CN115876901A - Method for determining high molecular polymer in clohexine dry suspension - Google Patents
Method for determining high molecular polymer in clohexine dry suspension Download PDFInfo
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- CN115876901A CN115876901A CN202111139642.6A CN202111139642A CN115876901A CN 115876901 A CN115876901 A CN 115876901A CN 202111139642 A CN202111139642 A CN 202111139642A CN 115876901 A CN115876901 A CN 115876901A
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- 229920000642 polymer Polymers 0.000 title claims abstract description 42
- 239000000725 suspension Substances 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 22
- 239000000243 solution Substances 0.000 claims abstract description 83
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 74
- 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 claims abstract description 56
- 229960005361 cefaclor Drugs 0.000 claims abstract description 56
- 238000001514 detection method Methods 0.000 claims abstract description 40
- 239000007788 liquid Substances 0.000 claims abstract description 28
- 239000004793 Polystyrene Substances 0.000 claims abstract description 20
- 229920002223 polystyrene Polymers 0.000 claims abstract description 20
- 239000012488 sample solution Substances 0.000 claims abstract description 20
- 238000005227 gel permeation chromatography Methods 0.000 claims abstract description 14
- 239000012490 blank solution Substances 0.000 claims abstract description 4
- 239000000872 buffer Substances 0.000 claims abstract 11
- 239000002904 solvent Substances 0.000 claims description 51
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 45
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 36
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 36
- 239000012085 test solution Substances 0.000 claims description 25
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 claims description 23
- 238000002360 preparation method Methods 0.000 claims description 22
- 238000005303 weighing Methods 0.000 claims description 19
- 238000001914 filtration Methods 0.000 claims description 14
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000000047 product Substances 0.000 claims description 8
- 239000000706 filtrate Substances 0.000 claims description 7
- 239000012088 reference solution Substances 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 238000006386 neutralization reaction Methods 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims 1
- -1 disodium hydrogen Chemical class 0.000 claims 1
- 229910052739 hydrogen Inorganic materials 0.000 claims 1
- 239000001257 hydrogen Substances 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical class O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 abstract 1
- 239000000945 filler Substances 0.000 abstract 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 24
- 239000003153 chemical reaction reagent Substances 0.000 description 24
- 239000012535 impurity Substances 0.000 description 23
- 239000013558 reference substance Substances 0.000 description 18
- 239000000523 sample Substances 0.000 description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 12
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 12
- 238000007865 diluting Methods 0.000 description 10
- 238000012360 testing method Methods 0.000 description 8
- 239000000539 dimer Substances 0.000 description 7
- 230000014759 maintenance of location Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 229940079593 drug Drugs 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 230000004807 localization Effects 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 229930186147 Cephalosporin Natural products 0.000 description 3
- 229940124587 cephalosporin Drugs 0.000 description 3
- 150000001780 cephalosporins Chemical class 0.000 description 3
- 230000001066 destructive effect Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 206010036790 Productive cough Diseases 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229960002335 bromhexine hydrochloride Drugs 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- YRSGDLIATOURQO-UHFFFAOYSA-N ethyl 4-acetyl-5-oxohexanoate Chemical compound CCOC(=O)CCC(C(C)=O)C(C)=O YRSGDLIATOURQO-UHFFFAOYSA-N 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 229920001730 Moisture cure polyurethane Polymers 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 229960003870 bromhexine Drugs 0.000 description 1
- OJGDCBLYJGHCIH-UHFFFAOYSA-N bromhexine Chemical compound C1CCCCC1N(C)CC1=CC(Br)=CC(Br)=C1N OJGDCBLYJGHCIH-UHFFFAOYSA-N 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 229940031703 low substituted hydroxypropyl cellulose Drugs 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000012489 system suitability test solution Substances 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The patent discloses a detection method for determining high molecular polymers in a clohexine dry suspension by gel chromatography, which comprises the following steps: the chromatographic column of the high performance liquid chromatograph is a chromatographic column taking spherical hydrophilic modified silica gel (the molecular weight application range is polymer 500-15000) as a filler; the mobile phase is 0.005mol/L buffer solution-acetonitrile with pH =6.8 to 7.2; the detector is an ultraviolet detector; detecting the wavelengths of 218nm to 222nm; the flow rate is 0.7ml/min to 0.9ml/min. Precisely measuring blank solution, system applicability solution, control solution and sample solution 20 μ l each, injecting into liquid chromatograph, recording chromatogram, and calculating high molecular polymer content in the sample solution according to main component self-control method. The method for determining the content of the cefaclor high molecular polymer in the cloxacin dry suspension by using the polystyrene gel chromatography has the advantages of good specificity, high sensitivity and strong durability, and can better control the quality of the cloxacin dry suspension.
Description
Technical Field
The invention relates to the technical field of analysis, in particular to a method for determining a high-molecular polymer in a clohexanew dry suspension.
Background
The kelouhexin dry suspension is a prescription preparation produced by Jiangsu Zhengda Qingjiang pharmaceutical Limited company according to the Chinese national drug standard, and is mainly used for treating patients suffering from respiratory tract infection caused by sensitive bacteria and having difficulty in expectoration of viscous sputum.
The preparation method of the cloxacin dry suspension comprises the following steps: the cefaclor (250 mg) and the bromhexine hydrochloride (8.77 mg) are respectively sieved by a 100-mesh sieve, and other auxiliary materials are respectively sieved by a 80-mesh sieve. Uniformly mixing bromhexine hydrochloride and cefaclor in an equivalent progressive manner, sequentially adding auxiliary materials such as sodium carboxymethylcellulose, low-substituted hydroxypropyl cellulose, sodium dodecyl sulfate, lactose, sucrose and the like, and uniformly mixing; adding 95% alcohol to make soft mass, sieving with 20 mesh sieve, granulating, and vacuum drying at 45 deg.C. Sieving with 18 mesh sieve, mixing, and packaging.
The main adverse reaction existing in clinical application of cephalosporin drugs is immediate anaphylactic reaction, and the main reason is caused by endogenous high molecular polymers generated by drug self polymerization, so that the research and improvement of the impurity level of the existing cephalosporin drug polymers are of great importance for the quality control of cephalosporin drugs.
Disclosure of Invention
The invention aims to establish a method for determining cefaclor high molecular polymer in a clohexine dry suspension, which can better control the high molecular polymer in the clohexine dry suspension and can better control the quality of the clohexine dry suspension.
The technical scheme of the invention is that a gel chromatography method is adopted to determine cefaclor high molecular polymer in the cloxacin dry suspension, which comprises the following steps:
preparation of a solvent: [ pH7.0 ] (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61));
preparing a test solution: taking a proper amount (about equivalent to cefaclor 50 mg) of the content of the product, precisely weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to a scale, shaking uniformly, filtering, and taking a subsequent filtrate as a test solution;
preparation of control solution: precisely measuring 1.0ml, placing in a 100ml measuring flask, adding solvent to dilute to scale, and shaking up to obtain a control solution;
preparation of system applicability solution: taking 10ml of sample solution, adding 1ml of 0.2 mol/L sodium hydroxide solution, standing at room temperature for 1 minute, adding 0.2 mol/L hydrochloric acid solution 1ml for neutralization, and shaking up to obtain a system applicability solution;
and (3) chromatographic column: polystyrene gel TSKgel G3000 SWXL 7.8 × 300mm 5um;
chromatographic conditions are as follows: the column temperature was 25 ℃, the detector was an ultraviolet detector, the detection wavelength was 220nm, the flow rate was 0.8ml/min, and the mobile phase was (ph 7.0) (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61)) -acetonitrile (97;
under the chromatographic conditions, 20 mul of blank solution, system applicability solution, control solution and sample solution are precisely measured and respectively injected into a liquid chromatograph, and the chromatogram is recorded. In the chromatogram of the system applicability solution, the appearance order of the solution is cefaclor polymer and cefaclor, and the separation degree of the cefaclor polymer and the cefaclor is not lower than 1.5; in the chromatogram of the test solution, the peak area of the impurity peak with relative retention time of 0.82-1.0 is not more than 1.0 times (1.0%) of the main peak area of the control solution.
Drawings
FIG. 1 is a specificity inspection chromatogram; FIG. 2 is a view for examining a linear relationship.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention will be described in further detail below by way of examples, but it should not be construed that the scope of the subject matter of the present invention is limited to the following examples. Such alterations and modifications are intended to be included herein within the scope of this disclosure as determined by those skilled in the art to which the invention pertains and by the annexed claims.
1. EXAMPLE 1 selection of solvent and Mobile phase System
Instruments and reagents:
the instrument comprises the following steps: ultraviolet detector for Dyan U3000 high performance liquid chromatograph
Reagent: water, disodium hydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid, acetonitrile
Chromatographic conditions are as follows:
a chromatographic column: polystyrene gel TSKgel G3000 SWXL 7.8X 300mm 5um chromatographic column
Column temperature: 25 ℃; detection wavelength: 220nm; the flow rate is 0.5ml/min; the mobile phase was (ph 7.0) (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61)) -acetonitrile (95: 20 μ l
Solution preparation:
solvent: and (3) water.
Polymer impurity localization solution: an appropriate amount of dimer impurity control was precisely weighed, dissolved with a solvent and quantitatively diluted to a concentration of 0.2mg/ml as a positioning solution.
Cefaclor positioning solution: and precisely weighing a proper amount of cefaclor reference substance, dissolving the cefaclor reference substance by using a solvent, and quantitatively diluting the cefaclor reference substance to a concentration of 0.1mg/ml to be used as a positioning solution.
Preparing a test solution: taking a proper amount of the content (about equivalent to cefaclor 50 mg), precisely weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to scale, shaking uniformly, filtering, and taking continuous filtration as a test solution.
And (3) sample determination:
precisely measuring 20 μ l of the sample solution, injecting into a liquid chromatograph, and recording chromatogram.
And (4) conclusion:
under this chromatographic condition, cefaclor is poorly soluble using water as solvent and it is not baseline separated from its pre-peak polymer impurities.
2. Example 2 selection of solvent and Mobile phase
Instruments and reagents:
the instrument comprises the following steps: ultraviolet detector for Dyan U3000 high performance liquid chromatograph
Reagent: water, disodium hydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid, acetonitrile
Chromatographic conditions are as follows:
a chromatographic column: polystyrene gel TSKgel G3000 SWXL 7.8 x 300mm 5um chromatographic column
Column temperature: 25 ℃; detection wavelength: 220nm; the flow rate is 0.5ml/min; the mobile phase was (ph 7.0) (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61)) -acetonitrile (99): 20 μ l
Solution preparation:
the same as in example 1.
And (3) sample determination:
precisely measuring 20 μ l of the sample solution, injecting into a liquid chromatograph, and recording chromatogram.
And (4) conclusion:
under this chromatographic condition, cefaclor is poorly soluble using water as solvent and it is not baseline separated from its pre-peak polymer impurities.
3. Example 3 selection of solvent and Mobile phase
Instruments and reagents:
the instrument comprises the following steps: ultraviolet detector for Dyan U3000 high performance liquid chromatograph
Reagent: water, disodium hydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid, acetonitrile
Chromatographic conditions are as follows:
a chromatographic column: polystyrene gel TSKgel G3000 SWXL 7.8X 300mm 5um chromatographic column
Column temperature: 25 ℃; detection wavelength: 220nm; the flow rate is 0.5ml/min; the mobile phase was (ph 7.0) (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61)) -acetonitrile (99): 20 μ l
Solution preparation:
solvent: (pH 4.0) [ 0.005mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61).
Polymer impurity localization solution: an appropriate amount of dimer impurity control was precisely weighed, dissolved with a solvent and quantitatively diluted to a concentration of 0.2mg/ml as a positioning solution.
Cefaclor positioning solution: and precisely weighing a proper amount of cefaclor reference substance, dissolving the cefaclor reference substance by using a solvent, and quantitatively diluting the cefaclor reference substance to a concentration of 0.1mg/ml to be used as a positioning solution.
Preparing a test solution: taking a proper amount of the content (about equivalent to cefaclor 50 mg), precisely weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to scale, shaking uniformly, filtering, and taking continuous filtration as a test solution.
And (3) sample determination:
precisely measuring 20 μ l of the sample solution, injecting into a liquid chromatograph, and recording chromatogram.
And (4) conclusion:
under the chromatographic condition, the change rate of the area of the main peak pre-polymer impurity in the cefaclor sample solution is more than 2% after 2 hours; the change rate of the peak area of the dimer impurity in the system applicability solution after 2 hours is more than 2%, and the stability is poor.
4. Example 4 selection of solvent and Mobile phase
Instruments and reagents:
the instrument comprises the following steps: ultraviolet detector for Dyan U3000 high performance liquid chromatograph
Reagent: water, disodium hydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid and acetonitrile
Chromatographic conditions are as follows:
and (3) chromatographic column: polystyrene gel TSKgel G3000 SWXL 7.8X 300mm 5um chromatographic column
Column temperature: 25 ℃; detection wavelength: 220nm; the flow rate is 0.5ml/min; the mobile phase was (ph 7.0) (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61)) -acetonitrile (97): 20 μ l
Solution preparation:
solvent: (pH 3.0) [ 0.005mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61).
Polymer impurity localization solution: an appropriate amount of dimer impurity control was precisely weighed, dissolved with a solvent and quantitatively diluted to a concentration of 0.2mg/ml as a positioning solution.
Cefaclor positioning solution: and precisely weighing a proper amount of cefaclor reference substance, dissolving the cefaclor reference substance by using a solvent, and quantitatively diluting the cefaclor reference substance to a concentration of 0.1mg/ml to be used as a positioning solution.
Preparing a test solution: taking a proper amount of the content (about equivalent to cefaclor 50 mg), precisely weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to scale, shaking uniformly, filtering, and taking continuous filtration as a test solution.
And (3) sample determination:
precisely measuring 20 μ l of the sample solution, injecting into a liquid chromatograph, and recording chromatogram.
And (4) conclusion:
under the chromatographic conditions, the cefaclor retention time is 23.055min; cefaclor separated well from its post-peak dimer impurities (R = 3.97); can be suitably optimized.
5. EXAMPLE 5 selection of solvent and Mobile phase
Instruments and reagents:
the instrument comprises: ultraviolet detector for Danan U3000 high-performance liquid chromatograph
Reagent: water, disodium hydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid, acetonitrile
Chromatographic conditions are as follows:
a chromatographic column: polystyrene gel TSKgel G3000 SWXL 7.8X 300mm 5um chromatographic column
Column temperature: 25 ℃; detection wavelength: 220nm; the flow rate is 0.5ml/min; the mobile phase was (ph 7.0) (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61)) -acetonitrile (97): 20 μ l
Solution preparation:
solvent: (pH 7.0) [ 0.005mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61).
Polymer impurity localization solution: an appropriate amount of dimer impurity control was precisely weighed, dissolved with a solvent and quantitatively diluted to a concentration of 0.2mg/ml as a positioning solution.
Cefaclor positioning solution: and precisely weighing a proper amount of cefaclor reference substance, dissolving the cefaclor reference substance by using a solvent, and quantitatively diluting the cefaclor reference substance to the concentration of 0.1mg/ml to be used as a positioning solution.
Preparing a test solution: taking a proper amount of the content (about equivalent to cefaclor 50 mg), precisely weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to scale, shaking uniformly, filtering, and taking continuous filtration as a test solution.
And (3) sample determination:
precisely measuring 20 μ l of the sample solution, injecting into a liquid chromatograph, and recording chromatogram.
And (4) conclusion:
under the chromatographic condition, the retention time of cefaclor is 14.072min, and no dimer impurity is detected in the sample solution; the cefaclor and the pre-peak polymer impurities thereof are well separated; the condition is drawn up as a method for detecting the polymer in the clohexine dry suspension, and the method is continuously verified.
6. Example 6 specificity examination
Instruments and reagents:
the instrument comprises the following steps: ultraviolet detector for Dyan U3000 high performance liquid chromatograph
Reagent: water, disodium hydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid and acetonitrile
Chromatographic conditions are as follows:
a chromatographic column: polystyrene gel TSKgel G3000 SWXL 7.8 x 300mm 5um chromatographic column
Column temperature: 25 ℃; detection wavelength: 220nm; the flow rate is 0.8ml/min; solvent: (pH 7.0) (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61)); the mobile phase was (ph 7.0) (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61)) -acetonitrile (97; sample introduction amount: 20 μ l
Solution preparation:
taking appropriate amount of clinohexan dry suspension, respectively adding into acid (1 mol/L hydrochloric acid solution), alkali (0.1 mol/L sodium hydroxide solution), heat (60 deg.C water bath), light (5000 LUX illumination for 3 days), and oxidation (1ml 0.2% 2 O 2 ) Under the conditions of (1), the product is accelerated to degrade, so as to investigate the specificity of the method.
And (3) sample determination:
precisely measuring 20 mul of the test solution under each destructive condition, injecting into a liquid chromatograph, and recording the chromatogram.
As a result:
material balance measurement results
Analysis of impurity detection in sample solution under respective destructive conditions
And (4) conclusion:
the test results show that: various destructive tests can enlarge the polymer peak, and the basic destruction and the acid destruction are most obvious; the separation degree of each peak is good, and blank solvent has no interference; the blank auxiliary materials have more impurities, but the detection of the impurities of the sample is not influenced; the bromhexine reference substance solution has no interference; the content of the product after being damaged is added with the content of impurities, the result is basically close to that of the product without being damaged, the material balance requirement is met, and the chromatographic condition and the system applicability established by the product are proved to be good, and the quality of the product can be effectively monitored.
7. Example 7 examination of precision
Instruments and reagents:
the instrument comprises the following steps: ultraviolet detector for Dyan U3000 high performance liquid chromatograph
Reagent: water, disodium hydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid, acetonitrile
Chromatographic conditions are as follows:
the same as in example 6.
Solution preparation:
control solution: taking a proper amount (about 12.5 mg) of cefaclor reference substance, precisely weighing, putting into a 50ml measuring flask, adding a solvent for dissolving, and diluting to a scale; precisely measuring 2.0ml, placing into a 10ml measuring flask, adding solvent to dilute to scale, and using as a reference solution.
Taking a proper amount of the content (about equivalent to cefaclor 50 mg), precisely weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to scale, shaking uniformly, filtering, and taking the subsequent filtrate as a test solution. 6 portions are prepared in parallel.
Control solution: precisely measuring 1.0ml of each sample solution, placing in a 100ml measuring flask, adding solvent to dilute to scale, and shaking to obtain control solution.
On different dates, different analysts take a proper amount of the content (about equivalent to cefaclor 50 mg), precisely weigh the content, put the content in a 10ml measuring flask, add a solvent to dissolve and dilute the content to a scale, shake the content evenly, filter the content, and take a subsequent filtrate as a test solution. 6 parts are prepared in parallel.
Control solution: precisely measuring 1.0ml of each sample solution, placing in a 100ml measuring flask, adding solvent to dilute to scale, and shaking to obtain control solution.
And (3) sample determination:
precisely measuring 20 mul of the solution, injecting the solution into a liquid chromatograph, recording a chromatogram, continuously injecting 6 needles of a reference solution, and inspecting the precision of the instrument; the sample and control solutions were examined for reproducibility.
As a result:
results of instrumental precision test
Results of precision test
And (4) conclusion:
cefaclor peak area RSD:0.58%, retention time RSD:0.06 percent, and good instrument precision;
polymer content in 6 parts of test solution: 0.53%, RSD:1.68 percent, good repeatability;
polymer content in 6 test solutions prepared by another person: 0.52%, RSD:1.72 percent, and good intermediate precision; the method has high precision.
8. Example 8 examination of the Linear relationship
Instruments and reagents:
the instrument comprises the following steps: ultraviolet detector for Dyan U3000 high performance liquid chromatograph
Reagent: water, disodium hydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid and acetonitrile
Chromatographic conditions are as follows:
the same as in example 6.
Solution preparation:
linear solution: taking a proper amount (about 12.5 mg) of cefaclor reference substance, precisely weighing, putting into a 50ml measuring flask, adding a solvent for dissolving, and diluting to a scale; precisely measuring 4.0ml, 2.0ml, 1.6ml, 1.0ml and 0.5ml respectively, placing in a 10ml measuring flask, adding solvent to dilute to scale, and preparing a series of linear solutions.
And (3) sample determination:
and precisely measuring 20 mul of each linear solution, injecting the linear solution into a liquid chromatograph, recording a chromatogram, and taking the linear solution concentration (mug/ml) as an X axis and the peak area (A) as a Y axis as a linear regression equation.
As a result:
results of the Linear test
And (4) conclusion:
cefaclor is within the range of 12.45 to 99.59 mu g/ml, the linearity is good, Y =0.799X +0.124, r = 1.000.
9. Example 9 examination of detection limits and quantitation limits
Instruments and reagents:
the instrument comprises the following steps: ultraviolet detector for Danan U3000 high-performance liquid chromatograph
Reagent: water, disodium hydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid, acetonitrile
Chromatographic conditions are as follows:
the same as in example 6.
Solution preparation:
taking a proper amount (about 12.5 mg) of cefaclor reference substance, precisely weighing, putting into a 50ml measuring flask, adding a solvent for dissolving, and diluting to a scale; respectively and precisely measuring appropriate amount, adding solvent, diluting step by step, and preparing a series of solutions.
And (3) sample determination:
precisely measuring 20 mul of each stepwise diluted solution, injecting the stepwise diluted solution into a liquid chromatograph, recording a chromatogram, and taking a later sample as a detection limit and a quantitative limit according to S/N =3.
As a result:
test results of detection limit and quantitative limit
And (4) conclusion:
the detection limit concentration is 0.10 mug/ml, the quantification limit concentration is 0.99 mug/ml, and the method has high sensitivity.
10. Example 10 solution stability Studies
Instruments and reagents:
the instrument comprises: ultraviolet detector for Dyan U3000 high performance liquid chromatograph
Reagent: water, disodium hydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid and acetonitrile
Chromatographic conditions are as follows:
the same as in example 6.
Solution preparation:
control solution: taking a proper amount (about 12.5 mg) of cefaclor reference substance, precisely weighing, putting into a 50ml measuring flask, adding a solvent for dissolving, and diluting to a scale; precisely measuring 2.0ml, placing into a 10ml measuring flask, adding solvent to dilute to scale, and using as a reference solution.
Test solution: taking a proper amount of the content (about equivalent to cefaclor 50 mg), precisely weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to scale, shaking uniformly, filtering, and taking the subsequent filtrate as a test solution.
And (3) sample determination:
placing the solution at 4 ℃ for 0h, 1h, 2h, 4h, 10h and 16h after preparation, precisely measuring 20 mul of the solution, respectively injecting the solution into a liquid chromatograph, and recording a chromatogram.
As a result:
solution stability results
And (4) conclusion:
the reference solution is placed for 10 hours at 4 ℃ after being prepared, the stability is good, and the peak area RSD is less than 2%; after the test solution is prepared, the polymer is placed for 16 hours at 4 ℃, the stability of the polymer is good, and the peak area RSD is less than 2%.
11. EXAMPLE 11 durability examination
Instruments and reagents:
the instrument comprises the following steps: ultraviolet detector for Danan U3000 high-performance liquid chromatograph
Reagent: water, disodium hydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid and acetonitrile
Chromatographic conditions are as follows:
in the same manner as in example 6, the flow rate was 0.7ml/min, the flow rate was 0.9ml/min, the column temperature was 30 ℃, 27 ℃, the column temperature was 20 ℃, the initial mobile phase ratio was 99, the initial mobile phase ratio was 95, the detection wavelength was 218nm, the detection wavelength was 222nm, the instrument was changed, the pH of the mobile phase a was 6.8, and the pH of the mobile phase a was 7.2.
Solution preparation:
control solution: taking a proper amount (about 12.5 mg) of cefaclor reference substance, precisely weighing, putting into a 50ml measuring flask, adding a solvent for dissolving, and diluting to a scale; precisely measuring 2.0ml, placing into a 10ml measuring flask, adding solvent to dilute to scale, and using as a reference solution.
Test solution: taking a proper amount of the content (about equivalent to cefaclor 50 mg), precisely weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to scale, shaking uniformly, filtering, and taking the subsequent filtrate as a test solution.
Control solution: precisely measuring 1.0ml of each sample solution, placing in a 100ml measuring flask, adding solvent to dilute to scale, and using as a control solution.
And (3) sample determination:
under the different chromatographic conditions, respectively, 20 μ l of each of the solvent blank, the system suitability test solution, the sample solution and the control solution is injected into a liquid chromatograph, and the chromatogram is recorded.
As a result:
durability test results
And (4) conclusion:
the proper changes of the flow rate, the detection wavelength and the concentration of the mobile phase A have no influence on the retention time of the main component and the measurement result of related substances; the proper change of the proportion of the mobile phase has no great influence on the retention time of impurities and main components and has no influence on the measurement result of related substances; when the column temperature is above 27 ℃, a blank auxiliary material solution can generate an impurity peak to influence the determination of the polymer, and the influence of the column temperature needs to be paid attention; when the pH value of the mobile phase exceeds 7.2, the chromatogram base line is poor, but the method has no influence on the measurement result of the polymer; the detection method has good durability.
12. EXAMPLE 12 sample determination
Instruments and reagents:
the instrument comprises the following steps: ultraviolet detector for Dyan U3000 high performance liquid chromatograph
Reagent: water, disodium hydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid, acetonitrile
Chromatographic conditions are as follows:
the same as in example 6.
Solution preparation:
test solution: taking a proper amount of the content (about equivalent to cefaclor 50 mg) of the product, accurately weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to a scale, shaking up, filtering, and taking the subsequent filtrate as a test solution.
Control solution: precisely measuring 1.0ml of each sample solution, placing in a 100ml measuring flask, adding solvent to dilute to scale, and using as a control solution.
And (3) sample determination:
the resulting solution was poured into a liquid chromatograph at 20. Mu.l each, and the chromatogram was recorded.
And (4) conclusion:
results of sample testing
As described above, the present invention can be preferably realized.
Claims (11)
1. The detection method for determining the high molecular polymer in the Clohexan dry suspension by the polystyrene gel chromatography is characterized by comprising the following steps:
preparation of a solvent: [ pH7.0 ] (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61));
preparation of a test solution: taking a proper amount (about equivalent to cefaclor 50 mg) of the content of the product, precisely weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to a scale, shaking uniformly, filtering, and taking a subsequent filtrate as a test solution;
preparation of control solution: precisely measuring 1.0ml, placing in a 100ml measuring flask, adding solvent to dilute to scale, and shaking up to obtain a control solution;
preparation of system suitability solution: taking 10ml of sample solution, adding 1ml of 0.2 mol/L sodium hydroxide solution, standing at room temperature for 1 minute, adding 0.2 mol/L hydrochloric acid solution 1ml for neutralization, and shaking up to obtain a system applicability solution;
a chromatographic column: polystyrene gel TSKgel G3000 SWXL 7.8 × 300mm 5um;
chromatographic conditions are as follows: the column temperature is 20-30 ℃, the detector is an ultraviolet detector, the detection wavelength is 218nm-222nm, the flow rate is 0.7 ml/min-0.9 ml/min, the mobile phase A is 0.005mol/L buffer solution with pH = 6.8-7.2 (0.005 mol/L disodium hydrogen phosphate-0.005 mol/L sodium dihydrogen phosphate (61;
under the chromatographic conditions, 20 mul of each of a blank solution, a system applicability solution, a reference solution and a sample solution is precisely measured, the blank solution, the system applicability solution, the reference solution and the sample solution are respectively injected into a liquid chromatograph, a chromatogram is recorded, the appearance sequence of the chromatogram is cefaclor polymer and cefaclor, and the resolution of the cefaclor polymer and the cefaclor is not lower than 1.5.
2. The detection method for determining cefaclor high molecular polymer in the cefaclor hexine dry suspension by polystyrene gel chromatography according to claim 1, is characterized by comprising the following steps: the column temperature was 25 ℃, the detector was an ultraviolet detector, the detection wavelength was 220nm, the flow rate was 0.8ml/min, the mobile phase a was 0.005mol/L ph7.2 buffer (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61): acetonitrile = 97.
3. The detection method for determining the high molecular polymer in the clohexine dry suspension by the polystyrene gel chromatography method according to claim 1, is characterized in that: the column temperature was 25 ℃, the detector was an ultraviolet detector, the detection wavelength was 220nm, the flow rate was 0.8ml/min, the mobile phase a was 0.005mol/L ph6.8 buffer (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61): acetonitrile = 97.
4. The detection method for determining the high molecular polymer in the clohexine dry suspension by the polystyrene gel chromatography method according to claim 1, is characterized in that: the column temperature was 25 ℃, the detector was an ultraviolet detector, the detection wavelength was 220nm, the flow rate was 0.8ml/min, the mobile phase a was 0.005mol/L ph7.0 buffer (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61): acetonitrile = 95.
5. The detection method for determining the high molecular polymer in the clohexine dry suspension by the polystyrene gel chromatography method according to claim 1, is characterized in that: the column temperature was 25 ℃, the detector was an ultraviolet detector, the detection wavelength was 220nm, the flow rate was 0.8ml/min, the mobile phase a was 0.005mol/L ph7.0 buffer (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61): acetonitrile = 99.
6. The detection method for determining the high molecular polymer in the clohexamine dry suspension by the polystyrene gel chromatography method according to claim 1, which is characterized in that: the column temperature was 25 ℃, the detector was an ultraviolet detector, the detection wavelength was 218nm, the flow rate was 0.8ml/min, the mobile phase a was 0.005mol/L ph7.0 buffer (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61): acetonitrile = 97.
7. The detection method for determining the high molecular polymer in the clohexine dry suspension by the polystyrene gel chromatography method according to claim 1, is characterized in that: the column temperature was 25 ℃, the detector was an ultraviolet detector, the detection wavelength was 222nm, the flow rate was 0.8ml/min, the mobile phase a was 0.005mol/L ph7.0 buffer (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61): acetonitrile = 97.
8. The detection method for determining the high molecular polymer in the clohexine dry suspension by the polystyrene gel chromatography method according to claim 1, is characterized in that: the column temperature was 20 ℃, the detector was an ultraviolet detector, the detection wavelength was 220nm, the flow rate was 0.8ml/min, the mobile phase a was 0.005mol/L ph7.0 buffer (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61): acetonitrile = 97.
9. The detection method for determining the high molecular polymer in the clohexine dry suspension by the polystyrene gel chromatography method according to claim 1, is characterized in that: the column temperature was 30 ℃, the detector was an ultraviolet detector, the detection wavelength was 220nm, the flow rate was 0.8ml/min, the mobile phase a was 0.005mol/L ph7.0 buffer (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61): acetonitrile = 97.
10. The detection method for determining the high molecular polymer in the clohexamine dry suspension by the polystyrene gel chromatography method according to claim 1, which is characterized in that: the column temperature was 25 ℃, the detector was an ultraviolet detector, the detection wavelength was 220nm, the flow rate was 0.7ml/min, the mobile phase a was 0.005mol/L ph7.0 buffer (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61: acetonitrile = 97.
11. The detection method for determining the high molecular polymer in the clohexine dry suspension by the polystyrene gel chromatography method according to claim 1, is characterized in that: the column temperature was 25 ℃, the detector was an ultraviolet detector, the detection wavelength was 220nm, the flow rate was 0.9ml/min, the mobile phase a was 0.005mol/L ph7.0 buffer (0.005 mol/L disodium hydrogenphosphate-0.005 mol/L sodium dihydrogenphosphate (61: acetonitrile = 97.
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