WO2020096056A1

WO2020096056A1 - Separation material for metabolome analysis and column for metabolome analysis

Info

Publication number: WO2020096056A1
Application number: PCT/JP2019/043995
Authority: WO
Inventors: 惠太櫻井; 道男佛願; 健史馬場; 自泰和泉; 航太中谷
Original assignee: 日立化成テクノサービス株式会社; 国立大学法人九州大学
Priority date: 2018-11-08
Filing date: 2019-11-08
Publication date: 2020-05-14
Also published as: JPWO2020096056A1; JP7341434B2

Abstract

Provided is a separation material for metabolome analysis having polymer particles and an organic group which is linked to the polymer particles and includes a quaternary ammonium group. Also provided is a separation material for metabolome analysis comprising the aforementioned kind of separation material for metabolome analysis.

Description

Separation material for metabolome analysis and column for metabolome analysis

The present invention relates to a metabolome analysis separation material and a metabolome analysis column.

Metabolomics is a research area applied to the diagnosis of diseases, etc., and is a research area for the purpose of comprehensively analyzing the metabolome, which is the total metabolites of cells, to understand life phenomena. As a metabolome analysis method, gas chromatography / mass spectrometry (GC / MS), liquid chromatography / mass spectrometry (LC / MS), capillary electrophoresis / mass spectrometry (CE / MS), etc. are used. (See Non-Patent Document 1).

GC / MS is suitable for measuring volatile metabolites such as aromatic compounds. However, derivatization is necessary when measuring non-volatile metabolites, which may cause problems in quantification. In addition, since most metabolites are non-volatile, measurement of volatile substances, which is the greatest merit of GC, tends to be limited to organic acids and the like.

LC / MS can measure metabolites of a wide range of chemical substances and is frequently used in metabolome research. However, there are problems in that the solvent selection is complicated, and that it is not compatible with MS in the analysis of ionic metabolites.

CE / MS is a useful analytical method because most of the metabolic intermediates contained in amino acid, nucleic acid metabolism, etc. have ionicity. However, compared to GC / MS and LC / MS, there are problems such as poor density sensitivity, difficulty in handling, and difficulty in use by the user.

In recent years, the measurement of metabolome components has also been investigated in separation methods such as ion exchange in the LC method, and for example, an example of separating nucleotides by ion exchange has been reported (see, for example, Patent Document 1). However, the separation of non-ionic components is insufficient, and if the eluent contains a large amount of organic solvent, the separation material swells and shrinks, and the column is damaged, making it impossible to confirm the measurement reproducibility. Etc. are worried as problems.

JP-A-2015-129775

Many biological components targeted by metabolome analysis are highly hydrophilic components, and it tends to be difficult to retain these components with an ODS column (octadecyl silica column) generally used in the LC method. Such a tendency is remarkable especially under basic conditions.

Therefore, the main object of the present invention is to provide a separation material for metabolome analysis capable of separating a metabolome from a sample containing the metabolome even when used under basic conditions.

As a result of intensive studies by the present inventors, they have found that the above problem can be solved by introducing a specific group into polymer particles, and have completed the present invention. That is, the present invention provides the separation material for metabolome analysis described in [1] to [4] below, and the column for metabolome analysis described in [5] below.

[1] A separation material for metabolome analysis, comprising polymer particles and an organic group containing a quaternary ammonium group, which is bonded to the polymer particles.
[2] The separation material for metabolome analysis according to [1], wherein the polymer particles contain a polymer having a structural unit derived from a (meth) acrylic acid ester.
[3] The separation material for metabolome analysis according to [1] or [2], wherein the separation material has an average particle diameter of 2 to 5 μm.
[4] The separation material for metabolome analysis according to any of [1] to [3], wherein the separation material has a quaternary ammonium group amount of 10 to 500 μeq / g.
[5] A metabolome analysis column comprising the metabolome analysis separation material according to any one of [1] to [4].

According to the present invention, there is provided a separation material for metabolome analysis, which is capable of separating a metabolome from a sample containing the metabolome even when used under basic conditions. Further, according to the present invention, there is provided a metabolome analysis column using such a separation material for metabolome analysis.

It is a graph which shows the gradient conditions of HPLC in the column evaluation of an Example. It is a score table of the chromatogram which shows the result of column evaluation of an example. (A) is a chromatogram of adenine under basic conditions when using the column of Example 1, and (b) is a chromatogram of adenine under basic conditions when using the column of Example 2. It is gram. (A) is a chromatogram of adenosine under basic conditions when using the column of Example 1, and (b) is a chromatogram of adenosine under basic conditions when using the column of Example 2. It is gram.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments. In the present specification, “(meth) acrylic acid” means “acrylic acid” or “methacrylic acid” corresponding thereto, and the same applies to other similar descriptions such as “(meth) acrylate”. Is.

<Separating material for metabolome analysis>
The separation material for metabolome analysis according to this embodiment has polymer particles and an organic group bonded to the polymer particles and containing a quaternary ammonium group. The metabolome-analyzing separation material can separate the metabolome from a sample containing the metabolome even when used under basic conditions.

The separation material for metabolome analysis includes, for example, a step of producing polymer particles (polymer particle producing step) and a step of introducing an organic group containing a quaternary ammonium group into the produced polymer particles (organic group introducing step). It can be manufactured by a method comprising.

(Polymer particles)
The polymer particles are generally considered to have excellent base resistance as compared with silica. Therefore, the separation material for metabolome analysis according to the present embodiment is considered to have excellent base resistance as compared with the separation material having ODS as a base material.

The polymer particles are not particularly limited, but since the base resistance is further improved, for example, a polymer ((meth) acrylate having a structural unit derived from a methacrylic acid ester or an acrylic acid ester ((meth) acrylic acid ester) is used. Based polymer). Examples of the (meth) acrylic acid ester include methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate, isobutyl acrylate, hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate, Dodecyl acrylate, lauryl acrylate, stearyl acrylate, 2-chloroethyl acrylate, phenyl acrylate, methyl α-chloroacrylate, methyl methacrylate, ethyl methacrylate, propyl methacrylate, n-butyl methacrylate, isobutyl methacrylate. , Monohexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, dodecyl methacrylate, lauryl methacrylate, stearyl methacrylate, etc. ) Acrylic ester, glycerol di (meth) acrylate, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, 1,3-butanediol diacrylate, 1,4-butane Diol di (meth) acrylate, 1,5-pentanediol di (meth) acrylate, 1,6-hexanediol di (meth) acrylate, 1,7-heptanediol di (meth) acrylate, 1,8-octanediol di (Meth) acrylate, 3-methyl-1,5-pentanediol di (meth) acrylate, 1,9-nonanediol di (meth) acrylate, 1,10-decanediol di (meth) acrylate, neopentyl glycol di ( (Meth) acrylate, Examples thereof include polyfunctional (meth) acrylic acid esters such as polyethylene glycol di (meth) acrylate and pentaerythritol tri (meth) acrylate. These (meth) acrylic acid esters may be used alone or in combination of two or more.

From the viewpoint of further improving the separation of metabolome, the polymer particles preferably include a polymer having a hydrophilic group such as a hydroxyl group, and the polymer particles are more preferably hydrophilic polymer particles. That is, the structural unit derived from the (meth) acrylic acid ester preferably contains a structural unit derived from the (meth) acrylic acid ester having a hydrophilic group such as a hydroxyl group.

(Polymer particle production process)
The polymer particle is not particularly limited, but it is preferably produced by using a seed polymerization method.

It is considered that the theoretical plate number of the column increases as the particle size of the polymer particles decreases. In general, polymer particles tend to form particles having a smaller particle size than silica, but polymer particles formed by a seed polymerization method easily form particles having a small particle size and have a high theoretical plate number. It tends to be easy to obtain the separating material.

The seed polymerization method can be performed according to a known method. A general method of the seed polymerization method will be described below as an example, but the seed polymerization method is not limited to this method.

The seed polymerization method includes, for example, preparing seed particles, swelling the seed particles in an emulsion containing a polymerizable monomer (after allowing the seed particles to absorb the polymerizable monomer), and then polymerizing the polymerizable monomer. It may be a method of making. That is, the polymer particles may be, for example, particles obtained by allowing the seed particles to absorb the polymerizable monomer and then polymerizing the polymerizable monomer. The polymer particle may be, for example, a polymer particle having a seed particle and a polymer having a structural unit derived from a polymerizable monomer, which is formed on the surface of the seed particle.

The seed particles can be produced by a known method such as an emulsion polymerization method, a soap-free emulsion polymerization method, or a dispersion polymerization method.

The seed particles may be particles obtained by polymerizing a monomer containing a (meth) acrylic acid ester, for example. That is, the seed particles may include a polymer having a structural unit derived from (meth) acrylic acid ester. The seed particles may be made of a polymer having a structural unit derived from (meth) acrylic acid ester.

Examples of the (meth) acrylic acid ester used for producing the seed particles include monofunctional (meth) acrylic acid esters exemplified as the (meth) acrylic acid ester of the polymer particles described above. These (meth) acrylic acid esters may be used alone or in combination of two or more.

The average particle size of the seed particles can be adjusted according to the designed particle size of the obtained polymer particles. The average particle diameter of the seed particles may be, for example, 2.0 μm or less or 1.5 μm or less because the absorption time of the polymerizable monomer can be shortened. The average particle size of the seed particles may be, for example, 0.1 μm or more or 0.5 μm or more, because it is easy to obtain seed particles that are uniform and close to a true sphere. From these viewpoints, the average particle size of the seed particles may be 0.1 to 2.0 μm, 0.5 to 2.0 μm, or 0.5 to 1.5 μm.

The coefficient of variation (CV) of the particle diameter (diameter) of the seed particles may be 10% or less or 7% or less because the homogeneity of the obtained polymer particles is difficult to decrease.

The average particle diameter and CV (coefficient of variation) of the particle diameter of the seed particles, the polymer particles, and the separating material can be determined by the following measuring method.
(1) The particles are dispersed in water using an ultrasonic dispersion device to prepare a dispersion liquid containing 1% by mass of the particles.
(2) Using a particle size distribution meter (MT-3300EX II, manufactured by Microtrac Bell Co., Ltd.), the above dispersion liquid is measured to calculate the average particle size and the CV of the particle size.

Since the interaction between the seed particles and the polymer formed from the polymerizable monomer is sufficient, the ratio of the particle diameter of the polymer particles finally obtained to the particle diameter of the seed particles is 3 to 10 times, 3 to 7 times. It is preferable to adjust so as to be four times or four to six times.

The following specifically describes an example of a method of absorbing the polymerizable monomer into the seed particles and then polymerizing the polymerizable monomer.

First, prepare an emulsion containing a polymerizable monomer and an aqueous medium, and add seed particles to the emulsion.

The emulsion can be prepared according to a known method. For example, an emulsion can be obtained by adding the polymerizable monomer to an aqueous medium and dispersing it in the aqueous medium with a fine emulsifying machine such as a homogenizer, an ultrasonic treatment machine, and a nanomizer. The emulsion may contain a polymerization initiator as the case requires. The addition of the polymerization initiator can be appropriately set depending on the type of the polymerizable monomer used. If the particle size of the polymerizable monomer droplets in the obtained emulsion is smaller than the particle size of the seed particles, the polymerizable monomer tends to be efficiently absorbed by the seed particles.

The method of adding the seed particles to the emulsion is not particularly limited, and may be a method of directly adding the seed particles to the emulsion. The seed particles are dispersed in an aqueous dispersion to give a dispersion, It may be a method of adding to the emulsion.

After adding seed particles to the emulsion, swell the seed particles to absorb the polymerizable monomer. The absorption of the polymerizable monomer can be usually performed by stirring the emulsion after adding the seed particles at room temperature (25 ° C.) for 0.5 to 24 hours. Further, by heating the emulsion to about 30 to 50 ° C., absorption of the polymerizable monomer can be promoted.

∙ Seed particles swell due to absorption of polymerizable monomers. If the mixing ratio of the polymerizable monomer to the seed particles is too small, the rate of increase in the particle size of the polymer particles formed will be small, and the productivity of the polymer particles tends to decrease. On the other hand, if the mixing ratio of the polymerizable monomer is too large, it may not be absorbed by the seed particles, and the polymerizable monomer itself may undergo suspension polymerization in an aqueous medium, and particles other than the intended particle size may be generated. is there. The absorption of the polymerizable monomer can be confirmed by, for example, observing the seed particles using an optical microscope and increasing the particle size, and it can be determined whether the absorption of the polymerizable monomer is completed or continued. it can.

The polymerizable monomer is not particularly limited as long as it has a polymerizable functional group, and examples thereof include a monofunctional (meth) acrylic acid ester and a polyfunctional (meth) acrylic acid ester exemplified as the (meth) acrylic acid ester of the polymer particles. ) Acrylic acid esters and the like can be mentioned. The polymerizable monomers may be used alone or in combination of two or more.

Examples of the monofunctional (meth) acrylic acid ester include methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate, isobutyl acrylate, hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate. , Dodecyl acrylate, lauryl acrylate, stearyl acrylate, 2-chloroethyl acrylate, phenyl acrylate, methyl α-chloroacrylate, methyl methacrylate, ethyl methacrylate, propyl methacrylate, n-butyl methacrylate, methacrylic acid Examples thereof include isobutyl, hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, dodecyl methacrylate, lauryl methacrylate and stearyl methacrylate.

Examples of polyfunctional (meth) acrylic acid esters include di (meth) acrylate compounds having two (meth) acryloyl groups such as alkanediol di (meth) acrylate.

The di (meth) acrylate compound may be, for example, a compound represented by the following formula (1).

In formula (1), R ¹ and R ² each independently represent a hydrogen atom or a methyl group, and L ¹ represents an alkylene group. The alkylene group may have, for example, 1 to 5 carbon atoms. The alkylene group may be substituted with a substituent, for example. Examples of the substituent include a hydroxyl group. Further, the alkylene group may be linear, branched or cyclic.

Examples of the di (meth) acrylate compound represented by the formula (1) include glycerol di (meth) acrylate, 1,3-butanediol diacrylate, 1,4-butanediol di (meth) acrylate, and 1,5. -Pentanediol di (meth) acrylate, 1,6-hexanediol di (meth) acrylate, 1,7-heptanediol di (meth) acrylate, 1,8-octanediol di (meth) acrylate, 3-methyl-1 Examples thereof include 1,5-pentanediol di (meth) acrylate, 1,9-nonanediol di (meth) acrylate, and 1,10-decanediol di (meth) acrylate.

Examples of the di (meth) acrylate compound include ethoxylated bisphenol A-based di (meth) acrylate, propoxylated ethoxylated bisphenol A-based di (meth) acrylate, tricyclodecane dimethanol di (meth) acrylate, 1,1,1. -Di (meth) acrylates such as trishydroxymethylethanedi (meth) acrylate, ethoxylated cyclohexanedimethanol di (meth) acrylate; (poly) ethylene glycol di (meth) acrylate, (poly) propylene glycol di (meth) acrylate It may be a (poly) alkylene glycol-based di (meth) acrylate such as (poly) tetramethylene glycol di (meth) acrylate.

Examples of the polyfunctional (meth) acrylic acid ester other than the di (meth) acrylate compound include trifunctional or higher functional (meth) acrylate. Trifunctional or higher functional (meth) acrylates include trimethylolpropane tri (meth) acrylate, tetramethylolmethane tri (meth) acrylate, tetramethylolpropane tetra (meth) acrylate, pentaerythritol tri (meth) acrylate, 1,1, Examples thereof include 1-trishydroxymethylethane tri (meth) acrylate and 1,1,1-trishydroxymethylpropane tri (meth) acrylate. Examples of commercially available trifunctional or higher functional (meth) acrylates include, for example, NK ester (A-TMPT-6P0, A-TMPT-3E0, A-TMM-3LMN, A-GLY series, manufactured by Shin-Nakamura Chemical Co., Ltd., A-9300, AD-TMP, AD-TMP-4CL, ATM-4E, A-DPH) and the like.

The polymerizable monomer may be a monofunctional (meth) acrylic acid ester and a polyfunctional (meth) acrylic acid ester, but in addition to these, other monofunctional monomers or polyfunctional monomers may be contained.

Examples of monofunctional monomers include styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, α-methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene and 2,4-dimethyl. Styrene, pn-butylstyrene, pt-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn-dodecylstyrene , P-methoxystyrene, p-phenylstyrene, p-chlorostyrene, 3,4-dichlorostyrene, and other styrenes and their derivatives; vinyl acetate, vinyl propionate, vinyl benzoate, vinyl butyrate, and other vinyl esters; N-vinyl Pyrrole, N-vinylcarbazole, N-vinylindole, N-vinylpyrrolidone, etc. N- vinyl compounds; vinyl fluoride, vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene, trifluoroethyl acrylate, fluorine-containing monomers such as acrylic acid tetrafluoropropyl; butadiene include conjugated dienes such as isoprene. These monofunctional monomers may be used alone or in combination of two or more. However, when the monofunctional monomer is a highly hydrophobic monofunctional monomer such as styrene, the content of the monofunctional monomer is such that hydrophilic polymer particles are easily obtained and hydrophobic adsorption with the analysis target component is unlikely to occur. Therefore, it is preferably 20% by mass or less based on the total mass of the polymerizable monomer.

Examples of polyfunctional monomers include divinyl compounds such as divinylbenzene, divinylbiphenyl and divinylnaphthalene; diallylphthalate and its isomers; triallyl isocyanurate and its derivatives. These polyfunctional monomers may be used alone or in combination of two or more.

The polymerizable monomer preferably contains (meth) acrylic acid ester, and includes glycerol di (meth) acrylate, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, 1, 3-butanediol diacrylate, 1,4-butanediol di (meth) acrylate, 1,5-pentanediol di (meth) acrylate, 1,6-hexanediol di (meth) acrylate, 1,7-heptanediol di (Meth) acrylate, 1,8-octanediol di (meth) acrylate, 3-methyl-1,5-pentanediol di (meth) acrylate, 1,9-nonanediol di (meth) acrylate, 1,10-decane Diol di (meth) acrylate, O neopentyl glycol di (meth) acrylate, more preferably contains polyethylene glycol di (meth) acrylate, and at least one selected from the group consisting of pentaerythritol tri (meth) acrylate.

Examples of the aqueous medium include water, a mixed medium of water and a water-soluble solvent (eg, lower alcohol), and the like. The aqueous medium may include a surfactant. As the surfactant, any of anionic, cationic, nonionic and zwitterionic surfactants can be used.

Examples of the anionic surfactant include fatty acid oils such as sodium oleate and potassium castor oil, sodium alkyl lauryl sulfate, ammonium lauryl sulfate, alkyl sulfate ester salts such as triethanolamine lauryl sulfate, and alkylbenzene sulfone such as sodium dodecylbenzenesulfonate. Acid salt, alkylnaphthalene sulfonate, alkane sulfonate, dialkyl sulfosuccinate such as sodium dioctyl sulfosuccinate, alkernyl succinate (dipotassium salt), alkyl phosphate ester salt, naphthalene sulfonic acid formalin condensate, polyoxy Ethylene alkyl phenyl ether sulfate, polyoxyethylene lauryl ether sodium sulfate, etc., polyoxyethylene alkyl ether sulfate, polyoxyethylene alky Le sulfuric acid ester salts.

Examples of cationic surfactants include alkylamine salts such as laurylamine acetate and stearylamine acetate, and quaternary ammonium salts such as lauryltrimethylammonium chloride.

Examples of the nonionic surfactant include hydrocarbon-based nonionic surfactants such as polyethylene glycol alkyl ethers, polyethylene glycol alkyl aryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, polyalkylene glycol alkylamines or amides. Agents, polyether modified silicone nonionic surfactants such as polyethylene oxide adducts of silicon and polypropylene oxide adducts, and fluorine nonionic surfactants such as perfluoroalkyl glycols.

Examples of zwitterionic surfactants include hydrocarbon surfactants such as lauryl dimethylamine oxide, phosphate ester surfactants, and phosphite ester surfactants.

The surfactants may be used alone or in combination of two or more. Among these, the anionic surfactant is preferable as the surfactant from the viewpoint of dispersion stability during polymerization of the polymerizable monomer.

Examples of the polymerization initiator added as necessary include benzoyl peroxide, lauroyl peroxide, benzoyl orthochloroperoxide, benzoyl orthomethoxyperoxide, 3,5,5-trimethylhexanoyl peroxide, and t-butylperoxide. Organic peroxides such as oxy-2-ethylhexanoate and di-t-butylperoxide; 2,2′-azobisisobutyronitrile, 1,1′-azobiscyclohexanecarbonitrile, 2,2 ′ -Azo-based compounds such as azobis (2,4-dimethylvaleronitrile) and the like can be mentioned. The content of the polymerization initiator may be 0.1 to 7.0 parts by mass with respect to 100 parts by mass of the total amount of polymerizable monomers.

Next, monodisperse polymer particles can be obtained by polymerizing the polymerizable monomer absorbed by the seed particles.

The polymerization temperature can be appropriately set according to the types of the polymerizable monomer and the polymerization initiator. The polymerization temperature may be 25-110 ° C or 50-100 ° C. The polymerization reaction is preferably carried out by raising the temperature after the seed particles have sufficiently swelled and the polymerizable monomer and any polymerization initiator have been sufficiently absorbed.

The polymer particles can be isolated by removing the aqueous medium from the polymerization solution by centrifugation or filtration as needed, washing with water and a solvent, and then drying.

In the above polymerization step, a polymer dispersion stabilizer may be added to the emulsion from the viewpoint of improving the dispersion stability of the seed particles.

Examples of the polymer dispersion stabilizer include polyvinyl alcohol, polycarboxylic acid, celluloses (hydroxyethyl cellulose, carboxymethyl cellulose, etc.), polyvinylpyrrolidone and the like. The polymer dispersion stabilizer may be used in combination with an inorganic water-soluble polymer compound such as sodium tripolyphosphate. Among these, the polymer dispersion stabilizer preferably contains polyvinyl alcohol or polyvinylpyrrolidone. The content of the polymer dispersion stabilizer may be 1 to 10 parts by mass based on 100 parts by mass of the total amount of the polymerizable monomers.

Prohibition of water-soluble polymerization of nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble vitamin Bs, citric acid, polyphenols, etc. in order to suppress emulsion polymerization of the polymerizable monomer itself in water Agents may be used.

Since the polymer particles have further improved base resistance, seed particles having a structural unit derived from methyl methacrylate and structural units derived from glycerol di (meth) acrylate formed on the surface of the seed particles are formed. And a polymer particle having.

The average particle size of the polymer particles may be, for example, 6 μm or less, 5 μm or less, or 4 μm or less because a high theoretical plate number is easily obtained.

The coefficient of variation of the particle diameter (diameter) of the polymer particles may be, for example, 25% or less, 20% or less, 15% or less, or 10% or less because a high theoretical plate number is easily obtained.

The polymer particles may be particles having a porous structure (porous polymer particles), for example.

(Organic group introduction step)
Then, an organic group containing a quaternary ammonium group is introduced into the produced polymer particles. When the polymer particles are produced using a seed polymerization method, organic groups containing quaternary ammonium groups can be introduced into the polymer having structural units derived from polymerizable monomers. The organic group containing a quaternary ammonium group may further contain an ionic group such as a sulfonic acid group or a phosphoric acid group. The organic group containing a quaternary ammonium group may be an alkyl group containing a quaternary ammonium group or an alkoxy group containing a quaternary ammonium group, and these alkyl groups or alkoxy groups may be a phosphoric acid group or a sulfonic acid group. It may further have an ionic group such as.

The method for introducing an organic group containing a quaternary ammonium group into polymer particles is not particularly limited, but for example, a polymer having an epoxy group by introducing an epoxy group by reacting a polymer particle with a compound having an epoxy group is introduced. A method of reacting particles with a tertiary amine, a method of reacting a polymer particle with a compound having an oxodioxophosphoryl group to introduce an oxodioxophosphoryl group, and a polymer particle having an oxodioxophosphoryl group and a tertiary Method of reacting with amine, method of reacting polymer particles having halogenated alkyl group obtained by copolymerizing (meth) acrylate having halogenated alkyl group such as 2-chloroethyl (meth) acrylate with tertiary amine , A secondary amino group such as 2-dimethylaminoethyl (meth) acrylate To (meth) reacting an alkyl halide such as methyl iodide in the polymer particles having a tertiary amino group obtained acrylates by copolymerization, and the like. Here, the organic group includes a compound having an epoxy group that reacts with polymer particles, a compound having an oxodioxophosphoryl group, a (meth) acrylate having a halogenated alkyl group, a (meth) acrylate having a tertiary amino group, and the like. Can be a group derived from

Examples of the method of introducing an epoxy group include a method of reacting a polymer particle having a hydroxyl group or the like with a halogen group-containing glycidyl compound such as epichlorohydrin.

Examples of the tertiary amine reacted with the epoxy group include trimethylamine, triethylamine, branched polyethyleneimine, and the like. Examples of commercially available polyethyleneimine include polyethyleneimine (weight average molecular weight 600) and polyethyleneimine (weight average molecular weight 1800) (trade name, manufactured by Wako Pure Chemical Industries, Ltd.). The tertiary amine may further have an ionic group such as a sulfonic acid group and a phosphoric acid group. For example, a separating material having an organic group containing a quaternary ammonium group and a sulfonic acid group can be obtained by reacting polymer particles having an epoxy group with a tertiary amine having a sulfonic acid group.

After the reaction with the tertiary amine, for example, a treatment such as sulfuric acid washing may be performed to open the unreacted epoxy group.

As a method for introducing an oxodioxophosphoryl group, for example, a polymer particle having a hydroxyl group or the like is reacted with a halogen group-containing dioxaphosphorane compound such as 2-chloro-oxo-1,3,2-dioxaphosphorane. The method of making it include.

Examples of the tertiary amine that reacts with the oxodioxophosphoryl group include the same ones as exemplified for the tertiary amine that reacts with the epoxy group. By reacting the polymer particles having an oxodioxophosphoryl group with a tertiary amine, a separating material having an organic group containing a quaternary ammonium group and a phosphoric acid group can be obtained.

The separation material for metabolome analysis thus obtained has polymer particles and an organic group containing a quaternary ammonium group, which is bonded to the polymer particles. The organic group may further contain an ionic group such as a sulfonic acid group and a phosphoric acid group in addition to the quaternary ammonium group, but it has high separability for metabolome and has a sharper chromatogram. From the viewpoint of obtaining a peak, it is preferable that no ionic group other than the quaternary ammonium group is contained.

The average particle size of the separation material for metabolome analysis may be, for example, 2 to 5 μm or less because a high theoretical plate number is easily obtained. The average particle size of the separating material may be 2.5 μm or more, 3 μm or more, or 3.2 μm or more, and may be 4.5 μm or less, 4 μm or less, or 3.8 μm or less.

The coefficient of variation of the particle size (diameter) of the separation material for metabolome analysis may be, for example, 25% or less, 20% or less, 15% or less, or 10% or less because a high theoretical plate number is easily obtained.

The quaternary ammonium group content (neutral salt decomposition capacity) of the separation material for metabolome analysis may be 10 to 500 μeq / g because it shows higher separability for ionic compounds. The amount of the quaternary ammonium group (neutral salt decomposition capacity) of the separating material may be 10 μeq / g or more, 30 μeq / g or more, or 60 μeq / g or more, and 500 μeq / g or less, 300 μeq / g or less, or It may be 100 μeq / g or less.

The separation material for metabolome analysis is a separation material for separating the metabolome from the sample containing the metabolome.

The metabolome may be at least one selected from the group consisting of amino acids, nucleobases, nucleosides, nucleotides, and organic acids.

Examples of amino acids include glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), cysteine (Cys), valine (Val), isoleucine (Ile), methionine (Met), proline (Pro). , Phenylalanine (Phe), tyrosine, tryptophan (Trp), aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), glutamine (Gln), histidine (His), lysine (Lys), arginine (Arg), glutathione. (GSH), and at least one selected from the group consisting of acidic glutathione (GSSG). The amino acid is more preferably alanine (Ala), threonine (Thr), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), proline (Pro), phenylalanine (Phe), tyrosine (Tyr). , Tryptophan (Trp), aspartic acid (Asp), asparagine (Asn), and arginine (Arg).

Examples of the nucleobase include at least one selected from the group consisting of adenine, guanine, thymine, uracil, and cytosine.

Examples of the nucleoside include at least one selected from the group consisting of adenosine (Adenosine), guanosine (Guanosine), thymidine (Thymidine), uridine (Uridine), and cytidine (Cytidine).

Examples of nucleotides include adenosine-1-phosphate (AMP), guanosine-1-phosphate (GMP), thymidine-1-phosphate (TMP), uridine-1-phosphate (UMP), and cytidine-1. -At least one selected from the group consisting of phosphoric acid (CMP).

Examples of the organic acid include at least one selected from the group consisting of citric acid (Cit), isocitric acid (IsoCit), fumaric acid (Fum), maleic acid (Mal), and succinic acid (Suc).

The separation material for metabolome analysis according to this embodiment can be suitably used as a packing material for column chromatography (for example, liquid chromatography).

<Column for metabolome analysis>
The metabolome analysis column according to the present embodiment includes the above-described metabolome analysis separation material. The column for metabolome analysis can be manufactured, for example, by packing the separating material for metabolome analysis. The method of filling the column with the separating material is not particularly limited, and a known method can be appropriately adopted. The metabolome analysis column can be preferably used even under basic conditions.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[Preparation of separation material for metabolome analysis]
(Example 1: Separation material having an organic group containing a quaternary ammonium group)
<Preparation of seed particles>
To a 500 mL separable flask, 70 g of methyl methacrylate, 2.1 g of octanethiol, and 370 g of ion-exchanged water were added, and while bubbling with nitrogen, the temperature was kept at 30 ° C. for 1 hour while stirring with a stirring blade. Then, 0.875 g of potassium peroxodisulfate and 30 g of ion-exchanged water were added, and the mixture was reacted at 70 ° C. for 6 hours to obtain a reaction liquid containing seed particles. After cooling the obtained reaction liquid, the lumps and fine particles in the reaction liquid were removed to obtain a slurry of seed particles (solid content concentration: 3.5 mass%). Here, the lumps were removed using a sieve having openings of 150 μm. Further, the fine particles were removed by treating the reaction liquid (slurry that passed through the sieve) after removing the lumps with a centrifugal dehydrator and discarding the supernatant liquid by decantation. The average particle size and the CV (coefficient of variation) of the particle size of the seed particles in the obtained slurry are calculated by measuring the particle size distribution with a particle size distribution measuring device (Microtrac Bell Co., Ltd. (MT-3300EX II)). The average particle size of the seed particles was 750 nm, and the CV was 6.4%.

<Preparation of polymer particles>
To a 3 L separable flask, 80.6 g of glycerol di (meth) acrylate, 72.5 g of butyl acetate, and 48.4 g of isoamyl alcohol were added, and 0.4 g of 2,2′-azobisisobutyronitrile was added. And dissolved. Next, to the separable flask, 11.6 g of an aqueous solution containing 40% by mass of triethanolamine lauryl sulfate and 1529.7 g of ion-exchanged water were further added, and ultrasonically dispersed with an ultrasonic horn for 10 minutes to obtain an emulsion. To the obtained emulsion, 14.0 g of the slurry of seed particles prepared above and 122 g of ion-exchanged water were added with stirring with a stirring blade, and the mixture was kept at 30 ° C. (temperature inside the flask) for 1 hour to obtain seed particles. Glycerol di (meth) acrylate was absorbed into. Next, 121 g of an aqueous polyvinyl alcohol solution (polyvinyl alcohol concentration: 6% by mass) was added, polymerization was carried out at 78 ° C. (temperature inside the flask) for 5 hours while bubbling with nitrogen, and the mixture was cooled. The particles produced by the polymerization were washed with ion-exchanged water, an ion-exchanged water / methanol mixed solution, and methanol in this order, and then wet-classified with a sieve having openings of 5 μm to remove aggregates. Polymer particles were obtained by filtering and drying particles generated by polymerization from the slurry after removal of aggregates. The average particle size and the CV (coefficient of variation) of the particle size of the obtained polymer particles were calculated by measuring the particle size distribution with a particle size distribution measuring device (MT-3300EX II, manufactured by Microtrac Bell Co., Ltd.). The average particle size of the particles was 3.5 μm, and the CV was 6.8%.

<Preparation of polymer particles having an epoxy group>
In a 300 mL three-necked flask, 15 g of dried polymer particles, 112.5 g of ion-exchanged water, 22.5 g of chloromethyloxirane, and 8.8 g of sodium hydroxide aqueous solution (sodium hydroxide concentration: 30% by mass) are ultrasonically dispersed for 1 minute. Then, the mixture was reacted at 30 ° C. for 1 hour while stirring with a stirring blade. The polymer particles after the reaction were separated by filtration and washed with ion-exchanged water and methanol in this order to obtain polymer particles having an epoxy group.

<Preparation of separation material for metabolome analysis>
To a 2 L separable flask, the total amount of the epoxy group-containing polymer particles prepared above, 300 g of ion-exchanged water, and 750 g of branched polyethyleneimine (weight average molecular weight 1800, manufactured by Wako Pure Chemical Industries, Ltd.) were added, and stirring blades were added. The reaction was carried out at 30 ° C. (temperature inside the flask) for 3 hours while stirring at. After the reaction, the polymer particles are separated by filtration and washed with ion-exchanged water and methanol in this order, and then the polymer particles after the reaction, 300 g of ion-exchanged water, and 1.2 g of a sulfuric acid aqueous solution (sulfuric acid concentration: 47 mass%) are added. While stirring with a stirring blade, washing was performed at 40 ° C. (temperature inside the flask) for 3 hours. The polymer particles after washing were separated by filtration, washed with ion-exchanged water and methanol in this order, and the polymer particles were dried to obtain a separation material having an organic group containing a quaternary ammonium group. The average particle size of the obtained separation material was calculated by measuring the particle size distribution with a particle size distribution analyzer (MT-3300EX II, manufactured by Microtrac Bell Co., Ltd.) The average particle size was 3.58 μm. ..

<Measurement of ion exchange capacity>
The ion exchange capacity of the obtained separation material was measured by the following method. Into a 100 mL beaker, 1 g of the separating material was weighed, 1 mol / L sodium hydroxide aqueous solution was added and stirred, and the separating material was filtered by suction filtration with a funnel. The particles separated by filtration were dispersed in ion-exchanged water, filtered by suction filtration with a funnel, and the separation material was washed with ion-exchanged water until the filtrate became neutral. The washed separation material was transferred to a 100 mL beaker, 0.1 mol / L hydrochloric acid aqueous solution was added and stirred, and the separation material was filtered by suction filtration with a funnel. The particles separated by filtration were dispersed in ion-exchanged water, filtered by suction filtration with a funnel, and the separation material was washed with ion-exchanged water until the filtrate became neutral. The filtrate (containing the ion-exchanged water used for washing) obtained at this time was transferred to a 500 mL beaker and titrated with a 0.1 mol / L sodium hydroxide aqueous solution to measure the ion-exchange capacity. The ion exchange capacity of the separating material was 1.7 meq / g.

<Measurement of quaternary ammonium group content (neutral salt decomposition capacity)>
The amount of quaternary ammonium group (neutral salt decomposition capacity) of the obtained separating material was measured by the following method. Into a 100 mL beaker, 1 g of the separating material was weighed, 1 mol / L sodium hydroxide aqueous solution was added and stirred, and the separating material was filtered by suction filtration with a funnel. The particles separated by filtration were dispersed in ion-exchanged water, filtered by suction filtration with a funnel, and the separation material was washed with ion-exchanged water until the filtrate became neutral. The washed separation material was transferred to a 100 mL beaker, 1 mol / L sodium chloride aqueous solution was added and stirred, and the separation material was filtered by suction filtration with a funnel. The particles separated by filtration were dispersed in ion-exchanged water, filtered by suction filtration with a funnel, and the separation material was washed with ion-exchanged water until the filtrate became neutral. The filtrate (including ion-exchanged water used for washing) obtained at this time was transferred to a 500 mL beaker and titrated with a 0.1 mol / L hydrochloric acid aqueous solution to determine the amount of quaternary ammonium group (neutral salt decomposition capacity). ) Was measured. The amount of quaternary ammonium groups (neutral salt decomposition capacity) of the separating material was 70 μeq / g.

(Example 2: Separation material having an organic group containing a quaternary ammonium group and a sulfonic acid group)
<Synthesis of 2-dimethylaminoethanesulfonic acid (DMAES)>
To a 250 mL flask, 10.9 g of a sodium salt of 2-ethylsulfonic acid 2-bromide was added, and 100 mL of water was added and dissolved therein. Further, 12.4 g of dimethylamine was added, the mixture was allowed to stand at room temperature for 45 minutes, and the resulting mixed liquid was reacted under heating at reflux at 70 to 80 ° C. for 18 hours. After the reaction, the reaction solution was cooled to 40 ° C., about 2 g of activated carbon was added, and then boiled for 15 minutes. Then, the reaction solution was cooled to room temperature, activated carbon was precipitated, and the supernatant of the reaction solution was filtered by suction filtration. DMAES was obtained by recrystallizing twice with water / ethanol and drying in a vacuum oven at 50 ° C. for 24 hours.

<Preparation of separation material for metabolome analysis>
To a 500 mL separable flask, the total amount of the polymer particles having an epoxy group produced under the conditions of Example 1, 16 g of DMAES, and 200 mL of a 0.2 mM phosphate buffer solution (pH 8) were added, and the internal temperature was 50 ° C. while stirring with a stirring blade. And reacted for 90 hours. After the reaction, the polymer particles are separated by filtration and washed with ion-exchanged water and methanol in this order, and then the polymer particles after the reaction, 300 g of ion-exchanged water, and 1.2 g of a sulfuric acid aqueous solution (sulfuric acid concentration: 47 mass%) are added. While stirring with a stirring blade, washing was performed at 40 ° C. (temperature inside the flask) for 3 hours. After washing, the polymer particles were filtered, washed with ion-exchanged water and methanol in that order, and dried to obtain a separating material having an organic group containing a quaternary ammonium group and a sulfonic acid group. The average particle size of the obtained separation material was calculated by measuring the particle size distribution with a particle size distribution analyzer (MT-3300EX II, manufactured by Microtrac Bell Co., Ltd.) The average particle size was 3.6 μm. When the amount of quaternary ammonium groups was measured by the same method as in Example 1, the amount of quaternary ammonium groups (neutral salt decomposition capacity) of the separating material was 50 μeq / g. The amount of sulfonic acid group determined from the amount was 50 μeq / g.

(Example 3: Separation material having organic group containing quaternary ammonium group and phosphoric acid group)
<Preparation of separation material for metabolome analysis>
To a 500 mL three-necked flask, 15 g of the polymer particles produced under the conditions of Example 1 and 200 mL of dry tetrahydrofuran were added, and the mixture was cooled to −5 ° C. under a nitrogen atmosphere while stirring. While stirring, 100 g of 2-chloro-oxo-1,3,2-dioxaphospholane was added dropwise under a nitrogen atmosphere over 1 hour, and the mixture was reacted at 5 ° C. for 48 hours. The solvent was distilled off under reduced pressure. 70 g of triethylamine and 200 mL of tetrahydrofuran were mixed to prepare a tetrahydrofuran solution of triethylamine, and the residue after distillation was added without taking time, and the mixture was reacted at 40 ° C. for 24 hours. The polymer particles after the reaction were filtered off and washed with ion-exchanged water and methanol in this order to obtain a separation material having an organic group containing a quaternary ammonium group and a phosphoric acid group. The average particle size of the obtained separation material was calculated by measuring the particle size distribution with a particle size distribution analyzer (MT-3300EX II, manufactured by Microtrac Bell Co., Ltd.) The average particle size was 3.6 μm. When the amount of quaternary ammonium groups was measured by the same method as in Example 1, the amount of quaternary ammonium groups (decomposition capacity of neutral salt) of the separating material was 40 μeq / g. The amount of phosphate group determined from the amount was 40 μeq / g.

[Column evaluation]
<Preparation of metabolome analysis column (packing of metabolome analysis separation material)>
A column was prepared using the separating materials of Examples 1 to 3. A filling slurry was prepared by adding 0.8 g of the separating material and a mixed solution of ultrapure water and acetonitrile at a ratio of 25:75 (volume ratio) to a 100 mL beaker and dispersing and mixing while applying ultrasonic waves. Then, the slurry is poured into a stainless steel packer equipped with a 2.1 mmφ × 150 mm stainless steel column (manufactured by Sugiyama Shoji Co., Ltd.) to seal it, and a plunger type filling pump (uniflows, uf-20020SZWP2 pump) pressurizes. Filled. After the filling, a 0.1 mol / L sodium hydroxide aqueous solution was passed through at 0.1 mL / min for 3 hours to replace the inside of the column with a basic solution. After passing an aqueous solution of sodium hydroxide, a mixture of ultrapure water and acetonitrile at a ratio of 25:75 (volume ratio) was passed through to wash off excess basic components (aqueous sodium hydroxide solution) to form a column. did.

<Column evaluation>
By using the produced columns of Examples 1 to 3 and attaching them to liquid chromatography (HPLC), column evaluation was performed by separation of metabolome from a sample containing metabolome under acidic conditions, neutral conditions, and basic conditions. It was

(HPLC conditions (acidic))
Detection: LC / MS / MS (Nexera UHPLC / HPLC System ultra-high speed triple quadrupole LC / MS / MS system LCMS-8060 manufactured by Shimadzu Corporation)
Mobile phase A: 95% by volume of 5 mM ammonium bicarbonate aqueous solution-5% by volume of acetonitrile (adjusted to pH 3.6 with acetic acid)
Mobile phase B: 5 mM aqueous solution of 5 mM ammonium bicarbonate-95% by volume of acetonitrile
Flow rate: 0-15 minutes, 0.25 mL / min, 25-35 minutes, 0.40 mL / min Gradient conditions: Figure 1

(HPLC conditions (neutral))
Temperature: 40 ° C
Detection: LC / MS / MS (Nexera UHPLC / HPLC System ultra-high speed triple quadrupole LC / MS / MS system LCMS-8060 manufactured by Shimadzu Corporation)
Mobile phase A: 95% by volume of 5 mM ammonium bicarbonate aqueous solution-5% by volume of acetonitrile (adjusted to pH 6.8 with acetic acid)
Mobile phase B: 5 mM aqueous solution of 5 mM ammonium bicarbonate-95% by volume of acetonitrile
Flow rate: 0-15 minutes, 0.25 mL / min, 25-35 minutes, 0.40 mL / min Gradient conditions: Figure 1

(HPLC conditions (basic))
Temperature: 40 ° C
Detection: LC / MS / MS (Nexera UHPLC / HPLC System ultra-high speed triple quadrupole LC / MS / MS system LCMS-8060 manufactured by Shimadzu Corporation)
Mobile phase A: 95% by volume of 5 mM ammonium bicarbonate aqueous solution-5% by volume of acetonitrile (adjusted to pH 9.8 with ammonia)
Mobile phase B: 5 mM aqueous solution of 5 mM ammonium bicarbonate-95% by volume of acetonitrile
Flow rate: 0-15 minutes, 0.25 mL / min, 25-35 minutes, 0.40 mL / min Gradient conditions: Figure 1

(Preparation of sample)
One kind of the following metabolome was mixed with ultrapure water to prepare 28 kinds of samples containing a metabolome of 10 μM (valine and adenosine were 1 μM).

1. Amino Acids Alanine (Ala), Threonine (Thr), Valine (Val), Leucine (Leu), Isoleucine (Ile), Methionine (Met), Proline (Pro), Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp) , Aspartic acid (Asp), asparagine (Asn), arginine (Arg)

2. Nucleotide bases Adenine, Guanine, Thymine, Uracil, Cytosine

3. Nucleoside Adenosine (Adenosine), Guanosine (Guanosine), Thymidine (Thymidine), Uridine (Uridine), Cytidine (Cytidine)

4. Nucleotides adenosine-1-phosphate (AMP), guanosine-1-phosphate (GMP), uridine-1-phosphate (UMP), cytidine-1-phosphate (CMP)

5. Organic acid fumaric acid (Fum)

Regarding nucleotides and organic acids, only HPLC conditions (basic) were examined.

(Evaluation criteria)
The chromatogram obtained for each metabolome was scored according to the following criteria. A result of +1 or more was evaluated as good.
(1) Hold in void volume range (1.88 or 2.06 minutes) +1 point (2) Hold for 5 minutes or more +1 point (3) Peak width is 1 minute or less +1 point (4) Peak Width is 3 minutes or more and less than 5 minutes -1 point (5) Peak width is 5 minutes or more -1 point

FIG. 2 is a chromatogram score table showing the results of column evaluation of the examples. As shown in FIG. 2, the metabolome analysis column of the Example showed good results even when used under basic conditions.

FIG. 3 (a) is a chromatogram of adenine under the basic conditions when using the column of Example 1, and FIG. 3 (b) is under the basic conditions when using the column of Example 2. Is a chromatogram of adenine of. FIG. 4 (a) is a chromatogram of adenosine under the basic conditions when using the column of Example 1, and FIG. 4 (b) is under the basic conditions when using the column of Example 2. Is a chromatogram of adenosine. As shown in FIGS. 3 and 4, the column of Example 1 was found to give a sharper peak than the column of Example 2.

As shown by these results, it was confirmed that the separation material for metabolome analysis of the present invention can separate metabolomes even when used under basic conditions.

Claims

Polymer particles,
An organic group containing a quaternary ammonium group bonded to the polymer particles,
A separating material for metabolome analysis, comprising:
The separation material for metabolome analysis according to claim 1, wherein the polymer particles include a polymer having a structural unit derived from (meth) acrylic acid ester.
The separation material for metabolome analysis according to claim 1 or 2, wherein the average particle diameter of the separation material is 2 to 5 µm.
The separation material for metabolome analysis according to any one of claims 1 to 3, wherein the separation material has a quaternary ammonium group amount of 10 to 500 µeq / g.
A metabolome analysis column comprising the metabolome analysis separation material according to any one of claims 1 to 4.