CN111351886B - Method for determining impurity and main medicine content in phenol sulfoethylamine medicine - Google Patents
Method for determining impurity and main medicine content in phenol sulfoethylamine medicine Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 241
- 238000000034 method Methods 0.000 title claims abstract description 75
- WMOTVIKCLHJGCF-UHFFFAOYSA-N 2-aminoethanesulfonic acid phenol Chemical compound S(=O)(=O)(O)CCN.C1(=CC=CC=C1)O WMOTVIKCLHJGCF-UHFFFAOYSA-N 0.000 title claims abstract description 59
- 239000003814 drug Substances 0.000 title claims abstract description 41
- 238000001514 detection method Methods 0.000 claims abstract description 58
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 42
- 229940079593 drug Drugs 0.000 claims abstract description 26
- VDZOOKBUILJEDG-UHFFFAOYSA-M tetrabutylammonium hydroxide Chemical compound [OH-].CCCC[N+](CCCC)(CCCC)CCCC VDZOOKBUILJEDG-UHFFFAOYSA-M 0.000 claims abstract description 24
- 239000007864 aqueous solution Substances 0.000 claims abstract description 12
- 238000010828 elution Methods 0.000 claims abstract description 11
- 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 7
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 97
- 238000007865 diluting Methods 0.000 claims description 66
- 239000003085 diluting agent Substances 0.000 claims description 64
- 239000012488 sample solution Substances 0.000 claims description 59
- 238000005303 weighing Methods 0.000 claims description 57
- 239000011259 mixed solution Substances 0.000 claims description 42
- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 claims description 25
- 239000013558 reference substance Substances 0.000 claims description 23
- 239000000523 sample Substances 0.000 claims description 21
- 239000011550 stock solution Substances 0.000 claims description 18
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 13
- 238000012360 testing method Methods 0.000 claims description 11
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- 239000000126 substance Substances 0.000 claims description 10
- -1 2, 5-dihydroxybenzene-1, 4-disulfonic acid diethylamine Chemical compound 0.000 claims description 7
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 claims description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- 238000004811 liquid chromatography Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 229940044652 phenolsulfonate Drugs 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 13
- 238000010812 external standard method Methods 0.000 abstract description 6
- 231100000024 genotoxic Toxicity 0.000 abstract description 4
- 230000001738 genotoxic effect Effects 0.000 abstract description 4
- 238000012544 monitoring process Methods 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 53
- 239000010413 mother solution Substances 0.000 description 21
- 238000012937 correction Methods 0.000 description 20
- 238000004364 calculation method Methods 0.000 description 17
- 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 14
- 238000011084 recovery Methods 0.000 description 14
- 239000007788 liquid Substances 0.000 description 11
- 230000014759 maintenance of location Effects 0.000 description 11
- 239000012452 mother liquor Substances 0.000 description 11
- 238000010790 dilution Methods 0.000 description 10
- 239000012895 dilution Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 238000000926 separation method Methods 0.000 description 6
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 description 5
- 229960003151 mercaptamine Drugs 0.000 description 5
- 239000007791 liquid phase Substances 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 208000032843 Hemorrhage Diseases 0.000 description 3
- 239000012470 diluted sample Substances 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000012088 reference solution Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- IMLAIXAZMVDRGA-UHFFFAOYSA-N 2-phenoxyethanamine Chemical compound NCCOC1=CC=CC=C1 IMLAIXAZMVDRGA-UHFFFAOYSA-N 0.000 description 2
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 2
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 2
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- WHOZNOZYMBRCBL-OUKQBFOZSA-N (2E)-2-Tetradecenal Chemical compound CCCCCCCCCCC\C=C\C=O WHOZNOZYMBRCBL-OUKQBFOZSA-N 0.000 description 1
- 241000993291 Armillariella Species 0.000 description 1
- 206010008111 Cerebral haemorrhage Diseases 0.000 description 1
- 101100136092 Drosophila melanogaster peng gene Proteins 0.000 description 1
- HBGOLJKPSFNJSD-UHFFFAOYSA-N Etamsylate Chemical compound CC[NH2+]CC.OC1=CC=C(O)C(S([O-])(=O)=O)=C1 HBGOLJKPSFNJSD-UHFFFAOYSA-N 0.000 description 1
- 208000012671 Gastrointestinal haemorrhages Diseases 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 206010073391 Platelet dysfunction Diseases 0.000 description 1
- 208000037486 Postoperative Hemorrhage Diseases 0.000 description 1
- 206010071229 Procedural haemorrhage Diseases 0.000 description 1
- 239000005607 Pyroxsulam Substances 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- 238000001237 Raman spectrum Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- XYUSESXYUHHDPC-UHFFFAOYSA-N diethylazanium;2,5-dihydroxybenzene-1,4-disulfonate Chemical compound CC[NH2+]CC.CC[NH2+]CC.OC1=CC(S([O-])(=O)=O)=C(O)C=C1S([O-])(=O)=O XYUSESXYUHHDPC-UHFFFAOYSA-N 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229960004817 etamsylate Drugs 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000004401 flow injection analysis Methods 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- GLBLPMUBLHYFCW-UHFFFAOYSA-N n-(5,7-dimethoxy-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)-2-methoxy-4-(trifluoromethyl)pyridine-3-sulfonamide Chemical compound N1=C2N=C(OC)C=C(OC)N2N=C1NS(=O)(=O)C1=C(OC)N=CC=C1C(F)(F)F GLBLPMUBLHYFCW-UHFFFAOYSA-N 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- 229940044654 phenolsulfonic acid Drugs 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 1
- 229940001584 sodium metabisulfite Drugs 0.000 description 1
- 235000010262 sodium metabisulphite Nutrition 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001635 urinary tract Anatomy 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- 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
-
- 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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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention discloses a method for determining impurities and main medicine content in a phenol sulfoethylamine drug, which adopts a chromatographic column with octadecylsilane chemically bonded silica as a filler, uses tetrabutyl ammonium hydroxide aqueous solution as a mobile phase A and methanol as a mobile phase B to perform gradient elution. All impurities of the phenol sulfoethylamine can be rapidly and effectively separated, an effective detection method is provided for monitoring the phenol sulfoethylamine and the medicine thereof, and the product quality and the medication safety of the phenol sulfoethylamine are ensured; the quantitative determination of the impurities is carried out by adopting a self-contrast method, the determination of the genotoxic impurities and the main drug content is carried out by adopting an external standard method, the operation is simple and convenient, the detection cost is low, and the method has good sensitivity, linearity, specificity, precision, accuracy, stability and durability, and is an effective detection method for the controlled phenol sulfoethylamine and the drugs thereof.
Description
Technical Field
The invention relates to the technical field of medicine quality detection, in particular to a method for measuring the content of impurities and main drugs thereof in a phenol sulfoethylamine medicine.
Background
Trifenesulfonate (Etamsylate), also known as hemostatic sensitivity, is clinically used for various hemorrhages such as hemorrhage due to platelet dysfunction, gastrointestinal hemorrhage, cerebral hemorrhage, urinary tract hemorrhage and the like, and is used for preventing and treating intraoperative and postoperative hemorrhages. The structural formula is as follows:
the phenol sulfoethylamine is unstable when meeting alkali, the illumination is easy to change color, and related substances can also change.
There is no patent literature concerning substances related to the phenol-sulfoethylamine and methods for detecting the content thereof. In the non-patent literature, lanvin et al have studied substances related to domestic phenol sulfoethylamine (10 th edition of volume 12 of 10 of 2014, middle and south pharmacy) by using an HPLC method, using an AgilentZOBAXNH2 (4.6X105 mm,5 μm) column, and subjecting known impurities to qualitative and quantitative analysis by using a methanol-0.2% potassium dihydrogen phosphate solution (10:90) as a mobile phase at a detection wavelength of 220nm and a column temperature of 35℃at a flow rate of 1.0 ml/min. The results showed that hydroquinone had a peak time of about 3.5min and cysteamine had a peak time of about 9.3min.
There are many non-patent documents on measurement of the content of substances related to the phenol-sulfoethylamine. The method for measuring the content of the active ingredients of the phenol sulfoethylamine injection by adopting the liquid phase detection is about two methods (the method is about the same as the research method of Lanwen et al except that the column temperature is slightly modified) of the method for measuring the content of the active ingredients of the phenol sulfoethylamine injection by adopting an HPLC method of Liu Jinrong et al (the 8 th phase 1 of the 8 th volume of the traditional medicine of the asia); the content of the phenol sulfoethylamine tablet (Anhui medicine 2009May13 (5)) is measured by a high performance liquid chromatography of Armillariella tabilis et al, wherein a C18 (4.6X250 mm,5 μm) chromatographic column is adopted, and methanol-0.05 mol/L potassium dihydrogen phosphate is used as a mobile phase (10:90); the flow rate is 1.0ml/min; the detection wavelength was 300nm at room temperature, and the result showed that the peak was formed at 4.2min for the phenol sulfonic acid ethylamine.
As for the method for measuring the content of the phenolsulfoethylamine, in addition to the above-mentioned HPLC method, there is also a method for measuring the content of the drug phenolsulfoethylamine by a visible spectrophotometry of Yang Fengzhen et al (Royal society of robusta 2015, volume 31, 1), which uses the characteristic that phenolsulfoethylamine is easily oxidized by oxygen in the air to a colored compound under alkaline conditions and has a maximum absorption at 470nm, and quantitatively analyzes it; he Jian et al Raman spectrum for rapid detection of the injection of cysteamine (China modern applied pharmaceutical 2015, 1 month, 32 vol 1 st phase), determination conditions of the invention: the microscopic Raman spectrometer has the excitation wavelength of 785nm, the objective lens of 50×, the laser power of 3mW and the signal acquisition time of 60s, and the method can identify the content of the phenol sulfoethylamine; cai Peng et al ion chromatography was used to determine the content of the injection of cysteamine (Chinese pharmaceutical Standard 2017, volume 18, phase 4), which used ion chromatography with CS17A (4 mm. Times.250 mm) column and CG17A (4 mm. Times.50 mm) column, with DIONEXCSRS3004-mm suppressor conductivity detector with 5mmol/L methanesulfonic acid solution as mobile phase. The flow rate is 1ml/min, and the column temperature is 30 ℃; finally, the contents of the phenol sulfoethylamine and the sodium metabisulfite in the injection of Yang Fengzhen et al are simultaneously measured (chemical research and application of the volume 28 of the month 11 of 2016, 11 th period), and the method is used for measuring the content of the phenol sulfoethylamine by using an IFIS-D intelligent flow injection sample injector and an MPI-A capillary electrophoresis chemiluminescence detector and utilizing the characteristic that the phenol sulfoethylamine generates chemiluminescence in an acidic image-text 80-rhodamine 6G system.
The above is about the incomplete statistics of the non-patent literature of the phenoxyethylamine, and the national standard phenoxyethylamine (WS 1- (YH-001) -1995-2010) of the national food and drug administration refers to the method for detecting the content of the phenoxyethylamine hydroquinone, which is a chromatographic column using amino-bonded silica gel as a filler and methanol-0.2% potassium dihydrogen phosphate (10:90) as a mobile phase, wherein the detection wavelength is 223nm.
Because the polarity of the phenol sulfoethylamine is large, the impurities generated by the synthesis process and degradation are also substances with large polarity, and in addition, in recent years, the detection of related substances of the phenol sulfoethylamine by the liquid phase detection method cannot be well separated and detected, and the detection equipment is not popularized by other methods, and the research condition of related substances is not mentioned, so that a detection method with high sensitivity, effective separation of related substances and wider popularization range is urgently needed; the chromatographic column used in the phenol sulfoethylamine content determination HPLC method is an amino column, the amino column has no common C18 repeatability and durability, other detection methods have low popularity, and the common mobile phase and the C18 phenol sulfoethylamine are not reserved, so that a method which can be widely used and is convenient is urgently needed.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for measuring the content of impurities and main drugs thereof in a phenol sulfoethylamine drug.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
the method for determining the impurity and the main medicine content of the phenol sulfoethylamine drug comprises the following steps:
s1, precisely weighing a proper amount of each impurity and the phenol sulfoethylamine respectively, and dissolving and diluting the impurities and the phenol sulfoethylamine to a required concentration by using a diluent to prepare an impurity stock solution;
s2, precisely measuring the same amount of each impurity stock solution, uniformly mixing, and diluting to the required concentration by using a diluent to prepare an impurity mixed solution;
s3, weighing a proper amount of phenol sulfoethylamine, and dissolving and diluting the phenol sulfoethylamine with the impurity mixed solution to a required concentration to prepare a system applicability solution;
s4, precisely weighing a proper amount of a phenolsulfoethylamine sample to be measured, and dissolving and diluting the phenolsulfoethylamine sample to be measured to a required concentration by using a diluent to prepare a sample solution;
s5, precisely measuring a certain amount of the sample solution, diluting the sample solution to a required concentration by using a diluent, and preparing a self control solution;
s6, precisely measuring a proper amount of the impurity stock solution, the impurity mixed solution, the system applicability solution, the sample solution and the self control solution, measuring by adopting a high performance liquid chromatography, recording a chromatogram, and calculating the impurity contained in the phenolsulfoethylamine sample to be measured and the content of the impurity contained in the phenolsulfoethylamine sample to be measured by the data measured on the chromatogram.
The invention solves the problems of no retention, low sensitivity and difficult separation of impurities contained in the phenol sulfoethylamine by using common liquid phase conditions through adopting a high performance liquid chromatography method, and obtains the analysis method which has high sensitivity, good repeatability and common detection conditions and can accurately quantify each impurity and content in the phenol sulfoethylamine.
Preferably, the measuring method of the present invention further comprises detection limits and quantitative limits for detecting the pyroxsulam and each impurity, and the specific detection steps are as follows:
a. precisely weighing a proper amount of each impurity and the phenol sulfoethylamine, and dissolving and diluting the impurities and the phenol sulfoethylamine to a set concentration by using a diluent to prepare an impurity mother solution;
b. precisely measuring a proper amount of the impurity mother solution and the phenol-sulfoethylamine mother solution respectively, and diluting the impurity mother solution and the phenol-sulfoethylamine mother solution to a required concentration by using a diluent to prepare a detection limiting solution;
c. precisely measuring a proper amount of the impurity mother solution and the phenol-sulfoethylamine mother solution respectively, and diluting the impurity mother solution and the phenol-sulfoethylamine mother solution to a required concentration by using a diluent to prepare a quantitative limiting solution;
d. and measuring the detection limit solution and the quantitative limit solution by adopting a high performance liquid chromatography, and calculating the detection limit and the quantitative limit of each impurity and the phenol sulfoethylamine according to measured data so as to characterize the detection sensitivity.
The detection limit and the quantitative limit of the fensulfoethylamine and each impurity are measured, the detection data are analyzed and calculated, and the detection result and the limit concentration are compared and analyzed, so that the sensitivity of the measuring method can be characterized. The detection limit and the quantitative limit of the phenol sulfoethylamine and the impurities are far smaller than the limit concentration, which shows that the method has high detection sensitivity, and the average S/N of the phenol sulfoethylamine and the impurities is about 10 after the quantitative limit is continuously injected for 6 times, thereby meeting the requirements.
Preferably, the diluent is methanol: the water is 5:95 methanol-water mixture.
Preferably, the concentration of the test solution in the step S4 is 2mg-20mg of the cysteamine-containing solution per 1ml, and the test solution is prepared for clinical use.
Preferably, the high performance liquid chromatography described in step S6 is performed under the following conditions:
a chromatographic column using octadecylsilane chemically bonded silica as filler, with specification of 250mm×4.6mm,5 μm;
mobile phase, including mobile phase A and mobile phase B, wherein mobile phase A adopts 0.1% -0.5% tetrabutylammonium hydroxide aqueous solution, mobile phase B adopts methanol, mobile phase A and mobile phase B elute according to the gradient program set;
the elution gradient is:
at 0 min, mobile phase A is 95% -70% and mobile phase B is 5% -30%; at 18 minutes, mobile phase A was 30% -45% and mobile phase B was 70% -55%; at 23 minutes, mobile phase A is 10% -20% and mobile phase B is 90% -80%;25 minutes, mobile phase A is 95% -70% and mobile phase B is 5% -30%; at 40 minutes, mobile phase a was 95% -70% and mobile phase B was 5% -30%.
Preferably, the elution gradient is:
the flow rate is 0.8ml/min-1.2ml/min;
column temperature, 20-40 ℃;
detection wavelength, 200nm-300nm (detector is ultraviolet detector);
the sample injection amount is 5-20. Mu.l.
Preferably, the specific configuration method of the mobile phase a is as follows:
12ml of 25% tetrabutylammonium hydroxide aqueous solution is weighed, 1000ml of water is added, and the PH is adjusted to 3.0-6.0 by phosphoric acid, thus obtaining mobile phase A.
Preferably, the pH is 4.0.
Preferably, the column temperature is 30 ℃.
Preferably, the detection wavelength is 200nm-240nm.
Preferably, the mobile phase A is 0.3% tetrabutylammonium hydroxide aqueous solution.
The beneficial effects of the invention are as follows:
1. the invention solves the problems of no retention, low sensitivity and difficult separation of impurities contained in the phenol sulfoethylamine by using common liquid phase conditions through adopting a high performance liquid chromatography method, and obtains the analysis method which has high sensitivity, good repeatability and common detection conditions and can accurately quantify each impurity and content in the phenol sulfoethylamine.
2. The detection limit and the quantitative limit of the phenol sulfoethylamine and the impurities are far smaller than the limit concentration, which shows that the method has high detection sensitivity, and the average S/N of the phenol sulfoethylamine and the impurities is about 10 after the quantitative limit is continuously injected for 6 times, thereby meeting the requirements.
3. According to the data measured on the chromatogram, the invention carries out linear regression by taking the concentration as an abscissa and the peak area as an ordinate, and the linear result shows that: the linear relation of each impurity in the phenol sulfoethylamine is good in the corresponding range, and the R value is more than 0.999; the correction factor calculation result shows that the condition meets the calculation of each impurity according to the self-comparison method added with the correction factor.
4. The measuring method has good accuracy, the average recovery rate of each impurity is between 95.0% and 105.0%, and the RSD% of each impurity meets the requirement of less than 6.0%.
5. In the determination method, each impurity can be effectively separated, and the separation degree meets the requirements.
6. The determination method of the invention has the advantages that after the column temperature, the pH of the mobile phase and the chromatographic column are changed, the content of impurities is not greatly changed, and other unknown impurities are not detected, so the condition durability is good; the external standard method is consistent with the self-comparison method added with the correction factors, so that the self-comparison method added with the correction factors is feasible to calculate.
Detailed Description
The following describes embodiments of the present invention in detail.
Example 1
Detection limit and quantitative limit detection
The method specifically comprises the following steps:
s11, preparing each impurity mother solution;
accurately weighing 20mg of each impurity A, B, C, D, P, respectively placing into 100ml measuring bottles, dissolving with diluent, diluting to scale, and shaking;
accurately weighing 20mg of each impurity L, J, placing into a 100ml measuring flask, firstly dissolving with 1ml of methanol, then dissolving with a diluent, diluting to scale, and shaking uniformly;
accurately weighing 10mg of the fensulfoethylamine, placing the obtained mixture into a 10ml measuring flask, dissolving the obtained mixture by using a diluent, diluting the obtained mixture to a scale, and shaking the obtained product uniformly.
S12, preparing each detection limit solution;
respectively precisely measuring 0.25ml of the impurity P, J, L, A, B mother liquor, 0.5ml of the impurity C mother liquor, 0.1ml of the impurity D mother liquor and 1ml of the fensulfoethylamine mother liquor, placing into a 10ml measuring flask, dissolving with a diluent, diluting to scale, and shaking uniformly.
S13, preparing quantitative limiting solutions;
precisely measuring 1ml of each of the above-mentioned phenolsulfoethylamine, impurity J and C mother liquor, 0.75ml of impurity A and P mother liquor, 0.5ml of impurity B and L mother liquor and 0.4ml of impurity D mother liquor, placing them into 10ml measuring flask, using diluent to make dissolution and dilution until the scale is uniformly shaken.
S14, detecting;
and precisely measuring 10 mu l of each of the quantitative limit solution and the detection limit solution, injecting into a liquid chromatograph for detection, and recording a chromatogram. Signal to noise ratio S/n=10: 1 is quantitative limit concentration; signal-to-noise ratio S/n=3: 1 is the detection concentration.
The concentration of each of the quantitative limit and the detection limit in this example is shown in the following table:
under the concentration, the detection limit and the quantitative limit are far smaller than the limit concentration, which shows that the detection sensitivity is very high, and the average S/N of the cysteamine and each impurity is about 10 after the quantitative limit is continuously injected for 6 times, thereby meeting the requirements.
Example 2
Detection of standard curve
The method specifically comprises the following steps:
s21, preparing mother liquor of each impurity reference substance;
accurately weighing 20mg of each impurity A, B, C, D, P, respectively placing into 100ml measuring bottles, dissolving with diluent, diluting to scale, and shaking;
accurately weighing 20mg of each impurity L, J, placing into a 100ml measuring flask, firstly dissolving with 1ml of methanol, then dissolving with a diluent, diluting to scale, and shaking uniformly;
accurately weighing 10mg of the fensulfoethylamine, placing the obtained mixture into a 50ml measuring flask, dissolving the obtained mixture by using a diluent, diluting the obtained mixture to a scale, and shaking the obtained product uniformly.
S22, preparing each linear solution;
linear solution 1: respectively precisely measuring 1.0ml of each impurity reference substance mother solution, placing into a 100ml measuring flask, dissolving with a diluent, diluting to scale, shaking to obtain an impurity mixed solution 1, then precisely measuring 0.1ml of the phenol sulfoethylamine, placing into a 10ml measuring flask, dissolving with the impurity mixed solution 1, diluting to scale, shaking to obtain a linear solution 1;
linear solution 2: respectively precisely measuring mother solution of each impurity reference substance in a measuring flask of 0.75ml to 100ml, dissolving and diluting with a diluent to scale and shaking uniformly to obtain an impurity mixed solution 2, then precisely measuring 0.075ml of the phenol sulfoethylamine in a measuring flask of 10ml, dissolving and diluting with the impurity mixed solution 2 to scale and shaking uniformly to obtain a linear solution 2;
linear solution 3: precisely measuring mother liquor of each impurity reference substance in a measuring flask with 0.5ml to 100ml respectively, dissolving with a diluent, diluting to scale, and shaking to obtain impurity mixed solution 3; precisely measuring 0.05ml of the fensulfoethylamine, placing the obtained solution into a 10ml measuring flask, dissolving and diluting the obtained solution to a scale by using the impurity mixed solution 3, and shaking the solution uniformly to obtain a linear solution 3;
linear solution 4: diluting the linear solution 3 by 2 times with a diluent to obtain a linear solution 4;
linear solution 5: diluting the linear solution 4 by 2 times with a diluent to obtain a linear solution 5;
linear solution 6: mixing the linear solution 4 and the linear solution 5 according to a ratio of 1:1 to obtain a linear solution 6;
linear solution 7: the respective quantitative limiting solutions in example 1 were employed.
S23, detecting;
precisely measuring 10 μl of each of the linear solutions 1-7, injecting into a liquid chromatograph, recording the chromatogram, and performing linear regression with the concentration as abscissa and the peak area as ordinate, wherein the linear results are shown in the following table:
the correction factor results calculated by the linear relation of 2 groups of data and 4 groups of data are respectively measured on two instruments in the same method as follows:
| name of the name | Correction factor 1 | Correction factor 2 | Correction factor 3 | Correction factor 4 | Average value of |
| D | 0.49 | 0.49 | 0.49 | 0.50 | 0.49 |
| C | 1.23 | 1.19 | 1.27 | 1.25 | 1.23 |
| P | 0.82 | 0.76 | 0.74 | 0.73 | 0.76 |
| L | 0.53 | 0.54 | 0.61 | 0.59 | 0.57 |
| J | 0.88 | 0.89 | 0.92 | 0.95 | 0.91 |
| A | 1.01 | 0.99 | 0.96 | 0.98 | 0.99 |
| B | 0.86 | 0.81 | 0.84 | 0.83 | 0.83 |
The linear relation of the impurities of the phenol sulfoethylamine in the corresponding range is good, and the R value is more than 0.999; the correction factor calculation result shows that the condition satisfies the calculation of each impurity according to the self-comparison method of the correction factor.
Example 3
Detection of accuracy
The method specifically comprises the following steps:
s31, preparing each impurity mother solution;
accurately weighing 20.0mg of each impurity A, B, C, D, P, respectively placing into 100ml measuring flask, dissolving with diluent, diluting to scale, and shaking;
20.0mg of each impurity L, J is precisely weighed, placed in a 100ml measuring flask respectively, dissolved by 1ml of methanol, then dissolved by a diluent, diluted to a scale and shaken uniformly.
S32, preparing a recovery rate impurity mixed solution;
50% impurity mixed solution: precisely measuring 0.25ml of each impurity mother solution, placing the mother solution into a 100ml measuring flask, diluting the mother solution to a scale with a diluent, and shaking the mother solution uniformly to obtain a 50% impurity mixed solution;
100% impurity mixed solution: precisely measuring 0.5ml of each impurity mother solution, placing the mother solution into a 100ml measuring flask, diluting the mother solution to a scale with a diluent, and shaking the mother solution uniformly to obtain a 100% impurity mixed solution;
150% impurity mixed solution: precisely measuring the mother liquor of each impurity in a measuring flask with 0.75ml to 100ml, diluting to the scale with a diluent, and shaking uniformly to obtain 150% impurity mixed solution.
S33, preparing a recovery rate sample solution;
impurity control solution: mixing the solution with the 100% impurity;
background solution: respectively precisely weighing two parts of the fensulfoethylamine 20.0mg, respectively placing the two parts in 10ml measuring flasks, diluting to a scale with a diluent, and shaking uniformly to obtain a background solution;
sample solution with 50% recovery: respectively precisely weighing three parts of the fensulfuron-ethylamine (20.0 mg), respectively placing in 10ml measuring flask, respectively diluting to scale with 50% impurity mixed solution, and shaking to obtain 50% recovery rate sample solution;
sample solution with 100% recovery rate: respectively precisely weighing three parts of the fensulfuron-ethylamine (20.0 mg), respectively placing in 10ml measuring flask, respectively diluting to scale with 100% impurity mixed solution, and shaking to obtain 100% recovery rate sample solution;
sample solution with 150% recovery rate: and respectively precisely weighing three parts of the fensulfoethylamine (20.0 mg), respectively placing the three parts in 10ml measuring flasks, respectively diluting the three parts to the scale with 150% of impurity mixed solution, and shaking the three parts uniformly to obtain the sample solution with 150% recovery rate.
S34, detecting;
precisely measuring 10 μl of each of the recovery rate test sample solutions, injecting into a liquid chromatograph, recording a chromatogram, and calculating by using peak area according to an external standard method to obtain tax rate results of each sample, wherein the tax rate results are shown in the following table:
the average recovery rate of each impurity is between 95.0% and 105.0%, the RSD% of each impurity meets the requirement of less than 6.0%, and the accuracy of the condition is good.
Example 4
Detection of degree of separation
The method specifically comprises the following steps:
s41, preparing impurity positioning solution;
and respectively taking a proper amount of each impurity reference substance A, B, C, D, P, L, J, precisely weighing, dissolving by using a diluent, and diluting to a solution with the concentration of 20 mug/ml to obtain an impurity positioning solution.
S42, preparing a system applicability solution;
and (3) taking a proper amount of each impurity reference substance A, B, C, D, P, L, J and the proper amount of the phenol sulfoethylamine, precisely weighing, dissolving with a diluent, and diluting to a solution with the concentration of 1 mug/ml to obtain the system applicability solution.
S43, detecting;
and precisely measuring 10 mu l of each impurity positioning solution and each system applicability solution, injecting into a liquid chromatograph, recording a chromatogram, and calculating according to the retention time.
Impurity localization specificity results are shown in the following table:
the experimental results show that: the impurity positioning is basically consistent with the retention time of each impurity peak in the system applicability, each impurity can be effectively separated, and the separation degree meets the requirements.
Example 5
The method for determining the impurity and the main medicine content of the phenol sulfoethylamine drug comprises the following steps:
s51, preparing an impurity stock solution;
20.37mg of impurity D, 20.46mg of impurity C, 22.92mg of impurity P, 20.14mg of impurity L, 20.57mg of impurity J, 30.10mg of impurity A and 20.03mg of impurity B are respectively precisely weighed, respectively placed in a 100ml volumetric flask, dissolved by a diluent and diluted to scale, wherein the impurity L and the impurity J are dissolved by 1ml of methanol and then diluted, and uniformly shaken to obtain an impurity stock solution.
S52, preparing an impurity mixed solution;
respectively precisely weighing each impurity stock solution 0.5ml, placing into a 100ml measuring flask, diluting with diluent to scale, and shaking to obtain impurity mixed solution.
S53, preparing a system applicability solution;
2mg (qualitatively and quantitatively) of the fensulfoethylamine is taken and placed in a 10ml measuring flask, and the mixed solution of the impurities is dissolved and diluted to scale and shaken uniformly to obtain the system applicability solution.
S54, preparing a sample solution;
21.56mg of the to-be-measured phenol sulfoethylamine sample is weighed, precisely weighed, placed in a 10ml volumetric flask, dissolved and diluted to a scale by a diluent, and shaken uniformly to obtain a test sample solution, wherein the test sample solution is freshly prepared.
S55, preparing self-control solution;
precisely weighing 0.1ml of the sample solution, placing in a 100ml measuring flask, diluting to scale with diluent, shaking, precisely weighing 5ml of the diluted sample solution, placing in a 10ml measuring flask, diluting to scale with diluent, and shaking to obtain self control solution.
S56, detecting;
and precisely measuring 10 mu l of each of the impurity stock solution, the impurity mixed solution, the system applicability solution, the test sample solution and the self control solution, injecting into a liquid chromatograph, and recording a chromatogram.
The liquid chromatography conditions are controlled as follows:
the chromatographic column adopts octadecylsilane chemically bonded silica gel as filler, and has specification of 250mm×4.6mm and 5 μm.
The mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A adopts 0.3% tetrabutylammonium hydroxide aqueous solution, and the preparation process is as follows: weighing 12ml of 25% tetrabutylammonium hydroxide aqueous solution, adding 1000ml of water, and adjusting the pH to 4.0 by phosphoric acid to obtain a mobile phase A; the mobile phase B adopts methanol.
Mobile phase a and mobile phase B were gradient eluted according to the following table elution procedure.
The flow rate was 1.0ml per minute, the column temperature was 30℃and the detection wavelength was 220nm.
The elution procedure is as follows:
according to the data on the measured chromatograms, the calculation is carried out according to the following method:
the chromatogram of the sample solution has impurity peaks, the retention time of the impurity peaks is consistent with that of each known impurity in the solution with system applicability, the impurity peaks are calculated according to the main component self-comparison method of the correction factors, other unknown impurities are calculated according to the main component self-comparison method, and the relative retention time, the correction factors and the limit of each impurity peak are shown in the following table:
the calculation method comprises the following steps:
wherein:
aimp: the peak area of impurities is known in the sample solution;
a control: self-contrast peak area;
f: a correction factor;
s: self-control dilution.
Wherein:
a single impurity: unknown single-impurity peak area in the sample solution;
a control: is the self-contrast peak area;
s: self-control dilution.
Wherein:
a control: is the self-contrast peak area;
Σa other single impurities: is the sum of the peak areas of unknown total impurities in the sample solution;
s: self-control dilution.
Total impurity% = sum of known impurities% + other total impurities%
The results obtained according to the above calculation method are as follows:
| name of the name | Impurity A% | Impurity D% | Maximum unknown single impurity% | Total impurity% |
| Sample of | 0.044 | 0.006 | 0.078 | 0.14 |
In this example, the unknown impurities appear after the stability of the room temperature sample solution is 2 hours, and the unknown impurities appear after the stability of the 7 ℃ temperature control sample solution is 2 hours, so that the phenol sulfoethylamine is stable within 2 hours under the solvent.
The self-control solution has a retention time of each impurity and a peak area RSD% of less than 6.0% within 7 days, so that the self-control is stable in the solvent for 7 days.
Example 6
The method for determining the impurity and the main medicine content of the phenol sulfoethylamine drug comprises the following steps:
s61, preparing an impurity stock solution;
respectively precisely weighing 20.32mg of impurity D, 20.45mg of impurity C, 22.90mg of impurity P, 20.13mg of impurity L, 20.59mg of impurity J, 30.08mg of impurity A and 20.03mg of impurity B, respectively placing into a 100ml volumetric flask, dissolving with a diluent and diluting to scale, wherein the impurity L and the impurity J are dissolved with 1ml of methanol, then diluting, and shaking uniformly to obtain an impurity stock solution.
S62, preparing an impurity mixed solution;
respectively precisely weighing each impurity stock solution 0.5ml, placing into a 100ml measuring flask, diluting with diluent to scale, and shaking to obtain impurity mixed solution.
S63, preparing a system applicability solution;
2mg (qualitatively and quantitatively) of the fensulfoethylamine is taken and placed in a 10ml measuring flask, and the mixed solution of the impurities is dissolved and diluted to scale and shaken uniformly to obtain the system applicability solution.
S64, preparing a sample solution;
21.55mg of the to-be-measured phenol sulfoethylamine sample is weighed, precisely weighed, placed in a 10ml volumetric flask, dissolved and diluted to a scale by a diluent, and shaken uniformly to obtain a test sample solution, wherein the test sample solution is freshly prepared.
S65, preparing self-control solution;
precisely weighing 0.1ml of the sample solution, placing in a 100ml measuring flask, diluting to scale with diluent, shaking, precisely weighing 5ml of the diluted sample solution, placing in a 10ml measuring flask, diluting to scale with diluent, and shaking to obtain self control solution.
S66, detecting;
precisely measuring 5 μl of the above impurity stock solution, impurity mixed solution, system applicability solution, test solution and self control solution, and injecting into a liquid chromatograph, and recording the chromatogram.
The liquid chromatography conditions are controlled as follows:
the chromatographic column adopts octadecylsilane chemically bonded silica gel as filler, and has specification of 250mm×4.6mm and 5 μm.
The mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A adopts 0.1% tetrabutylammonium hydroxide aqueous solution, and the preparation process is as follows: weighing 4ml of 25% tetrabutylammonium hydroxide aqueous solution, adding 1000ml of water, and adjusting the pH to 3.0 with phosphoric acid to obtain a mobile phase A; the mobile phase B adopts methanol.
Mobile phase a and mobile phase B were gradient eluted according to the following table elution procedure.
The flow rate was 0.8ml per minute, the column temperature was 20℃and the detection wavelength was 210nm.
The elution procedure is as follows:
according to the data on the measured chromatograms, the calculation is carried out according to the following method:
the chromatogram of the sample solution has impurity peaks, the retention time of the impurity peaks is consistent with that of each known impurity in the solution with system applicability, the impurity peaks are calculated according to the main component self-comparison method of the correction factors, other unknown impurities are calculated according to the main component self-comparison method, and the relative retention time, the correction factors and the limit of each impurity peak are shown in the following table:
the calculation method comprises the following steps:
wherein:
aimp: the peak area of impurities is known in the sample solution;
a control: self-contrast peak area;
f: a correction factor;
s: self-control dilution.
Wherein:
a single impurity: unknown single-impurity peak area in the sample solution;
a control: is the self-contrast peak area;
s: self-control dilution.
Wherein:
a control: is the self-contrast peak area;
Σa other single impurities: is the sum of the peak areas of unknown total impurities in the sample solution;
s: self-control dilution.
Total impurity% = sum of known impurities% + other total impurities%
The results obtained according to the above calculation method are as follows:
| name of the name | Impurity A% | Impurity D% | Maximum unknown single impurity% | Total impurity% |
| Sample of | 0.041 | 0.006 | 0.075 | 0.13 |
Example 7
The method for determining the impurity and the main medicine content of the phenol sulfoethylamine drug comprises the following steps:
s71, preparing an impurity stock solution;
20.37mg of impurity D, 20.46mg of impurity C, 22.92mg of impurity P, 20.14mg of impurity L, 20.57mg of impurity J, 30.10mg of impurity A and 20.03mg of impurity B are respectively precisely weighed, respectively placed in a 100ml volumetric flask, dissolved by a diluent and diluted to scale, wherein the impurity L and the impurity J are dissolved by 1ml of methanol and then diluted, and uniformly shaken to obtain an impurity stock solution.
S72, preparing an impurity mixed solution;
respectively precisely weighing each impurity stock solution 0.5ml, placing into a 100ml measuring flask, diluting with diluent to scale, and shaking to obtain impurity mixed solution.
S73, preparing a system applicability solution;
2mg (qualitatively and quantitatively) of the fensulfoethylamine is taken and placed in a 10ml measuring flask, and the mixed solution of the impurities is dissolved and diluted to scale and shaken uniformly to obtain the system applicability solution.
S74, preparing a sample solution;
21.56mg of the to-be-measured phenol sulfoethylamine sample is weighed, precisely weighed, placed in a 10ml volumetric flask, dissolved and diluted to a scale by a diluent, and shaken uniformly to obtain a test sample solution, wherein the test sample solution is freshly prepared.
S75, preparing self-control solution;
precisely weighing 0.1ml of the sample solution, placing in a 100ml measuring flask, diluting to scale with diluent, shaking, precisely weighing 5ml of the diluted sample solution, placing in a 10ml measuring flask, diluting to scale with diluent, and shaking to obtain self control solution.
S76, detecting;
and precisely measuring 20 mu l of each of the impurity stock solution, the impurity mixed solution, the system applicability solution, the test sample solution and the self control solution, injecting into a liquid chromatograph, and recording a chromatogram.
The liquid chromatography conditions are controlled as follows:
the chromatographic column adopts octadecylsilane chemically bonded silica gel as filler, and has specification of 250mm×4.6mm and 5 μm.
The mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A adopts 0.5% tetrabutylammonium hydroxide aqueous solution, and the preparation process is as follows: weighing 20ml of 25% tetrabutylammonium hydroxide aqueous solution, adding 1000ml of water, and adjusting the pH to 5.0 with phosphoric acid to obtain a mobile phase A; the mobile phase B adopts methanol.
Mobile phase a and mobile phase B were gradient eluted according to the following table elution procedure.
The flow rate was 0.9ml per minute, the column temperature was 40℃and the detection wavelength was 230nm.
The elution procedure is as follows:
according to the data on the measured chromatograms, the calculation is carried out according to the following method:
the chromatogram of the sample solution has impurity peaks, the retention time of the impurity peaks is consistent with that of each known impurity in the solution with system applicability, the impurity peaks are calculated according to the main component self-comparison method of the correction factors, other unknown impurities are calculated according to the main component self-comparison method, and the relative retention time, the correction factors and the limit of each impurity peak are shown in the following table:
the calculation method comprises the following steps:
wherein:
aimp: the peak area of impurities is known in the sample solution;
a control: self-contrast peak area;
f: a correction factor;
s: self-control dilution.
Wherein:
a single impurity: unknown single-impurity peak area in the sample solution;
a control: is the self-contrast peak area;
s: self-control dilution.
Wherein:
a control: is the self-contrast peak area;
Σa other single impurities: is the sum of the peak areas of unknown total impurities in the sample solution;
s: self-control dilution.
Total impurity% = sum of known impurities% + other total impurities%
The results obtained according to the above calculation method are as follows:
| name of the name | Impurity A% | Impurity D% | Maximum unknown single impurity% | Total impurity% |
| Sample of | 0.047 | 0.007 | 0.071 | 0.14 |
Example 8
Detection of the content of Triphenolsulfonate
The method specifically comprises the following steps:
s81, preparing a control solution;
respectively precisely weighing 12.91mg and 12.75mg of reference substances, respectively placing into 25ml measuring flask, dissolving with diluent, diluting to scale, shaking, precisely weighing 2ml, placing into 10ml measuring flask, dissolving with diluent, diluting to scale, shaking to obtain reference solution, and preparing two parts in parallel.
S82, preparing a sample solution;
respectively precisely weighing 26.12mg and 25.10mg of reference substances, respectively placing into 50ml measuring flask, dissolving with diluent, diluting to scale, shaking, precisely weighing 2ml, placing into 10ml measuring flask, dissolving with diluent, diluting to scale, shaking, and preparing two parts in parallel.
S83, detecting;
respectively precisely weighing 10 μl of each of the control solution and the sample solution, injecting into a liquid chromatograph, and recording the chromatograms.
According to the data on the chromatogram, the method is calculated as follows:
the chromatogram is recorded, and the peak area is calculated according to an external standard method, and the content of the phenol sulfoethylamine is 99.0% -101.0%.
Wherein:
asamp: the main peak area of the sample solution;
astd: controlling the main peak area of the solution;
wsamp: weighing the sample, and mg;
wstd: weighing reference substance, and mg;
pstd: purity of control,%;
w water: the water content of the test sample is percent.
The result of the calculation method shows that the content of the phenol sulfoethylamine is 100.9 percent, which meets the regulations.
Example 9
Detection of genotoxic impurity D, H detection limit and quantitative limit
The method specifically comprises the following steps:
s91, preparing quantitative limiting solution;
precisely weighing a proper amount of the reference substances of the impurity D and the impurity H, dissolving and diluting the reference substances by using a diluent to prepare a mixed solution containing 0.12 mug of the impurity D and 0.23 mug of the impurity H per 1ml, and preparing 6 parts by the same method;
s92, preparing a detection limit solution;
precisely weighing a proper amount of the reference substances of the impurity D and the impurity H, and dissolving and diluting the reference substances by using a diluent to prepare a mixed solution containing 0.0615 mug of the impurity D and 0.119 mug of the impurity H per 1 ml;
s93, detecting;
and precisely weighing 10 mu l of each of the quantitative limit solution and the detection limit solution, injecting into a liquid chromatograph, recording a chromatogram, and calculating according to the signal-to-noise ratio S/N value.
The calculation results are as follows:
detection limit result
Quantitative limit results
Quantitative limit of 6 sample injection results
| Name of the name | Impurity DS/N | Impurity HS/N |
| Limit of ration 1 | 9.1 | 10.8 |
| Limit of ration 2 | 9.2 | 11.0 |
| Limit of ration 3 | 9.1 | 10.9 |
| Limit of ration 4 | 8.2 | 9.9 |
| Limit of ration 5 | 8.3 | 10.2 |
| Limit of ration 6 | 8.1 | 10.2 |
| Average value of | 8.7 | 10.5 |
The above results indicate that the detection limit and the quantitative limit of the impurity D and the impurity H are measured respectively, and the detection limit and the quantitative limit concentration are far smaller than the limit concentration, which means that the sensitivity is high.
Example 10
Detection of genotoxic impurity D, H accuracy
The method specifically comprises the following steps:
s101, preparing background solution;
precisely weighing 2000.0mg of the fensulfoethylamine, placing the obtained product into a 100ml measuring flask, dissolving the obtained product by using a diluent, diluting the obtained product to a scale, and shaking the obtained product uniformly to obtain a background solution;
s102, preparing a control solution;
respectively taking reference substances of the impurity D and the impurity H, precisely weighing, dissolving with a diluent, and diluting to prepare a mixed reference solution containing 0.5 mug of the impurity D and 4.8 mug of the impurity H per 1 ml;
s103, recovering a sample solution;
50% sample solution: respectively taking reference substances of impurities D and H, precisely weighing, dissolving and diluting with a diluent to prepare an impurity mixed solution 1 containing 0.25 mug of the impurity D and 2.4 mug of the impurity H per 1ml, precisely weighing 2000.0mg of the fensulfoethylamine, placing into a 100ml measuring flask, dissolving and diluting to a scale with the impurity mixed solution 1, and shaking uniformly to obtain a 50% sample solution;
100% sample solution: respectively taking reference substances of impurities D and H, precisely weighing, dissolving and diluting with a diluent to prepare an impurity mixed solution 2 containing 0.5 mug of the impurity D and 4.8 mug of the impurity H per 1ml, precisely weighing 2000.0mg of the fensulfoethylamine, placing into a 100ml measuring flask, dissolving and diluting to a scale with the impurity mixed solution 2, and shaking uniformly to obtain a 100% sample solution;
150% sample solution: respectively taking reference substances of impurities D and H, precisely weighing, dissolving and diluting with a diluent to prepare an impurity mixed solution 3 containing 1.0 mug of the impurity D and 9.2 mug of the impurity H per 1ml, precisely weighing 2000.0mg of the fensulfoethylamine, placing into a 100ml measuring flask, dissolving and diluting to a scale with the impurity mixed solution 3, and shaking uniformly to obtain a 150% sample solution;
s104, detecting;
and precisely sucking 10 mu l of each of the background solution, the control solution, the 50% sample solution, the 100% sample solution and the 150% sample solution, injecting into a liquid chromatograph, recording the chromatograms, and calculating by a peak area external standard method.
The calculation formula is as follows:
the above measured recovery rate results are shown in the following table:
| name of the name | Average percent recovery (n=9) | RSD％ |
| D | 102.7 | 1.3 |
| H | 92.2 | 1.3 |
The condition has good accuracy, the average recovery rate of each impurity is between 90.0% and 105.0%, the RSD% of each impurity meets the requirement of less than 2.0%, and the condition has good accuracy.
Example 11
Detection of the content of genotoxic impurities D and H
The method specifically comprises the following steps:
s111, preparing a sample solution;
precisely weighing 2000.0mg of the fensulfoethylamine, placing the obtained product into a 100ml measuring flask, diluting the obtained product to a scale by using a diluent solvent, and shaking the obtained product uniformly to obtain a sample solution;
s112, preparing a control solution;
respectively taking reference substances of the impurity D and the impurity H, precisely weighing, dissolving with a diluent, and diluting to prepare a mixed reference solution containing 0.5 mug of the impurity D and 4.8 mug of the impurity H per 1 ml;
s113, detecting;
and precisely weighing 10 mu l of each solution, injecting into a liquid chromatograph, recording a chromatogram, and calculating by a peak area external standard method.
The calculation formula is as follows:
wherein:
asamp: the area of the impurity peak in the sample solution;
astd: controlling the main peak area of the solution;
wsamp: weighing the sample, and mg;
wstd: weighing reference substance, and mg;
pstd: purity of control,%.
The detection results are as follows:
| name of the name | Impurity D content% | Impurity H content% |
| Phenol sulfoethylamine | 1.56 | Not detected |
The diluents used in examples 1-8 above were methanol: the water is 5:95, and 15% methanol water was used as the diluent used in examples 9 to 11.
The impurity A described in the above examples 1 to 11 is 2, 5-dihydroxybenzene-1, 4-disulfonic acid bisdiethylamine; the impurity B is 2, 4-dihydroxybenzene-1, 5-disulfonic acid diethylamine; impurity C is 2, 3-dihydroxybenzene-1-diethylamine sulfonate; the impurity D is 1, 4-hydroquinone; the impurity P is 2-hydroxy benzene sulfonic acid diethylamine; impurity L is 4-acetoxy-1-phenol; impurity J is 1, 4-terephthalic acid; the impurity H is aniline.
The foregoing examples merely illustrate specific embodiments of the invention, which are described in greater detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (6)
1. A method for measuring the content of impurities and main drugs in a phenol sulfoethylamine drug adopts high performance liquid chromatography, which is characterized in that a chromatographic column using octadecylsilane chemically bonded silica as a filler, tetrabutyl ammonium hydroxide aqueous solution as a mobile phase A and methanol as a mobile phase B are adopted for gradient elution,
the specific steps of gradient elution are as follows:
at 0 min, mobile phase A is 95% -70% and mobile phase B is 5% -30%; at 18 minutes, mobile phase A was 30% -45% and mobile phase B was 70% -55%; at 23 minutes, mobile phase A is 10% -20% and mobile phase B is 90% -80%;25 minutes, mobile phase A is 95% -70% and mobile phase B is 5% -30%; at 40 minutes, mobile phase A is 95% -70% and mobile phase B is 5% -30%,
the concentration of the tetrabutylammonium hydroxide aqueous solution is 0.1-0.5%, and the pH value is adjusted to 3.0-6.0 by phosphoric acid; the impurities are one or more of impurities A, B, C, D, P, L and J; wherein, the impurity A is 2, 5-dihydroxybenzene-1, 4-disulfonic acid diethylamine, the impurity B is 2, 4-dihydroxybenzene-1, 5-disulfonic acid diethylamine, the impurity C is 2, 3-dihydroxybenzene-1-sulfonic acid diethylamine, the impurity D is 1, 4-hydroquinone, the impurity P is 2-hydroxybenzenesulfonic acid diethylamine, the impurity L is 4-acetoxy-1-phenol, and the impurity J is 1, 4-terephthalic acid.
2. The method for determining the content of impurities and main drugs thereof contained in a drug containing phenolsulfonate according to claim 1, wherein an ultraviolet detector is used as the detector.
3. The method for determining the content of impurities and main drugs thereof in a drug containing a phenol-sulfoethylamine as defined in claim 1, wherein the liquid chromatography conditions of the high performance liquid chromatography are as follows: the flow rate of the mobile phase is 0.8ml-1.2ml/min, the column temperature of the chromatographic column is 20-40 ℃, and the detection wavelength is 200-300 nm.
4. The method for determining the content of impurities and main drugs thereof contained in a drug containing phenolsulfoethylamine as defined in claim 3, wherein the detection wavelength is 200nm to 240nm.
5. The method for determining the content of impurities and main drugs thereof contained in a drug containing a phenol-sulfoethylamine as defined in claim 1, further comprising the steps of:
s1, precisely weighing a proper amount of various impurities and the phenol sulfoethylamine respectively, and dissolving and diluting the various impurities and the phenol sulfoethylamine to a required concentration by using a diluent to prepare an impurity stock solution;
s2, precisely measuring the same amount of each impurity stock solution, uniformly mixing, and diluting to the required concentration by using a diluent to prepare an impurity mixed solution;
s3, weighing a proper amount of phenol sulfoethylamine, and dissolving and diluting the phenol sulfoethylamine with the impurity mixed solution to a required concentration to prepare a system applicability solution;
s4, precisely weighing a proper amount of a phenolsulfoethylamine sample to be measured, and dissolving and diluting the phenolsulfoethylamine sample to be measured to a required concentration by using a diluent to prepare a sample solution;
s5, precisely measuring a certain amount of the sample solution, diluting the sample solution to a required concentration by using a diluent, and preparing a self control solution;
s6, precisely weighing a proper amount of the reference substance of the phenol sulfoethylamine to be measured, dissolving and diluting the reference substance to a required concentration by using a diluent, and preparing a reference substance solution;
s7, precisely measuring a proper amount of the impurity stock solution, the impurity mixed solution, the system applicability solution, the reference substance solution, the test substance solution and the self-control solution, measuring by adopting a high performance liquid chromatography, recording a chromatogram, and calculating the amount of impurities contained in the phenolsulfoethylamine sample to be measured and the content of the main medicine contained in the phenolsulfoethylamine sample to be measured by the data measured on the chromatogram.
6. The method for determining the content of impurities and main drugs thereof in a drug containing phenolsulfoethylamine as defined in claim 5, wherein the concentration of the sample solution is 2mg to 20mg per 1ml of the drug containing phenolsulfoethylamine.
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