Blood inspection method

Abstract

This blood inspection method comprises a preparation step for preparing whole blood collected from a subject, a pretreatment step for pretreating the whole blood to prepare an analysis sample, a separation step for separating the analysis sample into individual components by liquid chromatography, and an analysis step for analyzing each of the separated components with a mass spectrometry device. The subject has undergone the administration of a first immunosuppressive agent and a second immunosuppressive agent, the first immunosuppressive agent comprising at least one component selected from the group consisting of tacrolimus, ciclosporin A, everolimus and sirolimus, and the second immunosuppressive agent comprising at least one component selected from the group consisting of mycophenolic acid and metabolites thereof. The separated components include the first immunosuppressive agent and the second immunosuppressive agent. With respect to the method, in the analysis of the components with the mass spectrometry device, the analysis is performed in a positive mode when the first immunosuppressive agent is analyzed as a target component, and the analysis is performed in a negative mode when the second immunosuppressive agent is analyzed as a target component.

Description

Blood test method

[0001] The present invention relates to a blood testing method.

[0002] In organ or hematopoietic stem cell transplantation therapy, patients are usually administered immunosuppressants to suppress transplant rejection. However, excessive administration of immunosuppressants can suppress the patient's immune system more than necessary, which can lead to infections and other problems. Therefore, it is necessary to measure and monitor the drug concentration in the blood (also called "drug blood concentration") after administration and carefully manage the dosage.

[0003] Known methods for monitoring such drug blood concentrations (hereinafter sometimes abbreviated as "TDM") include separation and analysis methods using high performance liquid chromatography or gas chromatography, immunological methods such as fluorescence polarization immunoassay, enzyme immunoassay or radioimmunoassay, and atomic absorption spectrometry.

[0004] For example, Japanese Patent Application Laid-Open No. 2021-128005 (Patent Document 1) discloses a method for monitoring drug blood concentrations, which includes adding a deproteinized blood sample to a conditioned solid-phase extraction column, washing the solid-phase extraction column after the blood sample has passed through it, eluting the adsorbed material from the solid-phase extraction column with an elution solution, and analyzing the resulting eluate by LC-MS / MS.

[0005] Japanese Patent Application Laid-Open No. 2021-128005

[0006] Depending on the drug being monitored, whole blood or plasma may be used as the blood sample prepared for monitoring drug blood concentrations. For example, when monitoring tacrolimus, cyclosporine A, everolimus, and sirolimus, which are types of immunosuppressants, whole blood is used as the blood sample. On the other hand, when monitoring mycophenolic acid and its metabolites (e.g., mycophenolic acid β-D-glucuronide), which are another type of immunosuppressant, plasma is used as the blood sample. Therefore, when two or more types of drugs are being monitored and whole blood and plasma must be prepared separately, the amount of blood collected increases, imposing a significant burden on the patient.

[0007] The present invention has been made in view of the above circumstances, and has as its object to provide a blood testing method that can quantify two or more types of immunosuppressants in the same blood sample.

[0008] As a result of intensive research, the inventors have discovered that by using whole blood as a blood sample and changing the ionization mode in mass spectrometry depending on the components to be analyzed, it is possible to quantify two or more types of immunosuppressants in the same blood sample, and have completed the present invention.

[0009] A first aspect of the present invention relates to a blood testing method comprising: a preparation step of preparing whole blood collected from a subject; a pretreatment step of pretreating the whole blood to prepare an analytical sample; a separation step of separating the analytical sample into components using a liquid chromatograph; and an analysis step of analyzing the separated components using a mass spectrometer, wherein the subject is administered a first immunosuppressant and a second immunosuppressant; the first immunosuppressant comprises at least one selected from the group consisting of tacrolimus, cyclosporine A, everolimus, and sirolimus; the second immunosuppressant comprises at least one selected from the group consisting of mycophenolic acid and metabolites thereof; the separated components comprise the first immunosuppressant and the second immunosuppressant; and when analyzing with the mass spectrometer, analysis is performed in positive mode when analyzing the first immunosuppressant as the target component, and analysis is performed in negative mode when analyzing the second immunosuppressant as the target component.

[0010] According to the present invention, it is possible to provide a blood testing method that can quantify two or more types of immunosuppressants in the same blood sample.

[0011] An embodiment of the present invention (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited thereto. In this specification, the notation in the form of "A to Z" means the upper and lower limits of a range (i.e., A or more and Z or less), and when no unit is specified for A and a unit is specified only for Z, the unit of A and the unit of Z are the same.

[0012] <Blood Testing Method> A first aspect of the present invention relates to a blood testing method comprising: a preparation step of preparing whole blood collected from a subject; a pretreatment step of pretreating the whole blood to prepare an analysis sample; a separation step of separating the analysis sample into components using a liquid chromatograph; and an analysis step of analyzing the separated components using a mass spectrometer, wherein the subject is administered a first immunosuppressant and a second immunosuppressant, the first immunosuppressant comprises at least one selected from the group consisting of tacrolimus, cyclosporine A, everolimus, and sirolimus, the second immunosuppressant comprises at least one selected from the group consisting of mycophenolic acid and metabolites thereof, the separated components comprise the first immunosuppressant and the second immunosuppressant, and when analyzing with the mass spectrometer, analysis is performed in a positive mode when analyzing the first immunosuppressant as the target component, and analysis is performed in a negative mode when analyzing the second immunosuppressant as the target component.

[0013] <Preparation Step> In this step, whole blood collected from a subject is prepared. The subject is a subject who has been administered a first immunosuppressant and a second immunosuppressant. The term "subject" refers to a human being from whom the whole blood to be subjected to the blood testing method of this embodiment is derived, and there are no particular limitations on age, sex, or race. The subject may be a healthy individual or a patient with some kind of disease. In one aspect of this embodiment, the subject is preferably a patient undergoing organ or hematopoietic stem cell transplantation therapy.

[0014] In this embodiment, "whole blood" refers to blood collected from the subject as is, or a blood sample to which an anticoagulant has been added. The whole blood contains blood cell components, platelets, and plasma components. There are no particular limitations on the method for collecting whole blood, and it may be collected from the subject by a known method.

[0015] In this embodiment, the first immunosuppressant includes at least one selected from the group consisting of tacrolimus (generic name: tacrolimus hydrate), cyclosporin A, everolimus, and sirolimus.

[0016] In this embodiment, the second immunosuppressant comprises at least one selected from the group consisting of mycophenolic acid (MPA) and its metabolites (for example, mycophenolic acid β-D-glucuronide (MPAG)).

[0017] <Pretreatment Step> In this step, the whole blood is pretreated to prepare a sample for analysis.

[0018] The pretreatment method for the whole blood can be used without particular limitation as long as the analytical sample obtained by the pretreatment can be separated into components by liquid chromatography, as described below. In one aspect of this embodiment, the "analytical sample" can also be understood as a sample derived from the whole blood and subjected to the separation step, as described below. Examples of the pretreatment include a protein removal operation (sometimes referred to as a "deproteinization operation") and extraction by solid-phase extraction.

[0019] In one aspect of this embodiment, a predetermined amount of an internal standard substance may be added to the whole blood before the pretreatment is performed. As the internal standard substance, for example, a known internal standard substance can be used.

[0020] <Separation Step> In this step, the analytical sample is separated into individual components using a liquid chromatograph. Liquid chromatographs (LC) utilize the differences in the affinities of the components in the analytical sample for the mobile phase and the stationary phase to separate the components and elute them at different retention times. The type of liquid chromatograph is not limited as long as it can separate the components to be analyzed with the desired accuracy so that they can be separated and detected by a mass spectrometer. Examples of liquid chromatographs that can be used include nano-LC, micro-LC, high-performance liquid chromatographs (HPLC), and ultra-high-performance liquid chromatographs (UHPLC).

[0021] In this embodiment, the mobile phase is not particularly limited, but examples thereof include an ammonium formate aqueous solution as mobile phase A, a liquid containing acetonitrile and isopropanol in a predetermined ratio such as a volume ratio of 1:1 as mobile phase B, and mobile phases described in the examples below.

[0022] In this embodiment, the stationary phase is not particularly limited, but examples thereof include silanes to which linear hydrocarbons such as C8 and C18 are bonded, supported on a carrier such as silica gel, and stationary phases described in the examples below.

[0023] <Analysis Step> In this step, the separated components are analyzed using a mass spectrometer. The separated components include the first immunosuppressant and the second immunosuppressant.

[0024] In this embodiment, when analyzing the first immunosuppressant as a target component using a mass spectrometer, the analysis is performed in positive mode, and when analyzing the second immunosuppressant as a target component, the analysis is performed in negative mode. In one aspect of this embodiment, when the elution time of the target component is known in advance in separation by liquid chromatography, the ionization mode can be set to positive mode or negative mode based on the elution time.

[0025] Conventionally, whole blood has been used as a blood sample when monitoring immunosuppressants such as tacrolimus, cyclosporine A, everolimus, and sirolimus (first immunosuppressant). On the other hand, plasma has been used as a blood sample when monitoring another immunosuppressant, mycophenolic acid, and its metabolites (e.g., mycophenolic acid β-D-glucuronide) (second immunosuppressant). Therefore, when whole blood and plasma must be prepared separately to monitor the first and second immunosuppressants, the amount of blood collected increases, placing a significant burden on patients. Furthermore, the significant difference between the detection concentration ranges of the first and second immunosuppressants also contributes to the increased amount of blood collected. When measuring these immunosuppressants using LC-MS, the blood concentration of the second immunosuppressant is higher than that of the first immunosuppressant. Therefore, for example, if the sensitivity is optimized for detecting the first immunosuppressant, saturation occurs when detecting the second immunosuppressant, making it difficult to simultaneously analyze both components.

[0026] As a result of extensive research, the present inventors have discovered that by using whole blood as a blood sample and changing the ionization mode in mass spectrometry depending on the components to be analyzed, it is possible to quantify two or more immunosuppressants (a first immunosuppressant and a second immunosuppressant) in the same blood sample. Methods for reducing the sensitivity of mass spectrometry have traditionally been to use negative mode or to change the gas flow rate or voltage. However, the use of negative mode in the analysis of the second immunosuppressant, which allows for simultaneous analysis of the first immunosuppressant and the second immunosuppressant from the same blood sample, i.e., to align the dynamic range, has not previously been attempted.

[0027] The method of ionization using a mass spectrometer is not particularly limited, and may be electrospray ionization (ESI), nano-electrospray ionization (nano-LSI), etc. In this embodiment, the ionization method is preferably ESI.

[0028] Examples of the mass spectrometer include a Fourier transform mass spectrometer, a time-of-flight (Q-TOF) mass spectrometer, a triple quadrupole mass spectrometer, etc. In this embodiment, from the viewpoint of analyzing known components accurately in a short time, the mass spectrometer is preferably a triple quadrupole mass spectrometer.

[0029] In one aspect of this embodiment, the separation step and the analysis step may be performed consecutively. For example, the separation step and the analysis step may be performed consecutively by using a liquid chromatograph-tandem mass spectrometer. The liquid chromatograph-tandem mass spectrometer is typically a liquid chromatograph-triple quadrupole mass spectrometer or a liquid chromatograph-quadrupole / time-of-flight (Q-TOF) mass spectrometer. Typically, in such a liquid chromatograph-tandem mass spectrometer, analysis parameters are determined so as to maximize detection sensitivity, in other words, to achieve the best possible conditions, for each component in the eluate separated by the liquid chromatograph.

[0030] In one aspect of this embodiment, it is preferable that the analyzing step further includes quantifying the separated components. The method for quantifying the separated components is not particularly limited, and known methods can be used. Examples of such methods include the absolute calibration curve method and the internal standard method. In another aspect of this embodiment, it is preferable that the analyzing step further includes quantifying the separated components based on a calibration curve prepared in advance.

[0031] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0032] <<Creating a Calibration Curve>> <Preparing Whole Blood Calibration Solutions> Whole blood calibration solutions (L1 to L6) were prepared using the following procedure. First, 30 μl of each of a methanol solution of MPA (concentration: 0.01 to 5 mg / L) and a methanol solution of MPAG (concentration: 0.1 to 25 mg / L) was added to a microtube. The solvent, methanol, was then evaporated so that only MPA and MPAG remained in the microtube. In this way, six microtubes (L1 to L6) containing different amounts of MPA and MPAG were prepared.

[0033] To the microtube L1, 3 μl of the calibrator stock solution L1 was added. Here, the “calibrator stock solution” is a liquid sample in which tacrolimus, cyclosporin A, everolimus, and sirolimus are dissolved at the concentrations shown in Table 1 in a whole blood matrix.

[0034] Next, 3 μl of the internal standard sample included in the DOSIMMUNE immunosuppressant analysis kit (Shimadzu Corporation), 3 μl of the internal standard sample included in the DOSIMYCO immunosuppressant analysis kit (Shimadzu Corporation), and 200 μl of extraction solution (a 40:40:20 mixture of acetonitrile, methanol, and water) were added to the microtube L1 and mixed for 1 minute using a Bostex mixer. After mixing, the microtube L1 was centrifuged (15,000 rpm, 7 minutes) to separate the supernatant and precipitate (pretreatment step). 200 μl of this supernatant was collected and used as whole blood calibration solution L1.

[0035] The whole blood calibration solutions L2 to L6 were prepared in the same manner as above, except that 3 μl of the calibrator stock solutions L2 to L6 were added to microtubes L2 to L6, respectively. Table 1 shows the concentrations of each component contained in the calibrator stock solutions L1 to L6.

[0036]

[0037] <Separation and Analysis> The whole blood calibration solutions L1 to L6 were analyzed by LC-MS / MS using gradient elution under the following analytical conditions.

[0038] (Chromatography conditions) Trap solvent (Pump C): DOSIMMUNE MOBILE PHASE A Mobile phase A (Pump A): DOSIMMUNE MOBILE PHASE A Mobile phase B (Pump B): DOSIMMUNE MOBILE PHASE B Initial mobile phase flow rate: 0.2 mL / min Trap column: DOSIMMUNE Trap Column (S / N ALIMTCL000000155) Analytical column: DOSIMMUNE Analytical Column (S / N ALIMTCL000000155) Initial valve position: 0 (Pump C - Trap column - Drain) Time program : See Table 2 (time unit is "minutes") Column temperature: 65°C Injection volume: 20 μL

[0039]

[0040] (Mass spectrometry conditions) Ionization method: ESI-Negative MRM mode (0-1.25 min MPA, MPAG) ESI-Positive MRM mode (1.25-3.00 min everolimus, sirolimus, tacrolimus, cyclosporin A) Nebulizer gas flow rate: 3 L / min Heating gas flow rate: 10 L / min Interface temperature: 200°C Drying gas flow rate: 10 L / min DL temperature: 150°C Heat block temperature: 200°C CID gas pressure: 230 kPa Probe position: +3 mm

[0041] (MRM conditions) MPA (ESI-Negative): 319.2000>191.2500 (CE 30 V) MPA IS (ESI-Negative): 323.2000>191.2500 (CE 23 V) MPAG (ESI-Negative): 495.2000>191.3000 (CE 37 V) MPAG IS (ESI-Negative): 499.2000>191.2000 (CE 42 V) Everolimus (ESI-Positive): 975.6000>908.5500 (CE -17 V) Everolimus IS (ESI-Positive): 981.6000>914.7000 (CE -17 V) Sirolimus (ESI-Positive): 931.6000>864.6500 (CE -17 V) Sirolimus IS (ESI-Positive): 935.6000>864.5000 (CE -18 V) Tacrolimus (ESI-Positive): 821.5000>768.5500 (CE -22 V) Tacrolimus IS (ESI-Positive): 826.5000>773.6000 (CE -22 V) Cyclosporine A (ESI-Positive): 1220.0000>1202.9500 (CE -19 V) Cyclosporine A IS (ESI-Positive): 1232.0000>1215.0000 (CE -19 V)

[0042] The calibration curves for each component created based on the analysis results are shown in Tables 3 to 8. In Tables 3 to 8, "set concentration" is the concentration shown in the "calibrator stock solution" column in Table 1, and "concentration" indicates the concentration derived from the "area ratio" based on the obtained calibration curve. "Accuracy %" indicates the percentage of the concentration derived from the "area ratio" when the "set concentration" is used as the standard.

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049] The results in Tables 3 to 8 show that by ionizing everolimus, sirolimus, tacrolimus, and cyclosporine A (four types corresponding to the first immunosuppressant) in positive mode and ionizing MPA and MPAG (two types corresponding to the second immunosuppressant) in negative mode, it is possible to create calibration curves for each component from the same analytical sample (whole blood calibration solutions L1 to L6).

[0050] <Quantification of Blood Concentration of Immunosuppressant> <Preparation of Blood Samples> The following blood samples were prepared (preparation step): DOSIMMUNE control: manufactured by Shimadzu Corporation, product name "Immunosuppressant Analysis Kit DOSIMMUNE" (four concentrations); MPA MPAG QC whole blood: (three concentrations); DOSIMMUNE control is a sample in which four types corresponding to the first immunosuppressant described above are dissolved in a whole blood matrix. MPA MPAG QC whole blood is a sample in which two types corresponding to the second immunosuppressant described above are dissolved in a whole blood matrix. Both blood samples simulate blood samples (whole blood) collected from subjects administered immunosuppressants.

[0051] MPA MPAG QC whole blood (L1-L3) was prepared as follows. First, 5 μL of a methanol solution of MPA (concentration 4000 mg / L) and 50 μL of a methanol solution of MPAG (concentration 2000 mg / L) were added to 445 μL of whole blood, and the mixture was vortexed for 1 minute to obtain MPA MPAG QC whole blood (L3). Next, the obtained MPA MPAG QC whole blood (L3) was diluted 2-fold with whole blood to obtain MPA MPAG QC whole blood (L2). Finally, the obtained MPA MPAG QC whole blood (L2) was diluted 10-fold with whole blood to obtain MPA MPAG QC whole blood (L1). (MPA MPAG QC Concentration of each component in whole blood) L1: MPA: 2 mg / L, MPAG: 10 mg / L L2: MPA: 20 mg / L, MPAG: 100 mg / L L3: MPA: 40 mg / L, MPAG: 200 mg / L

[0052] <Pretreatment step> Each prepared blood sample (3 μl) was added to a microtube. Next, the internal standard sample (3 μl) included in the DOSIMMUNE immunosuppressant analysis kit, the internal standard sample (3 μl) included in the DOSIMYCO immunosuppressant analysis kit, and an extract (a mixture of acetonitrile, methanol, and water = 40:40:20) (200 μl) were added to the microtube and mixed in a Bostex mixer for 1 minute. After mixing, the microtube was centrifuged (15,000 rpm, 7 minutes) to separate the supernatant and precipitate (pretreatment step). 200 μl of this supernatant was collected and used as the analytical sample.

[0053] <Separation step and analysis step> The above analytical sample was analyzed by LC-MS / MS using gradient elution. The analytical conditions were the same as those used to create the calibration curve. The results are shown in Tables 9-1 and 9-2.

[0054]

[0055]

[0056] The results in Tables 9-1 and 9-2 confirmed that the quantitative values ​​of all components of the first immunosuppressant and the second immunosuppressant fell within the reference values ​​at all levels (L1 to L4).

[0057] <<Comparative Experiment: Analysis Results When a Second Immunosuppressant Was Ionized in Positive Mode>> As a comparative experiment, mass spectrometry was performed when a second immunosuppressant (MPA, MPAG) was ionized in positive mode. The analytical conditions were the same as those used to create the calibration curve described above, except that the ionization method was changed to positive mode and the MRM conditions were as follows: (MRM Conditions) MPA (ESI-Positive): 338.20 > 207.10 (CE -24V) MPA IS (ESI-Positive): 342.10 > 211.30 (CE -14V) MPAG (ESI-Positive): 514.10 > 207.15 (CE -37V) MPAG IS (ESI-Positive): 518.10 > 211.20 (CE -35V)

[0058] The approximate formula and contribution rate of the calibration curve obtained by the above analysis are shown below. In all results, it was found that a higher contribution rate was observed when ionizing in negative mode. From the above, it was considered necessary to ionize in negative mode when analyzing the second immunosuppressant. MPA calibration curve (positive mode) Approximate formula: Y = 0.0786918X + 0.0169183 R ２ (Contribution rate) = 0.8985811, R = 0.9479352 MPAG calibration curve (positive mode) Approximation formula: Y = 0.00586797X + 0.000886828 R ２ (Contribution rate) = 0.9924132, R = 0.9961994

[0059] From the above results, it was found that the blood test method according to the present invention is capable of quantifying two or more types of immunosuppressants in the same blood sample.

[0060] Aspects It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects.

[0061] (Item 1) A blood testing method according to one embodiment includes a preparation step of preparing whole blood collected from a subject, a pretreatment step of pretreating the whole blood to prepare an analytical sample, a separation step of separating the analytical sample into components using a liquid chromatograph, and an analysis step of analyzing the separated components using a mass spectrometer, wherein the subject is administered a first immunosuppressant and a second immunosuppressant, the first immunosuppressant comprises at least one selected from the group consisting of tacrolimus, cyclosporine A, everolimus, and sirolimus, the second immunosuppressant comprises at least one selected from the group consisting of mycophenolic acid and its metabolites, the separated components include the first immunosuppressant and the second immunosuppressant, and when analyzing with the mass spectrometer, analysis is performed in positive mode when analyzing the first immunosuppressant as the target component, and analysis is performed in negative mode when analyzing the second immunosuppressant as the target component. According to the blood test method described in item 1, two or more types of immunosuppressants can be quantified in the same blood sample.

[0062] (Item 2) In the blood test method described in Item 1, the analyzing step further includes quantifying the separated components based on a calibration curve prepared in advance. The blood test method described in Item 2 allows for more accurate quantification of the blood concentration of the administered immunosuppressant.

[0063] (Item 3) In the blood test method described in item 1 or 2, the liquid chromatograph is a high-performance liquid chromatograph. The blood test method described in item 3 allows for more accurate quantification.

[0064] (Item 4) In the blood testing method described in any one of Items 1 to 3, the mass spectrometer is a triple quadrupole mass spectrometer. The blood testing method described in Item 4 makes it possible to simultaneously detect multiple components in whole blood.

[0065] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0066] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims, not by the embodiments and examples described above, and is intended to include meanings equivalent to the claims and all modifications within the scope of the claims.

Claims

1. A blood testing method comprising: a preparation step of preparing whole blood collected from a subject; a pretreatment step of pretreating the whole blood to prepare an analytical sample; a separation step of separating the analytical sample into components using a liquid chromatograph; and an analysis step of analyzing the separated components using a mass spectrometer, wherein the subject is administered a first immunosuppressant and a second immunosuppressant, the first immunosuppressant comprises at least one selected from the group consisting of tacrolimus, cyclosporine A, everolimus, and sirolimus, the second immunosuppressant comprises at least one selected from the group consisting of mycophenolic acid and its metabolites, the separated components comprise the first immunosuppressant and the second immunosuppressant, and when analyzing with the mass spectrometer, the analysis is performed in positive mode when the first immunosuppressant is analyzed as the target component, and in negative mode when the second immunosuppressant is analyzed as the target component.

2. The blood test method according to claim 1, wherein the analyzing step further comprises quantifying the separated components based on a calibration curve prepared in advance.

3. The blood testing method according to claim 1 or 2, wherein the liquid chromatograph is a high performance liquid chromatograph.

4. The blood testing method according to claim 1 or 2, wherein the mass spectrometer is a triple quadrupole mass spectrometer.

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