CN113433237A - Method for separating and determining cefaclor bulk drug and impurities thereof by high performance liquid chromatography - Google Patents
Method for separating and determining cefaclor bulk drug and impurities thereof by high performance liquid chromatography Download PDFInfo
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- CN113433237A CN113433237A CN202110704959.3A CN202110704959A CN113433237A CN 113433237 A CN113433237 A CN 113433237A CN 202110704959 A CN202110704959 A CN 202110704959A CN 113433237 A CN113433237 A CN 113433237A
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- cefaclor
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- dihydrogen phosphate
- impurity
- sodium dihydrogen
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- QYIYFLOTGYLRGG-GPCCPHFNSA-N cefaclor Chemical compound C1([C@H](C(=O)N[C@@H]2C(N3C(=C(Cl)CS[C@@H]32)C(O)=O)=O)N)=CC=CC=C1 QYIYFLOTGYLRGG-GPCCPHFNSA-N 0.000 title claims abstract description 76
- 229960005361 cefaclor Drugs 0.000 title claims abstract description 76
- 239000012535 impurity Substances 0.000 title claims abstract description 72
- 239000003814 drug Substances 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 27
- 229940079593 drug Drugs 0.000 title claims abstract description 23
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 10
- 238000000926 separation method Methods 0.000 claims abstract description 47
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims abstract description 25
- 235000019799 monosodium phosphate Nutrition 0.000 claims abstract description 25
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 25
- 239000002994 raw material Substances 0.000 claims abstract description 22
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 238000010828 elution Methods 0.000 claims abstract description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 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
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 5
- 239000003643 water by type Substances 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 39
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- 239000012085 test solution Substances 0.000 claims description 20
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 18
- 239000002904 solvent Substances 0.000 claims description 18
- OOSZCNKVJAVHJI-UHFFFAOYSA-N 1-[(4-fluorophenyl)methyl]piperazine Chemical compound C1=CC(F)=CC=C1CN1CCNCC1 OOSZCNKVJAVHJI-UHFFFAOYSA-N 0.000 claims description 14
- 229940074545 sodium dihydrogen phosphate dihydrate Drugs 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 238000007865 diluting Methods 0.000 claims description 7
- 230000014759 maintenance of location Effects 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 11
- 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 8
- 230000000052 comparative effect Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 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 description 4
- KXDAEFPNCMNJSK-UHFFFAOYSA-N Benzamide Chemical compound NC(=O)C1=CC=CC=C1 KXDAEFPNCMNJSK-UHFFFAOYSA-N 0.000 description 4
- DTHMTBUWTGVEFG-QRPNPIFTSA-N [(1s)-2-methoxy-2-oxo-1-phenylethyl]azanium;chloride Chemical compound Cl.COC(=O)[C@@H](N)C1=CC=CC=C1 DTHMTBUWTGVEFG-QRPNPIFTSA-N 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- CVXXCIJNZNETDW-VIFPVBQESA-N (3s)-3-phenylpiperazine-2,5-dione Chemical compound N1C(=O)CNC(=O)[C@@H]1C1=CC=CC=C1 CVXXCIJNZNETDW-VIFPVBQESA-N 0.000 description 1
- 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 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- ZGUNAGUHMKGQNY-SSDOTTSWSA-N D-alpha-phenylglycine Chemical compound OC(=O)[C@H](N)C1=CC=CC=C1 ZGUNAGUHMKGQNY-SSDOTTSWSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000003936 benzamides Chemical class 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229940124588 oral cephalosporin Drugs 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic 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
-
- 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
- G01N30/14—Preparation by elimination of some components
-
- 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
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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
- 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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- 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
- 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
- G01N2030/8872—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 impurities
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Cephalosporin Compounds (AREA)
Abstract
The invention belongs to the technical field of analysis and detection, and particularly relates to a method for separating and determining cefaclor bulk drug and impurities thereof by high performance liquid chromatography, wherein the adopted chromatographic conditions are as follows: octadecylsilane chemically bonded silica was used as a filler, Waters Xbridge C18, 4.6mm × 250mm, 3.5 μm; the mobile phase A is sodium dihydrogen phosphate solution, the mobile phase B is sodium dihydrogen phosphate solution-acetonitrile, and linear gradient elution is carried out; the column temperature is 20 ℃; the flow rate is 1.0ml/min per minute; the detection wavelength was 220 nm. The method can realize the complete separation of the cefaclor peak and each impurity, the separation degree of the cefaclor peak and each impurity is more than 1.5, the separation degree of each impurity is more than 1.5, the separation efficiency is high, the analysis speed is high, the detection sensitivity is high, impurities possibly existing in the cefaclor raw material medicine can be better detected, and the quality of the cefaclor raw material medicine can be better controlled.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a method for separating and determining cefaclor bulk drug and impurities thereof by high performance liquid chromatography.
Background
Cefaclor, a semi-synthetic second generation oral cephalosporin antibiotic drug, has been approved by FDA in 1979 and successfully marketed in the united states in 1982, and has become one of the important drugs for the treatment of bacterial infections due to its broad spectrum, high efficacy and good clinical safety. The sterilization mechanism of cefaclor is to inactivate the transferase, interfere the synthesis of the final stage of the bacterial cell wall, prevent the cross-linking of the mucopeptide, and have strong killing effect on gram-positive bacteria and gram-negative bacteria such as staphylococcus, streptococcus, pneumococcus, escherichia coli and the like.
The starting raw materials, intermediates, final product analogs, degradation products and the like of the cefaclor raw material medicaments in the synthesis process can become impurities of the cefaclor raw material medicaments, so that the quality of the cefaclor raw material medicaments is influenced, and the final curative effect is further influenced. Therefore, quality control of bulk drugs is a key and difficult point in drug development.
At present, the impurities related to the cefaclor bulk drug include the following:
at present, no literature report on a separation and determination method of cefaclor and the impurities is found, so that the method for separating and determining cefaclor and the impurities is urgently found for controlling the quality of cefaclor raw material medicines, improving the curative effect of medicines and reducing toxic and side effects, and has important significance for quality control of cefaclor raw material medicines.
Disclosure of Invention
Aiming at the problems, the invention provides a method for separating and measuring cefaclor bulk drug and impurities thereof by high performance liquid chromatography, and the method has stronger impurity separation capability and detection capability.
The invention relates to a method for separating and measuring cefaclor bulk drug and impurities thereof by high performance liquid chromatography, which adopts the chromatographic conditions as follows: a chromatographic column: octadecylsilane chemically bonded silica was used as a filler, Waters Xbridge C18, 4.6mm × 250mm, 3.5 μm; the mobile phase A is sodium dihydrogen phosphate solution, the mobile phase B is sodium dihydrogen phosphate solution-acetonitrile, and the volume ratio of the sodium dihydrogen phosphate solution to the acetonitrile is 55:45, and the linear gradient elution is carried out; the column temperature is 20 ℃; the flow rate is 1.0ml/min per minute; the detection wavelength was 220 nm.
The preparation process of the sodium dihydrogen phosphate solution comprises the following steps: 7.8g of sodium dihydrogen phosphate dihydrate was taken, dissolved in water and diluted to 1000ml, and the pH was adjusted to 4.7 with phosphoric acid or 1mol/L sodium hydroxide.
The linear gradient elution procedure was:
| time (minutes) | Mobile phase A (%) | Mobile phase B(％) |
| 0 | 95 | 5 |
| 30 | 75 | 25 |
| 60 | 0 | 100 |
| 65 | 0 | 100 |
| 70 | 95 | 5 |
| 80 | 95 | 5 |
。
The method comprises the following steps:
(1) taking a proper amount of cefaclor raw material medicine, adding a solvent to dissolve and dilute the cefaclor raw material medicine to prepare a solution containing 5mg of cefaclor raw material medicine in every 1ml, filtering, and taking a subsequent filtrate to obtain a test solution;
(2) precisely measuring 1ml of a test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to a scale with a solvent, and shaking up to obtain a control solution;
(3) taking a proper amount of cefaclor and cefaclor delta-3 isomer, adding a solvent for dissolving and diluting to prepare a mixed solution containing 25 mu g of cefaclor and 50 mu g of cefaclor delta-3 isomer in each 1ml, and taking the mixed solution as a system applicability solution;
(4) injecting 20 mul of the system applicability solution into a liquid chromatograph to ensure that the separation degree between the cefaclor peak and the cefaclor 5-3 peak meets the requirement;
(5) precisely measuring 20 μ l of each of the test solution and the control solution, respectively injecting into a liquid chromatograph, and recording the chromatogram until the retention time of the main component peak is 5 times.
The solvent adopted in the steps (1) - (3) is a sodium dihydrogen phosphate solution with the pH value of 2.5, and the specific preparation process comprises the steps of taking 2.7g of sodium dihydrogen phosphate dihydrate, adding water to dissolve and dilute the sodium dihydrogen phosphate dihydrate to 1000ml, and adjusting the pH value to 2.5 by using 20% phosphoric acid.
The invention has the beneficial effects that: the method can realize the complete separation of the cefaclor peak and each impurity, the separation degree of the cefaclor peak and each impurity is more than 1.5, the separation degree of each impurity is more than 1.5, the separation efficiency is high, the analysis speed is high, the detection sensitivity is high, impurities possibly existing in the cefaclor raw material medicine can be better detected, and the quality of the cefaclor raw material medicine can be better controlled.
Drawings
Fig. 1 is a graph of separation of cefaclor bulk drug and impurities thereof in example 1.
Detailed Description
Example 1
A method for separating and measuring cefaclor bulk drug and impurities thereof by high performance liquid chromatography adopts the chromatographic conditions as follows: octadecylsilane chemically bonded silica was used as a filler, Waters Xbridge C18, 4.6mm × 250mm, 3.5 μm; the mobile phase A is sodium dihydrogen phosphate solution, the mobile phase B is sodium dihydrogen phosphate solution-acetonitrile, and the volume ratio of the sodium dihydrogen phosphate solution: acetonitrile 55:45, linear gradient elution; the column temperature is 20 ℃; the flow rate is 1.0ml/min per minute; the detection wavelength was 220 nm.
The preparation process of the sodium dihydrogen phosphate solution comprises the following steps: 7.8g of sodium dihydrogen phosphate dihydrate was taken, dissolved in water and diluted to 1000ml, and the pH was adjusted to 4.7 with phosphoric acid or 1mol/L sodium hydroxide.
The linear gradient elution procedure was:
| time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 95 | 5 |
| 30 | 75 | 25 |
| 60 | 0 | 100 |
| 65 | 0 | 100 |
| 70 | 95 | 5 |
| 80 | 95 | 5 |
。
The method specifically comprises the following steps:
(1) taking a proper amount of cefaclor raw material medicine, adding a solvent to dissolve and dilute the cefaclor raw material medicine to prepare a solution containing 5mg of cefaclor raw material medicine in every 1ml, filtering, and taking a subsequent filtrate to obtain a test solution;
(2) precisely measuring 1ml of a test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to a scale with a solvent, and shaking up to obtain a control solution;
(3) taking a proper amount of cefaclor and cefaclor delta-3 isomer, adding a solvent for dissolving and diluting to prepare a mixed solution containing 25 mu g of cefaclor and 50 mu g of cefaclor delta-3 isomer in each 1ml, and taking the mixed solution as a system applicability solution;
(4) injecting 20 mul of system applicability solution into a liquid chromatograph to ensure that the separation degree between the cefaclor peak and the cefaclor delta-3 peak meets the requirement;
(5) precisely measuring 20 μ l of each of the test solution and the control solution, respectively injecting into a liquid chromatograph, and recording the chromatogram until the retention time of the main component peak is 5 times.
The solvent adopted in the steps (1) - (3) is a sodium dihydrogen phosphate solution with the pH value of 2.5, and the specific preparation process comprises the steps of taking 2.7g of sodium dihydrogen phosphate dihydrate, adding water to dissolve and dilute the sodium dihydrogen phosphate dihydrate to 1000ml, and adjusting the pH value to 2.5 by using 20% phosphoric acid.
The map of the separation of cefaclor bulk drug and impurities thereof is shown in figure 1, and the result is as follows:
TABLE 1 impurity separation
| Name (according to retention time) | RT(min) | Degree of separation |
| BP impurity A | 3.682 | / |
| BP impurity B | 4.133 | 3.34732 |
| Cefaclor impurity A | 6.410 | 13.00047 |
| BP impurity G | 16.150 | 41.42427 |
| BP impurity C | 16.821 | 2.10335 |
| (3S) -3-phenylpiperazine-2, 5-dione | 17.803 | 2.77355 |
| D-phenylglycine methyl ester hydrochloride | 19.219 | 3.86724 |
| Cefaclor delta-3 isomer (BP impurity D) | 21.045 | 5.20145 |
| Benzamide derivatives | 22.887 | 4.68718 |
| Cefaclor | 24.951 | 5.13030 |
| BP impurity E | 26.303 | 3.91955 |
| BP impurity F | 39.729 | 48.24495 |
| BP impurity H | 40.497 | 4.58236 |
In order to ensure that the related substance inspection method is suitable for the detection of cefaclor bulk drugs and impurities thereof, the following methodological verification is carried out, and the verification results are shown in the following table:
comparative example 1
A method for separating and measuring cefaclor bulk drug and impurities thereof by high performance liquid chromatography adopts the chromatographic conditions as follows: octadecylsilane chemically bonded silica was used as a filler, Waters Xbridge C18, 4.6mm × 250mm, 3.5 μm; the mobile phase A is sodium dihydrogen phosphate solution, the mobile phase B is sodium dihydrogen phosphate solution-acetonitrile, and the volume ratio of the sodium dihydrogen phosphate solution: acetonitrile 55:45, linear gradient elution; the column temperature was 25 ℃; the flow rate is 1.0ml/min per minute; the detection wavelength was 220 nm.
The preparation process of the sodium dihydrogen phosphate solution comprises the following steps: 7.8g of sodium dihydrogen phosphate dihydrate was taken, dissolved in water and diluted to 1000ml, and the pH was adjusted to 4.0 with phosphoric acid or 1mol/L sodium hydroxide.
The linear gradient elution is as follows:
the method specifically comprises the following steps:
(1) taking a proper amount of cefaclor raw material medicine, adding a solvent to dissolve and dilute the cefaclor raw material medicine to prepare a solution containing 5mg of cefaclor raw material medicine in every 1ml, filtering, and taking a subsequent filtrate to obtain a test solution;
(2) precisely measuring 1ml of a test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to a scale with a solvent, and shaking up to obtain a control solution;
(3) taking a proper amount of cefaclor and cefaclor delta-3 isomer, adding a solvent for dissolving and diluting to prepare a mixed solution containing 25 mu g of cefaclor and 50 mu g of cefaclor delta-3 isomer in each 1ml, and taking the mixed solution as a system applicability solution;
(4) injecting 20 mul of system applicability solution into a liquid chromatograph to ensure that the separation degree between the cefaclor peak and the cefaclor delta-3 peak meets the requirement;
(5) precisely measuring 20 μ l of each of the test solution and the control solution, respectively injecting into a liquid chromatograph, and recording the chromatogram until the retention time of the main component peak is 5 times.
The solvent adopted in the steps (1) - (3) is a sodium dihydrogen phosphate solution with the pH value of 2.5, and the specific preparation process comprises the steps of taking 2.7g of sodium dihydrogen phosphate dihydrate, adding water to dissolve and dilute the sodium dihydrogen phosphate dihydrate to 1000ml, and adjusting the pH value to 2.5 by using 20% phosphoric acid.
As a result: the chromatographic peak separation degree of the BP impurity A and the BP impurity B (RT3.801min) and the chromatographic peak separation degree of the BP impurity C and the BP impurity G (RT15.784min) can not meet the requirement.
Comparative example 2
The column was replaced on the basis of comparative example 1, using a 3 μm column of AQ C18250 x 4.6mm, the remainder being the same as in comparative example 1.
As a result: the chromatographic peak separation degree of BP impurity A (RT4.451min) and BP impurity B (RT4.648min) is 1.57076, which meets the requirement, but the chromatographic peak separation degrees of BP impurity C (RT17.244min), BP impurity G (RT17.508min), cefaclor and benzamide (RT25.755min), D-phenylglycine methyl ester hydrochloride (RT19.230min), 3S) -3-phenylpiperazine-2, 5-dione (RT19.641min), BP impurity F (RT41.035min) and BP impurity H (RT41.125min) are all less than 1.0, and the chromatographic peak separation degrees are not satisfactory.
Comparative example 3
From the results of the experiments in comparative examples 1 and 2, it can be seen that the separation effect is not satisfactory when the experiments are performed with Xbridge C18250 × 4.6mm, 3.5 μm or AQ C18250 × 4.6mm, 3 μm chromatography columns, and the experiments are performed by first optimizing the pH of the mobile phase and adjusting the pH of the mobile phase to 4.5.
Chromatographic conditions
1) A chromatographic column: xbridge C18250 mm 4.6mm, 3.5 μm
2) A chromatographic column: AQ C18250 × 4.6mm, 3 μm
Detection wavelength: 220 nm;
flow rate: 1 ml/min;
column temperature: 25 ℃;
sample introduction amount: 20 mu l of the mixture;
mobile phase A: sodium dihydrogen phosphate solution (taking 7.8g of sodium dihydrogen phosphate dihydrate, adding water to dissolve and dilute to 1000ml, and adjusting pH value to 4.5 by using 1mol/L sodium hydroxide);
mobile phase B: mobile phase a-acetonitrile (55: 45);
elution gradient
As a result: when the pH value of the mobile phase is adjusted to 4.5, Xbridge C18250 x 4.6mm and 3.5 mu m are used, the chromatographic peak separation degree of BP impurity A (RT3.635min) and BP impurity B (RT3.900min) in the chromatogram is 2.20733, the requirement is met, the chromatographic peak separation degree of BP impurity C (RT15.383min) and BP impurity G (RT15.012min) is 1.23752, and the separation effect is obviously improved.
When the pH of the mobile phase is adjusted to 4.5, AQ C18250 x 4.6mm and 3 mu m is used, in a chromatogram of a mixed standard solution, chromatographic peaks of BP impurity C and BP impurity G (RT17.283min), D-phenylglycine methyl ester hydrochloride and (3S) -3-phenylpiperazine-2, 5-diketone (RT19.667min), cefaclor and benzamide (RT25.757min) and chromatographic peaks of BP impurity F and BP impurity H (RT41.009min) are overlapped, and the separation effect cannot meet the requirement.
From the above experimental results, it can be seen that when the mobile phase pH is adjusted to 4.5 and then the Xbridge C18250 × 4.6mm, 3.5 μm chromatographic column is used for the experiment, the separation effect of each component chromatographic peak is obviously improved, but the separation degree of the BP impurity G and the BP impurity C still cannot meet the requirement.
Experiments are carried out by adopting a 3-micron chromatographic column with AQ C18250 of 4.6mm, and the separation effect of other chromatographic peaks is not improved except that the separation degree of BP impurity A and BP impurity B is obviously improved.
For comprehensive consideration, Xbridge C18250 x 4.6mm, 3.5 μm chromatographic column was determined for further optimization of chromatographic conditions.
Comparative example 4
From the above experimental results, it can be seen that after the pH value is adjusted to 4.5, Xbridge C18250 × 4.6mm, 3.5 μm chromatography column is used for the experiment, the separation degree of BP impurity G and BP impurity C still cannot reach the requirement (not less than 1.5), and then the column temperature is further optimized, and the column temperature is adjusted to 20 ℃ and 40 ℃ respectively for the experiment.
Chromatographic conditions
Detection wavelength: 220 nm;
flow rate: 1 ml/min;
column temperature: 20 ℃/40 ℃;
sample introduction amount: 20 mu l of the mixture;
mobile phase A: sodium dihydrogen phosphate solution (taking 7.8g of sodium dihydrogen phosphate dihydrate, adding water to dissolve and dilute to 1000ml, and adjusting pH value to 4.5 by using 1mol/L sodium hydroxide);
mobile phase B: mobile phase a-acetonitrile (550: 450);
a chromatographic column: xbridge C18250 mm 4.6mm, 3.5 μm
Elution gradient
As a result: when the column temperature is adjusted to 20 ℃, chromatographic peaks of all components can be effectively separated, and the separation degrees of BP impurity A (RT3.647min) and BP impurity B (RT3.905min), BP impurity C (RT16.001min) and BP impurity G (RT15.499min) are all more than 1.5, so that the method meets the requirements. When the column temperature is adjusted to 40 ℃, the separation degree of BP impurity C and BP impurity G (RT13.983min) is not satisfactory.
The separation effect of each component chromatographic peak is the best at the column temperature of 20 ℃, the separation degree of each component meets the requirement, and the column temperature is determined to be 20 ℃. However, the chromatographic peak separation degree of the BP impurity G and the BP impurity C is only 1.66364, so that the separation effect is prevented from being poor after the column efficiency of a chromatographic column is reduced in the later period, and the chromatographic condition is further optimized to improve the separation degree.
Comparative example 5
After the pH value of the mobile phase is adjusted to be 4.5 and the column temperature is 20 ℃, the separation degree of chromatographic peaks of all components meets the requirement, but in order to further improve the separation degree of chromatographic peaks of impurities such as chromatographic peaks of BP impurity G, BP impurity C and the like, the pH value of the mobile phase is optimized again, the pH value of the mobile phase is adjusted to be 0.2 up and down on the basis of the optimized pH value of 4.5, and the mobile phase is adjusted to be 4.3 and 4.7 for investigation.
Chromatographic conditions
Detection wavelength: 220 nm;
flow rate: 1 ml/min;
column temperature: 20 ℃;
sample introduction amount: 20 mu l of the mixture;
mobile phase A: sodium dihydrogen phosphate solution (taking 7.8g of sodium dihydrogen phosphate dihydrate, adding water to dissolve and dilute to 1000ml, and adjusting pH to 4.3/4.7 with phosphoric acid or 1mol/L sodium hydroxide);
mobile phase B: mobile phase a-acetonitrile (550: 450);
a chromatographic column: xbridge C18250 mm 4.6mm, 3.5 μm
Elution gradient
As a result: the pH value is 4.3, and the chromatographic peak separation degrees of BP impurity A (RT3.663min) and BP impurity B (RT3.809min), BP impurity C (RT16.083min) and BP impurity G (RT15.666min) are less than 1.5, which is not satisfactory.
The pH value is 4.7, the chromatographic peak separation degree of each component meets the requirement, the chromatographic peak separation degree of BP impurity A (RT3.682min) and BP impurity B (RT4.133min), and the chromatographic peak separation degree of BP impurity C (RT16.821min) and BP impurity G (RT16.150min) are all larger than 2, the separation effect is better, but the BP impurity F and the BP impurity H which are reserved for about 40 minutes are close to the solvent impurity peak, and are easily interfered by the solvent impurity peak.
Claims (5)
1. The method for separating and measuring cefaclor bulk drug and impurities thereof by high performance liquid chromatography is characterized by adopting the chromatographic conditions as follows: octadecylsilane chemically bonded silica was used as a filler, Waters Xbridge C18, 4.6mm × 250mm, 3.5 μm; the mobile phase A is sodium dihydrogen phosphate solution, the mobile phase B is sodium dihydrogen phosphate solution-acetonitrile, and the volume ratio of the sodium dihydrogen phosphate solution: acetonitrile 55:45, linear gradient elution; the column temperature is 20 ℃; the flow rate is 1.0ml/min per minute; the detection wavelength was 220 nm.
2. The method for separating and determining cefaclor bulk drug and impurities thereof according to claim 1, wherein the preparation process of the sodium dihydrogen phosphate solution is as follows: 7.8g of sodium dihydrogen phosphate dihydrate was taken, dissolved in water and diluted to 1000ml, and the pH was adjusted to 4.7 with phosphoric acid or 1mol/L sodium hydroxide.
3. The method for separating and determining cefaclor bulk drug and impurities thereof according to claim 1, wherein the linear gradient elution procedure is as follows:
。
4. The method for separating and determining cefaclor bulk drug and impurities thereof by high performance liquid chromatography as claimed in claim 1, characterized by comprising the following steps:
(1) taking a proper amount of cefaclor raw material medicine, adding a solvent to dissolve and dilute the cefaclor raw material medicine to prepare a solution containing 5mg of cefaclor raw material medicine in every 1ml, filtering, and taking a subsequent filtrate to obtain a test solution;
(2) precisely measuring 1ml of a test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to a scale with a solvent, and shaking up to obtain a control solution;
(3) taking a proper amount of cefaclor and cefaclor delta-3 isomers, adding a solvent to dissolve and dilute the cefaclor and the cefaclor delta-3 isomers to prepare a mixed solution containing 25 mu g of cefaclor and 50 mu g of cefaclor 5-3 isomers in each 1ml of the mixed solution as a system applicability solution;
(4) injecting 20 mul of system applicability solution into a liquid chromatograph to ensure that the separation degree between the cefaclor peak and the cefaclor delta-3 peak meets the requirement;
(5) precisely measuring 20 μ l of each of the test solution and the control solution, respectively injecting into a liquid chromatograph, and recording the chromatogram until the retention time of the main component peak is 5 times.
5. The method for separating and determining cefaclor bulk drug and impurities thereof according to claim 2, wherein the solvent used in the steps (1) - (3) is a sodium dihydrogen phosphate solution with pH value of 2.5, and the specific preparation process comprises: 2.7g of sodium dihydrogen phosphate dihydrate was dissolved in water and diluted to 1000ml, and the pH was adjusted to 2.5 with 20% phosphoric acid.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115372498A (en) * | 2022-07-14 | 2022-11-22 | 石家庄四药有限公司 | Method for detecting residual impurities in cefaclor |
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2021
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Non-Patent Citations (5)
| Title |
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| L.J. LORENZ等: "Determination of Process-Related Impurities and Degradation Products in Cefaclor by High-Performance Liquid Chromatography", 《JOURNAL OF CHROMATOGRAPHIC SCIENCE》 * |
| 傅小英等: "头孢克洛胶囊有关物质考察", 《解放军药学学报》 * |
| 向世英等: "头孢克洛干混悬剂中有关物质的研究", 《化学分析计量》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115372498A (en) * | 2022-07-14 | 2022-11-22 | 石家庄四药有限公司 | Method for detecting residual impurities in cefaclor |
| CN115372498B (en) * | 2022-07-14 | 2023-05-05 | 石家庄四药有限公司 | Method for detecting residual impurities in cefaclor |
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