CN117269357A - Detection method for determining impurity C in Argatroban - Google Patents
Detection method for determining impurity C in Argatroban Download PDFInfo
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- CN117269357A CN117269357A CN202311227122.XA CN202311227122A CN117269357A CN 117269357 A CN117269357 A CN 117269357A CN 202311227122 A CN202311227122 A CN 202311227122A CN 117269357 A CN117269357 A CN 117269357A
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- 239000012535 impurity Substances 0.000 title claims abstract description 137
- 229960003856 argatroban Drugs 0.000 title claims abstract description 121
- KXNPVXPOPUZYGB-XYVMCAHJSA-N argatroban Chemical compound OC(=O)[C@H]1C[C@H](C)CCN1C(=O)[C@H](CCCN=C(N)N)NS(=O)(=O)C1=CC=CC2=C1NC[C@H](C)C2 KXNPVXPOPUZYGB-XYVMCAHJSA-N 0.000 title claims abstract description 120
- 238000001514 detection method Methods 0.000 title claims abstract description 73
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 87
- 239000000243 solution Substances 0.000 claims abstract description 83
- 239000013558 reference substance Substances 0.000 claims abstract description 32
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 23
- 238000002347 injection Methods 0.000 claims abstract description 17
- 239000007924 injection Substances 0.000 claims abstract description 17
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000005695 Ammonium acetate Substances 0.000 claims abstract description 16
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229940043376 ammonium acetate Drugs 0.000 claims abstract description 16
- 235000019257 ammonium acetate Nutrition 0.000 claims abstract description 16
- 238000010828 elution Methods 0.000 claims abstract description 15
- 238000002360 preparation method Methods 0.000 claims abstract description 14
- 239000000741 silica gel Substances 0.000 claims abstract description 4
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 46
- 238000000034 method Methods 0.000 claims description 41
- 239000000523 sample Substances 0.000 claims description 35
- 239000002904 solvent Substances 0.000 claims description 34
- 238000007865 diluting Methods 0.000 claims description 24
- 238000005303 weighing Methods 0.000 claims description 24
- 239000011550 stock solution Substances 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 239000012488 sample solution Substances 0.000 claims description 20
- 239000012046 mixed solvent Substances 0.000 claims description 19
- 238000012360 testing method Methods 0.000 claims description 13
- 239000007864 aqueous solution Substances 0.000 claims description 11
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 9
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 9
- 239000012490 blank solution Substances 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 7
- 230000004807 localization Effects 0.000 claims description 7
- 239000000377 silicon dioxide Substances 0.000 claims description 6
- 238000004090 dissolution Methods 0.000 claims description 5
- 238000010790 dilution Methods 0.000 claims description 4
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- 239000003814 drug Substances 0.000 description 26
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- 238000004458 analytical method Methods 0.000 description 12
- 229940079593 drug Drugs 0.000 description 11
- 230000000694 effects Effects 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 239000007791 liquid phase Substances 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 101000777134 Homo sapiens Ubiquitin carboxyl-terminal hydrolase 43 Proteins 0.000 description 4
- 102100031311 Ubiquitin carboxyl-terminal hydrolase 43 Human genes 0.000 description 4
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 4
- 239000000337 buffer salt Substances 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- REFMEZARFCPESH-UHFFFAOYSA-M sodium;heptane-1-sulfonate Chemical compound [Na+].CCCCCCCS([O-])(=O)=O REFMEZARFCPESH-UHFFFAOYSA-M 0.000 description 4
- 239000012085 test solution Substances 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 3
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- 238000006722 reduction reaction Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108090000190 Thrombin Proteins 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- FKCBLVCOSCZFHV-UHFFFAOYSA-N acetonitrile;ethanol Chemical compound CCO.CC#N FKCBLVCOSCZFHV-UHFFFAOYSA-N 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
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- 239000003085 diluting agent Substances 0.000 description 2
- 238000005984 hydrogenation reaction Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
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- 150000003839 salts Chemical class 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 229960004072 thrombin Drugs 0.000 description 2
- KXNPVXPOPUZYGB-IOVMHBDKSA-N (2R,4R)-1-[(2S)-5-(diaminomethylideneamino)-2-[(3-methyl-1,2,3,4-tetrahydroquinolin-8-yl)sulfonylamino]-1-oxopentyl]-4-methyl-2-piperidinecarboxylic acid Chemical compound OC(=O)[C@H]1C[C@H](C)CCN1C(=O)[C@H](CCCN=C(N)N)NS(=O)(=O)C1=CC=CC2=C1NCC(C)C2 KXNPVXPOPUZYGB-IOVMHBDKSA-N 0.000 description 1
- 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 description 1
- 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 description 1
- UVJPSTJAUUZEKC-UHFFFAOYSA-N 3-methyl-1,2,3,4-tetrahydroquinoline-8-sulfonamide Chemical group C1=CC=C(S(N)(=O)=O)C2=C1CC(C)CN2 UVJPSTJAUUZEKC-UHFFFAOYSA-N 0.000 description 1
- OUCSEDFVYPBLLF-KAYWLYCHSA-N 5-(4-fluorophenyl)-1-[2-[(2r,4r)-4-hydroxy-6-oxooxan-2-yl]ethyl]-n,4-diphenyl-2-propan-2-ylpyrrole-3-carboxamide Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@H]2OC(=O)C[C@H](O)C2)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 OUCSEDFVYPBLLF-KAYWLYCHSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- AUALQMFGWLZREY-UHFFFAOYSA-N acetonitrile;methanol Chemical compound OC.CC#N AUALQMFGWLZREY-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- -1 aminoiminomethyl Chemical group 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127217 antithrombotic drug Drugs 0.000 description 1
- 150000001483 arginine derivatives Chemical class 0.000 description 1
- 230000002902 bimodal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000007905 drug manufacturing Methods 0.000 description 1
- 229940126534 drug product Drugs 0.000 description 1
- 229940088679 drug related substance Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- UKVIEHSSVKSQBA-UHFFFAOYSA-N methane;palladium Chemical compound C.[Pd] UKVIEHSSVKSQBA-UHFFFAOYSA-N 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003586 protic polar solvent Substances 0.000 description 1
- 125000002943 quinolinyl group Chemical group N1=C(C=CC2=CC=CC=C12)* 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 125000000147 tetrahydroquinolinyl group Chemical group N1(CCCC2=CC=CC=C12)* 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
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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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- Physics & Mathematics (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a detection method for determining impurity C in Argatroban, which is high performance liquid chromatography and comprises the following steps: (1) preparation of a sample Argatroban solution; (2) preparing a reference argatroban impurity C solution; (3) Detecting, namely taking an argatroban solution as a sample and an argatroban impurity C solution as a reference substance, and carrying out sample injection detection according to the high performance liquid chromatography conditions: chromatographic column: monodisperse high-purity silica gel-based ball column, detection wavelength: 259nm; mobile phase: mobile phase a: ammonium acetate solution, mobile phase B: and (3) performing gradient elution on acetonitrile to obtain a chromatogram, and calculating the content of impurity C in the Argatroban serving as a sample according to the chromatogram.
Description
Technical Field
The invention belongs to the technical field of medicine analysis and detection. More specifically, it relates to a detection method for determining argatroban impurity C.
Background
Argatroban is a chemical of white crystalline powder. Chemical name (2R, 4R) -1- [ (2S) -5- (aminoiminomethyl) amino-2- [ (3-methyl-1, 2,3, 4-tetrahydro-8-quinolinyl) sulphonamide group]-1-oxopentyl group]-4-methylpiperidine-2-carboxylic acid monohydrate of formula C 23 H 36 N 6 O 5 S·H 2 O, molecular weight 526.65. Argatroban is an arginine derivative, and is a low-molecular drug synthesized for the first time by Mitsubishi chemical research institute at Honda. Argatroban binds to the catalytically active site of thrombin (including serine-histidine-arginine structures) inactivating thrombin. The drug was first marketed in japan in 1990, was approved for the treatment of peripheral thrombosis and acute cerebral stroke, and was marketed in the united states and china in 2000 and 2002.
The therapeutic effect of the medicine is not only to look at the activity of the compound, but also the impurity in the medicine has important influence on the safety and effectiveness of the compound. Drug impurities refer to substances which exist in the drug and have no therapeutic effect or influence on the stability and curative effect of the drug and are even harmful to human health, and main sources of impurities in the drug production process include two aspects: 1. the impurities in the raw materials of the medicines are uniformly called related substances because the raw materials are impure or the reaction is incomplete in the raw material reaction process, and the intermediate products of the reaction, byproducts of the reaction and the like exist; 2. impurities due to residues of solvents, catalysts, etc. reacted during the raw material production process. The relationship between the impurities in the medicine and the safety of the medicine is a complex relationship influenced by multiple factors, and usually, most of the impurities in the medicine have potential biological activity, and some of the impurities even interact with the medicine to influence the efficacy and safety of the medicine, so that serious toxic effects can be generated. The impurities in the medicine can affect the stability and safety of the medicine to different degrees, so that the purity of the medicine must be ensured in aspects of research, production, storage, clinical application and the like of the medicine, and the medicine can be effectively and safely ensured only by reducing the impurities of the medicine.
The argatroban bulk drug generates a plurality of by-product impurities (including impurity A, impurity C, impurity E, impurity G, impurity H, impurity IN2 and the like) and various degradation impurities (including impurity B, impurity D, impurity F, impurity I, impurity O and the like) IN the synthesis process, and if the related substances are excessively introduced into a finished drug product, serious medication safety problems can be caused. Therefore, strict control of these related substances is required in both the drug substance synthesis stage and the formulation stage.
Argatroban is typically a mixture of 21 (R) and 21 (S) Argatroban, and pharmaceutically acceptable Argatroban monohydrate needs to meet: 21 The ratio of the (R) to 21 (S) bits should be controlled within the range of R/S=63 to 67/37 to 33. The structural formula is as follows:
the methyl at the 21-position in the argatroban molecular structure is a racemization, the 21 (R) isomer and the 21 (S) isomer are diastereoisomers, and double peaks and larger peak width are frequently generated in the reversed phase liquid phase condition; because part of Argatroban impurities are similar to the Argatroban molecular structure, the molecular structure also contains the racemic methyl on the tetrahydroquinoline ring. Therefore, under the condition of conventional reversed-phase chromatography, part of impurities can have double peaks like argatroban, so that the peak width of the impurities is larger, and the separation effect of part of the impurities is poor. The liquid phase operation time of the common Argatroban related substance detection method is long, so that the effective separation of impurities with similar polarity from the main component can be ensured.
According to the published synthetic process route of argatroban, the reaction substrate (hydrolysate for short) is subjected to a series of reduction reactions such as nitro reduction, removal, quinoline ring reduction and the like under the action of high-pressure hydrogenation and palladium carbon, and finally the argatroban finished product is obtained, and the argatroban impurity C is an intermediate-state byproduct in the hydrogenation process generated in the process of synthesizing argatroban, has a chemical structure very similar to that of argatroban, is only one amino group more than that of argatroban molecular structure, has very similar physicochemical properties to that of the argatroban finished product, and cannot be effectively removed if the impurity C remains in a sample and is refined and purified through a methanol/water system and an ethanol/water system.
Deng Yu published in 2021 "synthesis of antithrombotic drug Argatroban" mentions liquid phase analysis methods for Argatroban related substances, chromatographic columns: agilent Eclipse XDB C18 (4.6 mm. Times.250 mm,5 μm); mobile phase a:0.1% trifluoroacetic acid +10mmol/L CH 3 COONH 4 Aqueous solution, mobile phase B: acetonitrile; a detection wavelength of 276nm; the flow rate is 1.0ml/min; sample injection amount is 10 μl; column temperature 40 ℃; running for 30min. Dilution (blank solution): acetonitrile-water=30:70 (% v/v). However, the analysis method has a short operation time, but the separation of each impurity is poor, and the detection wavelength is not suitable.
Analytical methods for detection of Argatroban-related substances as reported in United states pharmacopoeia USP43, column chromatography 4.6-mm X25-cm; 3- μm packing L1; mobile phase a, 10mM ammonium acetate with 5mM sodium heptanesulfonate aqueous solution, mobile phase B: acetonitrile-methanol (500:300); elution mode: gradient elution; detection wavelength 259nm; the flow rate is 0.6ml/min; sample injection amount is 10 μl; column temperature 50 ℃; running for 72min. The reproduction pharmacopoeia method finds that the double peaks and the main peaks of the impurity C coincide, and the impurity C cannot be accurately separated. Thus, the United states pharmacopoeia lists the detection methods separately for impurity C, column 4.6-mm x 25-cm;3- μm packing L1; mobile phase a: acetonitrile-ethanol-buffer salt (80:240:680) mobile phase B:10mM ammonium acetate and 5mM sodium heptanesulfonate in water; elution mode: gradient elution; detection wavelength 259nm; the flow rate is 0.6ml/min; sample injection amount is 10 μl; column temperature 50 ℃; running for 102min; when the pharmacopoeia method is reproduced, the impurity C and the Argatroban are both found to be bimodal, the first peak of the impurity C can be effectively separated from the Argatroban, but the second peak of the impurity C is completely overlapped with the Argatroban, so that only partial content of the impurity C can be detected, the detection result has larger deviation from the true value, the impurity detection quantity is lower, the evaluation of the quality of products is influenced, and the relative retention time and limit description of the impurity C in the USP43 pharmacopoeia are combined, wherein the relative retention time of the impurity C is only specified to be 0.94 compared with the relative retention time of one peak of the Argatroban, the relative retention time of the second peak is not provided, and the analysis method of the United states pharmacopoeia on the impurity C is only capable of effectively separating the first peak and is basically consistent with the reproduction result. In conclusion, the method for analyzing the recurring United states pharmacopoeia has the advantages that the separation degree of a plurality of impurities is poor, part of impurities coincide with main peaks, the operation time is long, and sodium heptanesulfonate is added in a mobile phase, so that the chromatographic column is damaged to a certain extent.
Analytical methods for detection of substances of interest, as embodied in Japanese pharmacopoeia JP17, chromatographic columns 4.6-mm X25-cm; 5- μm packing L1; buffer 0.25% acetic acid (ammonia adjusted pH to 5.0), mobile phase a: buffer-methanol (500:500), mobile phase B: buffer-methanol (200:800), elution mode: gradient elution; a detection wavelength of 254nm; the flow rate is 1.0ml/min; sample injection amount is 10 μl; column temperature 45 ℃; running for 50min. The reproduction of the japanese pharmacopoeia method found that impurity C was completely coincident with argatroban, with no distinguishing effect.
Disclosure of Invention
The invention aims to overcome the defect and the defect that the prior art cannot effectively and accurately measure the impurity C in Argatroban by using HPLC, and provides a detection method for measuring the impurity C in Argatroban.
The invention aims to solve the technical problems, and provides a detection method of Argatroban impurity C, which has the advantages of good separation degree, high detection efficiency and short operation time, wherein the detection method is a high performance liquid chromatography and comprises the following steps:
(1) Preparing a sample Argatroban solution; wherein the test argatroban comprises a bulk drug containing argatroban or a pharmaceutical preparation containing argatroban.
(2) Preparing a reference Argatroban impurity C solution;
(3) Detecting, namely taking an argatroban solution as a sample and an argatroban impurity C solution as a reference substance, and carrying out sample injection detection according to the high performance liquid chromatography conditions: chromatographic column: monodisperse high-purity silica gel-based ball column, detection wavelength: 259nm; mobile phase: mobile phase a: ammonium acetate solution, mobile phase B: acetonitrile, gradient elution, obtaining a chromatogram, wherein the gradient elution program is as follows:
and calculating the content of impurity C in the Argatroban serving as a sample according to the chromatogram.
The detection method of the present invention, wherein the chromatographic column is preferably a silica gel column Nano ChromCore Silica (250X 4.6mm,3 μm).
According to the detection method disclosed by the invention, the Argatroban solution to be tested is prepared by dissolving the Argatroban solution in a mixed solvent of acetonitrile and water, wherein the volume ratio of acetonitrile to water in the mixed solvent is 80:20.
The detection method of the invention, wherein the preparation method of the sample Argatroban solution comprises the following steps: taking Argatroban 20mg, placing into a 20ml measuring flask, adding a mixed solvent for dissolution, diluting to a scale, and shaking uniformly.
According to the detection method disclosed by the invention, the reference argatroban impurity C solution is prepared by dissolving acetonitrile and water in a mixed solvent, wherein the volume ratio of acetonitrile to water in the mixed solvent is 80:20.
The detection method of the invention, wherein, the preparation method of the reference Argatroban impurity C solution is as follows: weighing 5mg of impurity C reference substance, placing into a 50ml measuring flask, adding mixed solvent for dissolution and dilution to scale, shaking up, weighing 3.0ml of reference substance stock solution, placing into a 20ml measuring flask, diluting to scale with solvent, shaking up, weighing 2.0ml of reference substance stock solution, placing into a 20ml measuring flask, diluting to scale with solvent, and shaking up.
According to the detection method, the mobile phase A is an aqueous solution containing 0.05% -0.2% of ammonium acetate, and the pH is adjusted to 6.5-8.5 by ammonia water.
The detection method of the invention, wherein the chromatographic column temperature is 30-50 ℃, the mobile phase flow rate is 0.2-1.0 ml/min, and the sample injection amount is 2-10 μl.
The detection method also comprises the steps of preparing blank solution, adding a standard sample solution and other impurity positioning solutions, and injecting the blank solution, the standard sample solution and the impurity positioning solutions into a high performance liquid chromatograph. Wherein,
the preparation method of the blank solution comprises the following steps: acetonitrile and water with the volume ratio of 80:20 are uniformly mixed;
the preparation method of the labeled sample solution comprises the following steps: taking 20mg of Argatroban, precisely weighing, placing into a 20ml measuring flask, precisely adding 2.0ml of reference substance stock solution, adding solvent for dissolving and diluting to scale, and shaking uniformly;
the preparation method of other impurity locating solutions is as follows: weighing 1mg of each impurity A, B, D, E, F, G, H, I, J, M, N, O, SM-C, IN2 reference substance, respectively placing into 10ml measuring flask, adding methanol for dissolving and diluting to scale, shaking, precisely weighing 1ml, respectively placing into 20ml measuring flask, diluting to scale with solvent, and shaking.
The method for detecting Argatroban related substances provided by the invention, wherein the gradient elution program is preferably as follows:
in the method for detecting Argatroban related substances, in the high performance liquid chromatography condition, a mobile phase A is an ammonium acetate aqueous solution containing 0.05% -0.2%, ammonia water is used for adjusting the pH value to 6.5-8.5, and a mobile phase B is acetonitrile; preferably, the mobile phase A is 0.05 to 0.15 percent of ammonium acetate aqueous solution, and ammonia water is used for adjusting the pH value to 7.0 to 8.0; more preferably mobile phase a is an aqueous solution containing 0.1% ammonium acetate and the pH is adjusted to 7.5 with aqueous ammonia.
The detection method of Argatroban related substances provided by the invention, wherein in the high performance liquid chromatography condition, the temperature of a chromatographic column is 30-50 ℃; preferably, the column temperature of the chromatographic column is 35-45 ℃; more preferably the column temperature is 40 ℃.
The method for detecting Argatroban related substances provided by the invention comprises the steps of in the high performance liquid chromatography condition, enabling the flow rate of a mobile phase to be 0.2-1.0 ml/min; preferably, the flow rate of the mobile phase is 0.4-0.8 ml/min; more preferably, the mobile phase flow rate is 0.6ml/min.
The method for detecting Argatroban related substances provided by the invention comprises the steps of carrying out high performance liquid chromatography on a sample with a sample injection amount of 2-10 mu l; the sample loading is preferably 5. Mu.l.
The invention provides a detection method of Argatroban related substances, wherein in the high performance liquid chromatography condition, a detector used is a photodiode array detector, and the detection wavelength is 259nm.
The invention provides a detection method of Argatroban related substances, wherein the preparation method of the sample solution is to take Argatroban, precisely weigh, add a proper amount of solvent to dissolve and dilute to prepare a solution containing about 1mg per 1 ml. Because the sample injection volume is very small relative to the mobile phase volume, the appropriate amount of solvent can be selected from solvents that completely dissolve the sample and are suitable for chromatographic analysis, such as water, methanol, acetonitrile, methanol-water, acetonitrile-water.
The invention further provides a detection method of Argatroban related substances, which comprises the following steps:
(1) Preparing a sample solution: taking a proper amount of Argatroban, precisely weighing, adding a mixed solvent of acetonitrile and water for dissolving and diluting to prepare a solution containing about 0.5-4 mg per 1 ml; wherein the volume ratio of acetonitrile to water in the mixed solvent is 80:20;
(2) Chromatographic conditions: the column was Nano ChromCore Silica (250X 4.6mm,3 μm); the mobile phase A is an ammonium acetate aqueous solution containing 0.05 to 0.2 percent, the pH value is regulated to 6.5 to 8.5 by ammonia water, and the mobile phase B is acetonitrile; eluting in a gradient mode; the flow rate of the mobile phase is 0.2-1.0 ml/min, the column temperature of the chromatographic column is 30-50 ℃, the sample injection amount of the sample is 2-10 mu l, and a photodiode array detector is adopted to detect the wavelength of 259nm; wherein, the gradient elution procedure is:
(3) Measuring: sucking the sample solution, injecting into a liquid chromatograph, and measuring by high performance liquid chromatography.
The invention further provides a detection method for determining impurity C in Argatroban, which comprises the following preferable steps:
(1) Preparing a sample solution: taking a proper amount of Argatroban, precisely weighing, adding a mixed solvent of acetonitrile and water for dissolving and diluting to prepare a solution containing about 0.1mg per 1 ml; wherein the volume ratio of acetonitrile to water in the mixed solvent is 80:20;
(2) Chromatographic conditions: the column was Nano ChromCore Silica (250X 4.6mm,3 μm); the mobile phase A is an aqueous solution containing 0.1% of ammonium acetate, the pH is regulated to 7.5 by ammonia water, and the mobile phase B is acetonitrile; eluting in a gradient mode; the flow rate of the mobile phase is 0.6ml/min, the column temperature of the chromatographic column is 40 ℃, the sample injection amount of the sample is 5 mu l, and a photodiode array detector is adopted to detect the wavelength of 259nm; wherein, the gradient elution procedure is:
(3) Measuring: sucking the sample solution, injecting into a liquid chromatograph, and measuring by high performance liquid chromatography.
The invention has the following beneficial effects:
through a large number of experimental exploration, the inventor finally establishes a high-performance liquid chromatography method capable of efficiently separating and detecting the Argatroban impurity C on the premise of considering that other Argatroban impurities can be accurately detected. In the analysis method, the Argatroban is subjected to research, except for the impurity J, the Argatroban main component and other impurities are unimodal, and compared with the bimodality of the prior liquid phase detection method, the argatroban main component and other impurities have narrow peak width, ideal peak shape and separation degree, greatly shortened operation time, effectively reduced consumption materials and good economic value.
The analysis detection method has high sensitivity, higher recovery rate, precision and stability, good repeatability and high accuracy, can be used for monitoring the impurity C in the production process of the argatroban, and is beneficial to controlling and improving the quality of the argatroban; meanwhile, the method is simple to operate, short in detection time and very suitable for quality monitoring of the bulk drug in large-scale industrialized production.
Drawings
FIG. 1 is an HPLC chromatogram of Argatroban control solution under the Japanese pharmacopoeia JP17 method of comparative example 1.
FIG. 2 is an HPLC chromatogram of an impurity C localization solution under the Japanese pharmacopoeia JP17 method of comparative example 1.
Fig. 3 is a superimposed spectrum of argatroban and impurity C localization solution under the japanese pharmacopoeia JP17 method of comparative example 1.
FIG. 4 is an HPLC plot of a system applicability solution under the United states Pharmacopeia USP43 method of comparative example 2.
FIG. 5 is an HPLC plot of the impurity C localization solution under the United states Pharmacopeia USP43 method of comparative example 2.
FIG. 6 is an HPLC chart of a control solution of impurity C in example 1.
FIG. 7 is an HPLC chart of a sample labeling solution of impurity C and main component in example 1.
FIG. 8 is an HPLC chromatogram of the main component and each impurity localization solution in example 1.
Fig. 9 shows a linear relationship of impurity C.
Detailed Description
The following examples are presented only to aid in understanding the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims. The following description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Comparative example 1 (JP 17 Standard related substance method)
The diluents in the test sample preparation are as follows: methanol
The chromatographic detection conditions were as follows:
chromatographic column:XB-C18，250×4.6mm，5μm
column temperature: 45 DEG C
Mobile phase:
buffer solution: 0.25% acetic acid (ammonia pH 5.0)
A: buffer solution-methanol (500:500)
B: buffer-methanol (200:800)
Gradient procedure:
detection wavelength: 254nm
Flow rate: 1.0ml/min
Sample injection amount: 10 μl of
Solvent: methanol
Positioning solution: and respectively dissolving a proper amount of the Argatroban working reference substance and a proper amount of the impurity C reference substance by using a solvent to prepare a positioning solution containing 300 mug and 10 mug of the Argatroban reference substance and the impurity C in 1 ml.
Experimental data:
experimental results: the impurity peak separation conditions are shown in fig. 1 to 3, and argatroban and impurity C are completely overlapped without distinguishing force.
Comparative example 2 (method for detecting impurity C of substance of USP 43)
The diluents in the test sample preparation are as follows: methanol
The chromatographic detection conditions were as follows:
chromatographic column: ODS-3, 250X 4.6mm,3 μm
Column temperature: 50 DEG C
Mobile phase:
a: acetonitrile-ethanol-buffer salt (80:240:680)
B: buffer salt: 10mM ammonium acetate and 5mM sodium heptanesulfonate aqueous solution
Gradient procedure:
detection wavelength: 259nm of
Flow rate: 0.6ml/min
Sample injection amount: 10 μl of
Solvent: methanol
Impurity C localization solution: taking a proper amount of the reference substance of the impurity C, precisely weighing, adding methanol for dissolving and diluting to prepare a solution containing about 4 mug per 1ml, and taking the solution as an impurity C positioning solution.
System applicability solution: the argatroban and the impurity C are taken to be weighed with proper amount, and are dissolved and diluted by methanol to prepare a solution containing about 10mg of argatroban and 10 mug of impurity C per 1 ml.
Experimental data:
experimental results: the separation condition of the impurity C and the argatroban is shown in fig. 4-5, the first peak of the impurity C and the argatroban can be effectively separated, and the second peak of the impurity C and the argatroban completely coincide.
Example 1 detection method for determining impurity C in Argatroban
(1) Conditions of liquid chromatography
Detection wavelength: 259nm of
Chromatographic column: nano ChromCore Silica (250X 4.6mm,3 μm)
Column temperature: 40 DEG C
Flow rate: 0.6ml/min
Sample injection amount: 5 μl
Mobile phase a:0.1mol/L ammonium acetate (pH 7.5 adjusted with aqueous ammonia)
Mobile phase B: acetonitrile
Gradient procedure:
solvent: acetonitrile-water (80:20)
(2) Preparation of test sample and reference substance
Preparing a solution:
blank solution (solvent): acetonitrile-water (80:20).
Control stock solution: weighing about 5mg of the impurity C reference substance, precisely weighing, placing into a 50ml measuring flask, adding solvent to dissolve and dilute to scale, shaking up, precisely weighing 3.0ml of the reference substance stock solution, placing into a 20ml measuring flask, diluting to scale with solvent, and shaking up.
Control solution: precisely measuring 2.0ml of control stock solution, placing into a 20ml measuring flask, diluting to scale with solvent, and shaking.
Test solution: about 20mg of the product is taken, precisely weighed, placed in a 20ml measuring flask, dissolved by adding a solvent, diluted to a scale and shaken well.
Adding a standard test sample solution (system applicability solution): about 20mg of the product is taken, precisely weighed, placed in a 20ml measuring flask, precisely added with 2.0ml of reference substance stock solution, then dissolved and diluted to scale by adding solvent, and uniformly shaken.
Other impurity localization solutions: weighing about 1mg of each impurity A, B, D, E, F, G, H, I, J, M, N, O, SM-C, IN2 reference substance, respectively placing into 10ml measuring flask, adding methanol for dissolving and diluting to scale, shaking, precisely weighing 1ml, respectively placing into 20ml measuring flask, diluting to scale with solvent, and shaking.
Precisely measuring 5 μl of each of blank solution, reference solution, sample solution, labeled sample solution, and other impurity positioning solution, respectively injecting into liquid chromatograph, and recording chromatogram.
The separation of the impurity C from Argatroban and the respective impurities is shown in FIGS. 6 to 8.
(3) Detecting and obtaining the results of the labeled sample solution and the reference substance
TABLE 1 Argatroban and summary of detection data for each impurity
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(4) Detection limit and detection limit determination
Taking a reference substance solution, diluting the reference substance solution with a solvent until the signal to noise ratio S/N of the peak height is more than or equal to 10, taking the solution as a quantitative concentration limiting solution, and preparing 6 parts in parallel; and continuously diluting the solution until the signal to noise ratio S/N is more than or equal to 3, and taking the solution as a detection limit concentration solution. And precisely measuring 5 mu l of each of the quantitative limit solution and the detection limit solution, respectively injecting into a liquid chromatograph, and recording a chromatogram. The results are shown in tables 2 and 3.
TABLE 2 detection limit test results
TABLE 3 quantitative limit test results
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The quantitative limit concentration of impurity C is 0.3013 mug/ml, S/N is 12.48, and the concentration corresponds to 20.1% of the limit concentration; in the 6 parts of quantitative limiting solution chromatogram, the peak area RSD of the impurity C is 2.62 percent and is less than 20.0 percent. The detection limit concentration was 0.090. Mu.g/ml, S/N was 5.33, corresponding to 6.0% of the limit concentration. The result shows that the method has higher sensitivity and meets the detection requirement.
(5) Solution stability
Taking reference substance solution and adding standard sample solution, standing at room temperature, respectively sampling and analyzing at different times, and recording chromatogram. The results are shown in tables 4 and 5.
TABLE 4 stability results of control solutions
TABLE 5 stability results of test solutions
The reference substance solution is placed at room temperature for 34.5 hours, and the percentage of the area of the impurity C peak at each time point relative to 0 hour is in the range of 95.0-105.0%, which indicates that the reference substance solution is stable within 34.5 hours under the room temperature condition. The standard sample solution is placed at room temperature for 21.5 hours, and the percentage of the impurity C peak area relative to 0 hour at each time point is in the range of 95.0-105.0 percent, which indicates that the standard sample solution is stable in 21.5 hours under the room temperature condition.
(6) Linear and range
Linear stock: a proper amount of impurity C stock solution is precisely measured, and the stock solution is diluted by adding a solvent to prepare a solution with about 15 mug per 1 ml.
25% linear solution: 2.5ml of the linear stock solution is precisely measured, placed in a 100ml measuring flask, diluted to a scale by adding a solvent, and shaken well.
50% linear solution: precisely measuring 1ml of the linear stock solution, placing the stock solution into a 20ml measuring flask, adding a solvent to dilute the stock solution to a scale, and shaking the stock solution uniformly.
100% linear solution: 2ml of the linear stock solution is precisely measured, placed in a 20ml measuring flask, diluted to a scale by adding a solvent, and uniformly shaken.
150% linear solution: 3ml of the linear stock solution is precisely measured, placed in a 20ml measuring flask, diluted to a scale by adding a solvent, and uniformly shaken.
200% linear solution: 4ml of the linear stock solution is precisely measured, placed in a 20ml measuring flask, diluted to a scale by adding a solvent, and uniformly shaken.
Each linear solution was measured precisely at 5 μl, and was injected into a liquid chromatograph, and the chromatograms were recorded, and the detection results are shown in table 6.
TABLE 6 impurity C linearity and Range
The impurity C is in a concentration range of 0.3013-3.0133 mug/ml, the concentration and the peak area are in a linear relation, the linear equation is y= 6021.8x-200.57, the correlation coefficient r is 0.9999 and is more than 0.995, and the ratio of the absolute value of the Y-axis intercept to the 100% concentration response value is 2.3% and is less than 10.0%; each concentration response factor RSD was 5.0%, less than 10%. Indicating that the method has good linearity.
Example 2 detection method for determining impurity C in Argatroban
The detection method of example 2 is different from example 1 in that the column temperature of 40℃in the liquid chromatography condition of example 1 (1) is adjusted to 35℃and 45℃and the remaining parameters are referred to example 1. The results of the separation of the labeled test solutions are shown in Table 7.
TABLE 7 solution separation results for labeled test
| Conditions (conditions) | Argatroban | Impurity C |
| Initial conditions | / | 3.79 |
| Column temperature 35 DEG C | / | 3.31 |
| Column temperature 45 DEG C | / | 4.06 |
As can be seen from Table 7, the degree of separation of impurity C from Argatroban meets the requirements.
Example 3 detection method for determining impurity C in Argatroban
Example 3 the detection method differs from example 1 in that the buffer salt concentration in the mobile phase of example 1 (1) was adjusted to 0.09mol/L and 0.11mol/L, with the remaining parameters referring to example 1. The results of the separation of the labeled test sample solutions are shown in Table 8.
Table 8 results of degree of separation of solution of labeled test sample
| Conditions (conditions) | Argatroban | Impurity C |
| Initial conditions | / | 3.79 |
| Salt concentration-0.09 mol/L | / | 3.58 |
| Salt concentration-0.11 mol/L | / | 3.27 |
As can be seen from Table 8, the degree of separation of impurity C from Argatroban meets the requirements.
Example 4 detection method for determining impurity C in Argatroban
Example 4 the detection method differs from example 1 in that the mobile phase pH 7.5 in the liquid chromatography conditions of example 1 (1) is adjusted to 7.3 and 7.7, the remaining parameters being referred to example 1. The results of the separation of the labeled test solutions are shown in Table 9.
TABLE 9 solution separation results for labeled test
| Conditions (conditions) | Argatroban | Impurity C |
| Initial conditions | / | 3.79 |
| pH value-7.3 | / | 3.36 |
| pH value of 7.7 | / | 3.35 |
As can be seen from Table 9, the degree of separation of impurity C from Argatroban meets the requirements.
The chromatographic conditions, such as chromatographic columns and solvents, of the invention are obtained by screening by the inventor, and the screening process is as follows:
the Argatroban and each impurity have larger polarity and similar polarity, so that the conventional C18 chromatographic column has poorer separation effect, and the better separation effect can be obtained by adding an ion pair reagent or prolonging the liquid phase operation time.
Acetonitrile is generally stronger in eluting power than methanol in mobile phase selection, but is less effective in separation than the protic solvent methanol, which is determined by the chemical nature of the solvent itself. Therefore, in the analysis method of the pharmacopoeias of the various argatroban countries, the Japanese pharmacopoeias adopts methanol as a mobile phase, the operation time is short, but the main component and the impurity have almost no distinguishing force, the United states pharmacopoeias adopts acetonitrile as the mobile phase, and an ion pair reagent is needed to be added to improve the peak type, the operation time is long, and the separation effect of the impurity C is also not ideal. According to the invention, acetonitrile with stronger elution capacity is used as a mobile phase, an ammonium acetate solution is used as a water phase, and a series of adjustment, such as adjustment of pH value of the mobile phase and adjustment of elution gradient program, are carried out, so that chromatographic conditions in the invention are finally obtained, and good separation effect of Argatroban and impurity C is obtained.
The invention has been further described above in connection with specific embodiments, which are exemplary only and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
The invention belongs to the technical field of medicine analysis and detection, and particularly relates to a detection method for determining impurity C in Argatroban. The method can establish a high-performance liquid chromatography method capable of efficiently separating and detecting the Argatroban impurity C under the premise of considering that other Argatroban impurities can be accurately detected under one chromatography condition. In the analysis method, the Argatroban is subjected to research, except for the impurity J, the Argatroban main component and other impurities are unimodal, and compared with the bimodality of the prior liquid phase detection method, the argatroban main component and other impurities have narrow peak width, ideal peak shape and separation degree, greatly shortened operation time, effectively reduced consumption materials and good economic value. The analysis detection method has high sensitivity, higher recovery rate, precision and stability, good repeatability and high accuracy, can be used for monitoring the impurity C in the production process of the argatroban, and is beneficial to controlling and improving the quality of the argatroban; meanwhile, the method is simple to operate, short in detection time and very suitable for quality monitoring of the bulk drug in large-scale industrialized production.
Claims (10)
1. The method for detecting the argatroban impurity C is characterized by comprising the following steps of:
(1) Preparing a sample Argatroban solution;
(2) Preparing a reference Argatroban impurity C solution;
(3) Detecting, namely taking an argatroban solution as a sample and an argatroban impurity C solution as a reference substance, and carrying out sample injection detection according to the high performance liquid chromatography conditions: chromatographic column: monodisperse high-purity silica gel-based ball column, detection wavelength: 259nm; mobile phase: mobile phase a: ammonium acetate solution, mobile phase B: acetonitrile is subjected to gradient elution to obtain a chromatogram, and the content of impurity C in the argatroban to be tested is calculated according to the chromatogram;
wherein the gradient elution procedure is:
2. the method of claim 1, wherein the chromatographic column is Nano ChromCore Silica (250 x 4.6mm,3 μm).
3. The detection method according to claim 1, wherein the argatroban solution as the test sample is prepared by dissolving with a mixed solvent of acetonitrile and water, wherein the volume ratio of acetonitrile to water in the mixed solvent is 80:20.
4. The method according to claim 3, wherein the preparation method of the argatroban solution as a test product comprises the following steps: taking Argatroban 20mg, placing into a 20ml measuring flask, adding a mixed solvent for dissolution, diluting to a scale, and shaking uniformly.
5. The detection method according to claim 1, wherein the reference argatroban impurity C solution is prepared by dissolving acetonitrile and water in a mixed solvent, wherein the volume ratio of acetonitrile to water in the mixed solvent is 80:20.
6. The detection method according to claim 5, wherein the reference argatroban impurity C solution is prepared by the following steps: weighing 5mg of impurity C reference substance, placing into a 50ml measuring flask, adding mixed solvent for dissolution and dilution to scale, shaking up, weighing 3.0ml of reference substance stock solution, placing into a 20ml measuring flask, diluting to scale with solvent, shaking up, weighing 2.0ml of reference substance stock solution, placing into a 20ml measuring flask, diluting to scale with solvent, and shaking up.
7. The method according to claim 1, wherein the mobile phase A is an aqueous solution containing 0.05% to 0.2% of ammonium acetate, and the pH is adjusted to 6.5 to 8.5 with aqueous ammonia.
8. The method according to claim 1, wherein the column temperature of the chromatographic column is 30-50 ℃, the flow rate of the mobile phase is 0.2-1.0 ml/min, and the sample injection amount is 2-10 μl.
9. The method according to claim 1, further comprising the steps of preparing a blank solution, a labeled sample solution, and other impurity localization solutions, and injecting them into a high performance liquid chromatograph,
the preparation method of the blank solution comprises the following steps: acetonitrile and water with the volume ratio of 80:20 are uniformly mixed;
the preparation method of the labeled sample solution comprises the following steps: taking 20mg of Argatroban, precisely weighing, placing into a 20ml measuring flask, precisely adding 2.0ml of reference substance stock solution, adding solvent for dissolving and diluting to scale, and shaking uniformly;
the preparation method of other impurity locating solutions is as follows: weighing 1mg of each impurity A, B, D, E, F, G, H, I, J, M, N, O, SM-C, IN2 reference substance, respectively placing into 10ml measuring flask, adding methanol for dissolving and diluting to scale, shaking, precisely weighing 1ml, respectively placing into 20ml measuring flask, diluting to scale with solvent, and shaking.
10. The method of detection according to claim 1, comprising the steps of:
(1) Preparing a sample solution: taking a proper amount of Argatroban, precisely weighing, adding a mixed solvent of acetonitrile and water for dissolving and diluting to prepare a solution containing about 0.1mg per 1 ml; wherein the volume ratio of acetonitrile to water in the mixed solvent is 80:20;
(2) Preparing an argatroban impurity C solution as a reference substance: weighing 5mg of impurity C reference substance, placing into a 50ml measuring flask, adding mixed solvent for dissolution and dilution to scale, shaking up, weighing 3.0ml of reference substance stock solution, placing into a 20ml measuring flask, diluting to scale with solvent, shaking up, weighing 2.0ml of reference substance stock solution, placing into a 20ml measuring flask, diluting to scale with solvent, and shaking up;
(3) Measuring: sucking the sample solution, injecting the reference Argatroban impurity C solution into a high performance liquid chromatograph, and measuring according to the high performance liquid chromatography, wherein the chromatographic conditions are as follows: the column was Nano ChromCore Silica (250X 4.6mm,3 μm); the mobile phase A is an aqueous solution containing 0.1% of ammonium acetate, the pH is regulated to 7.5 by ammonia water, and the mobile phase B is acetonitrile; eluting in a gradient mode; the flow rate of the mobile phase is 0.6ml/min, the column temperature of the chromatographic column is 40 ℃, the sample injection amount of the sample is 5 mu l, and a photodiode array detector is adopted to detect the wavelength of 259nm; wherein, the gradient elution procedure is:
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