Method for quantifying analyte including mycophenolic acid and one or more other immunosuppressants in blood sample of subject

Abstract

Provided is a means for quantitative analysis of an analyte including mycophenolic acid and one or more other immunosuppressants using a whole blood fraction without requiring separation of a blood sample. One embodiment of the present invention relates to a method for quantifying an analyte including mycophenolic acid and one or more other immunosuppressants in a blood sample of a subject, the method comprising: an analyte concentration measurement step for measuring the concentration of the analyte; measuring the concentration of an analyte including mycophenolic acid and one or more other immunosuppressants in the whole blood fraction of the blood sample; a hematocrit value acquisition step for acquiring the hematocrit value of the target blood sample; and a mycophenolic acid concentration conversion step for converting the concentration of mycophenolic acid in the whole blood fraction of the blood sample into the concentration of mycophenolic acid in the plasma fraction of the blood sample on the basis of the hematocrit value.

Description

Technical Field

[0001] The present invention relates to a method for quantifying analytes including mycophenolic acid and one or more other immunosuppressants in a blood sample of a subject. Background Art

[0002] In transplantation medicine, postoperative immunosuppressant management is important for improving transplantation outcomes or long-term prognosis. In organ transplant patients, multiple immunosuppressants are usually used to control the survival of the transplanted organ. Examples of such immunosuppressants include mycophenolate mofetil (hereinafter also referred to as "mycophenolic acid"), tacrolimus, cyclosporine A, and everolimus.

[0003] To manage these immunosuppressants, therapeutic drug monitoring (TDM) is usually performed to measure and quantify the concentration of immunosuppressants in the blood samples of organ transplant patients. In TDM, the concentration of immunosuppressants in specific blood samples such as whole blood fractions or plasma fractions is usually measured using methods such as immunoassay or mass spectrometry, depending on the type of immunosuppressant being targeted. For example, for mycophenolic acid, the concentration in the plasma fraction is usually measured, while for tacrolimus, the concentration in the whole blood fraction is usually measured. Summary of the Invention

[0004] Problems to be Solved by the Invention

[0005] Among the immunosuppressants commonly used in organ transplant patients, there are not only immunosuppressants such as mycophenolic acid for which the plasma fraction concentration is usually measured, but also immunosuppressants such as tacrolimus, cyclosporine A, and everolimus for which the whole blood fraction concentration is usually measured. Therefore, in the TDM of these immunosuppressants in the blood samples of patients receiving these immunosuppressants, including organ transplant patients, it is difficult to handle by simultaneous analysis.

[0006] Among the immunosuppressants commonly used in organ transplant patients, the correlation between the trough concentration value of mycophenolic acid and the pharmacological effect is low. Therefore, in the TDM of mycophenolic acid, it is recommended to obtain the area under the plasma concentration-time curve (AUC) value. However, in actual clinical practice, frequent blood sampling is highly invasive and often difficult. In addition, timed blood sampling is difficult to implement in outpatient clinics. Therefore, if low-invasive blood sampling can be performed at the patient's home, it will be greatly beneficial to the patient.

[0007] At the patient's home, techniques for self - collecting blood using minimally invasive means and quantifying the concentration of analytes in blood samples, such as self - blood glucose measurement, have been put into practical use. However, when applying the techniques of self - blood collection and measurement in the TDM of immunosuppressants like mycophenolic acid, for which the plasma fraction of blood samples is commonly used in blood concentration measurement, there are technical problems in separating blood samples at the patient's home to obtain the plasma fraction. In addition, when self - collecting blood using minimally invasive means, there is a technical problem that the blood collection volume is usually small.

[0008] Therefore, an object of the present invention is to provide a means for quantitatively analyzing analytes including mycophenolic acid and one or more other immunosuppressants using the whole - blood fraction without separating the blood sample.

[0009] Solutions to the problems

[0010] The inventors have studied various solutions to solve the above - mentioned technical problems. The inventors have found that in the quantification of mycophenolic acid in a blood sample of an object, the concentration of mycophenolic acid in the whole - blood fraction of the blood sample can be accurately converted into the concentration of mycophenolic acid in the plasma fraction of the blood sample based on the hematocrit value of the blood sample. Based on the above - mentioned insight, the present invention has been completed.

[0011] That is, the present invention includes the following embodiments.

[0012] (Embodiment 1)

[0013] A method for quantifying analytes including mycophenolic acid and one or more other immunosuppressants in a blood sample of an object, comprising:

[0014] An analyte concentration measurement step of measuring the concentration of analytes including mycophenolic acid and one or more other immunosuppressants in the whole - blood fraction of the blood sample;

[0015] A hematocrit value acquisition step of acquiring the hematocrit value of the blood sample of the object;

[0016] A mycophenolic acid concentration conversion step of converting the concentration of mycophenolic acid in the whole - blood fraction of the blood sample into the concentration of mycophenolic acid in the plasma fraction of the blood sample based on the hematocrit value.

[0017] (Embodiment 2)

[0018] The method according to Embodiment 1, wherein the object is an organ transplant patient.

[0019] (Embodiment 3)

[0020] The method according to Embodiment 1 or 2, wherein one or more other immunosuppressants are selected from the group consisting of tacrolimus, cyclosporin A, and everolimus.

[0021] (Embodiment 4)

[0022] The method according to any one of Embodiments 1 to 3, wherein the hematocrit value acquisition step includes measuring the hematocrit value of a blood sample of the subject.

[0023] (Embodiment 5)

[0024] The method according to any one of Embodiments 1 to 4, wherein the mycophenolic acid concentration conversion step is carried out based on the following formula:

[0025] A p = A w × 100 / (100 - Ht)

[0026] (In the formula,

[0027] A p is the converted value of the concentration of mycophenolic acid in the plasma fraction of the blood sample,

[0028] A w is the concentration of mycophenolic acid in the whole blood fraction,

[0029] Ht is the hematocrit value of the blood sample.).

[0030] (Embodiment 6)

[0031] The method according to any one of Embodiments 1 to 5, wherein the analyte concentration measurement step is carried out by liquid chromatography-mass spectrometry (LC-MS).

[0032] Effects of the Invention

[0033] According to the present invention, there can be provided a means for quantitatively analyzing an analyte including mycophenolic acid and one or more other immunosuppressants using a whole blood fraction without separating a blood sample. Further, according to the present invention, it is possible to quantitatively analyze an analyte in a trace amount of blood sample.

[0034] This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2022-190246, which is the basis of the priority of this application. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a flowchart showing each step in a method for quantifying an analyte including mycophenolic acid and one or more other immunosuppressants in a blood sample of a subject in one embodiment of the present invention.

[0036] Figure 2 It is a graph showing the measurement and conversion results of mycophenolic acid concentration in blood samples of kidney transplant patients in Experiment I. A is a graph showing the correlation between the concentration of mycophenolic acid in the plasma fraction of each blood sample of a kidney transplant patient and the concentration of mycophenolic acid in the whole blood fraction of the same patient's blood sample. The horizontal axis represents the concentration of mycophenolic acid in the plasma fraction (μg / mL), and the vertical axis represents the concentration of mycophenolic acid in the whole blood fraction (μg / mL). B is a graph showing the correlation between the concentration of mycophenolic acid in the plasma fraction of each blood sample of a kidney transplant patient and the conversion value of the concentration of mycophenolic acid in the plasma fraction obtained by converting according to the concentration of mycophenolic acid in the whole blood fraction of the same patient's blood sample. The horizontal axis represents the concentration of mycophenolic acid in the plasma fraction (μg / mL), and the vertical axis represents the conversion value of the concentration of mycophenolic acid in the plasma fraction obtained by converting according to the concentration of mycophenolic acid in the whole blood fraction (μg / mL).

[0037] Figure 3 It is a graph showing the measurement and conversion results of mycophenolic acid concentration in blood samples obtained by collecting blood from different parts of kidney transplant patients in Experiment II. A is a graph showing the correlation between the concentration of mycophenolic acid in the whole blood fraction of a blood sample collected by venous blood sampling from a kidney transplant patient and the conversion value of the concentration of mycophenolic acid in the plasma fraction obtained by converting according to the concentration of mycophenolic acid in the whole blood fraction of the same patient's venous blood sampling. The horizontal axis represents the conversion value of the concentration of mycophenolic acid in the plasma fraction obtained by converting according to the concentration of mycophenolic acid in the whole blood fraction of a blood sample collected by venous blood sampling from a kidney transplant patient (μg / mL), and the vertical axis represents the concentration of mycophenolic acid in the whole blood fraction of the same patient's venous blood sampling (μg / mL). B is a graph showing the correlation between the concentration of mycophenolic acid in the whole blood fraction of a blood sample collected by fingertip blood sampling from a kidney transplant patient and the concentration of mycophenolic acid in the whole blood fraction of the same patient's venous blood sampling. The horizontal axis represents the concentration of mycophenolic acid in the whole blood fraction of the same patient's venous blood sampling (μg / mL), and the vertical axis represents the concentration of mycophenolic acid in the whole blood fraction of the same patient's fingertip blood sampling (μg / mL). C is a graph showing the correlation between the concentration of mycophenolic acid in the whole blood fraction of a blood sample collected by fingertip blood sampling from a kidney transplant patient and the conversion value of the concentration of mycophenolic acid in the plasma fraction obtained by converting according to the concentration of mycophenolic acid in the whole blood fraction of the same patient's venous blood sampling. The horizontal axis represents the conversion value of the concentration of mycophenolic acid in the plasma fraction obtained by converting according to the concentration of mycophenolic acid in the whole blood fraction of a blood sample collected by venous blood sampling from a kidney transplant patient (μg / mL), and the vertical axis represents the concentration of mycophenolic acid in the whole blood fraction of the same patient's fingertip blood sampling (μg / mL). Detailed Description of the Invention

[0038] <1. Method for Quantifying Analytes Including Mycophenolic Acid and One or More Other Immunosuppressants in a Blood Sample>

[0039] For immunosuppressants commonly used in organ transplant patients, there are not only immunosuppressants such as mycophenolic acid, for which the concentration in the plasma fraction is usually measured, but also immunosuppressants such as tacrolimus, cyclosporine A, and everolimus, for which the concentration in the whole blood fraction is usually measured. Therefore, in the therapeutic drug monitoring (TDM) of these immunosuppressants in the blood samples of patients receiving these immunosuppressants, including organ transplant patients, it is difficult to handle by simultaneous analysis.

[0040] In the TDM of immunosuppressants in organ transplant patients, it would be of great benefit to the patients if blood could be drawn by a minimally invasive method at home. However, when applying the technology of self-blood collection and measurement in the TDM of immunosuppressants such as mycophenolic acid, for which the plasma fraction of the blood sample is usually used in blood concentration measurement, there is a technical problem that it is difficult to separate the blood sample at home to obtain the plasma fraction. In addition, when drawing blood by a minimally invasive method, there is a technical problem that the amount of blood collected is usually small.

[0041] In response to this, the present inventors have found that in the quantification of mycophenolic acid in a blood sample of a subject, the concentration of mycophenolic acid in the whole blood fraction of the blood sample can be accurately converted into the concentration of mycophenolic acid in the plasma fraction of the blood sample based on the hematocrit value of the blood sample. Therefore, one embodiment of the present invention relates to a method for quantifying analytes including mycophenolic acid and one or more other immunosuppressants in a blood sample of a subject.

[0042] The subject to which the method of the present embodiment is applied may be a human or a non-human mammal. The method of the present embodiment can be applied, for example, to patients using mycophenolic acid in the uses of (1) treating refractory rejection after kidney transplantation, (2) suppressing rejection in kidney transplantation, heart transplantation, liver transplantation, lung transplantation, or pancreas transplantation, (3) treating lupus nephritis, or (4) suppressing graft-versus-host disease in hematopoietic stem cell transplantation. It is known that there are no significant differences in the pharmacokinetics of mycophenolic acid in these patients. Therefore, the method of the present embodiment can also be applied to any of the exemplified patients. The subject to which the method of the present embodiment is applied is preferably an organ transplantation patient of any solid organ, more preferably an organ transplantation patient of one or more selected from the group consisting of kidney, liver, lung, heart, small intestine, and pancreas, and further preferably an organ transplantation patient of kidney or liver. In the exemplified subjects, in order to manage the amount of drugs such as immunosuppressants used in the treatment in the blood, TDM needs to be implemented. Therefore, by applying the method of the present embodiment to the exemplified subjects, it is possible to quantitatively analyze analytes including mycophenolic acid and one or more other immunosuppressants using whole blood fractions without separating blood samples.

[0043] The analyte including mycophenolic acid and one or more other immunosuppressants to which the method of the present embodiment is applied is usually a drug used in the treatment of the exemplified subjects. For example, when the subject to which the method of the present embodiment is applied is an organ transplantation patient, the analyte including mycophenolic acid and one or more other immunosuppressants includes, in addition to mycophenolic acid, one or more other immunosuppressants selected from the group consisting of tacrolimus, cyclosporine A, and everolimus. For organ transplantation patients, it is important to implement TDM to manage postoperative immunosuppressants in order to improve transplantation results or long-term prognosis. Since it is important to suppress immunity both at the cellular and antibody levels for organ transplantation patients, the exemplified mycophenolic acid and one or more other immunosuppressants are usually administered in combination. Therefore, by applying the method of the present embodiment to the exemplified analyte including mycophenolic acid and one or more other immunosuppressants, it is possible to quantitatively analyze the analyte including mycophenolic acid and one or more other immunosuppressants using whole blood fractions without separating blood samples.

[0044] In the method of the present embodiment, a blood sample of an object can be obtained by using a blood collection means commonly used in this technical field. For the blood collection means to be used, those skilled in the art can appropriately select according to the age, gender and condition of the object to which the method of the present embodiment is applied (for example, the symptoms, diseases and / or severity of disorders that the object has), the blood collection site, the number of blood collections, the blood collection place, and the amount of the blood sample or its fraction used in each process described below. As the blood collection site, for example, the vein in the arm and the fingertip can be cited. As the blood collection means to be used, for example, a syringe, a blood collection tube, and a self-blood collection kit for micro blood collection can be cited. When obtaining a blood sample from the fingertip of an object using a self-blood collection kit for micro blood collection, the whole blood fraction of a micro amount (for example, in the range of 0.01 to 0.1 mL) of the blood sample can be used to quantitatively analyze an analyte including mycophenolic acid and one or more other immunosuppressants.

[0045] A flowchart showing each process in one embodiment of the method of the present embodiment is shown in Figure 1 as follows. As Figure 1 shown, the method of the present embodiment includes an analyte concentration measurement step (S1), a hematocrit value acquisition step (S2), and a mycophenolic acid concentration conversion step (S3). Hereinafter, each step will be described in detail.

[0046] [1-1. Analyte Concentration Measurement Step]

[0047] The method of the present embodiment includes an analyte concentration measurement step (step S1) for measuring the concentration of an analyte including mycophenolic acid and one or more other immunosuppressants in the whole blood fraction of a blood sample.

[0048] The blood sample used in this step can be obtained by using the blood collection means exemplified above.

[0049] The whole blood fraction of the blood sample used in this step represents the blood sample itself obtained by the blood collection means exemplified above (that is, an untreated blood sample).

[0050] In this step, as a means for measuring the concentration of an analyte including mycophenolic acid and one or more other immunosuppressants in the whole blood fraction of a blood sample, for example, immunoassay or instrumental analysis can be cited. The means for measuring the concentration of an analyte including mycophenolic acid and one or more other immunosuppressants in the whole blood fraction of a blood sample is preferably instrumental analysis, more preferably liquid chromatography, mass spectrometry or a combination thereof, further preferably liquid chromatography-mass spectrometry (LC-MS), and particularly preferably liquid chromatography-tandem mass spectrometry (LC-MS / MS or LC-MS / MS / MS, etc.). When measuring the concentration of one or more analytes containing mycophenolic acid using immunoassay, it is usually necessary to perform the measurement for each analyte. In addition, several mL of the whole blood fraction are required for each analyte. When measuring the concentration of an analyte including mycophenolic acid and one or more other immunosuppressants using the exemplified instrumental analysis, the analyte can usually be analyzed simultaneously. In addition, for one measurement, only a whole blood fraction in the range of 1 mL or less, for example, 0.01 to 0.1 mL, is sufficient. Especially when using LC-MS, preferably liquid chromatography-tandem mass spectrometry such as LC-MS / MS, the concentration of an analyte including mycophenolic acid and one or more other immunosuppressants can be measured even for a trace amount (for example, in the range of 0.01 to 0.1 mL) of the whole blood fraction. Therefore, by using the exemplified means, especially the exemplified instrumental analysis, preferably LC-MS to measure the concentration of an analyte including mycophenolic acid and one or more other immunosuppressants, it is possible to quantitatively analyze the analyte including mycophenolic acid and one or more other immunosuppressants with high sensitivity using a trace amount of the whole blood fraction of a blood sample.

[0051] [1-2. Hematocrit value acquisition step]

[0052] The method of the present embodiment includes a hematocrit value acquisition step (step S2) of obtaining the hematocrit value of a blood sample of an object.

[0053] In each embodiment of the present invention, the hematocrit value represents the percentage of the volume of the blood cell fraction relative to the total volume of the blood sample.

[0054] In this step, the hematocrit value can be obtained based on the measurement result of the hematocrit value of the blood sample of the object measured in advance, or can be obtained by measuring the hematocrit value of the blood sample of the object when implementing the method of the present embodiment. By obtaining the hematocrit value based on the measurement result of the hematocrit value of the blood sample of the object measured in advance, it is possible to quantitatively analyze mycophenolic acid in the plasma fraction without separating the blood sample.

[0055] [1-3. Mycophenolic acid concentration conversion step]

[0056] The method of the present embodiment includes a mycophenolic acid concentration conversion step (step S3) of converting the concentration of mycophenolic acid in the whole blood fraction of a blood sample into the concentration of mycophenolic acid in the plasma fraction of the blood sample based on the hematocrit value.

[0057] The hematocrit value is the percentage of the volume of the blood cell fraction relative to the total volume of the blood sample. Therefore, for an analyte that is substantially uniformly present in the blood sample of the subject, the concentration in the whole blood fraction of the blood sample can be converted into the concentration in the plasma fraction of the blood sample based on the hematocrit value. Thus, by performing this step, quantitative analysis of mycophenolic acid in the plasma fraction can be performed using the whole blood fraction without separating the blood sample.

[0058] This step is preferably performed based on the following formula.

[0059] A p = A w × 100 / (100 - Ht)

[0060] (In the formula,

[0061] A p is the conversion value of the concentration of mycophenolic acid in the plasma fraction of the blood sample,

[0062] A w is the concentration of mycophenolic acid in the whole blood fraction,

[0063] Ht is the hematocrit value of the blood sample.)

[0064] By converting the concentration of mycophenolic acid in the whole blood fraction of the blood sample into the concentration of mycophenolic acid in the plasma fraction based on the above formula, quantitative analysis of mycophenolic acid in the plasma fraction can be performed using the whole blood fraction. Thus, according to the method of the present embodiment, quantitative analysis of an analyte including mycophenolic acid and one or more other immunosuppressants can be performed using the whole blood fraction without separating the blood sample.

[0065] <2. Device for Quantifying Analyte in Blood Sample>

[0066] Another embodiment of the present invention relates to a device for quantifying an analyte including mycophenolic acid and one or more other immunosuppressants in a blood sample of a subject.

[0067] The device of the present embodiment can similarly be applied to the subject and analyte exemplified in the method of one embodiment of the present invention.

[0068] The device of the present embodiment includes an analyte concentration measurement unit, a hematocrit value acquisition unit, and a mycophenolic acid concentration conversion unit. Hereinafter, each unit will be described in detail.

[0069] [2-1. Analyte Concentration Measuring Unit]

[0070] The device of the present embodiment includes an analyte concentration measuring unit that measures the concentration of an analyte including mycophenolic acid and one or more other immunosuppressants in the whole blood fraction of a blood sample.

[0071] The analyte concentration measuring unit generally has a sample introduction part for introducing the whole blood fraction of a blood sample, and a concentration measuring part for measuring the concentration of an analyte including mycophenolic acid and one or more other immunosuppressants in the whole blood fraction of the blood sample. The sample introduction part and the concentration measuring part may be configured separately or integrally. As the concentration measuring part, for example, an immunoassay instrument or an instrumental analyzer can be cited. The concentration measuring part is preferably an instrumental analyzer, more preferably a liquid chromatograph, a mass spectrometer, or a combination thereof, further preferably a liquid chromatography-mass spectrometer (LC-MS), and particularly preferably a liquid chromatography-tandem mass spectrometer (LC-MS / MS or LC-MS / MS / MS, etc.). When the concentration measuring part is the instrumental analyzer exemplified above, it is usually configured integrally with the sample introduction part and can simultaneously measure one or more analytes including mycophenolic acid. In addition, for one measurement, as long as the whole blood fraction is 1 mL or less, for example, in the range of 0.01 to 0.1 mL. In particular, when the concentration measuring part is LC-MS, preferably a liquid chromatography-tandem mass spectrometer such as LC-MS / MS, the concentration of an analyte including mycophenolic acid and one or more other immunosuppressants can be measured even for a trace amount (for example, in the range of 0.01 to 0.1 mL) of the whole blood fraction. Therefore, by using the sample introduction part and the concentration measuring part exemplified above, particularly the instrumental analyzer exemplified above, preferably LC-MS, to measure the concentration of an analyte including mycophenolic acid and one or more other immunosuppressants, it is possible to quantitatively analyze an analyte including mycophenolic acid and one or more other immunosuppressants with high sensitivity using a trace amount of the whole blood fraction of a blood sample.

[0072] [2-2. Hematocrit Value Obtaining Unit]

[0073] The device of the present embodiment includes a hematocrit value obtaining unit that obtains the hematocrit value of a blood sample of an object.

[0074] In the hematocrit value acquisition unit, the hematocrit value can be obtained based on the measurement result of the hematocrit value of the blood sample of the subject measured in advance, or can be obtained by measuring the hematocrit value of the blood sample of the subject when using the device of the present embodiment. In the case of the former embodiment, the hematocrit value acquisition unit generally has a central control unit, a data storage unit, an input unit, and an output unit. The measurement result of the hematocrit value of the blood sample of the subject measured in advance is stored in the data storage unit. In the case of this embodiment, the hematocrit value acquisition unit is preferably in the form of a computer having a central processing unit, a storage device such as a hard disk, an input device, and an output device. In the case of the latter embodiment, the hematocrit value acquisition unit can be a measuring instrument for hematocrit values commonly used in this technical field. In the device of the present embodiment, the hematocrit value can be obtained based on the measurement result of the hematocrit value of the blood sample of the subject measured in advance, and mycophenolic acid in the plasma fraction can be quantitatively analyzed without separating the blood sample.

[0075] [2-3. Mycophenolic Acid Concentration Conversion Unit]

[0076] The device of the present embodiment includes a mycophenolic acid concentration conversion unit that converts the concentration of mycophenolic acid in the whole blood fraction of a blood sample into the concentration of mycophenolic acid in the plasma fraction of the blood sample based on the hematocrit value.

[0077] The mycophenolic acid concentration conversion unit generally has a central control unit, an analysis processing unit, a data storage unit, an input unit, and an output unit. The data storage unit generally contains a program for performing the concentration conversion of mycophenolic acid. The mycophenolic acid concentration conversion unit is preferably in the form of a computer having a central processing unit, a storage device such as a hard disk, an input device, and an output device, for example. In the device of the present embodiment, by converting the concentration of mycophenolic acid in the whole blood fraction of a blood sample into the concentration of mycophenolic acid in the plasma fraction of the blood sample, the whole blood fraction can be used to quantitatively analyze analytes including mycophenolic acid and one or more other immunosuppressants without separating the blood sample.

[0078] Examples

[0079] Hereinafter, the present invention will be described in more detail using examples. However, the scope of the present invention is not limited to these examples.

[0080] <Test I: Measurement and Conversion of Concentrations of Immunosuppressants in Blood Samples of Renal Transplant Patients>

[0081] Blood samples (2 mL) were obtained from 134 patients after kidney transplantation. A portion of each blood sample was collected as the whole blood fraction. The remaining portion was centrifuged to prepare the plasma fraction. The concentrations of mycophenolic acid (MPA) in the whole blood fraction (10 μL) and plasma fraction (10 μL) of each blood sample were measured by liquid chromatography-tandem mass spectrometry (LC-MS / MS). In addition, the hematocrit value of each blood sample was measured. Based on the hematocrit value of each blood sample and the following formula, the concentration of mycophenolic acid in the whole blood fraction of each blood sample was converted to the concentration of mycophenolic acid in the plasma fraction,

[0082] A(MPA) p = A(MPA) w ×100 / (100 - Ht)

[0083] (wherein,

[0084] A(MPA) p is the converted value of the concentration of mycophenolic acid in the plasma fraction of the blood sample,

[0085] A(MPA) w is the concentration of mycophenolic acid in the whole blood fraction,

[0086] Ht is the hematocrit value of the blood sample.).

[0087] The results of the measurement and conversion of the concentration of mycophenolic acid in the blood samples of kidney transplant patients are shown in Figure 2 . In the figure, A is a graph showing the correlation between the concentration of mycophenolic acid in the plasma fraction of each blood sample of kidney transplant patients and the concentration of mycophenolic acid in the whole blood fraction of the same patient's blood sample. The horizontal axis represents the concentration of mycophenolic acid in the plasma fraction (μg / mL), and the vertical axis represents the concentration of mycophenolic acid in the whole blood fraction (μg / mL). B is a graph showing the correlation between the concentration of mycophenolic acid in the plasma fraction of each blood sample of kidney transplant patients and the converted value of the concentration of mycophenolic acid in the plasma fraction obtained by converting based on the concentration of mycophenolic acid in the whole blood fraction of the same patient's blood sample. The horizontal axis represents the concentration of mycophenolic acid in the plasma fraction (μg / mL), and the vertical axis represents the converted value of the concentration of mycophenolic acid in the plasma fraction obtained by converting based on the concentration of mycophenolic acid in the whole blood fraction (μg / mL).

[0088] As Figure 2As shown in A of , a certain correlation was confirmed between the concentration of mycophenolic acid in the plasma fraction of the blood sample of the same patient and the concentration of mycophenolic acid in the whole blood fraction. Here, based on the hematocrit value of each blood sample and the above formula, the concentration of mycophenolic acid in the whole blood fraction of each blood sample was converted into the concentration of mycophenolic acid in the plasma fraction. As a result, it was in good agreement with the measured value of the concentration of mycophenolic acid in the plasma fraction of the blood sample of the same patient ( Figure 2 of B).

[0089] <Experiment II: Comparison of the determination and conversion of the concentration of immunosuppressive agents in blood samples collected from different parts of renal transplant patients>

[0090] 72 blood samples (2 mL) were obtained from the veins of the arms of a group of patients after renal transplantation. 72 blood samples (5.6 μL) were obtained from the fingertips of the same group of patients using a self-blood collection kit for micro-sampling (MSW2). The concentration of MPA in the whole blood fraction (5.6 μL) of each blood sample collected by venous blood collection and fingertip blood collection was measured by LC-MS / MS. In addition, the hematocrit value of each blood sample was measured. Based on the hematocrit value of each blood sample and the formula used in Experiment I, the concentration of mycophenolic acid in the whole blood fraction of each blood sample was converted into the concentration of mycophenolic acid in the plasma fraction. Among them, one case with a significantly outlying value of the concentration of mycophenolic acid in the whole blood fraction of the blood sample collected by fingertip blood collection was excluded from the statistical processing of the concentration of mycophenolic acid in the whole blood fraction of the blood sample collected by fingertip blood collection.

[0091] The results of the determination and conversion of the concentration of mycophenolic acid in blood samples collected from different parts of renal transplant patients are shown in Figure 3In the figure. In the figure, A is a graph showing the correlation between the concentration of mycophenolic acid in the whole blood fraction of a blood sample obtained by venous blood sampling from a renal transplant patient and the conversion value of the concentration of mycophenolic acid in the plasma fraction obtained by converting based on the concentration of mycophenolic acid in the whole blood fraction of the blood sample of the renal transplant patient's venous blood sampling. The horizontal axis represents the conversion value of the concentration of mycophenolic acid in the plasma fraction obtained by converting based on the concentration of mycophenolic acid in the whole blood fraction of the blood sample of the renal transplant patient's venous blood sampling (μg / mL), and the vertical axis represents the concentration of mycophenolic acid in the whole blood fraction of the blood sample of the renal transplant patient's venous blood sampling (μg / mL). B is a graph showing the correlation between the concentration of mycophenolic acid in the whole blood fraction of a blood sample obtained by fingertip blood sampling from a renal transplant patient and the concentration of mycophenolic acid in the whole blood fraction of the blood sample of the renal transplant patient's venous blood sampling. The horizontal axis represents the concentration of mycophenolic acid in the whole blood fraction of the blood sample of the renal transplant patient's venous blood sampling (μg / mL), and the vertical axis represents the concentration of mycophenolic acid in the whole blood fraction of the blood sample of the renal transplant patient's fingertip blood sampling (μg / mL). C is a graph showing the correlation between the concentration of mycophenolic acid in the whole blood fraction of a blood sample obtained by fingertip blood sampling from a renal transplant patient and the conversion value of the concentration of mycophenolic acid in the plasma fraction obtained by converting based on the concentration of mycophenolic acid in the whole blood fraction of the blood sample of the renal transplant patient's venous blood sampling. The horizontal axis represents the conversion value of the concentration of mycophenolic acid in the plasma fraction obtained by converting based on the concentration of mycophenolic acid in the whole blood fraction of the blood sample of the renal transplant patient's venous blood sampling (μg / mL), and the vertical axis represents the concentration of mycophenolic acid in the whole blood fraction of the blood sample of the renal transplant patient's fingertip blood sampling (μg / mL).

[0092] As Figure 3 shown in A, the conversion value of the concentration of mycophenolic acid in the plasma fraction obtained by converting based on the concentration of mycophenolic acid in the whole blood fraction of the blood sample of the renal transplant patient's venous blood sampling is very consistent with the measured value of the concentration of mycophenolic acid in the whole blood fraction of the blood sample of the same patient's venous blood sampling. In addition, as Figure 3 shown in B, the measured value of the concentration of mycophenolic acid in the whole blood fraction of the blood sample obtained by fingertip blood sampling from a renal transplant patient is very consistent with the measured value of the concentration of mycophenolic acid in the whole blood fraction of the blood sample of the same patient's venous blood sampling. Further, as Figure 3 shown in C, the measured value of the concentration of mycophenolic acid in the whole blood fraction of the blood sample obtained by fingertip blood sampling from a renal transplant patient is also very consistent with the conversion value of the concentration of mycophenolic acid in the plasma fraction obtained by converting based on the concentration of mycophenolic acid in the whole blood fraction of the blood sample of the same patient's venous blood sampling. From these results, it can be seen that even when using blood samples obtained by blood sampling from different sites, by applying the method of the present invention, quantitative analysis of mycophenolic acid can be performed using the whole blood fraction.

[0093] It is generally believed that after mycophenolic acid is absorbed into the body, in whole blood, the vast majority of it exists in the plasma fraction. However, there is no data to prove this yet. In addition, for tacrolimus, which is also used as an immunosuppressant, in whole blood, the vast majority of it exists in red blood cells, which are in the blood cell fraction. Thus, it is also considered possible that mycophenolic acid exists in the blood cell fraction. The inventors of the present invention first discovered that the vast majority of mycophenolic acid exists in the plasma fraction. Therefore, based on the fact that after mycophenolic acid is absorbed into the body, the vast majority of it exists in the plasma fraction in whole blood, the inventors of the present invention came up with using the value of the percentage of the volume of the plasma fraction calculated based on the hematocrit value, which is the percentage of the volume of the blood cell fraction relative to the total volume of the blood sample, relative to the total volume of the blood sample when converting the concentration of mycophenolic acid in whole blood to the concentration of mycophenolic acid existing in the plasma fraction, and completed the present invention.

[0094] It should be noted that the present invention is not limited to the described embodiments and includes various modifications. For example, the described embodiments are examples described in detail to illustrate the present invention in an easy-to-understand manner and are not limited to examples having all the configurations described. In addition, for the fraction configurations of each embodiment, other configurations can be added, deleted, and / or replaced.

[0095] The entire contents of all publications, patents, and patent applications cited in this specification are incorporated herein by reference.

Claims

1. A method for quantifying analytes including mycophenolic acid and one or more other immunosuppressants in a blood sample of a subject, comprising: An analyte concentration determination step of determining the concentration of analytes including mycophenolic acid and one or more other immunosuppressants in the whole blood fraction of the blood sample; A hematocrit value acquisition step of acquiring the hematocrit value of the blood sample of the subject; A mycophenolic acid concentration conversion step of converting the concentration of mycophenolic acid in the whole blood fraction of the blood sample into the concentration of mycophenolic acid in the plasma fraction of the blood sample based on the hematocrit value.

2. The method according to claim 1, wherein The subject is an organ transplant patient.

3. The method according to claim 1, wherein, One or more other immunosuppressants are selected from the group consisting of tacrolimus, cyclosporine A, and everolimus.

4. The method according to claim 1, wherein, The hematocrit value acquisition step includes measuring the hematocrit value of the blood sample of the subject.

5. The method according to claim 1, wherein The mycophenolic acid concentration conversion step is carried out based on the following formula: A p = A w × 100 / (100 - Ht) In the formula, A p is the converted value of the concentration of mycophenolic acid in the plasma fraction of a blood sample, A w is the concentration of mycophenolic acid in the whole blood fraction, Ht is the hematocrit value of the blood sample.

6. The method according to claim 1, wherein The analyte concentration determination step is carried out by liquid chromatography-mass spectrometry, i.e., LC-MS.

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