CN115308328A - Pretreatment kit for detecting multiple immunosuppressant drugs in human whole blood and use method thereof - Google Patents
Pretreatment kit for detecting multiple immunosuppressant drugs in human whole blood and use method thereof Download PDFInfo
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- CN115308328A CN115308328A CN202210946553.0A CN202210946553A CN115308328A CN 115308328 A CN115308328 A CN 115308328A CN 202210946553 A CN202210946553 A CN 202210946553A CN 115308328 A CN115308328 A CN 115308328A
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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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
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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
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Abstract
The invention discloses a pretreatment kit for detecting various immunosuppressant drugs in human whole blood, which is applied to the release of various immunosuppressant drugs such as cyclosporine A, tacrolimus, everolimus, rapamycin and the like in a human whole blood sample, and is convenient for the subsequent LC-MS/MS detection, the kit comprises a solution A and a solution C, wherein: the solution A comprises zinc sulfate which is used for breaking red blood cells in a whole blood sample and releasing a target compound, meanwhile, heavy metal salt can denature protein, and the solution C comprises methanol which is used for redissolving supernatant and reducing solvent effect. The zinc sulfate can break red blood cells in a whole blood sample and release a target compound, meanwhile, the heavy metal salt can denature proteins, the reagent can safely and effectively separate out and precipitate the proteins, the whole blood is simpler and more convenient to treat compared with a solid phase extraction method, and the treated supernatant is re-dissolved by adding methanol, so that the solvent effect can be effectively reduced, the detection time is shortened, and the detection flux is effectively improved.
Description
Technical Field
The invention relates to the technical field of trace compound detection, in particular to a pretreatment kit for detecting multiple immunosuppressant drugs in human whole blood and a using method thereof.
Background
Immunosuppressant drugs are a class of biological or chemical substances that alleviate tissue damage by suppressing cellular and humoral immune responses, and are currently widely used in the treatment of organ transplant rejection resistance and autoimmune diseases.
The defects of the prior art are as follows:
at present, various immunosuppressant drug detection kits based on a chemiluminescence method on the market mostly directly detect the content of immunosuppressant drugs in human whole blood, but are limited by the sensitivity and specificity of methodology, so that the detection result cannot accurately reflect the content of immunosuppressant drugs in human whole blood, and more effective information cannot be provided for clinical diagnosis.
Disclosure of Invention
The invention aims to provide a pretreatment kit for detecting various immunosuppressant drugs in human whole blood and a using method thereof, so as to solve the problems in the background technology.
The utility model provides a detect preliminary treatment kit of multiple immunosuppressant medicine in human whole blood, this preliminary treatment kit is applied to the release of multiple immunosuppressant medicine such as cyclosporine A, tacrolimus, everolimus, rapamycin in the human whole blood sample, is convenient for subsequent LC-MS/MS's detection, this kit includes liquid A and C, wherein:
the solution A comprises zinc sulfate which is used for breaking red blood cells in a whole blood sample, releasing a target compound and simultaneously enabling protein denaturation by heavy metal salt;
the solution C comprises methanol for redissolving the supernatant and reducing the solvent effect.
The invention also discloses a use method of the pretreatment kit for detecting various immunosuppressant drugs in human whole blood, and the method comprises the following steps: s1, uniformly mixing by primary oscillation; s2, centrifuging; s3, adding the solution C; s4, secondary oscillation, wherein:
s1, primary oscillation and uniform mixing: measuring a quantitative immunosuppressant marking 1-8 and quality control and internal standard stock solution, precisely absorbing 40-45 mu L of a whole blood sample or marking or quality control into a 1.5mL centrifuge tube, adding 180-220 mu L A solution and 380-420 mu L of internal standard working solution, oscillating for 5 minutes at room temperature, and rotating at 1000-1100rpm;
s2, centrifugal treatment: placing the mixture oscillated in the step S1 into a centrifugal device for centrifugation, wherein: the centrifugal temperature is 4-7 ℃, and the rotating speed is 1000-1400rpm;
s3, adding liquid C: sucking 90-120 mu L of supernatant of the mixed solution after centrifugation in the step S2 into a 96-well plate, and adding 100-110 mu L C solution;
s4, secondary oscillation: and (4) oscillating the mixed solution added with the solution C in the step S3 for 5 minutes at the rotating speed of 1000-1100rpm.
As a further improvement of the method, the immunosuppressant marked lines 1-8, the quality control and internal standard stock solutions measured in the step S1 of the method need to be recovered to room temperature first, and are subjected to oscillation and uniform mixing treatment.
As a further improvement of the invention, the internal standard working solution in step S1 of the method is prepared by diluting an internal standard storage solution with methanol.
As a further improvement of the method, 180-220 mu L A liquid and 380-420 mu L of internal standard working liquid are added in the step S1 of the method, and then the mixture is shaken up manually, treated by ultrasonic vibration for 30 seconds and then subjected to oscillation treatment.
As a further improvement of the invention, the oscillation treatment in steps S1 and S4 of the method adopts vortex oscillation of a vortex oscillator.
As a further improvement of the invention, in the method S4, after the oscillation is finished, the computer is used for detection by adopting an LC-MS method.
As a further improvement of the invention, the solution A and the solution C are both packaged by adopting a lightproof glass bottle.
As a further improvement of the invention, the standard curve C1-C8 is that the linear range of the cyclosporine A is as follows: 10-1000 ng/mL, wherein the linear range of everolimus is as follows: 2-200 ng/mL, and the linear range of tacrolimus is as follows: 2-200 ng/mL, wherein the linear range of sirolimus is as follows: 2-200 ng/mL;
the concentration of the quality control products LQC, MQC and HQC, which are cyclosporine A, is as follows: 20. 400 and 800ng/mL, and the concentration of everolimus is as follows: 4. 80 and 160ng/mL, and the concentration of tacrolimus is as follows: 4. 80 and 160ng/mL, wherein the concentration of sirolimus is as follows: 4. 80 and 160ng/mL;
the concentration of the internal standard working solution is that the concentration of the cyclosporine A internal standard is as follows: 200ng/mL, and the concentration of everolimus internal standard is as follows: 20ng/mL, tacrolimus internal standard concentration: 20ng/mL, and the sirolimus internal standard concentration is as follows: 20ng/mL.
Compared with the prior art, the invention has the beneficial effects that:
1. the solution A comprises zinc sulfate which is used for crushing red blood cells in a whole blood sample and releasing a target compound, and simultaneously heavy metal salt can denature protein, the reagent can safely and effectively separate out and precipitate protein, and the reagent is simpler and more convenient to treat the whole blood compared with a solid phase extraction method;
2. the solution C comprises methanol for redissolving the supernatant and reducing the solvent effect, is quick and simple, removes the previous complicated pretreatment steps, shortens the detection time, effectively improves the detection flux, has the remarkable advantages of less material consumption, low cost, good specificity, high sensitivity and lower detection limit, and has wider application prospect.
Drawings
FIG. 1 is a flow chart of a pretreatment kit for detecting various immunosuppressant drugs in human whole blood and a using method thereof.
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.
Example 1
Referring to fig. 1, the present invention provides the following technical solutions: the pretreatment kit is applied to the release of various immunosuppressant drugs such as cyclosporine A, tacrolimus, everolimus, rapamycin and the like in a human whole blood sample, and is convenient for the subsequent LC-MS/MS detection, the kit comprises a solution A and a solution C, wherein:
the solution A comprises zinc sulfate which is used for breaking red blood cells in a whole blood sample, releasing a target compound and simultaneously enabling protein denaturation by heavy metal salt;
the solution C comprises methanol for redissolving supernatant and reducing solvent effect;
and packaging the solution A and the solution C by adopting opaque glass bottles.
The invention also provides a use method of the pretreatment kit for detecting various immunosuppressant drugs in human whole blood, and the method comprises the following steps:
s1, oscillating and mixing uniformly for the first time,
measuring a quantitative immunosuppressant marked line 1-8, quality control and internal standard stock solution, firstly recovering to room temperature, oscillating and uniformly mixing, and diluting the internal standard stock solution with methanol to obtain an internal standard working solution;
wherein, the standard curve C1-C8 is that the linear range of the cyclosporine A is as follows: 10-1000 ng/mL, wherein the linear range of everolimus is as follows: 2-200 ng/mL, and the linear range of tacrolimus is as follows: 2-200 ng/mL, wherein the linear range of sirolimus is as follows: 2-200 ng/mL;
the concentrations of the quality control products LQC, MQC and HQC for cyclosporine A are as follows: 20. 400 and 800ng/mL, and the concentration of everolimus is as follows: 4. 80 and 160ng/mL, and the concentration of tacrolimus is as follows: 4. 80 and 160ng/mL, wherein the concentration of sirolimus is as follows: 4. 80 and 160ng/mL;
the concentration of the internal standard working solution is that the concentration of the cyclosporine A internal standard is as follows: 200ng/mL, and the concentration of everolimus internal standard is as follows: 20ng/mL, tacrolimus internal standard concentration: 20ng/mL, sirolimus internal standard concentration: 20ng/mL;
precisely absorbing 40 mu L of whole blood sample or marked line or quality control into a 1.5mL centrifuge tube, adding 180 mu L of LA liquid and 380 mu L of internal standard working solution, shaking up by hand, carrying out ultrasonic vibration treatment for 30 seconds, carrying out vortex vibration for 5 minutes by adopting a vortex oscillator at room temperature, wherein the rotating speed is 1000rpm;
s2, carrying out centrifugal treatment,
placing the mixture oscillated in the step S1 into a centrifugal device for centrifugation, wherein: the centrifugation temperature is 4 ℃, and the rotation speed is 1000rpm;
and S3, adding the solution C.
Sucking 90 mu L of supernatant of the mixed solution after centrifugation in the step S2 into a 96-well plate, and adding 100 mu L C solution;
s4, carrying out secondary oscillation,
oscillating the mixed solution added with the solution C in the step S3 for 5 minutes at the rotating speed of 1000rpm;
and after the oscillation is finished, the computer is used for detecting by adopting an LC-MS method.
Example 2
Referring to fig. 1, the present invention provides the following technical solutions: the pretreatment kit is applied to the release of various immunosuppressant drugs such as cyclosporine A, tacrolimus, everolimus, rapamycin and the like in a human whole blood sample, and is convenient for the subsequent LC-MS/MS detection, the kit comprises a solution A and a solution C, wherein:
the solution A comprises zinc sulfate which is used for breaking red blood cells in a whole blood sample, releasing a target compound and simultaneously enabling protein denaturation by heavy metal salt;
the solution C comprises methanol for redissolving supernatant and reducing solvent effect;
and both the solution A and the solution C are packaged by adopting opaque glass bottles.
The invention also provides a use method of the pretreatment kit for detecting various immunosuppressant drugs in human whole blood, and the method comprises the following steps:
s1, oscillating and mixing uniformly for the first time,
measuring a quantitative immunosuppressant marked line 1-8, quality control and internal standard stock solution, firstly recovering to room temperature, oscillating and uniformly mixing, and diluting the internal standard stock solution with methanol to obtain an internal standard working solution;
wherein, the standard curve C1-C8 is that the linear range of the cyclosporine A is as follows: 10-1000 ng/mL, wherein the linear range of everolimus is as follows: 2-200 ng/mL, and the linear range of tacrolimus is as follows: 2-200 ng/mL, wherein the linear range of sirolimus is as follows: 2-200 ng/mL;
the concentrations of the quality control products LQC, MQC and HQC for cyclosporine A are as follows: 20. 400 and 800ng/mL, and the concentration of everolimus is as follows: 4. 80 and 160ng/mL, and the concentration of tacrolimus is as follows: 4. 80 and 160ng/mL, wherein the concentration of sirolimus is as follows: 4. 80 and 160ng/mL;
the concentration of the internal standard working solution is that of the cyclosporine A internal standard: 200ng/mL, and the concentration of everolimus internal standard is as follows: 20ng/mL, tacrolimus internal standard concentration: 20ng/mL, and the sirolimus internal standard concentration is as follows: 20ng/mL;
precisely absorbing 45 mu L of a whole blood sample or a marked line or a quality control into a 1.5mL centrifuge tube, adding 180-220 mu L A liquid and 400 mu L of an internal standard working solution, shaking up by hand, carrying out ultrasonic vibration treatment for 30 seconds, carrying out vortex vibration for 5 minutes by adopting a vortex oscillator at room temperature and the rotating speed of 1100rpm;
s2, carrying out centrifugal treatment on the mixture,
placing the mixture oscillated in the step S1 into a centrifugal device for centrifugation, wherein: the centrifugal temperature is 7 ℃, and the rotating speed is 1400rpm;
and S3, adding the solution C.
Sucking 90 mu L of supernatant of the mixed solution after centrifugation in the step S2 into a 96-well plate, and adding 110 mu L C solution;
s4, carrying out secondary oscillation,
oscillating the mixed solution added with the solution C in the step S3 for 5 minutes at the rotating speed of 1100rpm;
and after the oscillation is finished, the computer is used for detecting by adopting an LC-MS method.
In summary, in the invention, the solution A comprises zinc sulfate which is used for crushing red blood cells in a whole blood sample and releasing a target compound, and simultaneously heavy metal salt can denature protein, the reagent can safely and effectively precipitate protein, compared with a solid phase extraction method, the whole blood is simpler and more convenient to process, the solution C comprises methanol which is used for redissolving a supernatant and reducing a solvent effect, the reagent is quick and simple, the previous complicated pretreatment steps are removed, the detection time is shortened, the detection flux is effectively improved, the used consumable materials are few, the cost is low, the reagent has the remarkable advantages of good specificity, high sensitivity and lower detection limit, and the reagent has a wider application prospect.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
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 (9)
1. The utility model provides a detect preliminary treatment kit of multiple immunosuppressant medicine in human whole blood, this preliminary treatment kit is applied to the release of multiple immunosuppressant medicine such as cyclosporine A, tacrolimus, everolimus, rapamycin in the human whole blood sample, is convenient for subsequent detection of LC-MS/MS, its characterized in that: the kit comprises a solution A and a solution C, wherein:
the solution A comprises zinc sulfate which is used for breaking red blood cells in a whole blood sample, releasing a target compound and simultaneously enabling protein denaturation by heavy metal salt;
the solution C comprises methanol for redissolving the supernatant and reducing the solvent effect.
2. The use method of the pretreatment kit for detecting multiple immunosuppressant drugs in human whole blood according to claim 1, characterized in that: the method comprises the following steps: s1, uniformly mixing by primary oscillation; s2, centrifuging; s3, adding the solution C; s4, secondary oscillation, wherein:
s1, primary oscillation and uniform mixing: measuring a quantitative immunosuppressant marking 1-8 and quality control and internal standard stock solution, precisely absorbing 40-45 mu L of a whole blood sample or marking or quality control into a 1.5mL centrifuge tube, adding 180-220 mu L A solution and 380-420 mu L of internal standard working solution, oscillating for 5 minutes at room temperature, and rotating at 1000-1100rpm;
s2, centrifugal treatment: placing the mixture oscillated in the step S1 into a centrifugal device for centrifugation, wherein: the centrifugal temperature is 4-7 ℃, and the rotating speed is 1000-1400rpm;
s3, adding liquid C: sucking 90-120 mu L of supernatant of the mixed solution after centrifugation in the step S2 into a 96-well plate, and adding 100-110 mu L C solution;
s4, secondary oscillation: and (4) oscillating the mixed solution added with the solution C in the step S3 for 5 minutes at the rotating speed of 1000-1100rpm.
3. The use method of the pretreatment kit for detecting multiple immunosuppressant drugs in human whole blood according to claim 2, characterized in that: in the method, the immunosuppressant marked lines 1-8 and the quality control and internal standard stock solutions measured in the step S1 need to be recovered to room temperature first, and are subjected to oscillation and uniform mixing treatment.
4. The use method of the pretreatment kit for detecting multiple immunosuppressant drugs in human whole blood according to claim 2, characterized in that: the internal standard working solution in the step S1 of the method is prepared by diluting an internal standard storage solution with methanol.
5. The use method of the pretreatment kit for detecting multiple immunosuppressant drugs in human whole blood according to claim 2, characterized in that: in the step S1 of the method, 180-220 mu L A liquid and 380-420 mu L of internal standard working solution are added, then the mixture is manually shaken up, ultrasonically vibrated for 30 seconds and then vibrated.
6. The pretreatment kit for detecting multiple immunosuppressant drugs in human whole blood according to claim 2 and the use method thereof, wherein: in the method, vortex oscillation of a vortex oscillator is adopted in the oscillation processing in the steps S1 and S4.
7. The use method of the pretreatment kit for detecting multiple immunosuppressant drugs in human whole blood according to claim 2, characterized in that: in the method S4, the computer is used for detecting by adopting an LC-MS method after the oscillation is finished.
8. The pretreatment kit for detecting a plurality of immunosuppressant drugs in human whole blood according to claim 1, characterized in that: and the solution A and the solution C are packaged by adopting opaque glass bottles.
9. The pretreatment kit for detecting multiple immunosuppressant drugs in human whole blood according to claim 2, characterized in that: the standard curve C1-C8 is that the linear range of the cyclosporine A is as follows: 10-1000 ng/mL, wherein the linear range of everolimus is as follows: 2-200 ng/mL, and the linear range of tacrolimus is as follows: 2-200 ng/mL, wherein the linear range of sirolimus is as follows: 2-200 ng/mL;
the concentration of the quality control products LQC, MQC and HQC, which are cyclosporine A, is as follows: 20. 400 and 800ng/mL, and the concentration of everolimus is as follows: 4. 80 and 160ng/mL, and the concentration of tacrolimus is as follows: 4. 80 and 160ng/mL, wherein the concentration of sirolimus is as follows: 4. 80 and 160ng/mL;
the concentration of the internal standard working solution is that the concentration of the cyclosporine A internal standard is as follows: 200ng/mL, and the concentration of everolimus internal standard is as follows: 20ng/mL, tacrolimus internal standard concentration: 20ng/mL, sirolimus internal standard concentration: 20ng/mL.
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| CN114509526A (en) * | 2021-12-31 | 2022-05-17 | 福建医科大学孟超肝胆医院(福州市传染病医院) | LC-MS/MS-based pure solvent type whole blood immunosuppressant drug concentration joint inspection kit and detection method |
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