JP7341434B2 - Separation materials for metabolome analysis and columns for metabolome analysis - Google Patents

Description

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

Metabolomics is a research field that is applied to disease diagnosis, etc., and aims to comprehensively analyze the metabolome, which is the total body of cellular metabolites, and to understand biological phenomena. Gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), capillary electrophoresis/mass spectrometry (CE/MS), etc. are used as metabolome analysis methods. (See Non-Patent Document 1).

GC/MS is suitable for measuring volatile metabolites such as aromatic compounds. However, when measuring nonvolatile metabolites, derivatization is necessary, which may cause problems in quantitative performance. Furthermore, since many metabolites are nonvolatile, measurement of volatile substances, which is the greatest advantage of GC, tends to be limited to organic acids and the like.

LC/MS is capable of measuring metabolites of a wide range of chemical substances and is frequently used in metabolome research. However, there are problems such as the complexity of solvent selection and the lack of compatibility with MS in ionic metabolite analysis.

CE/MS is a useful analytical method because most metabolic intermediates included in amino acid, nucleic acid metabolism, etc. have ionicity. However, compared to GC/MS and LC/MS, there are problems such as inferior concentration sensitivity and difficulty in handling and use by users.

In recent years, the measurement of metabolome components has been studied in separation methods such as ion exchange in LC methods, 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 too much organic solvent, the separation material will swell and contract, damaging the column and making it impossible to confirm measurement reproducibility. These are concerns as issues.

Japanese Patent Application Publication No. 2015-129775

Akiyoshi Hirayama, Jiyasu Izumi, Fumio Matsuda, Takamasa Ishikawa, Yuki Sugiura, Takashi Suzuki, Hydrophilic Metabolite Analysis in Metabolomics, Journal of the Mass Spectrometry Society of Japan, Vol. 65, No5, 2017

Many of the biological components targeted by metabolomic analysis are highly hydrophilic, and it tends to be difficult to retain these components with ODS columns (octadecyl silica columns) commonly used in LC methods. Such a tendency is particularly noticeable under basic conditions.

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

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

[1] A separation material for metabolomic analysis, comprising polymer particles and an organic group containing a quaternary ammonium group bonded to the polymer particles.
[2] The separation material for metabolome analysis according to [1], wherein the polymer particles include 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 one of [1] to [3], wherein the separation material has a quaternary ammonium group content of 10 to 500 μeq/g.
[5] A column for metabolome analysis, comprising the separation material for metabolome analysis according to any one of [1] to [4].

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

It is a graph showing HPLC gradient conditions in column evaluation of Examples. 2 is a chromatogram score table showing the results of column evaluation in Examples. (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. 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. 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 addition, in this specification, "(meth)acrylic acid" means "acrylic acid" or the corresponding "methacrylic acid", and the same applies to other similar descriptions such as "(meth)acrylate". It is.

<Separation material for metabolome analysis>
The separation material for metabolome analysis according to this embodiment has polymer particles and an organic group containing a quaternary ammonium group bonded to the polymer particles. Even when the separation material for metabolome analysis is used under basic conditions, it is possible to separate the metabolome from a sample containing the metabolome.

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

(polymer particles)
Polymer particles are generally considered to have superior base resistance compared to silica. Therefore, it is considered that the separation material for metabolome analysis according to the present embodiment has excellent base resistance compared to a separation material using ODS as a base material.

Although the polymer particles are not particularly limited, for example, polymers having structural units derived from methacrylic esters or acrylic esters ((meth)acrylic esters) can further improve base resistance. polymers). Examples of the (meth)acrylate 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 , monofunctional (meth)acrylic acid esters having a linear or branched alkyl group such as hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, dodecyl methacrylate, lauryl methacrylate, and stearyl methacrylate; 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 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, polyethylene Examples include polyfunctional (meth)acrylic acid esters such as glycol di(meth)acrylate and pentaerythritol tri(meth)acrylate. These (meth)acrylic esters may be used alone or in combination of two or more.

The polymer particles preferably contain a polymer having a hydrophilic group such as a hydroxyl group, and more preferably the polymer particles are hydrophilic polymer particles, from the viewpoint of further improving the separation of metabolomes. That is, the structural unit derived from a (meth)acrylic ester preferably includes a structural unit derived from a (meth)acrylic ester having a hydrophilic group such as a hydroxyl group.

(Polymer particle production process)
Although the polymer particles are not particularly limited, it is preferable to manufacture them using a seed polymerization method.

It is considered that the number of theoretical plates in a column increases as the particle diameter of the polymer particles becomes smaller. In general, polymer particles tend to have a tendency to form particles with a smaller particle size than silica, but polymer particles formed by seed polymerization tend to form particles with a small particle size and have a high theoretical plate number. separation material tends to be easily obtained.

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

In the seed polymerization method, for example, seed particles are prepared, the seed particles are swollen in an emulsion containing a polymerizable monomer (after the seed particles absorb the polymerizable monomer), and then the polymerizable monomer is polymerized. It may be a method of That is, the polymer particles may be particles obtained by, for example, making seed particles absorb a polymerizable monomer and then polymerizing the polymerizable monomer. The polymer particles may be, for example, polymer particles having a seed particle and a polymer having a structural unit derived from a polymerizable monomer formed on the surface of the seed particle.

Seed particles can be produced, for example, 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 contain 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 a (meth)acrylic acid ester.

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

The average particle diameter of the seed particles can be adjusted depending on the designed particle diameter of the resulting 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, since the absorption time of the polymerizable monomer can be shortened. The average particle diameter of the seed particles may be, for example, 0.1 μm or more or 0.5 μm or more, since uniform and nearly perfectly spherical seed particles can be easily obtained. From these viewpoints, the average particle diameter 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 size (diameter) of the seed particles may be 10% or less or 7% or less, since the uniformity of the obtained polymer particles is unlikely to deteriorate.

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

Since the interaction between the seed particles and the polymer formed from the polymerizable monomer is sufficient, the ratio of the particle size of the finally obtained polymer particles to the particle size of the seed particles is 3 to 10 times, 3 to 7 times. It is preferable to adjust the amount to 4 times or 4 to 6 times.

Hereinafter, an example of a method for absorbing a polymerizable monomer into seed particles and then polymerizing the polymerizable monomer will be specifically described.

First, an emulsion containing a polymerizable monomer and an aqueous medium is prepared, and seed particles are added to the emulsion.

An emulsion can be produced according to a known method. For example, an emulsion can be obtained by adding a polymerizable monomer to an aqueous medium and dispersing it in the aqueous medium using a microemulsifier such as a homogenizer, an ultrasonicator, or a nanomizer. The emulsion may contain a polymerization initiator if necessary. The addition of the polymerization initiator can be appropriately determined depending on the type of polymerizable monomer used. When 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 easily absorbed into the seed particles efficiently.

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, or a method of dispersing the seed particles in an aqueous dispersion to form a dispersion, and then adding the seed particles to the emulsion. It may also be added to an emulsion.

After adding the seed particles to the emulsion, the seed particles are allowed to swell and absorb the polymerizable monomer. Absorption of the polymerizable monomer can usually be carried out by stirring the emulsion after adding the seed particles at room temperature (25° C.) for 0.5 to 24 hours. Furthermore, absorption of the polymerizable monomer can be promoted by heating the emulsion to about 30 to 50°C.

The seed particles swell due to absorption of the polymerizable monomer. If the mixing ratio of the polymerizable monomer to the seed particles is too small, the rate of increase in the particle diameter of the formed polymer particles will be small, and the productivity of the polymer particles will tend to decrease. On the other hand, if the mixing ratio of the polymerizable monomer becomes too large, the polymerizable monomer itself will be suspended and polymerized in the aqueous medium without being absorbed by the seed particles, and particles with a particle size other than the desired size may be generated. be. In addition, the absorption of the polymerizable monomer can be confirmed by observing the seed particles using an optical microscope and observing the enlargement of the particle size, and it is possible to determine whether the absorption of the polymerizable monomer is to be terminated or continued. can.

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

Examples of monofunctional (meth)acrylic esters include methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate, isobutyl acrylate, hexyl acrylate, 2-ethylhexyl acrylate, and 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 include isobutyl, hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, dodecyl methacrylate, lauryl methacrylate, and stearyl methacrylate.

Examples of the polyfunctional (meth)acrylic ester 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 hydroxyl group. Further, the alkylene group may be linear, branched, or cyclic.

Examples of the di(meth)acrylate compound represented by formula (1) include glycerol 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-decanediol di(meth)acrylate and the like.

Di(meth)acrylate compounds include, for example, 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 trishydroxymethylethane di(meth)acrylate, ethoxylated cyclohexanedimethanol di(meth)acrylate; (poly)ethylene glycol di(meth)acrylate, (poly)propylene glycol di(meth)acrylate , (poly)alkylene glycol di(meth)acrylate such as (poly)tetramethylene glycol di(meth)acrylate.

Examples of polyfunctional (meth)acrylic esters other than di(meth)acrylate compounds include trifunctional or higher functional (meth)acrylates. Examples of 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 include 1-trishydroxymethylethane tri(meth)acrylate, 1,1,1-trishydroxymethylpropane tri(meth)acrylate, and the like. Commercially available trifunctional or higher functional (meth)acrylates include, for example, NK esters (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), etc.

The polymerizable monomer may be a monofunctional (meth)acrylic ester or a polyfunctional (meth)acrylic ester, but may also contain other monofunctional monomers or polyfunctional monomers.

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, pn-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 esters such as vinyl acetate, vinyl propionate, vinyl benzoate, and vinyl butyrate; N-vinyl N-vinyl compounds such as pyrrole, N-vinylcarbazole, N-vinylindole, N-vinylpyrrolidone; vinyl fluoride, vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene, trifluoroethyl acrylate, tetrafluoropropyl acrylate and conjugated dienes such as butadiene and 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 target component is difficult to occur. Therefore, it is preferably 20% by mass or less based on the total mass of the polymerizable monomer.

Examples of the polyfunctional monomer include divinyl compounds such as divinylbenzene, divinylbiphenyl, and divinylnaphthalene; diallyl phthalate 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, such as 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 (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 More preferably, it contains at least one selected from the group consisting of diol di(meth)acrylate, neopentyl glycol di(meth)acrylate, polyethylene glycol di(meth)acrylate, and 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 contain a surfactant. As the surfactant, any one of anionic, cationic, nonionic, and amphoteric surfactants can be used.

Examples of anionic surfactants include fatty acid oils such as sodium oleate and potassium castor oil, alkyl sulfate ester salts such as sodium lauryl sulfate, ammonium lauryl sulfate, and triethanolamine lauryl sulfate, and alkylbenzene sulfones such as sodium dodecylbenzenesulfonate. acid salts, alkylnaphthalene sulfonates, alkanesulfonates, dialkyl sulfosuccinates such as sodium dioctyl sulfosuccinate, alkenyl succinates (dipotassium salts), alkyl phosphate ester salts, naphthalene sulfonic acid formalin condensates, polyoxy Examples include polyoxyethylene alkyl ether sulfates such as ethylene alkyl phenyl ether sulfate, polyoxyethylene lauryl ether sodium sulfate, and polyoxyethylene alkyl sulfate.

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

Examples of nonionic surfactants include hydrocarbon nonionic surfactants such as polyethylene glycol alkyl ethers, polyethylene glycol alkylaryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, polyalkylene glycol alkyl amines, or amides. Examples include polyether-modified silicone-based nonionic surfactants such as polyethylene oxide adducts of silicone and polypropylene oxide adducts, and fluorine-based nonionic surfactants such as perfluoroalkyl glycols.

Examples of the amphoteric surfactant 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 surfactant is preferably an anionic 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, orthochlorobenzoyl peroxide, orthomethoxybenzoyl peroxide, 3,5,5-trimethylhexanoyl peroxide, and t-butyl peroxide. Organic peroxides such as oxy-2-ethylhexanoate and di-t-butyl peroxide; 2,2'-azobisisobutyronitrile, 1,1'-azobiscyclohexanecarbonitrile, 2,2' Examples include azo compounds such as -azobis(2,4-dimethylvaleronitrile). The content of the polymerization initiator may be 0.1 to 7.0 parts by weight based on 100 parts by weight of the total amount of polymerizable monomers.

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

The polymerization temperature can be appropriately set depending on the types of polymerizable monomer and 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 swollen and the polymerizable monomer and any polymerization initiator have been sufficiently absorbed.

Polymer particles can be isolated by removing the aqueous medium from the polymerization solution by centrifugation or filtration, washing with water and a solvent, and drying, if necessary.

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 acids, 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 weight based on 100 parts by weight of the total amount of polymerizable monomers.

In order to suppress emulsion polymerization of polymerizable monomers themselves in water, water-soluble polymerization of nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble vitamin B, citric acid, polyphenols, etc. is prohibited. Agents may also be used.

In order to further improve base resistance, the polymer particles contain seed particles having structural units derived from methyl methacrylate and structural units derived from glycerol di(meth)acrylate formed on the surface of the seed particles. It may be a polymer particle having a polymer having a

The average particle diameter of the polymer particles may be, for example, 6 μm or less, 5 μm or less, or 4 μm or less, since a high number of theoretical plates can be easily obtained.

The coefficient of variation of the particle size (diameter) of the polymer particles may be, for example, 25% or less, 20% or less, 15% or less, or 10% or less, since it is easy to obtain a high number of theoretical plates.

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

(Organic group introduction step)
Next, 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 phosphoric acid groups, sulfonic acid groups. It may further have an ionic group such as.

The method for introducing an organic group containing a quaternary ammonium group into a polymer particle is not particularly limited, but for example, a polymer particle and a compound having an epoxy group are reacted to introduce an epoxy group, and a polymer having an epoxy group is introduced into the polymer particle. A method of reacting particles with a tertiary amine, reacting a polymer particle with a compound having an oxodioxophosphoryl group to introduce an oxodioxophosphoryl group, and reacting the polymer particle with a tertiary amine having an oxodioxophosphoryl group. A method of reacting a tertiary amine with polymer particles having a halogenated alkyl group obtained by copolymerizing a (meth)acrylate having a halogenated alkyl group such as 2-chloroethyl (meth)acrylate. , 2-dimethylaminoethyl (meth)acrylate and other tertiary amino group-containing polymer particles obtained by copolymerizing (meth)acrylates having a tertiary amino group are reacted with an alkyl halide such as methyl iodide. For example, the method of Here, the organic group is a compound having an epoxy group to be reacted 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, etc. It can be a group derived from.

Examples of methods for introducing epoxy groups include a method in which polymer particles having hydroxyl groups and the like are reacted with a halogen group-containing glycidyl compound such as epichlorohydrin.

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

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

As a method for introducing an oxodioxophosphoryl group, for example, a halogen group-containing dioxaphosphorane compound such as 2-chloro-oxo-1,3,2-dioxaphosphorane is reacted with polymer particles having a hydroxyl group, etc. For example, how to

Examples of the tertiary amine to be reacted with the oxodioxophosphoryl group include those similar to those exemplified as the tertiary amine to be reacted with the epoxy group. A separation material having an organic group containing a quaternary ammonium group and a phosphoric acid group can be obtained by reacting a polymer particle having an oxodioxophosphoryl group with a tertiary amine.

The separation material for metabolomic analysis obtained in this way has polymer particles and an organic group containing a quaternary ammonium group bonded to the polymer particles. The organic group may further contain an ionic group such as a sulfonic acid group or a phosphoric acid group in addition to the quaternary ammonium group, but it has high separability with respect to the metabolome and has a sharper chromatogram. From the viewpoint of obtaining a peak, it is preferable that ionic groups other than quaternary ammonium groups are not included.

The average particle diameter of the separation material for metabolome analysis may be, for example, 2 to 5 μm or less, since it is easy to obtain a high number of theoretical plates. The average particle diameter of the separation material may be 2.5 μm or more, 3 μm or more, or 3.2 μm or more, or 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, since it is easy to obtain a high number of theoretical plates.

The amount of quaternary ammonium groups (neutral salt decomposition capacity) of the separation material for metabolome analysis may be 10 to 500 μeq/g since it shows higher separation performance for ionic compounds. The amount of quaternary ammonium groups (neutral salt decomposition capacity) of the separation 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.

A separation material for metabolome analysis is a separation material for separating metabolome from a sample containing 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), and 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 acidic glutathione (GSSG). Amino acids are more preferably alanine (Ala), threonine (Thr), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), proline (Pro), phenylalanine (Phe), and 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, guanosine, thymidine, uridine, and cytidine.

Examples of nucleotides include adenosine-1-phosphate (AMP), guanosine-1-phosphate (GMP), thymidine-1-phosphate (TMP), uridine-1-phosphate (UMP), and cytidine-1-phosphate. - 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 column for metabolome analysis according to this embodiment includes the separation material for metabolome analysis described above. A column for metabolome analysis can be manufactured, for example, by filling it with a separation material for metabolome analysis. The method of filling the column with the separation material is not particularly limited, and any known method can be employed as appropriate. The column for metabolome analysis can be suitably used even under basic conditions.

Hereinafter, the present invention will be described in more detail 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>
70 g of methyl methacrylate, 2.1 g of octanethiol, and 370 g of ion-exchanged water were added to a 500 mL separable flask, and the mixture was kept at 30° C. for 1 hour while bubbling with nitrogen and stirring with a stirring blade. Thereafter, 0.875 g of potassium peroxodisulfate and 30 g of ion-exchanged water were added and reacted at 70° C. for 6 hours to obtain a reaction solution containing seed particles. After cooling the obtained reaction liquid, lumps and fine particles in the reaction liquid were removed to obtain a slurry of seed particles (solid content concentration: 3.5% by mass). Here, the lumps were removed using a sieve with an opening of 150 μm. Furthermore, fine particles were removed by treating the reaction solution (slurry that passed through the sieve) with a centrifugal dehydrator after removing the lumps, and discarding the supernatant liquid by decantation. The average particle diameter and CV (coefficient of variation) of the particle diameter of the seed particles in the obtained slurry were calculated by measuring the particle size distribution with a particle size distribution analyzer (Microtrac Bell Co., Ltd. (MT-3300EX II)). The average particle diameter of the seed particles was 750 nm, and the CV was 6.4%.

<Preparation of polymer particles>
In a 3 L separable flask, add 80.6 g of glycerol di(meth)acrylate, 72.5 g of butyl acetate, and 48.4 g of isoamyl alcohol, and further add 0.4 g of 2,2'-azobisisobutyronitrile. and dissolved. Next, 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 to the separable flask, and the mixture was ultrasonically dispersed for 10 minutes using an ultrasonic horn to obtain an emulsion. To the obtained emulsion, while stirring with a stirring blade, 14.0 g of the seed particle slurry prepared above and 122 g of ion-exchanged water were added, and the mixture was kept at 30°C (temperature inside the flask) for 1 hour to form seed particles. was allowed to absorb glycerol di(meth)acrylate. Next, 121 g of a polyvinyl alcohol aqueous solution (polyvinyl alcohol concentration: 6% by mass) was added, polymerized at 78° C. (temperature inside the flask) for 5 hours while bubbling with nitrogen, and cooled. The particles produced by polymerization were washed with ion-exchanged water, an ion-exchanged water/methanol mixture, and methanol in this order, and then subjected to wet classification using a 5-μm sieve to remove aggregates. Polymer particles were obtained by filtering and drying particles generated by polymerization from the slurry after removing the aggregates. The average particle diameter and CV (coefficient of variation) of the particle diameter of the obtained polymer particles were calculated by measuring the particle size distribution with a particle size distribution analyzer (Microtrac Bell Co., Ltd. (MT-3300EX II). The average particle diameter of the particles was 3.5 μm, and the CV was 6.8%.

<Preparation of polymer particles having epoxy groups>
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 an aqueous sodium hydroxide solution (sodium hydroxide concentration: 30% by mass) were ultrasonically dispersed for 1 minute. The mixture was allowed to react at 30° C. for 1 hour while being stirred with a stirring blade. The polymer particles after the reaction were filtered and washed with ion-exchanged water and methanol in that order to obtain polymer particles having epoxy groups.

<Preparation of separation material for metabolome analysis>
Add the entire amount of the polymer particles having epoxy groups 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.) to a 2 L separable flask, and stir with a stirring blade. The mixture was reacted at 30°C (temperature inside the flask) for 3 hours while stirring. After the reaction polymer particles were filtered and washed with ion-exchanged water and methanol in that order, the reaction polymer particles, 300 g of ion-exchange water, and 1.2 g of a sulfuric acid aqueous solution (sulfuric acid concentration: 47% by mass) were added. The mixture was washed at 40° C. (temperature inside the flask) for 3 hours while stirring with a stirring blade. The washed polymer particles were filtered, washed with ion-exchanged water and methanol in that 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 (manufactured by Microtrac Bell Co., Ltd. (MT-3300EX II). 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. 1 g of the separation material was weighed into a 100 mL beaker, 1 mol/L aqueous sodium hydroxide solution was added thereto and stirred, and the separation material was filtered out by suction filtration using a funnel. The filtered particles were dispersed in ion-exchanged water, filtered through a funnel through suction filtration, 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 thereto and stirred, and the separation material was filtered out by suction filtration using a funnel. The filtered particles were dispersed in ion-exchanged water, filtered through a funnel through suction filtration, and the separation material was washed with ion-exchanged water until the filtrate became neutral. The filtrate obtained at this time (including the ion exchange water used for washing) was transferred to a 500 mL beaker, and the ion exchange capacity was measured by titrating with a 0.1 mol/L aqueous sodium hydroxide solution. The ion exchange capacity of the separation material was 1.7 meq/g.

<Measurement of quaternary ammonium group amount (neutral salt decomposition capacity)>
The amount of quaternary ammonium groups (neutral salt decomposition capacity) of the obtained separation material was measured by the following method. 1 g of the separation material was weighed into a 100 mL beaker, 1 mol/L aqueous sodium hydroxide solution was added thereto and stirred, and the separation material was filtered out by suction filtration using a funnel. The filtered particles were dispersed in ion-exchanged water, filtered through a funnel through suction filtration, 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 aqueous sodium chloride solution was added and stirred, and the separation material was filtered out by suction filtration using a funnel. The filtered particles were dispersed in ion-exchanged water, filtered through a funnel through suction filtration, and the separation material was washed with ion-exchanged water until the filtrate became neutral. The filtrate obtained at this time (including the ion-exchanged water used for washing) 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 groups (neutral salt decomposition capacity). ) was measured. The amount of quaternary ammonium groups (neutral salt decomposition capacity) of the separation 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)>
10.9 g of Na salt of 2-ethylbromide sulfonic acid was added to a 250 mL flask, and 100 mL of water was added thereto to dissolve it. Furthermore, 12.4 g of dimethylamine was added, and the mixture was allowed to stand at room temperature for 45 minutes, and the resulting mixture was reacted at 70 to 80° C. for 18 hours under heating and reflux. 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. Thereafter, the reaction solution was cooled to room temperature, the 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>
Into a 500 mL separable flask, add the entire amount of the polymer particles having epoxy groups produced under the conditions of Example 1, 16 g of DMAES, and 200 mL of 0.2 mM phosphate buffer (pH 8), and while stirring with a stirring blade, increase the internal temperature to 50°C. , and reacted for 90 hours. After the reaction polymer particles were filtered and washed with ion-exchanged water and methanol in that order, the reaction polymer particles, 300 g of ion-exchange water, and 1.2 g of a sulfuric acid aqueous solution (sulfuric acid concentration: 47% by mass) were added. The mixture was washed at 40° C. (temperature inside the flask) for 3 hours while stirring with a stirring blade. After washing, the polymer particles were filtered, washed with ion-exchanged water and methanol in that order, and the polymer particles were dried to obtain a separation 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 (manufactured by Microtrac Bell Co., Ltd. (MT-3300EX II). The average particle size was 3.6 μm. When the amount of quaternary ammonium groups was measured in the same manner as in Example 1, the amount of quaternary ammonium groups (neutral salt decomposition capacity) of the separation material was 50 μeq/g. The amount of sulfonic acid groups determined from the amount was 50 μeq/g.

(Example 3: Separation material having an organic group containing a quaternary ammonium group and a phosphoric acid group)
<Preparation of separation material for metabolome analysis>
15 g of the polymer particles produced under the conditions of Example 1 and 200 mL of dry tetrahydrofuran were added to a 500 mL three-necked flask, 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-dioxaphosphorane was added dropwise in a nitrogen atmosphere over 1 hour, and the mixture was reacted at 5°C for 48 hours. The residue was distilled off under reduced pressure. A tetrahydrofuran solution of triethylamine was prepared by mixing 70 g of triethylamine and 200 mL of tetrahydrofuran, and the solution was quickly added to the residue after distillation, and reacted at 40° C. for 24 hours. The polymer particles after the reaction were filtered and washed with ion-exchanged water and then methanol 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 (manufactured by Microtrac Bell Co., Ltd. (MT-3300EX II). The average particle size was 3.6 μm. When the amount of quaternary ammonium groups was measured in the same manner as in Example 1, the amount of quaternary ammonium groups (neutral salt decomposition capacity) of the separation material was 40 μeq/g. The amount of phosphoric acid groups determined from the amount was 40 μeq/g.

[Column evaluation]
<Preparation of column for metabolome analysis (packing of separation material for metabolome analysis)>
Columns were prepared using the separation materials of Examples 1 to 3. A filling slurry was prepared by adding 0.8 g of the separation material and a 25:75 (volume ratio) mixture of ultrapure water and acetonitrile to a 100 mL beaker, and dispersing and mixing while applying ultrasound. After that, the filling slurry was poured into a stainless steel packer equipped with a 2.1 mmφ x 150 mm stainless steel column (manufactured by Sugiyama Shoji Co., Ltd.), sealed, and pressurized with a plunger-type filling pump (uf-20020SZWP2 pump, manufactured by Uniflows Co., Ltd.). Filled. After filling, a 0.1 mol/L aqueous sodium hydroxide solution was passed through the column at a rate of 0.1 mL/min for 3 hours to make the inside of the column basic. After passing a sodium hydroxide aqueous solution through the column, a 25:75 (volume ratio) mixture of ultrapure water and acetonitrile was passed through the column to wash off excess basic components (sodium hydroxide aqueous solution) to create a column. did.

<Column evaluation>
Using the prepared columns of Examples 1 to 3, the columns were attached to liquid chromatography (HPLC) and column evaluation was performed by separating metabolome from samples containing metabolome under acidic, neutral, and basic conditions. Ta.

(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 5mM ammonium bicarbonate aqueous solution - 5% by volume of acetonitrile (adjusted to pH 3.6 with acetic acid)
Mobile phase B: 5% by volume of 5mM ammonium bicarbonate aqueous solution - 95% by volume of acetonitrile
Flow rate: 0 to 15 minutes, 0.25 mL/min, 25 to 35 minutes, 0.40 mL/min Gradient conditions: Figure 1

(HPLC conditions (neutral))
Temperature: 40℃
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 5mM ammonium bicarbonate aqueous solution - 5% by volume of acetonitrile (adjusted to pH 6.8 with acetic acid)
Mobile phase B: 5% by volume of 5mM ammonium bicarbonate aqueous solution - 95% by volume of acetonitrile
Flow rate: 0 to 15 minutes, 0.25 mL/min, 25 to 35 minutes, 0.40 mL/min Gradient conditions: Figure 1

(HPLC conditions (basic))
Temperature: 40℃
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: 5mM ammonium bicarbonate aqueous solution 95% by volume-acetonitrile 5% by volume (adjusted to pH 9.8 with ammonia)
Mobile phase B: 5% by volume of 5mM ammonium bicarbonate aqueous solution - 95% by volume of acetonitrile
Flow rate: 0 to 15 minutes, 0.25 mL/min, 25 to 35 minutes, 0.40 mL/min Gradient conditions: Figure 1

(Sample preparation)
One of the following metabolomes was mixed with ultrapure water to prepare 28 types of samples containing 10 μM of metabolome (1 μM for valine and adenosine).

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. Nucleic acid bases Adenine, Guanine, Thymine, Uracil, Cytosine

3. Nucleosides Adenosine, Guanosine, Thymidine, Uridine, 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)

Note that for nucleotides and organic acids, only HPLC conditions (basic) were investigated.

(Evaluation criteria)
The chromatograms obtained for each metabolome were scored using the following criteria. A score of +1 or more was evaluated as a good result.
(1) Retained in the void volume region (1.88 or 2.06 minutes) +1 point (2) Retained for more than 5 minutes +1 point (3) Peak width less than 1 minute +1 point (4) Peak 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 in Examples. As shown in FIG. 2, the metabolome analysis column of the example showed good results even when used under basic conditions.

Figure 3(a) is a chromatogram of adenine under basic conditions using the column of Example 1, and Figure 3(b) is a chromatogram of adenine under basic conditions using the column of Example 2. This is a chromatogram of adenine. FIG. 4(a) is a chromatogram of adenosine under basic conditions using the column of Example 1, and FIG. 4(b) is a chromatogram of adenosine under basic conditions using the column of Example 2. This is a chromatogram of adenosine. As shown in FIGS. 3 and 4, it was found that the column of Example 1 gave 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 is capable of separating metabolomes even when used under basic conditions.

Claims (4)

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

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