CN112505183A - Method for detecting concentration of drug in tacrolimus mononuclear cell and application - Google Patents
Method for detecting concentration of drug in tacrolimus mononuclear cell and application Download PDFInfo
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- QJJXYPPXXYFBGM-SHYZHZOCSA-N tacrolimus Natural products CO[C@H]1C[C@H](CC[C@@H]1O)C=C(C)[C@H]2OC(=O)[C@H]3CCCCN3C(=O)C(=O)[C@@]4(O)O[C@@H]([C@H](C[C@H]4C)OC)[C@@H](C[C@H](C)CC(=C[C@@H](CC=C)C(=O)C[C@H](O)[C@H]2C)C)OC QJJXYPPXXYFBGM-SHYZHZOCSA-N 0.000 title claims abstract description 42
- QJJXYPPXXYFBGM-LFZNUXCKSA-N Tacrolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1\C=C(/C)[C@@H]1[C@H](C)[C@@H](O)CC(=O)[C@H](CC=C)/C=C(C)/C[C@H](C)C[C@H](OC)[C@H]([C@H](C[C@H]2C)OC)O[C@@]2(O)C(=O)C(=O)N2CCCC[C@H]2C(=O)O1 QJJXYPPXXYFBGM-LFZNUXCKSA-N 0.000 title claims abstract description 23
- 239000003814 drug Substances 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 22
- 229960001967 tacrolimus Drugs 0.000 title claims abstract description 22
- 229940079593 drug Drugs 0.000 title claims abstract description 20
- 210000005087 mononuclear cell Anatomy 0.000 title claims abstract description 18
- 238000001514 detection method Methods 0.000 claims abstract description 26
- 210000004369 blood Anatomy 0.000 claims abstract description 18
- 239000008280 blood Substances 0.000 claims abstract description 18
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims abstract description 11
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims abstract description 11
- 238000004885 tandem mass spectrometry Methods 0.000 claims abstract description 9
- 239000000126 substance Substances 0.000 claims abstract description 8
- 238000002054 transplantation Methods 0.000 claims abstract description 8
- 210000003734 kidney Anatomy 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 46
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 18
- 238000002156 mixing Methods 0.000 claims description 11
- 210000004027 cell Anatomy 0.000 claims description 10
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 9
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 9
- 239000005695 Ammonium acetate Substances 0.000 claims description 9
- 235000019257 ammonium acetate Nutrition 0.000 claims description 9
- 229940043376 ammonium acetate Drugs 0.000 claims description 9
- 235000019253 formic acid Nutrition 0.000 claims description 9
- 210000000265 leukocyte Anatomy 0.000 claims description 9
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 7
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- 210000000601 blood cell Anatomy 0.000 claims description 4
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims description 4
- 229910052786 argon Inorganic materials 0.000 claims description 3
- 230000005540 biological transmission Effects 0.000 claims description 3
- 230000000857 drug effect Effects 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 238000002474 experimental method Methods 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 239000012139 lysis buffer Substances 0.000 claims description 3
- 238000012806 monitoring device Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000007704 transition Effects 0.000 claims description 3
- 239000006285 cell suspension Substances 0.000 claims description 2
- 238000004807 desolvation Methods 0.000 claims description 2
- 238000011067 equilibration Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 229940126585 therapeutic drug Drugs 0.000 claims description 2
- 208000004605 Persistent Truncus Arteriosus Diseases 0.000 abstract description 24
- 208000037258 Truncus arteriosus Diseases 0.000 abstract description 24
- 230000000694 effects Effects 0.000 abstract description 5
- 238000004949 mass spectrometry Methods 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 19
- 239000011550 stock solution Substances 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 210000004698 lymphocyte Anatomy 0.000 description 5
- 239000003643 water by type Substances 0.000 description 5
- 239000012224 working solution Substances 0.000 description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 4
- 239000004472 Lysine Substances 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 210000003743 erythrocyte Anatomy 0.000 description 4
- 230000001506 immunosuppresive effect Effects 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- 229960001484 edetic acid Drugs 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
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- 239000002904 solvent Substances 0.000 description 1
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- 238000003260 vortexing Methods 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The invention relates to the field of biomedicine, and discloses a method for detecting the concentration of a drug in a tacrolimus mononuclear cell, which comprises the steps of extracting the mononuclear cell, carrying out UPLC (ultra performance liquid chromatography) condition, carrying out MS/MS (mass spectrometry) condition, preparing a standard substance, preparing a standard curve and detecting the concentration of tacrolimus in the mononuclear cell; the invention also provides application of the method in management products after kidney transplantation. The method realizes the detection of the peripheral blood mononuclear cell TAC concentration, compared with the whole blood trough concentration detection, the detection direction and the method can more accurately and truly reflect the effect of the TAC treatment medicine in the kidney transplant recipient, and the detection method is simple, rapid in detection, capable of realizing automation and more accurate in detection result.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a detection method and application of drug concentration in tacrolimus mononuclear cells.
Background
Concentration monitoring of Tacrolimus (Tacrolimus, TAC, also known as FK506, trade name plenilla) therapeutic drug has become a detection item which is routinely carried out after renal transplantation, and TAC therapy monitoring is mainly carried out clinically at present by using whole blood trough concentration (C0) detection. The method for detecting the concentration of the TAC mainly comprises an immunological method and a chromatographic method. The immunology method is established based on an anti-drug specific monoclonal antibody, and has the defects that drugs in a sample and metabolites of the drugs cannot be distinguished, so that detection results cannot truly reflect in-vivo pharmacological active drug components, and in addition, due to the fact that titer and specificity of antibodies used by different manufacturers are different, the detection results of TAC (TAC) obtained by different brands of reagents and different detection platforms are different. Although the chromatography method can accurately identify the drug and the metabolite thereof and has higher detection accuracy, the sample pretreatment step is complex and takes long time, which is not beneficial to the development of a conventional laboratory. At present, adverse reactions such as rejection or infection still occur in part of transplant recipients even if the whole blood TAC valley concentration meets the recommended reference range, and the situations suggest that: the currently widely adopted whole blood TAC valley concentration detection is not the best index for reflecting the TAC drug immunosuppressive treatment effect.
In peripheral blood, TAC is mainly distributed in erythrocytes (accounting for about 90% of the whole blood concentration) and plasma, TAC in lymphocytes accounts for only about 1% of the whole blood concentration, but TAC mainly plays an immunosuppressive role in lymphocytes, so that the whole blood TAC concentration cannot accurately reflect the drug concentration level in lymphocytes of a recipient, and the immunosuppressive role and the clinical treatment effect of the drug on the lymphocytes cannot be accurately known. Since lymphocytes are target cells of an immunosuppressant exerting pharmacological effects, and the immunosuppressive efficiency of TAC is most directly and closely related to the concentration of TAC in immune cells, the detection of the concentration of TAC in Peripheral Blood Mononuclear Cells (PBMC) is a new monitoring index capable of more accurately and truly reflecting the effect of TAC treatment drugs in vivo. Therefore, the inventor invents a detection method and application of the concentration of the tacrolimus mononuclear cell drug.
Disclosure of Invention
Based on the problems, the invention provides a detection method and application of drug concentration in tacrolimus mononuclear cells, and provides a new direction and method for TAC treatment drug concentration monitoring.
In order to solve the technical problems, the invention provides a method for detecting the concentration of a tacrolimus mononuclear cell drug, which comprises the following steps:
s1: extraction of mononuclear cells
Taking fresh EDTA anticoagulated whole blood, extracting to obtain leukocyte suspension, wherein the cell number in each 50uL leukocyte suspension is 1 × 106A plurality of;
s2: UPLC Condition
A chromatographic column: reverse phase Waters Acquity UPLC BEH C18 column, 1.7 μm, 50mm x 2.1mm,
column temperature: at a temperature of 60 c,
mobile phase: mobile phase A: a solution obtained by dissolving 2mM ammonium acetate and 0.1% formic acid in 500mL of pure water; mobile phase B: a solution obtained by dissolving 2mM ammonium acetate and 0.1% formic acid in 500mL of methanol; gradient elution with a flow rate of 0.4 mL/min;
s3: MS/MS conditions
Working in a positive ion mode, wherein the capillary voltage is 1.0kV, the source block temperature is 150 ℃, the desolvation temperature is 350 ℃, and the transmission speed is 900L/h; argon is adopted for collision;
s4: preparing standard substance
S5: preparation of a Standard Curve
S6: detection of the concentration of tacrolimus in mononuclear cells
Adding 30uL Lysis Buffer for hold blood into an EP tube filled with 50uL of the cell suspension in the step S1, mixing uniformly by vortex, and incubating for 1min to completely dissolve blood cells; adding 20uL internal standard, 40uL MagSiMUS-TDMprep Type I Particle Mix and 145uL OPR VI, vortex mixing, standing at room temperature for 2min, centrifuging at 12000rpm for 5min, and collecting filtrate; 200uL of the filtrate was transferred to an autosampler vial, a 10uL volume was injected into the LC-MS/MS system, and the concentration in the sample was obtained according to the fitted standard curve, and this concentration was multiplied by 50 times to calculate the tacrolimus content containing 100 ten thousand PBMCs in a 50uL system.
Further, the initial conditions of UPLC are: 45% mobile phase A and 55% mobile phase B, balance for 10 min; sample running conditions: mobile phase B increased to 70% at 0.6min, then rapidly changed to 90% at 0.7min, then increased to 100% at 0.8min, and the final mobile phase a to mobile phase B ratio was 45:55, i.e. 2.2min of equilibration at initial conditions for a total run time of 3 min.
Further, the collision cell pressure in the MS/MS condition was 3.4X 10-3mbar。
Further, the residence time of each transition in the MS/MS condition is 50 MS; data acquisition by Multiple Reaction Monitoring (MRM); to mix 1ug/mL of Tac and Tac in methanol13C-2H4The injection experiment resulted in the best settings.
In order to solve the technical problems, the invention also provides the application of the method for detecting the concentration of the tacrolimus mononuclear cell drug in the management product after the kidney transplantation.
Further, the product is a TAC treatment drug effect monitoring device or system after kidney transplantation.
Compared with the prior art, the invention has the beneficial effects that: the method realizes the detection of the peripheral blood mononuclear cell TAC concentration, compared with the whole blood trough concentration detection, the detection direction and the method can more accurately and truly reflect the effect of the TAC treatment medicine in the kidney transplant recipient, and the detection method is simple, rapid in detection, capable of realizing automation and more accurate in detection result.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not used as limitations of the present invention.
Example (b):
the chemicals and reagents used in this example were as follows: tacrolimus (TAC) and ammonium acetate were obtained fromSigma-Aldrich Chemie B.V. (Netherlands), tacrolimus13C-2H4From Alsa Chim (france), methanol and formic acid from Fisher (usa); MagSiMUS-TDMprep Type I, Organic Precipitation Reagent VI, lysine Buffer for white blood kit purchased from MagnaMedics Diagnostics B.V. (the Netherlands).
The analytical instruments and software used in this example were as follows:
(1) using a Waters Acquity UPLC-MS/MS system (Waters corp., Milford, MA, USA), consisting of an Acquity binary solvent manager, a sample manager, a column manager, and a sample manager;
(2) the UPLC is connected to a triple quadrupole Waters TQ-S miniature mass spectrometer;
(3) the data processing uses the software MasslynxTM V4.1 and Targetlynx V4.1.
The mobile phase was prepared as follows:
(1) preparation of a water phase: taking 10ml of 0.1mol/L ammonium acetate, adding 490ml of I-grade pure water to prepare 2mmol/L NH4Ac·H2O + 0.1% formic acid;
(2) preparing an organic phase: taking 10ml of 0.1mol/L ammonium acetate, adding 490ml of HPLC-grade methanol to prepare 2mmol/L NH4Ac·CH3OH + 0.1% formic acid.
Preparing a standard product: A. preparing a stock solution: adding Tac13C-2H4(purity 98%) 1.0204mg is fully dissolved by methanol and completely transferred into a 25mL volumetric flask, the methanol is used for constant volume, and stock solution with the concentration of 0.04mg/mL is prepared; weighing 0.02577g of Tac standard substance (with the purity of 97%) in a 25mL volumetric flask, fixing the volume with methanol, preparing stock solution with the concentration of 1mg/mL, and subpackaging the stock solution at-80 ℃; B. preparing a working solution: tac is taken13C-2H450uL of 0.04mg/ml stock solution is put in a 50ml volumetric flask, methanol is added to a constant volume, and working solution with the concentration of 40ug/L is prepared; taking 50uL of Tac 1mg/mL stock solution into a 50mL volumetric flask, carrying out constant volume on methanol, and preparing working solution with the concentration of 1000 ug/L; C. preparing a standard substance: tac is taken13C-2H4100uL of 40ug/L working solution and 300uL of methanol are added to obtain the internal standard concentration of 10 ug/L; taking 50uL of Tac 1000ug/L working solution, adding450uL of methanol to obtain the concentration of Tac of 100 ug/L; taking 7 EP tubes, numbering A-G, and preparing the EP tubes with the concentration of 50ug/L, 25ug/L, 12.5ug/L, 6.25ug/L, 3.125ug/L, 1.56ug/L and 0.78ug/L respectively; taking 3 EP tubes with numbers of QCH, QCM and QCL, and preparing the EP tubes with the concentration of 25ug/L, 10ug/L and 1ug/L respectively; obtaining standard substance concentration 2500pg/1 x 10 by using healthy human PBMCs6cells(A)、1250pg/1*106cells(B)、625pg/1*106cells(C)、312.5pg/1*106cells(D)、156.25pg/1*106cells(E)、78pg/1*106cells(F)、39pg/1*106cells (G), quality control concentration 1250pg/1 x 106cells(QCH)、500pg/1*106cells(QCM)、50pg/1*106Cells (QCL); the prepared standard substance and quality control system containing the PBMCs of the healthy people are 50uL, and subsequent tests can be immediately carried out, and if the test needs to be waited for, the PBMCs can be frozen and stored at minus 80 ℃.
Preparing a standard curve: preparing a kit MagSiMUS-TDMprep Type I Particle Mix, Organic Precipitation Reagent VI (OPR VI) and lysine Buffer for hold blood; taking a set of standard tubes A-G, QCH, QCM and QCL, adding 30uL Lysis Buffer for hold blood into each EP tube (50uL), mixing uniformly by vortex, and incubating for 1 minute to completely dissolve blood cells; adding 20uL internal standard (10ug/L Tac)13C-2H4) Vortex mixing 40uL MagSiMUS-TDMprep Type I Particle Mix, 145uL OPR VI, standing for 2min at room temperature, and centrifuging for 5min at 12000 rpm; transferring 200uL of the filtrate into an autosampler vial; a10 uL volume was injected into the LC-MS/MS system and a standard curve was fitted and the measured quality control concentration was expressed as ug/L and this concentration was multiplied by a factor of 50 to calculate the tacrolimus content (pg) containing 100 million PBMCs in a 50uL system.
Extracting mononuclear cells: centrifuging 3-4mL of fresh EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood for 5min at 500g, removing the upper part of a platelet-containing plasma layer, transferring the rest cell layer into a 15mL centrifuge tube, adding 12mL of erythrocyte lysate, and gently swirling or reversing and uniformly mixing; standing at room temperature for 15min, gently vortexing twice, after erythrocyte lysis is completed, the solution should be clear and transparent, centrifuging at 300g for 10min to precipitate leukocyte, carefully sucking off the supernatant(ii) a Adding 15mL of erythrocyte lysate into the leukocyte sediment, gently swirling and fully resuspending the leukocytes, standing at room temperature for 15min, gently swirling and uniformly mixing twice during the gentle swirling, centrifuging for 10min at 300g to precipitate the leukocytes, carefully and thoroughly sucking away supernatant, adding 500uL of PBS (phosphate buffer solution) to resuspend the cells, performing CDC (charge-discharge control) counting on the resuspended cells at room temperature in the whole separation process, and counting the cells according to the volume of 1 × 10 per bottle6cells were aliquoted and frozen at-80 ℃ with a cell count of 1X 10 per 50uL leukocyte suspension6And (4) respectively.
The UPLC conditions for this example are as follows:
1. the chromatographic separation was carried out on a reverse phase Waters Acquity UPLC BEH C18 column (1.7 μm, 50 mm. times.2.1 mm) at 60 ℃;
2. using gradient elution, mobile phase a: dissolving 2mM of ammonium acetate and 0.1% of formic acid in 500mL of pure water; mobile phase B: dissolving 2mM ammonium acetate and 0.1% formic acid in 500mL of methanol;
3. the flow rate is 0.4 mL/min;
4. initial conditions were 45% mobile phase a and 55% mobile phase B, equilibrated for 10 min;
5. conditions under which the samples run: mobile phase B increased to 70% at 0.6min, then rapidly changed to 90% at 0.7min, then increased to 100% at 0.8min, and the final mobile phase a to mobile phase B ratio was 45:55, i.e. 2.2min of equilibrium at initial conditions;
6. the total run time was 3 min.
The MS/MS conditions for this example are as follows:
1. the mass spectrometer works in a positive ion mode, the capillary voltage is 1.0kV, the source block temperature is 150 ℃, the desolventizing temperature is 350 ℃, and the transmission speed is 900L/h;
2. argon is adopted for collision, and the pressure of a collision groove is 3.4 multiplied by 10-3mbar；
3. The residence time of each transition is 50 ms;
4. data acquisition by Multiple Reaction Monitoring (MRM);
5. the best setting was obtained by mixing 1ug/mL Tac and Tac 13C-2H4 injection experiments in methanol;
6. the optimized settings for Multiple Reaction Monitoring (MRM) for each analyte are summarized in the following table:
Table I:MS/MS settings
detection of tacrolimus concentration in mononuclear cells: preparing a kit MagSiMUS-TDMprep Type I Particle Mix, Organic Precipitation Reagent VI (OPR VI) and lysine Buffer for hold blood, adding 30uL lysine Buffer for hold blood into an EP tube filled with 50uL of sample, mixing uniformly by vortex, and incubating for 1min to completely dissolve blood cells; adding 20uL internal standard (10ug/L Tac)13C-2H4) 40uL MagSiMUS-TDMprep Type I Particle Mix and 145uL OPR VI, evenly mixing in a vortex, standing for 2min at room temperature, and centrifuging for 5min at 12000 rpm; 200uL of the filtrate was transferred to an autosampler vial, a 10uL volume was injected into the LC-MS/MS system, and the concentration in the sample was obtained according to the fitted standard curve, and the concentration measured as ug/L was multiplied by 50 times to calculate the tacrolimus content (pg) containing 100 ten thousand PBMCs in a 50uL system.
The method for detecting the concentration of tacrolimus mononuclear cell drug in the embodiment can be applied to a management product after renal transplantation, and the product can be a TAC treatment drug effect monitoring device or system after renal transplantation, but is not limited thereto.
The above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the process of verifying the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all the equivalent structural changes made by applying the content of the specification of the invention should be covered by the scope of the invention.
Claims (6)
1. The method for detecting the concentration of the drug in the tacrolimus mononuclear cell is characterized by comprising the following steps of:
s1: extraction of mononuclear cells
Taking fresh EDTA anticoagulated whole blood, extracting to obtain leukocyte suspension, wherein the cell number in each 50uL leukocyte suspension is 1 × 106A plurality of;
s2: UPLC Condition
A chromatographic column: reversed phase WatersAcquity UPLC BEH C18 column, 1.7 μm, 50 mm. times.2.1 mm,
column temperature: at a temperature of 60 c,
mobile phase: mobile phase A: a solution obtained by dissolving 2mM ammonium acetate and 0.1% formic acid in 500mL of pure water; mobile phase B: a solution obtained by dissolving 2mM ammonium acetate and 0.1% formic acid in 500mL of methanol; gradient elution with a flow rate of 0.4 mL/min;
s3: MS/MS conditions
Working in a positive ion mode, wherein the capillary voltage is 1.0kV, the source block temperature is 150 ℃, the desolvation temperature is 350 ℃, and the transmission speed is 900L/h; argon is adopted for collision;
s4: preparing standard substance
S5: preparation of a Standard Curve
S6: detection of the concentration of tacrolimus in mononuclear cells
Adding 30uL Lysis Buffer for hold blood into an EP tube filled with 50uL of the cell suspension in the step S1, mixing uniformly by vortex, and incubating for 1min to completely dissolve blood cells; adding 20uL internal standard, 40uL MagSiMUS-TDMprep Type I Particle Mix and 145uL OPR VI, vortex mixing, standing at room temperature for 2min, centrifuging at 12000rpm for 5min, and collecting filtrate; 200uL of the filtrate was transferred to an autosampler vial, a 10uL volume was injected into the LC-MS/MS system, and the concentration in the sample was obtained according to the fitted standard curve, and this concentration was multiplied by 50 times to calculate the tacrolimus content containing 100 ten thousand PBMCs in a 50uL system.
2. The method of claim 1, wherein the initial conditions for UPLC are: 45% mobile phase A and 55% mobile phase B, balance for 10 min; sample running conditions: mobile phase B increased to 70% at 0.6min, then rapidly changed to 90% at 0.7min, then increased to 100% at 0.8min, and the final mobile phase a to mobile phase B ratio was 45:55, i.e. 2.2min of equilibration at initial conditions for a total run time of 3 min.
3. The method of claim 1, wherein the collision cell pressure in MS/MS conditions is 3.4 x 10- 3mbar。
4. The method according to claim 1, characterized in that the residence time per transition in MS/MS conditions is 50 MS; data acquisition by Multiple Reaction Monitoring (MRM); to mix 1ug/mL of Tac and Tac in methanol13C-2H4The injection experiment resulted in the best settings.
5. Use of the method according to any one of claims 1 to 4 in a product for the management of the kidney after transplantation.
6. The use according to claim 5, wherein the product is a TAC therapeutic drug effect monitoring device or system after renal transplantation.
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| CN113624883A (en) * | 2021-08-18 | 2021-11-09 | 中日友好医院(中日友好临床医学研究所) | Method for detecting concentration of tacrolimus in PBMC (peripheral blood mononuclear cell) with high sensitivity |
Citations (4)
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