CN113533569A - Method for detecting related substances in antibacterial eye drops - Google Patents
Method for detecting related substances in antibacterial eye drops Download PDFInfo
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- CN113533569A CN113533569A CN202110799936.5A CN202110799936A CN113533569A CN 113533569 A CN113533569 A CN 113533569A CN 202110799936 A CN202110799936 A CN 202110799936A CN 113533569 A CN113533569 A CN 113533569A
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- 239000003889 eye drop Substances 0.000 title claims abstract description 68
- 229940012356 eye drops Drugs 0.000 title claims abstract description 63
- 239000000126 substance Substances 0.000 title claims abstract description 60
- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 title claims abstract description 54
- 239000012535 impurity Substances 0.000 claims abstract description 172
- 238000001514 detection method Methods 0.000 claims abstract description 51
- 239000000243 solution Substances 0.000 claims description 105
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- 238000004128 high performance liquid chromatography Methods 0.000 claims description 29
- 239000008055 phosphate buffer solution Substances 0.000 claims description 24
- 239000011259 mixed solution Substances 0.000 claims description 21
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 20
- SHFJWMWCIHQNCP-UHFFFAOYSA-M hydron;tetrabutylazanium;sulfate Chemical compound OS([O-])(=O)=O.CCCC[N+](CCCC)(CCCC)CCCC SHFJWMWCIHQNCP-UHFFFAOYSA-M 0.000 claims description 20
- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 229910019142 PO4 Inorganic materials 0.000 claims description 12
- 239000010452 phosphate Substances 0.000 claims description 12
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 12
- 229910001868 water Inorganic materials 0.000 claims description 12
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 claims description 11
- 238000010828 elution Methods 0.000 claims description 11
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 10
- 239000007924 injection Substances 0.000 claims description 10
- 238000002347 injection Methods 0.000 claims description 10
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 10
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 10
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000000945 filler Substances 0.000 claims description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- PARWUHTVGZSQPD-UHFFFAOYSA-N phenylsilane Chemical group [SiH3]C1=CC=CC=C1 PARWUHTVGZSQPD-UHFFFAOYSA-N 0.000 claims description 3
- 230000015556 catabolic process Effects 0.000 abstract description 33
- 238000006731 degradation reaction Methods 0.000 abstract description 33
- 229960005112 moxifloxacin hydrochloride Drugs 0.000 abstract description 27
- IDIIJJHBXUESQI-DFIJPDEKSA-N moxifloxacin hydrochloride Chemical compound Cl.COC1=C(N2C[C@H]3NCCC[C@H]3C2)C(F)=CC(C(C(C(O)=O)=C2)=O)=C1N2C1CC1 IDIIJJHBXUESQI-DFIJPDEKSA-N 0.000 abstract description 27
- 229960003702 moxifloxacin Drugs 0.000 abstract description 15
- FABPRXSRWADJSP-MEDUHNTESA-N moxifloxacin Chemical compound COC1=C(N2C[C@H]3NCCC[C@H]3C2)C(F)=CC(C(C(C(O)=O)=C2)=O)=C1N2C1CC1 FABPRXSRWADJSP-MEDUHNTESA-N 0.000 abstract description 15
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- 238000011084 recovery Methods 0.000 description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 239000000463 material Substances 0.000 description 13
- 229910052717 sulfur Inorganic materials 0.000 description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 9
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- 238000010525 oxidative degradation reaction Methods 0.000 description 7
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- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 6
- 239000004327 boric acid Substances 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
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- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 238000001782 photodegradation Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- WEXQOLCYKFJAJZ-ZUZCIYMTSA-N 7-[(4as,7as)-1,2,3,4,4a,5,7,7a-octahydropyrrolo[3,4-b]pyridin-6-yl]-1-cyclopropyl-6,8-difluoro-4-oxoquinoline-3-carboxylic acid Chemical compound C12=C(F)C(N3C[C@H]4NCCC[C@H]4C3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 WEXQOLCYKFJAJZ-ZUZCIYMTSA-N 0.000 description 1
- BZICMGNIHHJMQF-UHFFFAOYSA-N 7-[3-(3-aminopropyl)pyrrol-1-yl]-1-cyclopropyl-6-fluoro-8-methoxy-4-oxoquinoline-3-carboxylic acid Chemical compound NCCCC1=CN(C=C1)C1=C(C=C2C(C(=CN(C2=C1OC)C1CC1)C(=O)O)=O)F BZICMGNIHHJMQF-UHFFFAOYSA-N 0.000 description 1
- HFMKHBDEIOYPRL-UHFFFAOYSA-N 7-amino-1-cyclopropyl-6-fluoro-8-methoxy-4-oxoquinoline-3-carboxylic acid Chemical compound COC1=C(N)C(F)=CC(C(C(C(O)=O)=C2)=O)=C1N2C1CC1 HFMKHBDEIOYPRL-UHFFFAOYSA-N 0.000 description 1
- 108010054814 DNA Gyrase Proteins 0.000 description 1
- 108010041052 DNA Topoisomerase IV Proteins 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 208000001860 Eye Infections Diseases 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 238000010268 HPLC based assay Methods 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- 101710183280 Topoisomerase Proteins 0.000 description 1
- 108010046308 Type II DNA Topoisomerases Proteins 0.000 description 1
- 102000007537 Type II DNA Topoisomerases Human genes 0.000 description 1
- 229940101006 anhydrous sodium sulfite Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
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- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
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- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000006213 oxygenation reaction Methods 0.000 description 1
- LISFMEBWQUVKPJ-UHFFFAOYSA-N quinolin-2-ol Chemical compound C1=CC=C2NC(=O)C=CC2=C1 LISFMEBWQUVKPJ-UHFFFAOYSA-N 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 1
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Images
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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
- G01N30/26—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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
-
- 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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- 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)
- Investigating Or Analysing Biological Materials (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a method for detecting related substances in antibacterial eye drops, and belongs to the technical field of medicines. According to the method provided by the invention, the related substances in the antibacterial eye drops and the related substances can be well separated, and the separation degree is more than 1.5; the method provided by the invention has good specificity, and the peak purity of the main peak in the sample map after acid, alkali, oxidation, high temperature and light degradation is more than 995, which meets the requirement of drug registration standard; the method provided by the invention has high sensitivity, and the impurity concentration which can be effectively detected is lower than the report limit; according to the method provided by the invention, the moxifloxacin, the known impurities and the main degradation impurities show a good linear relationship in a certain concentration range; the method provided by the invention has good repeatability and high accuracy, can be used for quality control of related substances of moxifloxacin hydrochloride eye drops, and is remarkably superior to detection methods in United states pharmacopoeia and European pharmacopoeia.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a method for detecting related substances in antibacterial eye drops.
Background
Moxifloxacin hydrochloride as a novel quinolone medicine has a wide antibacterial spectrum, maintains antibacterial power on gram-negative bacteria, enhances antibacterial power on gram-positive bacteria, has very strong antibacterial power, and can inhibit topoisomerase II (DNA gyrase) and topoisomerase IV. Topoisomerases are enzymes that control DNA topology and are critical in DNA replication, repair and transcription. In addition, the transfer into the eye tissue is good, and drug-resistant bacteria are not easily found, and this is considered to be a great advantage in treating ocular infections. The chemical name of moxifloxacin hydrochloride is as follows: 1-cyclopropyl-6-fluoro-7- ((4aS,7aS) -1H- [3,4-b ] hexahydropyrido-dihydropyrrole-6 (2H)) -8-methoxy-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid hydrochloride having the following structural formula:
at present, no related substance control method of moxifloxacin hydrochloride eye drops exists in Chinese pharmacopoeia, the imported drug registration standard (standard number: JX20160008) and the United states pharmacopoeia (USP41-NF36) adopts two methods to detect related substances in the moxifloxacin hydrochloride eye drops so as to control impurities, and the method is too complicated. The European pharmacopoeia (EP9.0) only collects the detection methods of the relevant substances of the raw material medicaments of moxifloxacin hydrochloride, and both the methods have the problems of small separation degree, low theoretical plate number, interference of solvent peaks on the measurement of the relevant substances and the like, and are also not suitable for the quality control of the moxifloxacin hydrochloride eye drops. Therefore, the development of a related substance detection method aiming at the moxifloxacin hydrochloride eye drops has important significance on the quality control of the moxifloxacin hydrochloride eye drops.
Chinese patent CN201810446597.0 discloses a method for detecting moxifloxacin hydrochloride related substances by high performance liquid chromatography, wherein a chromatographic column with phenyl bonded silica gel as a filler is selected, and the column temperature is set to be 37 +/-1 ℃; the mobile phase A is phosphate buffer solution-methanol with the volume ratio of 80:20, the mobile phase B is phosphate buffer solution-methanol with the volume ratio of 20:80, and gradient elution is adopted; the phosphate buffer solution contains tetrabutylammonium hydrogen sulfate, potassium dihydrogen phosphate and phosphoric acid, and a gradient elution mode is selected. However, the detection method has poor detection specificity for a system degraded by acid, alkali, light, oxidation, high temperature and the like.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for detecting a related substance in an antibacterial eye drop, which is excellent in specificity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for detecting related substances in antibacterial eye drops, which adopts high performance liquid chromatography to detect the antibacterial eye drops;
the detection conditions of the high performance liquid chromatography comprise: the chromatographic column is a chromatographic column taking phenyl silane bonded silica gel as a filler, and the column temperature is 42-48 ℃; the mobile phase A is a mixed solution of a first phosphate buffer solution and methanol, and the volume ratio of a phosphate solution to the methanol in the mixed solution of the first phosphate buffer solution and the methanol is 72: 28; the mobile phase B is a mixed solution of a second phosphate buffer solution and methanol, and the volume ratio of a phosphate solution to the methanol in the mixed solution of the second phosphate buffer solution and the methanol is 25: 75; the flow rate of the mobile phase A and the mobile phase B is 1-1.5 mL/min; the phosphate solution comprises tetrabutylammonium hydrogen sulfate, potassium dihydrogen phosphate, phosphoric acid and water; the procedure of gradient elution is as follows:
| time (min) | Mobile phase a (% by volume) | Mobile phase B (% by volume) |
| 0 | 100 | 0 |
| 25 | 100 | 0 |
| 65 | 0 | 100 |
| 66 | 100 | 0 |
| 75 | 100 | 0 |
Preferably, the related substances comprise one or more of impurities A, B, C, D, E, RC-3, ACB-9, RC-1, F, I, ACB-8 and ACB-10.
Preferably, the detection conditions of the high performance liquid chromatography further comprise a detection wavelength of 210 nm; the sample injection amount is 10-30 mu L.
Preferably, the concentration of tetrabutylammonium hydrogen sulfate in the phosphate buffer solution in the first phosphate buffer solution-methanol mixed solution and the second phosphate buffer solution-methanol mixed solution is 1.36g/L, the concentration of potassium dihydrogen phosphate is 1g/L, and the concentration of phosphoric acid is 2 mL/L;
the pH value of the phosphate buffer solution is 6-7.
The invention provides a method for detecting related substances in antibacterial eye drops. According to the method provided by the invention, the related substances in the antibacterial eye drops and the related substances can be well separated, and the separation degree is more than 1.5; the method provided by the invention has good specificity, and the peak purity of the main peak in the sample map after acid, alkali, oxidation, high temperature and light degradation is more than 995, which meets the requirement of drug registration standard; the method provided by the invention has high sensitivity, and the impurity concentration which can be effectively detected is lower than the report limit; according to the method provided by the invention, the moxifloxacin, the known impurities and the main degradation impurities show a good linear relationship in a certain concentration range; the method provided by the invention has good repeatability and high accuracy, can be used for quality control of related substances of moxifloxacin hydrochloride eye drops, and is remarkably superior to detection methods in United states pharmacopoeia and European pharmacopoeia.
Drawings
FIG. 1 is a high performance liquid chromatogram of a blank solvent;
FIG. 2 is a high performance liquid chromatogram of a blank adjuvant solution;
FIG. 3 is a high performance liquid chromatogram of a system suitability solution;
FIG. 4 is a high performance liquid chromatogram of a mixed control solution;
FIG. 5 is a high performance liquid chromatogram of a test solution;
FIG. 6 is a high performance liquid chromatogram of a blank adjuvant for strong acid degradation treatment;
FIG. 7 is a high performance liquid chromatogram of a sample solution subjected to strong acid degradation treatment;
FIG. 8 is a high performance liquid chromatogram of a blank adjuvant solution subjected to strong base degradation treatment;
FIG. 9 is a high performance liquid chromatogram of a test solution subjected to strong base degradation treatment;
FIG. 10 is a high performance liquid chromatogram of an oxidative degradation treatment blank adjuvant solution;
FIG. 11 is a high performance liquid chromatogram of a test solution subjected to oxidative degradation treatment;
FIG. 12 is a high performance liquid chromatogram of a blank adjuvant solution subjected to high temperature degradation;
FIG. 13 is a high performance liquid chromatogram of a high temperature degradation treatment sample solution;
FIG. 14 is a high performance liquid chromatogram of a blank adjuvant solution subjected to photodegradation treatment;
FIG. 15 is a high performance liquid chromatogram of a sample solution subjected to photodegradation treatment;
FIG. 16 is a high performance liquid chromatogram of relevant substances of commercially available moxifloxacin hydrochloride eye drops;
FIG. 17 is a high performance liquid chromatogram of the test solution in comparative example 1.
Detailed Description
The invention provides a method for detecting related substances in antibacterial eye drops, which adopts high performance liquid chromatography to detect the antibacterial eye drops.
In the present invention, all the raw material components are commercially available products well known to those skilled in the art unless otherwise specified.
The antibacterial eye drops of the present invention are not particularly limited, and are preferably commercially available moxifloxacin hydrochloride eye drops or home-made antibacterial eye drops. In the invention, the self-made antibacterial eye drops preferably comprise moxifloxacin, auxiliary materials and water; the auxiliary materials preferably comprise sodium chloride, boric acid and a pH regulator; the pH regulator preferably comprises sodium hydroxide and/or hydrochloric acid; the concentration of the moxifloxacin in the antibacterial eye drop is preferably 4-6 mg/mL, more preferably 4.5-5.5 mg/mL, and further preferably 5 mg/mL; the content of the sodium chloride is preferably 0.8-1.0 wt%, more preferably 0.85-0.95 wt%, and further preferably 0.9 wt%; the content of the boric acid is preferably 0.2-0.4 wt%, more preferably 0.25-0.35 wt%, and further preferably 0.3 wt%; the content of the pH regulator is not particularly limited, and the pH value of the antibacterial eye drop can be ensured to be 6.3-7.3, more preferably 6.5-7, and further preferably 6.7-6.8. In the present invention, the water is preferably water for injection.
In the invention, the detection of related substances in the antibacterial eye drops by adopting the high performance liquid chromatography specifically comprises the following steps: performing high performance liquid chromatography detection on related substances in the antibacterial eye drops to obtain related substance chromatograms, and obtaining peak areas of the related substances according to the related substance chromatograms; calculating the content of related substances in the antibacterial eye drops according to the peak area and the linear curve; the linear curve is a linear curve of the chromatographic peak area of the relevant substance and the concentration of the relevant substance.
According to the invention, related substances in the antibacterial eye drops are subjected to high performance liquid chromatography detection to obtain related substance chromatograms, and peak areas of the related substances are obtained according to the related substance chromatograms.
In the present invention, the detection conditions of the high performance liquid chromatography include: the chromatographic column is a chromatographic column taking Phenyl silane bonded silica gel as a filler, preferably Agilent Eclipse XDB-Phenyl, and the parameters of the chromatographic column are preferably 4.6mm multiplied by 250mm and 5 mu m; the column temperature is 42-48 ℃, preferably 43-47 ℃, more preferably 44-46 ℃, and further preferably 45 ℃; the mobile phase A is a mixed solution of a first phosphate buffer solution and methanol, and the volume ratio of a phosphate solution to the methanol in the mixed solution of the first phosphate buffer solution and the methanol is 72: 28; the mobile phase B is a mixed solution of a second phosphate buffer solution and methanol, and the volume ratio of a phosphate solution to the methanol in the mixed solution of the second phosphate buffer solution and the methanol is 25: 75; the flow rate of the mobile phase A and the mobile phase B is 1-1.5 mL/min, preferably 1.1-1.4 mL/min, and more preferably 1.3 mL/min; the phosphate solution in the mixed solution of the first phosphate buffer solution and the methanol and the mixed solution of the second phosphate buffer solution and the methanol comprises tetrabutylammonium hydrogen sulfate, monopotassium phosphate, phosphoric acid and water, the concentration of the tetrabutylammonium hydrogen sulfate in the phosphate buffer solution is preferably 1.36g/L, the concentration of the monopotassium phosphate is preferably 1g/L, and the concentration of the phosphoric acid is preferably 2 mL/L; the pH value of the phosphate buffer solution is preferably 6-7, and preferably 6.7; the detection wavelength is preferably 210 nm; the amount of the sample is preferably 10 to 30. mu.L, more preferably 15 to 25. mu.L, still more preferably 18 to 22. mu.L, and most preferably 20. mu.L.
In the present invention, the elution mode of the high performance liquid chromatography is gradient elution, and the procedure of the gradient elution is shown in table 1:
TABLE 1 procedure for gradient elution
| Time (min) | Mobile phase a (% by volume) | Mobile phase B (% by volume) |
| 0 | 100 | 0 |
| 25 | 100 | 0 |
| 65 | 0 | 100 |
| 66 | 100 | 0 |
| 75 | 100 | 0 |
In the invention, the related substances preferably comprise one or more of impurities A, B, C, D, E, I, RC-3, ACB-9, RC-1, F, ACB-8 and ACB-10; the impurity A is 1-cyclopropyl-7- { (S, S) -2, 8-diazabicyclo [4.3.0] non-8-yl } -6, 8-difluoro-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid; the impurity B is 1-cyclopropyl-7- { (S, S) -2, 8-diazabicyclo ] [4.3.0] non-8-yl } -6, 8-dimethoxy-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid; impurity C is 1-cyclopropyl-7- { (S, S) -2, 8-diazabicyclo ] [4.3.0] non-8-yl } -6-fluoro-8-ethoxy-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid; the impurity D is 1-cyclopropyl-7- { (S, S) -2, 8-diazabicyclo ] [4.3.0] non-8-yl } -6-methoxy-8-fluoro-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid; the impurity E is 1-cyclopropyl-7- { (S, S) -2, 8-diazabicyclo ] [4.3.0] non-8-yl } -6-fluoro-8-hydroxy-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid; the impurity F is 1-cyclopropyl-7- { (S, S) -2-methyl-2, 8-diazabicyclo ] [4.3.0] nonan-8-yl } -6-fluoro-8-methoxy-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid; the impurity I is 1-cyclopropyl-6-fluoro-8-methoxy-7- [3- (3-aminopropyl) -1H-pyrrol-1-yl ] -4-oxo-1, 4-dihydroquinoline-3-carboxylic acid; the impurity ACB-8 is 1-cyclopropyl-7- { (S, S-3-oxo-2, 8-diazabicyclo ] [4.3.0] non-8-yl) } -6-fluoro-8-methoxy-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid; the impurity ACB-9 is 1-cyclopropyl-7- { (S, S) -2, 8-diazabicyclo ] [4.3.0] nonan-8-yl } -6-fluoro-8-methoxy-1, 4-dihydro-4-oxo-quinoline; the impurity ACB-10 is 1-cyclopropyl-6-fluoro-7-amino-8-methoxy-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid; the impurity RC-1 is 1-cyclopropyl-6-fluoro-8-methoxy-7- { (4S,7S) -octahydro-6H-pyrrolo [3,4-b ] pyridin-6-yl } -4-oxo-1, 4-dihydroquinoline-3-carboxylic acid; the impurity RC-3 is 1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-7- { (4S,7S) -5-oxooctahydro-6H-pyrrolo [3,4-b ] pyridin-6-yl } -4-1, 4-dihydroquinoline-3-carboxylic acid.
After the peak area is obtained, the content of related substances in the antibacterial eye drops is calculated according to the peak area and the linear curve; the linear curve is a linear curve of the chromatographic peak area of the relevant substance and the concentration of the relevant substance.
In the present invention, the preparation method of the linear relationship graph preferably comprises the following steps: preparing solutions of related substance series reference substances with different concentrations, performing high performance liquid chromatography detection on the solutions of the related substance series reference substances with different concentrations to obtain peak areas of the related substances, performing linear fitting on the peak areas of the related substances and the concentrations of the related substances, and performing linear regression by taking the peak areas as vertical coordinates and the concentrations as horizontal coordinates to obtain a linear relation graph.
In the present invention, the concentrations of the related substance in the solutions of the related substance series control with different concentrations are preferably the limit concentration of quantitation, 0.5. mu.g/mL, 0.8. mu.g/mL, 1.0. mu.g/mL and 1.5. mu.g/mL.
In the present invention, the quantitative limit concentration is preferably obtained by: preparing a series of related substance reference substance solutions, and carrying out high performance liquid chromatography detection on the series of related substance reference substance solutions; the concentration of the reference substance of interest in the solution is preferably used as the limit concentration for quantitation at a signal-to-noise ratio of 10.
In the present invention, the linear curve results are shown in table 2:
TABLE 2 Linear Curve results for related substances
After the peak area and the linear curve are obtained, the content of related substances in the antibacterial eye drops is calculated according to the peak area and the linear curve. In the invention, when calculating the total impurity content, the known impurities are calculated according to an external standard method, other unknown impurities are calculated according to a main component self-comparison method, and the calculation formula is as follows:
wherein: a. theSample (A)Test articlePeak areas of known impurities in the solution chromatogram; wTo pair-impurity control weighing (mg); dr-dilution factor of the control; p is the content of impurity reference substance; a. theTo pair-peak area of the main peak in each control solution chromatogram; dt-dilution factor of the test article; a. theSingle impurity of unknown maximum-peak area of unknown maximum single impurity in chromatogram of test solution; sTo pair-peak area of the main peak in the control solution chromatogram; a. theGeneral assemblyAnd the sum of the areas of the peaks of the unknown impurities in the chromatogram of the test solution.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. Experimental sample
The control information is shown in table 3:
TABLE 3 control information
(1.1) blank solvent: adding 1.36g of tetrabutylammonium hydrogen sulfate, 1.0g of monopotassium phosphate and 0.02g of anhydrous sodium sulfite into 500mL of water for dissolving, adding 2mL of phosphoric acid, diluting with water to 1000mL, and uniformly mixing to obtain a blank solvent; the solvents mentioned in the examples are all the blank solvents.
(1.2) reference stock solution
Solution of impurity A: accurately weighing 10mg of the reference substance of the impurity A, placing the reference substance in a 100mL measuring flask, diluting the reference substance to the scale with a solvent, and shaking up to obtain a solution of the impurity A.
Solution of impurity F: accurately weighing 10mg of the impurity F reference substance, placing the reference substance in a 100mL measuring flask, diluting the reference substance to the scale with a solvent, and shaking up to obtain an impurity F solution.
Impurity a stock solution: accurately weighing 10mg of the reference substance of the impurity A, placing the reference substance in a 100mL measuring flask, diluting the reference substance with a solvent to a scale, shaking up, accurately weighing 5mL of the reference substance from the middle, placing the reference substance in a 20mL measuring flask, and shaking up to obtain a stock solution of the impurity A.
Impurity F stock solution: accurately weighing 10mg of the impurity F reference substance, placing the reference substance in a 100mL measuring flask, diluting the reference substance to the scale with a solvent, shaking up, accurately weighing 5mL of the reference substance from the middle, placing the reference substance in a 20mL measuring flask, and shaking up to obtain an impurity F stock solution.
Impurity RC-3 stock solution: accurately weighing 10mg of an impurity RC-3 reference substance, placing the reference substance in a 100mL measuring flask, dissolving and diluting the reference substance to a scale with a solvent, shaking up, accurately weighing 5mL of the reference substance from the middle, placing the reference substance in a 20mL measuring flask, adding the solvent to dissolve and dilute the reference substance to the scale, and taking the reference substance as a stock solution of the impurity RC-3.
Impurity ACB-9 stock solution: precisely weighing 5mg of an ACB-9 impurity reference substance, placing the reference substance in a 50mL measuring flask, dissolving and diluting the reference substance to scale with a solvent, shaking up, precisely weighing 5mL of the reference substance from the middle, placing the reference substance in a 20mL measuring flask, adding the solvent to dissolve and dilute the reference substance to scale, and taking the reference substance as a stock solution of the ACB-9 impurity.
Impurity RC-1 stock solution: accurately weighing 10mg of an impurity RC-1 reference substance, placing the reference substance in a 100mL measuring flask, dissolving and diluting the reference substance to a scale with a solvent, shaking up, accurately weighing 5mL of the reference substance from the middle, placing the reference substance in a 20mL measuring flask, adding the solvent to dissolve and dilute the reference substance to the scale, and taking the reference substance as a stock solution of the impurity RC-1.
Impurity B stock solution: accurately weighing 5mg of the reference substance of the impurity B, placing the reference substance in a 50mL measuring flask, dissolving and diluting the reference substance to the scale with a solvent, shaking up, accurately weighing 5mL of the reference substance from the middle, placing the reference substance in a 20mL measuring flask, adding the solvent to dissolve and dilute the reference substance to the scale, and taking the reference substance as a stock solution of the impurity B.
Impurity C stock solution: precisely weighing 5mg of the impurity C reference substance, placing the reference substance in a 50mL measuring flask, dissolving and diluting the reference substance to a scale with a solvent, shaking up, precisely weighing 5mL from the middle, placing the reference substance in a 20mL measuring flask, adding the solvent to dissolve and dilute the reference substance to the scale, and taking the reference substance as a stock solution of the impurity C.
Impurity D stock solution: accurately weighing 5mg of the impurity D reference substance, placing the reference substance in a 50mL measuring flask, dissolving and diluting the reference substance to the scale with a solvent, shaking up, accurately weighing 5mL of the reference substance from the middle, placing the reference substance in a 20mL measuring flask, adding the solvent to dissolve and dilute the reference substance to the scale, and taking the reference substance as a stock solution of the impurity D.
Impurity E stock solution: precisely weighing 5mg of the impurity E reference substance, placing the reference substance in a 50mL measuring flask, dissolving and diluting the reference substance to the scale with a solvent, shaking up, precisely weighing 5mL of the reference substance from the middle, placing the reference substance in a 20mL measuring flask, adding the solvent to dissolve and dilute the reference substance to the scale, and taking the reference substance as a stock solution of the impurity E.
Impurity I stock solution: accurately weighing 5mg of the reference substance of the impurity I, placing the reference substance in a 50mL measuring flask, dissolving and diluting the reference substance to the scale by using a solvent, shaking up, accurately weighing 5mL of the reference substance from the middle, placing the reference substance in a 20mL measuring flask, adding the solvent to dissolve and dilute the reference substance to the scale, and taking the reference substance as a stock solution of the impurity I.
Impurity ACB-10 stock solution: accurately weighing 5mg of an impurity ACB-10 reference substance, placing the reference substance in a 10mL measuring flask, dissolving and diluting the reference substance to scale with N, N-dimethylformamide, shaking up, accurately weighing 2mL from the middle, placing the reference substance in the 10mL measuring flask, diluting the reference substance to scale with a solvent, shaking up, accurately weighing 5mL from the middle, placing the reference substance in a 20mL measuring flask, adding the solvent, dissolving and diluting to scale, and taking the reference substance as a stock solution of the impurity ACB-10.
Impurity ACB-8 stock solution: accurately weighing 5mg of an impurity ACB-8 reference substance, placing the reference substance in a 50mL measuring flask, adding 5mL of N, N-dimethylformamide to dissolve, diluting the reference substance to a scale with a solvent, shaking up, accurately weighing 5mL of the reference substance from the measuring flask, placing the reference substance in a 20mL measuring flask, adding the solvent to dissolve and dilute the reference substance to the scale, and taking the reference substance as a stock solution of the impurity ACB-8.
(1.3) mixing the reference solution: precisely measuring 1mL of each impurity stock solution, placing the impurity stock solution into a 25mL measuring flask, dissolving the impurity stock solution with a solvent, diluting the impurity stock solution to a scale mark, and shaking up to obtain a mixed reference substance solution, wherein the concentration of each impurity is 1 mu g/mL.
(1.4) System suitability solution: precisely weighing 11mg of moxifloxacin hydrochloride reference substance, precisely weighing 1mL of impurity A solution and 1mL of impurity F solution, putting the two solutions in a 10mL measuring bottle together, diluting the solution to a scale with a solvent, and shaking up to obtain a system applicability solution.
(1.5) antibacterial eye drops: uniformly mixing moxifloxacin, sodium chloride, boric acid and water, and adding sodium hydroxide and/or hydrochloric acid to adjust the pH value to 6.3-7.3 to obtain the antibacterial eye drops; wherein, the concentration of the moxifloxacin is 5mg/mL, the content of the sodium chloride is 0.9 wt%, and the content of the boric acid is 0.3 wt%.
(1.6) test solution: precisely measuring 5mL of the antibacterial eye drops, placing the antibacterial eye drops into a 25mL measuring flask, diluting the antibacterial eye drops to a scale with a solvent, and shaking up to obtain a test solution, wherein the concentration of the moxifloxacin is 1 mg/mL.
(1.7) blank adjuvant solution: uniformly mixing sodium chloride, boric acid and water, and adding sodium hydroxide and/or hydrochloric acid to adjust the pH value to 6.3-7.3 to obtain a blank auxiliary material solution; wherein, the content of sodium chloride is 0.9wt percent, and the content of boric acid is 0.3wt percent.
(1.8) bulk drug solution: dissolving a moxifloxacin hydrochloride raw material medicinal solvent, diluting, fixing the volume, and shaking up to obtain a raw material medicine solution, wherein the concentration of moxifloxacin is 5 mg/mL.
(2) Experimental methods
(2.1) detection conditions of the high performance liquid chromatography: the instrument is a FISHEU 3000 high performance liquid chromatograph;
a chromatographic column: agilent Eclipse XDB-Phenyl (250 mm. times.4.6 mm, 5 μm); mobile phase A: first phosphate buffer solution-methanol mixed solution (volume ratio: 72:28), mobile phase B: a second phosphate buffer solution-methanol mixed solution (volume ratio is 25:75), wherein the preparation method of the phosphate solution in the first phosphate buffer solution-methanol mixed solution and the second phosphate buffer solution-methanol mixed solution comprises the following steps: dissolving 1.36g of tetrabutylammonium hydrogen sulfate and 1.0g of potassium dihydrogen phosphate in 500mL, adding 2mL of phosphoric acid, uniformly mixing, diluting with water to 1000mL, and uniformly mixing; detection wavelength: 210 nm; flow rate: 1.3 mL/min; sample introduction amount: 10 mu L of the solution; column temperature: 45 ℃; the elution procedure is shown in table 1.
(2.2) carrying out high performance liquid chromatography detection on the blank solvent, wherein the high performance liquid chromatogram of the blank solvent is shown in figure 1; performing high performance liquid chromatography detection on the blank auxiliary material solution, wherein a high performance liquid chromatogram is shown in figure 2; performing high performance liquid chromatography detection on the system applicability solution, and performing continuous sample injection for 6 times, wherein the high performance liquid chromatogram is shown in FIG. 3, and the detection results of the continuous sample injection for 6 times are shown in Table 4; performing high performance liquid chromatography detection on the mixed control solution, wherein the high performance liquid chromatogram is shown in FIG. 4.
TABLE 4 detection results of 6 times of continuous solution sample introduction for system applicability
As can be seen from FIGS. 1 to 4 and Table 4, the separation degree of the main moxifloxacin peak in the solution with system applicability, the impurity F and the impurity A meets the requirement of system applicability, the peak shape is good, and no interference exists in the blank solvent. The method provided by the invention has higher system applicability; and the separation degree of each impurity is more than 1.5, so that the effective separation of each impurity can be realized.
Example 2
And (3) specificity verification: respectively carrying out acid, alkali, oxygen and illumination damage on a test solution, a blank solvent, a bulk drug solution and an auxiliary material solution, inspecting the degradation path of the moxifloxacin hydrochloride eye drops, and verifying the specificity of the detection method disclosed by the invention.
(1) Experimental methods
(1.1) strong acid destruction: precisely measuring 2mL of the antibacterial eye drops, placing the antibacterial eye drops into a 10mL measuring flask, adding 1mL of 1mol/L hydrochloric acid aqueous solution, carrying out strong acid degradation treatment at 80 ℃ for 8h, taking out the antibacterial eye drops, adding 1mL of 1mol/L sodium hydroxide aqueous solution for neutralization, cooling to room temperature, diluting with a solvent to a scale, and shaking up to obtain a test solution for strong acid degradation treatment; and replacing the antibacterial eye drops with a blank solvent by adopting the same treatment method to obtain the blank solvent for strong acid degradation treatment.
(1.2) strong alkali destruction: precisely measuring 2mL of the antibacterial eye drops, placing the antibacterial eye drops into a 10mL measuring flask, adding 1mL of 1mol/L sodium hydroxide aqueous solution, carrying out strong base degradation treatment at 80 ℃ for 8h, taking out the antibacterial eye drops, adding 1mL of 1mol/L hydrochloric acid aqueous solution for neutralization, cooling to room temperature, diluting with a solvent to a scale, and shaking up to obtain a test solution for strong base degradation treatment; and replacing the antibacterial eye drops with a blank auxiliary material solution by adopting the same treatment method to obtain the blank auxiliary material solution subjected to strong alkali degradation treatment.
(1.3) oxidative destruction: precisely measuring 2mL of the antibacterial eye drops, placing the antibacterial eye drops into a 10mL measuring flask, adding 1mL of 30 wt% aqueous hydrogen peroxide solution, carrying out oxidative degradation treatment at 80 ℃ for 8h, taking out the antibacterial eye drops, adding 1mL of 1mol/L aqueous hydrochloric acid solution for neutralization, cooling to room temperature, diluting with a solvent to a scale, and shaking up to obtain a test solution for oxidative degradation treatment; replacing the antibacterial eye drops with a blank adjuvant solution by adopting the same treatment method to obtain the blank adjuvant solution subjected to oxidative degradation treatment.
(1.4) high temperature destruction: precisely measuring 2mL of sample of the antibacterial eye drop, placing the sample in a 10mL measuring flask, sealing, placing the measuring flask in a water bath at 99 ℃ for high-temperature degradation treatment for 8h, taking out the sample, cooling to room temperature, diluting with a solvent to a scale, and shaking up to obtain a sample solution for high-temperature degradation treatment; and replacing the antibacterial eye drops with a blank adjuvant solution by adopting the same treatment method to obtain the blank adjuvant solution subjected to high-temperature degradation treatment.
(1.5) photo-disruption: precisely measuring 2mL of sample of the antibacterial eye drop, placing the sample in a 10mL measuring flask, sealing, performing strong light degradation treatment under the irradiation of 5000xl light source for 10 days, cooling to room temperature, diluting with solvent to scale, and shaking uniformly to obtain a test solution for light degradation treatment; and replacing the antibacterial eye drops with a blank adjuvant solution by adopting the same treatment method to obtain the blank adjuvant solution subjected to light degradation treatment.
(2) Test method
Performing sample injection detection on each sample according to the high performance liquid chromatography conditions of the embodiment 1, wherein high performance liquid chromatograms are shown in fig. 5-15 and table 5, wherein fig. 5 is the high performance liquid chromatogram of the test solution, fig. 6 is the high performance liquid chromatogram of the blank auxiliary material subjected to strong acid degradation treatment, fig. 7 is the high performance liquid chromatogram of the test solution subjected to strong acid degradation treatment, fig. 8 is the high performance liquid chromatogram of the blank auxiliary material solution subjected to strong base degradation treatment, and fig. 9 is the high performance liquid chromatogram of the test solution subjected to strong base degradation treatment; fig. 10 is a high performance liquid chromatogram of an oxidative degradation-treated blank adjuvant solution, fig. 11 is a high performance liquid chromatogram of an oxidative degradation-treated test sample solution, fig. 12 is a high performance liquid chromatogram of a high temperature degradation-treated blank adjuvant solution, fig. 13 is a high performance liquid chromatogram of a high temperature degradation-treated test sample solution, fig. 14 is a high performance liquid chromatogram of an illumination degradation-treated blank adjuvant solution, and fig. 15 is a high performance liquid chromatogram of an illumination degradation-treated test sample solution.
TABLE 5 results of the specificity experiments
a, note: material conservation ═ (total peak area of degraded sample/concentration of degraded sample)/(total peak area of undegraded sample/concentration of undegraded sample); the conservation range is 95-105%;
b, note: degree of separation of the main peak from its pre-or post-impurities;
c, note: a main peak purity of more than 995 indicates that the chromatographic peak is a single chromatographic peak.
As can be seen from Table 5 and FIGS. 5 to 15, the blank solvent and the auxiliary materials do not interfere with the detection of the related substances of the moxifloxacin hydrochloride eye drops; impurities generated under various strong degradation conditions are mainly impurity F and impurity ACB-10, the degradation degree of impurity D is maximum under the alkali destruction condition, the impurities adjacent to the main peak can be completely separated from the main peak, and the purity of the main peak is qualified; through calculating the balance of the materials after the damage, the result shows that the materials are conserved under each damage condition, and the method provided by the invention has good specificity.
Example 3
Detection of quantitation limit and detection limit
Diluting each reference stock solution prepared in the step (1.2) of the example 1 with the solvent prepared in the step (1.1) step by step; according to the high performance liquid chromatography condition sample injection detection of the embodiment 1, when the signal to noise ratio is 10, the concentrations of moxifloxacin hydrochloride, the impurity RC-3, the impurity ACB-9, the impurity RC-1, the impurity F, the impurity A, the impurity B, the impurity C, the impurity D, the impurity E, the impurity I, the impurity ACB-8 and the impurity ACB-10 are taken as quantitative limits; when the signal to noise ratio is 3, the concentrations of moxifloxacin, the impurity RC-3, the impurity ACB-9, the impurity RC-1, the impurity F, the impurity A, the impurity B, the impurity C, the impurity D, the impurity E, the impurity I, the impurity ACB-8 and the impurity ACB-10 are used as detection limits, and detection results of the quantitative limit and the detection limits are shown in table 6.
TABLE 6 quantitation Limit and detection Limit results
Note: the percentage of the test sample is the ratio of the detection concentration or the limit concentration of the related substances to the concentration of the test sample (the concentration of the moxifloxacin is 1 mg/mL).
As can be seen from Table 6, the quantitative limit concentrations of moxifloxacin hydrochloride and the related substances are both less than 0.2% of the concentration of the test sample, which indicates that the method provided by the invention can accurately and quantitatively detect the content of the related substances, thereby realizing the control of the quality of the antibacterial eye drops.
Example 4
Linear test
Respectively dissolving a reference substance of the impurity RC-3, the impurity ACB-9, the impurity RC-1, the impurity F, the impurity A, the impurity B, the impurity C, the impurity D, the impurity E, the impurity I, the impurity ACB-8 and the impurity ACB-10 by using the solvent prepared in the step (1.1), diluting and fixing the volume to obtain linear solutions of the impurities respectively; the linear solutions of each impurity were at the quantitation limit, 0.5. mu.g/mL, 0.8. mu.g/mL, 1.0. mu.g/mL, 1.5. mu.g/mL, respectively.
The linear solutions of each impurity were subjected to sample injection detection according to the conditions of high performance liquid chromatography of example 1, and a standard curve was drawn with the concentration as abscissa and the peak area of the high performance liquid chromatogram as ordinate to obtain a regression equation and a linear range, with the results shown in table 2. As can be seen from Table 2, when the method provided by the invention is used for detection, each impurity shows a good linear relationship within a certain concentration range.
Example 5
Accuracy test
(1) A sample to be tested:
(1.1) the test solution, wherein the concentration of the moxifloxacin is 1 mg/mL.
(1.2) solution preparation:
solution of impurity A: accurately weighing 5.08mg of the impurity A reference substance, placing the reference substance in a 50mL measuring flask, dissolving the reference substance with a solvent, diluting the reference substance to a scale, and shaking up;
solution of impurity B: accurately weighing 5.16mg of the reference substance of the impurity B, placing the reference substance in a 50mL measuring flask, dissolving the reference substance with a solvent, diluting the reference substance to a scale, and shaking up;
solution of impurity C: accurately weighing 5.10mg of impurity C reference substance, placing the reference substance in a 50mL measuring flask, dissolving the reference substance with a solvent, diluting the reference substance to a scale, and shaking up;
solution of impurity D: accurately weighing 5.19mg of the impurity D reference substance, placing the impurity D reference substance in a 50mL measuring flask, dissolving the impurity D reference substance by using a solvent, diluting the impurity D reference substance to a scale, and shaking up;
solution of impurity E: accurately weighing 5.21mg of the impurity E reference substance, placing the reference substance in a 50mL measuring flask, dissolving the reference substance by using a solvent, diluting the reference substance to a scale, and shaking up;
solution of impurity F: accurately weighing 4.91mg of impurity F reference substance, placing the reference substance in a 50mL measuring flask, dissolving the reference substance with a solvent, diluting the reference substance to a scale, and shaking up;
solution of impurity I: accurately weighing 5.03mg of the reference substance I as impurity, placing the reference substance I in a 50mL measuring flask, dissolving the reference substance I with a solvent, diluting the reference substance I to a scale, and shaking up;
impurity ACB-10 solution: accurately weighing 4.72mg of an ACB-10 reference substance serving as an impurity, placing the reference substance into a 10mL measuring flask, and dissolving and diluting the reference substance by using N, N-dimethylformamide to prepare a solution containing about 500 mu g of the reference substance in each 1 mL;
impurity RC-3 solution: accurately weighing 9.96mg of an impurity RC-3 reference substance, placing the reference substance in a 100mL measuring flask, dissolving the reference substance by using a solvent, diluting the reference substance to a scale, and shaking up;
impurity RC-1 solution: accurately weighing 9.91mg of an impurity RC-1 reference substance, placing the reference substance in a 100mL measuring flask, dissolving the reference substance with a solvent, diluting the reference substance to a scale, and shaking up;
impurity ACB-9 solution: accurately weighing 5.19mg of an ACB-9 impurity reference substance, placing the reference substance in a 50mL measuring flask, dissolving the reference substance with a solvent, diluting the reference substance to a scale, and shaking up;
impurity ACB-8 solution: accurately weighing 4.93mg of an ACB-8 impurity reference substance, placing the reference substance in a 50mL measuring flask, dissolving the reference substance with a solvent, diluting the reference substance to a scale, and shaking up;
moxifloxacin solution: precisely weighing 10.58mg of moxifloxacin hydrochloride reference substance, placing the moxifloxacin hydrochloride reference substance into a 100mL measuring flask, dissolving the moxifloxacin hydrochloride reference substance by using a solvent, diluting the moxifloxacin hydrochloride reference substance to a scale, and shaking up;
(1.3) quantitative limiting accuracy solution: according to the quantitative limit concentration of the embodiment 3, respectively measuring an appropriate amount of each impurity solution and 2mL of the antibacterial eye drops, respectively placing the impurity solutions and the antibacterial eye drops into 10mL measuring bottles, adding a solvent to dilute to a scale, shaking up to obtain a quantitative limit accuracy solution of each impurity, and preparing 3 parts by the same method.
(1.4) 80% accuracy solution: respectively measuring 0.8mL of each impurity solution and 2mL of the antibacterial eye drops, respectively placing the impurity solutions and the antibacterial eye drops into 10mL measuring bottles, adding a solvent to dilute to a scale, uniformly shaking to obtain an 80% accuracy solution of each impurity, and preparing 3 parts by the same method.
(1.5) 100% accuracy solution: respectively measuring 1.0mL of each impurity solution and 2mL of the antibacterial eye drops, respectively placing the impurity solutions and the antibacterial eye drops into 10mL measuring bottles, adding a solvent to dilute to a scale, uniformly shaking to obtain 100% accuracy solutions of each impurity, and preparing 3 parts by the same method.
(1.6) 120% accuracy solution: respectively measuring 1.2mL of each impurity solution and 2mL of the antibacterial eye drops, respectively placing the impurity solutions and the antibacterial eye drops into 10mL measuring bottles, adding a solvent to dilute to a scale, uniformly shaking to obtain a 120% accuracy solution of each impurity, and preparing 3 parts by the same method.
(3) Test results
The sample injection detection is carried out on each sample according to the detection condition method of the high performance liquid chromatography in the embodiment 1, and the recovery rate detection results of each related substance are shown in tables 7-19:
TABLE 7 experimental results on the recovery rate of impurity A
TABLE 8 impurity B recovery results
TABLE 9 impurity C recovery results
TABLE 10 recovery of impurity D
TABLE 11 recovery of impurity E
TABLE 12 recovery of impurity F
TABLE 13 recovery of impurity I
TABLE 14 recovery of RC-1 impurity
TABLE 15 recovery of RC-3 impurity
TABLE 16 recovery of ACB-8 as impurity
TABLE 17 recovery of ACB-10 as impurity
TABLE 18 recovery of ACB-9 impurity
TABLE 19 Moxifloxacin hydrochloride control recovery
As can be seen from tables 7 to 19, the average recovery rate of each reference substance in each accuracy solution is within the range of 80 to 120%, and the recovery rate RSD of each impurity is less than 10%, which indicates that the method provided by the invention has good accuracy.
Example 6
Repeatability test
Taking 6 parts of the test solution prepared in the step (1.6) of example 1, carrying out sample injection detection according to the detection conditions of the high performance liquid chromatography in example 1, wherein the test results are shown in table 20:
TABLE 20 repeatability test results
As can be seen from Table 20, the number of impurities with the content of 6 measurement results of the same batch of samples above the quantitative limit is consistent, and the RSD meets the corresponding regulations, so that the method provided by the invention has good precision. Indicating that the method has good repeatability.
Example 7
Stability test
Diluting each reference substance stock solution prepared in the step (1.2) in the example 1 to the concentration of 1 mu g/mL by using a solvent to obtain each reference substance solution to be detected;
and (3) respectively placing the reference substance solution to be detected and the sample solution to be detected for 0h, 6h, 12h, 18h and 24h at room temperature, and then carrying out sample injection detection according to the high performance liquid chromatography conditions of the embodiment 1. And (3) testing results: the peak area RSD of each known impurity reference substance is less than 2 percent, and the content of moxifloxacin hydrochloride and known impurities in the test solution has no obvious change, which indicates that the stability of each reference substance solution to be tested and the test solution to be tested is good within 24 hours.
Example 8
Durability test
Changing chromatographic columns of different batches, wherein the temperature of the chromatographic columns changes by +/-5 ℃, the flow rate changes by 0.1mL/min, and the composition of mobile phases is as follows: the system suitability solution was examined under the same conditions as those of the HPLC assay of example 1 under the conditions of 70:30 in terms of the volume ratio of phosphate buffer solution to methanol in mobile phase A and 74:26 in terms of the volume ratio of phosphate buffer solution to methanol in mobile phase B, and the results are shown in Table 21.
TABLE 21 durability test results
As is clear from Table 21, the results showed that the separation of the respective substances and the peak area were not significantly different from the separation degree of the system-compatible solution and the number of main peak plates in example 1, indicating that the method of the present invention is excellent in durability.
Example 9
The relevant substances were detected in commercially available moxifloxacin hydrochloride eye drops (lot No. 316788F), and the results of high performance liquid chromatography detection are shown in FIG. 16 and Table 22.
TABLE 22 detection results of related substances in moxifloxacin hydrochloride eye drops
Comparative example 1
Performing high performance liquid chromatography detection on a test solution (the concentration of moxifloxacin is 1mg/mL), wherein the detection conditions of the high performance liquid chromatography are as follows: phenyl bonded silica gel as filler, Agilent Poroshell 120Phenyl Hexyl, 4.6mm × 100mm, 2.7 μm; phosphate buffer solution: respectively taking 0.5g of tetrabutylammonium hydrogen sulfate, 1.0g of monopotassium phosphate and 2mL of phosphoric acid into a beaker, adding 1000mL of water for dissolution, adjusting the pH value to 3.0 by using triethylamine, and performing suction filtration; mobile phase A: phosphate buffer-methanol (80: 20); mobile phase B: phosphate buffer-methanol (20: 80); flow rate: 0.8 mL/min; column temperature: 37 ℃; detection wavelength: 293 nm; sample introduction amount: 20 mu L of the solution; the gradient elution procedure is shown in table 21:
TABLE 21 gradient elution procedure
The test results are shown in fig. 17 and table 22:
TABLE 22 test results
As can be seen from fig. 17 and table 22, in the method provided by the present comparative example, the separation degree of moxifloxacin as the main peak from impurity a does not reach 1.5, and the specificity does not meet the requirement; and the separation degree of the impurity D and the impurity C does not reach 1.5, and simultaneously, the special requirement is not met.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (4)
1. A method for detecting related substances in antibacterial eye drops is characterized in that the antibacterial eye drops are detected by adopting high performance liquid chromatography;
the detection conditions of the high performance liquid chromatography comprise: the chromatographic column is a chromatographic column taking phenyl silane bonded silica gel as a filler, and the column temperature is 42-48 ℃; the mobile phase A is a mixed solution of a first phosphate buffer solution and methanol, and the volume ratio of a phosphate solution to the methanol in the mixed solution of the first phosphate buffer solution and the methanol is 72: 28; the mobile phase B is a mixed solution of a second phosphate buffer solution and methanol, and the volume ratio of a phosphate solution to the methanol in the mixed solution of the second phosphate buffer solution and the methanol is 25: 75; the flow rate of the mobile phase A and the mobile phase B is 1-1.5 mL/min; the phosphate solution comprises tetrabutylammonium hydrogen sulfate, potassium dihydrogen phosphate, phosphoric acid and water; the procedure of gradient elution is as follows:
2. The method of claim 1, wherein the related substances comprise one or more of impurity A, impurity B, impurity C, impurity D, impurity E, impurity RC-3, impurity ACB-9, impurity RC-1, impurity F, impurity I, impurity ACB-8 and impurity ACB-10.
3. The method according to claim 1 or 2, wherein the detection conditions of the high performance liquid chromatography further comprise a detection wavelength of 210 nm; the sample injection amount is 10-30 mu L.
4. The method according to claim 1, wherein the concentration of tetrabutylammonium hydrogen sulfate in the phosphate buffer solution in the first and second phosphate buffer solution-methanol mixed solutions is 1.36g/L, the concentration of monopotassium phosphate is 1g/L, and the concentration of phosphoric acid is 2 mL/L;
the pH value of the phosphate buffer solution is 6-7.
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