JPH1183825A - Separating agent and separation method using it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To permit direct injection analysis of a compound in a biological component such as serum with a reversed-phase high-speed liquid chromatography by using a separator in which a hydrophilic polymer layer is formed on a surface of a polymer particle having a casting structure of a compound target for separation. SOLUTION: In a column in which a separator is a fixed phase, a biological component containing a compound targeted for separation (for example, human serum, bovine serum albumin or the like) is directly injected, and a compound is separated by a reversed-phase high-speed liquid chromatography method. The separator forms a hydrophilic polymer layer on the surface of polymeric particles having a casting structure of the compound targeted for separation. Among them, when a polymeric vinyl monomer having a mutual action with the compound is used, in continuation of polymeric reaction, a hydrophilic vinyl monomer, for example, a polymeric vinyl monomer having a functional group capable of producing an unsaturated carboxylic acid such as (meta) acrylic acid and a hydrophilic group is added.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel separating agent suitable for a packing material for liquid chromatography and a method for separating compounds in serum using the same. More specifically, the separating agent of the present invention can be used to reverse the drug or metabolite in a biological component containing a large amount of protein such as serum by direct injection of the biological component without performing pretreatment such as deproteinization. It enables separation and analysis by high performance liquid chromatography.

[0002]

2. Description of the Related Art Conventionally, immunoassays using natural antibodies or immobilized antibody methods have been used for the analysis and concentration of trace amounts of macromolecules, biological components and drugs. However, these methods require immunization of an animal with an antigen, selection of a single coulomb antibody, necessity of preparing an antibody including isolation of the antibody, stability of the prepared antibody, time and labor required for preparing the antibody, And so on. In order to overcome these problems, methods using artificial antibodies have been studied. As a method for producing an artificial antibody, a compound (guest compound) of interest is mixed with a polymerizable monomer that interacts complementarily with the compound, and the polymerizable monomer is polymerized in the interaction state. A method of forming a polymer having a template structure for a guest compound, mixing a guest compound and a polymer that interacts complementarily, and subjecting the polymer to a cross-linking reaction in an interacting state to form a template structure for the guest compound. A so-called “template polymerization method” such as a method of forming a crosslinked polymer having a polymer is known (for example, G. Wolff).
Et al., Angew. Chem. , Vol. 84, p36
4,1972).

However, the object of the conventional template polymerization method is
Limited to the template polymer for relatively fat-soluble guest compound, when applying the immobilized antibody method using the template polymer as an artificial antibody to chromatographic analysis,
Most of the analysis was performed in a normal phase system (B. Sellergr).
en, J. et al. Chromatogr. A, Vol. 67
3, p133, 1994). Therefore, direct injection analysis of serum is impossible in normal phase chromatography analysis, and when the above analysis method is applied to the analysis and separation of compounds in serum, there is a problem in efficiency due to the necessity of sample preparation work. was there. Also, for those that can be analyzed in reversed-phase systems,
It is difficult to perform an accurate analysis by direct injection of serum, because there is an influence on the separation performance and the life of the separating agent due to the adsorption of serum proteins and the like.

[0004]

SUMMARY OF THE INVENTION The present invention provides a separating agent for solving the problems when the conventional immobilized antibody method using the above-mentioned natural antibody and artificial antibody is applied to chromatographic analysis. It is another object of the present invention to provide a method for separating a compound in a biological component using the separating agent.

[0005]

The present inventor has studied and found that:
The present inventors have found that such an object can be achieved by forming a hydrophilic polymer layer on the surface of a polymer having a template structure, and completed the present invention. That is, the gist of the present invention is to provide a separating agent in which a hydrophilic polymer layer is formed on the surface of a polymer particle having a template structure of a compound to be separated and a reversed-phase high-performance liquid chromatography method using such a separating agent. In the separation method.

[0006]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
As the polymer having a template structure in the separating agent of the present invention, a polymer produced by a known method is used. For example, it is mixed with a polymerizable monomer having an interaction complementary to the compound to be separated and polymerized together with another monomer if necessary, or has a complementary interaction with the compound to be separated. After reacting the crosslinking agent in the state of being mixed with the polymer having the above, the separation target compound introduced from the obtained polymer is removed to form polymer particles having a template structure.

[0007] The composition of the polymer particles is not particularly limited as long as the template of the compound to be separated can be formed by polymerization or cross-linking reaction. When used, silica gel, inorganic such as hydroxyapatite,
Natural polymer systems such as dextran, cellulose and chitosan, and synthetic polymer systems such as styrene and (meth) acrylate are preferred.

[0008] The presence or absence of the porosity of the polymer particles is not particularly limited. However, when a large number of compounds to be separated are separated and purified, a porous structure is required to increase the adsorption capacity of the separating agent. It is preferred to have. Further, the form of the polymer particles may be either a crushed form or a spherical form, but a spherical form is preferable in order to obtain high resolution. The average particle diameter of the polymer particles is selected from 1 μm or more and 1000 μm or less, preferably 1 μm or more and 200 μm or less,
More preferably, spherical polymer particles having a particle size of 1 μm or more and 20 μm or less and a variation coefficient of the particle size of 10% or less are suitably used as a separating agent for high performance liquid chromatography. As a method for producing spherical polymer particles having a particle size of 1 μm or more and 20 μm or less and a variation coefficient of the particle size of 10% or less, seed particles having a narrow particle size distribution (monodispersed) are used, and further polymerized. Polymerized by impregnating a reactive monomer,
A method using a so-called seed polymerization method can be used.

Hereinafter, a method for producing polymer particles having a template structure by a seed polymerization method will be described, but the method for producing the separating agent of the present invention is not limited to such a method. In the seed polymerization method, polymer particles used as seed particles can be produced by generally well-known ball-forming polymerization such as emulsion polymerization, soap-free emulsion polymerization, dispersion polymerization, and suspension polymerization. Among them, emulsion polymerization, soap-free emulsion polymerization,
The polymer particles obtained by dispersion polymerization have a narrow particle size distribution as compared with those produced by suspension polymerization, and are preferable as seed particles.
A method of performing seed polymerization using polymer particles of 0 μm as seed particles (JP-A-64-26617) can be suitably used.

The polymer particles serving as seed particles are preferably a polymer comprising an aromatic vinyl monomer and / or an aliphatic vinyl monomer. These may be either homopolymers or copolymers of two or more monomers, and may be copolymers with 1% by weight or less of a polymerizable polyvinyl monomer. Preferred examples include polymer particles made of polystyrene or polymethacrylate. The particle diameter is preferably 0.1 μm or more and 10 μm or less, and the coefficient of variation of the particle diameter is preferably 10% or less.

Next, the seed particles are impregnated with a polymerizable monomer. Examples of the polymerizable monomer include a polymerizable vinyl monomer and / or a polymerizable polyvinyl monomer. Examples of the polymerizable vinyl monomer include unsaturated carboxylic acids such as (meth) acrylic acid, itaconic acid, and maleic acid;
Methyl (meth) acrylate, alkyl (meth) acrylate such as ethyl (meth) acrylate, hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, phenyl (meth) acrylate, (meth)
3-chloro-2-hydroxypropyl acrylate, 2,3-dihydroxypropyl (meth) acrylate, 2-chloroethyl (meth) acrylate, glycidyl (meth) acrylate, dimethylaminoethyl (meth) acrylate, (meth) ) (Meth) acrylic esters such as diethylaminoethyl acrylate and tetrahydrofurfuryl (meth) acrylate; (meth) acrylamide, N, N
-(Meth) acrylamide derivatives such as dimethyl (meth) acrylamide; styrene such as styrene, methylstyrene, α-methylstyrene, chlorostyrene, chloromethylstyrene and alkyl- or halogen-substituted products thereof;
Vinyl esters such as vinyl acetate and vinyl propionate; vinyl ethers such as methyl vinyl ether and ethyl vinyl ether; allyl alcohol and its esters or ethers; (meth) acrylonitrile, vinyl sulfonic acid, p-styrene sulfonic acid, vinyl pyridine, vinyl Other vinyl compounds such as pyrrolidone and the like can be mentioned, and these polymerizable vinyl monomers can be used alone or in combination of two or more.

Further, as the polymerizable polyvinyl monomer,
Aromatic polyvinyl monomers and aliphatic polyvinyl monomers are preferred. As the aromatic polyvinyl monomers, divinylbenzene and bisvinylphenylalkane are used, and as the aliphatic polyvinyl monomers, polyhydric alcohol poly (meta) is used. ) Acrylates and alkylene poly (meth) acrylamides are preferred. Specifically, (poly) ethylene glycol di (meth) acrylate, glycerin di (meth) acrylate, trimethylolpropane tri (meth) acrylate, tetrahydroxybutanedi (meth) acrylate,
Pentaerythritol di (meth) acrylate, methylene bisacrylamide, piperazine diacrylamide,
Diallyl tartaric acid diamide and the like may be mentioned, and these may be used alone or as a mixture. Particularly, ethylene glycol di (meth) acrylate and glycerin di (meth) acrylate are preferable.

These polymerizable monovinyl monomers and / or
Or the type and amount of the polymerizable polyvinyl monomer is appropriately determined in consideration of the size of the seed particles to be used and the size of the target particles, but imparts solvent resistance to the separating agent in the present invention, In order to increase the pressure resistance when used as a separating agent for high performance liquid chromatography, 1 to 100% by weight, preferably 20 to 95% by weight of a polymerizable polyvinyl monomer in the total polymerizable vinyl monomer to be impregnated is used. It is preferable to include a monomer.

When producing porous polymer particles by seed polymerization, it is desirable to add a porosifying agent. As a porogen for that purpose, that is, as a porous solvent for impregnating the seed particles, an aliphatic or aromatic hydrocarbon which is an organic solvent that acts as a phase separator during seed polymerization and promotes the porosity of the particles. , Esters, ketones, ethers and alcohols. Specifically, for example, toluene, xylene, cyclohexane, octane,
Butyl acetate, dibutyl phthalate, methyl ethyl ketone,
Examples thereof include dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol, cyclohexanol and the like, and these can be used alone or as a mixture. In some cases, desired characteristics can be imparted to the polymer particles obtained by selecting the type of the porous solvent, for example, whether the solvent is an aromatic hydrocarbon or an alcohol.

Radical polymerization initiators for seed polymerization include peroxide initiators such as benzoyl peroxide and butylperoxyhexanoate, and azo initiators such as azobisisobutyronitrile and azobisisovaleronitrile. Is preferred.
These polymerization initiators are dissolved in a vinyl monomer or a porous solvent, and are impregnated into the seed particles simultaneously with or before and after the impregnation of the vinyl monomer. In addition, when the seed particles are impregnated with the polymerizable vinyl monomer and / or the polymerizable polyvinyl monomer, the porosifying solvent, the radical polymerization initiator, and the like, the affinity for the seed particles may be high in some cases. It can be diluted with a solvent and impregnated. Examples of such a solvent include water-miscible solvents such as alcohol and acetone, and halogenated hydrocarbons such as dichloroethane and methylene chloride.

When a polymer having a size of 0.1 to 1.5 μm produced by an emulsion polymerization method or a soap-free emulsion polymerization method is used as seed particles, first, a swelling aid such as 1-chlorododecane or dibutyl phthalate is used. After the primary swelling, a polymerizable vinyl monomer and / or a polymerizable polyvinyl monomer is impregnated and seed-polymerized to produce porous particles having a uniform particle size of up to about 100 μm (for example, J. Pharm. U
gelstad et al., Die Makromolekul
are Chemie, Vol. 180, p737, 1
979) is preferably used.

The polymer particles serving as seed particles are impregnated with a polymerizable vinyl monomer and / or a polymerizable polyvinyl monomer, a porous solvent, and a polymerization initiator to enlarge the seed particles. The seed polymerization is carried out by suspending in an aqueous medium.
The aqueous medium preferably contains a dispersion stabilizer in order to prevent aggregation, deformation, and fusion during seed polymerization and to increase the dispersion stability. Examples of the dispersion stabilizer include known anionic and nonionic surfactants and polyvinylpyrrolidone, polyethyleneimine, vinyl alcohol /
Vinyl acetate copolymers and the like are preferred. Seed polymerization is started by raising the temperature to the polymerization temperature. The polymerization temperature depends on the type of polymerization initiator used, but is 50 ° C to 80 ° C.
Is preferred. Further, the polymerization time of the seed polymerization is preferably before or after the half-life of the polymerization initiator or longer.
Hours to 48 hours are preferred.

These methods are taken as an example during the production of polymer particles or by introducing a template structure for a compound to be separated into the obtained polymer particles, thereby selecting and separating the compound to be separated. Introduce the nature. As a method for introducing a template structure, a method of polymerizing a compound to be separated and a polymerizable monomer in a mixed state, a method of mixing a compound to be separated and a linear polymer, A method of reacting a crosslinking agent between molecules,
Anal. Sci. , Vol. 8, p749, 1992
Using a surfactant having a coordinating functional group in the hydrophilic part as shown in, polymerize the polymerizable monomer present in the oil phase while interacting with the compound to be separated at the oil-water interface After obtaining a polymer into which a compound to be separated is introduced by a method or the like, a method of removing the compound may be mentioned.

When a method for polymerizing a polymerizable monomer is used as a method for introducing a template structure into polymer particles, in order to maintain the template structure, a cross-linked structure is introduced using a polyfunctional polymerizable monomer. Is preferred. When a polymerizable polyvinyl monomer is used as the polyfunctional polymerizable monomer, 1 to 100% by weight, preferably 20%, of the total polymerizable vinyl monomer is used.
Preferably, it contains about 95% by weight of a polymerizable polyvinyl monomer.

In order to improve the performance of separating the compound to be separated from other compounds, it is preferable that the polymer particles contain a functional group having an interaction with the compound to be separated. As the functional group having an interaction with the compound to be separated, for example, an amino group for a metal ion,
Ionic functional groups such as carboxyl and sulfone groups,
Chelating groups such as iminodiacetic acid groups and phosphate groups, complex forming groups such as porphyrins, and crown ethers; ionic functional groups such as amino groups, carboxyl groups and sulfone groups for compounds having an ionic dissociation group; Inclusion compounds such as cyclodextrin for various organic compounds such as glandin, phenobarbital, vitamin A and vitamin D; borate groups for sugars; metal chelate groups for proteins;

When a method for polymerizing a polymerizable monomer is used as a method for introducing a template structure into polymer particles, a method for mixing and polymerizing a polymerizable monomer having an interaction with a compound to be separated is used. preferable. When a polymerizable vinyl monomer is used as the polymerizable monomer, the polymerizable monomer has an interaction with 0 to 99% by weight, preferably 5 to 80% by weight of the compound to be separated in the total polymerizable vinyl monomer. It preferably contains a polymerizable vinyl monomer.

As an example, when a compound to be separated is a phenylacetic acid derivative (including a naphthylacetic acid derivative) such as (S) -naproxen, ibuprofen, flurbiprofen, ketoprofen, etc., the compound has a carboxyl group 4- having electrostatic interaction with the group
It is preferable to mix vinyl pyridine or the like as a polymerizable monomer and polymerize in an interactive state. Further, (S) -propranolol, (S)-
When a β-blocker such as alprenolol and pindolol is used, methacrylic acid or the like having an electrostatic interaction with an amino group of the compound is mixed as a polymerizable monomer and polymerized in an interaction state. Preferably. By washing the obtained polymer, the compound to be separated is removed together with the initiator and other additives to form a template. The washing may be performed after forming the hydrophilic polymer layer on the surface. Specific examples of a method for introducing a template structure for a compound to be separated into polymer particles produced by a seed polymerization method include Chemistry Let.
ters, p555, 1997 and the like.

The separating agent in the present invention is obtained by forming a hydrophilic polymer layer on the surface of polymer particles having a template structure of the compound to be separated. The type of the hydrophilic polymer layer and the method for forming the same are not particularly limited. For example, JP-A-1-310744 and JP-A-3-17925.
9, JP-A-6-262070, JP-A-6-262070
Japanese Patent Application Laid-Open No. 265533 and JP-A-7-088366 disclose coating the surface of polymer particles with a polymer capable of generating a hydrophilic group such as a hydroxyl group by hydrolysis or the like, as disclosed in JP-A-7-088366. Alternatively, a method of polymerizing a polymer particle using a polymerizable vinyl monomer capable of producing a hydrophilic group or a hydrophilic group by hydrolysis or the like and, if necessary, producing a hydrophilic group by hydrolysis or the like is used. No. Above all, in the case of producing a polymer particle having a template structure by performing a polymerization reaction of a polymerizable vinyl monomer having an interaction with a compound to be separated, the hydrophilic vinyl monomer is continuously produced during the polymerization reaction. And / or a method of introducing a hydrophilic polymer layer on the surface of polymer particles by adding a polymerizable vinyl monomer having a functional group capable of forming a hydrophilic group is preferably used.

As the hydrophilic vinyl monomer, for example,
Unsaturated carboxylic acids such as (meth) acrylic acid, itaconic acid and maleic acid; hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, (meth)
Polyethylene glycol acrylate, 2,3-dihydroxypropyl (meth) acrylate, glycosylethyl (meth) acrylate, dimethylaminoethyl (meth) acrylate, diethylaminoethyl (meth) acrylate,
(Meth) acrylic esters having a hydroxyl group, an amino group, an ether group or the like such as tetrahydrofurfuryl (meth) acrylate; (meth) acrylamide, N, N
-(Meth) acrylamides such as dimethyl (meth) acrylamide, dimethylaminopropyl (meth) acrylamide, diethylaminopropyl (meth) acrylamide; vinyl ethers such as methyl vinyl ether and ethyl vinyl ether; allyl alcohol; vinyl pyrrolidone; Among them, hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, polyethylene glycol (meth) acrylate, 2,3-dihydroxypropyl (meth) acrylate, glycosylethyl (meth) acrylate, allyl alcohol and the like Those having high hydrophilicity such as (meth) acrylic acid ester having a hydroxyl group, (meth) acrylamide, and vinylpyrrolidone are preferred. Examples of the polymerizable vinyl monomer having a functional group capable of forming a hydrophilic group include alkyl (meth) acrylates such as methyl (meth) acrylate and ethyl (meth) acrylate; (Meth) acrylic esters such as chloro-2-hydroxypropyl, 2-chloroethyl methacrylate, and glycidyl (meth) acrylate; (meth) acrylamide, N, N
-(Meth) acrylamides such as dimethyl (meth) acrylamide and isopropyl (meth) acrylamide; vinyl esters such as vinyl acetate and vinyl propionate;
Vinyl ethers such as 2-chloroethyl vinyl ether; esters and ethers of allyl alcohol such as diallyl phthalate; vinylene carbonate; and the like, among which glycidyl (meth) acrylate, vinyl acetate, vinyl propionate, vinylene carbonate, and the like. Those which can generate a hydroxyl group are preferably used.

When a hydrophilic vinyl monomer and / or a polymerizable vinyl monomer having a functional group capable of forming a hydrophilic group are added during the continuation of the polymerization reaction, the hydrophilic vinyl monomer may be added if necessary. It is also possible to use polymerizable polyvinyl monomers. Such hydrophilic polymerizable polyvinyl monomers include (poly) ethylene glycol di (meth) acrylate, glycerol di (meth) acrylate, pentaerythritol di (meth) acrylate, tetramethylolmethane di (meth) acrylate, (Poly) ethylene glycol divinyl ether, methylene bis (meth) acrylamide and the like are preferable, and glycerol di (meth) acrylate is preferably used.

The proportion of the hydrophilic polymerizable polyvinyl monomer in the hydrophilic vinyl monomer and / or the polymerizable vinyl monomer capable of forming a hydrophilic group added during the continuation of the polymerization reaction is from 0 to 0. 60% by weight is preferred. If it exceeds 60% by weight, the surface of the polymer particles becomes insufficiently hydrophilic. The amount of the hydrophilic vinyl monomer and / or the polymerizable vinyl monomer having a functional group capable of forming a hydrophilic group is 1 to 30% by weight, preferably 2 to 30% by weight, based on the polymer being reacted. 20% by weight.

The hydrophilic vinyl monomer and / or the polymerizable vinyl monomer having a functional group capable of forming a hydrophilic group may be used 0.5 to 24 hours after the initiation of the polymerization reaction, preferably 1 to 12 hours.
After the time, add in batches or in portions. In the case of divisional addition, for example, a method of adding every 10 to 60 minutes is preferable. Further, the hydrophilic vinyl monomer and / or the polymerizable vinyl monomer having a functional group capable of forming a hydrophilic group may be added as it is, or may be added after being diluted or dissolved with a solvent. When adding a hydrophilic vinyl monomer and / or a polymerizable vinyl monomer having a functional group capable of forming a hydrophilic group, a polymerization initiator, particularly a water-soluble initiator such as potassium persulfate or ammonium persulfate is added simultaneously. This is also preferable for forming a hydrophilic polymer layer on the surface of the polymer particles.

The separating agent of the present invention having a hydrophilic polymer layer on the surface of polymer particles having a template structure is suitable as a stationary phase for liquid chromatography. As will be apparent from the examples (analysis examples) described later, the use of the separating agent of the present invention makes it possible to obtain a serum albumin recovery rate of 90% or more and 100% under the conditions for analyzing low-molecular organic compounds in the reversed-phase distribution mode. , And separation and analysis by direct injection of compounds in biological components such as serum by reverse-phase high-performance liquid chromatography are possible. When the compound used as a template in the present invention is a drug, it is possible to separate the drug and its metabolite in the component by direct injection of a biological component such as serum. For separation and analysis by liquid chromatography using the separating agent of the present invention, an apparatus and an analysis method usually used for high performance liquid chromatography analysis can be applied.

[0029]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist thereof. Example 1 An aqueous suspension of seed particles composed of spherical polystyrene particles having an average particle diameter of 1.0 μm (solid concentration: 0.497 ml / m
l) 0.17 ml was mixed with a microemulsion prepared by dispersing 0.48 ml of dibutyl phthalate as a swelling aid and 0.02 g of sodium dodecyl sulfate as a surfactant in 5.0 ml of water, and slowly mixed at room temperature. For 15 hours (primary swelling). This primary swelling was performed until no added microemulsion droplets could be seen in an optical microscope.

Next, toluene 3.0, which is a porous solvent, is used.
1 ml of azobisdimethyl valeronitrile as an initiator, 10 ml of a 4.8% polyvinyl alcohol (polymerization degree 500, saponification degree 86.5 to 89 mol%) solution as a suspension stabilizer, and water 12. The microdispersed liquid suspended in a solution obtained by mixing 5 ml with the mixture was added to the above-mentioned suspension after the primary swelling, followed by gentle stirring at room temperature for 2 hours (secondary swelling).

To the suspension after the completion of the secondary swelling, 7.0 ml of ethylene glycol dimethacrylate as a polymerizable polyvinyl monomer, 0.46 g of a phenylacetic acid derivative (S) -naproxen as a compound to be separated,
0.63 g of 4-vinylpyridine as a polymerizable vinyl monomer having an electrostatic interaction with the carboxyl group of (S) -naproxen and 0.02 g of sodium dodecyl sulfate as a surfactant are suspension stabilizers. 4.8% polyvinyl alcohol (polymerization degree 500, saponification degree 86.5)
(-89 mol%) A fine dispersion suspended in a solution obtained by mixing 10 ml of a solution and 12.5 ml of water was added to the suspension after the secondary swelling, followed by gentle stirring at room temperature for 2 hours ( Tertiary swelling).

After the tertiary swelling of the suspension, the temperature was raised to 50 ° C. with gentle stirring under an argon atmosphere to initiate polymerization. Four hours after the start of the polymerization, 0.5 ml of 2,3-dihydroxypropyl methacrylate as a hydrophilic vinyl monomer, 0.5 ml of glycerol dimethacrylate as a hydrophilic polymerizable polyvinyl monomer, and water solubility 0.02 g of potassium persulfate, which is a polymerization initiator, was added all at once, and the temperature was raised to 70 ° C. to carry out polymerization for another 20 hours. The suspension after polymerization was poured into 250 ml of water, and after the particles had settled, the supernatant was discarded to remove the suspension stabilizer. Further, the particles were washed again by redispersing the particles in methanol and removing the supernatant liquid after sedimentation of the particles. This operation was performed once more with methanol and twice with tetrahydrofuran to wash the particles and dissolve and remove (S) -naproxen added for forming the template structure.
The washed particles were filtered and dried under reduced pressure at room temperature. The average particle diameter of the produced particles was 5.0 μm.

Analysis Example 1 The particles obtained in Example 1 were prepared with an inner diameter of 4.6 mm and a length of 100.
The sample was packed in a 25 mm stainless steel column, and human serum containing 25 μl of human serum and (S) -naproxen at a concentration of 100 μg / ml was subjected to direct injection analysis by reversed-phase high performance liquid chromatography. 20 mM phosphate buffer (pH = 6.04) / acetonitrile = 80 /
The mixture of No. 20 was flowed at a flow rate of 1.0 ml / min. The detection was performed with a 220 nm ultraviolet detector.

As an example of serum protein, 1 mg of bovine serum albumin was previously injected into the column, and the eluted bovine serum albumin was quantified. The recovery was 100%. FIG. 1 shows a chromatogram obtained by injecting and analyzing 25 μl of human serum, and a concentration of (S) -naproxen of 100 μg / ml.
FIG. 2 shows a chromatogram obtained by injecting and analyzing 25 μl of the human serum contained in the above. From FIG. 1 and FIG. 2, it was found that the separating agent prepared in Example 1 enables direct injection analysis of (S) -naproxen in serum.

Comparative Example 1 In exactly the same manner as in Example 1, the temperature of the suspension after the tertiary swelling was raised to 50 ° C. with gentle stirring under an argon atmosphere to initiate polymerization. After a lapse of 4 hours from the start of the polymerization, the temperature was raised to 70 ° C., and the polymerization was further performed for 20 hours.
Particles having a template structure were obtained from the suspension after the polymerization in the same manner as in Example 1. Using these particles (having no hydrophilic polymer layer), reverse-phase high performance liquid chromatography analysis was performed under the same conditions as in Analysis Example 1, and 1 mg of bovine serum albumin was injected as an example of a serum protein, and the eluted bovine serum albumin was injected. When the serum albumin was quantified, the recovery was 40%. From these results, when performing direct injection analysis of serum,
It is clear that the adsorption of the serum protein causes contamination of the surface of the separating agent, which is not preferable in durability.

Comparative Example 2 In exactly the same manner as in Example 1, the suspension after the secondary swelling was mixed with 7.0 ml of ethylene glycol dimethacrylate as a polymerizable polyvinyl monomer and sodium dodecyl sulfate as a surfactant. 0.02 g of 4.8% polyvinyl alcohol (polymerization degree: 500, saponification degree: 86.5 to 89 mol%) solution as suspension stabilizer and water (12.5 m)
and a fine dispersion liquid suspended in a solution obtained by mixing 1 and 1 was slowly stirred at room temperature for 2 hours (tertiary swelling).

The temperature of the suspension after the tertiary swelling was raised to 50 ° C. with gentle stirring in an argon atmosphere to initiate polymerization. Four hours after the start of the polymerization, 0.5 ml of 2,3-dihydroxypropyl methacrylate as a hydrophilic vinyl monomer, 0.5 ml of glycerol dimethacrylate as a hydrophilic polymerizable polyvinyl monomer, and water solubility 0.02 g of potassium persulfate, which is a polymerization initiator, was added all at once, and the temperature was raised to 70 ° C. to carry out polymerization for another 20 hours. Particles were obtained in the same manner as in Example 1 from the suspension after the polymerization. These particles are obtained by forming a hydrophilic polymer layer on the surface of polymer particles having no template structure.

Using these particles, reverse-phase high-performance liquid chromatography analysis was performed under the same conditions as in Analysis Example 1, 1 mg of bovine serum albumin was injected as an example of serum protein, and the eluted bovine serum albumin was quantified. The recovery was 100%. FIG. 3 shows a chromatogram obtained by injecting and analyzing 25 μl of human serum, and FIG. 4 shows a chromatogram obtained by injecting and analyzing 25 μl of human serum containing (S) -naproxen at a concentration of 100 μg / ml. As is clear from FIGS. 3 and 4, the peak derived from (S) -naproxen and the peak derived from serum protein overlap, indicating that separation and quantification of (S) -naproxen is impossible.

Example 2 In the same manner as in Example 1, the suspension after secondary swelling was mixed with 7.0 ml of ethylene glycol dimethacrylate as a polymerizable polyvinyl monomer and a phenylacetic acid derivative as a compound to be separated. Ibuprofen 0.41
g, 0.63 g of 4-vinylpyridine as a polymerizable vinyl monomer having an electrostatic interaction with the carboxyl group of ibuprofen and 0.02 g of sodium dodecyl sulfate as a surfactant, 0.8% polyvinyl alcohol (degree of polymerization 500, degree of saponification 86.5)
(-89 mol%) A fine dispersion suspended in a solution obtained by mixing 10 ml of a solution and 12.5 ml of water was added to the suspension after the secondary swelling, followed by gentle stirring at room temperature for 2 hours ( Tertiary swelling).

The temperature of the suspension after the tertiary swelling was raised to 50 ° C. with gentle stirring under an argon atmosphere to initiate polymerization. Four hours after the start of the polymerization, 0.5 ml of 2,3-dihydroxypropyl methacrylate as a hydrophilic vinyl monomer, 0.5 ml of glycerol dimethacrylate as a hydrophilic polymerizable polyvinyl monomer, and water solubility 0.02 g of potassium persulfate, which is a polymerization initiator, was added all at once, and the temperature was raised to 70 ° C. to carry out polymerization for another 20 hours. Particles were obtained in the same manner as in Example 1 from the suspension after the polymerization. The average particle size of the produced particles is 5.0 μm
Met.

Analysis Example 2 Using the particles obtained in Example 2, reverse phase high performance liquid chromatography analysis was performed under the same conditions as in Analysis Example 1. 1 mg of bovine serum albumin was injected, and the eluted bovine serum albumin was quantified. The recovery was 100%. Further, in the same manner as in Analysis Example 1, 25 μl of human serum was used.
1 and 25 μl of human serum containing ibuprofen at a concentration of 100 μg / ml were measured. The elution in minutes indicated that direct injection analysis of ibuprofen in serum was possible.

Example 3 In the same manner as in Example 1, the suspension after secondary swelling was mixed with 7.0 ml of ethylene glycol dimethacrylate as a polymerizable polyvinyl monomer and β-blocked as a compound to be separated. 0.59 g of (S) -propranolol hydrochloride as a drug, 0.63 g of methacrylic acid as a polymerizable vinyl monomer having an electrostatic interaction with an amino group of (S) -propranolol hydrochloride, and dodecyl as a surfactant 10 ml of a 4.8% polyvinyl alcohol (polymerization degree: 500, saponification degree: 86.5 to 89 mol%) solution as a suspension stabilizer, 10 ml of water and 0.1N-sodium hydroxide aqueous solution are used. The fine dispersion suspended in a solution obtained by mixing 5 ml with the suspension was added to the suspension after the secondary swelling, followed by gentle stirring at room temperature for 2 hours ( Next swelling).

The temperature of the suspension after the tertiary swelling was raised to 50 ° C. with gentle stirring in an argon atmosphere to initiate polymerization. Four hours after the start of the polymerization, 0.5 ml of 2,3-dihydroxypropyl methacrylate as a hydrophilic vinyl monomer, 0.5 ml of glycerol dimethacrylate as a hydrophilic polymerizable polyvinyl monomer, and water solubility 0.02 g of potassium persulfate, which is a polymerization initiator, was added all at once, and the temperature was raised to 70 ° C. to carry out polymerization for another 20 hours. Particles were obtained in the same manner as in Example 1 from the suspension after the polymerization. The average particle size of the produced particles is 5.0 μm
Met.

Analysis Example 3 The particles obtained in Example 3 were prepared with an inner diameter of 4.6 mm and a length of 100.
mm stainless steel column, and a 50 mM phosphate buffer (pH = 3.20) / acetonitrile = 80 /
The mixed solution of No. 20 was flowed at a flow rate of 1.0 ml / min as a mobile phase, and reverse phase high performance liquid chromatography analysis was performed.
The detection was performed with a 220 nm ultraviolet detector. 1 mg of bovine serum albumin was injected, and the eluted bovine serum albumin was quantified. The recovery was 100%.

When a chromatogram of 25 μl of human serum containing (S) -propranolol hydrochloride at a concentration of 100 μg / ml was measured, the peak derived from human serum protein was eluted without being retained, and (S) -hydrochloride was eluted. The peak derived from propranolol eluted at 15.0 minutes, indicating that direct injection analysis of (S) -propranolol hydrochloride in serum was possible.

Example 4 In the same manner as in Example 1, the suspension after secondary swelling was mixed with 7.0 ml of ethylene glycol dimethacrylate as a polymerizable polyvinyl monomer and β-blocked as a compound to be separated. 0.80 g of alprenolol (S) -alprenolol tartrate, 0.63 g of methacrylic acid as a polymerizable vinyl monomer having an electrostatic interaction with the amino group of (S) -alprenolol tartrate and surface activity 0.02 g of sodium dodecyl sulfate as a dispersing agent, 10 ml of a 4.8% polyvinyl alcohol (polymerization degree 500, saponification degree 86.5 to 89 mol%) solution as a suspension stabilizer and 10 ml of water
And a 2.5N 0.1N aqueous solution of sodium hydroxide were added to the above-mentioned suspension after the secondary swelling, and the mixture was gently stirred at room temperature for 2 hours (tertiary Swelling).

The temperature of the suspension after the tertiary swelling was raised to 50 ° C. with gentle stirring in an argon atmosphere to initiate polymerization. Four hours after the start of the polymerization, 0.5 ml of 2,3-dihydroxypropyl methacrylate as a hydrophilic vinyl monomer, 0.5 ml of glycerol dimethacrylate as a hydrophilic polymerizable polyvinyl monomer, and water solubility 0.02 g of potassium persulfate, which is a polymerization initiator, was added all at once, and the temperature was raised to 70 ° C. to carry out polymerization for another 20 hours. Particles were obtained in the same manner as in Example 1 from the suspension after the polymerization. The average particle size of the produced particles is 5.0 μm
Met.

Analysis Example 4 The particles obtained in Example 4 were prepared by using the particles having an inner diameter of 4.6 mm and a length of 100 mm.
mm stainless steel column, and a 20 mM phosphate buffer (pH = 3.95) / acetonitrile = 90 /
Reversed-phase high-performance liquid chromatography analysis was performed using the mixed solution of No. 10 as a mobile phase at a flow rate of 1.0 ml / min.
The detection was performed with a 220 nm ultraviolet detector. 1 mg of bovine serum albumin was injected, and the eluted bovine serum albumin was quantified. The recovery was 100%.

When a chromatogram obtained by injecting 25 μl of human serum containing (S) -alprenolol tartrate at a concentration of 100 μg / ml was measured, the peak derived from human serum protein was eluted without being retained. The peak derived from alprenolol tartrate eluted at 27.0 minutes, respectively, indicating that direct injection analysis of (S) -alprenolol tartrate in serum was possible.

[0050]

The separation agent of the present invention enables direct injection analysis of compounds in biological components such as serum by reversed-phase high-performance liquid chromatography.

[Brief description of the drawings]

FIG. 1 Chromatogram of 25 μl of human serum by the reversed-phase high-performance liquid chromatography method using the separating agent of Example 1.

FIG. 2 shows a chromatogram of 25 μl of human serum containing (S) -naproxen at a concentration of 100 μg / ml using the separating agent of Example 1 by reversed-phase high-performance liquid chromatography.

FIG. 3 Chromatogram of 25 μl of human serum using the separating agent of Comparative Example 2 by reversed-phase high performance liquid chromatography

FIG. 4 shows 25 μl of human serum containing (S) -naproxen at a concentration of 100 μg / ml using the separating agent according to Comparative Example 2.
Of high-performance liquid chromatographic chromatograms

Claims (5)

[Claims] 1. A separating agent comprising a polymer particle having a template structure of a compound to be separated and a hydrophilic polymer layer formed on the surface of the polymer particle. 2. The separating agent according to claim 1, wherein the compound to be separated is a phenylacetic acid derivative. 3. The separating agent according to claim 1, wherein the compound to be separated is a β-blocker. 4. A biological component containing a compound to be separated is directly injected into a column using the separating agent according to claim 1 as a stationary phase, and the column is subjected to reverse phase high performance liquid chromatography. A method for separating a compound in a biological component, comprising separating the compound. 5. The method according to claim 4, wherein the biological component is serum.