CN117368390B - Method for determining impurity substances of loteprednol etabonate bulk drug by high performance liquid chromatography - Google Patents
Method for determining impurity substances of loteprednol etabonate bulk drug by high performance liquid chromatography Download PDFInfo
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- CN117368390B CN117368390B CN202311678272.2A CN202311678272A CN117368390B CN 117368390 B CN117368390 B CN 117368390B CN 202311678272 A CN202311678272 A CN 202311678272A CN 117368390 B CN117368390 B CN 117368390B
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- 239000012535 impurity Substances 0.000 title claims abstract description 136
- DMKSVUSAATWOCU-HROMYWEYSA-N loteprednol etabonate Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(=O)OCCl)(OC(=O)OCC)[C@@]1(C)C[C@@H]2O DMKSVUSAATWOCU-HROMYWEYSA-N 0.000 title claims abstract description 82
- 229960003744 loteprednol etabonate Drugs 0.000 title claims abstract description 82
- 239000003814 drug Substances 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 22
- 229940079593 drug Drugs 0.000 title claims abstract description 21
- 239000000126 substance Substances 0.000 title claims abstract description 20
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 11
- 238000012937 correction Methods 0.000 claims abstract description 24
- 238000000926 separation method Methods 0.000 claims abstract description 12
- 238000010828 elution Methods 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 88
- 239000013558 reference substance Substances 0.000 claims description 51
- 238000007865 diluting Methods 0.000 claims description 25
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 24
- 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 18
- 238000001514 detection method Methods 0.000 claims description 17
- 239000000523 sample Substances 0.000 claims description 17
- 230000035945 sensitivity Effects 0.000 claims description 17
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 15
- 238000005303 weighing Methods 0.000 claims description 14
- 239000012488 sample solution Substances 0.000 claims description 13
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 12
- 238000012360 testing method Methods 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 8
- 230000014759 maintenance of location Effects 0.000 claims description 8
- 238000004364 calculation method Methods 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 7
- 239000002994 raw material Substances 0.000 claims description 6
- ZYQPZAGCFHVLIY-UHFFFAOYSA-N 6-phenylhexylsilane Chemical group [SiH3]CCCCCCC1=CC=CC=C1 ZYQPZAGCFHVLIY-UHFFFAOYSA-N 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- AUALQMFGWLZREY-UHFFFAOYSA-N acetonitrile;methanol Chemical compound OC.CC#N AUALQMFGWLZREY-UHFFFAOYSA-N 0.000 claims description 5
- 239000011258 core-shell material Substances 0.000 claims description 5
- -1 ethoxycarbonyloxy Chemical group 0.000 claims description 5
- 239000000945 filler Substances 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 238000004090 dissolution Methods 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- 150000008064 anhydrides Chemical class 0.000 claims 1
- CQDGTJPVBWZJAZ-UHFFFAOYSA-N monoethyl carbonate Chemical compound CCOC(O)=O CQDGTJPVBWZJAZ-UHFFFAOYSA-N 0.000 claims 1
- 239000012071 phase Substances 0.000 abstract description 15
- 239000002253 acid Substances 0.000 abstract description 5
- 239000012074 organic phase Substances 0.000 abstract description 5
- 238000010812 external standard method Methods 0.000 abstract description 3
- 238000003908 quality control method Methods 0.000 abstract description 3
- 230000001105 regulatory effect Effects 0.000 abstract 2
- 239000002904 solvent Substances 0.000 description 21
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- 239000011550 stock solution Substances 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 5
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 description 4
- OUCSEDFVYPBLLF-KAYWLYCHSA-N 5-(4-fluorophenyl)-1-[2-[(2r,4r)-4-hydroxy-6-oxooxan-2-yl]ethyl]-n,4-diphenyl-2-propan-2-ylpyrrole-3-carboxamide Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@H]2OC(=O)C[C@H](O)C2)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 OUCSEDFVYPBLLF-KAYWLYCHSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 210000001072 colon Anatomy 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 1
- 230000003266 anti-allergic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 150000001875 compounds Chemical group 0.000 description 1
- 229940088679 drug related substance Drugs 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 206010023332 keratitis Diseases 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000011003 system suitability test Methods 0.000 description 1
- 238000012795 verification Methods 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
-
- 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 Analysing Biological Materials (AREA)
Abstract
The invention relates to the field of analytical chemistry, in particular to a method for measuring impurity substances of loteprednol etabonate bulk drug by utilizing a high performance liquid chromatography. According to the method, the chromatographic column is accurately screened, the types of acid added to the mobile phase are accurately regulated, the types and the proportion of the organic phase in the mobile phase and the gradient elution conditions are accurately regulated, the effective separation of impurities A, B, C, D, E, F, G, H, I, J, K, M, N, O is realized, the impurities are accurately quantified by using a main component external standard method added with correction factors, the correction factors are accurately measured, and the impurity substances of loteprednol etabonate can be simultaneously, rapidly and effectively monitored, so that the quality control of the loteprednol etabonate bulk drug is improved.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for measuring impurity substances of loteprednol etabonate bulk drug by utilizing a high performance liquid chromatography.
Background
Loteprednol etabonate is a novel glucocorticoid medicine, has the functions of resisting keratitis, various intraocular inflammations and antiallergic, can be rapidly metabolized into inactive products in vivo, has high fat solubility, can enhance the permeability to cells, has low toxicity and strong anti-inflammatory effect, and is widely applied to the treatment of ocular inflammations.
Loteprednol etabonate chemical name 17α- (ethoxycarbonyloxy) -11β-hydroxy-3-oxoandrosta-1, 4-diene-17-carboxylic acid chloromethyl ester having the formula:
in the production and storage process of the loteprednol etabonate bulk drug, starting material impurities, process impurity intermediate impurities, degradation impurities and the like are easy to introduce, the impurities of the loteprednol etabonate bulk drug and the detection method thereof are not carried in main stream pharmacopoeias such as Chinese pharmacopoeias, united states pharmacopoeias and European pharmacopoeias, the impurities of the bulk drug are not comprehensively analyzed in the literature at home and abroad, the impurity content in a product is strictly controlled in order to better ensure the product quality, the impurities introduced by the applicant from the bulk drug, byproducts generated in the production process and impurities generated by product degradation evaluate the impurity spectrum of the bulk drug, and the impurity spectrum of the loteprednol etabonate bulk drug is determined as follows:
；
；
；
。
the prior art does not provide the same detection method, and can accurately and quantitatively detect the impurity A, B, C, D, E, F, G, H, I, J, K, M, N, O of the loteprednol etabonate raw material medicine at the same time, so that the loteprednol etabonate raw material medicine can be controlled better in quality, and the technical problem which is not solved by the person skilled in the art is solved. It is therefore highly desirable to develop a method for detecting loteprednol etabonate raw material and its impurity substances which solves the above-mentioned problems.
Disclosure of Invention
The invention aims to control known impurities in loteprednol etabonate by using a high performance liquid chromatography, the method accurately adjusts the types of acid added to a mobile phase by accurately screening a chromatographic column, accurately adjusts the types and the proportion of an organic phase in the mobile phase and gradient elution conditions, realizes effective separation of impurities A, B, C, D, E, F, G, H, I, J, K, M, N, O, accurately measures correction factors, accurately and quantitatively analyzes the impurities by using a main component external standard method added with the correction factors, and can simultaneously, rapidly and effectively monitor impurity substances in bulk drugs, thereby improving the quality control of the loteprednol etabonate bulk drugs.
The impurity in the impurity substance detection method of the present invention includes impurity A, B, C, D, E, F, G, H, I, J, K, M, N, O, in particular, impurity C, D, E, F, H, I, M, N.
The chromatographic column is a core-shell column which is obtained by accurate screening and takes phenylhexyl silane bonded silica gel as a filler, and compared with a full-porous chromatographic column used in the prior art, the chromatographic column has excellent chromatographic separation degree, higher peak capacity and higher sensitivity, so that all impurities can be completely eluted within 50 minutes, and the effective separation can be realized, thereby greatly accelerating the analysis speed.
The types and the proportion of the organic phase in the mobile phase are acetonitrile-methanol (the volume ratio is 85:15), the mixed solution of acetonitrile and methanol is determined to be used as the mobile phase B according to the tiny difference between different compound structures and the difference reserved in different organic phases, and the volume ratio of acetonitrile to methanol is determined to be 85:15.
The invention is realized by the following technical scheme: a method for measuring loteprednol etabonate bulk drug substances by using high performance liquid chromatography comprises the following steps:
step one: configuration System applicability solution
Taking loteprednol etabonate reference substance and impurity D reference substance, adding acetonitrile-water with the volume ratio of 75:25 to dissolve and quantitatively diluting to prepare a mixed solution containing 0.3mg of loteprednol etabonate and 0.5 mug of impurity D in each 1 ml;
step two: preparing a sample solution
Taking loteprednol etabonate bulk drug, precisely weighing, adding acetonitrile-water with the volume ratio of 75:25 for dissolution and quantitatively diluting to prepare a solution containing 0.3mg of loteprednol etabonate bulk drug in each 1 ml;
step three: preparing reference substance solution
Taking loteprednol etabonate reference substance, precisely weighing, adding acetonitrile-water with the volume ratio of 75:25 for dissolution and quantitatively diluting to prepare a solution containing 0.3 mug loteprednol etabonate reference substance in each 1 ml;
step four: preparing a sensitivity solution
Precisely measuring the reference substance solution, quantitatively diluting with acetonitrile-water with the volume ratio of 75:25 to prepare a solution containing 0.15 mug of loteprednol etabonate reference substance in each 1 ml;
step five: chromatographic condition and System applicability test
A core-shell column using phenylhexyl silane bonded silica gel as a filler; taking 0.09% -0.11% phosphoric acid solution as a mobile phase A and acetonitrile-methanol (volume ratio is 85:15) as a mobile phase B; linear gradient elution was performed as follows; the flow rate is 1.0ml per minute; the column temperature is 25-35 ℃; the detection wavelength is 244nm; the sample injection volume is 10 μl; the temperature of the sample injector is 5+/-2 ℃; precisely measuring the applicability solution and the sensitivity solution of the system, respectively injecting into a liquid chromatograph, and recording a chromatogram;
；
step six: assay
Precisely measuring the solution of the sample and the solution of the reference substance, respectively injecting into a liquid chromatograph, and recording chromatograms; the detector is an ultraviolet detector; the calculation mode of the impurity A, B, C, D, E, F, G, H, I, J, K, M, N, O is defined as a main component self-comparison method added with correction factors, a regression line of the main component concentration and the impurity concentration to the peak area is drawn, the correction factors are obtained according to the ratio of the slope of the main component regression line to the slope of the impurity regression line, the correction factors are loaded into the impurity substance detection method of the variety, the actual measurement peak area of the impurities is corrected, the impurities are positioned by adopting relative retention time, and the values are loaded into the impurity substance detection method of the method together, so that the quantitative accuracy of the impurities reaches 90% -108%;
the relative retention times and correction factors for the components are shown in the following table:
；
the calculation formula is as follows:
；
；
wherein: a is that Sample Peak area of corresponding impurity in the sample solution;
A for a pair of The peak area of the main peak in the reference substance solution;
C for a pair of The concentration (mg/ml) of loteprednol etabonate in the control solution;
C sample Is the concentration (mg/ml) of the sample solution;
fcorrection factors for each impurity.
Preferably, in step five, the chromatographic column is Phenomenex Kinetex ® Phenyl-Hexyl (4.6mm.times.150mm, 2.6 μm) or a column with comparable performance.
Preferably, in step five, the concentration of the phosphoric acid solution is 0.10%.
Preferably, in step five, the column temperature is 30 ℃.
Preferably, in the fifth step, in the system applicability solution chromatogram, the separation degree between loteprednol etabonate and the impurity D peak is not less than 2.0; in the sensitivity solution chromatogram, the signal-to-noise ratio of the main component chromatogram peak height is not less than 10.
The types of acid added to the mobile phase are 0.09% -0.11% phosphoric acid solution, and compared with other acids (such as glacial acetic acid), the phosphoric acid has the advantages of lower ultraviolet background, lower background noise and higher sensitivity, as shown in figure 7.
The method is determined by high performance liquid chromatography (China pharmacopoeia 2020 edition four general rules 0512). And (5) operating in a dark place.
Compared with the prior art, the invention has the following advantages:
according to the invention, through accurately screening chromatographic columns, accurately adjusting the types of acid added to a mobile phase, accurately adjusting the types and the proportion of an organic phase in the mobile phase and gradient elution conditions, effective separation of impurities A, B, C, D, E, F, G, H, I, J, K, M, N, O is realized, the impurities are accurately quantified by using a main component external standard method added with correction factors, and the correction factors are accurately measured, so that the impurity substances of loteprednol etabonate can be simultaneously, rapidly and effectively monitored, and the quality control of the loteprednol etabonate bulk drug is improved;
drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a hollow white solvent chromatogram of example 2;
FIG. 2 is a chromatogram of the system applicability solution of example 2;
FIG. 3 is a chromatogram of the sensitivity solution in example 2;
FIG. 4 is a chromatogram of the control solution in example 2;
FIG. 5 is a chromatogram of the sample solution in example 2;
FIG. 6 is a chromatogram of the mixed solution in example 2;
FIG. 7 is a baseline noise plot of phosphoric acid and glacial acetic acid.
Detailed Description
The invention will be described in further detail with reference to the drawings and the specific examples.
The instrument, reagent, and model and manufacturer information of the reference substance used in the embodiment of the invention are as follows:
instrument: siemens flying Multimate 3000 high performance liquid chromatograph
Reagent: phosphoric acid (chromatographic purity, fisher)
Acetonitrile (chromatographic purity, chengdu Colon)
Methanol (chromatographic purity, chengdu Colon)
Control: loteprednol etabonate (99.6%, curia Spain S.A.U.)
Impurity A (98.70%, guangzhou meaning bath)
Impurity B (91.3%, TLC)
Impurity C (99.5%, TLC)
Impurity D (98.9%, TLC)
Impurity E (95.3%, TLC)
Impurity F (97.9%, TLC)
Impurity G (99.5%, TLC)
Impurity H (95.4%, TLC)
Impurity I (96.65%, guangzhou meaning bath)
Impurity J (99.8%, TLC)
Impurity K (98.9%, TLC)
Impurity M (98.8%, TLC)
Impurity N (98.9%, TLC)
Impurity O (99.1%, TLC).
Example 1
Experimental procedure
Step one: configuration System applicability solution
Taking a loteprednol etabonate reference substance and an impurity D reference substance, adding acetonitrile-water with the volume ratio of 75:25 to dissolve and quantitatively dilute to prepare a mixed solution containing 0.3mg of loteprednol etabonate and 0.5 mug of impurity D in each 1 ml;
step two: preparing a sample solution
Taking the product, precisely weighing the loteprednol etabonate bulk drug, adding acetonitrile-water with the volume ratio of 75:25 to dissolve and quantitatively diluting to prepare a solution containing 0.3mg of loteprednol etabonate bulk drug in each 1 ml;
step three: preparing reference substance solution
Taking loteprednol etabonate reference substance, precisely weighing, adding acetonitrile-water with the volume ratio of 75:25 for dissolving and quantitatively diluting to prepare a solution containing 0.3 mug loteprednol etabonate reference substance in each 1 ml;
step four: preparing a sensitivity solution
Precisely measuring the reference substance solution, quantitatively diluting with acetonitrile-water (75:25) to prepare a solution containing 0.15 mu g of loteprednol etabonate reference substance in each 1 ml;
step five: chromatographic condition and System applicability test
Core-shell column (Phenomenex Kinetex) with phenylhexyl silane bonded silica gel as filler ® Phenyl-Hexyl4.6 mm. Times.150 mm,2.6 μm); taking 0.09% -0.11% phosphoric acid solution as a mobile phase A, optimally 0.1%, and acetonitrile-methanol with the volume ratio of 85:15 as a mobile phase B; linear gradient elution was performed as follows; the flow rate is 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength is 244nm; the sample injection volume is 10 μl; the temperature of the sample injector is 5+/-2 ℃; the separation degree between the loteprednol etabonate and the impurity D peak is not less than 2.0; in the sensitivity solution chromatogram, the signal-to-noise ratio of the main component chromatogram peak height is not less than 10;
；
step six: assay
Precisely measuring the solution of the sample and the solution of the reference substance, respectively injecting into a liquid chromatograph, and recording chromatograms; the detector is an ultraviolet detector; the calculation mode of the impurity A, B, C, D, E, F, G, H, I, J, K, M, N, O is defined as a main component self-comparison method added with correction factors, a regression line of the main component concentration and the impurity concentration to the peak area is drawn, the correction factors are obtained according to the ratio of the slope of the main component regression line to the slope of the impurity regression line, the correction factors are loaded into the impurity substance detection method of the variety, the actual measurement peak area of the impurities is corrected, the impurities are positioned by adopting relative retention time, and the values are loaded into the impurity substance detection method of the method together, so that the quantitative accuracy of the impurities reaches 90% -108%;
the relative retention times and correction factors for the components are shown in the following table:
；
the calculation formula is as follows:
；
；
wherein: a is that Sample Peak area of corresponding impurity in the sample solution;
A for a pair of The peak area of the main peak in the reference substance solution;
C for a pair of The concentration (mg/ml) of loteprednol etabonate in the control solution;
C sample Is the concentration (mg/ml) of the sample solution;
fcorrection factors for each impurity.
The method is determined by high performance liquid chromatography (China pharmacopoeia 2020 edition four general rules 0512). And (5) operating in a dark place.
Example 2
Experimental procedure
Chromatographic column: core-shell column (Phenomenex Kinetex) with phenylhexyl silane bonded silica gel as filler ® Phenyl-Hexyl 4.6mm×150mm，2.6μm）；
Taking 0.1% phosphoric acid solution as a mobile phase A, acetonitrile-methanol (85:15) as a mobile phase B,
linear gradient elution was performed as follows; the flow rate is 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength is 244nm; the sample injection volume is 10 μl; the temperature of the sample injector is 5 ℃;
；
solvent: acetonitrile-water (75:25).
Sensitivity solution: taking a proper amount of loteprednol etabonate reference substance, precisely weighing, dissolving in a solvent, and quantitatively diluting to prepare a solution containing 0.15 mu g of loteprednol etabonate reference substance in 1 ml.
System applicability solution: taking proper amounts of loteprednol etabonate reference substance and impurity D reference substance, dissolving with solvent, and diluting to obtain solution containing loteprednol etabonate 0.3mg and impurity D0.5 μg per 1 ml.
Control solution: taking a proper amount of loteprednol etabonate reference substance, precisely weighing, dissolving with a solvent, diluting to prepare a solution containing a proper amount of loteprednol etabonate reference substance of 0.3 mug per 1ml, and shaking uniformly.
Test solution: taking 15mg of loteprednol etabonate, precisely weighing, placing into a 50ml measuring flask, dissolving with a solvent, diluting to a scale, and shaking uniformly.
Loteprednol etabonate positioning solution: taking 15mg of loteprednol etabonate reference substance, placing in a 50ml measuring flask, dissolving with solvent, diluting to scale, and shaking.
Impurity A, B, C, D, E, F, G, H, I, J, K, M, N, O localization solution: weighing impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, impurity N and impurity O respectively, dissolving in solvent, and diluting to obtain solutions containing 10 μg in each 1 ml.
Mixing solution: taking proper amounts of loteprednol etabonate, taking proper amounts of impurity positioning solutions, dissolving in a solvent, and diluting to prepare solutions containing 0.3mg of loteprednol etabonate, wherein each 1ml of the solutions contains 0.5 mug of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, impurity N and impurity O.
And respectively injecting a blank solvent, a system applicability solution, a sensitivity solution, a reference substance solution, a test sample solution, a loteprednol etabonate positioning solution, each impurity positioning solution and a mixed solution into a liquid chromatograph, analyzing according to the chromatographic conditions, and recording a chromatogram. The results are shown in the accompanying figures 1-6.
FIG. 1 is a chromatogram of a blank solvent that does not interfere with detection of various impurities;
FIG. 2 is a chromatogram of a system-applicable solution with retention time 13.705 min peak of loteprednol etabonate, retention time 14.380 min peak of impurity D, and separation degree of the two of 2.87 and 2.0, and good system applicability;
FIG. 3 is a chromatogram of a sensitive solution, wherein the signal to noise ratio of the peak height of the main component is 39.5, which is more than 10, and the sensitivity is good;
FIG. 4 is a chromatogram of a control solution with a retention time of 13.712 minutes with a peak of loteprednol etabonate;
FIG. 5 is a chromatogram of a sample solution, wherein the separation degree between detected impurities and between a main component peak and known impurity peaks is equal to or greater than 1.5, the separation degree is good, and the impurity detection capability is high; the test results of the loteprednol etabonate raw material medicine are shown in the following table:
fig. 6 shows a chromatogram of a mixed solution, wherein impurities a, B, C, D, E, F, G, H, I, J, K, M, N, and O can be effectively separated (the separation degree is greater than 1.5) under gradient elution conditions.
The results of the positioning test of the loteprednol etabonate main component and each impurity are shown in the following table:
example 3 durability test
The difference from example 2 is that: the durability inspection conditions were different.
Solvent: acetonitrile-water (75:25).
Sensitivity solution: taking a proper amount of loteprednol etabonate reference substance, precisely weighing, dissolving in a solvent, and quantitatively diluting to prepare a solution containing 0.15 mu g of loteprednol etabonate reference substance in 1 ml.
System applicability solution: taking proper amounts of loteprednol etabonate reference substance and impurity D reference substance, dissolving with solvent, and diluting to obtain solution containing loteprednol etabonate 0.3mg and impurity D0.5 μg per 1 ml.
Control solution: taking a proper amount of loteprednol etabonate reference substance, dissolving the loteprednol etabonate reference substance with a solvent, and quantitatively diluting to prepare a solution containing 0.3 mu g of loteprednol etabonate reference substance in each 1 ml.
Test solution: taking 15mg of loteprednol etabonate, placing in a 50ml measuring flask, dissolving with a solvent, diluting to a scale, and shaking uniformly.
Precisely measuring 10 μl of each solution, injecting into a liquid chromatograph under standard conditions, phosphoric acid water concentration + -10%, column temperature change + -5deg.C and different batch chromatographic column conditions of the same model in the examples, and recording chromatogram. In this example, the standard conditions for column 2 and column 1 are the same, and only the column model is changed.
TABLE 1 sensitivity test results
Table 2 results of System suitability test
TABLE 3 comparison of impurity detection amount results
Conclusion: under various chromatographic conditions, the minimum separation degree between loteprednol etabonate and an impurity D peak in a system applicability solution is 2.73 and is more than 2.0; in the sensitivity solution chromatogram, the minimum signal-to-noise ratio of the peak height of the main component chromatogram peak is 39.5, which is more than 10; in the sample solution, the number of impurities is the same, the impurities A, H, N, I, D, M and O are not detected, the RSD of other known impurity detection contents is less than 30%, the RSD of other maximum single impurity detection contents is 5.00%, less than 30%, and the RSD value of the total impurities is 2.43% and less than 20%.
In conclusion, the fine tuning chromatographic conditions (phosphoric acid water concentration.+ -. 10%, column temperature change.+ -. 5 ℃ and different lot number chromatographic columns) are excellent in durability.
The invention carries out linear verification on the analysis method for effectively separating the impurity A, B, C, D, E, F, G, H, I, J, K, M, N, O in loteprednol etabonate:
solvent: acetonitrile-water (75:25).
Impurity control stock solution (1): taking the appropriate amounts of the reference substances of the impurity A, the impurity B, the impurity C, the impurity E, the impurity F and the impurity G, precisely weighing, respectively adding solvents for dissolving and quantitatively diluting to prepare solutions with the concentration of 0.045mg in each 1 ml.
Impurity control stock solution (2): taking the right amounts of the reference substances of the impurity H, the impurity I, the impurity J, the impurity K, the impurity M, the impurity N and the impurity O, precisely weighing, respectively adding a solvent for dissolving and quantitatively diluting to prepare solutions containing 0.03mg of each 1 ml.
Impurity D control stock solution: taking a proper amount of impurity D reference substance, precisely weighing, adding acetonitrile for dissolving and quantitatively diluting to prepare a solution containing 0.045mg of each 1 ml.
Impurity control linear stock solution: precisely measuring the above impurity reference substance stock solution (1), impurity reference substance stock solution (2) and impurity D reference substance stock solution 10ml each, placing into 100ml measuring flask, fixing volume to scale with solvent, and shaking.
Loteprednol etabonate linear stock: precisely measuring 10ml of loteprednol etabonate reference stock solution, placing into a 100ml measuring flask, fixing volume to scale with solvent, and shaking.
Linear relation solution: the respective right amounts of the impurity reference substance linear stock solution and the loteprednol etabonate linear stock solution are precisely measured and placed in the same measuring flask, and the specific preparation modes are shown in the following table and are respectively used as linear relation solutions 1, 2, 3, 4 and 5.
TABLE 4 analysis method of loteprednol etabonate impurity substance verification-Linear relationship solution preparation method
TABLE 5 loteprednol etabonate impurity substance analysis method validation-Linear test results
Correction factor
The calculation formula is as follows:
correction factor = principal component slope/impurity slope
The correction factor calculation results are shown in Table 6.
TABLE 6 analysis method of loteprednol etabonate impurity substance-test results of various impurity correction factors
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Claims (3)
1. A method for measuring impurity substances of loteprednol etabonate bulk drug by using high performance liquid chromatography is characterized in that: the method comprises the following steps:
step one: configuration System applicability solution
Taking loteprednol etabonate reference substance and impurity D-17 alpha- (ethoxycarbonyloxy) -11 beta-hydroxy-3-oxo-androstane-1, 4-diene-17-carboxylic acid (ethylcarbonic acid) anhydride reference substance, adding acetonitrile-water with the volume ratio of 75:25 to dissolve and quantitatively diluting to prepare a mixed solution containing 0.3mg of loteprednol etabonate and 0.5 mug of impurity D in each 1 ml;
step two: preparing a sample solution
Taking loteprednol etabonate bulk drug, precisely weighing, adding acetonitrile-water with the volume ratio of 75:25 for dissolution and quantitatively diluting to prepare a solution containing 0.3mg of loteprednol etabonate bulk drug in each 1 ml;
step three: preparing reference substance solution
Taking loteprednol etabonate reference substance, precisely weighing, adding acetonitrile-water with the volume ratio of 75:25 for dissolution and quantitatively diluting to prepare a solution containing 0.3 mug loteprednol etabonate reference substance in each 1 ml;
step four: preparing a sensitivity solution
Precisely measuring the solution amount of the reference substance, and quantitatively diluting with acetonitrile-water with the volume ratio of 75:25 to prepare a solution containing 0.15 mug of loteprednol etabonate reference substance in each 1 ml;
step five: chromatographic condition and System applicability test
A core-shell column using phenylhexyl silane bonded silica gel as a filler; taking 0.09% -0.11% phosphoric acid solution as a mobile phase A, and acetonitrile-methanol with the volume ratio of 85:15 as a mobile phase B; linear gradient elution was performed as follows; the flow rate is 1.0ml per minute; the column temperature is 25-35 ℃; the detection wavelength is 244nm; the sample injection volume is 10 μl; the temperature of the sample injector is 5+/-2 ℃; precisely measuring the applicability solution and the sensitivity solution of the system, respectively injecting into a liquid chromatograph, and recording a chromatogram;
step six: assay
Precisely measuring the solution of the sample and the solution of the reference substance, respectively injecting into a liquid chromatograph, and recording chromatograms; the detector is an ultraviolet detector; drawing a regression line of the main component concentration and the impurity concentration to the peak area of the main component concentration, obtaining a correction factor according to the ratio of the slope of the main component regression line to the slope of the impurity regression line, and loading the correction factor into an impurity substance detection method in the product for correcting the actual measurement peak area of the impurity; the relative retention times and correction factors for the components are shown in the following table:
the calculation formula is as follows:
total impurities (%) = Σimpurities (%);
wherein: a is that Sample Peak area of corresponding impurity in the sample solution;
A for a pair of The peak area of the main peak in the reference substance solution;
C for a pair of The concentration of loteprednol etabonate in the reference substance solution is mg/ml;
C sample Is the concentration mg/ml of the sample solution;
f is the correction factor of each impurity.
2. The method for determining the impurity substances of loteprednol etabonate pharmaceutical raw material by using high performance liquid chromatography according to claim 1, wherein the method comprises the following steps: in the fifth step, in a system applicability solution chromatogram, the separation degree between loteprednol etabonate and an impurity D peak is not less than 2.0; in the sensitivity solution chromatogram, the signal-to-noise ratio of the main component chromatogram peak height is not less than 10.
3. The method for determining the impurity substances of loteprednol etabonate pharmaceutical raw material by using high performance liquid chromatography according to claim 1, wherein the method comprises the following steps: in the fifth step, the chromatographic column is PhenomenexPhenyl-Hexyl 4.6mm.times.150mm, 2.6 μm, phosphoric acid solution concentration of 0.1%, column temperature of 30deg.C.
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