CN116026949A - Method for detecting four immunosuppressants in whole blood - Google Patents

Abstract

The invention relates to the technical field of therapeutic drug monitoring, in particular to a detection method of four immunosuppressants in whole blood. The invention provides a detection method for accurately measuring the concentration of four immunosuppressant drugs in whole blood, which is simple to operate, has simple operation processes of sample adding, sample moving, uniform mixing, magnetic separation and the like, can obtain clean treatment liquid without complex purification steps, can simultaneously finish the extraction of the four immunosuppressant drugs in one sample treatment, can be directly used for liquid chromatography tandem mass spectrometry detection, realizes simultaneous detection of the concentration of the four immunosuppressant drugs in one sample injection, and improves detection flux; meanwhile, the method has better phospholipid and protein removal effects, can reduce the background and improve the detection sensitivity.

Description

Method for detecting four immunosuppressants in whole blood

Technical Field

The invention relates to the technical field of therapeutic drug monitoring, in particular to a detection method of four immunosuppressants in whole blood.

Background

Immunosuppressant is a kind of medicine for inhibiting proliferation and function of cells related to immune response, mainly T cells and B cells, and can reduce immune response, so that the immune response is in proper strength and time, and thus, the disease can be prevented and treated, and the internal environment of organism is maintained stable. Rejection is one of the most significant obstacles after solid organ transplantation. Immunosuppressants play an important role in organ transplant rejection, and in addition, host versus graft diseases, diseases caused by hypersensitivity reactions, autoimmune diseases such as rheumatoid arthritis, rheumatic fever, lupus erythematosus and the like are mainly treated by immunosuppressants.

Tacrolimus, cyclosporine A, sirolimus and everolimus are four immunosuppressant drugs most commonly used clinically at present, and have narrow treatment window and large individual differences of pharmacokinetics, so that treatment drug monitoring is needed. Different combined immunosuppressant therapeutic administration schemes are commonly used in clinic, so that four immunosuppressant drug concentrations are detected simultaneously by one sample injection, thereby greatly helping clinicians to obtain more complete therapeutic administration information and guiding the clinicians to adjust individual administration schemes in time.

The method for measuring the concentration of the immunosuppressant drug in the whole blood mainly comprises three technologies of immunoassay, chromatography (high performance liquid chromatography) and mass spectrometry (liquid chromatography tandem mass spectrometry).

Immunoassays are based on the principle of antigen, antibody reactions, and detect unknown antibodies with known antigens or unknown antigens with known antibodies. The disadvantage is that it is generally very difficult and labor-intensive to prepare a sufficient amount of purified enzyme as antigen and an antiserum having immunochemical properties, the number of measurement steps is large, and the operation is complicated. In addition, the non-specific binding of drugs to antibodies often results in excessive or abnormal immunoassay concentrations and only one drug can be assayed at a time, however, the combined use for different populations is disadvantageous because of the large cost and efficiency of immunoassay monitoring multiple drug concentrations.

The chromatography is a method for detecting each component and the content thereof according to a certain sequence after separating each component from a mobile phase and a stationary phase by utilizing the difference of acting forces (such as difference in adsorption, distribution, exchange and other performances) of each component in a sample, and is essentially a separation analysis method. The disadvantage is that the characterization of the separated components is difficult, the analysis time is long and the required sample size is large. In addition, cyclosporine A and sirolimus can be measured by liquid chromatography-ultraviolet spectrometry (HPLC-UV), but it is difficult to measure the concentration of drugs having low blood concentration or no chromophore such as tacrolimus. In addition, the chromatographic method adopts an external standard method for quantification, and a correction factor is not used, so that the accuracy is high, but the influence of the operation condition change on the accuracy of a result is large, and the accuracy control requirement on the sample injection amount is high.

The combination of the high separation capacity of liquid chromatography and the high selectivity and high sensitivity of mass spectrometry can eliminate the need for deliberate entanglement in chromatographic separation conditions, since the difference in mass spectrometry information can distinguish a large number of different substances. Mass spectrometry, such as liquid chromatography tandem mass spectrometry, is the qualitative and quantitative analysis of a compound under test by analyzing the mass-to-charge ratio of an ionized sample. Sample processing typically involves simple protein precipitation, adding several reagents from the group consisting of internal standard solution, zinc sulfate solution, methanol, acetonitrile to a whole blood sample, mixing by vortexing, centrifuging to obtain a supernatant, and subjecting the supernatant to mass spectrometry. The method is simple, quick and convenient, but the protein is required to be centrifugally precipitated by a centrifugal machine, so that automation is difficult to realize, in addition, the flux is influenced by a rotor of the centrifugal machine, the problems of low efficiency, personal errors and the like can occur, and the automation and large-batch operation are difficult to realize. The patent CN2018112488118 discloses a kit and a detection method for accurately measuring the concentration of four immunosuppressant drugs in human whole blood, wherein liquid-liquid extraction is added after simple protein precipitation, and the method can further purify a sample, but has the advantages of complicated operation, long time consumption and difficult realization of automatic operation.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for detecting four immunosuppressants in whole blood.

The invention provides a method for detecting immunosuppressant in whole blood, which comprises the steps of preprocessing a whole blood sample to be detected and detecting by adopting liquid chromatography-tandem mass spectrometry;

the pretreatment of the whole blood sample to be detected comprises the steps of uniformly mixing the whole blood sample to be detected with an internal standard solution, sequentially adding a red blood cell lysate, a protein precipitant and a magnetic bead solution, carrying out magnetic separation, and taking a supernatant for liquid chromatography tandem mass spectrometry detection quantitative analysis;

further, the internal standard is an isotope or a structural analogue of the object to be detected, which changes according to the change of the object to be detected. In some embodiments of the present invention, the internal standard solution comprises cyclosporine A-d ₁₂ Sirolimus-d ₃ Everolimus-d ₄ And ascomycin. Further, in some embodiments, the internal standard solution of the present invention is 1000ng/ml cyclosporin A-d12, 100ng/ml sirolimus-d ₃ Everolimus-d 100ng/ml ₄ And 100ng/ml ascomycin.

In the invention, the erythrocyte lysate is zinc sulfate solution; the solvent of the zinc sulfate solution is water, wherein the concentration of zinc sulfate is 0.1-0.3 mol/L;

the protein precipitant is methanol acetonitrile solution, wherein the volume fraction of methanol is 30%;

the magnetic bead solution consists of water, methanol and magnetic beads, wherein the volume fraction of the methanol is 1% -20%, and the concentration of the magnetic beads is 0.5-10 mg/mL.

Further, in the invention, the magnetic beads in the magnetic bead solution are carboxyl functional group magnetic beads; the hydroxyl functional group comprises 1-18 alkyl, octyl, phenyl, cyclohexyl and other types of functional groups.

Furthermore, the volume ratio of the whole blood sample to be tested, the internal standard solution, the erythrocyte lysate, the protein precipitant and the magnetic bead solution is 10:1:20:30:10.

in the detection method of the invention, the immunosuppressant comprises cyclosporine A, tacrolimus, sirolimus and everolimus;

wherein the chromatographic conditions of the liquid chromatography tandem mass spectrometry include:

mobile phase a: consists of deionized water, ammonium acetate and formic acid, wherein the volume fraction of the formic acid is 0.1 percent, and the concentration of the ammonium acetate is 2mM;

mobile phase B: consists of methanol, ammonium acetate and formic acid, wherein the volume fraction of the formic acid is 0.1 percent, and the concentration of the ammonium acetate is 2mM;

the elution procedure was:

0 to 0.2min, the volume fraction of the mobile phase B is 50%;

0.2 to 1.0min, the volume fraction of the mobile phase B is from 50% to 100%;

1.0 to 2.0min, the volume fraction of the mobile phase B is 100 percent;

2.0 to 2.1min, the volume fraction of the mobile phase B is from 100% to 50%;

2.1 to 3.0min, the volume fraction of the mobile phase B is 50%.

Further, the chromatographic conditions of the liquid chromatography tandem mass spectrometry are as follows: the analytical column is a C18 or phenyl column; flow rate: 0.5ml/min; column temperature: 50 ℃; sample introduction chamber temperature: 2-8 ℃; sample injection volume: 10 μl. The mass spectrum conditions of the liquid chromatography tandem mass spectrum are as follows: electrospray ion source (ESI), positive ion MRM scan; the positive ion MRM scanning specific parameters are as follows: ionization voltage 5500 (V), temperature 550 ℃, air curtain air 35psi, collision air 7psi, spray air 50psi, auxiliary heating air 50psi.

In some specific embodiments, the invention uses a C18 column for the liquid chromatography analysis.

In the invention, the liquid chromatography tandem mass spectrometry condition is used for detecting four immunosuppressants, the detection result is accurate, and the quantitative and qualitative analysis of detection substances can be realized; the liquid chromatography tandem mass spectrometry condition is combined with the pretreatment of specific step parameters, such as the selection of magnetic beads and the use of specific protein precipitants, so that the impurity proteins and macromolecular substances are precipitated, the background is reduced, the impurity interference is reduced, and the detection accuracy, precision and sensitivity are further improved. The invention eliminates partial complicated steps requiring manual operation, and only has simple operation processes of sample adding, sample moving, mixing, magnetic separation and the like, so that the detection efficiency is further improved, and the high-throughput processing of samples is more facilitated. Therefore, each technical feature in the detection method of the present invention affects the detection effect and the detection efficiency, and therefore, should be protected as a whole.

The invention provides a kit for rapidly detecting four immunosuppressants in whole blood, which comprises a calibrator, a quality control product, an internal standard solution, a red blood cell lysate, a protein precipitant, a magnetic bead solution, a mobile phase A and a mobile phase B;

the calibrator comprises tacrolimus, sirolimus, everolimus and cyclosporine A;

the quality control product comprises tacrolimus, sirolimus, everolimus and cyclosporine A;

the internal standard solution comprises cyclosporine A-d ₁₂ Sirolimus-d ₃ Everolimus-d ₄ And ascomycin;

the erythrocyte lysate is zinc sulfate solution;

the protein precipitant is methanol acetonitrile solution;

mobile phase a consisted of deionized water, ammonium acetate and formic acid, wherein the volume fraction of formic acid was 0.1% and the ammonium acetate concentration was 2mM;

mobile phase B consisted of methanol, ammonium acetate and formic acid, the volume fraction of formic acid being 0.1% and the ammonium acetate concentration being 2mM.

Further, in the kit of the present invention,

the calibrator is as follows: tacrolimus 2 ng/ml-100 ng/ml, sirolimus 2 ng/ml-100 ng/ml, everolimus 2 ng/ml-100 ng/ml and cyclosporin A10 ng/ml-2000 ng/ml;

the quality control product is as follows: tacrolimus 5 ng/ml-30 ng/ml, sirolimus 5 ng/ml-30 ng/ml, everolimus 5 ng/ml-30 ng/ml and cyclosporin A50 ng/ml-800 ng/ml;

the internal standard solution is as follows: 1000ng/ml cyclosporin A-d ₁₂ Sirolimus-d 100ng/ml ₃ Everolimus-d 100ng/ml ₄ And 100ng/ml ascomycin;

the erythrocyte lysate is zinc sulfate solution, and the solvent water of the zinc sulfate solution, wherein the concentration of zinc sulfate is 0.1-0.3 mol/L;

the protein precipitant is methanol acetonitrile solution, wherein the volume fraction of methanol is 30%;

the magnetic bead solution consists of water, methanol and magnetic beads, wherein the volume fraction of the methanol is 1% -20%, and the concentration of the magnetic beads is 0.5-10 mg/mL; the magnetic beads in the magnetic bead solution are carboxyl functional magnetic beads.

The volume ratio of the whole blood sample to be detected, the internal standard solution, the erythrocyte lysate, the protein precipitant and the magnetic bead solution is 10:1:20:30:10.

the invention provides application of the kit in detection of four immunosuppressants, namely cyclosporin A, tacrolimus, everolimus and sirolimus.

The invention provides a detection method for accurately measuring four immunosuppressants in whole blood, which is simple to operate, has simple operation processes of sample adding, sample moving, uniform mixing, magnetic separation and the like, can obtain clean treatment liquid without complex purification steps, can simultaneously finish extraction of four immunosuppressant drugs in one sample treatment, can be directly used for liquid chromatography tandem mass spectrometry detection, realizes simultaneous detection of four immunosuppressant drug concentrations in one sample injection, and improves detection flux; meanwhile, the method has better phospholipid and protein removal effects, can reduce the background and improve the detection sensitivity.

Drawings

Fig. 1 shows methanol: a protein precipitation system of acetonitrile;

FIG. 2 shows total ion flow chromatograms of four immunosuppressant drugs;

figure 3 shows a cyclosporine a calibration curve, linear range: 10ng/ml to 2000ng/ml;

fig. 4 shows sirolimus calibration curve, linear range: 2ng/ml to 100ng/ml;

fig. 5 shows tacrolimus calibration curve, linear range: 2ng/ml to 100ng/ml;

fig. 6 shows everolimus calibration curve, linear range: 2ng/ml to 100ng/ml;

FIG. 7 shows a quantitative ion chromatogram of cyclosporine A;

FIG. 8 shows a qualitative ion chromatogram of cyclosporine A;

FIG. 9 shows a quantitative ion chromatogram of sirolimus;

FIG. 10 shows a sirolimus qualitative ion chromatogram;

FIG. 11 shows a Tacrolimus quantitative ion chromatogram;

FIG. 12 shows a tacrolimus qualitative ion chromatogram;

FIG. 13 shows an everolimus quantitative ion chromatogram;

fig. 14 shows everolimus qualitative ion chromatograms.

Detailed Description

The invention provides a method for detecting four immunosuppressants in whole blood, and a person skilled in the art can properly improve the process parameters by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1 preparation of solution and sample treatment

1. The kit composition is shown in table 1:

table 1 kit composition

2. Sample processing:

2.1 overview:

and respectively adding an internal standard solution, a red blood cell lysate, a protein precipitator and a magnetic bead solution into the whole blood sample to be measured, vibrating, and then performing magnetic separation to obtain a supernatant after the magnetic separation, thereby completing the sample treatment process. And finally, enabling the supernatant to enter liquid chromatography tandem mass spectrometry to finish final detection.

2.2 detailed operating steps:

absorbing 100 mu L of whole blood calibrator/quality control product/clinical sample into a 1.5mL centrifuge tube, adding 10 mu L of internal standard solution, mixing for 1min by vortex, adding 200 mu L of 0.1mol/L zinc sulfate solution, mixing for 1min by vortex, adding 300 mu L of methanol acetonitrile solution, mixing for 1min by vortex, adding 100 mu L of magnetic bead solution, magnetically absorbing for 2min, and taking 100 mu L of supernatant into a sample injection vial/96 Kong Jinyang plate for liquid chromatography tandem mass spectrometry detection. The specific steps are as follows:

the first step: 100 mu L of calibrator/quality control sample/whole blood sample to be tested are put into a 1.5mL centrifuge tube;

and a second step of: adding 10 mu L of internal standard solution, and mixing by vortex for 1min by a 2500rpm oscillator to fully mix the internal standard;

and a third step of: adding 200 mu L of zinc sulfate solution, and mixing for 1min by vortex with a 2500rpm oscillator to break red blood cells in whole blood, and releasing the medicine to be tested;

fourth step: adding 300 μl of methanol acetonitrile solution, mixing with a 2500rpm oscillator for 1min under vortex to form a stable system for facilitating magnetic attraction process

Fifth step: adding 100 mu L of magnetic bead solution, and mixing for 1min by vortex by a 2500rpm oscillator to enable the magnetic beads to fully bind impurities;

sixth step: placing on a magnetic rack, performing magnetic separation for 2min, and sucking 100 μL into sample injection vial/96 Kong Jinyang plate by a pipette for liquid chromatography tandem mass spectrometry detection.

2.3 preparation of solutions

2.3.1 Whole blood calibrator/Whole blood quality control:

whole blood series concentration calibrator is 6 concentration levels, wherein, tacrolimus, sirolimus, everolimus are: 2ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 50ng/ml, 100ng/ml; cyclosporin a is: 10ng/ml, 50ng/ml, 200ng/ml, 500ng/ml, 1000ng/ml, 2000ng/ml.

The whole blood quality control product has low, medium and high concentration levels of 3, wherein, tacrolimus, sirolimus and everolimus are as follows: 5ng/ml, 15ng/ml, 30ng/ml; cyclosporin A was 50ng/ml, 400ng/ml, 800ng/ml.

The specific preparation method of the whole blood calibrator comprises the following steps:

2.3.1.1 the intermediate working fluid of the calibrator series was prepared at the concentrations shown in table 2:

table 2 concentration of working fluid in the middle of the calibrator series

	1	2	3	4	5	6
							Tacrolimus (ng/mL)	40	100	200	400	1000	2000
Sirolimus (ng/mL)	40	100	200	400	1000	2000
							Everolimus (ng/mL)	40	100	200	400	1000	2000
Cyclosporin A (ng/mL)	200	1000	4000	10000	20000	40000

2.3.1.2 the intermediate working solution of the quality control series is prepared, and the concentration is shown in table 3:

TABLE 3 concentration of working fluid in middle of quality control series

	1	2	3
				Tacrolimus (ng/mL)	100	300	600
Sirolimus (ng/mL)	100	300	600
				Everolimus (ng/mL)	100	300	600
Cyclosporin A (ng/mL)	1000	8000	16000

2.3.1.3 Whole blood series concentration calibrator

Respectively sucking 950 mu L of whole blood into 6 1.5ml centrifuge tubes, respectively adding 50 mu L of intermediate working solution points of the calibrator, and uniformly mixing by vortex for 10min to obtain tacrolimus, sirolimus and everolimus with the concentration of: 2ng/ml, 5ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 100ng/ml; cyclosporin a concentration was: whole blood series concentration calibrators of 10ng/ml, 50ng/ml, 200ng/ml, 500ng/ml, 1000ng/ml, 2000ng/ml are shown in Table 4

TABLE 4 Whole blood series concentration calibrator

	Calibration material 1	Calibration material 2	Calibrator 3	Calibration material 4	Calibrator 5	Calibrator 6
							Tacrolimus (ng/mL)	2	5	10	20	50	100
Sirolimus (ng/mL)	2	5	10	20	50	100
							Everolimus (ng/mL)	2	5	10	20	50	100
Cyclosporin A (ng/mL)	10	50	200	500	1000	2000

2.3.1.4 Whole blood quality control product

Respectively sucking 950 mu L of whole blood into 3 1.5ml centrifuge tubes, respectively adding 50 mu L of medium working solution points of a quality control product, and uniformly mixing by vortex for 10 minutes to obtain tacrolimus, sirolimus and everolimus with the concentration of: 5ng/ml, 15ng/ml, 30ng/ml; cyclosporin a concentration was: whole blood quality control products of 50ng/ml, 400ng/ml and 800ng/ml are shown in Table 5

Table 5 Whole blood series concentration quality control product

2.3.2 internal standard solutions

The internal standard solution contains cyclosporine A-d ₁₂ Sirolimus-d ₃ Everolimus-d ₄ Methanol solution of ascomycin.

The preparation method of the internal standard solution comprises the following steps: 100. Mu.L of cyclosporine was pipettedA-d ₁₂ Mother liquor (10. Mu.g/mL), 100. Mu.L of sirolimus-d ₃ Mother liquor (1. Mu.g/mL), 100. Mu.L everolimus-d ₄ Adding 600 mu L of methanol into a sample bottle with 1.5mL of mother solution (1 mu g/mL) and 100 mu L of ascomycin mother solution (1 mu g/mL), and uniformly mixing for 1-3 minutes to prepare cyclosporine A-d ₁₂ At a concentration of 1000ng/ml, sirolimus-d ₃ At a concentration of 100ng/ml, everolimus-d ₄ An internal standard solution with a concentration of 100ng/ml and an ascomycin concentration of 100 ng/ml.

2.3.3 magnetic bead solution: the concentration is 3mg/ml

The preparation method of the magnetic bead solution comprises the following steps: absorbing 300 mu L of magnetic bead mother solution (100 mg/mL) to a 15mL glass bottle or a centrifuge tube, placing the glass bottle or the centrifuge tube on a magnetic rack for magnetic separation (magnetic beads in the glass bottle or the centrifuge tube can be adsorbed on the side wall by using magnetic substances under the condition of no magnetic rack), discarding supernatant, adding 10mL of aqueous solution containing 5% of methanol, and uniformly mixing by vortex for 1-3 minutes to obtain the magnetic bead solution with the concentration of 3mg/mL (remark: the magnetic bead mother solution is carboxyl magnetic beads with the particle size of 1 mu m, and manufacturers: merck).

2.3.4 methanol acetonitrile solution

Sucking methanol 15mL to 50mL volumetric flask, and fixing the volume of acetonitrile to the scale to obtain methanol acetonitrile solution. 2.3.5 Zinc sulfate solution (0.1 mol/L)

1.4378g of zinc sulfate is weighed by an electronic balance (ten thousandth) and placed in a 50mL volumetric flask, the volume is fixed to a scale by ultrapure water, the scale is shaken uniformly, and the ultrasonic wave is adopted to prepare the zinc sulfate solution with the concentration of 0.1 mol/L.

3. Liquid chromatography method:

liquid phase system: AB SCIEX

HPLC

Analytical column: c18 chromatographic column

Mobile phase: a:2mM ammonium acetate+0.1% formic acid+water; b:2mM ammonium acetate+0.1% formic acid+methanol

Flow rate: 0.5ml/min

Column temperature: 50 DEG C

Sample introduction chamber temperature: 2-8 DEG C

Sample injection volume: 10 μl of

The gradient elution procedure is shown in table 6:

TABLE 6 gradient elution procedure

Time (min)	Flow rate (mL/min)	Mobile phase a	Mobile phase B
				0.00	0.5	50	50
0.20	0.5	50	50
				1.00	0.5	0	100
2.00	0.5	0	100
				2.10	0.5	50	50
3.00	0.5	50	50

4. Mass spectrometry method:

positive ion MRM scan analysis, including ion source parameters and MRM ion channels, was performed using electrospray ion sources (ESI). Ion source parameters include ionization voltage and temperature, gas curtain gas, collision gas, spray gas, auxiliary heating gas, and specific information is shown in table 7:

TABLE 7 ion Source parameters

Ionization voltage (V)	5500
		Temperature (. Degree. C.)	550
Air curtain gas (psi)	35
		Collision gas	7
Spray gas (psi)	50
		Auxiliary heating (psi)	50

MRM ion channel: ammonia adduct ions were used, and specific ion pair information is shown in Table 8:

TABLE 8MRM quantitative and qualitative ion pair information

Note that: * The rest is qualitative ions. The corresponding ion chromatograms are shown in fig. 7 to 14.

5. And (3) calculating results:

and (3) adopting data analysis software of a liquid chromatograph tandem mass spectrometer, taking the ratio of the peak area of the calibrator to the peak area of the internal standard as an ordinate, taking the corresponding concentration of the calibrator as an abscissa, establishing linear regression, and automatically calculating four immunosuppressants in a whole blood sample to be detected according to a method set by the data analysis software.

6. Analysis of results:

6.1 experiments and systems adapted to different magnetic beads are different, and slight changes can lead to low collision efficiency of the magnetic beads, so that the impurity adsorption efficiency cannot be guaranteed, the original purpose of sample treatment is lost, and substances irrelevant to target objects, such as proteins, cells, salts and the like, are directly fed into a mass spectrum. On one hand, the ionization efficiency is affected, so that the detection result is inaccurate, and on the other hand, the column head of the chromatographic column is separated out, so that the column efficiency of the chromatographic column is rapidly reduced, even a system is blocked, and expensive instrument maintenance cost is generated. The detection method and the kit of the invention have conducted intensive researches on key reaction systems of the kit, such as protein precipitants. The common organic solvent methanol can not ensure the collision efficiency of the magnetic beads in a certain time, so that the efficiency of the magnetic beads for adsorbing impurities is affected, the supernatant of the processed sample is turbid, and the supernatant can not be detected on the machine. The acetonitrile which is an organic solvent can ensure the collision efficiency of magnetic beads, but has single component and high price, and cannot be compatible with various substances, so that the extracted substances are insufficient and the protein precipitation is incomplete. Methanol is adopted: the acetonitrile (3:7) protein precipitation system not only ensures the collision efficiency of the magnetic beads, but also has low price compared with acetonitrile, is compatible with various substances, and can ensure the sufficient extraction of substances and the complete protein precipitation, as shown in figure 1.

6.2 the detection method and the kit examine four main performance indexes of matrix effect, calibration curve linearity, precision and accuracy, and all meet the requirements of expected performance indexes. The whole blood sample purification and detection methods are described above. The linear relation is that the concentration range of the detection sample is narrow, the precision is reflected in the consistency between independent measurement results under the specified condition, and the accuracy is an index of whether the measurement result is accurate and reliable. 6.3 investigation of matrix Effect Using Mixed experiments

The specific method comprises the steps of adding a standard substance into blank whole blood, and measuring for 3 times, wherein the average response is A; patient whole blood (6 different patient samples), 3 times, mean response was B; a blank whole blood sample with standard added is mixed with an equivalent patient sample to 1:1, and the mean response is C. Then the matrix effect = C/((a+b)/2) x 100% and if the difference is <20% then the matrix effect is negligible. The matrix effect results show that tacrolimus is 86.87% -104.00%, sirolimus is 83.50% -100.61%, everolimus is 86.46% -106.43%, cyclosporine A is 89.30% -96.15%, the matrix effect is negligible, detailed data are shown in table 9, and the total ion flow chromatogram of the four immunosuppressant drugs is shown in fig. 2.

TABLE 9 matrix Effect

6.4 Linear investigation of calibration Curve

The method comprises the steps of processing 4 sets of calibration curves in parallel, substituting the first set of calibration curves into the first set of calibration curves to obtain corresponding concentration values, and calculating the deviation and CV of the average value and the theoretical value of three values of each concentration point, wherein the minimum point deviation is required to be less than 20%, and the deviation of the other points is required to be less than 15%; the lowest CV is less than 20%, and the rest CV is less than 15%. The results were examined as follows: cyclosporine a linear equation is y=0.013885 x+0.04211, r ² = 0.994488, linear in 10-2000 ng/mLThe detection requirements are met as shown in fig. 3. Sirolimus linear equation is y=0.12960x+0.04962, r ² = 0.998501, the detection requirement is linearly fulfilled within 2-100 ng/mL, as shown in fig. 4. Tacrolimus linear equation is y=0.12081x+0.02702, r ² = 0.99942, the detection requirement is linearly fulfilled within 2-100 ng/mL, as shown in fig. 5. Everolimus linear equation is y=0.09942x+0.02920, r ² = 0.997302, the detection requirement is linearly fulfilled within 2-100 ng/mL, as shown in fig. 6. The linearity is shown in Table 10, table 11, table 12, table 13

TABLE 10 cyclosporin A linearity conditions

TABLE 11 sirolimus linearity

TABLE 12 Tacrolimus linearity case

TABLE 13 everolimus linearity

6.5 accuracy requires that the average value should be within +/-15% of the quality control product marking value, which is to add standard products with known concentrations into clinical blank samples, prepare marked samples with 3 concentrations, detect each concentration at least 6 times, and examine the results as shown in table 14:

TABLE 14 accuracy results

6.6 precision requires CV within + -15%, the method is that standard substances with known concentrations are added into clinical blank samples, 3 marked samples with concentrations are prepared, each concentration is detected at least 6 times, and the investigation results are shown in Table 15:

TABLE 15 precision results

From the results, the liquid chromatography tandem mass spectrometry detection method and the kit thereof have good linearity, accuracy and precision, and completely meet the clinical examination requirements.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. The method for detecting the immunosuppressant in the whole blood comprises the steps of preprocessing a whole blood sample to be detected, and then adopting liquid chromatography-tandem mass spectrometry for detection;

pretreatment of the whole blood sample to be tested comprises: uniformly mixing a sample to be detected with an internal standard solution, sequentially adding erythrocyte lysate, a protein precipitant and a magnetic bead solution, and taking a supernatant after magnetic separation;

the internal standard solution comprises cyclosporine A-d ₁₂ Sirolimus-d ₃ Everolimus-d ₄ And ascomycin;

the erythrocyte lysate is zinc sulfate solution; the solvent of the zinc sulfate solution is water, wherein the concentration of zinc sulfate is 0.1-0.3 mol/L;

the protein precipitant is methanol acetonitrile solution, wherein the volume fraction of methanol is 30%;

the magnetic bead solution consists of water, methanol and magnetic beads, wherein the volume fraction of the methanol is 1% -20%, and the concentration of the magnetic beads is 0.5-10 mg/mL.

2. The method according to claim 1, wherein,

the magnetic beads in the magnetic bead solution are carboxyl functional group magnetic beads;

the internal standard solution is 1000ng/ml cyclosporine A-d ₁₂ Sirolimus-d 100ng/ml ₃ Everolimus-d 100ng/ml ₄ And 100ng/ml ascomycin.

3. The detection method according to claim 1, wherein the ratio of the total blood sample to be detected, the internal standard solution, the red blood cell lysate, the protein precipitant and the magnetic bead solution is 10:1:20:30:10.

4. the method according to claim 1, wherein,

the immunosuppressant comprises cyclosporine A, tacrolimus, sirolimus and everolimus;

the chromatographic conditions of the liquid chromatography tandem mass spectrometry include:

mobile phase a: consists of deionized water, ammonium acetate and formic acid, wherein the volume fraction of the formic acid is 0.1 percent, and the concentration of the ammonium acetate is 2mM;

mobile phase B: consists of methanol, ammonium acetate and formic acid, wherein the volume fraction of the formic acid is 0.1 percent, and the concentration of the ammonium acetate is 2mM;

the elution procedure was:

0 to 0.2min, the volume fraction of the mobile phase B is 50%;

0.2 to 1.0min, the volume fraction of the mobile phase B is from 50% to 100%;

1.0 to 2.0min, the volume fraction of the mobile phase B is 100 percent;

2.0 to 2.1min, the volume fraction of the mobile phase B is from 100% to 50%;

2.1 to 3.0min, the volume fraction of the mobile phase B is 50%.

5. The method according to claim 4, wherein the chromatographic conditions of the liquid chromatography tandem mass spectrometry are: the analytical column is a C18 column; flow rate: 0.5ml/min; column temperature: 50 ℃; sample introduction chamber temperature: 2-8 ℃; sample injection volume: 10 mu L.

6. The method according to claim 4, wherein the mass spectrometry conditions of the liquid chromatography tandem mass spectrometry are: electrospray ion source (ESI), positive ion MRM scan; the positive ion MRM scanning specific parameters are as follows: ionization voltage 5500 (V), temperature 550 ℃, air curtain air 35psi, collision air 7psi, spray air 50psi, auxiliary heating air 50psi.

7. The kit for rapidly detecting four immunosuppressants in whole blood comprises a calibrator, a quality control product, an internal standard solution, a red blood cell lysate, a protein precipitant, a magnetic bead solution, a mobile phase A and a mobile phase B;

the calibrator comprises tacrolimus, sirolimus, everolimus and cyclosporine A;

the quality control product comprises tacrolimus, sirolimus, everolimus and cyclosporine A;

the internal standard solution comprises cyclosporine A-d ₁₂ Sirolimus-d ₃ Everolimus-d ₄ And ascomycin;

the erythrocyte lysate is zinc sulfate solution;

the protein precipitant is methanol acetonitrile solution;

mobile phase a consisted of deionized water, ammonium acetate and formic acid, wherein the volume fraction of formic acid was 0.1% and the ammonium acetate concentration was 2mM;

mobile phase B consisted of methanol, ammonium acetate and formic acid, the volume fraction of formic acid being 0.1% and the ammonium acetate concentration being 2mM.

8. The kit according to claim 7, wherein,

the calibrator is as follows: tacrolimus 2 ng/ml-100 ng/ml, sirolimus 2 ng/ml-100 ng/ml, everolimus 2 ng/ml-100 ng/ml and cyclosporin A10 ng/ml-2000 ng/ml;

the quality control product is as follows: tacrolimus 5 ng/ml-30 ng/ml, sirolimus 5 ng/ml-30 ng/ml, everolimus 5 ng/ml-30 ng/ml and cyclosporin A50 ng/ml-800 ng/ml;

the internal standard solution is as follows: 1000ng/ml cyclosporin A-d ₁₂ 100ng/ml sirolimus-d ₃ Everolimus-d 100ng/ml ₄ And 100ng/ml ascomycin;

the erythrocyte lysate is zinc sulfate solution, and the solvent water of the zinc sulfate solution, wherein the concentration of zinc sulfate is 0.1-0.3 mol/L;

the protein precipitant is methanol acetonitrile solution, wherein the volume fraction of methanol is 30%;

the magnetic bead solution consists of water, methanol and magnetic beads, wherein the volume fraction of the methanol is 1% -20%, and the concentration of the magnetic beads is 0.5-10 mg/mL; the magnetic beads in the magnetic bead solution are carboxyl functional magnetic beads.

9. The kit according to claim 7 or 8, wherein the volume ratio of whole blood sample to be tested, internal standard solution, red blood cell lysate, protein precipitant to magnetic bead solution is 10:1:20:30:10.

10. use of the kit of claim 8 or 9 in the detection of four immunosuppressants, cyclosporin a, tacrolimus, everolimus and sirolimus.

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