CN116124936B - Detection method for related substances of tafluprost - Google Patents
Detection method for related substances of tafluprost Download PDFInfo
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- CN116124936B CN116124936B CN202310009391.2A CN202310009391A CN116124936B CN 116124936 B CN116124936 B CN 116124936B CN 202310009391 A CN202310009391 A CN 202310009391A CN 116124936 B CN116124936 B CN 116124936B
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- 229960004458 tafluprost Drugs 0.000 title claims abstract description 60
- 238000001514 detection method Methods 0.000 title claims abstract description 44
- WSNODXPBBALQOF-VEJSHDCNSA-N tafluprost Chemical compound CC(C)OC(=O)CCC\C=C/C[C@H]1[C@@H](O)C[C@@H](O)[C@@H]1\C=C\C(F)(F)COC1=CC=CC=C1 WSNODXPBBALQOF-VEJSHDCNSA-N 0.000 title claims abstract description 42
- 239000000126 substance Substances 0.000 title claims abstract description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 82
- 239000000243 solution Substances 0.000 claims abstract description 68
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000013558 reference substance Substances 0.000 claims abstract description 20
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 15
- 239000007864 aqueous solution Substances 0.000 claims abstract description 9
- 239000012488 sample solution Substances 0.000 claims abstract description 9
- 239000002253 acid Substances 0.000 claims abstract description 8
- 239000000945 filler Substances 0.000 claims abstract description 8
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000003960 organic solvent Substances 0.000 claims abstract description 5
- 238000010828 elution Methods 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 25
- 239000000523 sample Substances 0.000 claims description 21
- 239000012528 membrane Substances 0.000 claims description 11
- 238000010790 dilution Methods 0.000 claims description 10
- 239000012895 dilution Substances 0.000 claims description 10
- -1 tafluprost carboxylic acid Chemical class 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 9
- 238000007865 diluting Methods 0.000 claims description 8
- 239000002245 particle Substances 0.000 claims description 7
- 239000000377 silicon dioxide Substances 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 238000004090 dissolution Methods 0.000 claims description 6
- 238000005516 engineering process Methods 0.000 claims description 6
- 239000005977 Ethylene Substances 0.000 claims description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 2
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 claims description 2
- 235000019253 formic acid Nutrition 0.000 claims description 2
- KIQXRQVVYTYYAZ-VKVYFNERSA-N (z)-7-[(1r,2r,3r,5s)-2-[(e)-3,3-difluoro-4-phenoxybut-1-enyl]-3,5-dihydroxycyclopentyl]hept-5-enoic acid Chemical compound OC(=O)CCC\C=C/C[C@H]1[C@@H](O)C[C@@H](O)[C@@H]1\C=C\C(F)(F)COC1=CC=CC=C1 KIQXRQVVYTYYAZ-VKVYFNERSA-N 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 10
- 239000000741 silica gel Substances 0.000 abstract 2
- 229910002027 silica gel Inorganic materials 0.000 abstract 2
- 239000012535 impurity Substances 0.000 description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 230000014759 maintenance of location Effects 0.000 description 18
- 238000004458 analytical method Methods 0.000 description 10
- 229920001450 Alpha-Cyclodextrin Polymers 0.000 description 9
- HFHDHCJBZVLPGP-RWMJIURBSA-N alpha-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO HFHDHCJBZVLPGP-RWMJIURBSA-N 0.000 description 9
- 229940043377 alpha-cyclodextrin Drugs 0.000 description 9
- 150000002500 ions Chemical class 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 7
- 239000003085 diluting agent Substances 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- QRVYABWJVXXOTN-UHFFFAOYSA-N 4-methylsulfanylbenzaldehyde Chemical compound CSC1=CC=C(C=O)C=C1 QRVYABWJVXXOTN-UHFFFAOYSA-N 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000004811 liquid chromatography Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 229920000858 Cyclodextrin Polymers 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 239000003889 eye drop Substances 0.000 description 4
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 4
- 150000003180 prostaglandins Chemical class 0.000 description 4
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 229960002368 travoprost Drugs 0.000 description 4
- MKPLKVHSHYCHOC-AHTXBMBWSA-N travoprost Chemical compound CC(C)OC(=O)CCC\C=C/C[C@H]1[C@@H](O)C[C@@H](O)[C@@H]1\C=C\[C@@H](O)COC1=CC=CC(C(F)(F)F)=C1 MKPLKVHSHYCHOC-AHTXBMBWSA-N 0.000 description 4
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000012088 reference solution Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000009079 Epoprostenol Receptors Human genes 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 1
- 206010030043 Ocular hypertension Diseases 0.000 description 1
- 206010030348 Open-Angle Glaucoma Diseases 0.000 description 1
- 108091006335 Prostaglandin I receptors Proteins 0.000 description 1
- BDAHDQGVJHDLHQ-UHFFFAOYSA-N [2-(1-hydroxycyclohexyl)phenyl]-phenylmethanone Chemical compound C=1C=CC=C(C(=O)C=2C=CC=CC=2)C=1C1(O)CCCCC1 BDAHDQGVJHDLHQ-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000004410 intraocular pressure Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- OJZYRQPMEIEQFC-UAWLTFRCSA-N limaprost Chemical compound CCCC[C@H](C)C[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1CCCC\C=C\C(O)=O OJZYRQPMEIEQFC-UAWLTFRCSA-N 0.000 description 1
- 229950009365 limaprost Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000005220 pharmaceutical analysis Methods 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000000018 receptor agonist Substances 0.000 description 1
- 229940044601 receptor agonist Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 238000001323 two-dimensional chromatography Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
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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)
- Engineering & Computer Science (AREA)
- Quality & Reliability (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a detection method of related substances of tafluprost, which uses phosphoric acid, water and acetonitrile to prepare a sample solution, a reference substance solution (a), a reference substance solution (B) and a system applicability solution, wherein chromatographic conditions adopt a bonded silica gel chromatographic column, octadecylsilane chemically bonded silica gel is selected as a filler, a mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is selected as a weak acid aqueous solution, the mobile phase B is selected as an acetonitrile organic solvent, and the volume ratio of the mobile phase A to the mobile phase B is 55:45-65:35 when a gradient elution program is carried out.
Description
Technical Field
The invention relates to the technical field of medicine analysis and detection, in particular to a detection method of related substances of tafluprost.
Background
Tafluprost (Tafluprost), one of the prostaglandins analogs, is a selective FP prostacyclin receptor agonist that reduces intraocular pressure by increasing aqueous outflow through the uveoscleral pathway. Tafluprost has the formula C 25 H 34 F 2 O 5 The molecular weight is 452.53, and the structural formula is as follows:
presently, tafluprost has been used in eye drop formulations for lowering elevated ocular pressure in open angle glaucoma and ocular hypertension patients. Whether the medicine is a bulk drug or a pharmaceutical preparation, the analysis and detection of related substances in the medicine are important analysis and detection items in the medicine quality research.
The disclosed method for detecting a substance related to tafluprost is a detection method specified in the standard for eye drop entrance registration of tafluprost (JX 20130135), and the detection method for a substance related specified in the standard comprises: preparing a system applicability solution 1, preparing a system applicability solution 2, preparing a test sample solution and preparing a control solution. The preparation method comprises the following specific steps:
preparation of System applicability solution 1: a. taking 0.29g of alpha-cyclodextrin, adding 3000mL of water to dissolve the alpha-cyclodextrin, taking 400mL of the alpha-cyclodextrin, adjusting the pH value to 2.5 by using a phosphoric acid solution (1 mL of phosphoric acid is dissolved in 199mL of water), adding 400mL of acetonitrile, and shaking uniformly to obtain a diluent; b. weighing a proper amount of 4- (methylthio) benzaldehyde, adding acetonitrile for dissolving and diluting to prepare a solution containing about 1mg of 4- (methylthio) benzaldehyde per 1 mL; c. measuring a proper amount of 4- (methylthio) benzaldehyde solution, and diluting the solution with the diluent prepared in the step a to prepare a solution with the concentration of about 0.15 mug in each 1mL to be used as a 4- (methylthio) benzaldehyde stock solution; d. taking 2.5mL of the product, placing the product in a 5mL measuring flask, adding 0.15mL of 1mol/L sodium hydroxide solution, standing at room temperature for 8 minutes, adding 0.15mL of 1mol/L hydrochloric acid solution for neutralization, standing and cooling, diluting to a scale (5 mL) by using a 4- (methylthio) benzaldehyde stock solution, and shaking uniformly.
Preparation of System applicability solution 2: 1-hydroxy cyclohexyl benzophenone is taken as proper amount, acetonitrile is added for dissolution and dilution to prepare a solution containing about 1mg of 1mL, proper amount is taken as well, mobile phase A is used for dilution to prepare a solution containing about 0.1 mug of 1 mL.
Preparing a test solution: e. taking 0.29g of alpha-cyclodextrin, adding 3000mL of water to dissolve the alpha-cyclodextrin, taking 400mL of the alpha-cyclodextrin, adjusting the pH value to 2.5 by using a phosphoric acid solution (1 mL of phosphoric acid is dissolved in 200mL of water), adding 400mL of acetonitrile, and shaking uniformly to obtain a diluent; f. taking a proper amount of tafluprost, diluting the tafluprost product by using the diluent prepared in the step e to prepare a solution containing 7.5 mug of tafluprost in 1mL, and shaking uniformly.
Control solutions were prepared: precisely measuring 1mL of the sample solution, placing the sample solution into a 100mL measuring flask, diluting to a scale by using a mobile phase A, and shaking uniformly.
Chromatographic conditions: octadecylsilane chemically bonded silica was used as a filler (Imtakt Cadenza CD-C18,4.6 mm. Times.250 mm,3 μm); 0.29g of a cyclodextrin solution [ alpha-cyclodextrin (alpha 25D: +148 degrees to +152 degrees; content: 97.0 percent or more) is taken, 3000mL of water is added to dissolve the cyclodextrin solution, 2600mL of phosphoric acid solution (1 mL of phosphoric acid is dissolved in 200mL of water) is taken as a mobile phase A, pH value is regulated to 4.5-acetonitrile (60:40), 0.29g of the cyclodextrin solution [ alpha-cyclodextrin (alpha 25D: +148 degrees to +152 degrees; content: 97.0 percent or more) is taken as a mobile phase B, 3000mL of water is added to dissolve the cyclodextrin solution, 2600mL of phosphoric acid solution (1 mL of phosphoric acid is dissolved in 200mL of water) is taken as a mobile phase B, and gradient elution is carried out according to the table 1; the flow rate is 1.0mL per minute; the column temperature was about 50 ℃; the detection wavelength is 220nm; the sample volume was 100. Mu.l.
TABLE 1 gradient elution procedure
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 55 | 0 | 100 |
| 75 | 0 | 100 |
| 75.1 | 100 | 0 |
The liquid chromatography used by the detection method specified in the standard for eye drop import of tafluprost (JX 20130135) is a separation and analysis technology, and is one of the most widely used analysis methods in the modern chromatographic technology, and the analysis and detection technology can be used in pharmaceutical analysis and biochemical analysis. Currently, liquid chromatography techniques have been widely used in the detection of prostaglandins analogs (e.g., travoprost, rimoprost, etc.).
Chinese patent application (CN 104297352 a) discloses a method for determining travoprost content and related substances, which uses an ultra-high performance liquid chromatograph, and employs a Aquity UPLC BEH phenyl chromatographic column; detecting by an ultraviolet detector, wherein the detection wavelength is 265nm-285nm; the mobile phase is composed of a phase A and a phase B, the phase A of the mobile phase is an aqueous solution with pH of 2.0-3.0 regulated by buffer salt, the phase B of the mobile phase is acetonitrile, and the mobile phase is measured by a gradient elution method, so that the content of travoprost in the travoprost eye drops and various related substances can be measured simultaneously.
Chinese patent application (CN 107607636 a) discloses a method for quantitatively determining prostaglandins in biological samples, which uses an ultra-high performance liquid chromatography-ion mobility-tandem mass spectrometry detection system to determine prostaglandins in biological samples; an ion mobility cell is additionally arranged between the high performance liquid chromatography and the mass spectrum; the ion mobility pool is arranged between the ion source of the mass spectrum and the first four-stage rod, and the interference of the limaprost in the plasma is further removed through the separation mode of the ion mobility, so that the separation difficulty of liquid chromatography is reduced, the two-dimensional chromatography is changed into one-dimensional chromatography, the sample analysis time is shortened, and the sample analysis flux is improved.
Disclosure of Invention
The inventors of the present application have found through studies that, using the detection method specified in the inlet registration standard (JX 20130135), both the mobile phase and the diluent contain ion-pair reagents, the ion-pair reagents are irreversibly damaged to the chromatographic column, and the column efficiency of the chromatographic column decreases faster with increasing number of injections than when using no ion-pair reagent as the mobile phase. In addition, the use of ion pair reagents requires more time in terms of column equilibration and column maintenance after the test is completed. Meanwhile, the detection method disclosed in the prior art adopts a self-comparison calculation mode, which means that the detection of a sample needs to prepare a sample solution and a self-comparison solution at the same time, the time is 180 minutes, the time is too long, and the detection method is particularly time-consuming in the aspect of verification of related substance methodology.
Aiming at the technical problems that a chromatographic column is extremely vulnerable to a mobile phase in the prior art, and the defects of complicated preparation process, long time consumption and the like of a system applicability solution, the invention provides a detection method of tafluprost related substances, which has the advantages of mild mobile phase, short running time and simple system applicability solution preparation.
In order to solve the technical problems, the invention provides a detection method of related substances of tafluprost, wherein chromatographic conditions adopt a chromatographic column with octadecylsilane chemically bonded silica as a filler, a mobile phase consists of a mobile phase A and a mobile phase B, wherein the mobile phase A is a weak acid aqueous solution, the mobile phase B is an acetonitrile organic solvent, and the volume ratio of the mobile phase A to the mobile phase B is 55:45-65:35 when a gradient elution program is carried out; the main impurities detected were tafluprost carboxylic acid, 5-trans tafluprost, 16F-form, 16E-diene.
According to certain embodiments of the invention, the gradient elution procedure is: the elution time is 0-10 minutes, and the volume ratio of the mobile phase A to the mobile phase B is 60:40; the elution time is 10-15 minutes, and the volume ratio of the mobile phase A to the mobile phase B is changed from 60:40 to 57:43; the elution time is 15-45 minutes, and the volume ratio of the mobile phase A to the mobile phase B is 57:43; the elution time is 45-45.1 minutes, and the volume ratio of the mobile phase A to the mobile phase B is changed from 57:43 to 60:40; the elution time is 45.1-50 minutes, and the volume ratio of the mobile phase A to the mobile phase B is 60:40.
According to certain embodiments of the invention, the filler of the octadecylsilane chemically bonded silica has a particle size of less than 2 μm.
According to certain embodiments of the invention, the weak acid aqueous solution is selected from one or more of phosphoric acid solution, phosphate solution, formic acid solution, trifluoroacetic acid solution, methanesulfonic acid solution, acetic acid solution.
According to certain embodiments of the invention, the detection wavelength in the gradient elution procedure is in the range of 208nm to 212nm.
According to certain embodiments of the invention, the gradient elution procedure has a column temperature in the range of 40 ℃ to 55 ℃.
According to certain embodiments of the invention, the temperature of the sample tray in the gradient elution procedure ranges from 5 ℃ to 15 ℃.
According to certain embodiments of the invention, the gradient elution procedure has a flow rate of 0.2 to 0.4mL per minute and a sample volume of 5. Mu.L.
According to some embodiments of the invention, the method comprises the steps of: taking a tafluprost sample, adding acetonitrile for dissolution and dilution, and filtering by using a filter membrane to prepare a sample solution; taking a tafluprost reference substance, adding acetonitrile for dissolution and dilution, and filtering by a filter membrane to prepare a reference substance solution (a); taking a reference substance solution (a), adding acetonitrile for dilution, and filtering by using a filter membrane to obtain a reference substance solution (b); taking a tafluprost reference substance, a tafluprost carboxylic acid reference substance and a 5-trans-tafluprost reference substance, respectively adding acetonitrile for dissolving and diluting, and filtering by using a filter membrane to prepare a system applicability solution.
According to certain embodiments of the invention, the filter pore size is 0.22 μm.
Compared with the prior art, the technical scheme of the embodiment of the invention has the following beneficial effects:
firstly, according to the detection method provided by the invention, the mobile phase and the diluent do not contain ion pair reagents, and the pH value does not need to be regulated, so that irreversible damage of the ion pair reagents to the chromatographic column is avoided. With the increase of the sample injection times, the column efficiency of the chromatographic column is not reduced by the detection method.
Secondly, the detection method provided by the invention has short time consumption no matter the balance and operation of the chromatographic column or the maintenance of the chromatographic column after the test is finished. According to the detection method disclosed by the prior art, the running time of the chromatographic column is 90 minutes, and according to the detection method provided by the invention, the running time of the gradient elution program is 50 minutes, so that the running time is greatly shortened.
According to the detection method provided by the invention, the solution of the sample and the self control solution do not need to be configured at the same time, the process of preparing the solution with the system applicability is simple, and the detection efficiency is improved.
Drawings
Fig. 1 is a liquid chromatogram obtained according to an embodiment of the present invention.
Fig. 2 is a graph of liquid chromatography obtained according to an embodiment of the present invention.
Detailed Description
Some embodiments according to the present invention will be described in more detail below with reference to the accompanying drawings. It is to be understood that other various embodiments can be devised and modifications to the embodiments by those skilled in the art based on the teachings herein without departing from the scope or spirit of this invention.
All numbers expressing feature sizes, amounts, and physical properties used in the specification and claims are to be understood as being modified by the term "about" unless otherwise indicated. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that can be varied by one of ordinary skill in the art to obtain the desired properties according to the teachings of the present invention. The recitation of numerical ranges by endpoints includes all numbers subsumed within that range including, for example, 1, 1.1, 1.3, 2, 2.75, 3, 3.84, 4, 5, etc. between 1 and 5.
Unless otherwise indicated, the starting materials used in the examples and comparative examples in this specification were commercial industrial products and were commercially available.
For drug analysis and detection by liquid chromatography, mobile phases and dilutions of ion pair-containing reagents are typically used, mobile phase a is typically a phosphate or formate buffer system, and in this application, a mixture of a weak acid aqueous solution (e.g., phosphoric acid) and acetonitrile is used directly without pH adjustment (mobile phase pH is around 2). In order to replace ion pair reagents (such as alpha-cyclodextrin) used in the prior art, the inventor of the application realizes the detection effect by changing technical means such as chromatographic conditions (filling agent of chromatographic column, mobile phase solution, gradient elution program) and the like, not only reduces damage caused by flowing relative to the chromatographic column, but also simplifies the preparation flow of solution with system applicability, shortens the running time and improves the detection efficiency.
The important principle of chromatography is that each substance to be detected is separated by a mobile phase, and chemical structure difference leads to obvious difference of physicochemical properties of the compound to be detected and the impurity to be detected due to different chemical structure difference. Thus, the difference in detection targets will directly affect the determination of chromatographic conditions, while parameters such as the shape, particle size, pore diameter, carbon content and bonding type, elution time, etc. of the filler are critical, and will directly affect the separation of the main components and impurities.
In the present application, the column uses BEH ethylene bridge hybrid particle technology (Ethylene Bridged Hybrid), octadecylsilane chemically bonded silica having a particle size of about 1.7 μm (Waters ACQUITY UPLC BEH C 18 Column) is a packing material, and the chromatographic column has the characteristics of peak symmetry, column effect and chemical stability.
Regarding the choice of mobile phase, comprehensive analysis is required based on the properties of the test sample, the polarity of the chromatographic column, and the like. ACQUITY UPLC BEH C in BEH ethylene bridge hybrid particle technology 18 The column is a reverse phase column, and the mobile phase can be prepared from weak acid aqueous solution with larger polarity and organic solvent with smaller polarity (such as acetonitrile and methanol) according to a certain proportion. However, methanol is highly reactive and may react with some samples, and methanol absorbs ultraviolet at low wavelengths, which reduces the sensitivity of the assay. Acetonitrile has stronger eluting power than methanol, less reaction with the sample, more stability in detection, the pressure of acetonitrile used as a mobile phase system is much lower than that of methanol, and the cut-off wavelength is 20nm lower than that of methanol, thereby increasingThe possibility of having absorbed impurities at low wavelengths is detected.
In the application, weak acid aqueous solution (such as phosphoric acid) with pH value of about 2 is selected as mobile phase A, acetonitrile organic solvent is selected as mobile phase B, and based on the compound to be separated, the inventor of the application designs a gradient elution time program according to the selected chromatographic column and mobile phase. In actual operation, the detection effect is affected by factors such as detection wavelength, elution flow rate, flow velocity, column temperature, sample tray temperature, sample injection volume and the like.
The range of detection wavelength variation is different due to the nature of the compound to be detected. The detection wavelength that is feasible is approximately in the range of 208nm to 212nm, since 210nm is already the (ultraviolet) end absorption. The larger the fluctuation, the more disturbing peaks of the impurities in the solvent (the closer the absorption spectrum wavelength of the impurities are).
The chromatographic column temperature is not too high, the temperature is too high, the chromatographic column tolerance problem is affected, the temperature is not too low and is not too high exceeding 55 ℃, the peak is late, the detection time is prolonged, and the temperature is not lower than 40 ℃.
Regarding the sample tray temperature, the tafluprost needs to be stored at a low temperature, and in order to meet the requirement of good stability of the compound (tafluprost), the sample tray temperature is lower than room temperature.
Regarding the flow rate, too large a flow rate may generate too high a pressure, not exceeding 0.4mL, and too small a flow rate may extend the detection time, not falling below 0.2mL.
Example 1
Preparation method
Preparing a test solution: the proper amount of the tafluprost product is taken, acetonitrile is added for dissolution and dilution to prepare a solution containing about 1.0mg of tafluprost in 1mL, and the solution is filtered by a 0.22 mu m filter membrane.
Preparing a reference solution (a): taking a proper amount of tafluprost reference substance, precisely weighing, adding acetonitrile for dissolving and diluting to prepare a solution containing about 1.0mg of tafluprost in 1mL, and filtering by a 0.22 mu m filter membrane.
Preparing a reference substance solution (b): a proper amount of the control solution (a) was precisely measured, acetonitrile was added to prepare a solution containing about 1. Mu.g of tafluprost per 1mL, and the solution was filtered through a 0.22 μm filter.
Preparing a system applicability solution: the tafluprost control, tafluprost carboxylic acid control and 5-trans tafluprost control were weighed separately and dissolved and diluted with acetonitrile to prepare solutions containing about 1.0mg of tafluprost per 1mL, 1.0 μg of each of tafluprost carboxylic acid and 5-trans tafluprost, and filtered through a 0.22 μm filter membrane.
Chromatographic conditions
Chromatographic column instrument: waters AQUITY UPLC.
Chromatographic column: octadecylsilane chemically bonded silica is used as filler (BEH C) 18 ，2.1mm×150mm，1.7μm)。
Mobile phase a: 2mL of a phosphoric acid solution of 0.0245% (w/v) was precisely measured, and water was added to 1000mL to prepare an aqueous phosphoric acid solution.
Mobile phase B: acetonitrile.
Gradient elution was performed according to table 2; the flow rate is 0.3ml per minute; column temperature is 50 ℃; the temperature of the sample tray is 10 ℃; the sample volume was 5. Mu.l.
TABLE 2 gradient elution procedure for example 1
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 60 | 40 |
| 10 | 60 | 40 |
| 15 | 57 | 43 |
| 45 | 57 | 43 |
| 45.1 | 60 | 40 |
| 50 | 60 | 40 |
System applicability requirements: precisely measuring the applicability solution of the system and the reference substance solution (a), respectively injecting into a liquid chromatograph, and recording a chromatogram; in the chromatogram of the reference substance solution (a), the theoretical plate number is not less than 3000 according to the tafluprost peak, the tailing factor is not more than 2.0, the sample is repeatedly injected for 5 times, and the tafluprost peak area RSD is not more than 2.0%; in the system applicability solution chromatogram, the separation degree between the tafluprost peak and the 5-trans tafluprost peak is not less than 2.0; in addition, the detection method of the present application has confirmed that the peak order of each impurity and tafluprost in the chromatogram obtained by the detection method of the present application is consistent with the corresponding peak order obtained by using the above-mentioned import registration standard method: as shown in FIG. 1, the second peak from the left corresponds to tafluprost carboxylic acid, the third peak corresponds to tafluprost, the fourth peak corresponds to 5-trans-tafluprost, the fifth peak corresponds to form 16F-and the sixth peak corresponds to 16E-diene.
A detector: an ultraviolet detector detects a wavelength of 210nm.
Assay: precisely measuring the sample solution and the reference solution (b), respectively injecting into a liquid chromatograph, and recording chromatograms, such as figure 1 and figure 2. In FIG. 1, "AU" means "chromatographic peak response value", "minerals" means "retention time unit (Minutes)". The relative retention time is the retention time of a single impurity divided by the main peak retention time. In FIG. 2, "Peak Results" means "Peak Results", "RT" means "retention time", "Area" means "Peak Area", "Height" means "Peak Height", "USP Resolution" means "USP Resolution", "USP" means "United states Pharmacopeia standard", "s/n" means "signal to noise ratio", i.e. the ratio of Peak signal to instrument noise Peak Height ".
Limit: the chromatogram of the sample solution contains impurity peaks, the peak area is calculated according to the external standard method of main components, the concentration of the tafluprost carboxylic acid, the 5-trans-tafluprost, the 16F-type and the 16E-diene is less than 0.10%, the concentration of other single impurities is less than 0.10%, the total impurity amount is less than 0.50%, and the relative retention time of each impurity peak is shown in table 3.
TABLE 3 relative retention times of the impurity peaks for example 1
| Impurity name | Relative retention time |
| Tafluprost carboxylic acid | About 0.18 |
| 5-trans-tafluprost | About 1.07 |
| 16F-type | About 1.15 |
| 16E-dienes | About 1.19 |
Example 2
The difference from example 1 is that: the flow rate was varied to 0.4ml per minute, the column temperature was 55℃and the other conditions were unchanged. The relative retention times of the impurities are shown in table 4.
TABLE 4 relative retention times of the impurity peaks for example 2
| Impurity name | Relative retention time |
| Tafluprost carboxylic acid | About 0.17 |
| 5-trans-tafluprost | About 1.05 |
| 16F-type | About 1.1 |
| 16E-dienes | About 1.15 |
Example 3
The difference from example 1 is that: the flow rate was changed to 0.2ml/min, the column temperature was 40℃and the other conditions were unchanged. The relative retention times of the impurities are shown in table 5.
TABLE 5 relative retention times of the impurity peaks for example 3
| Impurity name | Relative retention time |
| Tafluprost carboxylic acid | About 0.18 |
| 5-trans-tafluprost | About 1.07 |
| 16F-type | About 1.14 |
| 16E-dienes | About 1.18 |
In example 3, when the column temperature was changed to 55 ℃, the other conditions were not changed, the influence of the experimental results was relatively small, and the relative retention time of each impurity was almost unchanged, and the relative retention time of each impurity peak was within the error allowable range (2 bits after the decimal point) as shown in table 5 in example 3.
Example 4
The difference from example 1 is that: other conditions were unchanged when the initial ratio was changed to 55:45 and 45.1 minutes started to a regression value ratio of 55:45 as in table 6. The relative retention times of the impurities are shown in table 7.
TABLE 6 gradient elution procedure for example 4
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 55 | 45 |
| 10 | 55 | 45 |
| 15 | 57 | 43 |
| 45 | 57 | 43 |
| 45.1 | 55 | 45 |
| 50 | 55 | 45 |
TABLE 7 relative retention times of the impurity peaks for example 4
Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.
Claims (6)
1. A method for detecting a substance related to tafluprost, wherein,
the chromatographic conditions adopt a chromatographic column with octadecylsilane chemically bonded silica (BEH) ethylene bridge hybridized particle technology as a filler, a mobile phase consists of a mobile phase A and a mobile phase B, wherein the mobile phase A adopts weak acid aqueous solution, the mobile phase B adopts acetonitrile organic solvent, and when a gradient elution program is carried out, the gradient elution program is as follows:
the elution time is 0-10 minutes, and the volume ratio of the mobile phase A to the mobile phase B is 60:40;
the elution time is 10-15 minutes, and the volume ratio of the mobile phase A to the mobile phase B is changed from 60:40 to 57:43;
the elution time is 15-45 minutes, and the volume ratio of the mobile phase A to the mobile phase B is 57:43;
the elution time is 45-45.1 minutes, and the volume ratio of the mobile phase A to the mobile phase B is changed from 57:43 to 60:40;
the elution time is 45.1-50 minutes, and the volume ratio of the mobile phase A to the mobile phase B is 60:40;
in the gradient elution procedure, the detection wavelength range is 208 nm-212 nm;
the particle size of the octadecylsilane chemically bonded silica is smaller than 2 mu m;
the weak acid aqueous solution is selected from one or more of phosphoric acid solution, phosphate solution, formic acid solution, trifluoroacetic acid solution, methylsulfonic acid solution and acetic acid solution;
the related substances of the tafluprost are tafluprost acid, 5-trans-tafluprost, 16E-diene and 16F-type.
2. The method according to claim 1, wherein the column temperature in the gradient elution procedure is in the range of 40 ℃ to 55 ℃.
3. The method according to claim 1, wherein the temperature of the sample tray in the gradient elution procedure is in the range of 5 ℃ to 15 ℃.
4. The method according to claim 1, wherein in the gradient elution procedure, the flow rate is 0.2ml to 0.4ml per minute, and the sample injection volume is 5 μl.
5. The detection method according to claim 1, comprising the steps of:
taking a tafluprost sample, adding acetonitrile for dissolution and dilution, and filtering by using a filter membrane to prepare a sample solution;
taking a tafluprost reference substance, adding acetonitrile for dissolution and dilution, and filtering by a filter membrane to prepare a reference substance solution (a);
taking a reference substance solution (a), adding acetonitrile for dilution, and filtering by using a filter membrane to obtain a reference substance solution (b);
taking a tafluprost reference substance, a tafluprost carboxylic acid reference substance and a 5-trans-tafluprost reference substance, respectively adding acetonitrile for dissolving and diluting, and filtering by using a filter membrane to prepare a system applicability solution.
6. The method according to claim 5, wherein the pore size of the filter is 0.22. Mu.m.
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| WO2018033854A1 (en) * | 2016-08-15 | 2018-02-22 | Santen Pharmaceutical Co., Ltd. | Ophthalmic composition and a method for treating ocular hypertension and glaucoma |
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| WO2022162967A1 (en) * | 2021-01-27 | 2022-08-04 | Ａｇｃ株式会社 | Method for purifying tafluprost |
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| WO2018033854A1 (en) * | 2016-08-15 | 2018-02-22 | Santen Pharmaceutical Co., Ltd. | Ophthalmic composition and a method for treating ocular hypertension and glaucoma |
| CN109053452A (en) * | 2018-08-23 | 2018-12-21 | 扬子江药业集团有限公司 | A kind of preparation method of tafluprost bulk pharmaceutical chemicals |
| CN112209863A (en) * | 2020-07-07 | 2021-01-12 | 浙江尖峰药业有限公司 | Large-scale preparation method of tafluprost |
| WO2022162967A1 (en) * | 2021-01-27 | 2022-08-04 | Ａｇｃ株式会社 | Method for purifying tafluprost |
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