CN112557550A - Method for determining related substances in metronidazole sodium chloride injection - Google Patents
Method for determining related substances in metronidazole sodium chloride injection Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 35
- 239000000126 substance Substances 0.000 title claims abstract description 34
- 239000008354 sodium chloride injection Substances 0.000 title claims abstract description 23
- LKRWBLLGEAXPRY-UHFFFAOYSA-N metronidazole sodium Chemical compound [Na+].CC1=NC=C([N+]([O-])=O)N1CC[O-] LKRWBLLGEAXPRY-UHFFFAOYSA-N 0.000 title claims abstract description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 104
- 239000012535 impurity Substances 0.000 claims abstract description 89
- 238000001514 detection method Methods 0.000 claims abstract description 16
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 8
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims abstract description 8
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 7
- 239000000945 filler Substances 0.000 claims abstract description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 4
- 238000010828 elution Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 94
- 238000007865 diluting Methods 0.000 claims description 47
- 239000000523 sample Substances 0.000 claims description 36
- 239000012085 test solution Substances 0.000 claims description 23
- 239000013558 reference substance Substances 0.000 claims description 20
- 239000012488 sample solution Substances 0.000 claims description 18
- VAOCPAMSLUNLGC-UHFFFAOYSA-N metronidazole Chemical compound CC1=NC=C([N+]([O-])=O)N1CCO VAOCPAMSLUNLGC-UHFFFAOYSA-N 0.000 claims description 16
- 229960000282 metronidazole Drugs 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 15
- 238000005303 weighing Methods 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 10
- FFYTTYVSDVWNMY-UHFFFAOYSA-N 2-Methyl-5-nitroimidazole Chemical compound CC1=NC=C([N+]([O-])=O)N1 FFYTTYVSDVWNMY-UHFFFAOYSA-N 0.000 claims description 8
- 238000012937 correction Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 5
- 230000014759 maintenance of location Effects 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 4
- DCEBDJYBLDHZRV-UHFFFAOYSA-L CO.P(=O)([O-])([O-])O.[K+].[K+] Chemical compound CO.P(=O)([O-])([O-])O.[K+].[K+] DCEBDJYBLDHZRV-UHFFFAOYSA-L 0.000 claims description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 1
- 239000011780 sodium chloride Substances 0.000 claims 1
- 238000004458 analytical method Methods 0.000 abstract description 5
- 238000000926 separation method Methods 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 238000004587 chromatography analysis Methods 0.000 abstract description 2
- 239000000741 silica gel Substances 0.000 abstract 1
- 229910002027 silica gel Inorganic materials 0.000 abstract 1
- 239000002904 solvent Substances 0.000 description 15
- 238000012360 testing method Methods 0.000 description 14
- 239000011550 stock solution Substances 0.000 description 12
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 description 10
- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 description 10
- 238000003860 storage Methods 0.000 description 9
- 238000011084 recovery Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000001954 sterilising effect Effects 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
-
- 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
- G01N30/26—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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
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Abstract
The invention discloses a method for determining related substances in metronidazole sodium chloride injection, belonging to the technical field of drug analysis. The HPLC detection conditions were as follows: octadecylsilane chemically bonded silica gel as filler (ACE Excel C)18-AR 5 μm 250 × 4.6mm, or equivalent performance chromatography column); gradient elution was carried out using 0.01mol/L dipotassium hydrogen phosphate (pH adjusted to 6.5 with phosphoric acid) -methanol (85: 15) as mobile phase A and methanol as mobile phase B. The invention has the advantages of good separation degree among impurities, short analysis time and low cost, can effectively control related substances of the metronidazole sodium chloride injection and ensure the safety and the effectiveness of the product.
Description
Technical Field
The invention relates to the technical field of drug analysis, in particular to a method for determining related substances in metronidazole sodium chloride injection.
Background
The metronidazole and sodium chloride injection is an antibacterial drug and is mainly used for treating anaerobic infection.
The China pharmacopoeia 2020 edition collects the quality standard of metronidazole sodium chloride injection, and adopts high performance liquid chromatography to control the content of related substances, but the method only controls the content of impurity I, other impurities are determined in the mode of total amount of impurities, and the attribution and determination of each impurity are not carried out. Other pharmacopoeias such as the us pharmacopoeia 40 and the european pharmacopoeia 9.0 also do not control the content of each impurity. In order to more effectively control the content of the related substances in the metronidazole sodium chloride injection and ensure the safety and effectiveness of the medication by combining the above conditions, the applicant develops and develops a related substance inspection method suitable for the product on the basis of a related substance inspection method of metronidazole raw materials in European pharmacopoeia 9.0. The method can effectively separate the impurities A to G, is quick, simple and convenient, and has better accuracy, specificity and linearity.
Disclosure of Invention
In view of the serious defects of the prior art, the applicant searches a quality control method of the metronidazole sodium chloride injection through a large number of experiments on the basis of the European pharmacopoeia 9.0, strictly performs methodology verification, ensures the scientific and rigorous method, and meets the requirements of research, development and production. Therefore, the invention aims to provide an HPLC (high performance liquid chromatograph) determination method for related substances of metronidazole and sodium chloride injection.
TABLE 1 optimization of the substance analysis method
In order to achieve the purpose, the invention provides the following technical scheme: a method for determining related substances in metronidazole sodium chloride injection adopts High Performance Liquid Chromatography (HPLC) detection, and comprises the following steps:
the method comprises the following steps: taking a proper amount of samples, and quantitatively diluting with 15% methanol to prepare a metronidazole solution;
step two: taking dipotassium phosphate-methanol solution as a mobile phase A and taking methanol as a mobile phase B;
step three: and detecting the sample by using a high performance liquid chromatography with octadecylsilane chemically bonded silica as a filler and an ultraviolet detector as a detector.
Preferably, the volume ratio of the dipotassium hydrogen phosphate to the methanol is 85: mobile phase a is 15.
Preferably, the pH of the dipotassium phosphate is adjusted to 6.5 with phosphoric acid.
Preferably, in step three, the HPLC is ACE Excel C18-AR 5 μm 250 × 4.6mm column, volume ratio of dipotassium hydrogenphosphate to methanol of 0.01mol/L, pH of 6.5 85: mobile phase a at 15 and mobile phase B at methanol, the gradient elution was performed according to the procedure below.
Preferably, the detection method of the high performance liquid chromatograph is as follows: the sample injection volume of the sample injection chromatograph is 20 mul, and the detection wavelength is 315 nm.
Preferably, the sample is a test solution, a control solution, a sensitivity solution, or a system suitability solution.
Preferably, the method comprises the following steps:
1) sample preparation
Preparation of a test solution: taking a proper amount of the product, and quantitatively diluting the product with 15% methanol solution to prepare a solution containing 0.5mg of metronidazole in each 1 ml;
preparation of control solution: precisely measuring 1ml of a sample solution, placing the sample solution in a 100ml measuring flask, diluting the sample solution to a scale with a 15% methanol solution, shaking up, precisely measuring 5ml of the sample solution, placing the sample solution in a 50ml measuring flask, diluting the sample solution to a scale with a 15% methanol solution, and shaking up;
preparation of a sensitive solution: precisely measuring 5ml of the control solution, placing the control solution in a 25ml measuring flask, diluting the control solution to a scale with 15% methanol solution, and shaking up;
preparation of system suitability solution: taking a proper amount of a metronidazole impurity A, B, C, D, E, F, G reference substance, precisely weighing, adding a small amount of methanol to dissolve, quantitatively diluting with a 15% methanol solution to prepare a mixed solution containing 5 micrograms of the impurities A-G in each 1ml, precisely weighing 5ml and 5ml of a sample, placing in the same 50ml measuring flask, diluting with the 15% methanol solution to a scale, and shaking uniformly;
2) and detecting the test solution, the control solution and the system applicability solution by adopting the detection condition of the high performance liquid chromatography, and qualitatively analyzing and quantitatively analyzing related substances of the sample to be detected.
Preferably, metronidazole impurity a is 2-methyl-5 nitroimidazole.
Preferably, step 2) is specifically as follows: injecting the system applicability solution into a liquid chromatograph, recording a chromatogram, precisely measuring 20 mu l of the test solution and the control solution, respectively injecting into the liquid chromatograph, and recording the chromatogram; if a chromatographic peak with the relative retention time consistent with that of the impurities A-G exists in the chromatogram of the test solution, the product of the peak area and the relative correction factor is not more than 0.1 percent of the area of the main peak of the control solution according to the self-control method of the main component added with the correction factor; the peak area of other single impurities is not more than 0.1% of the main peak area of the control solution; the total amount of impurities is not more than 0.5%; the relative retention time with metronidazole in the chromatogram of the test solution is 0.22, and the previous peak is ignored.
The invention has the technical effects and advantages that: the separation degree of each impurity is good, the operation is simple, convenient and quick, the analysis time is short, the cost is low, the related substances of the metronidazole sodium chloride injection can be effectively controlled, and the safety and the effectiveness of the product are ensured.
Drawings
FIG. 1 is a diagram of a sample related substance examination-test article (20040216) according to the present invention;
FIG. 2 is a diagram of a sample related substance examination-test article (20040316) according to the present invention;
FIG. 3 is a diagram of a sample related substance examination-test article (20040416) according to the present invention;
FIG. 4 is a specificity test profile of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
As shown in fig. 1-3, the invention provides a method for determining related substances in metronidazole sodium chloride injection, comprising the following steps:
the method comprises the following steps: taking a proper amount of samples, and quantitatively diluting with 15% methanol to prepare a metronidazole solution;
step two: taking dipotassium phosphate-methanol solution as a mobile phase;
step three: a high performance liquid chromatograph using octadecylsilane chemically bonded silica as a filler;
step four: an ultraviolet detector is used as a detector.
Apparatus and conditions
Waters2695 high performance liquid chromatograph using octadecylsilane chemically bonded silica as filler (ACE Excel C)18-AR 5 μm 250 × 4.6mm, or equivalent performance chromatography column); gradient elution was performed according to the following procedure using 0.01mol/L dipotassium hydrogen phosphate (pH adjusted to 6.5 with phosphoric acid) -methanol (85: 15) as mobile phase A and methanol as mobile phase B; the column temperature was 30 ℃, the flow rate was 1.0ml/min, and the sample volume was 25. mu.l.
The experimental steps are as follows:
preparation of a test solution: taking a proper amount of the product, and quantitatively diluting the product with 15% methanol solution to prepare a solution containing about 0.5mg of metronidazole in each 1 ml.
Preparation of control solution: precisely measuring 1ml of the test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to the scale with 15% methanol solution, shaking up, precisely measuring 5ml of the test solution, placing the test solution in a 50ml measuring flask, diluting the test solution to the scale with 15% methanol solution, and shaking up.
Preparation of a sensitive solution: precisely measuring 5ml of the control solution, placing the control solution into a 25ml measuring flask, diluting the control solution to the scale with 15% methanol solution, and shaking up.
Preparation of system suitability solution: taking a proper amount of metronidazole impurity A (2-methyl-5 nitroimidazole) and B, C, D, E, F, G reference substances, precisely weighing, adding a small amount of methanol to dissolve, quantitatively diluting with 15% methanol solution to prepare mixed solution containing 5 micrograms of impurities A-G respectively per 1ml, precisely weighing 5ml and 5ml of sample, placing in a same 50ml measuring flask, diluting to scale with 15% methanol solution, and shaking uniformly.
Precisely measuring the test solution and the reference solution, respectively injecting into a chromatograph, and recording the chromatogram.
The method is used for detecting the related substances in the test samples of 3 batches of metronidazole sodium chloride injection, the detection results are shown in table 2, the spectra are shown in fig. 1-3, under the chromatographic conditions, the separation degree of the related substances of the metronidazole sodium chloride injection is good, the number of theoretical plates of each impurity is greater than 2000, and the tailing factors meet the requirements.
Table 2: the results of detection of the substances concerned (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G)
Correlation verification
1. Specificity
Auxiliary material test solution: preparing 100ml of each single adjuvant solution without metronidazole according to the prescription proportion of the single preparation, subpackaging in penicillin bottles, sterilizing at 121 ℃ for 12 minutes, taking 1ml of each single adjuvant solution, placing in the same 10ml measuring bottle, diluting to scale with a diluent solvent, and shaking uniformly;
impurity control solution: taking a proper amount of reference substances of impurities A, B, C, D, E, F and G, precisely weighing, quantitatively diluting with a diluting solvent to prepare solutions (single impurity stock solutions) containing 100 micrograms of impurities per 1ml, precisely weighing 1ml of the reference substance stock solutions, placing the solutions in a same 20ml measuring flask, diluting to scale with the diluting solvent, shaking up (stock solutions), precisely weighing 1ml, placing the solutions in a 10ml measuring flask, diluting to scale with the diluting solvent, and shaking up;
test article (self-grinding) solution: precisely measuring 1ml of a sample, placing the sample in a 10ml measuring flask, diluting the sample to a scale with a diluting solvent, and shaking up;
test (reference) solution: precisely measuring 1ml of a sample, placing the sample in a 10ml measuring flask, diluting the sample to a scale with a diluting solvent, and shaking up;
mixing (self-grinding) the solution: precisely measuring 2ml of a sample and 2ml of a mixed impurity reference substance storage solution, placing the samples and the mixed impurity reference substance storage solution into the same 20ml measuring flask, diluting the samples and the mixed impurity reference substance storage solution to a scale by using a diluting solvent, and shaking up the samples and the mixed impurity reference substance storage solution;
mix (reference) solution: precisely measuring 2ml of the sample and 2ml of the mixed impurity reference substance storage solution, placing the samples and the mixed impurity reference substance storage solution into the same 20ml measuring flask, diluting the samples and the mixed impurity reference substance storage solution to the scale with a diluting solvent, and shaking up the samples and the mixed impurity reference substance storage solution.
Taking each solution, injecting 20 μ l of sample under the preliminarily determined chromatographic conditions, and recording the chromatogram, which is shown in FIG. 4.
The test results show that:
(1) the blank solution and the blank auxiliary material test solution peak before 3min, and the main component peak and each impurity peak are not reserved, so that the detection of impurities is not influenced;
(2) after blank auxiliary material and solvent peaks are deducted from the sample solution, the separation degrees between a main component peak and adjacent impurity peaks and between impurities are all more than 1.5, and all component peaks are pure peaks;
(3) after blank auxiliary material and solvent peaks are deducted from the mixed solution, the separation degrees between the main component peak and the adjacent impurity peaks and between the impurities are all more than 1.5.
The above results show that the detection method has good specificity.
2. Quantitative limit and detection limit
Taking appropriate amount of metronidazole, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G reference substances, precisely weighing, respectively adding a diluting solvent to dissolve and quantitatively dilute to prepare a solution containing about 5 mu G of metronidazole, 0.5 mu G of impurity A, 0.5 mu G of impurity B, 0.5 mu G of impurity C, 0.5 mu G of impurity D, 0.5 mu G of impurity E, 0.5 mu G of impurity F and 0.5 mu G of impurity G in each 1ml, diluting the solution step by step, when the ratio S/N of the peak height of a main peak to the noise is not less than 10, calculating the concentration at the moment to be the quantitative limit concentration, and when the ratio S/N of the peak height of the main peak to the noise is not less than 3, calculating the concentration at the moment to be the detection limit concentration. Injecting 20 mul sample according to the detection method, recording chromatogram, and obtaining experimental results shown in the following table
Table 3: quantitative limit and detection limit test results of related substances (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G)
The test results show that: 6 parts of quantitative limiting solution are repeatedly injected, the RSD of the retention time is not more than 2.0 percent, and the RSD of the peak area is not more than 10 percent; meeting the acceptance criteria.
3. Linearity and range
Taking appropriate amount of metronidazole, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G as reference substances, precisely weighing, respectively adding a diluting solvent to dissolve and quantitatively dilute to prepare a solution containing about 2.5 mu G of metronidazole, 2.5 mu G of impurity A, 2.5 mu G of impurity B, 2.5 mu G of impurity C, 2.5 mu G of impurity D, 2.5 mu G of impurity E, 2.5 mu G of impurity F and 2.5 mu G of impurity G per 1ml, and taking the solution as a reference substance storage solution. Taking a proper amount of a reference substance stock solution, and preparing a solution with a quantitative limit concentration; precisely measuring 1ml, 2ml, 3 ml, 4 ml, 5ml, 6 ml, 7 ml and 8ml of the reference stock solutions, respectively placing in a 20ml measuring flask, diluting to scale with a diluent solvent, and shaking up to obtain reference stock solutions. Taking the reference substance solution, injecting 20 mu l of the reference substance solution respectively, recording a chromatogram, drawing a linear regression equation by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate, and obtaining the result shown in the following table:
table 4: results of the linearity and range tests of the substances (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G) involved
The test results show that:
(1) the linear correlation coefficient R of each component is more than 0.990;
(2) the y-axis intercept is within 25% of the 100% response value with a relative standard deviation of less than 10%.
4. Accuracy of
When the concentrations of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E, the impurity F and the impurity G were determined to be within the quantitative limits of 100% and 200% with the concentration (0.5. mu.g/ml) of the control solution as 100%, the recovery rate of the sample solution was examined.
Reference stock solution: respectively taking appropriate amount of reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G, precisely weighing, adding a diluting solvent to dissolve, and quantitatively diluting to obtain a solution containing about 5 μ G of impurity in 1 ml.
Quantification limit control stock solution: taking a proper amount of the reference stock solution, and preparing according to the concentration which is 10 times of the limit of quantitation.
Test solution: precisely measuring 1ml of the sample, placing the sample in a 10ml measuring flask, diluting the sample to a scale with a diluting solvent, and shaking up.
Sample solution to be tested was dosed (in triplicate): precisely measuring 1ml of sample and 1ml of quantitative limit reference stock solution, placing in the same 10ml measuring flask, diluting with water to scale, and shaking.
Sample solutions were loaded 100% (in triplicate): precisely measuring 1ml of sample and 1ml of reference stock solution, placing in the same 10ml measuring flask, diluting with water to scale, and shaking.
Sample solution was loaded at 200% (in triplicate): precisely measuring 1ml of sample and 2ml of reference stock solution, placing in the same 10ml measuring flask, diluting with water to scale, and shaking.
Taking the above sample solution, introducing sample of 20 μ l, recording chromatogram, calculating recovery rate and RSD according to main component self-contrast method with correction factor (relative correction factor of 1.00), and finding results in Table 5
Table 5: the result of the accuracy test of the related substances (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G)
The test results show that:
(1) the recovery rate of the quantitative limit concentration impurities is 70.0-130.0%;
(2) except for the quantitative limit, the impurity recovery rate is 90.0-108.0%, and the RSD of the recovery rate is not more than 10%.
It should be noted that:
firstly, the invention adopts the prior art without specific description; secondly, the method comprises the following steps: in the drawings of the disclosed embodiments of the invention, only the structures related to the disclosed embodiments are referred to, other structures can refer to common designs, and the same embodiment and different embodiments of the invention can be combined with each other without conflict; and finally: the above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that are within the spirit and principle of the present invention are intended to be included in the scope of the present invention.
Claims (9)
1. A method for determining related substances in metronidazole sodium chloride injection is characterized by comprising the following steps: the method comprises the following steps:
the method comprises the following steps: taking a proper amount of samples, and quantitatively diluting with 15% methanol to prepare a metronidazole solution;
step two: taking dipotassium phosphate-methanol solution as a mobile phase A and taking methanol as a mobile phase B;
step three: and detecting the sample by using a high performance liquid chromatography with octadecylsilane chemically bonded silica as a filler and an ultraviolet detector as a detector.
2. The method for determining related substances in metronidazole sodium chloride injection as claimed in claim 1, characterised in that: volume ratio of dipotassium hydrogenphosphate to methanol at 0.01mol/L, pH of 6.5, 85: mobile phase a is 15.
3. The method for determining related substances in metronidazole sodium chloride injection as claimed in claim 2, characterised in that: the pH of the dipotassium phosphate was adjusted to 6.5 with phosphoric acid.
4. Metronidazole sodium chloride according to claim 3A method for measuring a substance contained in an injection, comprising the steps of: in the third step, the high performance liquid chromatograph uses ACE Excel C18-AR 5 μm 250 × 4.6mm column, volume ratio of dipotassium hydrogenphosphate to methanol of 0.01mol/L, pH of 6.5 85: mobile phase a at 15 and mobile phase B in methanol, the gradient elution was carried out according to the following procedure:
5. the method for determining related substances in metronidazole sodium chloride injection as claimed in claim 1, characterised in that: the detection method of the high performance liquid chromatograph comprises the following steps: the sample injection volume of the sample injection chromatograph is 20 mul, and the detection wavelength is 315 nm.
6. The method for determining related substances in metronidazole sodium chloride injection as claimed in claim 1, characterised in that: the sample is a test solution, a control solution, a sensitivity solution or a system suitability solution.
7. The method for determining related substances in metronidazole sodium chloride injection as claimed in claim 6, characterised in that it comprises the following steps:
1) sample preparation
Preparation of a test solution: taking a proper amount of the product, and quantitatively diluting the product with 15% methanol solution to prepare a solution containing 0.5mg of metronidazole in each 1 ml;
preparation of control solution: precisely measuring 1ml of a sample solution, placing the sample solution in a 100ml measuring flask, diluting the sample solution to a scale with a 15% methanol solution, shaking up, precisely measuring 5ml of the sample solution, placing the sample solution in a 50ml measuring flask, diluting the sample solution to a scale with a 15% methanol solution, and shaking up;
preparation of a sensitive solution: precisely measuring 5ml of the control solution, placing the control solution in a 25ml measuring flask, diluting the control solution to a scale with 15% methanol solution, and shaking up;
preparation of system suitability solution: taking a proper amount of a metronidazole impurity A, B, C, D, E, F, G reference substance, precisely weighing, adding a small amount of methanol to dissolve, quantitatively diluting with a 15% methanol solution to prepare a mixed solution containing 5 micrograms of the impurities A-G in each 1ml, precisely weighing 5ml and 5ml of a sample, placing in the same 50ml measuring flask, diluting with the 15% methanol solution to a scale, and shaking uniformly;
2) and detecting the test solution, the control solution and the system applicability solution by adopting the detection condition of the high performance liquid chromatography, and qualitatively analyzing and quantitatively analyzing related substances of the sample to be detected.
8. The method for determining related substances in metronidazole sodium chloride injection as claimed in claim 7, wherein metronidazole impurity A is 2-methyl-5 nitroimidazole.
9. The method for determining related substances in metronidazole sodium chloride injection as claimed in claim 7, characterised in that step 2) is as follows: injecting the system applicability solution into a liquid chromatograph, recording a chromatogram, precisely measuring 20 mu l of the test solution and the control solution, respectively injecting into the liquid chromatograph, and recording the chromatogram; if a chromatographic peak with the relative retention time consistent with that of the impurities A-G exists in the chromatogram of the test solution, the product of the peak area and the relative correction factor is not more than 0.1 percent of the area of the main peak of the control solution according to the self-control method of the main component added with the correction factor; the peak area of other single impurities is not more than 0.1% of the main peak area of the control solution; the total amount of impurities is not more than 0.5%; the relative retention time with metronidazole in the chromatogram of the test solution is 0.22, and the previous peak is ignored.
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| CN106937944A (en) * | 2017-01-22 | 2017-07-11 | 北京圣传创世科技发展有限公司 | A kind of injection metronidazole freeze-dried powder and preparation method thereof |
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| CN106937944A (en) * | 2017-01-22 | 2017-07-11 | 北京圣传创世科技发展有限公司 | A kind of injection metronidazole freeze-dried powder and preparation method thereof |
| CN109115932A (en) * | 2018-07-31 | 2019-01-01 | 湖北省宏源药业科技股份有限公司 | A kind of related substance detecting method of 2- 5-nitro imidazole |
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