Chromatography analysis device and analysis method

Abstract

The main purpose of this technology is to provide a technique that can separate and detect multiple compounds from an analysis target that contains multiple compounds with similar physical properties and chemical structures, such as molecular weight, HLB value, logP value, and molecular skeleton. [Solution] As a result of extensive research, the inventors have discovered that by using two or more types of physicochemical separation columns with different separation patterns and performing independent measurements on each physicochemical separation column, it is possible to separate and detect multiple types of compounds from an analysis sample, such as a biological sample, that contains multiple types of compounds with similar physical properties and chemical structures, such as molecular weight, HLB value, logP value, and molecular skeleton.

Description

[Technical Field]

[0001] The present technology relates to a chromatography analysis device and an analysis method, and more particularly to a chromatography analysis device and an analysis method that can suitably separate and detect a sample containing multiple compounds with similar physicochemical properties. [Background technology]

[0002] BACKGROUND ART Techniques relating to chromatography analysis devices and analytical methods for separating and detecting a sample containing a plurality of types of compounds have been known for some time.

[0003] For example, Patent Document 1 below discloses a technique for continuously separating and analyzing a sample containing multiple types of analytes by two-stage separation using two types of columns: a pretreatment column and an analytical column. [Prior art documents] [Non-patent literature]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2018-169257 Summary of the Invention [Problem to be solved by the invention]

[0005] The main purpose of this technology is to provide a technique that can separate and detect multiple compounds from an analysis target that contains multiple compounds with similar physical properties and chemical structures, such as molecular weight, HLB value, logP value, and molecular skeleton. [Means for solving the problem]

[0006] As a result of extensive research, the present inventors have found that by using two or more types of physicochemical separation columns with different separation patterns and performing independent measurements for each physicochemical separation column, it is possible to separate and detect multiple types of compounds from an analysis target sample, such as a biological sample, that contains multiple types of compounds with similar physical properties and chemical structures, such as molecular weight, HLB value, logP value, and molecular skeleton.

[0007] Specifically, the present technology provides a chromatography analyzer comprising two or more physicochemical separation columns for separating a sample, a detector for detecting components of the sample, a flow path for each of the physicochemical separation columns, which supplies the sample to the physicochemical separation column and further supplies the detector with a liquid or gas as a mobile phase, and a switching mechanism for switching the flow paths, such that the two or more physicochemical separation columns independently separate and detect the sample by switching the switching mechanism. In the chromatography analyzer of the present technology, the sample may contain multiple compounds similar in molecular weight, HLB value, logP value, or molecular skeleton, and the sample may be a biological sample. Furthermore, the biological sample may contain a steroidal compound. The steroidal compound may be a vitamin D metabolite. The physicochemical separation column included in the analytical device of the present technology may be a column packed with silica gel particles having a fully porous or core-shell structure, or may be a reversed-phase column. Furthermore, the two or more types of physicochemical separation columns provided in the chromatography analyzer of the present technology may include an alkyl column packed with silica gel particles modified with an alkyl group and a mixed-mode column packed with silica gel particles modified with an alkyl group and an aromatic group. In this case, the alkyl groups in the alkyl column and the mixed-mode column may each have 8 to 30 carbon atoms, and each alkyl group in the alkyl column and the mixed-mode column may be an octadecyl group. Furthermore, the aromatic group may be an aromatic group substituted with one or more halogen atoms, and may be any of a pentafluorophenyl group, a pentachlorophenyl group, a pentabromophenyl group, and a pentaiodophenyl group. Furthermore, the detector included in the chromatography analysis apparatus of the present technology may be a tandem mass spectrometer.

[0008] Next, this technology provides a method for analyzing a sample, in which two or more types of physicochemical separation columns are used to separate components of a sample containing multiple compounds with similar molecular weights, HLB values, logP values, or molecular skeletons, independently for each column, and the separated components are detected using a detector. In the analysis method of the present technology, the sample may be a biological sample. Furthermore, the biological sample may contain a steroid compound. Furthermore, the steroid compound may be a vitamin D metabolite. In the analytical method of the present technology, before separating the components of the sample, the vitamin D metabolite contained in the sample may be subjected to a Diels-Alder reaction with a derivatization reagent at a diene moiety of the vitamin D to synthesize a derivative of the vitamin D metabolite. In this case, the derivatization reagent may be a compound having a triazoline skeleton, and the compound having a triazoline skeleton may be DAPTAD or its equivalent, DAP-PA. [Brief explanation of the drawings]

[0009] [Figure 1]1 is a diagram illustrating an example of the overall configuration of a chromatography analysis device according to the present technology. [Figure 2] FIG. 1 is a diagram showing an analysis flow using a chromatography analysis device according to the present technology. [Figure 3] FIG. 1 is a conceptual diagram showing the flow path of a sample when the chromatography analysis device of the present technology is equipped with two types of physicochemical separation columns. [Figure 4] 1 shows an SRM chromatogram obtained when a vitamin D mixed standard sample was analyzed by LC / MS / MS using a first physicochemical separation column (column 1) in an example. [Figure 5] 1 shows an SRM chromatogram obtained when a vitamin D mixed standard sample was analyzed by LC / MS / MS using a second physicochemical separation column (column 2) in an example. [Figure 6] 1 shows an SRM chromatogram obtained when a serum sample was analyzed by LC / MS / MS using a first physicochemical separation column (column 1) in an example. [Figure 7] 1 shows an SRM chromatogram obtained when a serum sample was analyzed by LC / MS / MS using a second physicochemical separation column (column 2) in an example. DETAILED DESCRIPTION OF THE INVENTION

[0010] Preferred embodiments of the present technology will be described below. However, the embodiments shown below are examples of typical embodiments of the present technology, and the present technology is not limited to only the preferred embodiments below and can be freely modified within the scope of the present technology.

[0011] [sample] This technology can separate and detect multiple compounds from an analysis target that contains multiple compounds with similar physical properties and chemical structures, such as molecular weight, HLB value (hydrophilic-lipophilic balance), logP value (octanol / water partition coefficient), and molecular skeleton.

[0012] Biological samples often contain multiple metabolites produced during metabolic pathways. Metabolites produced during metabolic pathways often have similar physical properties and chemical structures, such as molecular weight, HLB value, logP value, and molecular skeleton. Identifying these metabolites and analyzing their ratios makes it possible to analyze the state of the living body involved in the analysis. However, because these compounds have similar physical properties and chemical structures, they are not easy to separate.

[0013] Although it is possible to specifically separate specific compounds using an antibody extraction column, it is necessary to prepare an antibody extraction column for each compound, which is costly. Furthermore, the compounds that can be used for analysis are limited to those that can be separated using the antibody extraction column, which may prevent comprehensive analysis.

[0014] In general, compounds that cannot be specifically separated using an antibody extraction column are separated and quantified using, for example, analysis using a liquid chromatography tandem mass spectrometer (hereinafter sometimes referred to as "LC / MS / MS") equipped with a tandem mass spectrometer (hereinafter sometimes referred to as "MS / MS").

[0015] However, when an analyte containing multiple compounds with similar physical properties and chemical structures is separated by a conventional method using a physicochemical separation column used in LC / MS / MS, the separation may not be successful depending on the separation ability characteristics of the physicochemical separation column (the separation pattern of the physicochemical separation column), and compounds may appear with overlapping peaks in the chromatogram.

[0016] In contrast, this technology uses two or more types of physicochemical separation columns with different physicochemical separation capabilities, separates the components of the sample independently using each column, and detects the separated components using a detector, thereby enabling multifaceted analysis of the components contained in the sample, even if the sample contains multiple compounds with similar physical properties or chemical structures.Furthermore, depending on the combination of physicochemical separation columns used, it may be possible to comprehensively analyze metabolites and other substances contained in the sample.

[0017] Although the subject of analysis of this technology is not particularly limited, because this technology can analyze components contained in a sample from multiple perspectives, the subject of analysis of this technology can be suitably analyzed as a sample containing multiple compounds with similar physical properties and chemical structures. Here, physical properties include, but are not limited to, molecular weight, HLB value, logP value, etc. In this specification, compounds with similar physical properties and chemical structures can be defined as compounds with similar molecular weight, HLB value, logP value, or molecular skeleton. Examples of such compounds include steroid compounds such as 7-dehydrocholesterol, ergosterol, vitamin D, androgens, and estrogen, as well as polyene compounds such as conjugated linoleic acid and vitamin A.

[0018] Examples of samples containing multiple compounds with similar molecular weights, HLB values, logP values, or molecular skeletons that can be suitable analysis targets of the present technology include, but are not limited to, biological samples. Here, a biological sample refers to an organism such as an animal, plant, or microorganism that performs vital activities such as metabolism, growth, reaction, and reproduction.

[0019] When using animal biological samples, examples of the biological sample that can be used include, but are not limited to, blood, serum, plasma, cerebrospinal fluid, urine, saliva, and feces. It is desirable to pretreat the biological sample to remove as many contaminants as possible other than the target compound before analysis using this technology. This pretreatment can be performed using any method and under any conditions, depending on the purpose of the analysis and the target compound. Examples of pretreatment methods include, but are not limited to, deproteinization, liquid-liquid extraction (LLE), solid-liquid extraction (SLE), and solid-phase extraction (SPE).

[0020] For example, in living animals, the metabolism of vitamin D, which is produced from the steroid compound 7-dehydrocholesterol or introduced into the body through food intake, is involved in the maintenance of bones, muscles, etc. Therefore, for example, comprehensive analysis of vitamin D metabolites in a biological sample would enable the analysis of the biological condition of the biological sample being analyzed. More specifically, comprehensive analysis of vitamin D metabolites in a biological sample is expected to be applicable, for example, to the prevention of diseases such as cancer, arteriosclerosis, diabetes, aggravation of respiratory infections, and osteoporosis, as well as to the regulation of calcium levels related to bone metabolism, and as an indicator for maintaining biological homeostasis such as lipid metabolism, vitamin D metabolic activity, glucose metabolism, and insulin sensitivity.

[0021] In particular, vitamin D metabolites are secosteroids, which are formed by opening the steroid skeleton. They have a basic structure in which a six-membered ring and a five-membered ring share one edge, and this ring is further connected to another six-membered ring via a diene structure. During its metabolic pathway, vitamin D can produce multiple metabolites with different side chain structures, including positional isomers, stereoisomers, and structural isomers, which differ in the position, stereochemistry, and structure of the hydroxyl group substituents on these rings. These metabolites have similar physical properties, such as molecular weight, HLB value, logP value, and molecular skeleton, making their analysis difficult, even using LC / MS / MS.

[0022] Vitamin D metabolites include, but are not limited to, vitamin D that has been hydroxylated or oxidized by enzymes in the body, such as hydroxyvitamin D, dihydroxyvitamin D, trihydroxyvitamin D, vitamin D lactone, and calcitronic acid. More specifically, these include 25-hydroxyvitamin D3 (25(OH)D3), 25-hydroxyvitamin D2 (25(OH)D2), 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), 1α,25-dihydroxyvitamin D2 (1α,25(OH)2D2), 1β,25-dihydroxyvitamin D3 (1β,25(OH)2D3), 23,25-dihydroxyvitamin D3 (23,25(OH)2D3), 25,26-dihydroxyvitamin D3 (25,26(OH)2D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), and 4β,25-dihydroxyvitamin D3 (4β,25(OH)2 D3), ​​25-hydroxyvitamin D3-23,26-lactone (25(OH)D3 lactone), 1α,25-dihydroxyvitamin D3-23,26-lactone (1α,25(OH)2D3 lactone), 4β,25-dihydroxyvitamin D3-23,26-lactone (4β,25(OH)2D3 lactone), 1α,4α,25-trihydroxyvitamin D3-23,26-lactone (1α,4α,25(OH)3D3 lactone), 1α,4β,25-trihydroxyvitamin D3-23,26-lactone (1α,4β,25(OH)3D3 lactone), calcioic acid, calcitronic acid, etc. Vitamin D metabolites may be isomers of the aforementioned compounds, such as 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3), etc. These vitamin D metabolites may also be conjugated with sugars or sulfate groups.

[0023] Furthermore, when analyzing multiple metabolites contained in a biological sample, sensitivity and resolution during analysis can sometimes be improved by synthesizing derivatives of the metabolites of interest using a derivatization reagent appropriate for the metabolite before separating the sample components. When analyzing vitamin D metabolites in a biological sample, sensitivity and resolution can also be improved by synthesizing derivatives of vitamin D metabolites contained in the sample by subjecting the diene moiety of the vitamin D metabolite to a Diels-Alder reaction using a derivatization reagent before separating the sample components. The synthesis of the derivatives can also be performed after appropriate pretreatment to remove contaminants.

[0024] Derivatization reagents that can synthesize vitamin D metabolite derivatives by Diels-Alder reaction of the diene moiety of such vitamin D metabolites include compounds having a triazoline skeleton. Examples of compounds having a triazoline skeleton include 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), 4-[4-dimethylaminophenyl]-1,2,4-triazoline-3,5-dione (DAPTAD), 4-[4-(1-piperidinyl)phenyl]-1,2,4-triazoline-3,5-dione (PIPTAD), 4-(2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl)-1,2,4-triazoline-3,5-dione (DMEQ-TAD), and equivalents of these compounds, such as protected derivatives of DAPTAD. Examples include 14-(4-(dimethylamino)phenyl)-9-phenyl-9,10-dihydro-9,10-[1,2]epitriazoloanthracene-13,15-dione (DAP-PA). Of the above, DAP-PA is an equivalent of DAPTAD, with the dienophile portion of DAPTAD protected with 4-phenylanthracene (PA), improving storage stability in solution. Heating DAP-PA deprotects it to produce DAPTAD, which then reacts with the diene portion of vitamin D.

[0025] Even when analyzing metabolites such as vitamin D in biological samples using the analytical method of the present technology, sensitivity and resolution can be improved by synthesizing derivatives of vitamin D metabolites by subjecting the vitamin D metabolites contained in the sample to a Diels-Alder reaction with the diene moiety of the vitamin D using a derivatization reagent before separating the components of the sample. The derivatization reagents described above can be suitably used as derivatization reagents that can synthesize derivatives of vitamin D metabolites in this case. Among these derivatization reagents, the analytical method of the present technology can more suitably improve sensitivity and resolution by using DAPTAD or its equivalent, DAP-PA.

[0026] [Physicochemical separation column] In this technology, the "physicochemical separation column" is a combination of two or more arbitrary physicochemical separation columns that are tailored to the physical properties and chemical structure of the components to be measured, each column having different physicochemical separation capabilities. The physicochemical separation columns used in this technology can be combined arbitrarily, but the greater the difference in physicochemical separation capabilities between the combined physicochemical separation columns, the more versatile the separation of metabolites contained in the sample can be, which is preferable. Furthermore, in this technology, more physicochemical separation columns, such as three or more types, can also be used to achieve a more versatile separation of metabolites contained in the sample.

[0027] The physicochemical separation columns that can be used in this technology are columns that separate components contained in a sample based on the physical or chemical properties of the components, and do not include antibody extraction columns that specifically extract specific antigens or antibodies using antigens or antibodies. For example, reverse-phase columns, hydrophilic interaction chromatography (HILIC) columns, ion-exchange columns, size-exclusion columns, etc. can be suitably used as physicochemical separation columns in this technology. Any physicochemical separation column that can suitably separate the analyte components contained in the sample can be selected depending on the physical or chemical properties of the components.

[0028] Furthermore, as a form of physicochemical separation column that can be used in the present technology, for example, a column packed with silica gel particles can be preferably used, because it can shorten the diffusion distance of the sample during separation and improve separation efficiency. The silica gel particles that can be preferably used are, for example, fully porous or have a core-shell structure.

[0029] For example, when analyzing metabolites of steroid compounds such as vitamin D in a biological sample, a reversed-phase column can be used as a physicochemical separation column based on the properties of steroid compounds such as vitamin D. Examples of reversed-phase columns in this case include alkyl columns packed with silica gel particles modified with alkyl groups, aryl columns packed with silica gel particles modified with aromatic groups, and mixed-mode columns packed with silica gel particles modified with multiple types of hydrophobic substituents, such as a combination of alkyl and aromatic groups.

[0030] The number of carbon atoms in the alkyl group of the column is, for example, 8 to 30. Examples of alkyl columns include a C8 column (octylsilyl column), a C18 column (octadecylsilyl column), a C22 (docosylsilyl column), and a C30 column (triacontylsilyl column).

[0031] The aryl column may also be, for example, a halogenated aryl column in which the aromatic group is substituted with one or more halogen atoms. In this case, the aromatic group of the halogenated aryl column may be any of a pentafluorophenyl group, a pentachlorophenyl group, a pentabromophenyl group, and a pentaiodophenyl group. Furthermore, the aromatic group may be directly or indirectly bonded to the silica gel particles. When the aromatic group is indirectly bonded to the silica gel particles, the bond may be via a short alkyl chain such as methyl, ethyl, propyl, butyl, pentyl, or hexyl.

[0032] Furthermore, examples of the combination of hydrophobic substituents constituting a mixed-mode column include, but are not limited to, a combination of an alkyl group and an aromatic group. When the combination of hydrophobic substituents constituting a mixed-mode column is a combination of an alkyl group and an aromatic group, the alkyl group and aromatic group constituting the mixed-mode column can preferably be the alkyl group that can be used for the alkyl column and the aromatic group that can be used for the aryl column, as described above.

[0033] For example, when analyzing metabolites of steroid compounds in a biological sample using the analytical method of the present technology, as described above, by combining two or more physicochemical separation columns with different physicochemical separation capabilities, selected from among those suited to the physical properties and chemical structures of the components being measured, it is possible to achieve multifaceted separation of metabolites such as steroid compounds contained in the biological sample. Examples of such combinations of two or more physicochemical separation columns include, for example, a combination of an alkyl column packed with silica gel particles modified with an alkyl group and a mixed-mode column packed with silica gel particles modified with an alkyl group and an aromatic group, a combination of an alkyl group and an ion-exchange group, or a combination of an aromatic group and an ion-exchange group. The reversed-phase columns exemplified above can be suitably used as physicochemical separation columns for these combinations.

[0034] In particular, the characteristics of vitamin D metabolites, which are a type of steroid compound, differ depending on the reversed-phase column used due to the diversity of metabolites. For example, differences in the position and stereochemistry of substituents, such as hydroxyl groups, on the vitamin D skeleton can often result in metabolites that can be separated on one reversed-phase column but not on another. This makes it difficult to comprehensively analyze metabolites using a single reversed-phase column, such as conventional LC / MS / MS analysis. Therefore, the analytical method of this technology combines two or more reversed-phase columns with different physicochemical separation capabilities, which are physicochemical separation columns capable of separating vitamin D metabolites, enabling multifaceted separation of vitamin D metabolites.

[0035] In the analytical method of the present technology, when separating vitamin D metabolites, among the combinations listed above, a combination of an alkyl column packed with silica gel particles modified with an alkyl group and a mixed-mode column packed with silica gel particles modified with an alkyl group and an aromatic group is preferred. Each alkyl group in these columns preferably has 8 to 30 carbon atoms, and more preferably each alkyl group is an octadecyl group. Furthermore, the aromatic group in the silica gel particles packed in the mixed-mode column is preferably an aromatic group substituted with one or more halogen atoms. More specifically, it is preferably any one of a pentafluorophenyl group, a pentachlorophenyl group, a pentabromophenyl group, and a pentaiodophenyl group.

[0036] [Detector] The detector that can be used in the analytical method of the present technology is not particularly limited as long as it can detect sample components. For example, any detector can be suitably used, such as a mass spectrometer (MS), a tandem mass spectrometer (MS / MS), an ultraviolet detector, an ultraviolet-visible detector, a photodiode array detector, a fluorescence detector, a differential refractive index detector, a conductivity detector, an electrochemical detector, an evaporative light scattering detector, or a corona charged aerosol detector.

[0037] [Switching mechanism] The chromatography analyzer that realizes the analytical method of the present technology includes, in addition to the two or more types of physicochemical separation columns and detectors described above, a flow path provided for each physicochemical separation column, which supplies the sample to the physicochemical separation column and further supplies the liquid or gas to the detector as a mobile phase, and a switching mechanism for switching the flow paths. Thus, the chromatography analyzer of the present technology can independently separate and detect the sample using the two or more types of physicochemical separation columns by switching the switching mechanism.

[0038] The switching mechanism that can be used in the chromatography analyzer of the present technology is not particularly limited, and any switching mechanism can be suitably used. Examples of switching mechanisms include a switching valve that can switch flow paths, and a flow path control system that controls flow paths by switching on and off mobile phase supply units, such as a liquid pump or gas cylinder, in a flow path network equipped with multiple mobile phase supply units that supply mobile phase. When the chromatography analyzer of the present technology employs a combination of switching valves as the switching mechanism, for example, a switching valve provided upstream of each physicochemical separation column to switch the flow path that supplies the sample to the physicochemical separation column and then to the detector may be combined with a switching valve provided downstream of the physicochemical separation column to switch the flow path so that the sample separated by the physicochemical separation column flows downstream to the detector. The switching valve provided downstream of the physicochemical separation column may discharge fractions other than the fraction containing the target metabolite to the outside of the chromatography analyzer. This may enable more efficient detection of metabolites by the detector.

[0039] [Other configurations] The chromatography analyzer of the present technology may further include any components that known chromatography analyzers can include, as necessary, other than the components described above, as long as the desired physical properties are not significantly impaired. Examples of other components include, but are not limited to, a mobile phase supply unit such as a liquid pump used when the mobile phase is liquid or a gas cylinder used when the mobile phase is gas, a sample introduction unit for introducing a sample into the chromatography analyzer, a sampler or other component of the sample introduction unit, a prefilter for filtering the introduced sample before it enters the physicochemical separation column, and a guard column.

[0040] Specific embodiments of the chromatography analyzer of the present technology will be described below with reference to the drawings. Note that the embodiments described below are examples of the present technology, and the present technology should not be interpreted as being limited to the contents of these embodiments.

[0041] Fig. 1 is a diagram schematically illustrating an example of the overall configuration of a chromatography analyzer 100 according to the present technology. In the chromatography analyzer 100 illustrated in the example of Fig. 1, a sample introduced into a sample introduction unit S connected to a mobile phase supply unit F of the chromatography analyzer is first switched between flow paths (1) 102 and (2) 104 provided for a physicochemical separation column (column 1) 101 and a physicochemical column (column 2) 103 by a switching valve (corresponding to a switching mechanism) 106 provided upstream of these two physicochemical separation columns. After that, the sample separated in one of the physicochemical separation columns has its flow path switched by a switching valve 107 provided downstream of the physicochemical separation column (column 1) 101 and the physicochemical column (column 2) 103, and flows into a detector 105 for analysis.

[0042] As described above, after the sample separated in one of the physicochemical separation columns is flowed into the detector 105, the flow path provided for each of these two physicochemical separation columns is switched to the other flow path by the switching valve 106 provided upstream. Thereafter, the sample separated in the other physicochemical separation column has its flow path switched by a switching valve 107 provided downstream of the two physicochemical separation columns, and flows into a detector 105 for analysis.

[0043] In the chromatography analyzer 100 shown in the example of Figure 1, metabolites contained in a sample can be separated in a multifaceted manner by combining two or more types of arbitrary physicochemical separation columns with different physicochemical separation capabilities that are suited to the physical properties and chemical structures of metabolites, etc., contained in a sample. Furthermore, the chromatography analyzer shown in Figure 1 is not limited to a configuration equipped with two types of physicochemical separation columns, and may be configured to use more physicochemical separation columns, such as three or more types, as indicated by the dotted line in Figure 1, in order to separate metabolites contained in a sample in a more multifaceted manner.

[0044] FIG. 2 is a diagram showing an analysis flow using the chromatography analyzer 100 shown in the example of FIG. 1. The chromatography analyzer 100 switches the physicochemical separation column to be used according to the analysis flow and repeats the cycle of analysis. As the number of physicochemical separation columns used in the chromatography analyzer 100 shown in the example of FIG. 1 increases (1, 2, 3, ...), the analysis start command shown in the flow also increases accordingly (1, 2, 3, ...). FIG. 3 is a conceptual diagram showing the flow path of a sample within the chromatography analyzer 100 shown in the example of FIG. 1 or FIG. 2 when the number of physicochemical separation columns is two. Specific flow paths of the chromatography analyzer 100 of FIG. 3 will be described below in accordance with the analysis flow shown in FIG. 2.

[0045] As in the description of FIG. 1 , a sample introduced into the chromatography analyzer 100 is first switched between flow paths (1) 102 and (2) 104 provided for a physicochemical separation column (column 1) 101 and a physicochemical column (column 2) 103, respectively, by a switching valve 106 provided upstream of these two physicochemical separation columns in accordance with a first analysis start command shown in FIG. 2 . In the example shown in FIG. 2 , the physicochemical separation column (column 1) 101 is first selected from the two physicochemical separation columns by the first analysis start command, but the physicochemical separation column (column 2) 103 may also be selected first. Thereafter, the flow path of the sample separated in (column 1) 101 is switched by a switching valve provided downstream of the physicochemical separation column, and the sample flows into a detector (MS) 105 for analysis. Note that the valve switching may already be completed at the time of the first analysis start command.

[0046] After the sample separated in the physicochemical separation column (column 1) 101 flows into the detector 105, the flow path is switched again to flow path (2) 104 connecting the other physicochemical separation column (column 2) 103 by a switching valve 106 provided upstream of the physicochemical separation column in accordance with the valve switching command shown in Figure 2. Thereafter, the flow path of the sample separated in (column 2) 103 is switched by a switching valve provided downstream of the physicochemical separation column, and the sample flows into the detector (MS) 105 for analysis. After the measurement is completed, the analysis results are output.

[0047] 3 shows an example in which a sampler 108, which is a component for introducing a sample into the chromatography analyzer 100, and a prefilter 109 that filters the introduced sample before it enters the physicochemical separation column, but the configuration is not limited to this. Similarly, the configuration that can be used in the example of FIG. 1 can also be suitably used in the chromatography analyzer shown in FIG. 3.

[0048] The present technology can have the following configurations. [1] Two or more types of physicochemical separation columns for separating a sample; a detector for detecting components of the sample; a flow path provided for each of the physicochemical separation columns for supplying the sample to the physicochemical separation column and then to the detector; a switching mechanism for switching the flow path, A chromatography analysis device in which the two or more types of physicochemical separation columns independently separate and detect the sample by switching the switching mechanism. [2] The chromatography analyzer according to [1], wherein the sample contains a plurality of compounds that are similar in any of molecular weight, HLB value, logP value, or molecular skeleton. [3] The analytical device according to [2], wherein the sample is a biological sample. [4] The chromatography analyzer according to [3], wherein the biological sample contains a steroid compound. [5] The chromatography analyzer according to [4], wherein the steroid compound is a vitamin D metabolite. [6] The chromatography analyzer according to any one of [1] to [5], wherein the physicochemical separation column is a column packed with silica gel particles having a fully porous or core-shell structure. [7] The chromatography analyzer according to [4] or [5], wherein the physicochemical separation column is a reversed-phase column. [8] The chromatography analyzer according to [4] or [5], wherein the two or more types of physicochemical separation columns include an alkyl column packed with silica gel particles modified with alkyl groups, and a mixed-mode column packed with silica gel particles modified with alkyl groups and aromatic groups. [9] The chromatography analyzer according to [8], wherein the alkyl column and the mixed-mode column each have an alkyl group having 8 to 30 carbon atoms.

[10] The chromatography analyzer according to [8] or [9], wherein the alkyl group of each of the alkyl column and the mixed-mode column is an octadecyl group.

[11] The chromatography analyzer according to any one of [8] to

[10] , wherein the aromatic group is an aromatic group substituted with one or more halogen atoms.

[12] The chromatography analyzer according to any one of [8] to

[11] , wherein the aromatic group is any one of a pentafluorophenyl group, a pentachlorophenyl group, a pentabromophenyl group, and a pentaiodophenyl group.

[13] The chromatography analyzer according to any one of [1] to

[12] , wherein the detector is a tandem mass spectrometer.

[14] Using two or more types of physicochemical separation columns, A sample containing multiple compounds with similar molecular weights, HLB values, logP values, or molecular skeletons is Separating the components of the sample independently for each physicochemical separation column; A method of analyzing a sample in which the separated components are detected by a detector.

[15] The analytical method according to

[14] , wherein the sample is a biological sample.

[16] The analytical method according to

[15] , wherein the biological sample contains a steroid compound.

[17] The analytical method according to

[16] , wherein the steroid compound is a vitamin D metabolite.

[18] The analytical method described in

[17] , wherein, before separating the components of the sample, the vitamin D metabolites contained in the sample are subjected to a Diels-Alder reaction with a derivatization reagent at the diene portion of the vitamin D to synthesize a derivative of the vitamin D metabolite.

[19] The analytical method according to

[18] , wherein the derivatization reagent is a compound having a triazoline skeleton.

[20] The analytical method according to

[19] , wherein the compound having a triazoline skeleton is DAPTAD or its equivalent DAP-PA. [Example]

[0049] The present technology will be described in detail below based on specific examples, but the present technology is not limited to the contents of the examples shown below.

[0050] <Measurement Sample 1: Preparation of Vitamin D Mixed Standard Sample> (1) 25(OH)D3, 25(OH)D2, 24,25(OH)2D3, 3-epi-25(OH)D3, 1α,25(OH)2D3, 1β,25(OH)2D3, 4β,25(OH)2D3, 1α,25(OH)2D3-lactone, 4β,25(OH)2D3-lactone, 25(OH)D3-lactone, 1α,4α,25(OH)3D3-lactone, and 1α,4β,25(OH)3D3-lactone preparations were prepared in acetonitrile to a final concentration of 500 pg / mL. (2) After evaporating the solvent from 100 μL of the standard sample under a nitrogen stream, 100 μL of a derivatization reagent, DAP-PA 0.5 mg / mL (ethyl acetate solution), was added and heated at 80°C for 15 minutes to derivatize the sample. After evaporating the solvent under a nitrogen stream, the sample was redissolved in 50 μL of 50% acetonitrile aqueous solution (v / v) to prepare a measurement sample.

[0051] <Measurement Sample 2: Preparation of Serum Sample> Serum samples were prepared according to the following procedure. (1) As a biological sample, 150 μL of human serum (Human Serum Pool, Cosmo Bio) was mixed with 150 μL of acetonitrile and stirred. (2) After centrifugation at 10,000×G for 5 minutes, the supernatant was diluted with 450 μL of water to prepare a measurement sample. (3)Oasis PRiME HLB μElution Plate(Waters The entire amount of the sample was loaded into a container (USA) and then pressurized. (4) 200 μL of water was passed through to wash the solid phase. (5) 200 μL of 20% aqueous ethanol solution (v / v) was passed through to wash the column. (6) 200 μL of ethanol was passed through the column, and the fraction containing vitamin D metabolites was collected. (7) The solvent was distilled off (removed) from the collected fractions under a nitrogen stream. (8) A derivatization reagent, DAP-PA, 0.5 mg / mL (ethyl acetate solution) was heated at 80°C for 15 minutes. (9) 100 μL of the heated DAP-PA was added to the collected fraction from which the solvent had been distilled off, and the mixture was reacted for 15 minutes to synthesize a derivative of a vitamin D metabolite. (10) After the solvent was removed from the reaction solution under a nitrogen stream, 30 μL of 50% aqueous acetonitrile solution (v / v) was added to redissolve the residue, and the resulting solution was used as a measurement sample.

[0052] <Physicochemical separation column> The chromatography analysis device used in the examples was equipped with the following two types of physicochemical separation columns (columns 1 and 2). The chromatography analysis device was equipped with the mechanism shown in FIG. Column 1: SunShell PFP-C18 2.1 mm id × 100 mm (ChromaNik Technologies, Japan) Column 2: Capcellcore C18 2.1 mm id × 75 mm (Osaka Soda, Japan)

[0053] [Column 1 elution conditions] Apparatus: Shimadzu LC-30AD (Shimadzu, Japan) Mobile phase: 0.1% formic acid water (A), 0.1% formic acid acetonitrile (B) Flow rate: 0.4mL / min Column temperature: 50℃ The elution conditions are shown in Table 1.

[0054] [Table 1]

[0055] [Column 2 elution conditions] The apparatus, mobile phase, flow rate, and column temperature were the same as those for Column 1 described above, and the elution conditions are shown in Table 2.

[0056] [Table 2]

[0057] <LC / MS / MS analysis> As described above, the prepared measurement samples (1 μL for both columns 1 and 2 for measurement sample 1, 20 μL and 1 μL for columns 1 and 2 respectively for measurement sample 2) were injected into the chromatography analyzer equipped with the above columns 1 and 2, and analysis was performed according to the analysis flow shown in FIG. 2. The MS / MS analysis conditions for columns 1 and 2 are as follows.

[0058] [MS / MS analysis conditions for column 1] The measurement samples separated by column 1 were detected under the following conditions. Apparatus: Shimadzu LCMS8060 Ionization method: Positive ion detection ESI (1) SRM: m / z 635.3 → 357.1 (1α,25(OH)2D3 - DAPTAD, 1β,25(OH)2D3 - DAPTAD, 4β,25(OH)2D3 - DAPTAD) Collision activation energy: -24 eV (2) SRM: m / z 647.3 → 341.1 (25(OH)D3 - Lactone - DAPTAD) Collision activation energy: -27 eV (3) SRM: m / z 663.4 → 357.2 (1α,25(OH)2D3 - Lactone - DAPTAD, 4β,25(OH)2D3 - Lactone - DAPTAD) Collision activation energy: -27 eV (4) SRM: m / z 679.3 → 357.1 (1α,4α,25(OH)3D3 - Lactone, 1α,4β,25(OH)3D3 - Lactone - DAPTAD) Collision activation energy: -25 eV

[0059] [MS / MS analysis conditions for column 2] The measurement samples separated by column 2 were detected under the following conditions. Apparatus: Shimadzu LCMS8060 Ionization method: Positive ion detection ESI (5)SRM: m / z 619.2 → 341.2(25(OH)D3-DAPTAD, 3-epi-25(OH)D3-DAPTAD) Collision activation energy: -26 eV (6)SRM: m / z 631.2 → 341.2(25(OH)D2-DAPTAD) Collision activation energy: -26 eV (7)SRM: m / z 635.2 → 341.2(24,25(OH)2D3-DAPTAD) Collision activation energy: -25 eV

[0060] Figures 4 and 5 show SRM chromatograms obtained when measurement sample 1 (a vitamin D mixed standard sample) was analyzed by LC / MS / MS using columns 1 and 2. Figure 4 shows the results obtained using column 1, and Figure 5 shows the results obtained using column 2. Similarly, Figures 6 and 7 show SRM chromatograms obtained when measurement sample 2 (a serum sample) was analyzed by LC / MS / MS. Figure 6 shows the results obtained using column 1, and Figure 7 shows the results obtained using column 2.

[0061] The SRM chromatograms of LC / MS / MS analysis using column 2 shown in Figures 5 and 7 show that 25(OH)D3 and 3-epi-25(OH)D, which cannot be separated and detected by LC / MS / MS analysis using column 1, are 3、 It was confirmed that vitamin D metabolites such as 25(OH)D2 and 24,25(OH)2D3 could be detected. Furthermore, the SRM chromatograms shown in Figures 6 and 7 also confirm peaks thought to be derived from vitamin D metabolites, which were not observed in the results of Measurement Sample 1 using a standard sample. Specifically, by using two types of physicochemical separation columns with different separation patterns and switching the switching valve, which serves as a switching mechanism, independent measurements can be performed on each of the two types of physicochemical separation columns. This allows for the separation and detection of multiple compounds from a target sample, such as a biological sample, that contains multiple compounds with similar physical properties and chemical structures, such as molecular weight, HLB value, logP value, and molecular structure. This allows for comprehensive analysis of multiple compounds with similar physical properties and chemical structures in a biological sample.

[0062] Furthermore, when comparing the SRM chromatograms of measurement sample 1 (a vitamin D mixed standard sample) and measurement sample 2 (a serum sample), it was confirmed that peaks that could be measured in one sample were sometimes not measurable in the other. This demonstrates that this technology can be used to comprehensively analyze the metabolic status of multiple compounds with similar physical properties and chemical structures, such as vitamin D, contained in the sample being measured, using the SRM chromatogram of the standard sample as a reference. [Explanation of symbols]

[0063] 100 Chromatography analyzer 101 Physicochemical Column (Column 1) 102 Flow path (1) 103 Physicochemical Column (Column 2) 104 Flow path (2) 105 Detector (MS) 106 Switching mechanism (switching valve) (upstream side) 107 Switching mechanism (switching valve) (downstream side) 108 Sampler 109 Pre-filter S Sample introduction section (for introducing biological samples, etc.) F Mobile phase supply unit (supplies liquid or gas as mobile phase)

Claims

1. two or more types of physicochemical separation columns for separating a sample; a detector for detecting components of the sample; a flow path provided for each of the physicochemical separation columns, through which the sample is supplied to the physicochemical separation column and further supplied to the detector, the flow path using a liquid or gas as a mobile phase; a switching mechanism for switching the flow path, A chromatography analysis device in which the two or more types of physicochemical separation columns independently separate and detect the sample by switching the switching mechanism.

2. The chromatography analysis apparatus according to claim 1 , wherein the sample contains a plurality of compounds that are similar in any of molecular weight, HLB value, log P value, or molecular skeleton.

3. The chromatography analyzer according to claim 2 , wherein the sample is a biological sample.

4. The chromatography analyzer according to claim 3 , wherein the biological sample contains a steroid compound.

5. The chromatography analyzer according to claim 4 , wherein the steroid compound is a vitamin D metabolite.

6. 2. The chromatography analysis apparatus according to claim 1, wherein the physicochemical separation column is a column packed with silica gel particles having a fully porous or core-shell structure.

7. The chromatography analysis device according to claim 4 , wherein the physicochemical separation column is a reversed-phase column.

8. 5. The chromatography analyzer according to claim 4, wherein the two or more types of physicochemical separation columns include an alkyl column packed with silica gel particles modified with an alkyl group, and a mixed-mode column packed with silica gel particles modified with an alkyl group and an aromatic group.

9. 9. The chromatography analyzer according to claim 8, wherein the alkyl group of each of the alkyl column and the mixed-mode column has 8 to 30 carbon atoms.

10. 9. The chromatography analyzer according to claim 8, wherein the alkyl group of each of the alkyl column and the mixed-mode column is an octadecyl group.

11. The chromatography analyzer according to claim 8 , wherein the aromatic group is an aromatic group substituted with one or more halogen atoms.

12. The chromatography analyzer according to claim 8 , wherein the aromatic group is any one of a pentafluorophenyl group, a pentachlorophenyl group, a pentabromophenyl group, and a pentaiodophenyl group.

13. The chromatography analyzer of claim 1 , wherein the detector is a tandem mass spectrometer.

14. Using two or more types of physicochemical separation columns, A sample containing a plurality of compounds similar in molecular weight, HLB value, log P value, or molecular skeleton is Separating the components of the sample independently for each physicochemical separation column; A method of analyzing a sample in which the separated components are detected by a detector.

15. The analytical method according to claim 14 , wherein the sample is a biological sample.

16. The analytical method according to claim 15 , wherein the biological sample contains a steroid compound.

17. The analytical method according to claim 16, wherein the steroid compound is a vitamin D metabolite.

18. The analytical method of claim 17, wherein, before separating the components of the sample, the vitamin D metabolites contained in the sample are subjected to a Diels-Alder reaction with a derivatization reagent at the diene portion of the vitamin D to synthesize a derivative of the vitamin D metabolite.

19. The analytical method according to claim 18 , wherein the derivatization reagent is a compound having a triazoline skeleton.

20. 20. The analytical method according to claim 19, wherein the compound having a triazoline skeleton is DAPTAD or its equivalent DAP-PA.

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