Thyroid hormone analysis method, analysis system, and detection system

Abstract

To provide a technique of accurately measuring a thyroid hormone.SOLUTION: An analysis method for analyzing a thyroid hormone contained in a biological sample, includes the steps of: mixing the biological sample with a solvent and controlling a temperature to 20°C or higher and 80°C or lower for 0.1 hour or longer and 12 hours or shorter, to prepare an extract: separating an analytical component in the extract by liquid chromatography; and subjecting the separated analytical component to mass spectrometry.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method, an analysis system, and a detection system for thyroid hormones. [Background technology]

[0002] There is a known technology for predicting or diagnosing health conditions or diseases by detecting specific components contained in blood, tissues, etc. collected from living organisms using a mass spectrometer (MS) as indicators of health or disease (biomarkers). Among these biological components, thyroid hormones secreted by the thyroid gland play an important role in regulating metabolism throughout the body and are known to be associated with various diseases such as Graves' disease, Hashimoto's disease, and thyroid cancer. Therefore, it would be advantageous to be able to efficiently analyze thyroid hormones.

[0003] As a method for analyzing thyroid hormone secretion, Non-Patent Documents 1 to 3 describe techniques for measuring thyroid hormone levels from human hair. In the techniques described in Non-Patent Documents 1 to 3, components extracted from human hair are separated by liquid chromatography, and the separated components are measured using a mass spectrometer. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] N. Grova et. al., Journal of Chromatography A, Volume 1612, 8 February 2020, 460648 [Non-patent document 2] Wei Gao et al., Psychoneuroendocrinology, Volume 106, August 2019, Pages 129-137 [Non-patent document 3] Feng-Jiao Peng et al., Eur J Endocrinol, 2022 May 1; 186(5): K9-K15 Summary of the Invention [Problem to be solved by the invention]

[0005] Hair, which is used for analysis in the techniques described in Non-Patent Documents 1 to 3, is easy to collect and minimally invasive to collect, making it an excellent biological sample for measuring thyroid hormones. However, the amount of thyroid hormone contained in hair is so small that it is difficult to measure it accurately. Therefore, there is a need for the development of a technique for measuring thyroid hormones with high accuracy.

[0006] One aspect of the present invention has been made to solve the above-mentioned problems, and aims to realize a technology for measuring thyroid hormones from hair with high accuracy. [Means for solving the problem]

[0007] In order to solve the above-mentioned problems, one embodiment of the present invention provides an analytical method for analyzing thyroid hormones contained in a biological sample, which comprises the steps of: mixing the biological sample with a solvent and controlling the temperature at 20°C or higher and 80°C or lower for 0.1 hour or longer and 12 hours or shorter to prepare an extract; separating the analyte components in the extract by liquid chromatography; and subjecting the separated analyte components to mass spectrometry.

[0008] An analytical system according to one embodiment of the present invention is an analytical system for analyzing thyroid hormones contained in a biological sample, and includes an apparatus for separating the analytical components in an extract by liquid chromatography, which is prepared by adding a solvent to the biological sample and controlling the temperature to 20°C or higher and 80°C or lower for 0.1 hours or longer and 12 hours or shorter, and a mass spectrometer for performing mass analysis of the separated analytical components.

[0009] A detection system according to one embodiment of the present invention is a detection system for detecting thyroid diseases, and includes an apparatus for separating analytical components in an extract by liquid chromatography, which is prepared by adding a solvent to the biological sample and controlling the temperature to 20°C or higher and 80°C or lower for 0.1 hours or longer and 12 hours or shorter, and a mass spectrometer for performing mass analysis of the separated analytical components. [Effects of the Invention]

[0010] According to one aspect of the present invention, a technique for measuring thyroid hormones with high accuracy can be realized. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a block diagram showing a configuration of a main part of an analysis system according to an embodiment of the present invention. [Figure 2] FIG. 1 shows the results of an analysis of T3 and T4 extracted from hair in an example, comparing subjects who were not affected by thyroid disease with subjects who were affected by thyroid disease. DETAILED DESCRIPTION OF THE INVENTION

[0012] [Analysis method] An analytical method according to one embodiment of the present invention is a method for analyzing thyroid hormones, which are analytes contained in a biological sample. This analytical method includes the steps of preparing an extract for extracting the analytes from the biological sample, separating the analytes in the extract, and subjecting the separated analytes to mass spectrometry. Examples of thyroid hormones analyzed in this analytical method include triiodothyronine (T3), reverse T3, and thyroxine (T4). The thyroid hormones analyzed in this analytical method may be free thyroid hormones (FT3, FT4, etc.) that are not bound to proteins. Furthermore, in this analytical method, thyroid hormones may be analyzed by analyzing iodine, which constitutes the thyroid hormones.

[0013] <Sample> The biological sample to be analyzed by this analysis method is not particularly limited, but may be a sample that may contain thyroid hormones. The biological sample may be a sample collected or prepared from a human or an animal. The biological sample may be selected from the group consisting of hair, nails, skin, organs, tissues, cells, blood, urine, cerebrospinal fluid, feces, and cecal contents, and an example is hair or nails. Hair is also called hair and includes scalp hair and body hair. Hair also includes the hair shaft portion outside the skin and the hair root portion inside the skin, as well as parts involved in hair production, such as the root sheath and hair matrix that are present around the hair root within the hair follicle (or hair follicle).

[0014] (Step of preparing extract) In the step of preparing an extract, the biological sample is mixed with a solvent to prepare an extract, which may be prepared by extracting thyroid hormones from the biological sample by a known method such as solvent extraction or solid-phase extraction.

[0015] The solvent added to the biological sample is preferably a solvent that can dissolve thyroid hormones, and examples include methanol, heptane, ethyl acetate, butanol, chloroform, etc. An extract can be prepared by suspending and stirring the biological sample in such a solvent.

[0016] When the biological sample is a solid sample, the amount of the biological sample suspended in the solvent is not particularly limited, but is, for example, 1 mg or more and 10 mg or less.

[0017] The amount of the solvent in which the biological sample is suspended is, for example, 50 μL or more and 200 μL or less, 50 μL or more and 150 μL or less, or 50 μL to 100 μL. When the biological sample is a solid sample, the biological sample may be cut or crushed and suspended in the solvent.

[0018] In the step of preparing an extract, the temperature of the biological sample mixed with the solvent is controlled to 20°C or higher and 80°C or lower. The temperature of the biological sample mixed with the solvent may be controlled to 40°C or higher and 70°C or lower, or 50°C or higher and 60°C or lower. In the step of preparing an extract, the temperature of the biological sample mixed with the solvent is controlled for 0.1 hours or higher and 12 hours or lower. The temperature control time of the biological sample mixed with the solvent may be 1 hour or higher and 8 hours or lower, or 2 hours or higher and 5 hours or lower.

[0019] The extract prepared in the extract preparation step can be directly subjected to the subsequent liquid chromatography without supernatant recovery and drying treatment. In the extract preparation step, a small amount of solvent is used to extract thyroid hormones from a biological sample, and an extract with a high concentration of extracted components can be obtained. Therefore, an extract suitable for analysis can be prepared without concentrating the extracted components by supernatant recovery and drying treatment. The prepared extract may be further mixed with a solvent and distilled water for liquid chromatography to adjust the solvent concentration, and used as a sample for liquid chromatography.

[0020] (Step of separating the analyte components) In the step of separating the analytes, the analytes in the extract are separated by liquid chromatography. In the step of separating the analytes, the extract prepared in the step of preparing the extract is injected into a purification column for liquid chromatography to separate the thyroid hormones, which are the analytes in the extract. In the step of separating the analytes, the extract prepared in the step of preparing the extract is used as is and subjected to liquid chromatography. One example of the liquid chromatography is reversed-phase liquid chromatography.

[0021] The purification column used in liquid chromatography is, for example, a reversed-phase column (C8). The flow rate of the mobile phase in liquid chromatography is, for example, 100 to 400 μL / min. The solvent used in liquid chromatography can be a mixed solvent of water and a solvent. Suitable solvents include, for example, methanol, acetonitrile, and 2-propanol. The amount of extract injected into the purification column is, for example, 50 μL to 200 μL; 50 μL to 150 μL; or 50 μL to 100 μL.

[0022] In the step of separating the analyte, an eluate is obtained by eluting the analyte from the purification column. Then, in the step of performing mass spectrometry described below, the obtained eluate is subjected to mass spectrometry. The amount of solvent used to elute the analyte from the purification column is, for example, 20 μL to 150 μL; 20 μL to 120 μL; or 20 μL to 100 μL.

[0023] In the step of separating the analytes, the eluate from which the analytes are eluted from the purification column can be directly subjected to mass spectrometry, as described below. Because the amount of solvent used to elute the analytes is small, an eluate with a high concentration of the eluted components can be obtained. Therefore, an eluate suitable for mass spectrometry can be prepared without concentrating the analytes by a drying treatment. The obtained eluate can also be dried and then redissolved in a solvent for mass spectrometry.

[0024] (Mass spectrometry step) In the mass spectrometry step, the analytes separated in the analyte separation step are subjected to mass spectrometry. In the mass spectrometry step, the analytes, thyroid hormones, are analyzed using a known mass spectrometer such as a triple quadrupole MS. In the mass spectrometry step, the presence or absence of thyroid hormones in the analytes can be detected. In addition, in the mass spectrometry step, the amount of thyroid hormones in the analytes can also be measured.

[0025] (Step of presenting an indicator of thyroid disease) The analysis method may further include a step of presenting the measured values ​​of thyroid hormones obtained by mass spectrometry as an index of thyroid disease. In the step of presenting the index of thyroid disease, a predetermined relationship between the measured values ​​of thyroid hormones and the index of thyroid disease is referenced, and the index of thyroid disease is obtained from the measured values ​​of thyroid hormones obtained by mass spectrometry. The obtained index of thyroid disease is then presented to a user. The step of presenting the index of thyroid disease may be performed by an information processing device.

[0026] In the step of presenting an indicator of thyroid disease, for example, when a measured value of thyroid hormone is outside a predetermined reference range, an indicator indicating that the thyroid disease level is outside the reference range is presented. Here, for example, the thyroid disease level may indicate a staged possibility of suffering from a thyroid disease. Examples of thyroid diseases include Graves' disease and Hashimoto's disease.

[0027] The method of presenting the thyroid disease indicator to the user is not particularly limited. For example, the thyroid hormone measurement value and the thyroid disease indicator may be displayed on a computer display, a display of a mobile device such as a smartphone, or the like. Alternatively, the thyroid disease indicator may be presented to the user by providing the user with a medium on which the thyroid hormone measurement value and the thyroid disease indicator are printed.

[0028] [Analysis System] An analytical system according to one embodiment of the present invention is an analytical system for analyzing thyroid hormones contained in a biological sample, and includes an apparatus for separating analytes in an extract by liquid chromatography, the extract being prepared by mixing the biological sample with a solvent and controlling the temperature to 20°C or higher and 80°C or lower for 0.1 to 12 hours, and a mass spectrometer for performing mass spectrometry on the separated analytes. That is, the analytical system according to one embodiment of the present invention is one embodiment of a system for executing the analytical method according to the above-described one embodiment of the present invention.

[0029] Fig. 1 is a block diagram showing the main components of an analytical system according to one embodiment of the present invention. As shown in Fig. 1, analytical system 100 includes a liquid chromatograph (a device that separates) 10, a mass spectrometer 20, and a computing device 30. In analytical system 100, liquid chromatograph 10 and mass spectrometer 20 may be independent devices, or may be an integrated device that has the functions of both devices. A known reversed-phase liquid chromatography-mass spectrometer (LC-MS) can be used as an integrated device that has the functions of liquid chromatograph 10 and mass spectrometer 20.

[0030] (liquid chromatograph) The liquid chromatograph 10 is an apparatus that separates analytes by reversed-phase liquid chromatography. The liquid chromatograph 10 may be a conventionally known liquid chromatograph apparatus, and commercially available apparatuses may be suitably used. The analytes separated by the liquid chromatograph 10 may be a sample prepared in a state suitable for separation in the apparatus, such as an extract obtained by extracting the analytes with a solvent. The liquid chromatograph 10 can separate thyroid hormones, which are analytes in the extract. The eluate containing the analytes separated by the liquid chromatograph 10 is introduced into a mass spectrometer 20.

[0031] (Mass spectrometer) Mass spectrometer 20 is a device that performs MS analysis of thyroid hormones in a biological sample. Mass spectrometer 20 has an ionization unit 21 that ionizes molecules contained in the eluate obtained in liquid chromatograph 10, and a detection unit 22 that performs MS analysis of the ionized molecules and detects thyroid hormones in the eluate. Mass spectrometer 20 can be a conventionally known mass spectrometer, and commercially available devices can be suitably used.

[0032] The eluate introduced into the mass spectrometer 20 is first introduced into the ionization unit 21, where molecules in the eluate are ionized. The detection unit 22 separates and detects the molecules ionized in the ionization unit 21 according to their mass-to-charge ratio (m / z ratio). The detection results in the mass spectrometer 20 are output to the calculation device 30.

[0033] (computing device) The computing device 30 generates MS and MS / MS spectra of the thyroid hormones in the eluate based on the detection results output from the mass spectrometer 20, and measures the peak areas to quantify the thyroid hormone molecules in the eluate.

[0034] The calculation device 30 may be, for example, a computer in which software for executing calculations related to MS analysis is installed, and which is equipped with a memory for storing the calculation results, a display for displaying the calculation results, and the like.

[0035] <Software implementation example> The calculations performed by the calculation device 30 may be realized by a logic circuit (hardware) formed on an integrated circuit (IC chip) or the like, or may be realized by software using a CPU (Central Processing Unit).

[0036] In the latter case, the computing device 30 includes a CPU that executes instructions of a program, which is software that realizes each function; a ROM (Read Only Memory) or storage device (these are referred to as "recording media") on which the program and various data are recorded so as to be readable by a computer (or CPU); and a RAM (Random Access Memory) on which the program is deployed. The object of the present invention is achieved when the computer (or CPU) reads and executes the program from the recording media. The recording media may be "non-transitory tangible media," such as tapes, disks, cards, semiconductor memories, and programmable logic circuits. The program may also be supplied to the computer via any transmission medium capable of transmitting the program (such as a communications network or broadcast waves). The present invention may also be realized in the form of a data signal embedded in a carrier wave, in which the program is embodied by electronic transmission.

[0037] Detection Systems and Methods A detection system according to one embodiment of the present invention is a detection system for detecting thyroid disease, and includes an apparatus for separating analytes in an extract by liquid chromatography, the extract being prepared by adding a solvent to a biological sample and controlling the temperature to 20°C or higher and 80°C or lower for 0.1 hours or longer and 12 hours or shorter, and a mass spectrometer for performing mass analysis of the separated analytes. The detection system according to one embodiment of the present invention is the analytical system according to the above-described embodiment of the present invention, which is used for detecting thyroid disease. The detection system according to one embodiment of the present invention measures thyroid hormone levels in a biological sample and detects thyroid disease based on the measurement results. This detection system can detect whether a subject has a thyroid disease by measuring thyroid hormone levels in a biological sample collected from the subject.

[0038] The present invention also encompasses a method for detecting thyroid disease, which comprises the steps of: mixing a biological sample with a solvent and controlling the temperature at 20°C to 80°C for 0.1 to 12 hours to prepare an extract; separating analyte components in the extract by liquid chromatography; and subjecting the separated analyte components to mass spectrometry. According to this detection method, the presence or absence of a thyroid disease in a subject can be detected by measuring the thyroid hormone level in a biological sample collected from the subject.

[0039] [Appendix 1] The analytical method according to aspect 1 of the present invention is a method for analyzing thyroid hormones contained in a biological sample, and includes the steps of: mixing the biological sample with a solvent and controlling the temperature at 20°C or higher and 80°C or lower for 0.1 to 12 hours to prepare an extract; separating the analyte components in the extract by liquid chromatography; and subjecting the separated analyte components to mass spectrometry.

[0040] In the analytical method according to aspect 2 of the present invention, in the separating step of the first aspect, the extract is injected into a purification column to separate the analyte components in the extract.

[0041] In the analytical method according to aspect 3 of the present invention, in the first or second aspect, in the separating step, an eluate from which the analyte component is eluted on a purification column is obtained, and in the mass spectrometry step, the eluate is subjected to mass spectrometry.

[0042] A fourth aspect of the present invention relates to the analytical method of any one of the first to third aspects, wherein the biological sample is hair or nail.

[0043] The analytical method according to a fifth aspect of the present invention is the method according to any one of the first to fourth aspects, further comprising the step of presenting the measured value of the thyroid hormone obtained by the mass spectrometry as an index of a thyroid disease.

[0044] The analytical system according to aspect 6 of the present invention is an analytical system for analyzing thyroid hormones contained in a biological sample, and includes an apparatus for separating the analytical components in an extract by liquid chromatography, which is prepared by adding a solvent to the biological sample and controlling the temperature to 20°C or higher and 80°C or lower for 0.1 hours or longer and 12 hours or shorter, and a mass spectrometer for performing mass analysis of the separated analytical components.

[0045] The detection system according to aspect 7 of the present invention is a detection system for detecting thyroid diseases, and includes an apparatus for separating analytical components in an extract by liquid chromatography, which is prepared by adding a solvent to a biological sample and controlling the temperature to 20°C or higher and 80°C or lower for 0.1 hours or longer and 12 hours or shorter, and a mass spectrometer for performing mass analysis of the separated analytical components.

[0046] [Appendix 2] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Example]

[0047] Thyroid hormones in hair were measured as follows: Hair samples were collected from healthy individuals and patients with Graves' disease.

[0048] (Biological sample acquisition) Hair was cut and collected from the head and scalp of each subject. The cut hair was separated from the root and used. The collected hair was washed and allowed to dry naturally at room temperature before being used for analysis.

[0049] (Preparation of extract) The hair that had been pretreated as described above was placed in an extraction container, 100 μL of methanol water was added, and extraction was carried out for 4 hours at 60° C. To adjust the methanol concentration, further methanol water was added to prepare an extract.

[0050] (Preparation of eluate) First, the purification column was conditioned by applying methanol to the column and pushing it out with air using a syringe. Then, distilled water was applied to the column and pushed out in the same manner.

[0051] 100 μL of the extract prepared as described above was injected into the conditioned column and extruded. The remaining extract was then extruded in the same manner. After injecting and extruding hexane for washing, the column was dried by extruding only air.

[0052] Then, 100 μL of methanol was injected, and the liquid that was pushed out was collected in a new container as the eluate. The collected eluate was dried by blowing nitrogen gas over it. After drying, methanol was added to the container to redissolve the eluate. Distilled water was then added to the container, and the mixture was mixed well before being subjected to mass spectrometry.

[0053] (analysis) The extracted samples were separated by reversed-phase liquid chromatography using an ultra-high-performance liquid chromatograph NEXERA X2 (Shimadzu Corporation) as follows. A reversed-phase packing material for ultra-high-performance liquid chromatography was used as the packing material for the reversed-phase liquid chromatography column. Gradient elution was performed using two mobile phases for reversed-phase liquid chromatography under acidic conditions. The separated molecules were ionized using electrospray ionization (ESI) on a triple quadrupole LCMS-8060 system (Shimadzu Corporation), and MS spectra of T3 and T4 were generated and measured.

[0054] The results of measuring T3 and T4 in hair collected from nine healthy controls and eight Graves' disease patients are shown in Figure 2. Figure 2 shows the results of comparing the analysis results of T3 and T4 extracted from hair between subjects with and without thyroid disease. In the graph shown in Figure 2, the vertical axis represents the intensity of T4, and the horizontal axis represents the intensity of T3.

[0055] As shown in Figure 2, thyroid hormone levels were measurable in both healthy individuals and patients with Graves' disease. Furthermore, as shown in Figure 2, the T3 and T4 levels in hair were significantly different between healthy individuals and patients with Graves' disease. Therefore, by setting standard values ​​for thyroid hormones, such as normal, abnormal, and cautionary values, and comparing the subjects' thyroid hormone levels with these standard values, it was suggested that they could be used as markers for thyroid disease. [Industrial Applicability]

[0056] The present invention can be used in the medical field to predict or diagnose health conditions or diseases. [Explanation of symbols]

[0057] 10 Liquid chromatograph (analytical equipment) 20 Mass spectrometer 21 Ionization section 22 Detection unit 30 Arithmetic unit 100 Analysis Systems

Claims

1. 1. A method for providing an indication for the diagnosis of a thyroid disease, comprising: A method comprising the step of measuring thyroid hormones T3 and / or T4 in an amount of 1 mg or more and 10 mg or less of cut hair from a subject.

2. The method of claim 1, further comprising a step of comparing the measured levels of thyroid hormones T3 and / or T4 in the cut hair of the subject with the levels of thyroid hormones T3 and / or T4 in cut hair of a healthy individual.

3. 2. The method of claim 1, wherein the thyroid disease is Graves' disease.

4. The measuring step mixing the cut hair with a solvent and controlling the temperature to 20°C or higher and 80°C or lower for 0.1 hours or longer and 12 hours or shorter to prepare an extract, wherein the amount of the solvent is 200 μL or less; separating the analytes in the extract by liquid chromatography; and 2. The method of claim 1, comprising measuring the thyroid hormones T3 and / or T4 by mass spectrometry of the separated analytical components.

5. 5. The method of claim 4, wherein the separating step comprises injecting the extract into a purification column to separate the analytes in the extract.

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