CN115236216A

CN115236216A - Kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, preparation method and detection method thereof

Info

Publication number: CN115236216A
Application number: CN202210638740.2A
Authority: CN
Inventors: 韦弢; 倪君君; 贾永娟; 刘春冉; 张同兰
Original assignee: Hefei Hehe Medical Technology Co ltd
Current assignee: Hefei Hehe Medical Technology Co ltd
Priority date: 2022-06-07
Filing date: 2022-06-07
Publication date: 2022-10-25
Anticipated expiration: 2042-06-07
Also published as: CN115236216B

Abstract

The invention relates to the technical field of medicine detection, in particular to a kit for detecting an immunosuppressant in whole blood, a preparation method and a detection method thereof. The kit comprises a calibrator, a quality control product, an internal standard product, a mobile phase stock solution and a protein precipitator; the calibrator and the quality control product contain immunosuppressant and pharmaceutical excipients with series concentrations; immunosuppressants include tacrolimus, cyclosporine a, rapamycin, and mycophenolic acid; the pharmaceutical adjuvant contains at least one of whole blood, red blood cell and serum albumin. The kit has the advantages of no need of low-temperature freezing storage, difficult degradation and convenient long-distance transportation, and is simple to operate and convenient to use and dissolve after being used. The kit ensures the stability of the immunosuppressant, thereby ensuring the accuracy of the detection result, and simultaneously having the technical advantages of strong specificity and high sensitivity. The detection method has few pretreatment steps and can adapt to manual treatment and automatic treatment schemes.

Description

Kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, preparation method and detection method thereof

Technical Field

The invention relates to the technical field of medicine detection, in particular to a kit for detecting an immunosuppressant in whole blood, a preparation method and a detection method thereof.

Background

Immunosuppressants are chemical or biological substances that reduce tissue damage by suppressing cellular and humoral immune responses, can suppress abnormal immune responses in the body, and are mainly used in the treatment of organ transplant rejection resistance and autoimmune diseases. Immunosuppressive agents currently in clinical use can be basically divided into five major groups: the first is cytokine inhibitor, such as cyclosporine A, tacrolimus, sirolimus, everolimus, etc.; the second is a DNA synthesis inhibitor such as azathioprine, mycophenolic acid, mizoribine, etc.; the third is glucocorticoids such as prednisone, methylprednisolone, etc.; the fourth type is anti-lymphocyte antibody, such as anti-lymphocyte globulin, anti-thymoglobulin etc.; the fifth group is other immunosuppressants, such as AEB071, FTY720, etc. The immunosuppressant can inhibit the immune function of an organism by influencing the immune response reaction and immunopathological reaction of the organism, different immunosuppressants can play a synergistic role when acting on different lymphocyte activation periods, and the current clinical application adopts a combined medication method to improve the overall effectiveness and safety of a treatment scheme. Due to the narrow therapeutic window of immunosuppressive agents, individual variations in pharmacokinetics are large. When the amount thereof is excessively large, serious toxic reactions may be caused; when the dosage is insufficient, the patient will have immunological rejection, so Therapeutic Drug Monitoring (TDM) is necessary to obtain more complete therapeutic drug information and adjust the individualized dosing schedule.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is the current major method for quantitative analysis of immunosuppressive agents. However, due to the instability of the immunosuppressant, the related diagnostic reagent for detecting the concentration of the immunosuppressant in whole blood by the liquid chromatography tandem mass spectrometry at present usually needs low-temperature freezing preservation, ultralow-temperature preservation and even nitrogen-filled preservation; not only has poor stability, but also consumes energy and electricity. In addition, in the low-temperature storage kit, repeated freezing and thawing for a long time easily degrades the immunosuppressants in the standard substances and the quality control substances, and the quality stability of the detection products is difficult to ensure.

Disclosure of Invention

In order to solve the technical problems, the invention provides a kit for detecting an immunosuppressant in whole blood, a preparation method and a detection method thereof, which ensure the stability of the immunosuppressant and further ensure the accuracy of a detection result.

The invention provides a kit for detecting an immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, which comprises a calibrator, an internal standard, a mobile phase stock solution and a protein precipitator; the calibrator contains serial concentrations of immunosuppressant and pharmaceutical excipients; immunosuppressants include tacrolimus, cyclosporine a, rapamycin, and mycophenolic acid; the pharmaceutical adjuvant contains at least one of whole blood, erythrocyte and serum albumin, wherein the whole blood, erythrocyte and serum albumin are derived from human or mammal, and the mammal is preferably cattle, sheep or pig.

Optionally, the kit further comprises a quality control.

Optionally, the calibrator and the quality control product are both freeze-dried powder; the freeze-dried powder contains immunosuppressant and pharmaceutic adjuvant with series concentrations, and the pharmaceutic adjuvant also contains excipient and buffer salt; the excipient is at least one selected from serum albumin, trehalose, mannitol, PVP and sucrose, and the buffer salt is phosphate buffer salt with pH of 7.0-7.4.

Optionally, the mobile phase comprises a mobile phase A stock solution and a mobile phase B stock solution, wherein the mobile phase A stock solution is an aqueous solution containing 2-8% by volume of formic acid and 0.5-5 mM of ammonium acetate; the mobile phase B stock solution is a methanol solution containing 2-8% of formic acid by volume percentage; the internal standard substance is acetonitrile solution containing the internal standard substance, and the internal standard substance is ascomycin, cyclosporine A-d4, rapamycin-d 3 and mycophenolic acid-d 3; the protein precipitant comprises a protein precipitant I and a protein precipitant II, wherein the protein precipitant I is an aqueous solution of zinc sulfate with the concentration of 0.1-0.3 mol/L; the protein precipitant II is acetonitrile solution containing 0-5% methanol by volume percentage.

The invention provides a preparation method of the kit, which comprises the steps of preparing a standard substance and a quality control substance, and at least comprises the following steps:

s1, taking an immunosuppressant and pharmaceutic adjuvant according to a proportion, and preparing to obtain a solution;

s2, subpackaging the solution;

s3, freeze drying, wherein the freeze drying at least comprises the following steps:

s31, precooling: pre-cooling for 1-3 hours by equipment at the temperature of-20 ℃ to-50 ℃;

s32, pre-freezing: pre-freezing for 2-5 hours at-30 to-50 ℃;

s33, sublimation: sublimating for 8 to 12 hours at the temperature of minus 20 ℃ to minus 35 ℃;

s34, analysis and drying: the time is 3 to 5 hours, and the temperature is 0 to 20 ℃;

s35, enhanced drying: the time is 3 to 10 hours, and the temperature is 15 to 25 ℃;

and S36, packaging.

The invention provides a method for detecting an immunosuppressant in whole blood by using the kit, which at least comprises the following steps:

s1, preparing a solution: the method comprises the following steps:

respectively dissolving the calibrator and the quality control material in water for later use; diluting the mobile phase stock solution A with water to obtain a mobile phase A; diluting the mobile phase stock solution B with methanol to obtain a mobile phase B for later use;

s2, preparing a standard curve equation, which comprises the following steps: pretreating the calibration substance, and detecting by using a high performance liquid chromatography-mass spectrometer to obtain a standard curve equation;

s3, obtaining a sample to be detected, comprising the following steps: taking a whole blood sample or a quality control product to be detected, and performing pretreatment to obtain a sample to be detected;

s4, detecting a sample to be detected, comprising: detecting a sample to be detected by using a high performance liquid chromatography-mass spectrometer, substituting a detection result into a standard curve equation, and obtaining the concentration of the immunosuppressant in the sample to be detected;

in S2 and S3, the pretreatment includes: adding a sample solution into the internal standard, wherein the sample solution comprises a whole blood sample to be detected, a calibrator and a quality control material; adding a protein precipitant I, mixing uniformly, adding a protein precipitant II, mixing uniformly, centrifuging, taking supernate, and using the supernate as a sample to be detected for chromatographic analysis; the method also comprises the step of evaluating the precision of the detection method by adopting the quality control product.

Compared with the prior art, the technical scheme provided by the embodiment of the invention has the following advantages:

the kit disclosed by the invention is strong in universality, is suitable for all high performance liquid chromatography-tandem mass spectrometry detection systems, and provides a material and a method for clinically and rapidly detecting tacrolimus, cyclosporine A, rapamycin and mycophenolic acid in human whole blood.

In the preferred technical scheme, the calibrator or the quality control material adopts freeze-dried powder, so that the problems that tacrolimus, cyclosporine A, rapamycin and mycophenolic acid are easy to degrade and deteriorate in the process of placing are solved, and the possibility is provided for long-term stable storage of the kit. Has the advantages of no need of refrigeration storage, difficult degradation, convenient long-distance transportation, simple operation, convenient use and instant dissolution. The stability of the immunosuppressant is ensured, so that the accuracy of the detection result is ensured, and the method also has the technical advantages of strong specificity and high sensitivity.

In a preferred technical scheme, the kit provided by the invention adopts a substitute pharmaceutical adjuvant to substitute human whole blood, so that the possibility of disease transmission is reduced, and the biological safety is high.

The detection method has few pretreatment steps and can adapt to manual treatment and automatic treatment schemes.

Drawings

FIG. 1 is a standard curve equation obtained by an embodiment of the present invention;

FIG. 2 is a chromatogram of standard immunosuppressive agents and their intra-isotope standards in an example of the present invention;

FIG. 3 IS an enlarged map of FK-506 and FK-506-IS in FIG. 2;

FIG. 4 IS an enlarged map of CSA and CSA-IS of FIG. 2;

FIG. 5 IS an enlarged map of MPA and MPA-IS in FIG. 2;

FIG. 6 IS an enlarged map of RPM and RPM-IS of FIG. 2;

FIG. 7 is a chromatogram of a whole blood sample spectrogram immunosuppressant and its isotope internal standard in an embodiment of the present invention;

FIG. 8 IS an enlarged map of FK-506 and FK-506-IS in FIG. 7;

FIG. 9 IS an enlarged map of CSA and CSA-IS of FIG. 7;

FIG. 10 IS an enlarged map of MPA and MPA-IS in FIG. 7;

FIG. 11 IS an enlarged map of RPM and RPM-IS of FIG. 7.

Detailed Description

In order that the above objects, features and advantages of the present invention may be more clearly understood, a solution of the present invention will be further described below. It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the invention may be practiced otherwise than as described herein; it is to be understood that the embodiments described in this specification are only some embodiments of the invention, and not all embodiments.

The embodiment of the invention provides a kit for detecting an immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, which is suitable for high performance liquid chromatography tandem mass spectrometry and can be used for simultaneously detecting the concentrations of various immunosuppressant drugs in human whole blood and performing in-vitro diagnosis. The embodiment of the invention is complete in matching and comprises a calibrator, an internal standard, mobile phase stock solution and a protein precipitator. The calibrator contains immunosuppressant at a series of concentrations, and kits containing different immunosuppressant species can be provided according to requirements, and specifically, the immunosuppressant can comprise tacrolimus (FK-506), cyclosporine A (CSA), rapamycin (RPM), and mycophenolic acid (MPA). The medicinal auxiliary material contains at least one of whole blood, red blood cells and serum albumin, and the whole blood, red blood cells and serum albumin are from human or mammal.

The kit is used for treating a whole blood sample, and a clean treatment solution can be obtained by a simple protein precipitation-centrifugal extraction pretreatment method without performing a complicated purification step, so that the required sample amount is small; different consumables can be used in a matched manner, and the whole blood manual and automatic processing scheme can be flexibly matched; the kit calibrator is prepared by using a medicinal auxiliary material instead of whole blood, and the performance of the medicinal auxiliary material is consistent with that of a clinical fresh sample after evaluation. The kit can be used for simultaneously detecting four different immunosuppressants, can realize simultaneous detection of multiple indexes by a single sample, and has the advantages of good detection specificity, high sensitivity, small matrix effect, short detection time, high flux and low cost.

As an improvement of the technical scheme of the embodiment of the invention, the kit also comprises a quality control product, wherein the quality control product is an immunosuppressant and a pharmaceutic adjuvant which contain a series of concentrations, the pharmaceutic adjuvant contains at least one of whole blood, red blood cells and serum albumin, and the whole blood, red blood cells and serum albumin are from human or mammals. The quality control product can more effectively monitor the precision in the day and the day during the detection process, and ensure the stability and the accuracy during the process of testing the sample.

The calibrator and the quality control material in the embodiment of the invention may be liquid preparations. However, tacrolimus, cyclosporine A, rapamycin and mycophenolic acid are unstable, so that the tacrolimus, the cyclosporine A, the rapamycin and the mycophenolic acid are easily oxidized and deteriorated in a solution state, and the solution needs to be stored at a low temperature, is inconvenient to store and is repeatedly frozen and thawed to influence the accuracy of a subsequent detection result. In order to further improve the stability of the kit, the components containing the immunosuppressant in the kit are innovatively prepared into lyophilized powder, and meanwhile, medicinal auxiliary materials for ensuring the stability of the immunosuppressant are added into the lyophilized powder, so that the components do not need to be stored in a frozen manner, and can be stored at a low temperature of 2-8 ℃, and the stability is good. Compared with the common solution state, the kit is a freeze-dried product, has less water content (less than 3 percent), stable product quality and convenient transportation.

The embodiment of the invention finds that the sources of the whole blood, the red blood cells and the serum albumin are cattle, sheep or pigs, and further preferably sheep red blood cells and bovine serum albumin.

As an improvement of the technical scheme of the embodiment of the invention, the red blood cells are diluted into 20-80 percent by volume percentage concentration by adopting phosphate buffer salt with the pH value of 7.4, and the concentration is further preferably 50-80 percent. The concentration screening experiment of the red blood cells shows that the stability of 50-80% of the content of the red blood cells can meet the requirement, but in the process of experimental operation, 80% of red blood cell products are easy to generate a flocculation state, so that the red blood cells with the volume percentage concentration of 50-70% are more preferable.

As an improvement of the technical scheme of the embodiment of the invention, the freeze-dried powder contains immunosuppressant and pharmaceutic adjuvant with a series of concentrations, the pharmaceutic adjuvant is further added with excipient and buffer salt, the excipient is selected from at least one of serum albumin, trehalose, mannitol, PVP (polyvinylpyrrolidone) and sucrose, and the buffer salt is selected from phosphate buffer salt with pH of 7.0-7.4.

As an improvement of the technical scheme of the embodiment of the invention, the pharmaceutic adjuvant comprises:

7-12 parts of excipient;

100-800 parts of whole blood or red blood cells by volume;

100-700 parts by volume of phosphate buffer salt;

the excipient is selected from serum albumin and mannitol; wherein the weight ratio of the serum albumin to the mannitol is 0.1:1 to 10:1; parts by weight in g are in mL by volume. Preferably:

10 parts of excipient;

250-500 parts by volume of red blood cells;

250-500 parts by volume of phosphate buffer salt;

the excipient is selected from serum albumin and mannitol; wherein the weight ratio of the serum albumin to the mannitol is 0.5:1 to 5:1; parts by weight in g are in mL by volume.

As an improvement of the technical scheme of the embodiment of the invention, the specific concentrations of the auxiliary materials for the traditional Chinese medicines of the calibrator and the quality control material are as follows: bovine serum albumin 0.01g/mL, mannitol 0.01g/mL, erythrocytes 0.5mL, pH7.4 phosphate buffer salt 0.5mL.

As an improvement of the technical solution of the embodiment of the present invention, the serial concentrations of immunosuppressive agents in the calibrator include 6 concentrations of immunosuppressive agents, and the 6 concentrations of immunosuppressive agents are:

calibration product C1: tacrolimus 0.9-1.1 ng/mL, rapamycin 9-11 ng/mL, cyclosporine A0.9-1.1 ng/mL, mycophenolic acid 90-110 ng/mL;

a calibrator C2: 4.5-5.5 ng/mL of tacrolimus, 45-55 ng/mL of rapamycin, 4.5-5.5 ng/mL of cyclosporine A and 450-550 ng/mL of mycophenolic acid;

and (3) calibration product C3: 9-11 ng/mL of tacrolimus, 90-110 ng/mL of rapamycin, 9-11 ng/mL of cyclosporine A and 900-1100 ng/mL of mycophenolic acid;

calibrator C4: tacrolimus 18-22 ng/mL, rapamycin 180-220 ng/mL, cyclosporine A18-22 ng/mL, mycophenolic acid 1800-2200 ng/mL;

and (3) calibration product C5: 45-55 ng/mL of tacrolimus, 450-550 ng/mL of rapamycin, 45-55 ng/mL of cyclosporine A and 4500-5500 ng/mL of mycophenolic acid;

and (3) calibration product C6: 90-110 ng/mL of tacrolimus, 900-1100 ng/mL of rapamycin, 90-110 ng/mL of cyclosporine A and 9000-11000 ng/mL of mycophenolic acid.

The series of concentrations of immunosuppressant in the quality control product comprises 3 concentrations of immunosuppressant, including:

low-value quality control product Q7: 1.8-2.2 ng/mL of tacrolimus, 18-22 ng/mL of rapamycin, 1.8-2.2 ng/mL of cyclosporine A and 180-220 ng/mL of mycophenolic acid;

and (3) a median quality control product Q8: 9-11 ng/mL of tacrolimus, 180-220 ng/mL of rapamycin, 9-11 ng/mL of cyclosporine A and 900-1100 ng/mL of mycophenolic acid;

high-value quality control Q9: 72-88 ng/mL of tacrolimus, 720-880 ng/mL of rapamycin, 72-88 ng/mL of cyclosporine A and 7200-8800 ng/mL of mycophenolic acid.

As an improvement of the technical scheme of the embodiment of the invention, the kit also comprises an internal standard substance, a mobile phase stock solution and a protein precipitator;

the mobile phase stock solution comprises a mobile phase A stock solution and a mobile phase B stock solution: the mobile phase A stock solution is a methanol solution containing formic acid and ammonium acetate, the concentration of the formic acid in percentage by volume is 2-8 percent, the preference is 4 percent, and the concentration of the ammonium acetate is 0.5-5 mM, the preference is 0.1mM; the mobile phase B stock solution is a methanol solution containing formic acid, and the concentration of the formic acid in the mobile phase B stock solution is 2-8%, preferably 6%.

The internal standard substance is acetonitrile solution containing the internal standard substance; the internal standard substances are ascomycin, cyclosporins A-d4, rapamycin-d 3 and mycophenolic acid-d 3; wherein the inner standard substance of tacrolimus is ascomycin, the inner standard substance of cyclosporine A is cyclosporine A-d4, the inner standard substance of rapamycin is rapamycin-d 3 and the inner standard substance of mycophenolic acid is mycophenolic acid-d 3. Preferably, the concentration of the ascomycin in the internal standard is 0.009-0.011 mu g/mL, the concentration of the cyclosporine A-d4 is 0.4-0.6 mu g/mL, the concentration of the rapamycin-d 3 is 0.01-0.03 mu g/mL, and the concentration of the mycophenolic acid-d 3 is 0.8-1.2 mu g/mL; more preferably, the concentration of ascomycin in the internal standard is 0.01. Mu.g/mL, the concentration of cyclosporin A-d4 is 0.5. Mu.g/mL, the concentration of rapamycin-d 3 is 0.02. Mu.g/mL, and the concentration of mycophenolic acid-d 3 is 1. Mu.g/mL.

The protein precipitant comprises a protein precipitant I and a protein precipitant II; the protein precipitator I is zinc sulfate aqueous solution with the concentration of 0.1-0.3 mol/L, and the concentration of zinc sulfate is preferably 0.2mol/L; the protein precipitant II is acetonitrile solution containing 0-100% methanol by volume, preferably 100% acetonitrile.

The kit provided by the embodiment of the invention adopts two protein precipitants, has the technical advantages of simple pretreatment, small sample dosage and short detection time, and can effectively remove matrix interference.

As an improvement of the technical scheme of the embodiment of the invention, the kit also comprises an instruction book, a certificate, a lining and a packing box, and can also contain consumables such as EP tubes, 96-hole plates, heat sealing films and other products related to the using, storing and transporting processes of the kit.

The embodiment of the invention also relates to a preparation method of the kit, which comprises the step of preparing the calibrator freeze-dried powder or the quality control product freeze-dried powder and at least comprises the following steps:

s2, subpackaging the solution;

s3, freeze-drying, which at least comprises the following steps:

s31, precooling: pre-cooling for 1-3 hours by equipment at the temperature of-20 ℃ to-50 ℃, preferably for 2 hours at the temperature of-20 ℃;

s32, pre-freezing: pre-freezing for 2-5 hours at-30 ℃ to-50 ℃, preferably at-35 ℃ for 4 hours;

s33, sublimation: sublimating for 8 to 12 hours at the temperature of between 20 ℃ below zero and 35 ℃ below zero, preferably at the temperature of between 25 ℃ below zero for 10 hours;

s34, analysis and drying: the time is 3 to 5 hours, the temperature is 0 to 20 ℃, and the temperature is preferably 15 ℃ for drying for 3 hours;

s35, enhanced drying: the time is 3 to 10 hours, the temperature is 15 to 25 ℃, and the temperature is preferably 20 ℃ for drying for 4 hours;

and S36, packaging.

The freeze-drying process provided by the embodiment of the invention has the technical advantages of simple process, strong operability, low production cost, safety and reliability. The prepared freeze-dried product has good dissolution and redissolution properties, and can be quickly dissolved into dark red liquid by slight shaking after being redissolved by adding water.

The embodiment of the invention also relates to a method for detecting the immunosuppressant in the whole blood by adopting the kit, which comprises the step of recording a chromatogram of a sample to be detected, peak areas of tacrolimus, cyclosporine A, rapamycin and mycophenolic acid and peak areas of corresponding internal standard by using a high performance liquid chromatography-tandem mass spectrometer. And (3) preparing a calibration curve according to the ratio of the peak area of the FK-506, csA, RPM and MPA TICF (total ion current intensity) peak in the calibrator to the peak area of the corresponding internal standard and the calibration concentration, thereby calculating the contents of FK-506, csA, RPM and MPA in the sample to be detected. The embodiment of the invention adopts an isotope dilution high performance liquid tandem mass spectrometry method for detection, thereby reducing the analysis error caused by loss in the sample extraction process and the detection process to the maximum extent, and improving the accuracy of the determination result and the matrix interference resistance of the method.

At least comprises the following steps:

s1, preparing a solution: the method comprises the following steps:

dissolving the quality control product and the calibrator with water; diluting the mobile phase stock solution A with water; diluting the mobile phase stock solution B with methanol;

s2, preparing a standard curve equation, pretreating a calibration product, and detecting by using a high performance liquid chromatography-mass spectrometer to obtain the standard curve equation, wherein the standard curve equation comprises the following specific steps: taking the marked concentrations of the 6 calibrators as a horizontal coordinate (x), and taking the ratio of the actual detection peak area of the 6 calibrators to the peak area of each internal standard as a vertical coordinate (y) to draw a standard curve; performing linear regression on the labeled concentration (x) by using the peak area ratio (y) to obtain a regression equation: y = ax + b, where y is the ordinate, x is the abscissa, a is the slope of the curve, and a fitted correlation coefficient (r) is calculated ² ) Requires r ² Should not be less than 0.990;

s3, processing to obtain a sample to be detected, comprising: pre-treating blood plasma to be detected to obtain a sample to be detected;

s4, detecting a sample to be detected, comprising the following steps: and detecting the sample to be detected by using a high performance liquid chromatography-mass spectrometer, and substituting the detection result into a standard curve equation to obtain the concentration of the immunosuppressant in the sample to be detected.

In S2 and S3, the pretreatment includes: adding a sample solution into the internal standard, wherein the sample solution comprises a whole blood sample to be detected, a calibrator and a quality control material; adding protein precipitant I, mixing, adding protein precipitant II, mixing, centrifuging, collecting supernatant, centrifuging, and collecting supernatant as sample to be tested for chromatographic analysis.

As an improvement of the technical scheme of the embodiment of the invention, in S1, the mobile phase A obtained by dilution is an aqueous solution containing 0.05-0.2%, preferably 0.1% by volume of formic acid and 0.5-2 mM, preferably 1mM of ammonium acetate; the mobile phase B is a methanol solution containing formic acid at a concentration of 0.05 to 0.2% by volume, preferably 0.1%.

The preparation method can be specifically adopted as follows: adding 5mL of mobile phase stock solution A into 200mL of deionized water, fully and uniformly mixing, and performing ultrasonic treatment for 3-5 minutes; mobile phase B phase: and adding 5mL of the mobile phase stock solution B into 300mL of methanol, fully and uniformly mixing, and performing ultrasonic treatment for 3-5 minutes to obtain the product.

As an improvement of the technical scheme of the embodiment of the invention, in the pretreatment process, the volume ratio of the sample solution to the internal standard substance is 1:0.5 to 2, preferably 1:1; the volume ratio of the sample solution to the protein precipitant I is 1:0.5 to 2, preferably 1:1; the volume ratio of the sample solution to the protein precipitant II is 1:1 to 3, preferably 1:2.

as an improvement of the technical scheme of the embodiment of the invention, in the pretreatment process, the specific whole blood sample implementation process is as follows:

the 96-well plate treatment method comprises the following steps: precisely sucking 100 mu L of sample solution into a 96-well plate → adding 100 mu L of internal standard substance → adding 100 mu L of protein precipitant I → mixing for 1 minute at 500r/min → adding 200 mu L of protein precipitant II → laminating → mixing for 5 minutes at 2000r/min → centrifuging at 4000r/min for 10 minutes → taking supernatant 300 mu L → laminating → centrifuging at 4000r/min for 10 minutes → centrifuging the supernatant for chromatographic analysis.

The EP tube treatment method comprises the following steps: precisely sucking 100 μ L of sample solution into a 1.0mL centrifuge tube → adding 100 μ L of internal standard substance → adding 100 μ L of protein precipitant I → 2000r/min and mixing for 2 minutes → adding 200 μ L of protein precipitant II → 2000r/min and mixing for 5 minutes → 14000r/min for 10 minutes → taking supernatant fluid 300 μ L → 14000r/min for 5 minutes → taking 100 μ L for testing on a machine.

As an improvement of the technical scheme of the embodiment of the invention, the detection system is a high performance liquid chromatography tandem mass spectrometry detection system, the detection method is a liquid chromatography tandem mass spectrometry, triple quadrupole tandem mass spectrometry is used, an ESI positive ion detection mode is used, and a multi-reaction detection MRM scanning mode is adopted. Specific examples are as follows: SCIEX TripleQuad 4500 series liquid chromatography tandem mass spectrometry system.

As an improvement of the technical scheme of the embodiment of the invention, the specific conditions of the high performance liquid chromatography are as follows: the chromatographic column is selected from a C18 column, and the particle size is 2-5 mu m; specific optional chromatographic columns include: waters, such as AWaters, xbridge C18 column, (2.1X 100mm,5 μm); waters, atlantis dC18 column, (2.1X 50mm,5 μm); waters, atlantis T3 column (2.1X 50mm,3 μm); preferably a 2.1X 100mm,5 μm or equivalent chromatography column.

As an improvement of the technical scheme of the embodiment of the invention, the conditions of the high performance liquid chromatography are as follows:

the column temperature is 25-45 ℃, and preferably 40 ℃;

the flow rate is 0.3-0.5 mL/min, preferably 0.4mL/min;

the amount of the sample is 10 to 30. Mu.L, preferably 20. Mu.L.

As an improvement of the technical scheme of the embodiment of the invention, the elution of the high performance liquid chromatography adopts gradient elution:

0-0.5 min, mobile phase A: 30-50%, mobile phase B:50 to 70 percent;

0.51-2 minutes, mobile phase a: 1-5%, mobile phase B:5 to 99 percent;

2.1-4 minutes, mobile phase a: 30-50%, mobile phase B:50 to 70 percent;

as an improvement of the technical scheme of the embodiment of the invention, the elution conditions of the high performance liquid chromatography are shown in Table 1:

TABLE 1

Time per minute	Water (0.1% formic acid +1mM ammonium acetate)/%	Methanol (0.1% formic acid)%	Flow rate/(mL/min)
				0	40	60	0.4
0.5	3	97	0.4
				2.0	3	97	0.4
2.1	40	60	0.4
				3.0	40	60	0.4
4.0	40	60	0.4

The conditions of the mass spectrum are as follows:

ionspray voltage is 0-5500V, and 4000V is preferred; gas 1:40 to 60psi, preferably 50psi; gas 2:0 to 90psi, preferably 50psi; air curtain air: 20 to 50psi, preferably 25psi; the heating gas temperature is 150-550 ℃, preferably 400 ℃.

ESI ion source recommended parameters, ion pair information are shown in Table 2:

TABLE 2

The technical scheme of the embodiment of the invention is further explained by the following specific embodiment, and the reagents used in the specific embodiment are all commercially available.

Example 1

The specific composition of the kit is shown in the following table 3:

table 3: kit components (96 persons/box)

Wherein, the sheep red blood cell has a volume percentage concentration of 50%, and is diluted by phosphate buffer salt with pH = 7.4.

Example 2

This example is used to illustrate the method of use and detection of the kit:

s1, preparing a solution:

1. dissolving the calibrator freeze-dried powder and the quality control product freeze-dried powder in the kit respectively by using 0.5mL of purified water for later use;

2. mobile phase a phase: adding 5mL of mobile phase stock solution A in the kit into 200mL of deionized water, fully and uniformly mixing, and performing ultrasonic treatment for 3 minutes to obtain the reagent; mobile phase B phase: adding 5mL of methanol into 300mL of mobile phase stock solution B in the kit, fully and uniformly mixing, and performing ultrasonic treatment for 3 minutes to obtain the reagent;

s2, preparing a standard curve equation: pre-treating the calibrator, precisely sucking 100 μ L calibrator into a 1.0mL centrifuge tube → adding 100 μ L internal standard → adding 100 μ L protein precipitant I → mixing uniformly for 2 minutes at 2000r/min → adding 200 μ L protein precipitant II → mixing uniformly for 5 minutes at 2000r/min → centrifuging at 14000r/min for 10 minutes → taking supernatant 300 μ L → centrifuging at 14000r/min for 5 minutes → taking 100 μ L to carry out on-machine inspection.

Detecting by using a high performance liquid chromatography-mass spectrometer, and drawing a standard curve by taking the marked concentration of 6 calibrators as a horizontal coordinate (x) and the ratio of the actual detection peak area of the 6 calibrators to the peak area of each internal standard as a vertical coordinate (y); performing linear regression on the labeled concentration (x) by using the peak area ratio (y) to obtain a regression equation: y = ax + b, where y is the ordinate, x is the abscissa, a is the slope of the curve, andfitting the correlation coefficient (r) ² ) Require r to ² Should not be less than 0.990; obtaining a standard curve equation as shown in figure 1;

s3, obtaining a sample to be detected: precisely sucking 100 mu L of whole blood sample into a 1.0mL centrifuge tube → adding 100 mu L of internal standard substance → adding 100 mu L of protein precipitant I → mixing at 2000r/min for 2 minutes → adding 200 mu L of protein precipitant II → mixing at 2000r/min for 5 minutes → 14000r/min for 10 minutes → taking supernatant 300 mu L → 14000r/min for 5 minutes → taking 100 mu L for machine inspection;

and S4, detecting a sample to be detected, injecting the treated sample supernatant into a high performance liquid tandem mass spectrometer for detection, and recording a chromatogram, peak areas of tacrolimus, cyclosporine A, rapamycin and mycophenolic acid in the sample and peak areas of isotope internal standard thereof. And substituting the ratio of the actual peak area of the sample to be detected to the peak area of the internal standard into the equation, and calculating the concentration of each immunosuppressant in the sample to be detected.

The specific chromatographic conditions were as follows:

a SCIEX tripleQuad 4500 series liquid chromatography-tandem mass spectrometry system is adopted, and a chromatographic column comprises: waters Xbridge C18 column (2.1X 100mm,5 μm); column temperature: 40 ℃; sample introduction amount: 20 mu L of the solution; the gradient elution mode is the same as that in Table 1; the Ionspray voltage is 4000V; gas 1:50psi; gas 2:50psi; air curtain air: 25psi; the heating gas temperature is 400 ℃; the ion pair information is as in table 2.

And (2) arranging a liquid chromatograph-mass spectrometer according to the chromatographic conditions and the mass spectrum conditions, injecting a sample into the instrument for detection and analysis, and obtaining a chromatogram of the standard substance immunosuppressant and the isotope internal standard substance thereof, wherein the chromatogram is shown in figures 2-6, the main information of the chromatogram is shown in table 4, four immunosuppressants and isotope internal standard peaks thereof can be seen, the peak shape is symmetrical, the response is moderate, the interference of impurities is basically avoided, and conditions are provided for accurate quantification of the immunosuppressant to be detected.

Table 4: chromatogram information of standard

Target substance	Area(cps)	RT(min)	IS Area
				FK-506	1565000	2.37	972600
CsA	3030000	2.5	8768000
				RPM	3546000	2.01	1566000
MPA	327900	2.37	58680

The chromatograms of the four immunosuppressive agents in the whole blood sample are shown in fig. 7 to fig. 11, and the main information of the chromatograms is shown in table 5, so that the impurities are few, some impurities exist, and the target peak is not influenced.

Table 5: chromatogram information of whole blood sample

Target substance	Area(cps)	RT(min)	IS Area
				FK-506	936200	2.4	530400
CsA	60710	2.5	4373000
				RPM	80640	2.01	1100000
MPA	10010	2.4	28810

Example 3

This example illustrates the preparation of the kit:

1. preparation method of calibrator and quality control product

(1) Preparation: and uniformly mixing tacrolimus, cyclosporine A, rapamycin, mycophenolic acid and pharmaceutical excipients in the calibrator and the quality control products with the final concentrations according to the table 3 in the example 1, wherein the concentrations of the tacrolimus and the rapamycin are sequentially 1ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL and 100ng/ML, the concentration of the cyclosporine A is sequentially 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 500ng/mL and 1000ng/mL, and the concentration of the mycophenolic acid is sequentially 100ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL, 5000ng/mL and 10000ng/mL. And keeping out of the sun and stirring in a sealed way during the period to obtain the semi-finished product solution of the calibrator and the quality control product.

(2) Subpackaging: using a yellow light lamp in a clean area environment, subpackaging each calibrator and quality control semi-finished product solution in a brown ampoule, adding a stopper in the latter half, and putting into a freeze dryer.

(3) And (3) freeze drying:

and (3) freeze-drying the semi-finished product to obtain a freeze-dried powder reagent in the kit: the method comprises the following steps of (1) calibrating and quality control products, and the freeze-drying procedure is as follows:

pre-cooling: pre-cooling for 2 hours by equipment at the temperature of-20 ℃;

pre-freezing: putting the sample into equipment, pre-freezing for 4 hours at-35 ℃;

sublimation: the sublimation temperature is minus 25 ℃ and the time is 10h;

and (3) resolving and drying: maintaining the temperature at 15 ℃ for 3 hours;

enhanced drying: the temperature was maintained at 20 ℃ for 4 hours.

(4) Packaging: and (3) completing the freeze-drying stage, starting the processes of corking, capping and packaging, wherein the whole process is completed in a dark or yellow light environment.

Experimental example 1

This experimental example serves to illustrate the stability of the kit of the invention:

(1) Placing the semi-finished product prepared in the embodiment 3 in a refrigerator at the temperature of 2-8 ℃ for stability investigation of the semi-finished product; randomly taking a semi-finished product sample after subpackaging; the relative deviation between the actual concentration and the theoretical feed concentration was calculated using the mean value of three samples at each concentration point at five time points of 0 day, 1 day, 3 days, 5 days, and 7 days, and the results are shown in tables 8 to 11:

table 8: tacrolimus semi-finished product stability investigation

C1

C2

C3

C4

C5

C6

Q7

Q8

Q9

Day

0

101％

100％

102％

101％

102％

99％

98％

101％

102％

1 day

103％

102％

97％

100％

99％

103％

3 days

104％

98％

101％

103％

100％

98％

99％

103％

102％

5 days

104％

102％

103％

102％

97％

100％

99％

103％

7 days

106％

103％

104％

97％

102％

100％

101％

106％

Table 9: stability investigation of cyclosporin A semi-finished product

C1

C2

C3

C4

C5

C6

Q7

Q8

Q9

Day

0

98％

102％

101％

102％

100％

99％

103％

101％

1 day

98％

101％

103％

102％

98％

96％

104％

100％

3 days

100％

104％

103％

104％

102％

98％

101％

104％

103％

5 days

99％

102％

101％

102％

99％

101％

105％

100％

7 days

103％

104％

103％

101％

103％

102％

101％

103％

104％

Table 10: stability study of rapamycin intermediates

C1

C2

C3

C4

C5

C6

Q7

Q8

Q9

Day

0

102％

101％

98％

102％

98％

101％

100％

1 day

103％

104％

102％

101％

99％

98％

3 days

103％

99％

101％

102％

100％

99％

5 days

102％

105％

101％

100％

99％

100％

99％

7 days

106％

103％

102％

97％

102％

101％

96％

97％

Table 11: stability survey of mycophenolic acid semi-finished product

C1

C2

C3

C4

C5

C6

Q7

Q8

Q9

Day

0

103％

100％

101％

103％

99％

101％

100％

97％

101％

1 day

102％

98％

100％

99％

98％

99％

101％

3 days

103％

100％

101％

104％

102％

103％

98％

102％

5 days

105％

99％

103％

100％

99％

100％

7 days

103％

101％

100％

101％

103％

99％

98％

102％

Statistics shows that the relative deviation of the tacrolimus, the cyclosporine A, the rapamycin and the mycophenolic acid of the semi-finished product is not more than 3% after the semi-finished product is placed in a refrigerator at the temperature of between 2 and 8 ℃ for 5 days, and the relative deviation of the tacrolimus, the cyclosporine A, the rapamycin and the mycophenolic acid of the semi-finished product is not more than 5% after the semi-finished product is placed for 7 days, so that the semi-finished product has better stability.

(2) Inspecting the moisture content and appearance of the freeze-dried product;

4 boxes of the kit prepared in example 3 were randomly sampled and the lyophilized product was measured for moisture by volumetric titration using a Karl Fischer moisture meter. The measurement was repeated 2 times for each sample, and the mean value thereof was calculated. The results of the experiment are shown in table 12:

table 12: investigation of stability of water content of freeze-dried finished product

C1

C2

C3

C4

C5

C6

Q7

Q8

Q9

0 month

1.45％

1.50％

1.38％

1.48％

1.45％

1.52％

1.45％

1.58％

1.56％

1 month

1.50％

1.52％

1.40％

1.43％

1.44％

1.48％

1.49％

1.55％

3 month

1.46％

1.52％

1.47％

1.50％

1.48％

1.53％

1.52％

1.54％

1.58％

Month

5

1.62％

1.58％

1.52％

1.54％

1.59％

1.56％

1.52％

1.58％

1.62％

6 month

1.58％

1.62％

1.60％

1.57％

1.55％

1.61％

1.58％

1.61％

1.65％

The finished product is stored in a dark environment at 2-8 ℃ for 6 months, 5 boxes are respectively sampled in 0, 1, 3, 5 and 6 months, the appearance change is inspected, and the experimental result is shown in table 13:

table 13: appearance inspection of freeze-dried finished product

The moisture content of the packaged finished product is not more than 3 percent when the packaged finished product is stored at room temperature in a dark place for 6 months, the packaging sealing performance is good, and the stability of the finished product is good.

(3) And (3) inspecting the stability of the finished product: and (3) storing the finished product in a dark environment at 2-8 ℃ for 6 months, sampling 5 boxes in 0, 1, 3, 5 and 6 months respectively, processing the samples according to the requirements on the specification of the kit, and calculating the relative deviation between each concentration point and the theoretical concentration. The results of the experiment are shown in tables 14 to 17:

table 14: tacrolimus finished product stability investigation

Table 15: stability survey of cyclosporin A finished product

C1

C2

C3

C4

C5

C6

Q7

Q8

Q9

Day

0

100％

99％

100％

99％

98％

101％

104％

100％

1 month

101％

102％

96％

95％

104％

99％

97％

3 month

103％

105％

103％

100％

102％

100％

Month

5

100％

98％

99％

97％

100％

102％

99％

6 month

98％

96％

98％

97％

96％

95％

100％

97％

Table 16: rapamycin Final product stability study

C1

C2

C3

C4

C5

C6

Q7

Q8

Q9

Day

0

103％

99％

100％

98％

99％

103％

102％

1 month

101％

102％

99％

98％

96％

99％

100％

97％

3 month

99％

101％

99％

98％

97％

98％

103％

100％

102％

Month

5

100％

98％

99％

96％

99％

100％

98％

6 month

100％

97％

98％

97％

100％

99％

Table 17: investigation of stability of finished mycophenolic acid product

C1

C2

C3

C4

C5

C6

Q7

Q8

Q9

Day

0

103％

99％

101％

98％

99％

101％

104％

1 month

97％

102％

98％

100％

97％

96％

100％

101％

Month 3

97％

104％

100％

99％

97％

98％

99％

98％

104％

Month

5

97％

95％

97％

96％

99％

97％

98％

100％

6 month

96％

95％

94％

96％

95％

97％

98％

The experimental results show that the freeze-dried powder has good stability at the temperature of 2-8 ℃.

Experimental example 2:

the experimental example is used for explaining the linear range and the lower limit of quantification of the kit and the detection method of the embodiment of the invention:

the chromatograms obtained by performing the calibration samples of 6 concentrations shown in table 3 were obtained by the method of example 2. The peak area-concentration of the chromatogram was plotted to obtain a standard curve, and the results showed that the linear range and the quantitative limit of the 4 immunosuppressive agents are shown in table 18.

Watch 18

Compound (I)	Detection limit	Limit of quantification	Linear range of
				FK-506(ng/mL)	0.1	0.2	1～100
CsA(ng/mL)	2	5	10～1000
				RPM(ng/mL)	0.1	0.3	1～100
MPA(ng/mL)	1	3	100～1000

The above experimental results show that the kit and the detection method of the embodiment of the invention have wide linear range and low detection limit.

Experimental example 3:

the experimental example is used for explaining the uniformity of the kit and the detection method in the embodiment of the invention:

the experimental method comprises the following steps: randomly extracting 15 boxes of the kit prepared in example 3, testing three samples at each concentration point of each box of calibrator according to a certain sequencing rule, and testing the in-bottle Coefficient of Variation (CV) of each concentration point of C1, C2, C3, C4, C5 and C6 _{In the bottle} ) And Coefficient of Variation (CV) between bottles _{Bottle room} )。

Table 19: uniformity of calibrator

From the above experimental results, it can be seen that (CV) _{In the bottle} ) And coefficient of variation between bottles (CV) _{Bottle room} ) Are less than 6 percent and less than 15 percent specified by the standard, so the kit has better uniformity.

Experimental example 4:

the experimental example is used for explaining the precision of the kit and the detection method of the embodiment of the invention:

the experimental method comprises the steps of randomly drawing 1 batch of the kit and a normal human whole blood sample, synchronously processing the human whole blood sample and a quality control product for 10 times at a time, measuring the concentrations of four immunosuppressive agents in the whole blood and the quality control product according to the quantitative method, wherein the quality control product is in a given target value range, and the precision range in the calculation batch is 1.10-2.54%, as shown in Table 20; in addition, 3 batches of the kit and normal human whole blood samples are randomly extracted, and the calibrator, the quality control material and the human whole blood samples are synchronously processed in batches;

table 20: precision in batch

Table 21: inter-batch precision

The concentrations of the immunosuppressive agents in the quality control material and the serum sample are determined according to the quantitative method, the quality control material is in the given target value range, and the precision range between calculation batches is within 10 percent.

Experimental example 5:

the experimental example is used for explaining the recovery rate of the kit and the detection method in the embodiment of the invention:

the experimental method comprises the steps of selecting a normal whole blood sample, dividing the normal whole blood sample into 4 equal parts, adding standard solutions of objects to be detected with different concentrations and the same volume into 3 samples to prepare 3 samples to be recovered and analyzed with different added concentrations, and ensuring that the final concentration of the sample does not exceed a linear range; the same volume of the solvent without the analyte was added to the remaining sample to prepare a base sample. The recovery rate was calculated using equation (1).

In the formula:

r: recovery rate;

v: adding the volume of the standard solution or the solvent without the substance to be detected;

V ₀ : the volume of the sample;

c: detecting the concentration of the sample after the sample is added into the standard solution;

C _{sign board} : concentration of standard solution.

Note: addition concentration = standard solution concentration x [ standard solution addition volume/(sample volume + standard solution volume) ]

Table 22: recovery rate

According to the experimental results, the recovery rates of the kit and the experimental method are both 90-110%.

Experimental example 6:

the experimental example is used for illustrating the technical effect of the addition type of the pharmaceutical excipients in the embodiment of the invention.

A kit was prepared according to the method of example 3, except that:

comparative example 1: the stability of the freeze-dried product was tested according to the method of experimental example 1 without addition of sheep red blood cells to the calibrator C4 to obtain the contents of each immunosuppressant (obtained by converting the percentage of the test result and the theoretical value with the theoretical charge of 100%), and the results are shown in table 23:

TABLE 23

As can be seen from the data in Table 23, the stability of the sample without sheep red blood cells was poor.

Comparative example 2: adopt ox whole blood to replace sheep erythrocyte preparation calibrator C4, discover in the experiment, adopt sample solution after the freeze-drying of ox whole blood redissolves to appear the flocculation state easily, influence the application of sample degree of accuracy.

Comparative example 3: the pharmaceutical excipients of the calibrator C4 are prepared by matching other substances with bovine serum albumin, and are specifically shown in Table 24. Then, the appearance, moisture content and content (obtained by converting the percentage of the theoretical charge to 100% and the detection result to the theoretical value) of the freeze-dried product were measured according to the method of experimental example 1, and the experimental results are shown in table 24:

watch 24

As shown in table 24, sucrose, PVP, and trehalose were used instead of mannitol, and the product was in an atrophied state in appearance and had a high water content and a poor freeze-drying effect after freeze-drying. If mannitol is not added, only serum albumin is used as a filling agent, and the appearance of the product is cracked after freeze-drying.

Experimental example 7:

the experimental example is used for illustrating the technical effect of the addition ratio of the pharmaceutical excipients in the embodiment of the invention.

Comparative example 4: lyophilized powder was prepared using the pharmaceutical excipients shown in table 25, and calibrator C4 was prepared according to the same formulation as in example 1. The content (calculated by the theoretical charge of 100%, the percentage of the detection result and the theoretical value converted, and stored in a dark place at 2-8 ℃ for 72 hours) and the solution state of the lyophilized product after reconstitution were tested according to the method of experimental example 1, and the experimental results are shown in table 25:

TABLE 25

Wherein, the red blood cells are diluted by phosphate buffer salt with pH7.4, and the red blood cells with the volume percentage concentration of 80 percent, 50 percent and 20 percent are respectively obtained.

As is clear from the data in Table 25, the red blood cell contents of 50% and 80% and the stability were satisfactory, and 50% red blood cells were more preferable from the viewpoints of cost and experimental operability (80% red blood cell products are likely to be flocculated).

Comparative example 5: the same formulation as in example 1 was followed using the excipients shown in Table 26, and then a calibrator C4 was prepared according to the method of example 3: the content (obtained by converting the percentage of the detection result and the theoretical value by taking the theoretical charge as 100%), appearance (state after reconstitution of the lyophilized product), and moisture were measured according to the method of experimental example 1, and the experimental results are shown in table 26:

watch 26

As shown in Table 26, if the ratio of bovine serum albumin to mannitol is too high, which affects the excessive water content of lyophilization, the lyophilization and the desolvation drying should be performed for a prolonged period of time during the lyophilization process, which increases the lyophilization cost. And after freeze-drying and redissolving, the sample is easy to generate a flocculation state, and the accuracy of sample adding is influenced.

Example 8:

the experimental example is used for illustrating the technical effect of the freeze-drying curve of the embodiment of the invention;

calibrator C4 was prepared according to the procedure of example 3, except that the lyophilization profile shown in Table 27 was used.

Comparative example 6: and (3) precooling, namely putting the product into a freeze-drying box at-35 ℃ for 2h.

Comparative example 7: analytical drying, temperatures and conditions are shown in table 27.

The contents of each immunosuppressant (based on 100% of the theoretical charge, the percentage of the detection result and the theoretical value is converted), and the experimental results in the prefreezing state are shown in table 27:

watch 27

As can be seen from Table 27, the prefreezing effect of the rapid rate freeze-drying is not as good in appearance and content as the slow freezing effect. The temperature of the desorption drying is increased, the moisture is not improved due to the time extension, and the freeze-drying cost is increased.

Example 9:

this example is used to illustrate the technical effects of the protein precipitant and the pretreatment method:

comparative example 9: when the volume ratio of the calibrator C4 to the protein precipitant II is 1: 1. 1:2. 1: and 3, detecting the content (obtained by converting the percentage of a detection result and a theoretical value by taking the theoretical feeding as 100%) according to the method of the experimental example 1 under the same other conditions as the example 2. The results are shown in Table 28:

watch 28

As can be seen from the experimental results in table 28, the volume ratio of the sample solution to the protein precipitant II was 1:2, the red blood cells are fully cracked, and the recovery rate of the substance to be detected is better.

Comparative example 10: the content of each immunosuppressant in the calibrator C4 (obtained by converting the theoretical feeding of 100% and the detection result value and the percentage of the theoretical value) was measured by the method of experimental example 1 while only changing the concentration of the protein precipitant I. The results of the experiment are shown in Table 29.

Watch 29

As can be seen from the results in Table 29, the concentration of the protein precipitant I is 0.2mol/L, red blood cells are sufficiently lysed, and the recovery rate of the analyte is high.

Comparative example 11: the effect of centrifugation on the calibrator C4 samples during the treatment was examined by changing only the type of the protein precipitant II, as shown in Table 30.

Watch 30

Protein precipitantClass II	Effect of centrifugal precipitation
		Acetonitrile (ACN)	Complete precipitation
N-hexane	Poor settling effect, floating small particles

From the experimental results in Table 30, it is found that acetonitrile has a better precipitation effect than n-hexane.

Comparative example 12: the content of each immunosuppressant (obtained by converting the percentage of a detection result value and a theoretical value by taking the theoretical feeding as 100%) in the calibrator C4 was detected according to the method of experimental example 1 only by changing the volume ratio of the calibrator C4 sample solution to the protein precipitant II. The results of the experiment are shown in Table 31.

Watch 31

As can be seen from the experimental results in table 31, the volume ratio of the sample solution to the protein precipitant II was 1:2, the content of the object to be detected is better, and the sample is cleaner after the type treatment.

The above description is merely illustrative of particular embodiments of the invention that enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A kit for detecting an immunosuppressant in whole blood by high performance liquid chromatography-tandem mass spectrometry is characterized by comprising a calibrator, an internal standard, a mobile phase stock solution and a protein precipitator;

the calibrator contains serial concentrations of immunosuppressants and pharmaceutical excipients; the immunosuppressant comprises tacrolimus, cyclosporine A, rapamycin and mycophenolic acid; the pharmaceutical auxiliary material contains at least one of whole blood, red blood cells and serum albumin, the source of the whole blood, red blood cells and serum albumin is human or mammal, and the mammal is preferably cattle, sheep or pig.

2. The kit of claim 1, further comprising a quality control product, wherein the quality control product contains low, medium and high concentrations of immunosuppressant and pharmaceutical excipients; the immunosuppressant comprises tacrolimus, cyclosporine A, rapamycin and mycophenolic acid; the pharmaceutical auxiliary material contains at least one of whole blood, erythrocytes and serum albumin, the sources of the whole blood, the erythrocytes and the serum albumin are human or mammals, and the mammals are preferably cattle, sheep or pigs.

3. The kit according to claim 1 or 2, wherein the calibrator and the quality control material are both freeze-dried powders; the freeze-dried powder contains the immunosuppressant and pharmaceutic adjuvant with a series of concentrations, and the pharmaceutic adjuvant also contains an excipient and buffer salt; the excipient is at least one selected from serum albumin, trehalose, mannitol, PVP and sucrose, and the buffer salt is phosphate buffer salt with pH of 7.0-7.4.

4. The kit according to claim 3, wherein the pharmaceutical excipients comprise:

7-12 parts of excipient;

100-800 parts by volume of whole blood or red blood cells;

100-700 parts by volume of phosphate buffer salt;

the volume percentage concentration of the red blood cells is 50 to 70 percent;

when parts by weight are given in g, parts by volume are given in mL.

5. The kit according to any one of claims 1 to 4,

the series of concentrations of immunosuppressant in the calibrator comprises 6 concentrations of immunosuppressant, the 6 concentrations of immunosuppressant being:

calibration product C1: tacrolimus 0.9-1.1 ng/mL, cyclosporine A9-11 ng/mL, rapamycin 0.9-1.1 ng/mL, mycophenolic acid 90-110 ng/mL;

and (3) calibration product C2: 4.5-5.5 ng/mL of tacrolimus, 45-55 ng/mL of cyclosporine A, 4.5-5.5 ng/mL of rapamycin and 450-550 ng/mL of mycophenolic acid;

and (3) calibration product C3: 9-11 ng/mL of tacrolimus, 90-110 ng/mL of cyclosporine A, 9-11 ng/mL of rapamycin and 900-1100 ng/mL of mycophenolic acid;

and (3) calibration product C4: tacrolimus 18-22 ng/mL, cyclosporine A180-220 ng/mL, rapamycin 18-22 ng/mL, mycophenolic acid 1800-2200 ng/mL;

a calibrator C5: 45-55 ng/mL of tacrolimus, 450-550 ng/mL of cyclosporine A, 45-55 ng/mL of rapamycin and 4500-5500 ng/mL of mycophenolic acid;

and (3) calibration product C6: 90-110 ng/mL of tacrolimus, 900-1100 ng/mL of cyclosporine A, 90-110 ng/mL of rapamycin A and 9000-11000 ng/mL of mycophenolic acid;

the series of concentrations of immunosuppressant in the quality control product comprises 3 concentrations of immunosuppressant, comprising:

low-value quality control product Q7: 1.8-2.2 ng/mL of tacrolimus, 18-22 ng/mL of cyclosporine A, 1.8-2.2 ng/mL of rapamycin and 180-220 ng/mL of mycophenolic acid;

and (3) a median quality control product Q8: 9-11 ng/mL of tacrolimus, 180-220 ng/mL of cyclosporine A, 9-11 ng/mL of rapamycin and 900-1100 ng/mL of mycophenolic acid;

high-value quality control Q9: 72-88 ng/mL of tacrolimus, 720-880 ng/mL of cyclosporine A, 72-88 ng/mL of rapamycin and 7200-8800 ng/mL of mycophenolic acid.

6. The kit according to claim 1,

the mobile phase comprises a mobile phase A stock solution and a mobile phase B stock solution, wherein the mobile phase A stock solution is an aqueous solution containing 2-8% of formic acid and 0.5-5 mM of ammonium acetate in percentage by volume; the mobile phase B stock solution is a methanol solution containing 2-8% of formic acid by volume percentage;

the internal standard substance is an acetonitrile solution containing the internal standard substance, and the internal standard substance is ascomycin, cyclosporine A-d4, rapamycin-d 3 and mycophenolic acid-d 3; the concentration of the ascomycin in the inner standard product is 0.009-0.011 mu g/mL, the concentration of cyclosporine A-d4 is 0.4-0.6 mu g/mL, the concentration of rapamycin-d 3 is 0.01-0.03 mu g/mL, and the concentration of mycophenolic acid-d 3 is 0.8-1.2 mu g/mL;

the protein precipitator comprises a protein precipitator I and a protein precipitator II, wherein the protein precipitator I is an aqueous solution of zinc sulfate with the concentration of 0.1-0.3 mol/L; the protein precipitator II is acetonitrile solution containing 0-5% methanol by volume percentage.

7. A method for preparing the kit according to any one of claims 1 to 6, comprising the steps of preparing the standard substance and the quality control substance, comprising at least:

s1, taking the immunosuppressant and the pharmaceutic adjuvant according to a proportion, and preparing to obtain a solution;

s2, subpackaging the solution;

s3, freeze-drying, wherein the freeze-drying at least comprises the following steps:

s32, pre-freezing: pre-freezing for 2-5 hours at-30 to-50 ℃;

and S36, packaging.

8. A method for detecting an immunosuppressive agent in whole blood using the kit of claim 6, comprising at least the steps of:

s1, preparing a solution: the method comprises the following steps:

respectively dissolving the calibrator and the quality control material with water for later use; diluting the mobile phase stock solution A with water to obtain a mobile phase A; diluting the mobile phase stock solution B with methanol to obtain a mobile phase B for later use;

s2, preparing a standard curve equation, which comprises the following steps:

pretreating the calibrator, and detecting by using a high performance liquid chromatography-mass spectrometer to obtain a standard curve equation;

s3, obtaining a sample to be detected, comprising: taking a whole blood sample or a quality control product to be detected, and performing pretreatment to obtain a sample to be detected;

s4, detecting the sample to be detected, wherein the method comprises the following steps: detecting the sample to be detected by using a high performance liquid chromatography-mass spectrometer, substituting the detection result into the standard curve equation, and obtaining the concentration of the immunosuppressant in the sample to be detected;

in S2 and S3, the preprocessing includes: adding a sample solution into an internal standard substance, wherein the sample solution comprises a whole blood sample to be detected, a calibrator and a quality control product; adding the protein precipitant I, mixing, adding the protein precipitant II, mixing, centrifuging, collecting the supernatant as a sample to be detected for chromatographic analysis;

the method also comprises the precision of the quality control evaluation detection method.

9. The method according to claim 8, wherein, in S2 and S3,

the volume ratio of the sample solution to the internal standard is 1:0.5 to 2; the volume ratio of the sample solution to the protein precipitant I is 1:0.5 to 2; the volume ratio of the sample solution to the protein precipitant II is 1:1 to 3.

10. The method according to claim 8, wherein the conditions of the high performance liquid chromatography are: the chromatographic column is selected from a C18 column, and the particle size is 2-5 mu m; the column temperature is 25-45 ℃; the flow rate is 0.3-1 mL/min;

the gradient elution conditions were:

0-0.5 min, mobile phase A: 30-50%, mobile phase B:50 to 70 percent;

0.51-2 minutes, mobile phase a: 1-5%, mobile phase B:5 to 99 percent;

2.1-4 minutes, mobile phase a: 30-50%, mobile phase B:50 to 70 percent;

the conditions of the mass spectrum are as follows:

the Ionspray voltage; 0-5500V; gas 1: 40-60 psi; gas 2:0 to 90psi; air curtain air: 20-50 psi; heating gas temperature: 150 to 550 ℃;

the ion pair information is:

tacrolimus: the parent ion is 821.6, the daughter ion is 768.5, and the collision energy is 85eV;

rapamycin: parent ion 931.7, daughter ion 864.5, collision energy 80eV;

cyclosporin A: parent ion 1220, daughter ion 1202.9, collision energy 40eV;

mycophenolic acid: parent ion 321.1, daughter ion 207, collision energy 75eV.