CN117405784A - Method for detecting related impurities in levofloxacin preparation by HPLC - Google Patents
Method for detecting related impurities in levofloxacin preparation by HPLC Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 174
- 238000000034 method Methods 0.000 title claims abstract description 70
- GSDSWSVVBLHKDQ-JTQLQIEISA-N Levofloxacin Chemical compound C([C@@H](N1C2=C(C(C(C(O)=O)=C1)=O)C=C1F)C)OC2=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-JTQLQIEISA-N 0.000 title claims abstract description 59
- 229960003376 levofloxacin Drugs 0.000 title claims abstract description 59
- 238000002360 preparation method Methods 0.000 title claims abstract description 36
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 29
- 238000001514 detection method Methods 0.000 claims abstract description 108
- 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 abstract description 18
- OUCSEDFVYPBLLF-KAYWLYCHSA-N 5-(4-fluorophenyl)-1-[2-[(2r,4r)-4-hydroxy-6-oxooxan-2-yl]ethyl]-n,4-diphenyl-2-propan-2-ylpyrrole-3-carboxamide Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@H]2OC(=O)C[C@H](O)C2)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 OUCSEDFVYPBLLF-KAYWLYCHSA-N 0.000 claims abstract description 18
- 238000010828 elution Methods 0.000 claims abstract description 17
- 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 abstract description 14
- 239000012488 sample solution Substances 0.000 claims abstract description 13
- 239000007788 liquid Substances 0.000 claims abstract description 10
- 230000002829 reductive effect Effects 0.000 claims abstract description 10
- 230000007423 decrease Effects 0.000 claims abstract description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 89
- 238000000926 separation method Methods 0.000 claims description 39
- 239000000523 sample Substances 0.000 claims description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical group [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 claims description 12
- 229910001488 sodium perchlorate Inorganic materials 0.000 claims description 12
- KCLCTHVLYXRXIC-UHFFFAOYSA-M azanium sodium acetate perchlorate Chemical compound Cl(=O)(=O)(=O)[O-].[Na+].C(C)(=O)[O-].[NH4+] KCLCTHVLYXRXIC-UHFFFAOYSA-M 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- GJFNLGKXCWQHIZ-UHFFFAOYSA-N acetic acid;azane;sodium Chemical compound N.[Na].CC(O)=O GJFNLGKXCWQHIZ-UHFFFAOYSA-N 0.000 claims description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 4
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 239000000243 solution Substances 0.000 description 69
- 230000000052 comparative effect Effects 0.000 description 36
- 239000011550 stock solution Substances 0.000 description 28
- 238000012360 testing method Methods 0.000 description 22
- 238000011084 recovery Methods 0.000 description 19
- 238000001228 spectrum Methods 0.000 description 17
- 230000014759 maintenance of location Effects 0.000 description 16
- 238000007865 diluting Methods 0.000 description 12
- 238000004811 liquid chromatography Methods 0.000 description 11
- 239000000203 mixture Substances 0.000 description 9
- 239000013558 reference substance Substances 0.000 description 9
- 238000009472 formulation Methods 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 238000002372 labelling Methods 0.000 description 8
- GSDSWSVVBLHKDQ-UHFFFAOYSA-N 9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid Chemical compound FC1=CC(C(C(C(O)=O)=C2)=O)=C3N2C(C)COC3=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-UHFFFAOYSA-N 0.000 description 6
- 229960001699 ofloxacin Drugs 0.000 description 6
- 239000012085 test solution Substances 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 3
- 239000005695 Ammonium acetate Substances 0.000 description 3
- 229940043376 ammonium acetate Drugs 0.000 description 3
- 235000019257 ammonium acetate Nutrition 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 108020000946 Bacterial DNA Proteins 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000011056 performance test Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000606161 Chlamydia Species 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000606768 Haemophilus influenzae Species 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 241000588653 Neisseria Species 0.000 description 1
- 241000588769 Proteus <enterobacteria> Species 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- RFHAOTPXVQNOHP-UHFFFAOYSA-N fluconazole Chemical compound C1=NC=NN1CC(C=1C(=CC(F)=CC=1)F)(O)CN1C=NC=N1 RFHAOTPXVQNOHP-UHFFFAOYSA-N 0.000 description 1
- 229960004884 fluconazole Drugs 0.000 description 1
- 229940018978 levofloxacin injection Drugs 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 229940072132 quinolone antibacterials Drugs 0.000 description 1
- IXGNPUSUVRTQGW-UHFFFAOYSA-M sodium;perchlorate;hydrate Chemical compound O.[Na+].[O-]Cl(=O)(=O)=O IXGNPUSUVRTQGW-UHFFFAOYSA-M 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229960004295 valine Drugs 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
-
- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- 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
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
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Abstract
The application relates to the technical field of analytical chemistry, and particularly discloses a method for detecting related impurities in a levofloxacin preparation by using HPLC. The detection method provided by the application comprises the following steps: preparing a sample solution, injecting the sample solution into a liquid chromatograph, and separating and detecting by adopting a mobile phase gradient elution method, wherein the mobile phase gradient elution method comprises the following steps: 0-42min, mobile phase A is 100%, mobile phase B is 0%;42-60min, the mobile phase A is linearly reduced from 100% to 0%, and the mobile phase B is linearly increased from 0% to 100%;60-70min, mobile phase A is 0%, mobile phase B is 100%;70-70.1min, mobile phase A increases from 0% to 100%, mobile phase B decreases from 100% to 0%;70.1-80min, mobile phase A is 100%, mobile phase B is 0%. The detection method provided by the application can realize simultaneous detection of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E, the impurity F, the impurity G, the impurity H and the impurity J in the levofloxacin preparation, and has the advantages of high detection speed, high detection sensitivity and high accuracy.
Description
Technical Field
The application relates to the technical field of analytical chemistry, in particular to a method for detecting related impurities in levofloxacin preparations by HPLC.
Background
Levofloxacin belongs to a third-generation quinolone antibacterial agent, has antibacterial activity about 2 times that of ofloxacin, and has a main action mechanism of inhibiting bacterial DNA gyrase activity, thereby inhibiting bacterial DNA replication. The levofloxacin has the characteristics of strong antibacterial efficacy, high safety, low drug resistance and the like, has stronger antibacterial activity on most enterobacteriaceae bacteria such as klebsiella pneumoniae, proteus, salmonella typhi, shigella, part of escherichia coli and the like, and also has good antibacterial effect on part of staphylococcus, streptococcus pneumoniae, influenza bacillus, pseudomonas aeruginosa, gonococcus, chlamydia and the like.
Because of the complex composition of levofloxacin, the safety and effectiveness of levofloxacin preparations are adversely affected by related impurities in the levofloxacin raw material. At present, high performance liquid chromatography is mainly adopted for detecting relevant impurities of the levofloxacin preparation, but with the increase of the relevant impurities, the detection needs to be carried out at a plurality of detection wavelengths, and the problems that the baseline separation of a detection spectrogram is difficult, the separation degree difference between peaks (the separation degree is less than 1.5), part of impurities are not eluted and the like are likely to exist. Therefore, it is highly desirable to provide a method for detecting relevant impurities (impurity a, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and impurity J) in levofloxacin preparations with high detection speed, high detection sensitivity and high accuracy.
Disclosure of Invention
In order to rapidly and accurately detect relevant impurities (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and impurity J) in a levofloxacin preparation, the application provides a method for detecting relevant impurities in the levofloxacin preparation by HPLC.
In a first aspect, the present application provides a method for detecting relevant impurities in a levofloxacin preparation by using HPLC, which adopts the following technical scheme:
a method for detecting related impurities in a levofloxacin preparation by HPLC, the detection method comprising the steps of: preparing a sample solution, injecting the sample solution into a liquid chromatograph, and separating and detecting by adopting a chromatographic column filled with octadecylsilane chemically bonded silica gel and a mobile phase gradient elution method;
the mobile phase gradient elution method comprises the following steps: 0-42min, mobile phase A is 100%, mobile phase B is 0%;42-60min, the mobile phase A is linearly reduced from 100% to 0%, and the mobile phase B is linearly increased from 0% to 100%;60-70min, mobile phase A is 0%, mobile phase B is 100%;70-70.1min, mobile phase A increases from 0% to 100%, mobile phase B decreases from 100% to 0%;70.1-80min, mobile phase A is 100%, mobile phase B is 0%;
the related impurities are selected from the group consisting of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and impurity J.
In the present application, in order to achieve simultaneous detection of impurity a, impurity B, impurity E, impurity F, impurity G and impurity J in a levofloxacin preparation, improvement was made based on the method provided in the United States Pharmacopeia (USP) 43, and the relevant detection conditions in the relevant substance detection method in a levofloxacin preparation were newly determined through a large number of experimental studies. Therefore, the detection method provided by the application can rapidly and accurately detect the impurities A, B, C, D, E, F, G, H and J in the levofloxacin preparation simultaneously by adopting a specific mobile phase gradient elution program and controlling the mobile phase flow rate to be 1.2-1.4mL/min, and has the advantages of strong detection specificity, high sensitivity and good separation degree; the detection method is used for the production of the levofloxacin preparation, and can realize effective control of the quality of the preparation.
Preferably, in the separation and detection process, the mobile phase A is ammonium acetate sodium perchlorate solution-acetonitrile, and the mobile phase B is sodium perchlorate solution-acetonitrile-methanol.
Further preferably, in the separation and detection process, the volume ratio of the ammonium acetate sodium perchlorate solution to acetonitrile in the mobile phase A is (88-92): 8-12; in the mobile phase B, the volume ratio of the sodium perchlorate solution to the acetonitrile to the methanol is (40-60): 25-35): 15-25.
In a specific embodiment, the volume ratio of the ammonium acetate sodium perchlorate solution to acetonitrile in mobile phase a is 90:10; in the mobile phase B, the volume ratio of the sodium perchlorate solution to the acetonitrile to the methanol is 50:30:20.
Preferably, in the separation detection process, the detection wavelength is 230-300nm.
Preferably, the column temperature is 58-60 ℃.
Preferably, the sample injection amount is 10-15 mu L.
Preferably, the pH of the ammonium acetate sodium perchlorate solution is in the range of 2.0 to 2.4.
Preferably, the octadecylsilane chemically bonded silica packing chromatographic column is a C18Inertsil ODS-3 chromatographic column with a specification of 4.6mm by 250mm and 5 μm.
In summary, the present application has the following beneficial effects:
1. the application adopts HPLC to detect 9 kinds of relevant impurities (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and impurity J) in the levofloxacin preparation simultaneously, and has the advantages of high detection sensitivity, high detection accuracy, short detection time, simple operation and the like.
2. The method for detecting the relevant impurities in the levofloxacin preparation by using the HPLC can detect the relevant impurities (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and impurity J) possibly existing in the fluconazole at the same time, and the quantitative limit of the impurity A is 0.5029 mug/mL and the detection limit is 0.2514 mug/mL; the quantitative limit of the impurity B is 0.1004 mug/mL, and the detection limit is 0.0502 mug/mL; the quantitative limit of impurity C is 0.1508 mug/mL, and the detection limit is 0.0754 mug/mL; the quantitative limit of the impurity D is 0.2990 mug/mL, and the detection limit is 0.1495 mug/mL; the quantitative limit of the impurity E is 0.649 mug/mL, and the detection limit is 0.2325 mug/mL; the quantitative limit of the impurity F is 0.1167 mug/mL, and the detection limit is 0.0584 mug/mL; the quantitative limit of the impurity G is 0.2260 mug/mL, and the detection limit is 0.1130 mug/mL; the quantitative limit of the impurity H is 0.4972 mug/mL, and the detection limit is 0.2486 mug/mL; the quantitative limit of the impurity J is 0.7697 mug/mL, and the detection limit is 0.3848 mug/mL; the quantitative limit of the levofloxacin is 0.2448 mug/mL, and the detection limit is 0.1224 mug/mL, which shows that the detection method provided by the application has excellent detection sensitivity.
3. Under 3 standard adding concentrations, the recovery rate of the impurity A is 97.83% -117.96%, and the RSD is 7.75% (n=9); the recovery rate of the impurity B is 97.45-100.23%, and the RSD is 0.98% (n=9); the recovery rate of the impurity C is 97.83% -101.68%, and the RSD is 1.18% (n=9); the recovery rate of the impurity D is 101.17% -107.08%, and the RSD is 1.67% (n=9); the recovery rate of the impurity E is 94.56% -102.35%, and the RSD is 2.67% (n=9); the recovery rate of the impurity F is 98.59-101.81%, and the RSD is 0.96% (n=9); the recovery rate of the impurity G is 98.00% -102.35%, and the RSD is 1.76% (n=9); the recovery rate of the impurity H is 89.69-104.40%, and the RSD is 5.50% (n=9); the recovery rate of the impurity J is between 90.33% and 102.34%, and the RSD is 4.81% (n=9). Therefore, the method for detecting the relevant impurities in the levofloxacin preparation by using the HPLC provided by the application can detect the impurities A, B, C, D, E, F, G, H and J, and the detection accuracy is high.
Drawings
Fig. 1 is a graph showing the linear relationship between the concentration of impurity a and the peak area.
Fig. 2 is a graph of the concentration of impurity B versus peak area.
Fig. 3 is a graph showing the linear relationship between the concentration of impurity C and the peak area.
Fig. 4 is a graph showing the linear relationship between the concentration of impurity D and the peak area.
Fig. 5 is a graph of the concentration of impurity E versus peak area.
Fig. 6 is a graph showing the linear relationship between the concentration of impurity F and the peak area.
Fig. 7 is a graph showing a linear relationship between the concentration of impurity G and the peak area.
Fig. 8 is a graph showing the linear relationship between the concentration of impurity H and the peak area.
Fig. 9 is a graph of the concentration of impurity J versus peak area.
FIG. 10 is a graph of the concentration of levofloxacin versus peak area.
FIG. 11 is a spectrum obtained by detecting the labeling solution of the test sample by the method of example 1.
FIG. 12 is a graph showing the results of the test sample labeling solution by the method of comparative example 1.
FIG. 13 is a spectrum obtained by detecting the labeled solution of the test sample by the method of comparative example 2.
FIG. 14 is a spectrum obtained by detecting the labeled solution of the test sample by the method of comparative example 3.
FIG. 15 is a spectrum obtained by detecting the labeled solution of the test sample by the method of comparative example 4.
FIG. 16 is a graph showing the results of the test sample labeling solution by the method of comparative example 5.
Detailed Description
The application provides a method for detecting related impurities in levofloxacin preparation by HPLC, which specifically comprises the following steps:
(1) Preparing a sample solution;
(2) Injecting the sample solution into a liquid chromatograph, and separating and detecting by adopting a C18Inertsil ODS-3 chromatographic column (specification is 4.6mm multiplied by 250mm,5 mu m) and a mobile phase gradient elution method; in the separation and detection process, the column temperature is 58-60 ℃, the detection wavelength is 230-300nm, the flow rate of a mobile phase is 1.2-1.4mL/min, and the sample injection amount is 10-15 mu L;
the mobile phase gradient elution method comprises the following steps: 0-42min, mobile phase A is 100%, mobile phase B is 0%;42-60min, the mobile phase A is linearly reduced from 100% to 0%, and the mobile phase B is linearly increased from 0% to 100%;60-70min, mobile phase A is 0%, mobile phase B is 100%;70-70.1min, mobile phase A increases from 0% to 100%, mobile phase B decreases from 100% to 0%;70.1-80min, mobile phase A is 100%, mobile phase B is 0%.
Wherein the mobile phase A is ammonium acetate sodium perchlorate solution-acetonitrile, and the mobile phase B is sodium perchlorate solution-acetonitrile-methanol; further, in the mobile phase A, the volume ratio of the ammonium acetate sodium perchlorate solution to acetonitrile is (88-92): 8-12, and in the mobile phase B, the volume ratio of the sodium perchlorate solution, acetonitrile and methanol is (40-60): 25-35): 15-25.
The sources of the raw materials used in the present application are shown in table 1 below, and the remaining raw materials, reagents, solvents, etc. are commercially available.
TABLE 1 sources of raw materials in this application
The remaining materials, reagents, solvents, and the like are commercially available.
The present application will be described in further detail with reference to the following preparations, examples, performance test and accompanying drawings.
Preparation example 1
Solution preparation
10% acetonitrile solution: measuring 400mL of acetonitrile, adding 3600mL of water, and uniformly mixing to obtain the final product.
Impurity a stock solution: taking a proper amount of the impurity A reference substance, precisely weighing, adding 6mol/L ammonia solution for dissolution, and quantitatively diluting with 10% acetonitrile solution to prepare a solution containing about 100 mug of the impurity A in each 1 mL.
Each impurity stock solution (impurity B, C, D, E, F, G, H, J): and taking a proper amount of each impurity B, C, D, E, F, G, H, J reference substance, precisely weighing, respectively adding 10% acetonitrile solution for dissolving and quantitatively diluting to prepare a solution containing about 100 mug of impurities in each 1 mL.
A storage solution of a levofloxacin reference substance: taking a proper amount of levofloxacin reference substance, precisely weighing, adding 10% acetonitrile solution for dissolving and quantitatively diluting to prepare a solution containing about 50 mug of levofloxacin in each 1 mL.
Mixing an impurity reference substance solution: precisely measuring the left ofloxacin reference stock solution and the impurity stock solutions, placing the right amounts of the left ofloxacin reference stock solution and the impurity stock solutions into the same measuring flask, and quantitatively diluting the mixed solution with 10% acetonitrile solution to prepare a mixed solution containing 1 mug of the left ofloxacin and 2 mug of each impurity in each 1 mL.
Mixing the reference substance solution: precisely measuring the sample and each appropriate amount of each impurity stock solution, placing the sample and each appropriate amount of each impurity stock solution into the same measuring flask, and quantitatively diluting the sample and each impurity stock solution by using a 10% acetonitrile solution to prepare a mixed solution containing 1mg of levofloxacin and 2 mug of each impurity in each 1 mL.
Test solution: precisely measuring a proper amount of levofloxacin injection, and quantitatively diluting with a 10% acetonitrile solution to prepare a solution containing about 1mg of levofloxacin in each 1 mL.
Each impurity localization solution: accurately measuring each appropriate amount of each impurity stock solution, respectively placing the impurity stock solutions into different measuring flasks, and quantitatively diluting the impurity stock solutions with 10% acetonitrile solution to prepare a solution containing about 2 mug of each impurity in each 1 mL.
Ammonium acetate sodium perchlorate solution: 8.43g of sodium perchlorate monohydrate and 3.08g of ammonium acetate are taken, 1000mL of water is added to dissolve, and the pH value is adjusted to 2.2 by phosphoric acid.
Example 1
Example 1 provides a method for HPLC detection of related impurities in levofloxacin formulations comprising the steps of:
(1) Preparing a sample solution: as in preparation example 1.
(2) Precisely measuring 10% acetonitrile solution, mixed reference substance solution, mixed impurity reference substance solution and each impurity positioning solution by 10 μl, and injecting into a liquid chromatograph; separating and detecting by adopting a mobile phase gradient elution method; in the separation and detection process, the liquid chromatography detection conditions are as follows: c18Inertsil ODS-3 chromatographic column (specification 4.6mm×250mm,5 μm), column temperature 58 ℃, detection wavelength 280nm, mobile phase flow rate 1.2mL/min, and sample injection amount 10 μl;
the mobile phase gradient elution method comprises the following steps: 0-42min, mobile phase A is 100%, mobile phase B is 0%;42-60min, the mobile phase A is linearly reduced from 100% to 0%, and the mobile phase B is linearly increased from 0% to 100%;60-70min, mobile phase A is 0%, mobile phase B is 100%;70-70.1min, mobile phase A increases from 0% to 100%, mobile phase B decreases from 100% to 0%;70.1-80min, mobile phase A is 100%, mobile phase B is 0%. Mobile phase a was sodium ammonium acetate solution-acetonitrile (volume ratio 90:10), and mobile phase B was sodium perchlorate solution-acetonitrile-methanol (volume ratio 50:30:20).
The mixed control solution was tested by the above test method, and the obtained liquid chromatography test results (i.e., the elution order of each impurity in the control solution and the degree of separation between each peak) are shown in table 2 below.
TABLE 2 elution order of related impurities and liquid chromatography detection results
According to the detection result, the levofloxacin and related impurities can be eluted, and the separation degree of the impurities and the levofloxacin and adjacent impurities is good, so that the requirements are met. Therefore, the method for detecting the related impurities in the levofloxacin preparation by using the HPLC provided by the application can rapidly and accurately detect the impurities A, B, C, D, E, F, G, H and J in the levofloxacin preparation simultaneously, and the detection spectrograms obtained by detection have good symmetry, proper peak height, centered peak positions and far difference of peak positions of substances, and have obvious separation effect.
Limit of detection and limit of quantification
1. Preparing a solution:
quantitative limit test solution: taking appropriate amounts of the left ofloxacin reference stock solution and the impurity A, B, C, D, E, F, G, H, J stock solution, placing the stock solution and the impurity A, B, C, D, E, F, G, H, J stock solution into a same measuring flask, and quantitatively diluting the stock solution and the impurity A, B, C, D, E, F, G, H, J stock solution by using a 10% acetonitrile solution until the S/N of each peak is about 10. 6 parts were prepared in parallel.
Detecting a limited test solution: the quantitative limiting test solution was taken and quantitatively diluted with a 10% acetonitrile solution to a peak S/N of about 3.
2. Precisely measuring and quantitatively limiting solution and detecting 10 mu L of the limiting solution respectively, and respectively injecting into a liquid chromatograph; then, the detection was carried out according to the liquid chromatography detection conditions of example 1, and the quantitative limit test results are shown in the following table 3; the detection limit test results are shown in the following table 4; the quantitative limit reproducibility (peak area) test results are shown in table 5 below;
TABLE 3 quantitative Limit (LOQ) test results
| Name of the name | Concentration μg/mL | S/N | Equivalent to the limit concentration |
| Impurity B | 0.1004 | 12.3 | 5.02％ |
| Impurity C | 0.1508 | 12.1 | 7.54％ |
| Impurity D | 0.2990 | 11.5 | 14.95％ |
| Impurity G | 0.2260 | 14.3 | 11.30％ |
| Impurity E | 0.4649 | 13.1 | 23.25％ |
| Levofloxacin | 0.2448 | 11.9 | 24.48％ |
| Impurity H | 0.4972 | 14.2 | 24.86％ |
| Impurity J | 0.7697 | 15.4 | 38.48％ |
| Impurity F | 0.1167 | 11.3 | 5.84％ |
| Impurity A | 0.5029 | 12.5 | 25.14％ |
TABLE 4 limit of detection (LOD) test results
TABLE 5 quantitative Limit (LOQ) repeatability test results
As can be seen from the detection results of tables 3 to 5, the quantitative limit of impurity A was 0.5029. Mu.g/mL, and the detection limit was 0.2514. Mu.g/mL; the quantitative limit of the impurity B is 0.1004 mug/mL, and the detection limit is 0.0502 mug/mL; the quantitative limit of impurity C is 0.1508 mug/mL, and the detection limit is 0.0754 mug/mL; the quantitative limit of the impurity D is 0.2990 mug/mL, and the detection limit is 0.1495 mug/mL; the quantitative limit of the impurity E is 0.649 mug/mL, and the detection limit is 0.2325 mug/mL; the quantitative limit of the impurity F is 0.1167 mug/mL, and the detection limit is 0.0584 mug/mL; the quantitative limit of the impurity G is 0.2260 mug/mL, and the detection limit is 0.1130 mug/mL; the quantitative limit of the impurity H is 0.4972 mug/mL, and the detection limit is 0.2486 mug/mL; the quantitative limit of the impurity J is 0.7697 mug/mL, and the detection limit is 0.3848 mug/mL; the limit of the quantification of the levofloxacin is 0.2448 mug/mL, the limit of the detection is 0.1224 mug/mL, and the limit of the detection, the limit of the quantification and the repeatability of the limit of the quantification all meet the requirement of detection sensitivity. Therefore, the sensitivity of the method for detecting levofloxacin-related substances by using the HPLC provided by the application is good.
Linearity and range
The linearity test of the levofloxacin-related impurity content in the range of 20% limit-200% limit was examined. The method comprises the following steps:
(1) Taking a proper amount of left ofloxacin reference stock solution and each impurity stock solution, diluting with 10% acetonitrile solution, and preparing a series of solutions with the concentration of 20-200% as a linear solution. The quantitative test solution and the linear solution were weighed precisely at 10. Mu.L each, poured into a liquid chromatograph, and detected by the liquid chromatograph method in example 1, respectively, and the chromatograms were recorded.
(2) Linear regression was performed with the concentration (. Mu.g/mL) on the abscissa and the peak area on the ordinate. The linear relationship of the impurities is shown in table 6. The linear relationship between the concentration of the impurity a and the peak area is shown in fig. 1, the linear relationship between the concentration of the impurity B and the peak area is shown in fig. 2, the linear relationship between the concentration of the impurity C and the peak area is shown in fig. 3, the linear relationship between the concentration of the impurity E and the peak area is shown in fig. 4, the linear relationship between the concentration of the impurity F and the peak area is shown in fig. 5, the linear relationship between the concentration of the impurity G and the peak area is shown in fig. 6, the linear relationship between the concentration of the impurity H and the peak area is shown in fig. 7, the linear relationship between the concentration of the impurity J and the peak area is shown in fig. 9, and the linear relationship between the concentration of the levofloxacin and the peak area is shown in fig. 10.
TABLE 6 results of Linear experiments on impurities
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As can be seen from the detection results in Table 6, the concentration of impurity A was in the range of 0.5029. Mu.g/mL to 4.0230. Mu.g/mL, and the concentration and the peak area were in good linear relationship; the concentration of the impurity B is in the concentration range of 0.1004 mu g/mL-4.0141 mu g/mL, and the concentration and the peak area are in good linear relation; the concentration of the impurity C is in the concentration range of 0.1508 mu g/mL-4.0200 mu g/mL, and the concentration and the peak area are in good linear relation; the impurity D is in the concentration range of 0.2990 mug/mL-3.9867 mug/mL, and the concentration and the peak area have good linear relation; the concentration of the impurity E is in the concentration range of 0.4649 mu g/mL-3.7192 mu g/mL, and the concentration and the peak area are in good linear relation; the concentration of the impurity F is in the concentration range of 0.1167-3.8916 mug/mL, and the concentration and the peak area are in good linear relation; the concentration of the impurity G is in the concentration range of 0.2260 mu G/mL-3.6160 mu G/mL, and the concentration and the peak area are in good linear relation; impurity H is in the concentration range of 0.4972 mug/mL-3.9774 mug/mL, and the concentration and the peak area have good linear relation; the impurity J is in the concentration range of 0.7697 mu g/mL-3.8484 mu g/mL, and the concentration and the peak area have good linear relation; the concentration of the levofloxacin is in a concentration range of 0.2448 mu g/mL-1.9586 mu g/mL, and the concentration and the peak area are in good linear relation. Therefore, the linearity of the method for detecting the related substances in the levofloxacin preparation by using the HPLC is good.
Accuracy of
1. The detection method comprises the following steps: preparing accuracy solutions (50% sample labeling solution, 100% sample labeling solution and 150% sample labeling solution); then, the above-mentioned accuracy solution was detected under the conditions of high performance liquid chromatography provided in example 1, and the labeled recovery rate and the relative standard deviation RSD value of the sample solution were calculated based on the labeled amount and the detection result.
The preparation method of each accuracy solution is as follows:
accuracy stock solution: precisely measuring 1mL of each impurity stock solution, placing the stock solutions into the same 20mL measuring flask, diluting to the scale with 10% acetonitrile solution, and shaking uniformly. (25. Mu.g/mL of each impurity)
50% of test sample adding standard solution: 1mL of accurate stock solution and 1mL of test sample solution are precisely measured, the solution is placed in a 25mL measuring flask, diluted to a scale by 10% acetonitrile solution, and uniformly shaken. (triplicate formulations of ACC50-1, ACC50-2, ACC 50-3) 100% test sample labeling solution: accurately measuring 2mL of accurate stock solution, 1mL of test sample solution, placing in a 25mL measuring flask, diluting to a scale with 10% acetonitrile solution, and shaking uniformly. (triplicate formulations of ACC100-1, ACC100-2, ACC 100-3) 150% test sample labeling solution: 3mL of accurate stock solution is precisely measured, 1mL of test sample solution is placed in a 25mL measuring flask, diluted to a scale with 10% acetonitrile solution, and shaken well. (triplicate preparation of ACC150-1, ACC150-2, ACC 150-3) 2. Each concentration of the sample was precisely weighed and 10. Mu.L of each of the labeled solutions was injected into a liquid chromatograph, and each of the samples was examined by the liquid chromatograph method in example 1, and the results of the accuracy test are shown in Table 7.
TABLE 7 accuracy test results for impurity A
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As can be seen from the detection results in table 7, the impurity a was within the 50% to 150% limit concentration range, the recovery rate was between 97.83% and 117.96%, and the RSD was 7.75% (n=9); the impurity B is in the limit concentration range of 50-150%, the recovery rate is 97.45-100.23%, and the RSD is 0.98% (n=9); impurity C is within 50% -150% limit, recovery rate is 97.83% -101.68%, RSD is 1.18% (n=9); impurity D is within 50% -150% limit concentration, recovery rate is 101.17% -107.08%, RSD is 1.67% (n=9); the impurity E is in the limit concentration range of 50-150%, the recovery rate is 94.56-102.35%, and the RSD is 2.67% (n=9); the impurity F is in the limit concentration range of 50-150%, the recovery rate is 98.59-101.81%, and the RSD is 0.96% (n=9); the impurity G is in the limit concentration range of 50-150%, the recovery rate is 98.00-102.35%, and the RSD is 1.76% (n=9); impurity H in the 50% -150% limit concentration range, recovery rate between 89.69% -104.40%, RSD 5.50% (n=9); the impurity J is in the limit concentration range of 50-150%, the recovery rate is between 90.33-102.34%, and the RSD is 4.81% (n=9). Therefore, the method for detecting the relevant impurities in the levofloxacin preparation by using the HPLC provided by the application can detect the impurities A, B, C, D, E, F, G, H and J, and the detection accuracy is high.
Comparative example 1
Comparative example 1 provides a method for detecting relevant impurities in levofloxacin formulations by HPLC.
The above comparative example is different from example 1 in that: the liquid chromatography detection conditions are specifically as follows: c18Inertsil ODS-3 chromatographic column (specification 4.6mm×250mm,5 μm), column temperature 38 ℃, detection wavelength 280nm, mobile phase flow rate 1.0mL/min, and sample injection amount 10 μl;
the mobile phase gradient elution method comprises the following steps: 0-5min, mobile phase A is 100%, mobile phase B is 0%;5-10min, the mobile phase A is linearly reduced from 100% to 82%, and the mobile phase B is linearly increased from 0% to 18%;10-15min, the mobile phase A is linearly reduced from 82% to 40%, and the mobile phase B is linearly increased from 18% to 60%;15-30min, 40% mobile phase A and 60% mobile phase B; 30-30.1min, mobile phase A increases from 40% to 100%, mobile phase B decreases from 60% to 0%;30.1-38min, mobile phase A is 100%, mobile phase B is 0%. Mobile phase a was sodium ammonium acetate solution-acetonitrile (volume ratio of 84:16), and mobile phase B was sodium perchlorate solution-acetonitrile-methanol (volume ratio of 50:30:20).
Comparative example 2
Comparative example 2 provides a method for detecting relevant impurities in levofloxacin formulations by HPLC.
The above comparative example is different from example 1 in that: the liquid chromatography detection conditions are specifically as follows: c18Inertsil ODS-3 chromatographic column (specification 4.6mm×250mm,5 μm), column temperature 38 ℃, detection wavelength 280nm, mobile phase flow rate 1.0mL/min, and sample injection amount 10 μl;
the mobile phase gradient elution method comprises the following steps: 0-23min, mobile phase A is 100%, mobile phase B is 0%;23-40min, mobile phase A is linearly reduced from 100% to 20%, mobile phase B is linearly increased from 0% to 80%;40-60min, 20% of mobile phase A and 80% of mobile phase B; 60-60.1min, mobile phase A increases from 20% to 100%, mobile phase B decreases from 80% to 0%;60.1-75min, mobile phase A is 100% and mobile phase B is 0%. Mobile phase a was sodium ammonium acetate solution-acetonitrile (volume ratio of 84:16), and mobile phase B was sodium perchlorate solution-acetonitrile-methanol (volume ratio of 50:30:20).
Comparative example 3
Comparative example 3 provides a method for detecting relevant impurities in levofloxacin formulations by HPLC.
The above comparative example is different from example 1 in that: the liquid chromatography detection conditions are specifically as follows: c18Inertsil ODS-3 chromatographic column (specification 4.6mm×250mm,5 μm), column temperature 55 ℃, detection wavelength 280nm, mobile phase flow rate 1.0mL/min, and sample injection amount 10. Mu.L;
the mobile phase gradient elution method comprises the following steps: 0-20min, mobile phase A is 100%, mobile phase B is 0%; for 20-30min, the mobile phase A is linearly reduced from 100% to 0%, and the mobile phase B is linearly increased from 0% to 100%;30-45min, mobile phase A of 0% and mobile phase B of 100%;45-45.1min, mobile phase A increases from 0% to 100%, mobile phase B decreases from 100% to 0%;45.1-55min, mobile phase A is 100% and mobile phase B is 0%. Mobile phase a was sodium ammonium acetate solution-acetonitrile (volume ratio 88:12), and mobile phase B was sodium perchlorate solution-acetonitrile-methanol (volume ratio 50:30:20).
Comparative example 4
Comparative example 4 provides a method for detecting related impurities in levofloxacin formulations by HPLC.
The method is a liquid chromatography detection method in the Chinese pharmacopoeia ChP2015, and the chromatographic conditions are as follows:
chromatographic column: kromasil 100-5-C8, 4.6mm.times.250 mm,5 μm; the flow rate is 1mL/min; detecting 294nm of wavelength and 238nm of impurity A; column temperature 40 ℃; the sample injection amount was 10. Mu.L.
The mobile phase gradient elution method comprises the following steps: 0-18min, mobile phase A is 100%, mobile phase B is 0%;18-25min, mobile phase A is linearly reduced from 100% to 70%, mobile phase B is linearly increased from 0% to 30%;25-39min, mobile phase A70% and mobile phase B30%; 39-40min, mobile phase A increased from 70% to 100%, mobile phase B decreased from 30% to 0%;40-50min, mobile phase A is 100%, mobile phase B is 0%. Mobile phase a: ammonium acetate sodium perchlorate solution (ammonium acetate 4.0g and sodium perchlorate 7.0g, dissolved by adding 1300mL of water, pH adjusted to 2.2 with phosphoric acid) -acetonitrile (85:15); mobile phase B: acetonitrile.
Comparative example 5
Comparative example 5 provides a method for detecting relevant impurities in levofloxacin formulations by HPLC.
The method is a liquid chromatography detection method in Japanese pharmacopoeia JP17, and the chromatographic conditions are as follows:
chromatographic column: symmetry@C18, 4.6mm.times.150mm, 5 μm; the flow rate is 0.5mL/min; the detection wavelength is 340nm; column temperature 45 ℃; the sample injection amount was 10. Mu.L.
The mobile phase is formulated by the following method: 1.76g of L-valine, 7.71g of ammonium acetate and 1.25g of copper (II) sulfate pentahydrate were weighed out, 1000mL of water was added to dissolve, and 250mL of methanol was added to the solution.
Performance test
1. Preparing a mixed reference substance solution: precisely measuring the sample and each impurity stock solution, placing the sample and each impurity stock solution in the same measuring flask, and quantitatively diluting with 10% acetonitrile solution to prepare a mixed solution containing 1mg of levofloxacin and 2ug of each impurity in each 1 mL.
2. The above mixed control solutions were examined by the methods of example 1 and comparative examples 1 to 3, respectively, and the examination spectra are shown in FIGS. 11 to 14. The detection spectrum obtained in example 1 is shown in FIG. 11, and the peak table is shown in Table 8; the detection spectrum obtained in comparative example 1 is shown in FIG. 12, and the peak table is shown in Table 9; the detection spectrum obtained in comparative example 2 is shown in FIG. 13, and the peak table is shown in Table 10; the detection spectrum obtained in comparative example 3 is shown in FIG. 14, and the peak table is shown in Table 11; the detection spectrum obtained in comparative example 4 is shown in FIG. 15, and the peak table is shown in Table 12; the detection spectrum obtained in comparative example 5 is shown in FIG. 16, and the peak table is shown in Table 13.
TABLE 8 Peak Table of detection spectrogram obtained in example 1
PDA Ch1 280nm
| Peak number | Retention time | Area of | Height | Area percent | Tailing factor | Theoretical plate number (USP) | Degree of separation (USP) |
| 1 | 11.126 | 80420 | 5987 | 0.275 | 1.04 | 14776 | -- |
| 2 | 14.810 | 1421 | 95 | 0.005 | 1.26 | 16759 | 8.95 |
| 3 | 20.864 | 87710 | 3815 | 0.300 | 1.02 | 18415 | 11.29 |
| 4 | 31.331 | 57112 | 1653 | 0.195 | 1.00 | 19530 | 13.85 |
| 5 | 34.780 | 100501 | 2672 | 0.344 | 1.01 | 19349 | 3.64 |
| 6 | 38.318 | 58240 | 1420 | 0.199 | 1.00 | 20181 | 3.40 |
| 7 | 42.567 | 2865736 | 605869 | 98.071 | 1.26 | 18153 | 3.63 |
| 8 | 50.497 | 60231 | 4696 | 0.206 | 1.00 | 331751 | 9.82 |
| 9 | 52.050 | 41118 | 1513 | 0.141 | 0.94 | 77919 | 2.83 |
| 10 | 52.915 | 53297 | 6177 | 0.182 | 1.03 | 751974 | 1.75 |
| 11 | 60.678 | 11253 | 1869 | 0.038 | 1.05 | 1893861 | 36.93 |
| 12 | 63.701 | 12620 | 1583 | 0.043 | 1.04 | 1304034 | 15.14 |
TABLE 9 Peak Table of the detection spectra obtained in comparative example 1
PDA Ch1 280nm
| Peak number | Retention time | Area of | Height | Area percent | Tailing factor | Theoretical plate number (USP) | Degree of separation (USP) |
| 1 | 6.759 | 96096 | 9573 | 0.364 | 1.08 | 10101 | -- |
| 2 | 9.050 | 2246 | 173 | 0.009 | 0.94 | 10494 | 7.36 |
| 3 | 11.409 | 101728 | 7696 | 0.386 | 1.04 | 16426 | 6.65 |
| 4 | 14.846 | 65048 | 6110 | 0.247 | 1.01 | 41129 | 10.59 |
| 5 | 15.283 | 113328 | 10822 | 0.430 | 1.05 | 44644 | 1.50 |
| 6 | 16.088 | 63838 | 6655 | 0.242 | -- | 45023 | 2.72 |
| 7 | 16.301 | 66861 | 7174 | 0.254 | -- | 50550 | 0.72 |
| 8 | 16.774 | 25770960 | 2340959 | 97.710 | 1.18 | 57262 | 1.66 |
| 9 | 18.063 | 19755 | 505 | 0.075 | 1.10 | 5835 | 2.10 |
| 10 | 18.945 | 61616 | 8386 | 0.234 | 1.04 | 143584 | 1.54 |
| 11 | 29.935 | 13437 | 724 | 0.051 | 1.06 | 61463 | 32.18 |
| Totals to | 26374911 | 2398776 | 100.000 |
Table 10 peak table of the detection spectrogram obtained in comparative example 2
PDA Ch1 280nm
| Peak number | Retention time | Area of | Height | Area percent | Tailing factor | Theoretical plate number (USP) | Degree of separation (USP) |
| 1 | 6738 | 96321 | 9882 | 0.289 | 1.11 | 10104 | -- |
| 2 | 9.004 | 2280 | 177 | 0.007 | 0.99 | 10288 | 7.27 |
| 3 | 11.416 | 101425 | 7064 | 0.304 | 1.06 | 13850 | 6.49 |
| 4 | 16.510 | 66456 | 3434 | 0.199 | 1.03 | 16073 | 11.21 |
| 5 | 17.426 | 115188 | 5639 | 0.345 | 1.05 | 16276 | 1.72 |
| 6 | 19.218 | G1061 | 2896 | 0.183 | 0.96 | 18163 | 3.21 |
| 7 | 20.010 | 61116 | 2759 | 0.183 | 1.05 | 17652 | 1.35 |
| 8 | 21.336 | 32728918 | 1271295 | 98.133 | 1.20 | 15631 | 2.06 |
| 9 | 25.904 | 26521 | 164 | 0.080 | 1.09 | 964 | 2.27 |
| 10 | 32.049 | 63677 | 3376 | 0.191 | 1.01 | 70446 | 3.22 |
| 11 | 45.768 | 13637 | 1449 | 0.041 | 1.06 | 492881 | 36.89 |
| 12 | 51.597 | 14997 | 810 | 0.045 | 1.06 | 176545 | 15.50 |
| Totals to | 33351598 | 1308944 | 100.000 |
TABLE 11 Peak Table of detection spectrogram obtained in comparative example 3
PDA Ch1 280nm
| Peak number | Retention time | Area of | Height | Area percent | Tailing factor | Theoretical plate number (USP) | Degree of separation (USP) |
| 1 | 8.568 | 96529 | 8652 | 0.321 | 1.08 | 12675 | -- |
| 2 | 10.508 | 2180 | 159 | 0.007 | 1.13 | 11438 | 5.56 |
| 3 | 14.837 | 103095 | 6099 | 0.342 | 1.03 | 17022 | 10.21 |
| 4 | 21.443 | 67179 | 2878 | 0.223 | 1.04 | 19120 | 12.29 |
| 5 | 23.461 | 117502 | 4616 | 0.390 | 1.02 | 19059 | 3.11 |
| 6 | 26.016 | 68442 | 2759 | 0.227 | 0.96 | 24287 | 3.79 |
| 7 | 27.489 | 29437947 | 2150931 | 97.773 | 1.19 | 95659 | 2.88 |
| 8 | 28.601 | 70432 | 9339 | 0.234 | 1.05 | 289113 | 3.91 |
| 9 | 30.038 | 115453 | 12475 | 0.383 | 0.70 | 372747 | 7.02 |
| 10 | 34.601 | 15180 | 2338 | 0.050 | 0.76 | 753121 | 25.61 |
| 11 | 38.228 | 14651 | 1613 | 0.049 | 1.02 | 360244 | 17.51 |
| Totals to | 30108590 | 2201860 | 100.000 |
Table 12 peak table of the detection spectrogram obtained in comparative example 4
PDA Ch1 294nm
| Peak number | Retention time | Area of | Height | Area percent | Tailing factor | Theoretical plate number (USP) | Degree of separation (USP) |
| 1 | 5.088 | 14241 | 2297 | 0.030 | 1.12 | 12507 | -- |
| 2 | 8.139 | 46291 | 4875 | 0.099 | 1.02 | 15181 | 13.67 |
| 3 | 8.693 | 57941 | 5877 | 0.124 | 1.05 | 16059 | 2.06 |
| 4 | 12.608 | 11589 | 847 | 0.025 | 1.02 | 18297 | 12.10 |
| 5 | 13.600 | 69296 | 4585 | 0.148 | 1.01 | 17880 | 2.54 |
| 6 | 15.008 | 58051 | 3586 | 0.124 | 0.99 | 18725 | 3.33 |
| 7 | 16.5S7 | 46541452 | 2099586 | 99.246 | 1.37 | 13342 | 3.12 |
| 8 | 25.504 | 72543 | 7677 | 0.155 | 0.97 | 148722 | 21.26 |
| 9 | 28.853 | 11005 | 2606 | 0.023 | 1.12 | 783151 | 16.96 |
| 10 | 35.061 | 12487 | 1156 | 0.027 | 1.07 | 224337 | 29.11 |
| Totals to | 46894895 | 2133090 | 100.000 |
TABLE 13 Peak Table of detection spectrogram obtained in comparative example 5
PDA Ch1 340nm
| Peak number | Retention time | Area of | Height | Area percent | Tailing factor | Theoretical plate number (USP) | Degree of separation (USP) |
| 1 | 10.251 | 25477 | 1080 | 0.077 | 1.29 | 3941 | -- |
| 2 | 11.072 | 17285 | 950 | 0.052 | 1.30 | 8075 | 1.43 |
| 3 | 13.109 | 26523 | 1034 | 0.080 | 1.55 | 7048 | 3.65 |
| 4 | 15.019 | 6045 | 356 | 0.018 | 1.04 | 16481 | 3.50 |
| 5 | 15.936 | 50408 | 1562 | 0.153 | 1.06 | 6151 | 1.43 |
| 6 | 20.075 | 26160 | 723 | 0.079 | 1.36 | 7248 | 4.71 |
| 7 | 23.755 | 32780962 | 512601 | 99.249 | 0.64 | 2585 | 2.62 |
| 8 | 28.896 | 48147 | 1029 | 0.146 | 1.38 | 8929 | 3.33 |
| 9 | 40.181 | 23643 | 379 | 0.072 | 1.30 | 10022 | 7.98 |
| 10 | 44.139 | 24460 | 336 | 0.074 | 1.27 | 10592 | 2.38 |
| Totals to | 33029111 | 520049 | 100.000 |
As can be seen from fig. 11 and table 8, the baseline separation was achieved between the peaks, with a minimum separation of 1.75 (retention time between the chromatographic peaks of 52.050min and 52.915min, respectively). Therefore, the method for detecting the relevant impurities in the levofloxacin preparation by using the HPLC provided in the embodiment 1 of the application can realize simultaneous elution and detection of the levofloxacin and the impurities A, B, C, D, E, F, G, H and J, and the obtained detection spectrogram base line is completely separated, and the separation degree between peaks is more than 1.5.
As can be seen from fig. 12 and table 9, the separation degree between chromatographic peaks with retention times of 16.088min and 16.301min respectively in the detection spectra is only 0.72 (separation degree < 1.5), which indicates that the method for detecting relevant impurities in levofloxacin preparation by HPLC provided in comparative example 1 has a problem of poor separation degree.
As can be seen from fig. 13 and table 10, the separation degree between chromatographic peaks with retention times of 19.218min and 20.010min respectively in the detection spectra is 1.35 (separation degree < 1.5), which indicates that the method for detecting relevant impurities in levofloxacin preparation by HPLC provided in comparative example 2 has a problem of poor separation degree.
As can be seen from fig. 14 and table 11, in the detection spectrum, the chromatographic peak with retention time of 30.038min has a significant peak inclusion phenomenon; and retention time > 34.601min, baseline is obviously unstable, and chromatographic peak is difficult to separate from baseline at retention time of 38.228 min. Therefore, it is explained that the method for detecting relevant impurities in levofloxacin preparation by HPLC provided in comparative example 3 has a problem of poor separation degree.
As can be seen from fig. 15 and table 12, under the detection conditions of comparative example 4, 10 target peaks were detected in the detection spectrum, while 11 theoretical target peaks of the mixed control solution indicate that 1 impurity peak may coincide or not be eluted. Analysis showed that the retention time of levofloxacin was 16.587min, impurity H was a localization solution and the retention time was 16.525min, so that the peak of impurity H and the peak of levofloxacin were coincident. Therefore, it is explained that the liquid chromatography detection method in the "chinese pharmacopoeia" ChP2015 provided in comparative example 4 is poor in separation degree, and it is difficult to separate impurity H from levofloxacin.
As can be seen from fig. 16 and table 13, under the detection conditions of comparative example 4, 10 target peaks were detected in the detection spectrum, while 11 theoretical target peaks of the mixed control solution indicate that 1 impurity peak may coincide or not be eluted. And the separation degree between the chromatographic peaks with retention time of 10.251min and 11.072min is 1.43 (separation degree < 1.5), respectively, and the separation degree between the chromatographic peaks with retention time of 15.019min and 15.936min is 1.43 (separation degree < 1.5). Therefore, it is explained that the liquid chromatography detection method in japanese pharmacopoeia JP17 provided in comparative example 5 is poor in separation degree.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (8)
1. A method for detecting related impurities in a levofloxacin preparation by using an HPLC (high performance liquid chromatography), which comprises the following steps: preparing a sample solution, injecting the sample solution into a liquid chromatograph, and separating and detecting by adopting a chromatographic column filled with octadecylsilane chemically bonded silica gel and a mobile phase gradient elution method; the flow rate of the mobile phase is 1.2-1.4mL/min;
the mobile phase gradient elution method comprises the following steps: 0-42min, mobile phase A is 100%, mobile phase B is 0%;42-60min, the mobile phase A is linearly reduced from 100% to 0%, and the mobile phase B is linearly increased from 0% to 100%;60-70min, mobile phase A is 0%, mobile phase B is 100%;70-70.1min, mobile phase A increases from 0% to 100%, mobile phase B decreases from 100% to 0%;70.1-80min, mobile phase A is 100%, mobile phase B is 0%;
the related impurities are selected from the group consisting of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and impurity J.
2. The method according to claim 1, wherein in the separation and detection process, the mobile phase A is sodium ammonium acetate solution-acetonitrile, and the mobile phase B is sodium perchlorate solution-acetonitrile-methanol.
3. The method according to claim 2, wherein in the separation and detection process, the volume ratio of the ammonium acetate sodium perchlorate solution to acetonitrile in the mobile phase A is (88-92): 8-12; in the mobile phase B, the volume ratio of the sodium perchlorate solution to the acetonitrile to the methanol is (40-60): 25-35): 15-25.
4. The method according to claim 1, wherein the detection wavelength is 230-300nm in the separation detection process.
5. The method according to claim 1, wherein the column temperature is 58-60 ℃ during the separation and detection.
6. The method according to claim 1, wherein the sample amount is 10-15 μl during the separation and detection process.
7. The method according to claim 2, wherein the pH of the ammonium acetate sodium perchlorate solution is 2.0-2.4.
8. The method according to claim 1, wherein the octadecylsilane chemically bonded silica filler is used as a C18Inertsil ODS-3 column having a size of 4.6 mm. Times.250 mm, 5. Mu.m.
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