CN116223655A - UPLC determination method of tiotropium bromide spray - Google Patents
UPLC determination method of tiotropium bromide spray Download PDFInfo
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- CN116223655A CN116223655A CN202211720210.9A CN202211720210A CN116223655A CN 116223655 A CN116223655 A CN 116223655A CN 202211720210 A CN202211720210 A CN 202211720210A CN 116223655 A CN116223655 A CN 116223655A
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- tiotropium bromide
- performance liquid
- ultra
- spray
- mobile phase
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- LERNTVKEWCAPOY-VOGVJGKGSA-N C[N+]1(C)[C@H]2C[C@H](C[C@@H]1[C@H]1O[C@@H]21)OC(=O)C(O)(c1cccs1)c1cccs1 Chemical compound C[N+]1(C)[C@H]2C[C@H](C[C@@H]1[C@H]1O[C@@H]21)OC(=O)C(O)(c1cccs1)c1cccs1 LERNTVKEWCAPOY-VOGVJGKGSA-N 0.000 title claims abstract description 46
- 229960000257 tiotropium bromide Drugs 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 40
- 239000007921 spray Substances 0.000 title claims abstract description 30
- 238000004704 ultra performance liquid chromatography Methods 0.000 title claims abstract 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 29
- 239000000243 solution Substances 0.000 claims description 24
- 239000007788 liquid Substances 0.000 claims description 23
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 21
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 18
- 238000001514 detection method Methods 0.000 claims description 17
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 16
- 238000001195 ultra high performance liquid chromatography Methods 0.000 claims description 13
- 239000000945 filler Substances 0.000 claims description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
- 230000002441 reversible effect Effects 0.000 claims description 4
- 239000000741 silica gel Substances 0.000 claims description 4
- 229910002027 silica gel Inorganic materials 0.000 claims description 4
- 229940111688 monobasic potassium phosphate Drugs 0.000 claims description 3
- -1 octadecyl silica gel Chemical compound 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
- 239000012071 phase Substances 0.000 abstract description 17
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 12
- 150000002500 ions Chemical class 0.000 abstract description 7
- 239000007791 liquid phase Substances 0.000 abstract description 3
- 239000011859 microparticle Substances 0.000 abstract 1
- 239000012488 sample solution Substances 0.000 description 13
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 12
- 239000000523 sample Substances 0.000 description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 239000003085 diluting agent Substances 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 5
- REFMEZARFCPESH-UHFFFAOYSA-M sodium;heptane-1-sulfonate Chemical compound [Na+].CCCCCCCS([O-])(=O)=O REFMEZARFCPESH-UHFFFAOYSA-M 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- LERNTVKEWCAPOY-DZZGSBJMSA-N tiotropium Chemical compound O([C@H]1C[C@@H]2[N+]([C@H](C1)[C@@H]1[C@H]2O1)(C)C)C(=O)C(O)(C=1SC=CC=1)C1=CC=CS1 LERNTVKEWCAPOY-DZZGSBJMSA-N 0.000 description 3
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000013065 commercial product Substances 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000010812 external standard method Methods 0.000 description 2
- 230000002427 irreversible effect Effects 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
- BKIMMITUMNQMOS-UHFFFAOYSA-N nonane Chemical compound CCCCCCCCC BKIMMITUMNQMOS-UHFFFAOYSA-N 0.000 description 2
- FPLYNRPOIZEADP-UHFFFAOYSA-N octylsilane Chemical group CCCCCCCC[SiH3] FPLYNRPOIZEADP-UHFFFAOYSA-N 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- HRQDCDQDOPSGBR-UHFFFAOYSA-M sodium;octane-1-sulfonate Chemical compound [Na+].CCCCCCCCS([O-])(=O)=O HRQDCDQDOPSGBR-UHFFFAOYSA-M 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- VDZOOKBUILJEDG-UHFFFAOYSA-M tetrabutylammonium hydroxide Chemical compound [OH-].CCCC[N+](CCCC)(CCCC)CCCC VDZOOKBUILJEDG-UHFFFAOYSA-M 0.000 description 2
- 229940110309 tiotropium Drugs 0.000 description 2
- MQLXPRBEAHBZTK-SEINRUQRSA-M tiotropium bromide hydrate Chemical compound O.[Br-].C[N+]1(C)[C@H]2C[C@@H](C[C@@H]1[C@H]1O[C@@H]21)OC(=O)C(O)(c1cccs1)c1cccs1 MQLXPRBEAHBZTK-SEINRUQRSA-M 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000009863 impact test Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- CAAULPUQFIIOTL-UHFFFAOYSA-N methyl dihydrogen phosphate Chemical compound COP(O)(O)=O CAAULPUQFIIOTL-UHFFFAOYSA-N 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 229910052611 pyroxene Inorganic materials 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229940067741 sodium octyl sulfate Drugs 0.000 description 1
- QWSZRRAAFHGKCH-UHFFFAOYSA-M sodium;hexane-1-sulfonate Chemical compound [Na+].CCCCCCS([O-])(=O)=O QWSZRRAAFHGKCH-UHFFFAOYSA-M 0.000 description 1
- WFRKJMRGXGWHBM-UHFFFAOYSA-M sodium;octyl sulfate Chemical compound [Na+].CCCCCCCCOS([O-])(=O)=O WFRKJMRGXGWHBM-UHFFFAOYSA-M 0.000 description 1
- ROBLTDOHDSGGDT-UHFFFAOYSA-M sodium;pentane-1-sulfonate Chemical compound [Na+].CCCCCS([O-])(=O)=O ROBLTDOHDSGGDT-UHFFFAOYSA-M 0.000 description 1
- JRMUNVKIHCOMHV-UHFFFAOYSA-M tetrabutylammonium bromide Chemical compound [Br-].CCCC[N+](CCCC)(CCCC)CCCC JRMUNVKIHCOMHV-UHFFFAOYSA-M 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
-
- 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
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- 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
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
- G01N2030/3007—Control of physical parameters of the fluid carrier of temperature same temperature for whole column
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
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Abstract
The invention provides a method for measuring ultra-high performance liquid phase (UPLC) of a tiotropium bromide inhalation spray, in particular to micro-particles of the tiotropium bromide inhalation spray. The method can accurately measure the content of the tiotropium bromide in a remarkably short time, does not use an ion pair reagent, can better shorten the balance time between a mobile phase and a chromatographic column, and can prolong the service life of the chromatographic column.
Description
Technical Field
The invention relates to a method for measuring ultra-high performance liquid chromatography (UPLC) of a tiotropium bromide inhalation spray, in particular to micro-fine particles of the tiotropium bromide inhalation spray, and belongs to the technical field of medicines.
Background
Tiotropium bromide (Tiotropium bromide, 1) having the chemical name (1α,2β,4β,5α,7β) -7- [ (hydroxy-2-thiopheneacetyl) oxy]-9, 9-dimethyl-3-oxa-9-azoxyion tricyclo [3.3.1.0 2,4 ]Nonane bromide, developed by the company Boringer John, is sold with the company of pyroxene, and was first marketed in the Netherlands and Philippines at month 6 of 2002.
In addition, we know the detection method of control method 3: chromatographic column: column chromatography with octyl silane bonded silica gel as filler (LiChrospher 60RP Select B,125 ×4mm,5.0 μm); mobile phase: 0.18% sodium heptanesulfonate solution (1.64 g sodium heptanesulfonate, 1000ml water was added to dissolve, pH was adjusted to 3.0 with phosphoric acid): acetonitrile (700:300). Detection wavelength: 240nm, column temperature: 25 ℃, flow rate: 2.0ml/min, sample injection amount: 100 μl.
The method uses the sodium octane sulfonate which is an ion pair reagent and can improve the retention and peak shape of a substance to be detected, the retention time can be changed after replacement, and the retention and peak shape can be influenced by the selection of the concentration of the ion pair reagent. Of course, similar ion pair reagents are trifluoroacetic acid, tetrabutylammonium hydroxide, tetrabutylammonium bromide, sodium pentanesulfonate, sodium hexanesulfonate, sodium heptanesulfonate, and the like.
The method in reference 2 also adopts a Diode Array Detector (DAD), and compared with the ultraviolet detector conventionally used at present, the method has higher cost, lower popularity of manufacturers and is not beneficial to popularization of the method.
However, there are some problems in using ion-pair reagents, such as irreversible adsorption caused by combination of ion-pair reagents and stationary phase, difficulty in washing, increased dead volume of the column, advanced retention time, reduced theoretical plates, and poor separation. The adsorption process is irreversible, and even if the chromatographic column is washed sufficiently, the performance of the chromatographic column can be partially recovered, and the scrapping time of the chromatographic column is delayed. Meanwhile, the balance time of the ion pair reagent between the mobile phase and the chromatographic column is long, so that the working efficiency is reduced.
In view of this, the present invention has been made.
Disclosure of Invention
Aiming at the problems existing in the determination of the tiotropium bromide, the invention provides a brand new ultra-high performance liquid phase analysis method, which can accurately determine the content of the tiotropium bromide in a remarkably short time, does not use an ion pair reagent, can better shorten the balance time between a mobile phase and a chromatographic column, and can prolong the service life of the chromatographic column.
The analysis method provided by the invention has been validated according to the 2015 edition of Chinese pharmacopoeia, and comprises a system applicability test, a linear test, a recovery test and a specificity test. Through the verification, the analysis method provided by the invention is practical and reliable and has good stability.
A method for measuring tiotropium bromide spray by using an ultra-high performance liquid chromatograph, which comprises the following steps:
a. the chromatographic column adopts a reverse ultra-high performance liquid chromatographic column with bonded octadecyl silica gel as a filler;
b. methanol and potassium dihydrogen phosphate buffer solution are used as mobile phases, the concentration of the potassium dihydrogen phosphate solution is 0.2-0.4%, and the volume ratio of the mobile phases is that the potassium dihydrogen phosphate solution is methanol=65-75:35-25;
c. the detection wavelength is selected from the range of 230-240 nm.
The method for measuring the tiotropium bromide spray by adopting the ultra-high performance liquid chromatograph is characterized in that the concentration of the monopotassium phosphate solution in the mobile phase is 0.23-0.27%.
The method for measuring the tiotropium bromide spray by adopting the ultra-high performance liquid chromatograph is characterized in that the concentration of the monopotassium phosphate solution in the mobile phase is 0.25%.
The method for measuring the tiotropium bromide spray by adopting the ultra-high performance liquid chromatograph is characterized in that the PH value of the monobasic potassium phosphate solution in the mobile phase is 2.5-4.
The method for measuring the tiotropium bromide spray by adopting the ultra-high performance liquid chromatograph is characterized in that the volume ratio of the mobile phase is potassium dihydrogen phosphate solution, namely methanol=68-72:32-28.
The method for measuring the tiotropium bromide spray by adopting the ultra-high performance liquid chromatograph is characterized in that the volume ratio of the mobile phase is potassium dihydrogen phosphate solution, namely methanol=70:30.
The method for measuring the tiotropium bromide spray by adopting the ultra-high performance liquid chromatograph is characterized in that the detection wavelength is selected from the range of 234-236 nm.
The method for measuring the tiotropium bromide spray by adopting the ultra-high performance liquid chromatograph is characterized in that the flow rate of the mobile phase is 0.5ml/min.
The method for measuring the tiotropium bromide spray by adopting the ultra-high performance liquid chromatograph is characterized in that the column temperature is 25-30 ℃.
The method for measuring the tiotropium bromide spray by adopting the ultra-high performance liquid chromatograph is characterized in that the quantitative limit concentration of the tiotropium bromide is 4-5 ng.mL < -1 >.
The method for measuring the tiotropium bromide spray by adopting the ultra-high performance liquid chromatograph is characterized in that the reverse chromatographic column adopts a filler with the size of 1.7 mu m and has an ethylene bridge hybridization particle technology.
The method for measuring the tiotropium bromide spray by adopting the ultra-high performance liquid chromatograph is characterized in that the reverse chromatographic column ACQUITY
BEH C18 column.
Compared with the reported literature method, the ultra-high performance liquid phase method of the tiotropium bromide provided by the invention has the following advantages:
1. the detection time is effectively shortened, and the detection efficiency is improved;
2. the separation capacity of the system is enhanced, and the accuracy of the sample detection result is improved;
3. ion pair reagents are not used, so that pollution to chromatographic columns is reduced, and the service life of consumable materials is prolonged;
4. greatly reduces the dosage of organic solvent related to detection, saves cost and is environment-friendly.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a UPLC spectrum of an external standard solution;
FIG. 2-1 is a chart of a sample solution (1) of tiotropium bromide UPLC in example 1;
FIG. 2-2 is a chart of a UPLC sample solution (2) of tiotropium bromide in example 1;
FIGS. 2-3 are graphs of sample solutions (3) of tiotropium bromide UPLC in example 1;
FIG. 3 is a UPLC spectrum of tiotropium bromide in example 2;
FIG. 4-1 is a graph of sample solution (1) of tiotropium bromide in comparative example 1;
FIG. 4-2 is a graph of sample solution (2) of tiotropium bromide in comparative example 1;
FIGS. 4-3 are graphs of sample solutions (3) of tiotropium bromide in comparative example 1;
FIG. 5 is a graph of a specific assay in methodology where diluent does not affect the detection of a sample.
Detailed Description
The invention will be further described by way of the following examples, which are not intended to limit the scope of the invention in any way. It will be understood by those skilled in the art that equivalent substitutions and corresponding modifications to the technical features of the present invention are included within the scope of the present invention.
The same reagents and reagents are used in the following examples in the same lot. The sample of tiotropium bromide inhalation spray was purchased as a commercial product (Siderurgh, neigible, lot number: B53507).
A diluent: 25mg of disodium ethylenediamine tetraacetate is taken, 950ml of water is added for dissolution, 10ml of 1mol/L hydrochloric acid solution is added, the mixture is diluted to 1000ml with water, and the mixture is evenly mixed, thus obtaining the product.
Method for selecting a commercially available product as a sample solution to be passed through an inhalation device to obtain sample solutions at different locations:
the collecting cups are placed in the tray, the tray is arranged on the bottom support, and the corresponding positions of the collecting cups corresponding to the bottom support are guaranteed. Closing the cover, pulling down the handle, and sealing the instrument. An L-shaped connecting pipe is inserted into the inlet end of the striker, and a proper suction nozzle adapter is arranged at the other end of the L-shaped connecting pipe. After the inhalation device of the commercial product is inserted, the end of the suction nozzle is on the horizontal axis of the L-shaped connecting pipe, and the port of the suction nozzle is flush with the opening of the L-shaped connecting pipe. The direction of placement of the inhalation device should be consistent with the actual direction of use. The outlet of the impactor is connected with a vacuum pump, the gas flow rate is regulated to ensure that the gas flow rate at the inlet of the L-shaped connecting pipe is 30L/min (+ -5%), and the vacuum pump is closed.
Removing the impactor, taking down the L-shaped connecting pipe and the suction nozzle adapter, loosening the handle, opening the impactor, taking down the tray and the collecting cup together, and fully washing the device to be extracted with 10ml of diluent to form a sample solution.
Sample solutions (1), (2) and (3) are sample solutions with different concentrations extracted from the L-shaped connecting pipe, the suction nozzle adapter and the collecting cup respectively, and the final concentration is calculated by a UPLC external standard method.
The above manner was used for inventive example 1 and comparative example 1.
The following examples and comparative examples all employ external standard methods, and external standard preparation methods are as follows:
preparing an external standard solution: tiotropium bromide monohydrate (purity greater than 98%) was precisely weighed, dissolved in a diluent and diluted to a solution containing 0.125 μg of tiotropium per 1 ml.
Example 1
Chromatographic conditions:
chromatographic column: ACQUITY
BEH C18 column (2.1 mm. Times.50 mm,1.7 μm);
mobile phase: potassium dihydrogen phosphate buffer (2.5 g of potassium dihydrogen phosphate, 1000ml of water are added to dissolve, and pH is adjusted to 3.0 by phosphoric acid) -methanol (70:30);
detection wavelength: 235nm;
column temperature: 30 ℃;
flow rate: 0.5ml/min
Sample injection amount: 10 μl.
And (3) injecting the sample solution to be tested into an ultra-high performance liquid chromatograph, wherein the chromatograms are shown in figures 2-1, 2-2 and 2-3 of the specification.
Example 2
Preparing quantitative limiting solution: the tiotropium bromide monohydrate (purity greater than 98%) was taken in appropriate amounts, precisely weighed, and diluted with a diluent to a concentration of about 5ng/ml.
Chromatographic conditions:
chromatographic column: ACQUITY
BEH C18 column (2.1 mm. Times.50 mm,1.7 μm);
mobile phase: potassium dihydrogen phosphate buffer (2.5 g of potassium dihydrogen phosphate, 1000ml of water are added to dissolve, and pH is adjusted to 3.0 by phosphoric acid) -methanol (70:30);
detection wavelength: 235nm;
column temperature: 30 ℃;
flow rate: 0.5ml/min
Sample injection amount: 10 μl.
And (3) injecting the quantitative limiting solution into an ultra-high performance liquid chromatograph, wherein a chromatogram is shown in figure 3 of the specification.
| Sample name | Retention time | Substance name | S/N signal to noise ratio |
| Quantitative limiting solution | 1.070 | |
10 |
Comparative example 1
Chromatographic conditions:
chromatographic column: column chromatography with octyl silane bonded silica gel as filler (LiChrospher 60RP Select B,125 ×4mm,5.0 μm);
mobile phase: 0.18% sodium heptanesulfonate solution (1.64 g sodium heptanesulfonate, 1000ml water was added to dissolve, pH was adjusted to 3.0 with phosphoric acid): acetonitrile (700:300);
detection wavelength: 240nm;
column temperature: 25 ℃;
flow rate: 2.0ml/min;
sample injection amount: 100 μl.
And (3) injecting the sample solution to be tested into an ultra-high performance liquid chromatograph, wherein the chromatograms are shown in figures 4-1, 4-2 and 4-3 of the specification.
Impact test on chromatographic column:
in view of the difference in tray numbers between UPLC and HPLC columns, the following relative values after use of the new column are used.
Comparison of organic reagent dosage in different detection methods
By comparison, the method of the embodiment 1 can be used for detecting the sample of the tiotropium inhalation spray, so that the dosage of the organic solvent can be greatly reduced, the cost is saved, and the environment is friendly.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. A method for measuring tiotropium bromide spray by using an ultra-high performance liquid chromatograph, which comprises the following steps:
a. the chromatographic column adopts a reverse ultra-high performance liquid chromatographic column with bonded octadecyl silica gel as a filler;
b. methanol and potassium dihydrogen phosphate buffer solution are used as mobile phases, the concentration of the potassium dihydrogen phosphate solution is 0.2-0.4%, and the volume ratio of the mobile phases is that the potassium dihydrogen phosphate solution is methanol=65-75:35-25;
c. the detection wavelength is selected from the range of 230-240 nm.
2. The method for measuring tiotropium bromide spray by using an ultra performance liquid chromatograph according to claim 1, wherein the concentration of the monobasic potassium phosphate solution in the mobile phase is 0.23-0.27%.
3. The method for determining tiotropium bromide spray using ultra performance liquid chromatography according to claim 1, wherein the concentration of potassium dihydrogen phosphate solution in the mobile phase is 0.25%.
4. The method for determining tiotropium bromide spray using ultra high performance liquid chromatography according to claim 1, wherein the PH of the monobasic potassium phosphate solution in the mobile phase is 2.5-4.
5. The method for determining tiotropium bromide spray by using an ultra performance liquid chromatograph according to claim 1, wherein the mobile phase volume ratio is a potassium dihydrogen phosphate solution:
methanol=68-72:32-28.
6. The method for determining the tiotropium bromide spray by using an ultra performance liquid chromatograph according to claim 1, wherein the volume ratio of the mobile phase is potassium dihydrogen phosphate solution, methanol=70:30.
7. The method for determining the tiotropium bromide spray using an ultra performance liquid chromatograph according to claim 1, wherein the detection wavelength is selected from the range of 234-236 nm.
8. The method for determining tiotropium bromide spray using ultra high performance liquid chromatography according to claim 1, wherein the mobile phase flow rate is 0.5ml/min.
9. The method for determining tiotropium bromide spray using ultra high performance liquid chromatography according to claim 1, wherein the column temperature is 25-30 ℃.
10. The method for measuring the tiotropium bromide spray by using the ultra performance liquid chromatograph according to claim 1, wherein the quantitative limit concentration of the tiotropium bromide is 4-5 ng.mL < -1 >.
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