 25mg of vinpocetine raw material, precisely weighing, dissolving in a solvent, diluting to prepare a solution containing 1mg of vinpocetine per 1mL, and shaking uniformly to obtain a sample solution;
(3) Preparing a control solution
Taking a vinpocetine reference substance, precisely weighing, dissolving in a solvent, diluting to obtain a solution containing 10 mug of vinpocetine per 1mL, and shaking uniformly to obtain a reference solution;
(4) Preparing a sensitivity solution
Precisely measuring a proper amount of control solution, diluting with a solvent to prepare a solution containing 0.2 mug of vinpocetine per 1mL, and shaking uniformly to obtain a sensitivity solution;
(5) Detection by HPLC
The conditions for detection were as follows:
chromatographic column: a column of island liquid VP-ODS 250X 4.6mm,5 μm or equivalent performance;
mobile phase a:15.4g/L ammonium acetate solution-acetonitrile, the volume ratio is 75:25;
mobile phase B: acetonitrile;
column temperature: 40 ℃;
detection wavelength: 280nm;
flow rate: 1.0mL/min;
sample injection amount: 20. Mu.L;
solvent: 0.1% tartaric acid solution-acetonitrile at a volume ratio of 75:25.
Example 2
The obtained patterns were examined by the method of example 1 and are shown in FIGS. 1 to 2.
As can be seen from fig. 1: 9 impurities of the vinpocetine are well separated by the method, so that the risk of missed detection is completely avoided, and the detection accuracy is improved;
as can be seen from fig. 2: the detection is carried out by the method, so that the interference of auxiliary materials in the injection can be avoided, and related substances in the vinpocetine can be controlled more effectively.
Example 3 quantitative limit and detection limit
(1) Solution preparation
Quantitative limiting solution: taking appropriate amounts of ethyl vincamine (impurity A), apovincamine (impurity B), methoxyvinpocetine (impurity C), dihydrovinpocetine (impurity D), vinpocetine, vincamine, vinblastine oxime, and vinblastine, diluting with solvent to obtain a solution containing impurity A0.1 μg, impurity B0.3 μg, and impurity C in 1mL
0.3 mug, 0.2 mug of impurity D, 0.1 mug of vinpocetine, 0.05 mug of vincamine acid, 0.05 mug of vincamine oxime, 0.05 mug of vincamine ketone;
detection limit solution: precisely measuring 5mL of quantitative limiting solution, placing in a 10mL volumetric flask, diluting to scale with solvent, and shaking.
(2) The operation is as follows: and precisely measuring 20 mu L of quantitative limit solution and detection limit solution, injecting into a liquid chromatograph, and recording a chromatogram.
(3) Results and conclusions
Quantitative limit and detection limit conditions of the respective components of Table 1
From the above table, it can be concluded that: the quantitative limit and the detection limit of each related impurity detection method can meet the detection requirement.
Example 4 accuracy detection
(1) Solution preparation
Impurity stock solution: taking a proper amount of each of ethyl vincamine (impurity A), apovincamine (impurity B), methoxyvinpocetine (impurity C), dihydrovinpocetine (impurity D), vinpocetine, vincamine, vinblastine oxime and vinblastine, and diluting with a solvent to prepare a solution containing about 0.6mg of impurity A, 0.2mg of impurity B, 0.2mg of impurity C, 0.25mg of impurity D, 0.25mg of vinpocetine, 0.25mg of vinblastine oxime and 0.25mg of vinblastine in 1 mL.
Stock solution (1): precisely measuring 5mL of each of impurity A, impurity B, impurity C, impurity D and vinpocetine stock solution, placing in a 100mL volumetric flask, diluting to scale with solvent, and shaking.
Stock solution (2): precisely measuring 5mL of each stock solution of vincamine acid, vincamine ketone and vincamine oxime, placing in a 100mL volumetric flask, diluting to scale with solvent, and shaking.
Impurity control solution: the stock solution (1. Mu.m.L and stock solution (2. Mu.m.L) were precisely measured, placed in a 25mL volumetric flask, diluted to scale with solvent, and shaken well.
Blank test solution: about 25mg of the medicine is taken, precisely weighed, placed in a 25mL volumetric flask, added with 4mL of acetonitrile, ultrasonically dissolved, diluted to a scale with a solvent, and shaken well.
Recovery rate solution (1): about 25mg of the medicine is taken, precisely weighed, placed in a 25mL volumetric flask, added with 4mL of acetonitrile for ultrasonic dissolution, precisely added with stock solution (1) (2.5 mL of stock solution (2) (2 mL), diluted to scale by solvent and shaken well).
Recovery solution (2): about 25mg of the medicine is taken, precisely weighed, placed in a 25mL volumetric flask, added with 4mL of acetonitrile for ultrasonic dissolution, precisely added with stock solution (1 5mL, 25mL, diluted to scale with solvent and shaken well).
Recovery rate solution (3): about 25mg of the medicine is taken, precisely weighed, placed in a 25mL volumetric flask, added with 4mL of acetonitrile for ultrasonic dissolution, precisely added with stock solution (1.5 mL of 7.5mL of stock solution (2) and 10mL of stock solution, diluted to scale by solvent and shaken well).
(2) Operation of
Precisely measuring 20 mu L of each solution, injecting into a liquid chromatograph, and recording a chromatogram.
(3) The results are shown in tables 2-3.
TABLE 2 recovery results of impurity A, impurity B, impurity C, impurity D, vinpocetine
TABLE 3 recovery results of vincamine, vincamine acid, vincristine oxime
As can be seen in the above table: the recovery rate of each relevant impurity is in the range of 90% -110%, and RSD is less than 5%, so that the method has good accuracy.
Example 5 durability
After the basic chromatographic conditions are kept unchanged and the flow rate (+ -0.1 mL/min), the column temperature (+ -5 ℃) and the initial proportion (+ -2%) of the mobile phase A are slightly changed, the influence on the content of 9 related impurities in the test sample and the related impurity separation degree result in the system applicability solution is examined.
Since the sample solution does not detect vinpocetine, vincamine acid, vincamine ketone and vincamine ketoxime, and the durability of the method is not easy to evaluate, the impurity reference solution is added into the sample as a standard sample solution, and the RSD of the content of 9 relevant impurities is calculated and measured, and the durability of the method is evaluated as follows:
(1) Solution preparation
System applicability solution: taking appropriate amounts of vinpocetine impurity A, impurity B, impurity C, impurity D and vinpocetine reference substance, adding solvent, dissolving, and diluting to obtain 1mL each
The solution containing vinpocetine 1 μg, impurity A6 μg, impurity B, impurity C and impurity D5 μg respectively is shaken uniformly.
Vinpocetine stock solution: 2mg of impurity vinpocetine acid is weighed, placed in a 20mL volumetric flask, dissolved by adding 2mL of acetonitrile, diluted to a scale by a solvent, and shaken well.
Impurity mixing stock solution: taking about 2.5mg of each of the impurities of vincamine, vincamine acid, vincamine ketone and vincamine oxime as reference, precisely weighing, placing into a same 25mL volumetric flask, adding 10mL of acetonitrile for 5min with ultrasound, adding 10mL of solvent for dissolving with ultrasound, diluting to scale with solvent, and shaking uniformly.
Impurity stock solution: 1mL of vinpocetine stock solution and 1mL of impurity mixed stock solution are precisely measured, placed in a 20mL volumetric flask, diluted to scale by a solvent, and shaken uniformly.
Control solution: taking 5mg of a reference substance of the medicine, precisely weighing, placing into a 25mL volumetric flask, adding solvent, carrying out ultrasonic treatment to dissolve and dilute to a scale, shaking uniformly, precisely weighing 5mL, placing into a 100mL volumetric flask, dissolving and diluting to the scale with the solvent, and shaking uniformly.
Sensitivity solution: precisely measuring 1mL of reference substance solution, placing in a 50mL volumetric flask, dissolving with a solvent, diluting to a scale, and shaking uniformly.
Test solution: taking 25mg of medicine, precisely weighing, placing into a 25mL measuring flask, adding 4mL of acetonitrile, performing ultrasonic treatment to dissolve, precisely adding 5mL of impurity stock solution, dissolving with a solvent, diluting to a scale, and shaking uniformly to obtain the medicine.
(2) The operation is as follows: precisely measuring 20 mu L of each solution, injecting into a liquid chromatograph, and recording a chromatogram.
(3) The test sample examination pattern after fine tuning the chromatographic conditions is shown in fig. 4.
(4) The results are shown in tables 4-5.
Table 4 durability inspection system applicability results
TABLE 5 durability test related impurity content results
Conclusions can be drawn in the above table: fine-tuning column temperature, flow rate and mobile phase A proportion, wherein the separation degree of impurities D and B in a system applicability solution is more than 2.0, and the S/N of a sensitivity solution is more than 10, which indicates that the system applicability is good;
RSD of the related impurity content in the test sample solution is less than 10%, which shows that the method has good durability.
Claims (6)
1. The detection method of 9 relevant impurities in the medicaments for dilating cerebral vessels and protecting nerves comprises the following steps:
(1) Preparing a System applicability solution
Adding solvent into impurities A, B, C and D and medicinal reference substance, dissolving and diluting to obtain solution containing medicine, impurity A, impurity B, impurity C and impurity D, and shaking; the medicine is a medicine for expanding cerebral vessels and protecting nerves;
(2) Preparing test solution
Taking the medicine, precisely weighing, adding a solvent for dissolving and diluting to prepare a solution containing the medicine, and shaking uniformly to obtain a sample solution;
(3) Preparing reference substance solution
Precisely measuring the solution of the sample, diluting with solvent to obtain solution containing the drug, and shaking to obtain reference solution;
(4) Preparing a sensitivity solution
Precisely measuring control solution, diluting with solvent to obtain solution containing the drug, and shaking to obtain sensitivity solution;
(5) Detection by HPLC
The conditions for detection were as follows:
chromatographic column: a column of island liquid VP-ODS 250X 4.6mm,5 μm or equivalent performance;
solvent: tartaric acid solution-acetonitrile;
mobile phase a:15.4g/L acetate solution: acetonitrile with the volume ratio of 75:25;
mobile phase B: acetonitrile;
column temperature: 40 ℃;
detection wavelength: 280nm;
flow rate: 1.0mL/min;
sample injection amount: 20. mu L.
2. The method for detecting 9 related impurities in the medicaments for dilating cerebral vessels and protecting nerves according to claim 1, wherein the method comprises the following steps of: (1) In the preparation of the system applicability solution, medicine impurities A, B, C and D and a medicine reference substance are taken, added with a solvent for dissolution and dilution to prepare a solution containing 1 mug of the medicine, 6 mug of the impurity A, 5 mug of the impurity B, the impurity C and the impurity D, and the solution is uniformly shaken to obtain the system applicability solution.
3. The method for detecting 9 related impurities in the medicaments for dilating cerebral vessels and protecting nerves according to claim 1, wherein the method comprises the following steps of: (2) In the step of preparing the sample solution, the medicine 25mg is taken, precisely weighed, dissolved and diluted by a solvent to prepare a solution containing 1mg of the medicine every 1mL, wherein the solvent is a mixed solution of 0.1% tartaric acid solution and acetonitrile, the volume ratio of the 0.1% tartaric acid solution to the acetonitrile is 75:25, and the mixed solution is uniformly shaken to obtain the sample solution.
4. The method for detecting 9 related impurities in the medicaments for dilating cerebral vessels and protecting nerves according to claim 1, wherein the method comprises the following steps of: (3) In the preparation of the control solution, a drug control is taken, precisely weighed, dissolved and diluted by a solvent to prepare a solution containing 10 mug of the drug per 1mL, wherein the solvent is prepared by the following steps of: 25, shaking up the mixed solution of 0.1% tartaric acid solution and acetonitrile to obtain a reference substance solution.
5. The method for detecting 9 related impurities in the medicaments for dilating cerebral vessels and protecting nerves according to claim 1, wherein the method comprises the following steps of: (4) In the preparation of the sensitivity solution, a proper amount of control solution is precisely measured, and the control solution is dissolved and diluted by a solvent to prepare a solution containing 0.2 mug of medicine per 1mL, wherein the solvent is prepared by the following steps of: 25, shaking up the mixed solution of 0.1% tartaric acid solution and acetonitrile to obtain a sensitivity solution.
6. The method for detecting 9 related impurities in the medicaments for dilating cerebral vessels and protecting nerves according to claim 1, wherein the method comprises the following steps of: (5) Wherein the solvent is a mixed solution of 0.1% tartaric acid solution and acetonitrile, and the volume ratio of the 0.1% tartaric acid solution to the acetonitrile is 75:25, the column temperature is 40 ℃, and the mobile phase A is ammonium acetate solution-acetonitrile; mobile phase B was acetonitrile.
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