CN111624293A - Method for measuring captopril concentration in human plasma - Google Patents
Method for measuring captopril concentration in human plasma Download PDFInfo
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- CN111624293A CN111624293A CN202010666288.1A CN202010666288A CN111624293A CN 111624293 A CN111624293 A CN 111624293A CN 202010666288 A CN202010666288 A CN 202010666288A CN 111624293 A CN111624293 A CN 111624293A
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- FAKRSMQSSFJEIM-RQJHMYQMSA-N captopril Chemical compound SC[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O FAKRSMQSSFJEIM-RQJHMYQMSA-N 0.000 title claims abstract description 95
- 229960000830 captopril Drugs 0.000 title claims abstract description 88
- 238000000034 method Methods 0.000 title claims abstract description 66
- 239000012224 working solution Substances 0.000 claims abstract description 66
- 239000000523 sample Substances 0.000 claims abstract description 47
- 230000000694 effects Effects 0.000 claims abstract description 24
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 22
- 239000011550 stock solution Substances 0.000 claims abstract description 21
- 239000013062 quality control Sample Substances 0.000 claims abstract description 20
- 239000011159 matrix material Substances 0.000 claims abstract description 10
- 238000011002 quantification Methods 0.000 claims abstract description 9
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 100
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- 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 50
- 235000019253 formic acid Nutrition 0.000 claims description 50
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 33
- 238000007865 diluting Methods 0.000 claims description 30
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 26
- 238000004458 analytical method Methods 0.000 claims description 19
- 150000002500 ions Chemical class 0.000 claims description 18
- 238000002360 preparation method Methods 0.000 claims description 18
- 238000003908 quality control method Methods 0.000 claims description 18
- 239000003085 diluting agent Substances 0.000 claims description 16
- 238000000703 high-speed centrifugation Methods 0.000 claims description 12
- 238000012544 monitoring process Methods 0.000 claims description 12
- 239000013558 reference substance Substances 0.000 claims description 12
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- 238000006243 chemical reaction Methods 0.000 claims description 7
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- 238000000926 separation method Methods 0.000 claims description 6
- BCAARMUWIRURQS-UHFFFAOYSA-N dicalcium;oxocalcium;silicate Chemical compound [Ca+2].[Ca+2].[Ca]=O.[O-][Si]([O-])([O-])[O-] BCAARMUWIRURQS-UHFFFAOYSA-N 0.000 claims description 5
- 230000008569 process Effects 0.000 abstract description 7
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- PQSUYGKTWSAVDQ-UHFFFAOYSA-N Aldosterone Natural products C1CC2C3CCC(C(=O)CO)C3(C=O)CC(O)C2C2(C)C1=CC(=O)CC2 PQSUYGKTWSAVDQ-UHFFFAOYSA-N 0.000 description 1
- 102000005862 Angiotensin II Human genes 0.000 description 1
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- CZGUSIXMZVURDU-JZXHSEFVSA-N Ile(5)-angiotensin II Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C([O-])=O)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=[NH2+])NC(=O)[C@@H]([NH3+])CC([O-])=O)C(C)C)C1=CC=C(O)C=C1 CZGUSIXMZVURDU-JZXHSEFVSA-N 0.000 description 1
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- ORWYRWWVDCYOMK-HBZPZAIKSA-N angiotensin I Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 ORWYRWWVDCYOMK-HBZPZAIKSA-N 0.000 description 1
- 229950006323 angiotensin ii Drugs 0.000 description 1
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000004531 blood pressure lowering effect Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
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- 230000002860 competitive effect Effects 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
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- 229940126586 small molecule drug Drugs 0.000 description 1
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- 125000003396 thiol group Chemical group [H]S* 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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- 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
- G01N2030/045—Standards internal
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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
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—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
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8895—Independent juxtaposition of embodiments; Reviews
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
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Abstract
The invention provides a method for measuring captopril concentration in human plasma, 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, pretreating the sample, analyzing by LC-MS/MS and verifying by a method; the beneficial effects are that: compared with the prior chemical inhibitor addition method, the method for determining the captopril concentration in human plasma is more sensitive, and the lower limit of the quantification of the captopril is reduced from 10.00ng/mL to 4.000 ng/mL; meanwhile, the method has the advantages of small sample usage amount, less mobile phase consumption, reduced pretreatment process, increased sensitivity, improved reliability and durability, good linearity, and elimination of matrix effect, and reduced harm to the subject and the environment in the test process.
Description
Technical Field
The invention relates to the technical field of blood plasma determination, in particular to a method for determining captopril concentration in human blood plasma.
Background
Captopril is a competitive angiotensin converting enzyme inhibitor, so that angiotensin I cannot be converted into angiotensin II, thereby reducing peripheral vascular resistance, and reducing water and sodium retention by inhibiting aldosterone secretion. Has obvious blood pressure lowering effect on various types of hypertension, and can improve the cardiac function of patients with congestive heart failure.
Currently, studies on drug bioequivalence and bioavailability are evaluated by measuring pharmacokinetic parameters of the drug. Therefore, the determination of the concentration of captopril in plasma is of great importance for the development of new captopril drugs. Because the captopril molecule contains a sulfhydryl group, it is readily conjugated to albumin and other plasma proteins. Meanwhile, the captopril molecules and the captopril and endogenous compounds (such as cysteine and glutathione) in blood plasma all react to generate disulfide compounds. Due to the above factors, captopril is extremely unstable in the plasma of healthy persons, and therefore, it is difficult to accurately measure the concentration of captopril. In order to keep the stability of captopril in the plasma of healthy people, the prior documents report two methods, one is to add a chemical stabilizer into the plasma in advance to inhibit the reaction of captopril, and directly measure the concentration of captopril by an LC-MS/MS method after pretreatment; the other method is that a molecular derivative agent is added into plasma to react with captopril to generate a stable derivative compound, and after pretreatment, the concentration of the captopril derivative is measured by an LC or GC method to indirectly calculate the concentration of the captopril.
In the prior art, the method for determining the concentration of captopril in human plasma by a chemical stabilizer addition method and a derivation method is reported; the derivatization method is more and more favored by researchers because the pretreatment process is complex and the investigation of the matrix effect and the recovery rate is difficult to operate; the LC-MS/MS method is the first choice method for the biological analysis of small molecule drugs due to the advantages of good specificity, high sensitivity and the like. The method adopts a chemical stabilizer adding method to stabilize the captopril in the blood plasma of healthy people, establishes a simple, rapid, durable, high-sensitivity and good-selectivity LC-MS/MS method for determining the blood concentration of the captopril in the blood plasma of EDTA-K2 anticoagulated people, and can be used for the research on the bioequivalence and the bioavailability of the captopril preparation; therefore, the invention provides a method for measuring the concentration of captopril in human plasma.
Disclosure of Invention
The present invention aims at providing a method for determining the concentration of captopril in human plasma, 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 the concentration of captopril in human plasma comprising the steps of:
the method comprises the following steps: preparation of stock solutions, acetonitrile with 0.1% formic acid: dissolving captopril reference substance in water (9:1, v: v) as diluent to obtain captopril standard curve Stock solution (STD Stock-KTPL) and captopril quality control Stock solution (QC Stock-KTPL) with the concentration of 400 mu g/mL;
step two: preparing a working solution, wherein the standard curve working solution takes STD Stock-KTPL as an initial solution, and 0.1% formic acid in acetonitrile: diluting the solution of water (9:1, v: v) as a diluting solvent step by step to obtain working solution of standard curves of captopril at all levels; the quality control working solution takes QC Stock-KTPL as an initial solution, and the mixture of 0.1% formic acid in acetonitrile: diluting the solution of water (9:1, v: v) as a diluting solvent step by step to obtain all-stage quality control working solution of captopril;
step three: preparing a standard curve and a quality control sample, namely sucking 95% of EDTA-K2 anticoagulant human blank plasma by volume, adding 200mM Dithiothreitol (DTT) physiological saline solution by volume of 5% to prepare blank plasma containing 10mM DTT, taking 950 mu L of the blank plasma containing 10mM DTT, and adding 50 mu L of the working solution to obtain a curve sample and a quality control sample;
step four: preparation of internal standard working solution, using acetonitrile of 0.1% formic acid: diluting captopril-d 3 reference substance with water (1:1, v: v) as diluent to obtain captopril-d 3 internal standard working solution with the concentration of 500 ng/mL;
step five: pretreating a sample, namely putting 100L of a plasma sample into a 96-well plate, adding 30uL of internal standard working solution, performing vortex for 3-8min, adding 300L of acetonitrile solution, performing vortex for 10-20min, performing high-speed centrifugation (4000rpm) for 15-30min, putting 180L of supernate into another 96-well plate which is added with 180L of 0.1% formic acid aqueous solution in advance, uniformly mixing for 5-10min, performing LC-MS/MS analysis after high-speed centrifugation (4000rpm) for 3-8min, and sampling the volume to be 6L;
step six: LC-MS/MS analysis, the temperature of the column incubator and the sample injector are respectively set to 40 ℃ and 4 ℃; the chromatographic column is used as a separation column; the needle wash was methanol with 0.1% formic acid: water (1:1, v: v), wherein the needle washing mode is cleaning before and after sample injection, and the needle washing volume is 450L; mobile phase: 0.05% formic acid in water-0.05% formic acid in methanol (62:38), isocratic conditions, flow rate of 0.40mL/min to separate the analytes, analysis time of 3.8 minutes; the polarity of the ion source is in a negative mode, and the ion monitoring mode is multi-reaction monitoring (MRM); the ion pairs of captopril and the internal standard (deuterated captopril) are 215.9/181.9 and 219.1/185.1 respectively; after the electrical parameter optimization, the cluster removing voltage (DP) is minus 80V, and the collision voltage (CE) is 19V; the data acquisition system is Analyst 1.6.3;
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 two, working solutions are prepared at concentrations of 30.00. mu.g/mL, 10.00. mu.g/mL, 4.000. mu.g/mL, 1.000. mu.g/mL, 0.4000. mu.g/mL, 0.1600. mu.g/mL, 0.08000. mu.g/mL, 24.00. mu.g/mL, 3.000. mu.g/mL, 0.2400. mu.g/mL, 0.08000. mu.g/mL, respectively.
Preferably, in the third step, the concentration of the curve sample and the concentration of the quality control sample are set to be multiple, and the multiple concentrations are 1500ng/mL, 500.0ng/mL, 200.0ng/mL, 50.00ng/mL, 20.00ng/mL, 8.000ng/mL, 4.000ng/mL, 1200ng/mL, 150.0ng/mL, 12.00ng/mL and 4.000ng/mL respectively.
Preferably, in step six, the LC-MS/MS system consists of a TRIPLE QUADU model 5500 TRIPLE quadrupole tandem mass spectrometer (AB Sciex), a high pressure pump (LC-30AD), an online degasser (DGU-20A-5R), an autosampler (SIL-30AC), a column oven (CTO-20A) (Shimadzu, Japan), and a controller CBM-20 Alite.
Preferably, in step six, a catalyst of type Welch,
XS-C18,3.0 μm,3.0 x 100mm column.
Compared with the prior art, the invention has the beneficial effects that:
1. compared with the prior chemical inhibitor addition method, the method for determining the captopril concentration in human plasma is more sensitive, and the lower limit of the quantification of the captopril is reduced from 10.00ng/mL to 4.000 ng/mL; meanwhile, the method has the advantages of small sample usage amount, less mobile phase consumption, reduced pretreatment process, increased sensitivity, improved reliability and durability, good linearity, and elimination of matrix effect, and reduced harm to the subject and the environment in the test process;
2. when the method for determining the captopril concentration in human plasma provided by the invention is used for preparing a working solution, the method adopts acetonitrile of 0.1 percent formic acid: water (9:1, v: v) is used as a diluent to prepare working solution and stock solution, which can stabilize captopril in the solution and eliminate the adsorption of the captopril in a polypropylene centrifugal tube, so that other suitable diluent can achieve the technical effect; human plasma with the concentration of 10mM dithiothreitol can stabilize captopril in the human plasma, so that other inhibitors with the same efficacy can reduce the degradation rate of captopril in the plasma after being added to human plasma samples, and can stabilize the captopril concentration.
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 the concentration of captopril in human plasma comprising the steps of:
the method comprises the following steps: preparation of stock solutions, acetonitrile with 0.1% formic acid: dissolving captopril reference substance in water (9:1, v: v) as diluent to obtain captopril standard curve Stock solution (STD Stock-KTPL) and captopril quality control Stock solution (QC Stock-KTPL) with the concentration of 400 mu g/mL;
step two: preparing a working solution, wherein the standard curve working solution takes STD Stock-KTPL as an initial solution, and 0.1% formic acid in acetonitrile: diluting the solution of water (9:1, v: v) as a diluting solvent step by step to obtain working solution of standard curves of captopril at all levels; the quality control working solution takes QC Stock-KTPL as an initial solution, and the mixture of 0.1% formic acid in acetonitrile: diluting the solution of water (9:1, v: v) as a diluting solvent step by step to obtain all-stage quality control working solution of captopril;
step three: preparing a standard curve and a quality control sample, namely sucking 95% of EDTA-K2 anticoagulant human blank plasma by volume, adding 200mM Dithiothreitol (DTT) physiological saline solution by volume of 5% to prepare blank plasma containing 10mM DTT, taking 950 mu L of the blank plasma containing 10mM DTT, and adding 50 mu L of the working solution to obtain a curve sample and a quality control sample;
step four: preparation of internal standard working solution, using acetonitrile of 0.1% formic acid: diluting captopril-d 3 reference substance with water (1:1, v: v) as diluent to obtain captopril-d 3 internal standard working solution with the concentration of 500 ng/mL;
step five: pretreating a sample, namely putting 100L of a plasma sample into a 96-pore plate, adding 30uL of internal standard working solution, performing vortex for 3min, adding 300L of acetonitrile solution, performing vortex for 10min, performing high-speed centrifugation (4000rpm) for 15min, taking 180L of supernate into another 96-pore plate which is added with 180L of 0.1% formic acid aqueous solution in advance, uniformly mixing for 5min, performing LC-MS/MS analysis after 3min of high-speed centrifugation (4000rpm), and sampling the volume of 6L;
step six: LC-MS/MS analysis, the temperature of the column incubator and the sample injector are respectively set to 40 ℃ and 4 ℃; the chromatographic column is used as a separation column; the needle wash was methanol with 0.1% formic acid: water (1:1, v: v), wherein the needle washing mode is cleaning before and after sample injection, and the needle washing volume is 450L; mobile phase: 0.05% formic acid in water-0.05% formic acid in methanol (62:38), isocratic conditions, flow rate of 0.40mL/min to separate the analytes, analysis time of 3.8 minutes; the polarity of the ion source is in a negative mode, and the ion monitoring mode is multi-reaction monitoring (MRM); the ion pairs of captopril and the internal standard (deuterated captopril) are 215.9/181.9 and 219.1/185.1 respectively; after the electrical parameter optimization, the cluster removing voltage (DP) is minus 80V, and the collision voltage (CE) is 19V; the data acquisition system is Analyst 1.6.3;
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.
Wherein, in the second step, working solutions with various concentrations are prepared, and the preparation concentrations are respectively 30.00 mu g/mL, 10.00 mu g/mL, 4.000 mu g/mL, 1.000 mu g/mL, 0.4000 mu g/mL, 0.1600 mu g/mL, 0.08000 mu g/mL, 24.00 mu g/mL, 3.000 mu g/mL, 0.2400 mu g/mL and 0.08000 mu g/mL; in the third step, the concentration of the curve sample and the concentration of the quality control sample are set to be various, and the various concentrations are respectively 1500ng/mL, 500.0ng/mL, 200.0ng/mL, 50.00ng/mL, 20.00ng/mL, 8.000ng/mL, 4.000ng/mL, 1200ng/mL, 150.0ng/mL, 12.00ng/mL and 4.000 ng/mL; in the sixth step, the LC-MS/MS system consists of a TRIPLE QUADU 5500 type TRIPLE quadrupole tandem mass spectrometer (AB Sciex), a high-pressure pump (LC-30AD), an online degasser (DGU-20A-5R), an autosampler (SIL-30AC), a column oven (CTO-20A) (Shimadzu corporation) and a controller CBM-20 Alite; in the sixth step, the model number Welch is used,
XS-C18,3.0 μm,3.0 x 100mm column.
Example two
The invention provides a technical scheme that: a method for determining the concentration of captopril in human plasma comprising the steps of:
the method comprises the following steps: preparation of stock solutions, acetonitrile with 0.1% formic acid: dissolving captopril reference substance in water (9:1, v: v) as diluent to obtain captopril standard curve Stock solution (STD Stock-KTPL) and captopril quality control Stock solution (QC Stock-KTPL) with the concentration of 400 mu g/mL;
step two: preparing a working solution, wherein the standard curve working solution takes STD Stock-KTPL as an initial solution, and 0.1% formic acid in acetonitrile: diluting the solution of water (9:1, v: v) as a diluting solvent step by step to obtain working solution of standard curves of captopril at all levels; the quality control working solution takes QC Stock-KTPL as an initial solution, and the mixture of 0.1% formic acid in acetonitrile: diluting the solution of water (9:1, v: v) as a diluting solvent step by step to obtain all-stage quality control working solution of captopril;
step three: preparing a standard curve and a quality control sample, namely sucking 95% of EDTA-K2 anticoagulant human blank plasma by volume, adding 200mM Dithiothreitol (DTT) physiological saline solution by volume of 5% to prepare blank plasma containing 10mM DTT, taking 950 mu L of the blank plasma containing 10mM DTT, and adding 50 mu L of the working solution to obtain a curve sample and a quality control sample;
step four: preparation of internal standard working solution, using acetonitrile of 0.1% formic acid: diluting captopril-d 3 reference substance with water (1:1, v: v) as diluent to obtain captopril-d 3 internal standard working solution with the concentration of 500 ng/mL;
step five: pretreating a sample, namely putting 100L of a plasma sample into a 96-pore plate, adding 30uL of internal standard working solution, performing vortex for 8min, adding 300L of acetonitrile solution, performing vortex for 20min, performing high-speed centrifugation (4000rpm) for 30min, taking 180L of supernate into another 96-pore plate which is added with 180L of 0.1% formic acid aqueous solution in advance, uniformly mixing for 10min, performing LC-MS/MS analysis after 8min of high-speed centrifugation (4000rpm), and sampling the volume of 6L;
step six: LC-MS/MS analysis, the temperature of the column incubator and the sample injector are respectively set to 40 ℃ and 4 ℃; the chromatographic column is used as a separation column; the needle wash was methanol with 0.1% formic acid: water (1:1, v: v), wherein the needle washing mode is cleaning before and after sample injection, and the needle washing volume is 450L; mobile phase: 0.05% formic acid in water-0.05% formic acid in methanol (62:38), isocratic conditions, flow rate of 0.40mL/min to separate the analytes, analysis time of 3.8 minutes; the polarity of the ion source is in a negative mode, and the ion monitoring mode is multi-reaction monitoring (MRM); the ion pairs of captopril and the internal standard (deuterated captopril) are 215.9/181.9 and 219.1/185.1 respectively; after the electrical parameter optimization, the cluster removing voltage (DP) is minus 80V, and the collision voltage (CE) is 19V; the data acquisition system is Analyst 1.6.3;
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.
Wherein, in the second step, working solutions with various concentrations are prepared, and the preparation concentrations are respectively 30.00 mu g/mL, 10.00 mu g/mL, 4.000 mu g/mL, 1.000 mu g/mL, 0.4000 mu g/mL, 0.1600 mu g/mL, 0.08000 mu g/mL, 24.00 mu g/mL and 3.000 μ g/mL, 0.2400 μ g/mL, 0.08000 μ g/mL; in the third step, the concentration of the curve sample and the concentration of the quality control sample are set to be various, and the various concentrations are respectively 1500ng/mL, 500.0ng/mL, 200.0ng/mL, 50.00ng/mL, 20.00ng/mL, 8.000ng/mL, 4.000ng/mL, 1200ng/mL, 150.0ng/mL, 12.00ng/mL and 4.000 ng/mL; in the sixth step, the LC-MS/MS system consists of a TRIPLE QUADU 5500 type TRIPLE quadrupole tandem mass spectrometer (AB Sciex), a high-pressure pump (LC-30AD), an online degasser (DGU-20A-5R), an autosampler (SIL-30AC), a column oven (CTO-20A) (Shimadzu corporation) and a controller CBM-20 Alite; in the sixth step, the model number Welch is used,
XS-C18,3.0 μm,3.0 x 100mm column.
EXAMPLE III
The invention provides a technical scheme that: a method for determining the concentration of captopril in human plasma comprising the steps of:
the method comprises the following steps: preparation of stock solutions, acetonitrile with 0.1% formic acid: dissolving captopril reference substance in water (9:1, v: v) as diluent to obtain captopril standard curve Stock solution (STD Stock-KTPL) and captopril quality control Stock solution (QC Stock-KTPL) with the concentration of 400 mu g/mL;
step two: preparing a working solution, wherein the standard curve working solution takes STD Stock-KTPL as an initial solution, and 0.1% formic acid in acetonitrile: diluting the solution of water (9:1, v: v) as a diluting solvent step by step to obtain working solution of standard curves of captopril at all levels; the quality control working solution takes QC Stock-KTPL as an initial solution, and the mixture of 0.1% formic acid in acetonitrile: diluting the solution of water (9:1, v: v) as a diluting solvent step by step to obtain all-stage quality control working solution of captopril;
step three: preparing a standard curve and a quality control sample, namely sucking 95% of EDTA-K2 anticoagulant human blank plasma by volume, adding 200mM Dithiothreitol (DTT) physiological saline solution by volume of 5% to prepare blank plasma containing 10mM DTT, taking 950 mu L of the blank plasma containing 10mM DTT, and adding 50 mu L of the working solution to obtain a curve sample and a quality control sample;
step four: preparation of internal standard working solution, using acetonitrile of 0.1% formic acid: diluting captopril-d 3 reference substance with water (1:1, v: v) as diluent to obtain captopril-d 3 internal standard working solution with the concentration of 500 ng/mL;
step five: pretreating a sample, namely putting 100L of a plasma sample into a 96-pore plate, adding 30uL of internal standard working solution, performing vortex for 5min, adding 300L of acetonitrile solution, performing vortex for 14min, performing high-speed centrifugation (4000rpm) for 20min, taking 180L of supernate into another 96-pore plate which is added with 180L of 0.1% formic acid aqueous solution in advance, uniformly mixing for 6min, performing LC-MS/MS analysis after 5min of high-speed centrifugation (4000rpm), and sampling the volume of 6L;
step six: LC-MS/MS analysis, the temperature of the column incubator and the sample injector are respectively set to 40 ℃ and 4 ℃; the chromatographic column is used as a separation column; the needle wash was methanol with 0.1% formic acid: water (1:1, v: v), wherein the needle washing mode is cleaning before and after sample injection, and the needle washing volume is 450L; mobile phase: 0.05% formic acid in water-0.05% formic acid in methanol (62:38), isocratic conditions, flow rate of 0.40mL/min to separate the analytes, analysis time of 3.8 minutes; the polarity of the ion source is in a negative mode, and the ion monitoring mode is multi-reaction monitoring (MRM); the ion pairs of captopril and the internal standard (deuterated captopril) are 215.9/181.9 and 219.1/185.1 respectively; after the electrical parameter optimization, the cluster removing voltage (DP) is minus 80V, and the collision voltage (CE) is 19V; the data acquisition system is Analyst 1.6.3;
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.
Wherein, in the second step, working solutions with various concentrations are prepared, and the preparation concentrations are respectively 30.00 mu g/mL, 10.00 mu g/mL, 4.000 mu g/mL, 1.000 mu g/mL, 0.4000 mu g/mL, 0.1600 mu g/mL, 0.08000 mu g/mL, 24.00 mu g/mL, 3.000 mu g/mL, 0.2400 mu g/mL and 0.08000 mu g/mL; in the third step, the concentration of the curve sample and the concentration of the quality control sample are set to be various, and the various concentrations are respectively 1500ng/mL, 500.0ng/mL, 200.0ng/mL, 50.00ng/mL, 20.00ng/mL, 8.000ng/mL, 4.000ng/mL, 1200ng/mL, 150.0ng/mL, 12 ng/mL00ng/mL, 4.000 ng/mL; in the sixth step, the LC-MS/MS system consists of a TRIPLE QUADU 5500 type TRIPLE quadrupole tandem mass spectrometer (AB Sciex), a high-pressure pump (LC-30AD), an online degasser (DGU-20A-5R), an autosampler (SIL-30AC), a column oven (CTO-20A) (Shimadzu corporation) and a controller CBM-20 Alite; in the sixth step, the model number Welch is used,
XS-C18,3.0 μm,3.0 x 100mm column.
Example four
The invention provides a technical scheme that: a method for determining the concentration of captopril in human plasma comprising the steps of:
the method comprises the following steps: preparation of stock solutions, acetonitrile with 0.1% formic acid: dissolving captopril reference substance in water (9:1, v: v) as diluent to obtain captopril standard curve Stock solution (STD Stock-KTPL) and captopril quality control Stock solution (QC Stock-KTPL) with the concentration of 400 mu g/mL;
step two: preparing a working solution, wherein the standard curve working solution takes STD Stock-KTPL as an initial solution, and 0.1% formic acid in acetonitrile: diluting the solution of water (9:1, v: v) as a diluting solvent step by step to obtain working solution of standard curves of captopril at all levels; the quality control working solution takes QC Stock-KTPL as an initial solution, and the mixture of 0.1% formic acid in acetonitrile: diluting the solution of water (9:1, v: v) as a diluting solvent step by step to obtain all-stage quality control working solution of captopril;
step three: preparing a standard curve and a quality control sample, namely sucking 95% of EDTA-K2 anticoagulant human blank plasma by volume, adding 200mM Dithiothreitol (DTT) physiological saline solution by volume of 5% to prepare blank plasma containing 10mM DTT, taking 950 mu L of the blank plasma containing 10mM DTT, and adding 50 mu L of the working solution to obtain a curve sample and a quality control sample;
step four: preparation of internal standard working solution, using acetonitrile of 0.1% formic acid: diluting captopril-d 3 reference substance with water (1:1, v: v) as diluent to obtain captopril-d 3 internal standard working solution with the concentration of 500 ng/mL;
step five: pretreating a sample, namely putting 100L of a plasma sample into a 96-pore plate, adding 30uL of internal standard working solution, performing vortex for 7min, adding 300L of acetonitrile solution, performing vortex for 17min, performing high-speed centrifugation (4000rpm) for 26min, taking 180L of supernate into another 96-pore plate which is added with 180L of 0.1% formic acid aqueous solution in advance, uniformly mixing for 8min, performing LC-MS/MS analysis after 6min of high-speed centrifugation (4000rpm), and sampling the volume of 6L;
step six: LC-MS/MS analysis, the temperature of the column incubator and the sample injector are respectively set to 40 ℃ and 4 ℃; the chromatographic column is used as a separation column; the needle wash was methanol with 0.1% formic acid: water (1:1, v: v), wherein the needle washing mode is cleaning before and after sample injection, and the needle washing volume is 450L; mobile phase: 0.05% formic acid in water-0.05% formic acid in methanol (62:38), isocratic conditions, flow rate of 0.40mL/min to separate the analytes, analysis time of 3.8 minutes; the polarity of the ion source is in a negative mode, and the ion monitoring mode is multi-reaction monitoring (MRM); the ion pairs of captopril and the internal standard (deuterated captopril) are 215.9/181.9 and 219.1/185.1 respectively; after the electrical parameter optimization, the cluster removing voltage (DP) is minus 80V, and the collision voltage (CE) is 19V; the data acquisition system is Analyst 1.6.3;
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.
Wherein, in the second step, working solutions with various concentrations are prepared, and the preparation concentrations are respectively 30.00 mu g/mL, 10.00 mu g/mL, 4.000 mu g/mL, 1.000 mu g/mL, 0.4000 mu g/mL, 0.1600 mu g/mL, 0.08000 mu g/mL, 24.00 mu g/mL, 3.000 mu g/mL, 0.2400 mu g/mL and 0.08000 mu g/mL; in the third step, the concentration of the curve sample and the concentration of the quality control sample are set to be various, and the various concentrations are respectively 1500ng/mL, 500.0ng/mL, 200.0ng/mL, 50.00ng/mL, 20.00ng/mL, 8.000ng/mL, 4.000ng/mL, 1200ng/mL, 150.0ng/mL, 12.00ng/mL and 4.000 ng/mL; in the sixth step, the LC-MS/MS system consists of a TRIPLE QUADU 5500 TRIPLE quadrupole tandem mass spectrometer (AB Sciex), a high pressure pump (LC-30AD), an online degasser (DGU-20A-5R), an autosampler (SIL-30AC), a column oven (CTO-20A) (Shimadzu corporation), and a controller CBM-20AlThe composition of the white iron; in the sixth step, the model number Welch is used,
XS-C18,3.0 μm,3.0 x 100mm column.
The four groups of embodiments can realize the determination of captopril in human plasma, and the invention has the advantages that: compared with the prior chemical inhibitor addition method, the method for determining the captopril concentration in human plasma is more sensitive, and the lower limit of the quantification of the captopril is reduced from 10.00ng/mL to 4.000 ng/mL; meanwhile, the method has the advantages of small sample usage amount, less mobile phase consumption, reduced pretreatment process, increased sensitivity, improved reliability and durability, good linearity, and elimination of matrix effect, and reduced harm to the subject and the environment in the test process; when the method for determining the captopril concentration in human plasma provided by the invention is used for preparing the working solution, the acetonitrile of 0.1% formic acid is adopted: water (9:1, v: v) is used as a diluent to prepare working solution and stock solution, which can stabilize captopril in the solution and eliminate the adsorption of the captopril in a polypropylene centrifugal tube, so that other suitable diluent can achieve the technical effect; human plasma with the concentration of 10mM dithiothreitol can stabilize captopril in the human plasma, so that other inhibitors with the same efficacy can reduce the degradation rate of captopril in the plasma after being added to human plasma samples, and can stabilize the captopril concentration.
Wherein:
TABLE 1 concentration setting table for working fluid
TABLE 2 concentration table of curve sample and quality control sample
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 (5)
1. A method for determining the concentration of captopril in human plasma, comprising the steps of:
the method comprises the following steps: preparation of stock solutions, acetonitrile with 0.1% formic acid: dissolving captopril reference substance in water (9:1, v: v) as diluent to obtain captopril standard curve Stock solution (STD Stock-KTPL) and captopril quality control Stock solution (QC Stock-KTPL) with the concentration of 400 mu g/mL;
step two: preparing a working solution, wherein the standard curve working solution takes STD Stock-KTPL as an initial solution, and 0.1% formic acid in acetonitrile: diluting the solution of water (9:1, v: v) as a diluting solvent step by step to obtain working solution of standard curves of captopril at all levels; the quality control working solution takes QC Stock-KTPL as an initial solution, and the mixture of 0.1% formic acid in acetonitrile: diluting the solution of water (9:1, v: v) as a diluting solvent step by step to obtain all-stage quality control working solution of captopril;
step three: preparing a standard curve and a quality control sample, namely sucking 95% of EDTA-K2 anticoagulant human blank plasma by volume, adding 200mM Dithiothreitol (DTT) physiological saline solution by volume of 5% to prepare blank plasma containing 10mM DTT, taking 950 mu L of the blank plasma containing 10mM DTT, and adding 50 mu L of the working solution to obtain a curve sample and a quality control sample;
step four: preparation of internal standard working solution, using acetonitrile of 0.1% formic acid: diluting captopril-d 3 reference substance with water (1:1, v: v) as diluent to obtain captopril-d 3 internal standard working solution with the concentration of 500 ng/mL;
step five: pretreating a sample, namely putting 100L of a plasma sample into a 96-well plate, adding 30uL of internal standard working solution, performing vortex for 3-8min, adding 300L of acetonitrile solution, performing vortex for 10-20min, performing high-speed centrifugation (4000rpm) for 15-30min, putting 180L of supernate into another 96-well plate which is added with 180L of 0.1% formic acid aqueous solution in advance, uniformly mixing for 5-10min, performing LC-MS/MS analysis after high-speed centrifugation (4000rpm) for 3-8min, and sampling the volume to be 6L;
step six: LC-MS/MS analysis, the temperature of the column incubator and the sample injector are respectively set to 40 ℃ and 4 ℃; the chromatographic column is used as a separation column; the needle wash was methanol with 0.1% formic acid: water (1:1, v: v), wherein the needle washing mode is cleaning before and after sample injection, and the needle washing volume is 450L; mobile phase: 0.05% formic acid in water-0.05% formic acid in methanol (62:38), isocratic conditions, flow rate of 0.40mL/min to separate the analytes, analysis time of 3.8 minutes; the polarity of the ion source is in a negative mode, and the ion monitoring mode is multi-reaction monitoring (MRM); the ion pairs of captopril and the internal standard (deuterated captopril) are 215.9/181.9 and 219.1/185.1 respectively; after the electrical parameter optimization, the cluster removing voltage (DP) is minus 80V, and the collision voltage (CE) is 19V; the data acquisition system is Analyst 1.6.3;
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 of claim 1, wherein the captopril concentration in human plasma is determined by: in the second step, working solutions with various concentrations are prepared, and the preparation concentrations are respectively 30.00 mu g/mL, 10.00 mu g/mL, 4.000 mu g/mL, 1.000 mu g/mL, 0.4000 mu g/mL, 0.1600 mu g/mL, 0.08000 mu g/mL, 24.00 mu g/mL, 3.000 mu g/mL, 0.2400 mu g/mL and 0.08000 mu g/mL.
3. The method of claim 1, wherein the captopril concentration in human plasma is determined by: in the third step, the concentration of the curve sample and the concentration of the quality control sample are set to be various, and the various concentrations are respectively 1500ng/mL, 500.0ng/mL, 200.0ng/mL, 50.00ng/mL, 20.00ng/mL, 8.000ng/mL, 4.000ng/mL, 1200ng/mL, 150.0ng/mL, 12.00ng/mL and 4.000 ng/mL.
4. The method of claim 1, wherein the captopril concentration in human plasma is determined by: in the sixth step, the LC-MS/MS system consisted of a TRIPLE QUADU 5500 TRIPLE quadrupole tandem mass spectrometer (AB Sciex), a high pressure pump (LC-30AD), an online degasser (DGU-20A-5R), an autosampler (SIL-30AC), a column oven (CTO-20A) (Shimadzu corporation), and a controller CBM-20 Alite.
5. The method of claim 1, wherein the captopril concentration in human plasma is determined by: in the sixth step, the model number Welch is used,
XS-C18,3.0 μm,3.0 x 100mm column.
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Application publication date: 20200904 |