CN111624292A - Method for determining concentration of indacaterol glycopyrronium bromide in serum - Google Patents
Method for determining concentration of indacaterol glycopyrronium bromide in serum Download PDFInfo
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- 229960002462 glycopyrronium bromide Drugs 0.000 title claims abstract description 72
- 238000000034 method Methods 0.000 title claims abstract description 70
- QZZUEBNBZAPZLX-QFIPXVFZSA-N indacaterol Chemical compound N1C(=O)C=CC2=C1C(O)=CC=C2[C@@H](O)CNC1CC(C=C(C(=C2)CC)CC)=C2C1 QZZUEBNBZAPZLX-QFIPXVFZSA-N 0.000 title claims abstract description 60
- 229960004078 indacaterol Drugs 0.000 title claims abstract description 58
- 210000002966 serum Anatomy 0.000 title claims abstract description 43
- VPNYRYCIDCJBOM-UHFFFAOYSA-M Glycopyrronium bromide Chemical compound [Br-].C1[N+](C)(C)CCC1OC(=O)C(O)(C=1C=CC=CC=1)C1CCCC1 VPNYRYCIDCJBOM-UHFFFAOYSA-M 0.000 title claims abstract description 21
- 239000012224 working solution Substances 0.000 claims abstract description 63
- 239000000523 sample Substances 0.000 claims abstract description 59
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 37
- 239000011550 stock solution Substances 0.000 claims abstract description 37
- 229940015042 glycopyrrolate Drugs 0.000 claims abstract description 16
- 239000013062 quality control Sample Substances 0.000 claims abstract description 14
- 238000011002 quantification Methods 0.000 claims abstract description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 108
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 36
- ANGKOCUUWGHLCE-HKUYNNGSSA-N [(3s)-1,1-dimethylpyrrolidin-1-ium-3-yl] (2r)-2-cyclopentyl-2-hydroxy-2-phenylacetate Chemical compound C1[N+](C)(C)CC[C@@H]1OC(=O)[C@](O)(C=1C=CC=CC=1)C1CCCC1 ANGKOCUUWGHLCE-HKUYNNGSSA-N 0.000 claims description 36
- ANGKOCUUWGHLCE-UHFFFAOYSA-N 2-cyclopentyl-2-hydroxy-2-phenylacetic acid (1,1-dimethyl-3-pyrrolidin-1-iumyl) ester Chemical compound C1[N+](C)(C)CCC1OC(=O)C(O)(C=1C=CC=CC=1)C1CCCC1 ANGKOCUUWGHLCE-UHFFFAOYSA-N 0.000 claims description 31
- 238000007865 diluting Methods 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 25
- 239000013558 reference substance Substances 0.000 claims description 24
- 238000004458 analytical method Methods 0.000 claims description 21
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- 229940020900 indacaterol and glycopyrronium bromide Drugs 0.000 claims description 17
- 238000003908 quality control method Methods 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 238000012795 verification Methods 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000005303 weighing Methods 0.000 claims description 12
- 238000000703 high-speed centrifugation Methods 0.000 claims description 10
- 238000002347 injection Methods 0.000 claims description 9
- 239000007924 injection Substances 0.000 claims description 9
- 238000004445 quantitative analysis Methods 0.000 claims description 7
- 239000012472 biological sample Substances 0.000 claims description 6
- 239000003085 diluting agent Substances 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 239000011159 matrix material Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 238000007781 pre-processing Methods 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 238000011084 recovery Methods 0.000 claims description 6
- 238000003860 storage Methods 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 4
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 238000001514 detection method Methods 0.000 abstract description 4
- 230000006920 protein precipitation Effects 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 210000002381 plasma Anatomy 0.000 description 4
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 description 2
- 208000006673 asthma Diseases 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
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- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 102000034337 acetylcholine receptors Human genes 0.000 description 1
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- 239000000048 adrenergic agonist Substances 0.000 description 1
- 210000005091 airway smooth muscle Anatomy 0.000 description 1
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- 102000014974 beta2-adrenergic receptor activity proteins Human genes 0.000 description 1
- 108040006828 beta2-adrenergic receptor activity proteins Proteins 0.000 description 1
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- 239000000843 powder Substances 0.000 description 1
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- 150000003242 quaternary ammonium salts Chemical group 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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- G—PHYSICS
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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- G01N2030/8822—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving blood
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8895—Independent juxtaposition of embodiments; Reviews
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Abstract
The invention provides a method for determining the concentration of indacaterol glycopyrrolate in serum, which comprises the following steps: preparing a stock solution, preparing a working solution, preparing a standard curve and a quality control sample, preparing an internal standard working solution, and pretreating an indacaterol sample; and (3) pretreating a glycopyrronium bromide sample, and verifying the method. The beneficial effects are that: the method for determining the concentration of indacaterol glycopyrronium bromide in serum provided by the invention has high sensitivity (the indacaterol LLOQ is 20.00pg/mL, and the glycopyrronium bromide LLOQ is 5.000pg/mL), the LC-MS/MS method is used for detecting the concentration of human serum blood serum by using the unknown report of glycopyrronium bromide, the lower limit of the quantification of the method is 5.000pg/mL, although the existing LC-MS/MS method reduces the lower limit of the indacaterol quantification to 3.000pg/mL and 10.00pg/mL respectively, and the method adopts a protein precipitation method to treat samples, so that the method is convenient to operate, stable and reliable, and can meet the requirement of clinical large-batch sample detection.
Description
Technical Field
The invention relates to the technical field of plasma determination, in particular to a method for determining the concentration of indacaterol glycopyrrolate in serum.
Background
Indacaterol is a novel, ultra-long-acting, fast-acting beta 2-adrenoceptor agonist, and is used for daily treatment of asthma and chronic obstructive pulmonary disease. Indacaterol acts by stimulating β 2-adrenoceptors in airway smooth muscle, which leads to muscle relaxation, increasing the diameter of the airway, and thereby contracting in asthma and chronic obstructive pulmonary disease. Indacaterol has a high affinity for the lipid raft domain in the respiratory membrane, slowly separates from the receptor, and therefore has a long duration of action. The indacaterol has high intrinsic efficacy and quick response. Glycopyrrolate is a synthetic long-acting acetylcholine receptor antagonist with a quaternary ammonium salt structure, has the effects of inhibiting gastric secretion and regulating gastrointestinal peristalsis, has 4 times higher selectivity on human body acetylcholine energy M3 receptors than M2 receptors, and can be specifically combined with and inhibit M3 type acetylcholine receptors distributed on bronchial smooth muscle to expand airways.
At present, the pharmacokinetics and bioequivalence of drugs are evaluated by measuring pharmacokinetic parameters of the drug. Therefore, the concentration determination of indacaterol and glycopyrronium bromide in serum is very important for the development of indacaterol glycopyrronium bromide drugs, and few analytical methods for detecting indacaterol and glycopyrronium bromide in human serum are reported at present. After the indacaterol glycopyrronium bromide is used for being inhaled into the powder inhalation capsule, the indacaterol and glycopyrronium bromide have low blood concentration in the serum of healthy people [1], the indacaterol is weak to be reserved, the signal response is poor, and the requirement on the detection sensitivity is high. At present, the quantitative analysis method for accurately measuring the plasma concentration of indacaterol in human plasma mainly adopts HPLC (high performance liquid chromatography) and LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry). Glycopyrrolate was detected mainly in equine urine by the LC-MS/MS method and in preclinical pharmacokinetic studies. The method adopts an LC-MS/MS method to measure the concentrations of indacaterol and glycopyrronium bromide in human serum.
In the prior art, the HPLC method and the LC-MS/MS method for determining the concentration of indacaterol in human plasma are reported, and the methods have low sensitivity and long analysis time, or have more samples and narrow linear range, and cannot meet the requirement of biological analysis of mass samples; the concentration of glycopyrronium bromide in human serum determined by the LC-MS/MS method is not reported, and the LC-MS/MS method is a preferred method for bioanalysis of small molecule drugs due to the advantages of good specificity, high sensitivity and the like. The LC-MS/MS method is simple, rapid, durable, high in sensitivity and good in selectivity, is used for determining the blood concentration of indacaterol and glycopyrronium bromide in human serum, and can be used for researching the bioequivalence of the indacaterol glycopyrronium bromide preparation.
Disclosure of Invention
The invention aims to provide a method for measuring the concentration of indacaterol glycopyrronium bromide in serum, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a method for determining indacaterol glycopyrrolate concentration in serum comprising the steps of:
the method comprises the following steps: preparing Stock solutions, namely accurately weighing 2 parts of indacaterol reference substance respectively, dissolving and shaking the indacaterol reference substance by using methanol to prepare the indacaterol Stock solution with the concentration of 20.00 mu g/mL, namely the Stock solutions indacaterol-STD-Stock and indacaterol-QC-Stock; precisely weighing 2 parts of glycopyrronium bromide reference substance respectively, dissolving with methanol, shaking up, and preparing into glycopyrronium bromide Stock solution with concentration of 20.00 μ g/mL, namely, the Stock solution glycopyrronium bromide-STD-Stock, glycopyrronium bromide-QC-Stock;
step two: preparing a working solution, namely preparing the standard curve working solution by taking indacaterol-STD-Stock and glycopyrronium bromide-STD-Stock as initial solutions and methanol: diluting the solution of water (50: 50, v: v) as a diluting solvent step by step to obtain working solutions of standard curves of indacaterol and glycopyrronium bromide at each stage; the quality control working solution takes indacaterol-QC-Stock and glycopyrronium bromide-QC-Stock as initial solutions, and methanol: diluting the solution of water (50: 50, v: v) as a diluting solvent step by step to obtain quality control working solutions of indacaterol and glycopyrronium bromide at different levels;
step three: preparing a standard curve and a quality control sample, namely sucking 95% of human blank serum by volume, and adding 5% of working solution with the concentration provided in the step two by volume to obtain a curve sample and a quality control sample;
step four: preparing an internal standard working solution, dissolving an indacaterol-d 3 and a glycopyrronium bromide-d 3 reference substance by using methanol, and diluting by using 50% methanol as a diluent to obtain the internal standard working solution with the concentrations of 8.000ng/mL and 2.000ng/mL respectively;
step five: preprocessing an indacaterol sample; taking 150.0mL of a serum sample into a 96-well plate, adding 50.00mL of internal standard working solution (indacaterol-d 3, 8.000ng/mL), adding 450.0mL of precipitant acetonitrile, performing vortex for 10-20min, performing high-speed centrifugation (4000-5000rpm) for 15-25min, taking 400.0mL of supernatant into another 96-deep-well plate, performing LC-MS/MS analysis, and taking 5.000mL of a sample;
step six: pretreating a glycopyrronium bromide sample, putting 50.00mL of a serum sample into a 96-well plate, adding 20.00mL of an internal standard working solution (glycopyrronium bromide-d 3, 2.000ng/mL), adding 250.0mL of a precipitant acetonitrile, performing vortex for 10-20min, performing high-speed centrifugation (4000 plus 5000rpm) for 15-25min, putting 150.0mL of a supernatant into another 96-well plate added with 150.0mL of ultrapure water, performing vortex mixing for 5-10min, performing centrifugation for 3-8min, performing LC-MS/MS analysis, and putting 10.00mL of a sample injection body;
step seven: the method is verified according to the requirements of the verification guiding principle of the quantitative analysis method of the biological samples in the Chinese pharmacopoeia 2015 edition, and the verification contents comprise selectivity, a standard curve, a lower limit of quantification, precision and accuracy, an extraction recovery rate, a matrix effect, a dilution effect, a residual effect, stability and sample capacity.
Preferably, in step six, the LC-MS/MS analysis process, the LC-MS/MS system consists of a QTRAP 6500 type triple quadrupole tandem mass spectrometer (AB Sciex), a high pressure pump (LC-30AD), an online degasser (DGU-20A-5R), an autosampler (SIL-30AC), and a column oven (CTO-20A) (Shimadzu, Japan).
Preferably, in step one, the storage temperature of the stock solution is 4 ℃.
Compared with the prior art, the invention has the beneficial effects that:
the method for determining the concentration of indacaterol glycopyrronium bromide in serum provided by the invention has high sensitivity (the indacaterol LLOQ is 20.00pg/mL, and the glycopyrronium bromide LLOQ is 5.000pg/mL), the LC-MS/MS method is used for detecting the concentration of human serum blood serum by using the unknown report of glycopyrronium bromide, the lower limit of the quantification of the method is 5.000pg/mL, although the existing LC-MS/MS method reduces the lower limit of the indacaterol quantification to 3.000pg/mL and 10.00pg/mL respectively, and the method adopts a protein precipitation method to treat samples, so that the method is convenient to operate, stable and reliable, and can meet the requirement of clinical large-batch sample detection.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
The invention provides a technical scheme that: a method for determining indacaterol glycopyrrolate concentration in serum comprising the steps of:
the method comprises the following steps: preparing Stock solutions, namely accurately weighing 2 parts of indacaterol reference substance respectively, dissolving and shaking the indacaterol reference substance by using methanol to prepare the indacaterol Stock solution with the concentration of 20.00 mu g/mL, namely the Stock solutions indacaterol-STD-Stock and indacaterol-QC-Stock; precisely weighing 2 parts of glycopyrronium bromide reference substance respectively, dissolving with methanol, shaking up, and preparing into glycopyrronium bromide Stock solution with concentration of 20.00 μ g/mL, namely, the Stock solution glycopyrronium bromide-STD-Stock, glycopyrronium bromide-QC-Stock;
step two: preparing a working solution, namely preparing the standard curve working solution by taking indacaterol-STD-Stock and glycopyrronium bromide-STD-Stock as initial solutions and methanol: diluting the solution of water (50: 50, v: v) as a diluting solvent step by step to obtain working solutions of standard curves of indacaterol and glycopyrronium bromide at each stage; the quality control working solution takes indacaterol-QC-Stock and glycopyrronium bromide-QC-Stock as initial solutions, and methanol: diluting the solution of water (50: 50, v: v) as a diluting solvent step by step to obtain quality control working solutions of indacaterol and glycopyrronium bromide at different levels;
step three: preparing a standard curve and a quality control sample, namely sucking 95% of human blank serum by volume, and adding 5% of working solution with the concentration provided in the step two by volume to obtain a curve sample and a quality control sample;
step four: preparing an internal standard working solution, dissolving an indacaterol-d 3 and a glycopyrronium bromide-d 3 reference substance by using methanol, and diluting by using 50% methanol as a diluent to obtain the internal standard working solution with the concentrations of 8.000ng/mL and 2.000ng/mL respectively;
step five: preprocessing an indacaterol sample; taking a serum sample of 150.0mL into a 96-well plate, adding 50.00mL of internal standard working solution (indacaterol-d 3, 8.000ng/mL), adding 450.0mL of precipitant acetonitrile, performing vortex for 10min, performing high-speed centrifugation (4000rpm) for 15min, taking 400.0mL of supernatant into another 96-deep-well plate, and performing LC-MS/MS analysis, wherein the sample injection is 5.000 mL;
step six: pretreating a glycopyrronium bromide sample, putting 50.00mL of a serum sample into a 96-well plate, adding 20.00mL of an internal standard working solution (glycopyrronium bromide-d 3, 2.000ng/mL), adding 250.0mL of a precipitant acetonitrile, performing vortex for 10min, performing high-speed centrifugation (4000rpm) for 15min, putting 150.0mL of a supernatant into another 96-well plate added with 150.0mL of ultrapure water, performing vortex mixing for 5min, performing centrifugation for 3min, performing LC-MS/MS analysis, and putting 10.00mL of a sample injection;
step seven: the method is verified according to the requirements of the verification guiding principle of the quantitative analysis method of the biological samples in the Chinese pharmacopoeia 2015 edition, and the verification contents comprise selectivity, a standard curve, a lower limit of quantification, precision and accuracy, an extraction recovery rate, a matrix effect, a dilution effect, a residual effect, stability and sample capacity.
In the sixth step, in the LC-MS/MS analysis process, an LC-MS/MS system consists of a QTRAP 6500 type triple quadrupole tandem mass spectrometer (AB Sciex), a high-pressure pump (LC-30AD), an online degasser (DGU-20A-5R), an automatic sample injector (SIL-30AC) and a column incubator (CTO-20A) (Shimadzu, Japan); in step one, the storage temperature of the stock solution was 4 ℃.
Example two
The invention provides a technical scheme that: a method for determining indacaterol glycopyrrolate concentration in serum comprising the steps of:
the method comprises the following steps: preparing Stock solutions, namely accurately weighing 2 parts of indacaterol reference substance respectively, dissolving and shaking the indacaterol reference substance by using methanol to prepare the indacaterol Stock solution with the concentration of 20.00 mu g/mL, namely the Stock solutions indacaterol-STD-Stock and indacaterol-QC-Stock; precisely weighing 2 parts of glycopyrronium bromide reference substance respectively, dissolving with methanol, shaking up, and preparing into glycopyrronium bromide Stock solution with concentration of 20.00 μ g/mL, namely, the Stock solution glycopyrronium bromide-STD-Stock, glycopyrronium bromide-QC-Stock;
step two: preparing a working solution, namely preparing the standard curve working solution by taking indacaterol-STD-Stock and glycopyrronium bromide-STD-Stock as initial solutions and methanol: diluting the solution of water (50: 50, v: v) as a diluting solvent step by step to obtain working solutions of standard curves of indacaterol and glycopyrronium bromide at each stage; the quality control working solution takes indacaterol-QC-Stock and glycopyrronium bromide-QC-Stock as initial solutions, and methanol: diluting the solution of water (50: 50, v: v) as a diluting solvent step by step to obtain quality control working solutions of indacaterol and glycopyrronium bromide at different levels;
step three: preparing a standard curve and a quality control sample, namely sucking 95% of human blank serum by volume, and adding 5% of working solution with the concentration provided in the step two by volume to obtain a curve sample and a quality control sample;
step four: preparing an internal standard working solution, dissolving an indacaterol-d 3 and a glycopyrronium bromide-d 3 reference substance by using methanol, and diluting by using 50% methanol as a diluent to obtain the internal standard working solution with the concentrations of 8.000ng/mL and 2.000ng/mL respectively;
step five: preprocessing an indacaterol sample; taking a serum sample of 150.0mL into a 96-well plate, adding 50.00mL of internal standard working solution (indacaterol-d 3, 8.000ng/mL), adding 450.0mL of precipitant acetonitrile, performing vortex for 20min, performing high-speed centrifugation (5000rpm) for 15-25min, taking 400.0mL of supernatant into another 96-deep-well plate, performing LC-MS/MS analysis, and taking 5.000mL of sample injection;
step six: pretreating a glycopyrronium bromide sample, putting 50.00mL of a serum sample into a 96-well plate, adding 20.00mL of an internal standard working solution (glycopyrronium bromide-d 3, 2.000ng/mL), adding 250.0mL of a precipitant acetonitrile, performing vortex 20min, performing high-speed centrifugation (5000rpm) for 25min, putting 150.0mL of a supernatant into another 96-well plate added with 150.0mL of ultrapure water, performing vortex mixing for 10min, performing centrifugation for 8min, performing LC-MS/MS analysis, and putting 10.00mL of a sample injection;
step seven: the method is verified according to the requirements of the verification guiding principle of the quantitative analysis method of the biological samples in the Chinese pharmacopoeia 2015 edition, and the verification contents comprise selectivity, a standard curve, a lower limit of quantification, precision and accuracy, an extraction recovery rate, a matrix effect, a dilution effect, a residual effect, stability and sample capacity.
In the sixth step, in the LC-MS/MS analysis process, an LC-MS/MS system consists of a QTRAP 6500 type triple quadrupole tandem mass spectrometer (AB Sciex), a high-pressure pump (LC-30AD), an online degasser (DGU-20A-5R), an automatic sample injector (SIL-30AC) and a column incubator (CTO-20A) (Shimadzu, Japan); in step one, the storage temperature of the stock solution was 4 ℃.
EXAMPLE III
The invention provides a technical scheme that: a method for determining indacaterol glycopyrrolate concentration in serum comprising the steps of:
the method comprises the following steps: preparing Stock solutions, namely accurately weighing 2 parts of indacaterol reference substance respectively, dissolving and shaking the indacaterol reference substance by using methanol to prepare the indacaterol Stock solution with the concentration of 20.00 mu g/mL, namely the Stock solutions indacaterol-STD-Stock and indacaterol-QC-Stock; precisely weighing 2 parts of glycopyrronium bromide reference substance respectively, dissolving with methanol, shaking up, and preparing into glycopyrronium bromide Stock solution with concentration of 20.00 μ g/mL, namely, the Stock solution glycopyrronium bromide-STD-Stock, glycopyrronium bromide-QC-Stock;
step two: preparing a working solution, namely preparing the standard curve working solution by taking indacaterol-STD-Stock and glycopyrronium bromide-STD-Stock as initial solutions and methanol: diluting the solution of water (50: 50, v: v) as a diluting solvent step by step to obtain working solutions of standard curves of indacaterol and glycopyrronium bromide at each stage; the quality control working solution takes indacaterol-QC-Stock and glycopyrronium bromide-QC-Stock as initial solutions, and methanol: diluting the solution of water (50: 50, v: v) as a diluting solvent step by step to obtain quality control working solutions of indacaterol and glycopyrronium bromide at different levels;
step three: preparing a standard curve and a quality control sample, namely sucking 95% of human blank serum by volume, and adding 5% of working solution with the concentration provided in the step two by volume to obtain a curve sample and a quality control sample;
step four: preparing an internal standard working solution, dissolving an indacaterol-d 3 and a glycopyrronium bromide-d 3 reference substance by using methanol, and diluting by using 50% methanol as a diluent to obtain the internal standard working solution with the concentrations of 8.000ng/mL and 2.000ng/mL respectively;
step five: preprocessing an indacaterol sample; taking a serum sample of 150.0mL into a 96-well plate, adding 50.00mL of internal standard working solution (indacaterol-d 3, 8.000ng/mL), adding 450.0mL of precipitant acetonitrile, vortexing for 15min, performing high-speed centrifugation (4300rpm) for 18min, taking 400.0mL of supernatant into another 96-deep-well plate, and performing LC-MS/MS analysis, wherein the sample injection is 5.000 mL;
step six: pretreating a glycopyrronium bromide sample, putting 50.00mL of a serum sample into a 96-well plate, adding 20.00mL of an internal standard working solution (glycopyrronium bromide-d 3, 2.000ng/mL), adding 250.0mL of a precipitant acetonitrile, swirling for 15min, centrifuging at a high speed (4300rpm) for 18min, putting 150.0mL of a supernatant into another 96-well plate added with 150.0mL of ultrapure water, swirling and uniformly mixing for 7min, centrifuging for 5min, performing LC-MS/MS analysis, and putting 10.00mL of a sample injection;
step seven: the method is verified according to the requirements of the verification guiding principle of the quantitative analysis method of the biological samples in the Chinese pharmacopoeia 2015 edition, and the verification contents comprise selectivity, a standard curve, a lower limit of quantification, precision and accuracy, an extraction recovery rate, a matrix effect, a dilution effect, a residual effect, stability and sample capacity.
In the sixth step, in the LC-MS/MS analysis process, an LC-MS/MS system consists of a QTRAP 6500 type triple quadrupole tandem mass spectrometer (AB Sciex), a high-pressure pump (LC-30AD), an online degasser (DGU-20A-5R), an automatic sample injector (SIL-30AC) and a column incubator (CTO-20A) (Shimadzu, Japan); in step one, the storage temperature of the stock solution was 4 ℃.
Example four
The invention provides a technical scheme that: a method for determining indacaterol glycopyrrolate concentration in serum comprising the steps of:
the method comprises the following steps: preparing Stock solutions, namely accurately weighing 2 parts of indacaterol reference substance respectively, dissolving and shaking the indacaterol reference substance by using methanol to prepare the indacaterol Stock solution with the concentration of 20.00 mu g/mL, namely the Stock solutions indacaterol-STD-Stock and indacaterol-QC-Stock; precisely weighing 2 parts of glycopyrronium bromide reference substance respectively, dissolving with methanol, shaking up, and preparing into glycopyrronium bromide Stock solution with concentration of 20.00 μ g/mL, namely, the Stock solution glycopyrronium bromide-STD-Stock, glycopyrronium bromide-QC-Stock;
step two: preparing a working solution, namely preparing the standard curve working solution by taking indacaterol-STD-Stock and glycopyrronium bromide-STD-Stock as initial solutions and methanol: diluting the solution of water (50: 50, v: v) as a diluting solvent step by step to obtain working solutions of standard curves of indacaterol and glycopyrronium bromide at each stage; the quality control working solution takes indacaterol-QC-Stock and glycopyrronium bromide-QC-Stock as initial solutions, and methanol: diluting the solution of water (50: 50, v: v) as a diluting solvent step by step to obtain quality control working solutions of indacaterol and glycopyrronium bromide at different levels;
step three: preparing a standard curve and a quality control sample, namely sucking 95% of human blank serum by volume, and adding 5% of working solution with the concentration provided in the step two by volume to obtain a curve sample and a quality control sample;
step four: preparing an internal standard working solution, dissolving an indacaterol-d 3 and a glycopyrronium bromide-d 3 reference substance by using methanol, and diluting by using 50% methanol as a diluent to obtain the internal standard working solution with the concentrations of 8.000ng/mL and 2.000ng/mL respectively;
step five: preprocessing an indacaterol sample; taking a serum sample of 150.0mL into a 96-well plate, adding 50.00mL of internal standard working solution (indacaterol-d 3, 8.000ng/mL), adding 450.0mL of precipitant acetonitrile, vortexing for 18min, performing high-speed centrifugation (4700rpm) for 22min, taking 400.0mL of supernatant into another 96-deep-well plate, and performing LC-MS/MS analysis, wherein the sample injection is 5.000 mL;
step six: pretreating a glycopyrronium bromide sample, putting 50.00mL of a serum sample into a 96-well plate, adding 20.00mL of an internal standard working solution (glycopyrronium bromide-d 3, 2.000ng/mL), adding 250.0mL of a precipitant acetonitrile, swirling for 18min, centrifuging at a high speed (4700rpm) for 22min, putting 150.0mL of a supernatant into another 96-well plate added with 150.0mL of ultrapure water, swirling and uniformly mixing for 8min, centrifuging for 6min, performing LC-MS/MS analysis, and putting 10.00mL of a sample;
step seven: the method is verified according to the requirements of the verification guiding principle of the quantitative analysis method of the biological samples in the Chinese pharmacopoeia 2015 edition, and the verification contents comprise selectivity, a standard curve, a lower limit of quantification, precision and accuracy, an extraction recovery rate, a matrix effect, a dilution effect, a residual effect, stability and sample capacity.
In the sixth step, in the LC-MS/MS analysis process, an LC-MS/MS system consists of a QTRAP 6500 type triple quadrupole tandem mass spectrometer (AB Sciex), a high-pressure pump (LC-30AD), an online degasser (DGU-20A-5R), an automatic sample injector (SIL-30AC) and a column incubator (CTO-20A) (Shimadzu, Japan); in step one, the storage temperature of the stock solution was 4 ℃.
The four groups of embodiments can measure the indacaterol glycopyrronium bromide in the blood plasma, and the advantages of the invention are as follows: the method for determining the concentration of indacaterol glycopyrronium bromide in serum provided by the invention has high sensitivity (the indacaterol LLOQ is 20.00pg/mL, and the glycopyrronium bromide LLOQ is 5.000pg/mL), the LC-MS/MS method is used for detecting the concentration of human serum blood serum by using the unknown report of glycopyrronium bromide, the lower limit of the quantification of the method is 5.000pg/mL, although the existing LC-MS/MS method reduces the lower limit of the indacaterol quantification to 3.000pg/mL and 10.00pg/mL respectively, and the method adopts a protein precipitation method to treat samples, so that the method is convenient to operate, stable and reliable, and can meet the requirement of clinical large-batch sample detection.
Wherein:
TABLE 1 Indacterol glycopyrrolate working solution concentration chart
TABLE 2 Indanterol glycopyrrolate Standard Curve sample and quality control sample concentration Table
TABLE 3 Indanterol glycopyrrolate chromatography conditions Table
TABLE 4 Indacterol glycopyrrolate Mass Spectrometry Condition Table
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (3)
1. A method for determining the concentration of indacaterol glycopyrrolate in serum, which is characterized by comprising the following steps:
the method comprises the following steps: preparing Stock solutions, namely accurately weighing 2 parts of indacaterol reference substance respectively, dissolving and shaking the indacaterol reference substance by using methanol to prepare the indacaterol Stock solution with the concentration of 20.00 mu g/mL, namely the Stock solutions indacaterol-STD-Stock and indacaterol-QC-Stock; precisely weighing 2 parts of glycopyrronium bromide reference substance respectively, dissolving with methanol, shaking up, and preparing into glycopyrronium bromide Stock solution with concentration of 20.00 μ g/mL, namely, the Stock solution glycopyrronium bromide-STD-Stock, glycopyrronium bromide-QC-Stock;
step two: preparing a working solution, namely preparing the standard curve working solution by taking indacaterol-STD-Stock and glycopyrronium bromide-STD-Stock as initial solutions and methanol: diluting the solution of water (50: 50, v: v) as a diluting solvent step by step to obtain working solutions of standard curves of indacaterol and glycopyrronium bromide at each stage; the quality control working solution takes indacaterol-QC-Stock and glycopyrronium bromide-QC-Stock as initial solutions, and methanol: diluting the solution of water (50: 50, v: v) as a diluting solvent step by step to obtain quality control working solutions of indacaterol and glycopyrronium bromide at different levels;
step three: preparing a standard curve and a quality control sample, namely sucking 95% of human blank serum by volume, and adding 5% of working solution with the concentration provided in the step two by volume to obtain a curve sample and a quality control sample;
step four: preparing an internal standard working solution, dissolving an indacaterol-d 3 and a glycopyrronium bromide-d 3 reference substance by using methanol, and diluting by using 50% methanol as a diluent to obtain the internal standard working solution with the concentrations of 8.000ng/mL and 2.000ng/mL respectively;
step five: preprocessing an indacaterol sample; taking 150.0mL of a serum sample into a 96-well plate, adding 50.00mL of internal standard working solution (indacaterol-d 3, 8.000ng/mL), adding 450.0mL of precipitant acetonitrile, performing vortex for 10-20min, performing high-speed centrifugation (4000-5000rpm) for 15-25min, taking 400.0mL of supernatant into another 96-deep-well plate, performing LC-MS/MS analysis, and taking 5.000mL of a sample;
step six: pretreating a glycopyrronium bromide sample, putting 50.00mL of a serum sample into a 96-well plate, adding 20.00mL of an internal standard working solution (glycopyrronium bromide-d 3, 2.000ng/mL), adding 250.0mL of a precipitant acetonitrile, performing vortex for 10-20min, performing high-speed centrifugation (4000 plus 5000rpm) for 15-25min, putting 150.0mL of a supernatant into another 96-well plate added with 150.0mL of ultrapure water, performing vortex mixing for 5-10min, performing centrifugation for 3-8min, performing LC-MS/MS analysis, and putting 10.00mL of a sample injection body;
step seven: the method is verified according to the requirements of the verification guiding principle of the quantitative analysis method of the biological samples in the Chinese pharmacopoeia 2015 edition, and the verification contents comprise selectivity, a standard curve, a lower limit of quantification, precision and accuracy, an extraction recovery rate, a matrix effect, a dilution effect, a residual effect, stability and sample capacity.
2. The method for determining the concentration of indacaterol glycopyrrolate in serum as claimed in claim 1, wherein: and in the sixth step, an LC-MS/MS analysis process, wherein an LC-MS/MS system consists of a QTRAP 6500 type triple quadrupole tandem mass spectrometer (ABSciex), a high-pressure pump (LC-30AD), an online degasser (DGU-20A-5R), an automatic sample injector (SIL-30AC) and a column oven (CTO-20A) (Shimadzu, Japan).
3. The method for determining the concentration of indacaterol glycopyrrolate in serum as claimed in claim 1, wherein: in step one, the storage temperature of the stock solution was 4 ℃.
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| CN109789126A (en) * | 2016-09-19 | 2019-05-21 | 墨西哥氟石股份公司 | Pharmaceutical composition |
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| CN108982712A (en) * | 2018-10-08 | 2018-12-11 | 山东越品检验检测有限公司 | Salmeterol, datro, the method for Ao Dateluo in a kind of measurement pork |
Non-Patent Citations (9)
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