CN114509526A - LC-MS/MS-based pure solvent type whole blood immunosuppressant drug concentration joint inspection kit and detection method - Google Patents
LC-MS/MS-based pure solvent type whole blood immunosuppressant drug concentration joint inspection kit and detection method Download PDFInfo
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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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- 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
- G01N30/88—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
- G01N2030/884—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 organic compounds
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Abstract
The invention provides a pure solvent type whole blood immunosuppressant drug concentration joint inspection kit based on LC-MS/MS and a detection method, wherein the kit comprises a pretreatment agent, a standard solution of cyclosporine A, tacrolimus and sirolimus, an internal standard mixed solution of cyclosporine A, tacrolimus and sirolimus, and a mobile phase. When the pure solvent type kit provided by the invention is used for treating a whole blood sample, the required sample amount is small, the treatment solution is clean, the kit can be matched with UPLC-MS/MS for analysis and detection, and three different immunosuppressant drugs can be detected simultaneously. The invention has the advantages of simple and quick operation, high sensitivity, accuracy and precision and small matrix effect, and can meet the requirement of clinical application on concentration monitoring of three immunosuppressive drugs.
Description
Technical Field
The invention relates to the technical field of medical inspection, in particular to a pure solvent type whole blood immunosuppressant drug concentration joint inspection kit based on LC-MS/MS and a detection method.
Background
Immunosuppressants are a class of drugs that inhibit or reduce the activity of the human immune system and play a critical role in preventing organ rejection in transplant recipients and in treating various autoimmune diseases. However, these drugs have narrow therapeutic window, large individual difference and need to be taken for a long time, so Therapeutic Drug Monitoring (TDM) is essential for guiding treatment and improving patient care.
The use of immunosuppressive agents is a trade-off between preventing rejection and gradually reducing the amount of immunosuppressive agent used, thereby allowing long-term coexistence of the recipient and the transplanted organ. Cyclosporine (CsA), Tacrolimus (TAC) and Sirolimus (SRL) are currently the most commonly used immunosuppressants. CsA and TAC are calpain inhibitors (CNI), SRL is a representative drug of mTOR (mammalian target of rapamycin) inhibitors, and mainly inhibits T lymphocyte proliferation and function and inhibits cell-mediated rejection.
In the blood of a patient, more than 50% of CsA, TAC or SRL enters erythrocytes, so the immunosuppressant drug concentration detection samples are all detected by using whole blood samples. However, the detection kits for relevant detection research and commercialization so far are all whole blood products, and have a plurality of problems in practical application: (1) the kit is expensive, taking the siemens syva tacrolimus detection kit as an example, the kit comprises an FK506 detection reagent, an FK506 calibration solution and an FK506 sample pretreatment reagent, the market price is up to about 4 million yuan of RMB, but only about 10 calibration curves are contained, the immunoassay system is only single drug detection, and the detection of CSA and SRL is provided with independent detection kits, because the immunoassay method is rapid in detection of the concentration of the drugs, but the price is expensive, and the detection result has larger deviation due to non-specific reaction; (2) the whole blood immunosuppressant calibration reagents which are sold in the market and researched in laboratories at present are all matrix type, so that the biological safety problem is solved, and the pretreatment of the whole blood immunosuppressant calibration reagents is also the speed-limiting step of detection; (3) the whole blood sample is pretreated by the conventional organic solvent method, although the method can be used for extracting cyclosporine A, tacrolimus and sirolimus in the whole blood, residual impurities in the protein precipitation method can still block a chromatographic column and pollute an instrument even if the impurities are filtered by a microporous filter membrane; the liquid-liquid extraction method adopts ether solvents for extraction, the operation is complicated, and the ether solvents have unpleasant smell, are inflammable and explosive; meanwhile, the effective concentration ranges of the tacrolimus and the sirolimus are both in the ng level, the detection limit is low, and a blood sample required by detection is large.
In order to solve the problems, the invention fully inspects the influence of matrix effect, develops a joint detection kit for the concentration of immunosuppressive drugs in pure solvent type whole blood, which is simple to operate and suitable for clinical drug concentration detection, and can be combined with an LC-MS/MS method to efficiently and quickly detect the concentration of immunosuppressive drugs. The standard solution in the pure solvent type kit can be directly loaded, so that the detection time is saved; the pretreatment reagent in the pure solvent type kit utilizes pure water to swell and break erythrocytes to release intracellular drugs, and combines an acetonitrile protein precipitation method to effectively extract cyclosporine A, tacrolimus and sirolimus in whole blood, so that the required blood sample is less, the method is simple and easy to implement, and the residual impurities are less.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provide a detection kit and a detection method for immunosuppressive drugs in whole blood based on LC-MS/MS, so that a pretreatment method which is simple to operate and relatively environment-friendly is adopted, and the concentrations of immunosuppressive drugs (including cyclosporine A, tacrolimus and sirolimus) in whole blood are detected by combining rapid, sensitive, accurate and high-precision ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS), thereby meeting the requirements and purposes of clinical application for detecting the concentrations of the immunosuppressive drugs.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention firstly provides a pure solvent type whole blood immunosuppressant drug concentration joint inspection kit based on LC-MS/MS, which is characterized in that: the kit comprises a whole blood sample pretreatment agent.
Furthermore, the LC-MS/MS-based pure solvent-based whole blood immunosuppressant drug concentration joint inspection kit further comprises a standard solution of cyclosporine A, tacrolimus and sirolimus, and a mobile phase.
Further, the whole blood sample pretreatment agent includes pure water and acetonitrile.
Further, each of the cyclosporine A, tacrolimus and sirolimus in the standard solution has 8 concentration gradients, wherein 8 concentration points of the tacrolimus and the sirolimus are the same and are respectively 6.25ng/mL, 12.5ng/mL, 25ng/mL, 50ng/mL, 75ng/mL, 100ng/mL, 150ng/mL and 200ng/mL, and 8 concentration points of the cyclosporine A are respectively 250ng/mL, 500ng/mL, 10000ng/mL, 2000ng/mL, 3000ng/mL, 400ng/mL, 6000ng/mL and 8000 ng/mL.
Furthermore, the internal standard mixed solution is a methanol solution containing cyclosporine A-d4, tacrolimus 13Cd2 and sirolimus-d 3, the concentration of the cyclosporine A-d4 in the internal standard mixed solution is 400ng/mL, the concentration of the tacrolimus 13Cd2 in the internal standard mixed solution is 50 mu g/mL, and the concentration of the sirolimus-d 3 in the internal standard mixed solution is 16 ng/mL.
Further, the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution, the mobile phase B is a methanol solution, the mobile phase A contains 5mM ammonium acetate and 0.01% by volume of formic acid, and the mobile phase B contains 500mM ammonium acetate and 0.01% by volume of formic acid.
The invention also provides a method for detecting the concentration of the immunosuppressant drug in human whole blood by LC-MS/MS by using the kit, which comprises the following steps:
1) pretreatment of a whole blood sample: adding 0.2 time volume of internal standard mixed solution into a whole blood sample to be detected, adding pure water 2-5 times the volume of the whole blood, performing vortex oscillation for 2-5 min, adding acetonitrile 12.5-20 times the volume of the whole blood, performing vortex oscillation for 2-5 min, centrifuging at 12000rpm for 5-10 min, filtering a supernatant by using a 0.22 mu m filter, and performing UPLC-MS/MS sample injection analysis, wherein the detected sample also comprises a standard solution;
2) and (3) calculation of detection results: and (3) taking the ratio of the standard substance peak area to the corresponding internal standard peak area as the ordinate and the concentration of the standard substance solution as the abscissa, fitting a linear equation to obtain a standard curve of cyclosporine A, tacrolimus and sirolimus, and calculating the concentrations of the three immunosuppressant drugs according to the standard curve.
Further, the chromatographic conditions are as follows: a chromatographic column: shim-pack XR-ODS III (2.0 mm. d.times.50 mm, 1.6 μm); pre-column: shim-pack GIST-HP (G) (2. mu. m C18, 2.1X 10 mm); mobile phase: mobile phase a and mobile phase B; column temperature: 60 ℃; sample injector temperature: 4 ℃; sample introduction amount: 10 mu L of the solution; the flow rate is 0.3 mL/min; gradient elution: 0min, 30% A +70% B; 0.10min, 30% A +70% B; 1.00min, 0% A +100% B; 2.00min, 0% A +100% B; 2.01min, 30+ 70; 4.00min, 30% A +70% B.
Further, the mass spectrometry conditions are as follows: in positive ion mode, an ESI source is adopted, 9psi of collision gas, 30psi of gas curtain gas, 50psi of atomization gas, 55psi of auxiliary heating gas, 5500V of spraying voltage and 250 ℃ of atomization temperature are adopted.
The invention has the beneficial effects that:
1. the whole blood immunosuppressant is detected by adopting the pure solvent type calibration reagent for the first time, the whole blood sample is taken as a reference, the pure water is used for replacing the whole blood, the calibration reagent can be directly used for detection on a computer without pretreatment, and the clinical rapid detection requirement can be met.
2. The invention establishes a whole blood pretreatment method which has simple steps, is relatively friendly to the environment and has less residual impurities after treatment, and combines a method for detecting the concentrations of 3 immunosuppressive drugs (including CsA, TAC and SRL) in whole blood by ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) which has the advantages of rapidness, high sensitivity, high accuracy and high precision.
3. The method reasonably sets the chromatographic conditions, so that the chromatogram of the object to be measured and the internal standard obtained by the method is stable and reliable, and the pretreatment method is adjusted to ensure that the solvent-based standard and the whole blood matrix standard have basically consistent results.
4. The invention is suitable for detecting the concentration of 3 immunosuppressive drugs (including CsA, TAC and SRL) in whole blood, but not limited to the whole blood immunosuppressive agent, and is suitable for detecting the concentration of a substance to be detected in whole blood when the substance to be detected enters erythrocytes in a large proportion.
Drawings
FIG. 1 is a chromatogram of three immunosuppressant drugs in a standard solution and an internal standard solution in example 1.
FIG. 2 is a standard curve of immunosuppressants (cyclosporin A, tacrolimus, sirolimus) plotted using neat solvent-based calibration reagents in example 2, linear range: cyclosporine A is (50-1600) ng/mL, and tacrolimus and sirolimus are both 2.5-40) ng/mL.
FIG. 3 is a graph showing the standard curve matching of immunosuppressants (cyclosporin A, tacrolimus, sirolimus) prepared from the pure solvent-based calibrator reagent and the whole blood matrix calibrator reagent in example 2.
FIG. 4 is a comparison of the results of detection of the sample treated with the divalent copper ion pretreatment reagent and pure water in example 5.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
The UPLC-MS/MS is short for the ultra-high performance liquid chromatography-mass spectrometry technology.
Example 1
1 preparing standard substance solutions of three immunosuppressant drugs
Methanol is used as a solvent, and the concentration ranges of the standard solution are respectively 250-8000 ng/mL (CsA); 6.25-200 ng/mL (TAC, SRL), specifically, the gradient concentration shown in the following table can be adopted in the invention.
TABLE 1 gradient concentration of standard solutions
| Standard curve sample | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 |
| CsA(ng/mL) | 250 | 500 | 10000 | 2000 | 3000 | 4000 | 6000 | 8000 |
| TAC(ng/mL) | 6.25 | 12.5 | 25 | 50 | 75 | 100 | 150 | 200 |
| SRL(ng/mL) | 6.25 | 12.5 | 25 | 50 | 75 | 100 | 150 | 200 |
2 preparing an internal standard mixed solution
Methanol is used as a solvent, and the internal standard mixed solution contains CsA-d 4400 ng/mL, TAC-13C d 250 μ g/mL and SRL-d 316 ng/mL.
3 preparation of the mobile phase
The mobile phase A is an aqueous solution, the mobile phase B is a methanol solution, 5mM ammonium acetate and 0.01% by volume of formic acid are contained in the mobile phase A, and 500mM ammonium acetate and 0.01% by volume of formic acid are contained in the mobile phase B.
4 operating machine detection
Chromatographic conditions are as follows: a chromatographic column: shim-pack XR-ODS III (2.0 mm. d.times.50 mm, 1.6 μm); pre-column: shim-pack GIST-HP (G) (2. mu. m C18, 2.1X 10 mm); mobile phase: mobile phase a and mobile phase B; column temperature: 60 ℃; sample injector temperature: 4 ℃; sample introduction amount: 10 mu L of the solution; gradient elution: the gradient elution procedure is shown in the table below.
TABLE 2 gradient elution procedure
Mass spectrum conditions: in positive ion mode, an ESI source is adopted, 9psi of collision gas, 30psi of gas curtain gas, 50psi of atomization gas, 55psi of auxiliary heating gas, 5500V of spraying voltage and 250 ℃ of atomization temperature are adopted. The mass spectrum parameters of the specific compounds are shown in the following table.
TABLE 3 Mass Spectrometry parameters for specific compounds
5 results of detection
The chromatographic peaks of the three standards and the internal standard are shown in FIG. 1.
Example 2
The degree of matching of standard curves of immunosuppressants (cyclosporine A, tacrolimus and sirolimus) drawn by the pure solvent-based scaling reagent and the whole blood matrix scaling reagent is compared.
1 preparing standard solution of three immunosuppressant drugs
The same as in example 1.
2 preparing an internal standard mixed solution
The same as in example 1.
Preparation of Whole blood sample pretreatment agent
The whole blood sample pretreatment agent comprises pure water and acetonitrile.
4 preparation of the mobile phase
The same as in example 1.
5 preparation of solvent-based calibration solution
Respectively taking 10 mu L of standard substance solutions with different concentrations, adding 10 mu L of internal standard mixed solution, adding 140 mu L of pure water, performing vortex oscillation at 1000-2000rpm for 2min, adding 500 mu L of acetonitrile, performing vortex oscillation at 1000-2000rpm for 2min, uniformly mixing, centrifuging at 12000rpm at 4 ℃ for 5min, filtering the obtained supernatant by a 0.22 mu m filter, and using the filtered supernatant for UPLC-MS/MS detection and analysis.
6 preparing the whole blood matrix type calibration solution
Taking 40 mu L of blank whole blood sample into a centrifuge tube, respectively adding 10 mu L of standard substance solutions with different concentrations into the centrifuge tube, carrying out vortex oscillation at 1000-2000rpm for 2min, adding 10 mu L of mixed internal standard working solution, then adding 100 mu L of pure water into the centrifuge tube, carrying out vortex oscillation at 1000-2000rpm for 2min, then adding 500 mu L of acetonitrile, carrying out vortex oscillation at 1000-2000rpm for 2min, centrifuging at 12000rpm at 4 ℃ for 5min after uniformly mixing, filtering the obtained supernatant by using a 0.22 mu m filter, and then using the filtered supernatant for UPLC-MS/MS detection and analysis.
7 on-machine detection
The detection conditions were the same as in example 1.
8 results of the detection
And (4) taking the concentration of the standard substance as an abscissa and taking the ratio of the peak area of the standard substance to the peak area of the internal standard substance as an ordinate to draw a standard curve. The calibration curve obtained for the solvent-based calibration solution is shown in FIG. 2. The standard curve obtained for the whole blood based calibration solutions is shown in FIG. 3.
Example 3
And (5) detecting matrix effect.
The applicability of the solvent-based calibration solution is based on the fact that the whole blood has no matrix effect or the matrix effect is in a controllable range, and the detection result is not influenced.
1 preparing quality control sample
The quality control sample contains four concentration points of high concentration QH, medium concentration QM, low concentration QL and lower limit of quantitation LLOQ of three kinds of immunosuppressants by using methanol as a solvent. In particular, the quality control samples for each concentration point can be shown in the following table.
TABLE 4 quality control sample for each concentration point
| Quality control sample | QH | QM | QL | LLOQ |
| CsA(ng/mL) | 6400 | 2560 | 640 | 500 |
| TAC(ng/mL) | 128 | 64 | 16 | 6.25 |
| SRL(ng/mL) | 128 | 64 | 16 | 6.25 |
2 preparing an internal standard mixed solution
The same as in example 2.
2 preparation of Whole blood sample pretreatment agent
The same as in example 2.
3 preparation of the mobile phase
The same as in example 1.
4 blank Whole blood sample preparation
Taking 40 mu L of blank whole blood sample, adding 100 mu L of pure water into the blank whole blood sample, carrying out vortex oscillation at 1000-2000rpm for 2min, then adding 500 mu L of acetonitrile, carrying out vortex oscillation at 1000-2000rpm for 2min, finally centrifuging at 12000r/min for 5min, taking all supernatant, adding 10 mu L of quality control sample with different concentration points (low and high) and 10 mu L of internal standard mixed solution into the blank whole blood sample, carrying out vortex oscillation at 1000-2000rpm for 2min, filtering by using a 0.22 mu m filter, using the mixture for UPLC-MS/MS detection and analysis, and parallelly configuring 6 parts.
Respectively taking 10 mu L of quality control sample (low and high) with different concentration points, adding 10 mu L of internal standard mixed solution, adding 140 mu L of pure water and 500 mu L of acetonitrile, carrying out vortex for 10s at 1000-2000rpm, configuring 6 parts in parallel, and taking 10 mu L for analysis.
5 operating machine detection
The detection conditions were the same as in example 1.
6 results of the detection
The results of the measurements are shown in the following table.
TABLE 5 results of matrix Effect detection
Example 4
1 preparing standard substance solutions of three immunosuppressant drugs
The same as in example 1.
2 preparing quality control sample
The same as in example 2.
3 preparing an internal standard mixture
The same as in example 1.
4 preparation of Whole blood sample pretreatment agent
The same as in example 2.
5 preparation of the mobile phase
The same as in example 1.
6 preparation of solvent-based calibration solution
The same as in example 2.
7 preparing the Whole blood matrix type calibration solution
The same as in example 2.
8 actual sample processing
Each of 10 whole blood samples from liver transplant patients or kidney transplant patients who were each administered cyclosporin A, tacrolimus, and sirolimus was collected and named samples 1 to 10(CsA), 11 to 20(TAC), and 21 to 30(SRL), respectively.
The collected whole blood sample was processed as follows: taking 50 mu L of whole blood sample, adding 10 mu L of internal standard mixed solution, then adding 100 mu L of pure water, and performing vortex oscillation at 1000-2000rpm for 2 min; then 500. mu.L of acetonitrile is added, vortex shaking is carried out at 1000-2000rpm for 2min, finally centrifugation is carried out at 12000r/min for 5min, and the supernatant is filtered by a 0.22 μm filter and then used for sample loading for analysis. Detecting cyclosporine A, tacrolimus and sirolimus in the sample, and calculating the content of each component according to the linear range of the standard curve.
9 on-machine detection
The detection conditions were the same as in example 1.
10 results of the detection
And (3) drawing a standard curve by taking the concentration of the standard substance as a horizontal coordinate and taking the ratio of the peak area of the standard substance to the peak area of the internal standard substance as a vertical coordinate to obtain a linear equation. The linear equations obtained for the solvent-based calibrator and the whole blood-based calibrator are shown in the following table.
TABLE 6 linear equation obtained for solvent-based calibration solutions
TABLE 7 Linear equation obtained for whole blood matrix-type calibration solutions
The results of the actual sample measurements are shown in the following table.
TABLE 8 actual sample test results
Example 5
Taking a whole blood sample of a patient taking cyclosporin A as an example, divalent copper ion (0.1mol L) was compared-1) The whole blood sample pretreatment reagent (the currently commercially available whole blood sample pretreatment reagent is an aqueous solution containing copper ions) of (1) and (2) the ability of pure water to disrupt erythrocytes.
1 preparing standard substance solutions of three immunosuppressant drugs
The same as in example 1.
2 preparing quality control sample
The same as in example 2.
3 preparing an internal standard mixture
The same as in example 1.
4 preparation of Whole blood sample pretreatment agent
The same as in example 2.
5 preparation of the mobile phase
The same as in example 1.
6 preparing solvent type calibration solution
The same as in example 2.
7 actual sample processing
Taking 50 mu L of whole blood sample, adding 10 mu L of internal standard mixed solution, then adding 100 mu L of pure water, and performing vortex oscillation at 1000-2000rpm for 2 min; and then adding 500 mu L of acetonitrile, performing vortex oscillation at 1000-2000rpm for 2min, finally centrifuging at 12000r/min for 5min, filtering the supernatant by using a 0.22 mu m filter, and then using the filtered supernatant for sample loading analysis to detect the substance to be detected (cyclosporine A) in the sample.
Taking 50 mu L of whole blood sample, adding 10 mu L of internal standard mixed solution, adding 100 mu L of bivalent copper ion-containing whole blood sample pretreatment agent (0.1mol/L copper sulfate aqueous solution), and performing vortex oscillation at 1000-2000rpm for 2 min; and then adding 500 mu L of acetonitrile, performing vortex oscillation at 1000-2000rpm for 2min, finally centrifuging at 12000r/min for 5min, filtering the supernatant by using a 0.22 mu m filter, and then using the filtered supernatant for sample loading analysis to detect the substance to be detected (cyclosporine A) in the sample.
8 on-machine detection
The detection conditions were the same as in example 1.
9 results of the detection
As shown in fig. 4, the detection results of the sample treated with the divalent copper ion pretreatment reagent and the sample treated with pure water were not significantly different. Therefore, the analyte enters the red blood cells in a large proportion, and the sample needing to detect the concentration of the analyte in the whole blood can be detected by using the pretreatment reagent and the pretreatment method provided by the invention.
The full-solvent kit disclosed by the invention replaces expensive pretreatment reagents by a physical method, and replaces the whole blood sample pretreatment reagents containing divalent copper ions of the whole blood matrix preparation kit by pure water, so that the cost is reduced, and meanwhile, the biological safety problem caused by whole blood products is avoided; and the detection results of the full-solvent type standard curve and the whole blood type standard curve are within the acceptable error range, so that the clinical sample can be accurately detected. Although the 5 embodiments of the present invention have been described in detail, the description is only a preferred embodiment of the present invention, and should not be considered as limiting the scope of the invention, and all equivalent variations and modifications made within the scope of the present invention should be covered by the claims.
Claims (9)
1. A pure solvent type whole blood immunosuppressant drug concentration joint inspection kit based on LC-MS/MS is characterized in that: the kit comprises a whole blood sample pretreatment agent.
2. The LC-MS/MS-based pure solvent-based whole blood immunosuppressant drug concentration joint inspection kit of claim 1, characterized in that: the kit also comprises a standard solution of cyclosporine A, tacrolimus and sirolimus, an internal standard mixed solution of cyclosporine A, tacrolimus and sirolimus, and a mobile phase.
3. The LC-MS/MS-based pure solvent-based whole blood immunosuppressant drug concentration joint inspection kit of claim 1, characterized in that: the whole blood sample pretreatment agent comprises pure water and acetonitrile.
4. The LC-MS/MS-based pure solvent-based whole blood immunosuppressant drug concentration joint inspection kit of claim 2, characterized in that: the cyclosporine A, the tacrolimus and the sirolimus in the standard solution all have 8 concentration gradients, wherein 8 concentration points of the tacrolimus and the sirolimus are the same and are respectively 6.25ng/mL, 12.5ng/mL, 25ng/mL, 50ng/mL, 75ng/mL, 100ng/mL, 150ng/mL and 200ng/mL, and 8 concentration points of the cyclosporine A are respectively 250ng/mL, 500ng/mL, 10000ng/mL, 2000ng/mL, 3000ng/mL, 400ng/mL, 6000ng/mL and 8000 ng/mL.
5. The LC-MS/MS-based pure solvent-based whole blood immunosuppressant drug concentration joint inspection kit of claim 2, characterized in that: the internal standard mixed solution is a methanol solution containing cyclosporine A-d4, tacrolimus 13Cd2 and sirolimus-d 3, the concentration of the cyclosporine A-d4 in the internal standard mixed solution is 400ng/mL, the concentration of the tacrolimus 13Cd2 in the internal standard mixed solution is 50 mu g/mL, and the concentration of the sirolimus-d 3 in the internal standard mixed solution is 16 ng/mL.
6. The LC-MS/MS-based pure solvent-based whole blood immunosuppressant drug concentration joint inspection kit of claim 2, characterized in that: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution, the mobile phase B is a methanol solution, the mobile phase A contains 5mM ammonium acetate and 0.01% by volume of formic acid, and the mobile phase B contains 500mM ammonium acetate and 0.01% by volume of formic acid.
7. A method for detecting the concentration of an immunosuppressant drug in human whole blood by LC-MS/MS using the kit as defined in any one of claims 1 to 6, wherein: the method comprises the following steps:
1) pretreatment of a whole blood sample: adding 0.2 time volume of internal standard mixed solution into a whole blood sample to be detected, adding pure water 2-5 times the volume of the whole blood, performing vortex oscillation for 2-5 min, adding acetonitrile 12.5-20 times the volume of the whole blood, performing vortex oscillation for 2-5 min, centrifuging at 12000rpm for 5-10 min, filtering a supernatant by using a 0.22 mu m filter, and performing UPLC-MS/MS sample injection analysis, wherein the detected sample also comprises a standard solution;
2) and (3) calculation of detection results: and (3) taking the ratio of the standard substance peak area to the corresponding internal standard peak area as the ordinate and the concentration of the standard substance solution as the abscissa, fitting a linear equation to obtain a standard curve of cyclosporine A, tacrolimus and sirolimus, and calculating the concentrations of the three immunosuppressant drugs according to the standard curve.
8. The method of claim 7 for detecting the concentration of an immunosuppressant drug in human whole blood, wherein: the chromatographic conditions are as follows: a chromatographic column: shim-pack XR-ODS III (2.0 mm. d.times.50 mm, 1.6 μm); pre-column: shim-pack GIST-HP (G) (2. mu. m C18, 2.1X 10 mm); mobile phase: mobile phase a and mobile phase B; column temperature: 60 ℃; sample injector temperature: 4 ℃; sample introduction amount: 10 mu L of the solution; the flow rate is 0.3 mL/min; gradient elution: 0min, 30% A +70% B; 0.10min, 30% A +70% B; 1.00min, 0% A +100% B; 2.00min, 0% A +100% B; 2.01min, 30+ 70; 4.00min, 30% A +70% B.
9. The method of claim 7 for detecting the concentration of an immunosuppressant drug in human whole blood, wherein: the mass spectrum conditions are as follows: in positive ion mode, an ESI source is adopted, 9psi of collision gas, 30psi of gas curtain gas, 50psi of atomization gas, 55psi of auxiliary heating gas, 5500V of spraying voltage and 250 ℃ of atomization temperature are adopted.
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