CN116482261A - HPLC detection method for polymer impurities in cefmetazole and preparation thereof - Google Patents
HPLC detection method for polymer impurities in cefmetazole and preparation thereof Download PDFInfo
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- CN116482261A CN116482261A CN202310400166.1A CN202310400166A CN116482261A CN 116482261 A CN116482261 A CN 116482261A CN 202310400166 A CN202310400166 A CN 202310400166A CN 116482261 A CN116482261 A CN 116482261A
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- cefmetazole
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- SNBUBQHDYVFSQF-HIFRSBDPSA-N cefmetazole Chemical compound S([C@@H]1[C@@](C(N1C=1C(O)=O)=O)(NC(=O)CSCC#N)OC)CC=1CSC1=NN=NN1C SNBUBQHDYVFSQF-HIFRSBDPSA-N 0.000 title claims abstract description 66
- 239000012535 impurity Substances 0.000 title claims abstract description 65
- 229960003585 cefmetazole Drugs 0.000 title claims abstract description 61
- 229920000642 polymer Polymers 0.000 title claims abstract description 53
- 238000001514 detection method Methods 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 22
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 6
- 239000007857 degradation product Substances 0.000 claims abstract description 3
- 239000000539 dimer Substances 0.000 claims abstract description 3
- 239000013638 trimer Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 64
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 51
- 239000012071 phase Substances 0.000 claims description 45
- 238000000034 method Methods 0.000 claims description 25
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 16
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 15
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 15
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 15
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 10
- 239000000499 gel Substances 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 7
- 239000012074 organic phase Substances 0.000 claims description 7
- 239000008363 phosphate buffer Substances 0.000 claims description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 239000000337 buffer salt Substances 0.000 claims description 6
- 239000000945 filler Substances 0.000 claims description 6
- 239000000741 silica gel Substances 0.000 claims description 6
- 229910002027 silica gel Inorganic materials 0.000 claims description 6
- 239000007974 sodium acetate buffer Substances 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 102000034238 globular proteins Human genes 0.000 claims description 2
- 108091005896 globular proteins Proteins 0.000 claims description 2
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 2
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 2
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical group [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 6
- 239000000047 product Substances 0.000 abstract description 13
- 229940079593 drug Drugs 0.000 abstract description 12
- 239000003814 drug Substances 0.000 abstract description 12
- 230000035945 sensitivity Effects 0.000 abstract description 10
- 238000000926 separation method Methods 0.000 abstract description 9
- 238000003908 quality control method Methods 0.000 abstract description 6
- 239000012085 test solution Substances 0.000 description 12
- 239000012488 sample solution Substances 0.000 description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000012360 testing method Methods 0.000 description 8
- 239000008055 phosphate buffer solution Substances 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 238000007865 diluting Methods 0.000 description 6
- 238000004090 dissolution Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- 230000000844 anti-bacterial effect Effects 0.000 description 5
- 229960002676 cefmetazole sodium Drugs 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 238000010812 external standard method Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 206010002198 Anaphylactic reaction Diseases 0.000 description 3
- 229930186147 Cephalosporin Natural products 0.000 description 3
- 108090000204 Dipeptidase 1 Proteins 0.000 description 3
- 208000003455 anaphylaxis Diseases 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 102000006635 beta-lactamase Human genes 0.000 description 3
- 229940124587 cephalosporin Drugs 0.000 description 3
- 150000001780 cephalosporins Chemical class 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 238000002270 exclusion chromatography Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 239000012088 reference solution Substances 0.000 description 3
- 239000013558 reference substance Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- XOHZHMUQBFJTNH-UHFFFAOYSA-N 1-methyl-2h-tetrazole-5-thione Chemical compound CN1N=NN=C1S XOHZHMUQBFJTNH-UHFFFAOYSA-N 0.000 description 2
- WFIYPADYPQQLNN-UHFFFAOYSA-N 2-[2-(4-bromopyrazol-1-yl)ethyl]isoindole-1,3-dione Chemical compound C1=C(Br)C=NN1CCN1C(=O)C2=CC=CC=C2C1=O WFIYPADYPQQLNN-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- 241000588769 Proteus <enterobacteria> Species 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 2
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 241000606125 Bacteroides Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 241000192001 Pediococcus Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 208000030961 allergic reaction Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000013557 residual solvent Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000001946 ultra-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 241001148471 unidentified anaerobic bacterium Species 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative 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
- G01N30/26—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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
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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
- 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
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- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
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- Pathology (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
An HPLC detection method for polymer impurities in cefmetazole and a preparation thereof comprises the steps of separating and measuring the polymer impurities in the cefmetazole and the preparation thereof by using a High Performance Liquid Chromatography (HPLC), wherein the polymer impurities are dimers, trimers and polymers formed by the polymerization of the cefmetazole and the polymerization between the cefmetazole and degradation products. The invention has the characteristics of good separation degree, simplicity, rapidness, strong specificity and high sensitivity, can perform better quality control on the cefmetazole and the polymer impurities of the preparation thereof, and ensures the quality of the whole product and the clinical medication safety.
Description
Technical Field
The invention relates to the technical field of chromatographic test or analysis, in particular to an HPLC detection method for polymer impurities in cefmetazole and a preparation thereof.
Background
Cefmetazole is a second-generation semi-synthetic cephalosporin antibiotic which is highly stable to beta-lactamase, so that the cefmetazole has strong antibacterial activity on sensitive bacteria producing beta-lactamase and non-beta-lactamase. The product has excellent antibacterial effect on Staphylococcus aureus, escherichia coli, klebsiella pneumoniae and indole negative Proteus, and also has strong antibacterial effect on indole positive Proteus which is generally insensitive to other cephalosporins and penicillin antibiotics. In addition, they also exhibit excellent antibacterial activity against anaerobic bacteria such as Bacteroides, pediococcus and Pediostreptococcus. The action mechanism of the product is to strongly inhibit the synthesis of cell walls of bacteria in the proliferation period, so as to play a bactericidal role.
The chemical structure of cefmetazole is shown as follows:
the quality control of cefmetazole sodium salt in the 2020 edition of Chinese pharmacopoeia comprises acidity, clarity, related substances (1-methyl-5-mercaptotetrazole impurity), cefmetazole polymer impurity, residual solvent, heavy metal, bacterial endotoxin and the like.
Wherein the quality control detection of cefmetazole polymer impurity is determined according to molecular exclusion chromatography (China pharmacopoeia general rule 0514). The chromatographic conditions are as follows: using sephadex G-10 (40-120 μm) as filler; the inner diameter of the glass column is 1.0-1.4 cm, and the column length is 30-40 cm; taking 0.1mol/L phosphate buffer solution (0.1 mol/L disodium hydrogen phosphate solution-0.1 mol/L sodium dihydrogen phosphate solution (95:5)) with pH of 8.0 as a mobile phase A and water as a mobile phase B; the flow rate is about 0.8ml per minute; the detection wavelength is 254nm; the sample injection volume is 100-200 mul. The specific measurement method comprises the following steps: taking the mobile phase A as a mobile phase, precisely measuring a sample solution to be tested, injecting the sample solution into a liquid chromatograph, and recording a chromatogram; taking the mobile phase B as a mobile phase, precisely measuring a control solution, injecting the control solution into a liquid chromatograph, and recording a chromatogram.
The quality control limit is: the content of the cefmetazole-containing polymer is less than 0.1 percent calculated by the peak area of the cefmetazole according to an external standard method.
The polymeric impurities in antibiotics are collectively referred to as polymers. A great deal of research shows that the allergen for inducing cephalosporin anaphylactic reaction is high molecular polymer impurity in the preparation, and the anaphylactic reaction can be controlled by controlling the polymer impurity. Therefore, the quality of the product and the safety of subsequent clinical medication are directly determined by the content level of the polymer impurities in the cefmetazole and the preparation thereof. Although the Chinese pharmacopoeia 2020 stipulates that the polymer impurities of cefmetazole sodium and the preparation thereof are controlled, the molecular exclusion chromatography method of the sephadex G10 is adopted, and the method can not effectively separate the polymer impurities from the main components, so that the detection result of the polymer impurities is low, the result can not accurately represent the actual content of the polymer impurities in a sample, the clinical medication safety can not be ensured, and a great deal of allergic reaction still occurs in the actual medication process.
Chinese patent application CN102627660a discloses a cefmetazole sodium sterile powder and a preparation method thereof, wherein it is mentioned that a crystallization process and a drying process may cause an increase of related substances such as a high molecular polymer, and the solution disclosed in the patent document can control the prepared high molecular polymer to be below 0.1% by avoiding the contact of cefmetazole sodium with water, selecting a proper poor solvent and crystallizing the good solvent at a proper temperature, and the like.
Chinese patent application CN104844627a discloses a cefmetazole sodium compound and a preparation method thereof, which discloses that the content of 5-mercapto-1-methyltetrazole impurity and polymer is reduced by avoiding the use of solvent water which is liable to cause degradation of cefmetazole sodium, wherein the content of polymer meets the pharmacopoeia requirement less than 0.1% within 24 months.
The schemes disclosed in the patent documents still follow the detection methods and standards of Chinese pharmacopoeia, and naturally cannot solve the problems. Other research institutions also adopt two-dimensional liquid chromatography-mass spectrometry detection methods to control polymer impurities in cefmetazole and preparations thereof, and although detection results can be improved to a certain extent, the methods have high cost, are only suitable for theoretical research and are not suitable for daily detection quality control of medicines.
Aiming at the current situation, a new more accurate and sensitive daily detection and analysis method for the content of the polymer impurities of the cefmetazole and the preparation thereof is necessary to be established, so that the cefmetazole polymer impurities can be effectively separated and detected, the content of the cefmetazole and the preparation thereof is effectively controlled, and the quality of subsequent products is ensured and the clinical medication safety is ensured especially in daily monitoring in the production, transportation and quality inspection processes.
Disclosure of Invention
The invention aims to establish an HPLC analysis method for the cefmetazole and the polymer impurities in the preparation thereof, which has the advantages of good separation degree, simplicity, rapidness, strong specificity and high sensitivity, and can better control the quality of the cefmetazole and the polymer impurities in the preparation thereof, thereby ensuring the quality of the product and the medication safety.
For this purpose, the invention adopts the following specific technical scheme:
an HPLC detection method for polymer impurities in cefmetazole and a preparation thereof comprises the steps of separating and measuring the polymer impurities in the cefmetazole and the preparation thereof by using a High Performance Liquid Chromatography (HPLC), wherein the polymer impurities are dimers, trimers and polymers formed by the polymerization of the cefmetazole and the polymerization between the cefmetazole and degradation products, and the HPLC takes spherical hydrophilic silica gel as a filling agent and takes buffer salt-organic phase solution as a mobile phase.
Preferably, the buffer system in the mobile phase is selected from disodium hydrogen phosphate-sodium dihydrogen phosphate, citric acid-sodium citrate, acetic acid-sodium acetate buffer systems. More preferably, the buffer system in the mobile phase is a mixed solution composed of 45-55% by volume of disodium hydrogen phosphate solution and 55-45% by volume of sodium dihydrogen phosphate solution. More preferably, the organic phase in the mobile phase is selected from acetonitrile, acetone, methanol or ethanol, in a ratio of 45-55:55-45 with the buffer salt solution.
Preferably, the filler is hydrophilic silica gel for spherical protein chromatography with molecular weight application range of 1000-10000. Preferably, the column temperature in the detection method is 20-30 ℃.
Preferably, the detection wavelength in the detection method is 254+ -5 nm.
Preferably, the flow rate of the mobile phase in the detection method is 0.4-0.8mL/min.
Most preferably, the invention adopts an Shimadzu LC-20AT chromatograph and a TSK-GEL SW chromatographic column, takes a mixed solution of 0.005mol/L phosphate buffer solution and acetonitrile 50:50 as a mobile phase, and is carried out under the conditions of a flow rate of 0.6mL/min, a column temperature of 25 ℃ and a detection wavelength of 254 nm.
Due to the adoption of the impurity analysis method, the cefmetazole preparation has the characteristics of good separation degree, simplicity, rapidness, strong specificity and high sensitivity, can better control the quality of the cefmetazole and the polymer impurities of the cefmetazole preparation, and ensures the quality of the whole product and the clinical medication safety. Therefore, the method can be used as a monitoring means for the polymer impurities in cefmetazole and the preparation thereof.
The beneficial effects of the invention are as follows:
the HPLC analysis method for the cefmetazole and the polymer impurities in the preparation thereof, which is established by the invention, has the advantages of good separation degree, simplicity, rapidness, strong specificity and high sensitivity, can better control the quality of the cefmetazole and the polymer impurities in the preparation thereof, and ensures the quality of the product and the medication safety.
The inventor groups find that the polymer quality standard controlled by the novel HPLC analysis method can conveniently test the content of the polymer impurities in real time and on site in the processes of preparation, storage, transportation, use and the like of cefmetazole and the preparation thereof, and the detected content of the polymer impurities is more accurate. Compared with the quality control of polymer impurities by the molecular exclusion chromatography of Chinese pharmacopoeia in the prior art, the preparation which is qualified according to the analysis method is more accurate, sensitive and convenient, and is beneficial to reducing adverse reactions such as allergy and the like in the use process.
Drawings
In order to more clearly illustrate and explain the technical solutions of the present invention, the following brief description will be given of the drawings that are required to be used in connection with the detailed description. It will be apparent to those skilled in the art that the accompanying drawings, which are included to illustrate and describe certain embodiments of the invention, are intended to provide a person skilled in the art with the benefit of the description and the claims, without affecting the practice of the invention.
FIG. 1 shows the correlation of cefmetazole polymer impurity and the like, which has a retention time of about 15min or more, as located by a system suitability profile of a mixed solution of 0.005mol/L phosphate buffer solution [0.005mol/L disodium hydrogen phosphate solution-0.005 mol/L sodium dihydrogen phosphate solution (50:50) ] and acetonitrile 50:50 as a mobile phase in example 1 of the present invention.
FIG. 2 shows a system suitability graph of example 2 of the present invention after adjusting the mobile phase to a mixture of 0.005mol/L phosphate buffer [0.005mol/L disodium hydrogen phosphate solution-0.005 mol/L sodium dihydrogen phosphate solution (45:55) ] and acetonitrile 50:50.
FIG. 3 shows a system suitability graph of example 3 of the present invention after adjusting the mobile phase to a mixture of 0.005mol/L phosphate buffer [0.005mol/L disodium hydrogen phosphate solution-0.005 mol/L sodium dihydrogen phosphate solution (55:45) ] and acetonitrile 50:50.
Detailed Description
In order to further illustrate the embodiments of the present invention, those skilled in the art will be able to more clearly understand the embodiments of the present invention in conjunction with the drawings provided above in the specification. It will be apparent to those skilled in the art that these examples are merely illustrative of specific embodiments of the invention and do not limit the scope of the invention in any way. Those skilled in the art will appreciate that other possible embodiments and various equivalent alternatives, combinations, etc. of the various devices, reagents, test parameters, etc. used in the following examples are included within the scope of the present invention without undue burden. The specific protection scope of the invention is subject to the claims.
The high performance liquid chromatograph of the invention preferably uses a product LC-20AT of Shimadzu corporation, the transfusion mode is serial double plungers, the plunger capacity is 47 Mul of the main pump head and 23 Mul of the auxiliary pump head, the maximum liquid discharge pressure is 40MPa, the flow setting range is 0.001mL/min-10.000mL/min, the flow accuracy is 2% or less than the larger value (0.01 mL/min-5 mL/min) in 2 MuL/min, and the flow precision is below 0.3. The invention may also be practiced using other high performance liquid chromatographs of comparable performance available to those skilled in the art.
As the column of the present invention, a TSK-GEL G2000SWxl column from Tosoh corporation, tosoh, japan is preferably used. The packing of TSK-GEL SW series chromatographic column is made up by using rigid spherical silica GEL as matrix, and chemically bonding hydrophilic group on its surface by means of covalent bond, and said packing possesses low adsorptivity and good pore size distribution, its pH is suitable for being 2.5-7.5, and can use organic solvent which is completely mutually soluble with water, such as acetonitrile, acetone, methyl alcohol or ethyl alcohol, etc.. The specification of the TSK-GEL G2000SWxl chromatographic column selected by the invention is 7.8mm multiplied by 30cm multiplied by 5 mu m, and the molecular weight application range of the filler spherical hydrophilic silica GEL is 1000-10000. The invention may also be practiced with other chromatographic columns of comparable performance available to those skilled in the art.
The invention specifically uses 0.005mol/L phosphate buffer solution (0.005 mol/L disodium hydrogen phosphate solution-0.005 mol/L sodium dihydrogen phosphate solution) as buffer solution of mobile phase, and mixes the buffer solution with organic phase as mobile phase. According to the technical scheme of the invention, buffer salt is required to be added into the mobile phase, the reason is that the polymer impurities contain carboxyl groups, and the buffer salt is added into the mobile phase to enhance ionization of the detected object and improve the solubility, so that the separation is facilitated. The buffer salt additive in the mobile phase may be anhydrous disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, acetic acid and sodium acetate buffer systems, or any other suitable buffer system available to those skilled in the art. Preferably, the buffer salt is a combination of disodium hydrogen phosphate and sodium dihydrogen phosphate.
According to one embodiment of the present invention, the concentration of the disodium hydrogen phosphate solution and the sodium dihydrogen phosphate solution is 0.001mol/L to 0.009mol/L. Preferably, the concentration of the disodium hydrogen phosphate solution and the sodium dihydrogen phosphate solution is 0.005mol/L, for example, obtained by mixing 0.005mol/L of the disodium hydrogen phosphate solution with 0.005mol/L of the sodium dihydrogen phosphate solution in a ratio of 50:50. According to another embodiment of the invention, the ratio of disodium hydrogen phosphate solution to sodium dihydrogen phosphate solution in the mobile phase may be 45-55:55-45.
The organic phase used in the mobile phase of the present invention may be selected from acetonitrile, acetone, methanol or ethanol. Preferably, the organic phase is acetonitrile. More preferably, the ratio of phosphate buffer to acetonitrile in the mobile phase is 45-55:55-45, most preferably 50:50.
In chromatographic conditions, the detection wavelength of the HPLC of the invention is 254+ -5 nm, preferably 254nm; the flow rate of the mobile phase is 0.4-0.8mL/min, preferably 0.6mL/min; the column temperature of the chromatographic column is 20-30deg.C, preferably 25deg.C.
Other operation conditions not specifically described are available, and those skilled in the art can select appropriate conditions according to the specifications and the specific conditions of the instruction manual or the instruction manual.
Unless otherwise indicated, all ratios or percentages referred to herein are by volume or percent by volume. When expressed in percent form, it is understood that the two materials are mixed in the stated ranges of volume percentages and the sum of the two volume percentages is 100%; when expressed in a proportional form, it is understood that the two materials are mixed in the stated range of parts by volume and the sum of the parts by volume is 100. The two terms are used interchangeably and are meant to be identical.
Example 1
Instrument and conditions
High performance liquid chromatograph: shimadzu LC-20AT
Chromatographic column: TSK-GEL G2000SWxl,7.8 mm. Times.30 cm. Times.5 μm
Flow rate: 0.6mL/min
Detection wavelength: 254nm
Column temperature: 25 DEG C
Sample injection amount: 20 mu L
Mobile phase: 0.005mol/L phosphate buffer [0.005mol/L disodium hydrogen phosphate solution-0.005 mol/L sodium dihydrogen phosphate solution (50:50), acetonitrile; the phosphate buffer solution and acetonitrile are mixed according to the proportion of 50:50 to obtain a mobile phase.
The experimental steps are as follows:
step 1: solution preparation
System applicability: taking 10ml of a sample solution, adding 1ml of 0.1mol/L sodium hydroxide solution, standing for 1 minute at room temperature, adding 1ml of 0.1mol/L hydrochloric acid solution, and shaking uniformly to obtain a system applicability solution. The separation degree between the cefmetazole peak and the front adjacent degradation impurity peak meets the requirement.
Test solution: taking a proper amount of the product, precisely weighing, adding a mobile phase for dissolution and quantitatively diluting to prepare a solution containing about 1mg of cefmetazole in each 1ml, and taking the solution as a test sample solution.
Control solution: taking a proper amount of cefmetazole reference substance, precisely weighing, adding a mobile phase for dissolution, and quantitatively diluting to prepare a solution containing about 15 mug in each 1 ml.
Sensitivity solution: a suitable amount of control solution was precisely measured and quantitatively diluted with mobile phase to give a solution of about 0.1. Mu.g per 1 ml.
Step 2: measurement
Precisely measuring 20 μl of each of the sample solution and the control solution, respectively injecting into a liquid chromatograph, and recording the chromatograms; if impurity peaks exist in the chromatogram of the test solution, the impurity peaks in the reference solution are calculated according to an external standard method, the retention time is less than 1.5% of the total amount of the impurity peaks of the cefmetazole peaks, and the peaks smaller than the main peak area of the sensitivity test solution in the chromatogram of the test solution are ignored.
A typical system applicability map of example 1 is shown in fig. 1. .
Example 2
Instrument and conditions
High performance liquid chromatograph: shimadzu LC-20AT
Chromatographic column: TSK-GEL G2000SWxl,7.8 mm. Times.30 cm. Times.5 μm
Flow rate: 0.6mL/min
Detection wavelength: 254nm
Column temperature: 25 DEG C
Sample injection amount: 20 mu L
Mobile phase: 0.005mol/L phosphate buffer [0.005mol/L disodium hydrogen phosphate solution-0.005 mol/L sodium dihydrogen phosphate solution (45:55), acetonitrile; the phosphate buffer solution and acetonitrile are mixed according to the proportion of 50:50 to obtain a mobile phase.
The experimental steps are as follows:
step 1 solution preparation
System applicability: taking 10ml of a sample solution, adding 1ml of 0.1mol/L sodium hydroxide solution, standing for 1 minute at room temperature, adding 1ml of 0.1mol/L hydrochloric acid solution, and shaking uniformly to obtain a system applicability solution. The separation degree between the cefmetazole peak and the front adjacent degradation impurity peak meets the requirement.
Test solution: taking a proper amount of the product, precisely weighing, adding a mobile phase for dissolution and quantitatively diluting to prepare a solution containing about 1mg of cefmetazole in each 1ml, and taking the solution as a test sample solution.
Control solution: taking a proper amount of cefmetazole reference substance, precisely weighing, adding a mobile phase for dissolution, and quantitatively diluting to prepare a solution containing about 15 mug in each 1 ml.
Sensitivity solution: a suitable amount of control solution was precisely measured and quantitatively diluted with mobile phase to give a solution of about 0.1. Mu.g per 1 ml.
Step 2: measurement
Precisely measuring 20 μl of each of the sample solution and the control solution, respectively injecting into a liquid chromatograph, and recording the chromatograms; if impurity peaks exist in the chromatogram of the test solution, the impurity peaks in the reference solution are calculated according to an external standard method, the retention time is less than 1.5% of the total amount of the impurity peaks of the cefmetazole peaks, and the peaks smaller than the main peak area of the sensitivity test solution in the chromatogram of the test solution are ignored.
A typical system applicability map of example 2 is shown in fig. 2. .
Example 3
Instrument and conditions
High performance liquid chromatograph: shimadzu LC-20AT
Chromatographic column: TSK-GEL G2000SWxl,7.8 mm. Times.30 cm. Times.5 μm
Flow rate: 0.6mL/min
Detection wavelength: 254nm
Column temperature: 25 DEG C
Sample injection amount: 20 mu L
Mobile phase: 0.005mol/L phosphate buffer [0.005mol/L disodium hydrogen phosphate solution-0.005 mol/L sodium dihydrogen phosphate solution (55:45), acetonitrile; the phosphate buffer solution and acetonitrile are mixed according to the proportion of 50:50 to obtain a mobile phase.
The experimental steps are as follows:
step one: solution preparation
System applicability: taking 10ml of a sample solution, adding 1ml of 0.1mol/L sodium hydroxide solution, standing for 1 minute at room temperature, adding 1ml of 0.1mol/L hydrochloric acid solution, and shaking uniformly to obtain a system applicability solution. The separation degree between the cefmetazole peak and the front adjacent degradation impurity peak meets the requirement.
Test solution: taking a proper amount of the product, precisely weighing, adding a mobile phase for dissolution and quantitatively diluting to prepare a solution containing about 1mg of cefmetazole in each 1ml, and taking the solution as a test sample solution.
Control solution: taking a proper amount of cefmetazole reference substance, precisely weighing, adding a mobile phase for dissolution, and quantitatively diluting to prepare a solution containing about 15 mug in each 1 ml.
Sensitivity solution: a suitable amount of control solution was precisely measured and quantitatively diluted with mobile phase to give a solution of about 0.1. Mu.g per 1 ml.
Step two: measurement
Precisely measuring 20 μl of each of the sample solution and the control solution, respectively injecting into a liquid chromatograph, and recording the chromatograms; if impurity peaks exist in the chromatogram of the test solution, the impurity peaks in the reference solution are calculated according to an external standard method, the retention time is less than 1.5% of the total amount of the impurity peaks of the cefmetazole peaks, and the peaks smaller than the main peak area of the sensitivity test solution in the chromatogram of the test solution are ignored.
A typical system applicability map of example 3 is shown in fig. 3.
Example 4
The polymer impurity detection results obtained after the acceleration test of a sample of the existing cefmetazole product marked to be qualified under the same condition are compared according to the molecular exclusion color-spill method recorded in the Chinese pharmacopoeia 2020 edition and the method described in the embodiment 1 of the present invention, and the results are shown in table 1.
TABLE 1 comparison of Polymer impurity detection results for accelerated test samples
| Detection method | 0 month | Accelerating for 3 months | Accelerating for 6 months |
| Method of Chinese pharmacopoeia 2020 edition | 0.007％ | 0.008％ | 0.009％ |
| Example 1 method of the invention | 0.67％ | 0.90％ | 1.00％ |
The result shows that the prior art method has too low separation degree of the cefmetazole and the polymer impurities of the preparation thereof, is not sensitive enough, can not accurately detect more polymer impurities actually existing, and has great hidden trouble on medication quality safety. The detection method can more sensitively separate and detect more polymer impurities actually existing, is beneficial to better controlling the quality of medicines and reduces side effects such as anaphylactic reaction of medicines.
The examples described above are only preferred embodiments of the present invention and do not limit the present invention in any way. Any modification, equivalent replacement, improvement, combination, etc. which can be made by those skilled in the art without creative efforts, are included in the protection scope of the present invention.
Claims (9)
1. An HPLC detection method for cefmetazole and polymer impurities in a preparation thereof is characterized by comprising the following steps: the polymer impurities in cefmetazole and the preparation thereof are separated and measured by using a High Performance Liquid Chromatography (HPLC), wherein the polymer impurities are dimers, trimers and polymers formed by cefmetazole self-polymerization and cefmetazole and degradation products polymerization, and the HPLC takes spherical hydrophilic silica gel as a filling agent and takes buffer salt-organic phase solution as a mobile phase.
2. The method for HPLC detection of cefmetazole and polymeric impurities in its formulations according to claim 1, wherein the buffer system in the mobile phase is selected from disodium hydrogen phosphate-sodium dihydrogen phosphate, citric acid-sodium citrate, acetic acid-sodium acetate buffer systems.
3. The method for HPLC detection of cefmetazole and polymeric impurities in its formulations according to claim 2, wherein the buffer system in the mobile phase is a mixture of 45-55% by volume of disodium hydrogen phosphate solution and 55-45% by volume of sodium dihydrogen phosphate solution.
4. A method for HPLC detection of cefmetazole and polymeric impurities in formulations thereof according to any of claims 1-3, characterized in that the organic phase in the mobile phase is selected from acetonitrile, acetone, methanol or ethanol in a ratio to buffer salt solution of 45-55:55-45.
5. The method for HPLC detection of cefmetazole and polymer impurity in its preparation according to any one of claims 1 to 4, wherein said filler is hydrophilic silica gel for spherical protein chromatography with molecular weight application range of 1000 to 10000.
6. The method for HPLC detection of cefmetazole and polymeric impurities in formulations thereof according to any of claims 1-5, wherein the column temperature in the detection method is 20-30 ℃.
7. The method for HPLC detection of cefmetazole and polymeric impurities in formulations thereof according to any of claims 1-6, wherein the detection wavelength in the detection method is 254±5nm.
8. The method for HPLC detection of cefmetazole and polymeric impurities in formulations thereof according to any of claims 1-7, wherein the flow rate of mobile phase in the detection method is 0.4-0.8mL/min.
9. The method for detecting the polymer impurities in cefmetazole and the preparation thereof according to any one of claims 1 to 8, wherein the detection is performed under conditions of a flow rate of 0.6mL/min, a column temperature of 25 ℃ and a detection wavelength of 254nm by using an island liquid LC-20AT chromatograph and a TSK-GEL SW chromatographic column and using a 50:50 mixture of 0.005mol/L phosphate buffer and acetonitrile as a mobile phase.
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