CN111208221B - Method for detecting lomefloxacin hydrochloride related substances - Google Patents
Method for detecting lomefloxacin hydrochloride related substances Download PDFInfo
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- WWJBDSBGLBEFSH-UHFFFAOYSA-N 2-(4-methoxyphenyl)azepane Chemical compound C1=CC(OC)=CC=C1C1NCCCCC1 WWJBDSBGLBEFSH-UHFFFAOYSA-N 0.000 title claims abstract description 65
- 229960003814 lomefloxacin hydrochloride Drugs 0.000 title claims abstract description 65
- 239000000126 substance Substances 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 30
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 102
- 239000000243 solution Substances 0.000 claims abstract description 72
- 239000012535 impurity Substances 0.000 claims abstract description 66
- 238000001514 detection method Methods 0.000 claims abstract description 31
- 239000012085 test solution Substances 0.000 claims abstract description 29
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- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 9
- ONQDAESGZUODFI-UHFFFAOYSA-N ethyl 6,7,8-trifluoro-4-oxo-1h-quinoline-3-carboxylate Chemical compound FC1=C(F)C=C2C(=O)C(C(=O)OCC)=CNC2=C1F ONQDAESGZUODFI-UHFFFAOYSA-N 0.000 claims abstract description 6
- LWLLHOVWIFISMG-UHFFFAOYSA-N ethyl 1-ethyl-6,7,8-trifluoro-4-oxoquinoline-3-carboxylate Chemical compound FC1=C(F)C=C2C(=O)C(C(=O)OCC)=CN(CC)C2=C1F LWLLHOVWIFISMG-UHFFFAOYSA-N 0.000 claims abstract description 5
- 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 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000000523 sample Substances 0.000 claims description 14
- 239000012088 reference solution Substances 0.000 claims description 11
- 238000012937 correction Methods 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 8
- 239000012488 sample solution Substances 0.000 claims description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 230000014759 maintenance of location Effects 0.000 claims description 6
- WSGYTJNNHPZFKR-UHFFFAOYSA-N 3-hydroxypropanenitrile Chemical compound OCCC#N WSGYTJNNHPZFKR-UHFFFAOYSA-N 0.000 claims description 5
- 238000004364 calculation method Methods 0.000 claims description 5
- 230000005526 G1 to G0 transition Effects 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 3
- 239000000945 filler Substances 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 238000000691 measurement method Methods 0.000 claims 2
- 239000000203 mixture Substances 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- 238000003786 synthesis reaction Methods 0.000 abstract description 5
- 238000003908 quality control method Methods 0.000 abstract description 4
- 239000011550 stock solution Substances 0.000 description 9
- 238000005303 weighing Methods 0.000 description 6
- 238000004811 liquid chromatography Methods 0.000 description 5
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- XXMFJKNOJSDQBM-UHFFFAOYSA-N 2,2,2-trifluoroacetic acid;hydrate Chemical compound [OH3+].[O-]C(=O)C(F)(F)F XXMFJKNOJSDQBM-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- OMOVVBIIQSXZSZ-UHFFFAOYSA-N [6-(4-acetyloxy-5,9a-dimethyl-2,7-dioxo-4,5a,6,9-tetrahydro-3h-pyrano[3,4-b]oxepin-5-yl)-5-formyloxy-3-(furan-3-yl)-3a-methyl-7-methylidene-1a,2,3,4,5,6-hexahydroindeno[1,7a-b]oxiren-4-yl] 2-hydroxy-3-methylpentanoate Chemical compound CC12C(OC(=O)C(O)C(C)CC)C(OC=O)C(C3(C)C(CC(=O)OC4(C)COC(=O)CC43)OC(C)=O)C(=C)C32OC3CC1C=1C=COC=1 OMOVVBIIQSXZSZ-UHFFFAOYSA-N 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 229960002422 lomefloxacin Drugs 0.000 description 3
- ZEKZLJVOYLTDKK-UHFFFAOYSA-N lomefloxacin Chemical compound FC1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCNC(C)C1 ZEKZLJVOYLTDKK-UHFFFAOYSA-N 0.000 description 3
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- 239000000543 intermediate Substances 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 241000204031 Mycoplasma Species 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007806 chemical reaction intermediate Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000006200 ethylation reaction Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 229940072132 quinolone antibacterials Drugs 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000002229 urogenital system Anatomy 0.000 description 1
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/065—Preparation using different phases to separate parts of sample
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention discloses a method for detecting lomefloxacin hydrochloride related substances, which comprises the following specific steps: a. dissolving lomefloxacin hydrochloride in acetonitrile water solution to obtain a test solution; b. dissolving a reference substance of related substances and a reference substance of lomefloxacin hydrochloride by using acetonitrile aqueous solution to obtain a system applicability solution; c. detecting by high performance liquid chromatography. The detection method can effectively separate the lomefloxacin hydrochloride from the impurity peak, accurately determine the content of the known impurity 6,7, 8-trifluoro-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester and 1-ethyl-6, 7, 8-trifluoro-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester of the lomefloxacin hydrochloride, has stable and reliable determination result and strong specificity, and provides possibility for quality control in the synthesis process of the lomefloxacin hydrochloride, thereby better controlling the product quality of the lomefloxacin hydrochloride.
Description
Technical Field
The invention relates to a method for detecting lomefloxacin hydrochloride related substances, belonging to the field of medicines.
Background
Lomefloxacin hydrochloride is a fourth-generation quinolone antibacterial agent, para G+,G-The medicament has strong antibacterial effect on infection caused by bacteria, mycoplasma and the like, is clinically used for treating infection of respiratory tract, urogenital system, gastrointestinal tract, skin soft tissue and the like caused by sensitive bacteria, has long half-life period and definite curative effect, and is widely applied.
The chemical name of the lomefloxacin hydrochloride is (+/-) -l-ethyl-6, 8-difluoro-1, 4-dihydro-7- (3-methyl-1-piperazinyl) -4-oxo-3-quinoline carboxylate, and the chemical formula is as follows: c17H19F2N3O3HCI, structural formula:
in the synthesis of lomefloxacin hydrochloride, impurities may be derived from starting materials and reaction intermediates, ethyl 6,7, 8-trifluoro-1, 4-dihydro-4-oxo-3-quinolinecarboxylate is used as a starting material, ethyl 1-ethyl-6, 7, 8-trifluoro-1, 4-dihydro-4-oxo-3-quinolinecarboxylate is used as an ethylation reaction product, the detection method of lomefloxacin hydrochloride related substances is improved and the separation of related substances is improved in the 2010 edition of the Chinese pharmacopoeia, but the known impurity A (ethyl 6,7, 8-trifluoro-1, 4-dihydro-4-oxo-3-quinolinecarboxylate), the impurity B (ethyl 1-6, 7, 8-trifluoro-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester), which may cause side effects of lomefloxacin hydrochloride, and therefore, providing a high performance liquid chromatography condition capable of detecting impurities a and B and more comprehensively detecting lomefloxacin hydrochloride related substances becomes a problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a method for detecting lomefloxacin hydrochloride related substances, which adopts a high performance liquid chromatography for detection and comprises the following specific steps:
a. dissolving lomefloxacin hydrochloride in acetonitrile water solution to obtain a test solution;
b. adding a lomefloxacin hydrochloride reference substance and a related substance reference substance into an acetonitrile aqueous solution for dissolving to obtain a system applicability solution;
c. the following chromatographic conditions are adopted to respectively detect the test solution and the system applicability solution:
stationary phase: octadecylsilane chemically bonded silica is used as a filling agent;
mobile phase: using a 0.01-1.0% trifluoroacetic acid aqueous solution as a mobile phase A, and using a methanol acetonitrile solution with a volume ratio of 40-60: 40-60 as a mobile phase B;
the gradient elution procedure was: 0.01-30 min, 75-85% → 72-82% A, 30-40 min, 72-82% → 30-40% A, 40-50 min, 30-40% A, 50.01-65min, 75-85% A;
detection wavelength: 260-300 nm;
the flow rate is 0.5-1.5 ml/min;
the column temperature is 30-40 ℃.
Further, the mass-to-volume ratio of the lomefloxacin hydrochloride to the acetonitrile aqueous solution in the step a) is 0.5-1.0 mg: 1ml, preferably: 1.0 mg: 1 ml.
Further, the volume ratio of acetonitrile to water in the acetonitrile water solution is 30: 70.
further, the related substances in the step B) are impurities A and/or impurities B; the system applicability solution contains 0.5-1.0 mg of lomefloxacin hydrochloride and 0.5-1.0ug of impurities in each 1 ml.
Further, the stationary phase is an Inertsil ODS-3 column; the specification of the chromatographic column is as follows: the inner diameter is 4.6X 250mm, 5 um.
Further, the detection wavelength in the step c) is 287nm, the flow rate is 1.0-1.1 ml/min, the column temperature is 30 ℃, and the sample injection amount is 20 mu L.
Further, step c) the gradient elution procedure is: 0.01-30 min, 80% → 77% A, 30-40 min, 77% → 35% A, 40-50 min, 35% A, 50.01-65min, 80% A.
Furthermore, the chromatogram of the test solution shows a chromatographic peak corresponding to the retention time of the chromatographic peak of the related substance in the chromatogram of the system applicability solution and the reference substance, namely, the test solution contains the related substance.
The invention also provides a method for measuring the content of lomefloxacin hydrochloride related substances, which comprises the following specific operation steps:
1) adding acetonitrile aqueous solution into the sample solution obtained in the step a to dilute the sample solution by 100-1000 times to obtain a reference solution; the volume ratio of acetonitrile to water in the acetonitrile water solution is 30: 70;
2) detecting according to the method;
3) and (3) calculating the content of related substances according to the following calculation formula:
wherein, XiPercent: the content of the relevant substance in the sample, Ai: peak area of the relevant substance in the test solution, A: peak area of lomefloxacin hydrochloride in control solution, NThe dilution factor of the control solution, and fi is the correction factor of the related substance.
Further, the acetonitrile water solution is added for dilution by 1000 times in the step a.
Further, the relevant substance is impurity a, the correction factor of which is 2.42, and the relevant substance is impurity B, the correction factor of which is 2.93.
The detection method can effectively separate the lomefloxacin hydrochloride from the impurity peak, accurately determine the content of the known impurity 6,7, 8-trifluoro-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester and 1-ethyl-6, 7, 8-trifluoro-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester of the lomefloxacin hydrochloride, has stable and reliable determination result and strong specificity, and provides possibility for quality control in the synthesis process of the lomefloxacin hydrochloride, thereby better controlling the product quality of the lomefloxacin hydrochloride.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 HPLC chart of sample solution in example 1
FIG. 2 example 2 HPLC chart of test solution
FIG. 3 HPLC chart of sample solution in example 3
FIG. 4 blank solution
FIG. 5 lomefloxacin hydrochloride isolation solution
FIG. 6 HPLC chart of reference solution (a)
FIG. 7 reference solution (b) HPLC chart
Detailed Description
1. Material
Lomefloxacin hydrochloride (supplied by Chengdu Vital pharmaceutical Co., Ltd., lots WB10300401, WB10300402, WB10300501)
Control known to be contaminated A (supplied by Chengdu-bang pharmaceutical Co., Ltd., lot No. WBZ001B)
Control sample of known impurity B (provided by Chengdu-bang pharmaceutical Co., Ltd., lot No. WBZ002B)
2. Main instrument
High performance liquid chromatograph: shimadzu of Japan
Example 1 measurement of the content of impurity A and impurity B in lomefloxacin hydrochloride
1) Lomefloxacin hydrochloride (batch number: WB10300401)25mg, adding 25ml acetonitrile water solution (acetonitrile to water volume ratio 30:70) to dissolve, preparing test solution with concentration of 1.0 mg/ml;
2) precisely transferring the test solution obtained in the step 1), and adding the volume ratio of 30:70 acetonitrile in water to form a control solution with the concentration of 1.0 ug/ml;
3) 10mg of each of the impurity A and the impurity B as reference substances are respectively taken and dissolved by 100ml of acetonitrile aqueous solution (the volume ratio of acetonitrile to water is 30:70) to prepare solution with the concentration of 100ug/ml as impurity stock solution.
4) Weighing 25mg of lomefloxacin hydrochloride reference substance, placing the reference substance into a 25ml measuring flask, adding 0.25ml of the impurity stock solution, and adding acetonitrile aqueous solution (the volume ratio of acetonitrile to water is 30:70) to dissolve and fix the solution to obtain the system applicability solution. (lomefloxacin hydrochloride 1.0mg/ml, impurities A, B each 1.0 ug/ml);
5) the following chromatographic conditions are adopted to respectively detect the test solution, the reference solution and the system applicability solution:
a chromatographic column: inertsil ODS-34.6X 250mm, 5 um;
mobile phase: taking 0.05% trifluoroacetic acid water solution as a mobile phase A, and taking a methanol acetonitrile solution with a volume ratio of 50:50 as a mobile phase B; detection wavelength: 287 nm; the flow rate is 1.0 ml/min; the column temperature is 30 ℃; the sample amount is 20 ul;
the gradient elution procedure was:
| time (min) | Mobile phase B (%) | Mobile phase A (%) |
| 0.01 | 20 | 80 |
| 30 | 23 | 77 |
| 40 | 65 | 35 |
| 50 | 65 | 35 |
| 50.01 | 20 | 80 |
| 65 | 20 | 80 |
6) Chromatogram of the test solution presents chromatographic peaks corresponding to retention time of chromatographic peaks of impurities A and B in the chromatogram of the system applicability solution, namely lomefloxacin hydrochloride contains related substances: the contents of related substances contained in the impurities A and B are calculated by the following formula:
wherein, XiPercent: the content of the relevant substance in the sample, Ai: peak area of the relevant substance in the test solution, A: the peak area of lomefloxacin in the control solution, N is the dilution multiple of the control solution, and fi is the correction factor of related substances (f)A＝2.42，fB＝2.93)。
7) As a result:
the detection spectrum of related substances with lomefloxacin hydrochloride batch number WB10300401 is shown in figure 1, and the calculation result is as follows:
| item | Related substance A | Related substance B | MAX unknown related substances |
| Content% | ND (not containing) | ND (not containing) | 0.079％ |
Example 2 detection of impurity a and impurity B content in lomefloxacin hydrochloride
1) Lomefloxacin hydrochloride (batch number: WB10300402)25mg, adding 25ml acetonitrile water solution (acetonitrile to water volume ratio 30:70) to dissolve, preparing to test solution with concentration of 1.0 mg/ml;
2) precisely transferring the test solution obtained in the step 1), and adding the test solution into the sample solution according to the volume ratio of 30:70 acetonitrile in water to form a control solution with a concentration of 1.0 ug/ml;
3) 10mg of each of the impurity A and the impurity B as reference substances are respectively taken and dissolved by 100ml of acetonitrile aqueous solution (the volume ratio of acetonitrile to water is 30:70) to prepare solution with the concentration of 100ug/ml as impurity stock solution.
4) Weighing 25mg of lomefloxacin hydrochloride reference substance, placing the reference substance into a 25ml measuring flask, adding 0.25ml of the impurity stock solution, and adding acetonitrile aqueous solution (the volume ratio of acetonitrile to water is 30:70) to dissolve and fix the solution to obtain the system applicability solution. (lomefloxacin hydrochloride 1.0mg/ml, impurities A, B each 1.0ug/ml)
5) The following chromatographic conditions are adopted to respectively detect the test solution, the reference solution and the system applicability solution:
a chromatographic column: inertsil ODS-34.6X 250mm, 5 um;
mobile phase: taking 0.05% trifluoroacetic acid water solution as a mobile phase A, and taking a methanol acetonitrile solution with a volume ratio of 50:50 as a mobile phase B; detection wavelength: 287 nm; the flow rate is 1.1 ml/min; the column temperature is 30 ℃; the sample amount is 20 ul;
the gradient elution procedure was:
| time (min) | Mobile phase B (%) | Mobile phase A (%) |
| 0.01 | 20 | 80 |
| 30 | 23 | 77 |
| 40 | 65 | 35 |
| 50 | 65 | 35 |
| 50.01 | 20 | 80 |
| 65 | 20 | 80 |
6) Chromatogram of the test solution presents chromatographic peaks corresponding to retention time of chromatographic peaks of impurities A and B in the chromatogram of the system applicability solution, namely lomefloxacin hydrochloride contains related substances: the contents of related substances contained in the impurities A and B are calculated by the following formula:
wherein, XiPercent: the content of the relevant substance in the sample, Ai: peak area of the relevant substance in the test solution, A: the peak area of the lomefloxacin in the control solution, N is the dilution multiple of the control solution, fi is the correction factor of the related substance (f)A＝2.42，fB＝2.93)。
7) As a result:
the detection spectrum of related substances with the lomefloxacin hydrochloride batch number WB10300402 is shown in figure 2, and the calculation result is as follows:
| item | Related substance A | Related substance B | MAX unknown related substances |
| Content% | ND (not containing) | ND (not containing) | 0.078％ |
Example 3 content measurement of impurity a and impurity B in lomefloxacin hydrochloride
1) Lomefloxacin hydrochloride (batch number: WB10300501)25mg, adding 25ml acetonitrile water solution (acetonitrile to water volume ratio 30:70) to dissolve, and preparing into 1.0mg/ml test solution;
2) precisely transferring the test solution obtained in the step 1), and adding the volume ratio of 30:70 acetonitrile in water to form a control solution with the concentration of 1.0 ug/ml;
3) 10mg of each of the impurity A and the impurity B as reference substances are respectively taken and dissolved by 100ml of acetonitrile aqueous solution (the volume ratio of acetonitrile to water is 30:70) to prepare solution with the concentration of 100ug/ml as impurity stock solution.
4) Weighing 25mg of lomefloxacin hydrochloride reference substance, placing the reference substance into a 25ml measuring flask, adding 0.25ml of the impurity stock solution, and adding acetonitrile aqueous solution (the volume ratio of acetonitrile to water is 30:70) to dissolve and fix the solution to obtain the system applicability solution. (lomefloxacin hydrochloride 1.0mg/ml, impurities A, B each 1.0ug/ml)
5) The following chromatographic conditions are adopted to respectively detect the test solution, the reference solution and the system applicability solution:
a chromatographic column: inertsil ODS-34.6X 250mm, 5 um;
mobile phase: taking 0.05% trifluoroacetic acid water solution as a mobile phase A, and taking a methanol acetonitrile solution with a volume ratio of 50:50 as a mobile phase B; detection wavelength: 287 nm; the flow rate is 1.0 ml/min; the column temperature is 30 ℃; the sample amount is 20 ul;
the gradient elution procedure was:
6) chromatogram of the test solution presents chromatographic peaks corresponding to retention time of chromatographic peaks of impurities A and B in the chromatogram of the system applicability solution, namely lomefloxacin hydrochloride contains related substances: the contents of related substances contained in the impurities A and B are calculated by the following formula:
wherein, XiPercent: the content of the relevant substance in the sample, Ai: peak area of the relevant substance in the test solution, A: the peak area of lomefloxacin in the control solution, N is the dilution multiple of the control solution, and fi is the correction factor of related substances (f)A＝2.42，fB＝2.93)
7) As a result:
the detection spectrum of the related substances with the lomefloxacin hydrochloride batch number WB10300501 is shown in figure 3, and the calculation result is as follows:
| item | Related substance A | Related substance B | MAX unknown related substances |
| Content% | ND (not containing) | ND (not containing) | 0.078％ |
The beneficial effects of the present invention are further illustrated by the following experimental examples:
experimental example 1 selection of detection wavelength
The Chinese pharmacopoeia has a related substance detection wavelength of 287nm, lomefloxacin hydrochloride, an impurity A and an impurity B solution are detected by DAD, the maximum absorption of the lomefloxacin hydrochloride is about 287nm, the difference with the pharmacopoeia is not large, the maximum absorption of the impurity A and the impurity B is about 310nm, the absorption of other impurities is weak at 310nm, the absorption of the impurities A and B is strong at 287nm, and the wavelength of a detector is selected to be 287nm by comprehensively considering.
Experimental example 2 System applicability
The instrument comprises the following steps: shimadzu LC-20AT
A chromatographic column: inertsil ODS-34.6X 250mm, 5um, flow rate: 1.0ml/min, column temperature: 30 ℃, sample size 20ul, detector wavelength: 287nm, mobile phase a: 0.05% aqueous trifluoroacetic acid mobile phase B: acetonitrile: methanol (50:50), gradient elution conditions were as follows:
| time (min) | Mobile phase B (%) | Mobile phase A (%) |
| 0.01 | 20 | 80 |
| 30 | 23 | 77 |
| 40 | 65 | 35 |
| 50 | 65 | 35 |
| 50.01 | 20 | 80 |
| 65 | 20 | 80 |
(1) Blank solution: acetonitrile: water ═ 30:70(v/v)
(2) Impurity stock solution: precisely weighing 10mg of each of the impurities A and B in a 100ml measuring flask, dissolving the diluent and fixing the volume (100 ug/ml of the impurity A and 100ug/ml of the impurity B).
(3) System applicability solution: precisely weighing 25mg of lomefloxacin hydrochloride reference substance in a 25ml measuring flask, precisely adding 0.25ml of impurity stock solution, and adding diluent for diluting to constant volume. (lomefloxacin hydrochloride 1.0mg/ml, impurities A, B each 1.0ug/ml)
(4) Impurity localization solution: 1.0mg of each of the impurities A and B is weighed and respectively placed in a 100ml measuring flask, and the diluent is added for dissolving and fixing the volume. (impurity A: 10ug/ml, impurity B: 10ug/ml)
(5) And (b) precisely weighing 25mg of lomefloxacin hydrochloride into a 25ml measuring flask, and adding the diluent solution to fix the volume.
(6) Reference solution (b): precisely measuring a proper amount of the reference solution (a), and diluting to 1.0ug/ml
Blank solution, system applicability solution, reference solution (a) (i.e. sample solution) and reference solution (b) (i.e. control solution) are injected according to chromatographic conditions, and the chromatographic process is recorded, and the results are shown in table 1 and fig. 4-7.
TABLE 1 System applicability
Experimental example 3 specificity
Experimental conditions, liquid chromatography method and solution preparation As in Experimental example 1
The method specifically inspects peak identification and selectivity, samples blank solution and impurity positioning solution respectively, records chromatogram, and the result is shown in Table 2
TABLE 2 specialization
| Name of solution | Name of substance | Concentration of solution | Retention time |
| Positioning solution for impurity A | Impurity A | 10.8ug/ml | 44.453 |
| Positioning solution for impurity B | Impurity B | 10.5ug/ml | 48.314 |
Experimental example 4 detection and quantitation limits
Experimental conditions, liquid chromatography method and solution preparation As in Experimental example 1
For known impurities, the limit of detection (LOD) and limit of quantitation (LOQ) are determined from the signal-to-noise ratio. Diluting an impurity stock solution with known concentration to low concentration, injecting sample, determining a detection signal-to-noise ratio S/N (signal-to-noise ratio) of more than or equal to 10 as a quantitative limit, and determining the S/N of 2-4 as a detection limit. The test results are shown in Table 3
TABLE 3 quantitation and detection limits
Experimental example 5 linearity and Range
Experimental conditions, liquid chromatography method and solution preparation As in Experimental example 1
For known impurities, 6 points are investigated from the range of LOQ concentration to the index concentration of not less than 150%, the linear relationship is plotted as a function of the measured response signal (peak area) against the analyte concentration, linear regression is carried out by the least squares method, and at least the correlation coefficient R is reported2To confirm a good linear relationship, the linear regression coefficient R is required2Should be not less than 0.990.
TABLE 4 Linearity and Range
Impurity correction factor
EXAMPLE 6 precision
Experimental conditions, liquid chromatography method and solution preparation As in Experimental example 1
Taking the separation degree solution as a test solution, preparing 6 parts in parallel, sequentially injecting samples, and obtaining the repeatability results shown in Table 5
TABLE 5 repeatability
| Sequence of | Impurity A | Impurity B | Max unknown simple hetero |
| 1 | 0.032 | 0.026 | 0.076 |
| 2 | 0.033 | 0.026 | 0.076 |
| 3 | 0.032 | 0.025 | 0.076 |
| 4 | 0.032 | 0.026 | 0.078 |
| 5 | 0.033 | 0.026 | 0.076 |
| 6 | 0.032 | 0.026 | 0.077 |
| RSD％ | 1.60 | 1.58 | 1.09 |
The intermediate precision was carried out by the same operating method and by different personnel on different instruments, the results are shown in Table 6
TABLE 6 intermediate precision
Experimental example 7 accuracy
Experimental conditions, liquid chromatography method and solution preparation As in Experimental example 1
The accuracy is obtained by adding three impurities with different concentrations of 80%, 100% and 120% of the index into a test sample and measuring the recovery rate of the impurities. The accuracy of the known impurities is determined by adding a known amount of the impurity and measuring the ratio between the amount of the known impurity in the sample to be added and the theoretical value (recovery), expressed as a percentage%. The accuracy of the unknown impurity was replaced by lomefloxacin hydrochloride.
TABLE 7 accuracy
Experimental examples 1-7 show that the method for measuring the known impurities of the lomefloxacin hydrochloride has good specificity, good precision, high accuracy and high sensitivity, and can be applied to quality control of related substances in the synthesis of raw material medicines.
In conclusion, the detection method can effectively separate the lomefloxacin hydrochloride from the impurity peak, accurately determine the content of the known impurity 6,7, 8-trifluoro-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester and the content of the known impurity 1-ethyl-6, 7, 8-trifluoro-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester of the lomefloxacin hydrochloride, has stable and reliable determination result and strong specificity, and provides possibility for quality control in the synthesis process of the lomefloxacin hydrochloride, thereby better controlling the product quality of the lomefloxacin hydrochloride.
Claims (11)
1. A method for detecting lomefloxacin hydrochloride related substances is characterized by comprising the following steps: the detection is carried out by adopting a high performance liquid chromatography, and the method comprises the following specific steps:
a. dissolving lomefloxacin hydrochloride in acetonitrile water solution to obtain a test solution;
b. adding a lomefloxacin hydrochloride reference substance and a related substance reference substance into an acetonitrile aqueous solution for dissolving to obtain a system applicability solution;
c. the following chromatographic conditions are adopted to respectively detect the test solution and the system applicability solution:
stationary phase: octadecylsilane chemically bonded silica is used as a filling agent;
mobile phase: using a 0.01-1.0% trifluoroacetic acid aqueous solution as a mobile phase A, and using a methanol acetonitrile solution with a volume ratio of 40-60: 40-60 as a mobile phase B;
the gradient elution procedure was: 0.01-30 min, 75-85% → 72-82% A, 30-40 min, 72-82% → 30-40% A, 40-50 min, 30-40% A, 50.01-65min, 75-85% A;
detection wavelength: 260-300 nm;
the flow rate is 0.5-1.5 ml/min;
the column temperature is 30-40 ℃;
the related substances are impurity A and/or impurity B;
the impurity A is 6,7, 8-trifluoro-1, 4-dihydro-4-oxo-3-quinoline carboxylic acid ethyl ester, and the impurity B is 1-ethyl-6, 7, 8-trifluoro-1, 4-dihydro-4-oxo-3-quinoline carboxylic acid ethyl ester.
2. The detection method according to claim 1, characterized in that: the mass-to-volume ratio of the lomefloxacin hydrochloride to the acetonitrile aqueous solution in the step a) is 0.5-1.0 mg: 1 ml.
3. The detection method according to claim 1 or 2, characterized in that: the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 30: 70.
4. the detection method according to claim 1, characterized in that: 0.5-1.0 mg of lomefloxacin hydrochloride and 0.5-1.0 mu g of impurities in each 1ml of the system applicability solution in the step b).
5. The detection method according to claim 1, characterized in that: the stationary phase is an Inertsil ODS-3 column; the specification of the chromatographic column is as follows: the inner diameter is 4.6 multiplied by 250mm, 5 um.
6. The detection method according to claim 1, characterized in that: and c) the detection wavelength is 287nm, the flow rate is 1.0-1.1 ml/min, the column temperature is 30 ℃, and the sample injection amount is 20 mu L.
7. The detection method according to claim 1, wherein: step c) the gradient elution procedure is: 0.01-30 min, 80% → 77% A, 30-40 min, 77% → 35% A, 40-50 min, 35% A, 50.01-65min, 80% A.
8. The detection method according to claim 1, wherein: and a chromatogram of the test solution shows a chromatographic peak corresponding to the retention time of a chromatographic peak of a related substance reference substance in a system applicability solution chromatogram, namely the test solution contains related substances.
9. A method for measuring the content of lomefloxacin hydrochloride related substances is characterized by comprising the following steps: the specific operation steps are as follows:
1) adding acetonitrile aqueous solution into the sample solution obtained in the step a to dilute the sample solution by 100-1000 times to obtain a reference solution; the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 30: 70;
2) detected according to the method of any one of claims 1 to 8;
3) and (3) calculating the content of related substances according to the following calculation formula:
wherein,
: the content of the relevant substances in the test sample,
: peak area of the relevant substance in the test solution, A: the peak area of lomefloxacin hydrochloride in the control solution, N is the dilution multiple of the control solution, fiCorrection factors for the substances of interest.
10. The content measurement method according to claim 9, characterized in that: adding the acetonitrile aqueous solution to dilute the mixture by 1000 times in the step 1).
11. The content measurement method according to claim 10, characterized in that: the related substance is impurity A, the correction factor of which is 2.42, and the related substance is impurity B, the correction factor of which is 2.93.
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