CN111879875A - Method for determining cytarabine and uridine arabinoside in blood plasma - Google Patents
Method for determining cytarabine and uridine arabinoside in blood plasma Download PDFInfo
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- CN111879875A CN111879875A CN202010784724.5A CN202010784724A CN111879875A CN 111879875 A CN111879875 A CN 111879875A CN 202010784724 A CN202010784724 A CN 202010784724A CN 111879875 A CN111879875 A CN 111879875A
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- G—PHYSICS
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- 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
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- G—PHYSICS
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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
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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
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Abstract
The invention relates to the technical field of medical detection, in particular to a method for determining cytarabine and uridine arabinoside in blood plasma, which comprises the following steps: preparing a cytarabine stock solution; step two: preparing an arabinouridine stock solution; step three: preparing a working solution; step four: preparing a standard curve and a quality control sample; step five: preparing an internal standard working solution; step six: sample pretreatment; step seven: LC-MS/MS analysis process; step eight: the comparison verifies that the beneficial effects are as follows: the detection method adopted by the invention can simultaneously determine the blood concentration in the plasma of cytarabine and uridine arabinoside, adopts a protein precipitation method to process a sample, and has the advantages of convenient operation, high sensitivity, accurate measurement and stable and reliable method; the mode through setting up synchronous survey can satisfy clinical big batch sample test demand, simultaneously through setting up standard curve and sample, realizes hierarchical contrast, can clearly audio-visual understanding survey scope.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a method for determining cytarabine and uridine arabinoside in blood plasma.
Background
Cytarabine is a pyrimidine antimetabolite acting mainly on the proliferation phase of cells S and is used for treating various types of leukemia, including acute granulocytic leukemia and meningeal leukemia, and at present, the research on pharmacokinetics and bioequivalence of drugs is evaluated by measuring the pharmacokinetic parameters of the drugs. Therefore, the simultaneous determination of the blood concentration of cytarabine and uridine in human plasma has important significance for reasonable use of cytarabine.
The LC-MS/MS method for determining the concentrations of cytarabine and uridine in human plasma has the advantages of low sensitivity, long analysis time, large sample amount, narrow linear range or only measuring the concentration of a single analyte of cytarabine, and cannot meet the requirement of biological analysis of a large batch of samples for simultaneously detecting the blood concentrations of cytarabine and uridine.
Therefore, the method for measuring cytarabine and uridine arabinoside in the plasma is provided, and the problems of sensitivity, convenience and cost of plasma measurement are solved.
Disclosure of Invention
The present invention aims to provide a method for measuring cytarabine and uridine in plasma, which solves the problems of the background art.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for determining cytarabine and uridine arabinoside in plasma, comprising the steps of:
the method comprises the following steps: preparing cytarabine Stock solutions, precisely weighing 2 parts of cytarabine reference substances respectively, dissolving and shaking up by using 50% acetonitrile, and preparing the cytarabine Stock solutions with the concentration of 1.000mg/mL, wherein the cytarabine Stock solutions are respectively marked as cytarabine-STD-Stock and cytarabine-QC-Stock;
step two: preparing an arabinouridine Stock solution, precisely weighing 2 parts of an arabinouridine reference substance respectively, dissolving and shaking the arabinouridine Stock solution by using 50% acetonitrile to prepare the arabinouridine Stock solution with the concentration of 1.000mg/mL, and marking the arabinouridine Stock solution as an arabinouridine-STD-Stock and an arabinouridine-QC-Stock respectively;
step three: preparing a working solution, wherein the working solution is divided into a standard curve working solution and a quality control working solution, the standard curve working solution takes cytarabine-STD-Stock and uridine-STD-Stock as initial solutions, and ultrapure water as a diluting solvent is used for stepwise dilution to obtain various levels of standard curve working solutions of cytarabine and uridine; taking cytarabine-QC-Stock and uridine-QC-Stock as initial solutions and ultrapure water as a diluting solvent to dilute the quality control working solution step by step to obtain all-level quality control working solutions of cytarabine and uridine;
step four: preparing a standard curve and a quality control sample, namely sucking 90% of human blank serum by volume, and adding 10% of standard curves of all levels and working solution of all levels of quality control concentration in the step III to obtain a curve sample and a quality control sample;
step five: preparing an internal standard working solution, namely diluting an ultrapure deuterium-substituted cytarabine reference substance by using ultrapure water as a diluent to obtain the internal standard working solution with the concentration of 100.0 ng/mL;
step six: pre-treating the sample, namely putting 150 mu L of plasma sample into a 96-well plate centrifuge tube, adding 30 mu L of the internal standard solution in the fifth step, adding 450 mu L of precipitator methanol, performing vortex for 10min, and performing high-speed centrifugation (4000rpm) for 15 min;
step seven: analyzing the supernatant extracted in the sixth step by an LC-MS/MS system to obtain a mass spectrum and a chromatographic analysis table of cytarabine and uridine;
step eight: and (4) contrast verification, comparing the chromatographic and mass spectrometric analysis results obtained in the step seven with samples of all levels obtained in the step four, and verifying the method according to the requirements of 'biological sample quantitative analysis method verification guiding principle' in the Chinese pharmacopoeia 2015 edition.
Preferably, the supernatant obtained in the seventh step is obtained after centrifugation in the fifth step, 250 μ L of the supernatant is taken to perform LC-MS/MS analysis in another 96-deep-well plate, and the injection volume is 0.5 μ L.
Preferably, the LC-MS/MS system in step seven consists of an AB SCIEX QTRAP 5500 type triple quadrupole tandem mass spectrometer, a high pressure pump, an in-line degasser, an autosampler, and a column oven.
Preferably, the contents of the verification in step eight include selectivity, standard curve, lower limit of quantitation, precision and accuracy, extraction recovery, matrix effect, dilution effect, residual effect, stability and sample volume.
Preferably, said cytarabine-STD-Stock, cytarabine-QC-Stock, uridine-STD-Stock and uridine-QC-Stock obtained in step one and step two are stored at 4 ℃.
Compared with the prior art, the invention has the beneficial effects that:
1. the detection method adopted by the invention can simultaneously determine the blood concentration in the plasma of cytarabine and uridine arabinoside, adopts a protein precipitation method to process a sample, and has the advantages of convenient operation, high sensitivity, accurate measurement and stable and reliable method;
2. the invention can meet the requirement of clinical large-scale sample detection by setting a synchronous measurement mode, and realizes grading comparison by setting a standard curve and a sample, thereby clearly and intuitively knowing the measurement range.
Drawings
FIG. 1 is a table of working fluid preparation concentrations of the present invention;
FIG. 2 is a standard sample concentration table of the present invention;
FIG. 3 is a table of chromatographic conditions according to the invention;
FIG. 4 is a table of mass spectrometry conditions according to the present invention;
FIG. 5 is a flow chart of a measurement method of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in 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.
Referring to fig. 1 to 5, the present invention provides a technical solution:
a method for determining cytarabine and uridine arabinoside in plasma, comprising the steps of:
the method comprises the following steps: preparing cytarabine Stock solutions, precisely weighing 2 parts of cytarabine reference substances respectively, dissolving and shaking up by using 50% acetonitrile, and preparing the cytarabine Stock solutions with the concentration of 1.000mg/mL, wherein the cytarabine Stock solutions are respectively marked as cytarabine-STD-Stock and cytarabine-QC-Stock;
step two: preparing an arabinouridine Stock solution, precisely weighing 2 parts of an arabinouridine reference substance respectively, dissolving and shaking the arabinouridine Stock solution by using 50% acetonitrile to prepare an arabinouridine Stock solution with the concentration of 1.000mg/mL, and respectively marking the arabinouridine Stock solution as an arabinouridine-STD-Stock and an arabinouridine Stock solution as a QC-Stock to obtain the arabinocytidine-STD-Stock, the arabinocytidine-QC-Stock, the arabinouridine-STD-Stock and the arabinouridine-QC-Stock which are all stored in an environment at 4 ℃;
step three: preparing a working solution, wherein the working solution is divided into a standard curve working solution and a quality control working solution, the standard curve working solution takes cytarabine-STD-Stock and uridine-STD-Stock as initial solutions, and ultrapure water as a diluting solvent is used for stepwise dilution to obtain various levels of standard curve working solutions of cytarabine and uridine; taking cytarabine-QC-Stock and uridine-QC-Stock as initial solutions and ultrapure water as a diluting solvent to dilute the quality control working solution step by step to obtain all-level quality control working solutions of cytarabine and uridine;
step four: preparing a standard curve and a quality control sample, namely sucking 90% of human blank serum by volume, and adding 10% of standard curves of all levels and working solution of all levels of quality control concentration in the step III to obtain a curve sample and a quality control sample;
step five: preparing an internal standard working solution, namely diluting an ultrapure deuterium-substituted cytarabine reference substance by using ultrapure water as a diluent to obtain the internal standard working solution with the concentration of 100.0 ng/mL;
step six: pre-treating the sample, namely putting 150 mu L of plasma sample into a 96-well plate centrifuge tube, adding 30 mu L of the internal standard solution in the fifth step, adding 450 mu L of precipitator methanol, performing vortex for 10min, and performing high-speed centrifugation at 4000rpm for 15 min;
step seven: an LC-MS/MS analysis process, wherein an LC-MS/MS system consists of an AB SCIEX QTRAP 5500 type triple quadrupole tandem mass spectrometer, a high-pressure pump, an online degasser, an automatic sample injector and a column incubator, the supernatant extracted in the sixth step is analyzed by the LC-MS/MS system, the supernatant is obtained after the fifth step is centrifuged, 250 mu L of the supernatant is taken to be placed in another 96 deep-well plate for LC-MS/MS analysis, and the sample injection volume is 0.5 mu L, so that the mass spectrum and the chromatographic analysis table of cytarabine and uridine are obtained;
step eight: and (3) contrast verification, comparing the chromatographic and mass spectrometric analysis results obtained in the step seven with samples of all levels obtained in the step four, and verifying the method according to the requirements of 'biological sample quantitative analysis method verification guiding principle' in the Chinese pharmacopoeia 2015 edition, wherein the verification contents comprise selectivity, a standard curve, a quantitative lower limit, precision and accuracy, an extraction recovery rate, a matrix effect, a dilution effect, a residual effect, stability and sample capacity.
The working principle is as follows: firstly, precisely weighing 2 parts of each reference substance of cytarabine and uridine respectively, dissolving and shaking up with 50% acetonitrile respectively to prepare a 1.000mg/mL stock solution of uridine arabinoside and uridine arabinoside, wherein the four stock solutions are respectively marked as: cytarabine-STD-Stock, cytarabine-QC-Stock, uridine-STD-Stock and uridine-QC-Stock.
The working solution is divided into a standard curve working solution and a quality control working solution, the standard curve working solution takes cytarabine-STD-Stock and cytarabine-STD-Stock as initial solutions, and ultrapure water as a diluting solvent to dilute step by step to obtain each-level standard curve working solution of cytarabine and cytarabine; and diluting the quality control working solution by taking cytarabine-QC-Stock and cytarabine-QC-Stock as initial solutions and ultrapure water as a diluting solvent step by step to obtain all levels of quality control working solutions of cytarabine and cytarabine, then sucking human blank serum with the volume of 90%, and adding the standard curves and the working solutions with the quality control concentrations of all levels with the volume of 10% to obtain curve samples and quality control samples.
And (3) diluting with ultrapure water deuterated cytarabine reference substance by using ultrapure water as a diluent to obtain an internal standard working solution with the concentration of 100.0ng/mL, putting 150 mu L of a plasma sample into a centrifuge tube of a 96-well plate, adding 30 mu L of the internal standard solution obtained in the fifth step, adding 450 mu L of precipitator methanol, performing vortex for 10min, performing high-speed centrifugation at 4000rpm for 15 min, putting 250 mu L of supernate into another 96-deep-well plate, performing LC-MS/MS analysis, and obtaining a sample injection volume of 0.5 mu L to obtain a mass spectrum and a chromatographic analysis table of cytarabine and uridine.
Comparing the obtained chromatographic and mass spectrometric analysis results with samples of all levels, verifying the method according to the requirements of the verification guiding principle of the quantitative analysis method of biological samples in the Chinese pharmacopoeia 2015 edition, wherein the verification contents comprise selectivity, a standard curve, a lower limit of quantitation, precision and accuracy, an extraction recovery rate, a matrix effect, a dilution effect, a residual effect, stability and sample capacity.
Through verification, the method meets the technical requirements of 'biological sample quantitative analysis method verification guiding principle' in the Chinese pharmacopoeia 2015 edition, and can be used for quantitative analysis of the concentrations of cytarabine and uridine in human plasma. The method has high sensitivity (cytarabine LLOQ is 2.000ng/mL, and uridine arabinoside is 20.00ng/mL), although the existing LC-MS/MS method reduces the lower limit of the quantitation of cytarabine and uridine arabinoside to 0.5000ng/mL and 2.08ng/mL respectively, the cytarabine (LLOQ 0.5000ng/mL) adopts the SPE method to process samples, the operation is complex, and only a single uridine arabinoside compound is detected, so that the requirement of processing large-batch samples is not met; uridine arabinoside (LLOQ 2.08ng/mL) was assayed as rat plasma. The method is a sample processing mode, is a detection method for simultaneously determining the blood concentration of human plasma containing cytarabine and uridine arabinoside, adopts a protein precipitation method to process samples, is convenient to operate, is stable and reliable, and can meet the detection requirement of clinical large-batch samples.
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 cytarabine and uridine arabinoside in plasma, comprising: the determination method comprises the following steps:
the method comprises the following steps: preparing cytarabine Stock solutions, precisely weighing 2 parts of cytarabine reference substances respectively, dissolving and shaking up by using 50% acetonitrile, and preparing the cytarabine Stock solutions with the concentration of 1.000mg/mL, wherein the cytarabine Stock solutions are respectively marked as cytarabine-STD-Stock and cytarabine-QC-Stock;
step two: preparing an arabinouridine Stock solution, precisely weighing 2 parts of an arabinouridine reference substance respectively, dissolving and shaking the arabinouridine Stock solution by using 50% acetonitrile to prepare the arabinouridine Stock solution with the concentration of 1.000mg/mL, and marking the arabinouridine Stock solution as an arabinouridine-STD-Stock and an arabinouridine-QC-Stock respectively;
step three: preparing a working solution, wherein the working solution is divided into a standard curve working solution and a quality control working solution, the standard curve working solution takes cytarabine-STD-Stock and uridine-STD-Stock as initial solutions, and ultrapure water as a diluting solvent is used for stepwise dilution to obtain various levels of standard curve working solutions of cytarabine and uridine; taking cytarabine-QC-Stock and uridine-QC-Stock as initial solutions and ultrapure water as a diluting solvent to dilute the quality control working solution step by step to obtain all-level quality control working solutions of cytarabine and uridine;
step four: preparing a standard curve and a quality control sample, namely sucking 90% of human blank serum by volume, and adding 10% of standard curves of all levels and working solution of all levels of quality control concentration in the step III to obtain a curve sample and a quality control sample;
step five: preparing an internal standard working solution, namely diluting an ultrapure deuterium-substituted cytarabine reference substance by using ultrapure water as a diluent to obtain the internal standard working solution with the concentration of 100.0 ng/mL;
step six: pre-treating the sample, namely putting 150 mu L of plasma sample into a 96-well plate centrifuge tube, adding 30 mu L of the internal standard solution in the fifth step, adding 450 mu L of precipitator methanol, performing vortex for 10min, and performing high-speed centrifugation (4000rpm) for 15 min;
step seven: analyzing the supernatant extracted in the sixth step by an LC-MS/MS system to obtain a mass spectrum and a chromatographic analysis table of cytarabine and uridine;
step eight: and (4) contrast verification, comparing the chromatographic and mass spectrometric analysis results obtained in the step seven with samples of all levels obtained in the step four, and verifying the method according to the requirements of 'biological sample quantitative analysis method verification guiding principle' in the Chinese pharmacopoeia 2015 edition.
2. The method for assaying cytarabine and uridine in plasma according to claim 1, wherein: and step seven, centrifuging the supernatant obtained in the step five, taking 250 mu L of the supernatant to perform LC-MS/MS analysis in another 96 deep-well plate, wherein the injection volume is 0.5 mu L.
3. The method for assaying cytarabine and uridine in plasma according to claim 1, wherein: and the LC-MS/MS system in the seventh step consists of an AB SCIEX QTRAP 5500 type triple quadrupole tandem mass spectrometer, a high-pressure pump, an online degasser, an automatic sample injector and a column incubator.
4. The method for assaying cytarabine and uridine in plasma according to claim 1, wherein: and step eight, verifying the contents of the verification, wherein the contents comprise selectivity, a standard curve, a lower quantitative limit, precision and accuracy, extraction recovery rate, matrix effect, dilution effect, residual effect, stability and sample volume.
5. The method for assaying cytarabine and uridine in plasma according to claim 1, wherein: and D, storing the cytarabine-STD-Stock, the cytarabine-QC-Stock, the cytarabine-STD-Stock and the cytarabine-QC-Stock obtained in the first step and the second step in an environment at 4 ℃.
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