JP2013011632A - Measurement method of hemoglobin a1c - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measurement method of hemoglobin A1c for reducing nonspecific adsorption of hemoglobins to a column packing, and accurately measuring the hemoglobin A1c.SOLUTION: In a measurement method of hemoglobin A1c by a cation exchange chromatography method, as a column packing, a cross-linked polymer of a (meth) acrylic ester in which a cation-exchange group and a sugar structure are introduced at least on a surface thereof is used.

Description

The present invention relates to a method for measuring hemoglobin A1c that reduces non-specific adsorption of hemoglobin to a column filler and enables highly accurate measurement.

Hemoglobin (also referred to as Hb), particularly hemoglobin A1c (hereinafter also referred to as HbA1c), which is a type of glycated hemoglobin, reflects the average sugar concentration in the blood during the past 1-2 months. Widely used as a screening item for diabetes and as a test item for grasping blood glucose control status of diabetic patients.

Conventionally, HbA1c has been measured by liquid chromatography (HPLC), immunization, electrophoresis, and the like. Of these, the liquid chromatography method is widely used in the clinical laboratory field. In the liquid chromatography method, measurement in 1 to 2 minutes per sample is possible, and measurement accuracy of about 1.0% CV value in the simultaneous reproducibility test is realized. As a measurement method for grasping the blood glucose control state of a diabetic patient, at least this level of performance is required.

As a column filler for measuring hemoglobin with high accuracy in a short time, for example, Patent Document 1 discloses a column filler for cation exchange based on a polymer of (meth) acrylic acid ester. Yes. Such a cation exchange column filler is advantageous in that it can be subjected to various modifications to optimize the separation performance because the raw material is inexpensive and easily available and has a wide range of selection.

However, when hemoglobins are measured using a conventional cation exchange column packing material, hemoglobin may cause non-specific adsorption on the part of the column packing material, that is, the part other than the cation exchange group. there were. Such non-specific adsorption causes various problems such as a decrease in peak separation performance, a decrease in measurement reproducibility, and a shortened column life.

As a method for preventing nonspecific adsorption, generally, a method of hydrophilizing a column filler is known (for example, Patent Documents 1 to 7). However, so far, no effective method for preventing nonspecific adsorption of hemoglobin has been known.

Japanese Patent Publication No. 08-007197 Japanese Patent Laid-Open No. 01-098965 JP 09-210981 A Japanese Patent Laid-Open No. 11-023551 Japanese Unexamined Patent Publication No. 63-068603 JP-A 63-159754 Japanese Patent Laid-Open No. 04-050765

In view of the above situation, an object of the present invention is to provide a method for measuring hemoglobin A1c that reduces non-specific adsorption of hemoglobin to a column filler and enables measurement with high accuracy.

The present invention relates to a method for measuring hemoglobin A1c by a cation exchange chromatography method, and uses a column packing material in which a cation exchange group and a sugar structure are introduced on at least the surface of a crosslinked polymer of (meth) acrylate ester. It is a measuring method of hemoglobin A1c.
The present invention is described in detail below.

As a result of intensive studies, the present inventors have conducted analysis by liquid chromatography using a column-filler comprising a cross-linked polymer of (meth) acrylate and having at least a cation exchange group and a sugar structure on the surface. In this case, it was found that not only non-specific adsorption can be suppressed but also the measurement accuracy of hemoglobin can be remarkably improved, and the present invention has been completed.

In the method for measuring hemoglobin of the present invention, the column filler is made of a cross-linked polymer obtained by polymerizing a cross-linkable monomer, and has at least the surface having a cation exchange group and a sugar structure.
Although it does not specifically limit as said crosslinkable monomer, Crosslinkable (meth) acrylic acid ester is suitable.

The crosslinkable (meth) acrylic acid ester is not particularly limited. For example, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, tetraethylene glycol di (meth) Polyethylene glycol di (meth) acrylates such as acrylate and polyethylene glycol di (meth) acrylate; propylene glycol di (meth) acrylate, dipropylene glycol di (meth) acrylate, tripropylene glycol di (meth) acrylate, tetrapropylene glycol di Polypropylene glycol di (meth) acrylates such as (meth) acrylate and polypropylene glycol di (meth) acrylate; polytetramethylene glycol di ( Acrylate), poly (propylene glycol-tetramethylene glycol) -di (meth) acrylate, polyethylene glycol polypropylene glycol polyethylene glycol-di (meth) acrylate, 1,3-butylene glycol di (meth) acrylate, 1,4-butylene Alkylene glycol di (meth) acrylates such as glycol di (meth) acrylate, 1,6-hexaglycol di (meth) acrylate, 1,9-nonanediol di (meth) acrylate, neopentyl glycol di (meth) acrylate; 2-hydroxy-1,3-di (meth) acryloxypropane, 2-hydroxy-1- (meth) acryloxy-3- (meth) acryloxypropane, 2-hydroxy-3- (meth) acryloyloxypropi (Meth) acryl, glycerol di (meth) acrylate, glycerol acrylate methacrylate, urethane (meth) diacrylate, isocyanuric acid di (meth) acrylate, isocyanuric acid tri (meth) acrylate, 1,10-di (meth) acryloxy-4 , 7-Dioxadecane-2,9-diol, 1,10-di (meth) acryloxy-5-methyl-4,7-dioxadecane-2,9-diol, 1,11-di (meth) acryloxy-4,8 -Hydroxyalkyl di (meth) acrylates such as dioxaoundegan-2,6,10-triol; trimethylolpropane tri (meth) acrylate, tetramethylolpropane tri (meth) acrylate, ditrimethylolpropane tetra (meth) acrylate , Tetramethylo And xalkalol alkane y (meth) acrylates such as methane methane tri (meth) acrylate, tetramethylol methane tetra (meth) acrylate, trimethylol ethane tri (meth) acrylate, and the like. These crosslinkable (meth) acrylic acid esters may be used alone or in combination of two or more.

The cross-linked polymer constituting the column filler may be a copolymer of the cross-linkable (meth) acrylic acid ester and a non-cross-linkable monomer.
Although it does not specifically limit as said non-crosslinkable monomer, Non-crosslinkable (meth) acrylic acid ester is suitable.
The non-crosslinkable (meth) acrylate esters are not particularly limited, and examples thereof include methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, and butyl (meth) acrylate. Examples include alkyl (meth) acrylates; acrylic acid derivatives such as (meth) acrylic acid-2-ethylhexyl and (meth) acrylic acid-2-ethylhexyl.
Ethylene glycol mono (meth) acrylate, diethylene glycol mono (meth) acrylate, triethylene glycol mono (meth) acrylate, tetraethylene glycol mono (meth) acrylate, polyethylene glycol mono (meth) acrylate, methoxytri (poly) ethylene glycol mono (meta) ) Acrylate, polyethylene glycol mono (meth) acrylates such as methoxypolyethylene glycol mono (meth) acrylate; propylene glycol mono (meth) acrylate, dipropylene glycol mono (meth) acrylate, tripropylene glycol mono (meth) acrylate, tetrapropylene Polypropylene such as glycol mono (meth) acrylate and polypropylene glycol mono (meth) acrylate Poly (ethylene glycol / propylene glycol) mono (meth) acrylate, poly (ethylene glycol / tetramethylene glycol) mono (meth) acrylate, poly (propylene glycol / tetramethylene glycol) mono (meth) Alkylene glycol mono (meth) acrylates such as acrylate and recall polypropylene glycol mono (meth) acrylate; glycerin mono (meth) acrylate, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, 2, 3 -(Meth) acrylates having a hydroxyl group such as dihydroxylethyl (meth) acrylate and 2,3-dihydroxylpropyl (meth) acrylate. These non-crosslinkable (meth) acrylic acid esters may be used alone or in combination of two or more.

When the cross-linked polymer constituting the column filler is a copolymer of the cross-linkable (meth) acrylic acid ester and a non-cross-linkable monomer, 100 wt% of the cross-linkable (meth) acrylic acid ester A preferred upper limit of the blending amount of the non-crosslinkable monomer with respect to parts is 50 parts by weight. If the amount exceeds 50 parts by weight, the cross-linked polymer constituting the base material has a low degree of cross-linking and tends to swell and shrink. Therefore, it takes time to equilibrate when the buffer solution is switched in the measurement by the liquid chromatography method. May take longer measurement time. A more preferred upper limit is 20 parts by weight.

The column filler has a cation exchange group on at least the surface.
In this specification, the cation exchange group means a functional group having cation exchange properties. The cation exchange group is not particularly limited, and examples thereof include a carboxyl group, a phosphoric acid group, and a sulfonic acid group. Of these, sulfonic acid groups are preferred.
The cation exchange group may be any structure associated with the functional group (carboxyl group, phosphoric acid group, sulfonic acid group, etc.). For example, “carboxyl group” includes all functional groups to which carboxyl groups such as carboxylethyl group and carboxylpropyl group are bonded.

The cation exchange group is preferably derived from a monomer having a cation exchange group (hereinafter also referred to as “cation exchange monomer”).
That is, in producing a cross-linked polymer constituting the column filler, a method of adding a cation exchange monomer to a monomer mixture and copolymerizing, or after forming the cross-linked polymer, It is preferable to use a method in which a cation exchange monomer is polymerized on the surface of the crosslinkable polymer.

The cation exchange monomer having a carboxyl group is not particularly limited, and examples thereof include (meth) acrylic acid, 2- (meth) acryloyloxyethyl succinate, 2- (meth) acryloyloxyethylphthalic acid, and the like ( (Meth) acrylic acid derivatives; crotonic acid, itaconic acid, citraconic acid, mesaconic acid, maleic acid, fumaric acid and the like.
The cation exchange monomer having a phosphoric acid group is not particularly limited. For example, ((meth) acryloyloxyethyl) acid phosphate, (2- (meth) acryloyloxyethyl) acid phosphate, (3- (meth) And (meth) acrylic acid derivatives such as (acryloyloxypropyl) acid phosphate.
The cation exchange monomer having a sulfonic acid group is not particularly limited, and examples thereof include (meth) acrylic acid such as 2- (meth) acrylamido-2-methylpropanesulfonic acid and 3-sulfopropyl (meth) acrylic acid. Derivatives; (3-sulfopropyl) -itaconic acid, allylsulfonic acid and the like.
These cation exchange monomers may be used independently and may use 2 or more types together. Among these, as the cation exchange monomer, (meth) acrylic acid derivatives are preferable.

As the cation exchange monomer, a monomer having a cation exchange group and a sugar structure (hereinafter also referred to as “cationic sugar monomer”) is also suitable.
Examples of the cationic sugar monomer include, for example, a monosaccharide in which a monosaccharide such as glucose or fructose, a functional group including a sugar structure such as a glucosylethyl group, or a polysaccharide such as cellulose is introduced into the cation exchange monomer. Monomers: monomers in which a cation exchange group such as a carboxyl group, a phosphate group, or a sulfonic acid group is introduced into the sugar structure portion of the sugar monomer; the cation exchange described above in a portion other than the sugar structure of the sugar monomer And monomers having a group introduced therein.

The cation exchange group may be one obtained by converting a functional group that can be converted into a cation exchange group of a monomer having a functional group that can be converted into a cation exchange group into a cation exchange group.
That is, in the production of the crosslinked polymer constituting the column filler, a monomer having a functional group that can be converted into a cation exchange group is added to the monomer mixture and copolymerized, and then the cation exchange group is added. A method of converting a functional group convertible into a cation exchange group by a known chemical reaction, or after forming the crosslinked polymer, polymerizing a cation exchange monomer on the surface of the crosslinkable polymer, The functional group that can be converted into the cation exchange group may be converted by a known chemical reaction into a cation exchange group.

The functional group that can be converted into the cation exchange group is not particularly limited. For example, a monomer having an ester group such as a carboxylic acid ester, a phosphoric acid ester, or a sulfonic acid ester; a monomer having an epoxy group or a glycidyl group Etc.
The ester group derived from the monomer having an ester group can be easily converted into a carboxyl group, a phosphoric acid group, and a sulfonic acid group, respectively, by a known method of hydrolysis in the presence of an acid or a base. .
In the monomer having an epoxy group or glycidyl group, a carboxyl group, a phosphoric acid group, or a sulfonic acid group can be introduced by a known method after the epoxy group is opened.
These monomers having a functional group that can be converted into a cation exchange group may be used alone or in combination of two or more.

As the blending amount of the cation exchangeable monomer or the monomer having a functional group convertible into a cation exchange group, the crosslinkable (meth) acrylic acid ester (when a non-crosslinkable monomer is added) Is preferably 10 parts by weight with respect to 100 parts by weight, and a preferred upper limit is 100 parts by weight. If the amount is less than 10 parts by weight, the introduction amount of the cation exchange group is too small and sufficient cation exchange reaction may not be performed, and the peak to be measured may not be sufficiently separated. Aggregates may be generated and a polymer may not be obtained. A more preferred lower limit is 20 parts by weight, and a more preferred upper limit is 80 parts by weight.

The column filler has a sugar structure on at least the surface.
In this specification, the sugar structure means a polyhydric alcohol having an aldehyde group or a ketone group, that is, a reducing monosaccharide structure having a polyhydroxyaldehyde (aldose) structure or a polyhydroxyketone (ketose) structure, and the monosaccharide structure. Means a disaccharide structure, an oligosaccharide structure, or a polysaccharide structure, which is a polymer or copolymer having a basic unit.

The sugar structure is preferably derived from a monomer having a sugar structure (hereinafter also referred to as “sugar monomer”).
That is, in producing the cross-linked polymer constituting the column filler, a method of copolymerization by adding a sugar monomer to a monomer mixture, or after forming the cross-linked polymer, It is preferable to use a method in which a sugar monomer is polymerized on the surface of the polymer.

Examples of the sugar monomer include monomers in which a sugar structure is introduced into a known polymerizable monomer. For example, a sugar structure is introduced into the crosslinkable monomer or the non-crosslinkable monomer. Monomers are preferred. Specifically, for example, glycoside (meth) acrylate, glycosylethyl (meth) acrylate, glycosylpropyl (meth) acrylate, glycosylbutyl (meth) acrylate, glycosylethoxyethyl (meth) acrylate, D-mannosylethyl (meth) acrylate, Saccharified (meth) acrylates such as D-fructosylethyl (meth) acrylate, galactosylethyl (meth) acrylate, xylosylethyl (meth) acrylate, maltosylethyl (meth) acrylate, and lactosylethyl (meth) acrylate Is preferred. The cationic sugar monomer is also suitable as the sugar monomer.

As the compounding amount of the sugar monomer, a preferable lower limit with respect to 100 parts by weight of the crosslinkable (meth) acrylic acid ester (the total when non-crosslinkable monomers are added) is 1 part by weight, and a preferable upper limit. Is 100 parts by weight. If the amount is less than 1 part by weight, the amount of sugar structure introduced is too small to allow sufficient interaction with hemoglobins, and the peak to be measured may not be sufficiently separated. In some cases, aggregates are generated and a polymer cannot be obtained. A more preferred lower limit is 5 parts by weight, and a more preferred upper limit is 80 parts by weight.

Examples of the method for producing the column filler include the crosslinkable (meth) acrylic acid ester, non-crosslinkable monomer, cation exchange monomer, sugar monomer, and cation exchange sugar monomer. And a method of copolymerizing a monomer mixture such as the above. Specifically, for example, the following one-step polymerization method, two-step polymerization method or three-step polymerization method may be mentioned. Of these, a two-stage polymerization method or a three-stage polymerization method is preferable.

The one-stage polymerization method will be described.
In the one-step polymerization method, a polymerization reaction is carried out by adding a polymerization initiator and various additives as necessary to the monomer mixture and heating.
Specific examples of the polymerization method include known polymerization methods such as an emulsion polymerization method, a soap-free polymerization method, a dispersion polymerization method, a suspension polymerization method, and a seed polymerization method.

The two-stage polymerization method will be described.
In the two-stage polymerization method, first, the crosslinkable (meth) acrylic acid ester (non-crosslinkable monomer as required) and a cation exchange monomer are mixed, and a polymerization initiator or necessary Accordingly, various additives are added and heated to conduct a polymerization reaction to obtain a crosslinked polymer having a cation exchange group. Next, a sugar monomer is polymerized on the surface of the obtained crosslinked polymer having a cation exchange group.
Alternatively, first, the crosslinkable (meth) acrylic acid ester (non-crosslinkable monomer if necessary) and a sugar monomer are mixed, and a polymerization initiator and various additives are added as necessary. Then, the polymerization reaction is carried out by heating to obtain a crosslinked polymer having a sugar structure. Next, a cation exchange monomer is polymerized on the surface of the obtained crosslinked polymer having a sugar structure.
Alternatively, first, a polymerization initiator and various additives as necessary are added to the crosslinkable (meth) acrylic acid ester (if necessary, a non-crosslinkable monomer), and the polymerization reaction is performed by heating. To obtain a crosslinked polymer. Next, a mixture of a cation exchange monomer and a sugar monomer is polymerized on the surface of the obtained crosslinked polymer.

The three-stage polymerization method will be described.
In the above three-stage polymerization method, first, a polymerization initiator and various additives as required are added to the crosslinkable (meth) acrylic acid ester (non-crosslinkable monomer as required) and added. A polymerization reaction is performed by heating to obtain a crosslinked polymer. Next, a cation exchange monomer is polymerized on the surface of the obtained crosslinked polymer to obtain a crosslinked polymer having a cation exchange group. Finally, a sugar monomer is polymerized on the surface of the obtained crosslinked polymer having a cation exchange group.
Alternatively, first, a polymerization initiator and various additives as necessary are added to the crosslinkable (meth) acrylic acid ester (if necessary, a non-crosslinkable monomer), and the polymerization reaction is performed by heating. To obtain a crosslinked polymer. Next, on the surface of the obtained crosslinked polymer, a sugar monomer is polymerized to obtain a crosslinked polymer having a sugar structure. Finally, a cation exchange monomer is polymerized on the surface of the obtained crosslinked polymer having a sugar structure.

In the two-stage polymerization method or the three-stage polymerization method, when the cation exchange monomer and / or sugar monomer is polymerized on the surface of the crosslinkable polymer, for example, after the formation of the crosslinkable monomer, A method in which after the obtained crosslinkable polymer is washed, a polymerization initiator is impregnated into the crosslinkable polymer, and then a cation exchange monomer and / or a sugar monomer is added to the reaction system for polymerization; In the middle of the polymerization reaction of the polymerizable monomer, a method of polymerizing by adding a cation exchange monomer and / or a sugar monomer to the reaction system may be performed. In addition, when adding a cation exchange monomer and / or a sugar monomer, you may dilute with water, various aqueous solution, an organic solvent, etc.

It does not specifically limit as a polymerization initiator used in the said polymerization method, A water-soluble or oil-soluble well-known radical polymerization initiator can be used.
Examples of the radical polymerization initiator include persulfates such as potassium persulfate, sodium persulfate, and ammonium persulfate; cumene hydroperoxide, benzoyl peroxide, lauroyl peroxide, octanoyl peroxide, o-chlorobenzoyl peroxide Acetyl peroxide, t-butyl hydroperoxide, t-butyl peroxyacetate, t-butyl peroxyisobutyrate, 3,5,5-trimethylhexanoyl peroxide, t-butylperoxy-2-ethylhexa Organic peroxides such as noate and di-t-butyl peroxide; 2,2-azobisisobutyronitrile, 2,2-azobis (2,4-dimethylvaleronitrile), 4,4-azobis (4 -Cyanopentanoic acid), 2,2-azobi (2-methylbutyronitrile), 2,2-azobis (2,4-dimethylvaleronitrile), azo compounds such as azo-bis-cyclohexane carbonitrile and the like.

As for the usage-amount of the said polymerization initiator, a preferable minimum is 0.05 weight part with respect to 100 weight part of monomer mixtures, and a preferable upper limit is 5 weight part. When the amount is less than 0.05 parts by weight, the polymerization reaction may be insufficient, or the polymerization reaction may take a long time. When the amount exceeds 5 parts by weight, an agglomerate is generated due to rapid progress of the reaction. Sometimes.

In addition to the above, the column filler is, for example, a method of adsorbing or binding a polysaccharide to the surface of a crosslinked polymer having a cation exchange group, or adsorbing or binding a polysaccharide having a cation exchange group to the surface of the crosslinked polymer. It can be manufactured by a method or the like.

As the polysaccharide, known polysaccharides can be used. Specifically, for example, neutral polysaccharides such as cellulose, mannan, starch, chitin, dextran, chondroitin are suitable.
Examples of the polysaccharide having a cation exchange group include polysaccharides having a carboxyl group such as hyaluronic acid, muramic acid, pectinic acid and alginic acid; polysaccharides having a phosphate group such as tycoic acid and phosphomannan; chondroitin sulfate; Examples include polysaccharides having a sulfonic acid group such as dermatan sulfate, keratan sulfate, heparan sulfate, heparin, and fucoidan; and cation exchange group-introduced derivatives of the above neutral polysaccharide. Of these, polysaccharides having a sulfonic acid group are preferred.

The method for adsorbing or binding the polysaccharide or the polysaccharide having a cation exchange group to the surface of the crosslinked polymer is not particularly limited. For example, a solution in which the polysaccharide or the polysaccharide having a cation exchange group is dissolved is crosslinked. Examples include a method in which the polymer is brought into contact, placed in a heated, vacuum, or dried state as necessary, and further washed or the like as necessary. In addition, a functional group such as a hydroxyl group, an ester group, an amino group, a cation exchange group or the like possessed by the polysaccharide or the polysaccharide having a cation exchange group is reacted with a similar functional group possessed by a crosslinked polymer, and bonded by chemical bonding. You may let them.

When the polysaccharide or the polysaccharide having a cation exchange group is adsorbed or bonded to the surface of the crosslinked polymer, it is preferable to react these polysaccharides with the crosslinkable polymer in a solution state. In this case, a preferable lower limit of the polysaccharide concentration is 0.01% by weight, and a preferable upper limit is 20% by weight. If the amount is less than 0.01% by weight, the amount of polysaccharide introduced may be small and a sufficient effect may not be obtained. If the amount exceeds 20% by weight, aggregates tend to occur during the reaction, and uniform adsorption or Sometimes it is not possible to perform a bond.

The method for measuring hemoglobin of the present invention is measured by liquid chromatography using the above column filler.
The measurement is performed by connecting a column packed with the column packing material to a known liquid chromatography system, that is, a system including a pump, a sampler, a detector and the like for feeding an eluent.

In the method for measuring hemoglobin of the present invention, a buffer solution containing a known salt compound can be used as an eluent. Examples of such buffers include organic acids such as citric acid, succinic acid, tartaric acid and malic acid and salts thereof; amino acids such as glycine, taurine and arginine; hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boric acid, Examples thereof include inorganic acids such as acetic acid and salts thereof, and Good's buffers. In addition, conventionally known additives such as surfactants, various polymers, and hydrophilic low molecular compounds may be appropriately added to the buffer solution.
The preferable lower limit of the salt concentration of the buffer solution is 10 mM, and the preferable upper limit is 1000 mM. If the concentration is less than 10 mM, the ion exchange reaction may not be performed, and hemoglobin may not be separated. If the concentration exceeds 1000 mM, salt may precipitate to adversely affect the system.

According to the liquid chromatography method using the column filler, in addition to the hemoglobin A1c, abnormal hemoglobins such as hemoglobin S and hemoglobin C, hemoglobin F (fetal hemoglobin), hemoglobin A2 and the like are measured with high accuracy. be able to.

ADVANTAGE OF THE INVENTION According to this invention, the nonspecific adsorption | suction to the column filler of hemoglobin can be reduced and the measuring method of hemoglobin A1c which can measure with high precision can be provided.

It is the chromatogram obtained when measuring hemoglobin A1c using the column packing material of Example 1. It is the chromatogram obtained when measuring hemoglobin A1c using the column filler of the comparative example 1. FIG. It is the chromatogram obtained when measuring hemoglobin A1c using the column packing material of the comparative example 3. FIG. It is the chromatogram obtained when measuring abnormal hemoglobin using the column packing material of Example 6. It is the chromatogram obtained when measuring hemoglobin A2 using the column packing material of Example 6.

Hereinafter, embodiments of the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

Example 1
300 g of triethylene glycol dimethacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical), 50 g of trimethylolethane triacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical), methacrylic acid (cation-exchangeable monomer: Wako Pure) 1.0 g of benzoyl peroxide (polymerization initiator: manufactured by Nacalai Tesque) was dissolved in a mixture of 100 g of drug and 50 g of glycosylethyl methacrylate (sugar monomer). The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous solution of polyvinyl alcohol (GOHSENOL GH-20: manufactured by Nippon Synthetic Chemical), heated to 80 ° C. in a nitrogen atmosphere with stirring, and polymerized for 12 hours. Was done. The obtained polymer was washed with ion-exchanged water and acetone to obtain a crosslinked polymer (column filler) having a cation exchange group and a sugar structure. The obtained packing material was packed into a stainless steel column having an inner diameter of 3 mm and a length of 30 mm to obtain a column.

The obtained column was connected to an HPLC system (LC-10A manufactured by Shimadzu Corporation). As a measurement sample, blood of healthy human blood collected from sodium fluoride was diluted 200 times with a phosphate buffer solution (pH 6.8) containing 0.05% by weight of Triton X-100. Two types of eluents, eluent A: 200 mM phosphate buffer (pH 5.3) and eluent B: 400 mM phosphate buffer (pH 7.5), were sent stepwise with a flow rate of 1.0 mL. Separated by a gradient method, the absorbance at 415 nm was measured. As a result of measuring the above sample, the chromatogram of FIG. 1 was obtained. In FIG. 1, peak 1 is hemoglobin A1c, and peak 2 is hemoglobin Ao. Hemoglobin A1c was well separated from other hemoglobin fractions within a short time.

(Example 2)
250 g of tetraethylene glycol dimethacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical), 50 g of 2-hydroxyethyl methacrylate (non-crosslinkable monomer: manufactured by Wako Pure Chemical Industries), 2-methacrylamide-2-methylpropanesulfonic acid 1.0 g of benzoyl peroxide was dissolved in a mixture of 100 g (cation exchangeable monomer: manufactured by Toagosei Co., Ltd.) and 50 g of glycosylethyl methacrylate (sugar monomer). The obtained monomer mixture was polymerized in the same manner as in Example 1 to obtain a column filler.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Example 3)
200 g of diethylene glycol dimethacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical), 100 g of tetramethylolmethane triacrylate (crosslinked monomer: manufactured by Shin-Nakamura Chemical) and glucosyl 3-sulfopropylacrylic acid (single cation exchange sugar) Body) was dissolved 1.0 g of benzoyl peroxide. The obtained monomer mixture was polymerized in the same manner as in Example 1 to obtain a column filler.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

Example 4
1.0 g of benzoyl peroxide was dissolved in a mixture of 300 g of tetraethylene glycol dimethacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 150 g of trimethylolpropane triacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical). .
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., a mixture of 100 g of methacrylic acid (cation exchange monomer: manufactured by Wako Pure Chemical Industries) and 50 g of maltosylethyl acrylate (sugar monomer) was added to the reaction system, and the temperature was again increased to 80 ° C. The polymerization reaction was carried out for 1 hour after heating. The obtained polymer was washed with ion-exchanged water and acetone to obtain a crosslinked polymer (column filler) having a cation exchange group and a sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Example 5)
250 g of triethylene glycol dimethacrylate, 100 g of tetramethylolmethane triacrylate, 50 g of glycerol dimethacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of 2-hydroxyethyl methacrylate (non-crosslinkable monomer: manufactured by Wako Pure Chemical Industries) In this mixture, 1.2 g of benzoyl peroxide was dissolved.
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., a mixture of 150 g of 2-methacrylamide-2-methylpropanesulfonic acid (cation exchange monomer) and 60 g of glycosylethyl methacrylate (sugar monomer) was added to the reaction system, and again The polymerization reaction was carried out for 1 hour by heating to 80 ° C. The obtained polymer was washed with ion-exchanged water and acetone to obtain a crosslinked polymer (column filler) having a cation exchange group and a sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Example 6)
1.2 g of benzoyl peroxide was dissolved in a mixture of 200 g of triethylene glycol dimethacrylate, 100 g of tetramethylolmethane triacrylate, 100 g of tetramethylolmethane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of glycerol dimethacrylate. .
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., a mixture of 140 g of 2-methacrylamido-2-methylpropanesulfonic acid (cation exchange monomer) and 60 g of glycosylethoxyethyl methacrylate (sugar monomer) is added to the reaction system, The mixture was heated again to 80 ° C. and polymerized for 1 hour. The obtained polymer was washed with ion-exchanged water and acetone to obtain a crosslinked polymer (column filler) having a cation exchange group and a sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Example 7)
1.0 g of benzoyl peroxide was dissolved in 450 g of tetraethylene glycol dimethacrylate. The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., a mixture of 160 g of (2-acryloyloxyethyl) acid phosphate (cation exchange monomer: manufactured by NOF Corporation) and 40 g of glycosylethyl methacrylate (sugar monomer) was added to the reaction system. Then, the mixture was heated again to 80 ° C., and a polymerization reaction was performed for 1 hour. The obtained polymer was washed with ion-exchanged water and acetone to obtain a crosslinked polymer (column filler) having a cation exchange group and a sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Example 8)
1.2 g of benzoyl peroxide was dissolved in a mixture of 250 g of triethylene glycol dimethacrylate, 100 g of tetramethylolmethane triacrylate, 50 g of glycerol dimethacrylate and 50 g of 2-hydroxyethyl methacrylate.
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., 150 g of glucosyl 3-sulfopropylacrylic acid (cation exchange sugar monomer) was added to the reaction system, and the mixture was heated again to 80 ° C. to conduct a polymerization reaction for 1 hour. The obtained polymer was washed with ion-exchanged water and acetone to obtain a crosslinked polymer (column filler) having a cation exchange group and a sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

Example 9
To a mixture of 250 g of triethylene glycol dimethacrylate, 50 g of tetramethylolmethane triacrylate, 50 g of glycerol dimethacrylate and 150 g of 2-methacrylamide-2-methylpropanesulfonic acid (cation exchange monomer), 1.2 g of benzoyl peroxide was added. Dissolved.
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., 150 g of glycosyl ethoxyethyl methacrylate (sugar monomer) was added to the reaction system, and the mixture was heated again to 80 ° C. to conduct a polymerization reaction for 1 hour. The obtained polymer was washed with ion-exchanged water and acetone to obtain a crosslinked polymer (column filler) having a cation exchange group and a sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Example 10)
1.2 g of benzoyl peroxide was dissolved in a mixture of 250 g of triethylene glycol dimethacrylate, 50 g of tetramethylolmethane triacrylate, 50 g of glycerol dimethacrylate and 150 g of glycosylethoxyethyl methacrylate (sugar monomer).
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., 150 g of 2-methacrylamideamido-2-methylpropane sulfonic acid (cation exchange monomer) is added to the reaction system, and the mixture is heated again to 80 ° C. to conduct a polymerization reaction for 1 hour. It was. The obtained polymer was washed with ion-exchanged water and acetone to obtain a crosslinked polymer (column filler) having a cation exchange group and a sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Example 11)
1.2 g of benzoyl peroxide was dissolved in a mixture of 200 g of triethylene glycol dimethacrylate, 100 g of tetramethylolmethane triacrylate, 100 g of tetramethylolmethane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of glycerol dimethacrylate. .
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., 140 g of 2-methacrylamideamido-2-methylpropanesulfonic acid (cation exchange monomer) was added to the reaction system, and the mixture was heated again to 80 ° C. to conduct a polymerization reaction. After 0.5 hour, 60 g of glycosyl ethoxyethyl methacrylate (sugar monomer) was added to the reaction system, and the polymerization reaction was further carried out for 1 hour. The obtained polymer was washed with ion-exchanged water and acetone to obtain a crosslinked polymer (column filler) having a cation exchange group and a sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Example 12)
1.2 g of benzoyl peroxide was dissolved in a mixture of 200 g of triethylene glycol dimethacrylate, 100 g of tetramethylolmethane triacrylate, 100 g of tetramethylolmethane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of glycerol dimethacrylate. .
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., 60 g of glycosylethoxyethyl methacrylate (sugar monomer) was added to the reaction system, and the temperature was again raised to 80 ° C. to carry out the polymerization reaction. After 0.5 hour, 140 g of 2-methacrylamide-2-methylpropanesulfonic acid (cation exchangeable monomer) was added to the reaction system, and the polymerization reaction was further performed for 1 hour. The obtained polymer was washed with ion-exchanged water and acetone to obtain a crosslinked polymer (column filler) having a cation exchange group and a sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Example 13)
200 g of triethylene glycol dimethacrylate, 100 g of tetramethylol methane triacrylate, 100 g of tetramethylol methane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical), 50 g of glycerol dimethacrylate and 200 g of methacrylic acid (cation exchange monomer) In the mixture, 1.2 g of benzoyl peroxide was dissolved.
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and subjected to a polymerization reaction for 12 hours. The obtained polymer was washed with ion-exchanged water and acetone, and then the polymer was immersed in an aqueous solution of 2% by weight dextran (polysaccharide) and heated at 40 ° C. for 24 hours to obtain a cation exchange group and a sugar structure. A cross-linked polymer (column filler) having was obtained.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Example 14)
In a mixture of 300 g of triethylene glycol dimethacrylate, 50 g of trimethylolethane triacrylate and 150 g of 2-methacrylamide-2-methylpropanesulfonic acid (cation exchange monomer), 1.0 g of benzoyl peroxide was dissolved.
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and subjected to a polymerization reaction for 12 hours. After the obtained polymer was washed with ion-exchanged water and acetone, the polymer was immersed in an aqueous solution of 1.5% by weight cellulose (polysaccharide) and heated at 40 ° C. for 24 hours, whereby cation exchange groups and A crosslinked polymer (column filler) having a sugar structure was obtained.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Example 15)
1.2 g of benzoyl peroxide was dissolved in a mixture of 200 g of triethylene glycol dimethacrylate, 100 g of tetramethylolmethane triacrylate, 100 g of tetramethylolmethane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of glycerol dimethacrylate. .
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., 150 g of 2-methacrylamideamido-2-methylpropanesulfonic acid (cation exchange monomer) was added to the reaction system, and the temperature was again raised to 80 ° C. to conduct a polymerization reaction. The obtained polymer was washed with ion-exchanged water and acetone, and then the polymer was immersed in an aqueous solution of 2% by weight dextran (polysaccharide) and heated at 40 ° C. for 24 hours to obtain a cation exchange group and a sugar structure. A cross-linked polymer (column filler) having was obtained.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Example 16)
1.2 g of benzoyl peroxide was dissolved in a mixture of 200 g of triethylene glycol dimethacrylate, 100 g of tetramethylolmethane triacrylate, 100 g of tetramethylolmethane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of glycerol dimethacrylate. .
The obtained polymer was washed with ion-exchanged water and acetone, then immersed in an aqueous solution of 2% by weight dextran (polysaccharide), and heated at 40 ° C. for 24 hours to obtain a polysaccharide-coated crosslinked polymer. Obtained.
300 g of the obtained polysaccharide-coated crosslinked polymer was dispersed in 1 L of an acetone solution containing 1% by weight of benzoyl peroxide, and stirred for 10 hours. The polymer in which benzoyl peroxide was absorbed was dispersed in 5 L of a 4 wt% aqueous polyvinyl alcohol solution, and 150 g of 2-methacrylamide-2-methylpropanesulfonic acid (cation exchange monomer) was further added to the reaction system. While stirring, the mixture was heated to 80 ° C. in a nitrogen atmosphere and subjected to a polymerization reaction for 3 hours to obtain a crosslinked polymer (column filler) having a cation exchange group and a sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Example 17)
1.2 g of benzoyl peroxide was dissolved in a mixture of 200 g of triethylene glycol dimethacrylate, 100 g of tetramethylolmethane triacrylate, 100 g of tetramethylolmethane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of glycerol dimethacrylate. .
The obtained polymer was washed with ion-exchanged water and acetone, and then the polymer was immersed in 1 L of a 2% by weight aqueous solution of chondroitin sulfate (manufactured by Wako Pure Chemicals: cation-exchangeable polysaccharide) and heated at 40 ° C. for 24 hours. As a result, a crosslinked polymer coated with a cationic polysaccharide, that is, a crosslinked polymer having a cation exchange group and a sugar structure (column filler) was obtained.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Example 18)
1.2 g of benzoyl peroxide was dissolved in a mixture of 200 g of triethylene glycol dimethacrylate, 100 g of tetramethylolmethane triacrylate, 100 g of tetramethylolmethane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of glycerol dimethacrylate. .
The obtained polymer was washed with ion-exchanged water and acetone, and then the polymer was immersed in 1 L of a 2% by weight dextran sulfate (cation-exchangeable polysaccharide) aqueous solution and heated at 40 ° C. for 24 hours to obtain a polysaccharide. A coated crosslinked polymer was obtained.
300 g of the obtained polysaccharide-coated crosslinked polymer was dispersed in 1 L of an acetone solution containing 1% by weight of benzoyl peroxide, and stirred for 10 hours. The polymer in which benzoyl peroxide was absorbed was dispersed in 5 L of a 4 wt% aqueous polyvinyl alcohol solution, and 150 g of 2-methacrylamide-2-methylpropanesulfonic acid (cation exchange monomer) was further added to the reaction system. While stirring, the mixture was heated to 80 ° C. in a nitrogen atmosphere and subjected to a polymerization reaction for 3 hours to obtain a crosslinked polymer (column filler) having a cation exchange group and a sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Example 19)
1.2 g of benzoyl peroxide was dissolved in a mixture of 200 g of triethylene glycol dimethacrylate, 100 g of tetramethylolmethane triacrylate, 100 g of tetramethylolmethane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of glycerol dimethacrylate. .
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., 150 g of 2-methacrylamideamido-2-methylpropane sulfonic acid (cation exchange monomer) is added to the reaction system, and the mixture is heated again to 80 ° C. to conduct a polymerization reaction for 1 hour. It was. After the obtained polymer was washed with ion-exchanged water and acetone, the polymer was immersed in 1 L of a 1.5% by weight aqueous solution of chondroitin sulfate (cation-exchangeable polysaccharide) and heated at 40 ° C. for 24 hours. Then, a crosslinked polymer (column filler) having a cation exchange group and a sugar structure was obtained.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 1 was obtained.

(Comparative Example 1)
A column filler was prepared in the same manner as in Example 1 except that no sugar monomer was used.
That is, 1.0 g of benzoyl peroxide (polymerization initiator: manufactured by Nacalai Tesque) was dissolved in a mixture of 300 g of triethylene glycol dimethacrylate, 50 g of trimethylolethane triacrylate and 100 g of methacrylic acid (cation exchange monomer).
The obtained monomer mixture was polymerized according to the method of Example 1 to obtain a crosslinked polymer (column filler) having a cation exchange group and having no sugar structure.
When human blood was measured by the same method as in Example 1, the chromatogram of FIG. 2 was obtained. In FIG. 2, peak 1 is hemoglobin A1c and peak 2 is hemoglobin Ao. Compared to Example 1, hemoglobin A1c separation was poor.

(Comparative Example 2)
A column filler was prepared in the same manner as in Example 2 except that no sugar monomer was used.
That is, 1.0 g of benzoyl peroxide was dissolved in a mixture of 250 g of tetraethylene glycol dimethacrylate, 50 g of 2-hydroxyethyl methacrylate, and 100 g of 2-methacrylamide-2-methylpropanesulfonic acid (cation exchange monomer).
The obtained monomer mixture was polymerized according to the method of Example 1 to obtain a crosslinked polymer (column filler) having a cation exchange group and having no sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 2 was obtained.

(Comparative Example 3)
A column filler was prepared in the same manner as in Example 2 except that no cation exchange monomer was used.
That is, 1.0 g of benzoyl peroxide was dissolved in a mixture of 250 g of tetraethylene glycol dimethacrylate, 50 g of 2-hydroxyethyl methacrylate and 50 g of glycosylethyl methacrylate (sugar monomer).
The obtained monomer mixture was polymerized according to the method of Example 1 to obtain a crosslinked polymer (column filler) having a sugar structure and having no cation exchange group.
When human blood was measured by the same method as in Example 1, the chromatogram of FIG. 3 was obtained. With the column packing material of this comparative example having no cation exchange group, hemoglobin A1c could not be separated.

(Comparative Example 4)
A column filler was prepared in the same manner as in Example 6 except that no sugar monomer was used.
That is, 1.2 g of benzoyl peroxide was added to a mixture of 200 g of triethylene glycol dimethacrylate, 100 g of tetramethylol methane triacrylate, 100 g of tetramethylol methane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of glycerol dimethacrylate. Dissolved.
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., 140 g of 2-methacrylamideamido-2-methylpropanesulfonic acid (cation exchange monomer) is added to the reaction system, and the mixture is heated again to 80 ° C. to conduct a polymerization reaction for 1 hour. It was. The obtained polymer was washed with ion exchange water and acetone to obtain a crosslinked polymer (column filler) having a cation exchange group and having no sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 2 was obtained.

(Comparative Example 5)
A column filler was prepared in the same manner as in Example 6 except that the cation exchange monomer was not used.
That is, 1.2 g of benzoyl peroxide was added to a mixture of 200 g of triethylene glycol dimethacrylate, 100 g of tetramethylol methane triacrylate, 100 g of tetramethylol methane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of glycerol dimethacrylate. Dissolved.
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., 60 g of glycosylethoxyethyl methacrylate (sugar monomer) was added to the reaction system, and the mixture was heated again to 80 ° C. to conduct a polymerization reaction for 1 hour. The obtained polymer was washed with ion exchange water and acetone to obtain a crosslinked polymer (column filler) having a sugar structure and having no cation exchange group.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 3 was obtained.

(Comparative Example 6)
A column filler was prepared in the same manner as in Example 14 except that the cation exchange monomer was not used.
That is, 1.0 g of benzoyl peroxide was dissolved in a mixture of 300 g of triethylene glycol dimethacrylate and 50 g of trimethylolethane triacrylate.
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and subjected to a polymerization reaction for 12 hours. After the obtained polymer was washed with ion-exchanged water and acetone, the polymer was immersed in an aqueous solution of 1.5% by weight cellulose (polysaccharide) and heated at 40 ° C. for 24 hours, whereby cation exchange groups and A crosslinked polymer (column filler) having a sugar structure was obtained.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 3 was obtained.

(Comparative Example 7)
A column filler was prepared in the same manner as in Example 6 except that a monomer having a non-sugar polyhydroxyl group was used in place of the sugar monomer.
That is, 1.2 g of benzoyl peroxide was added to a mixture of 200 g of triethylene glycol dimethacrylate, 100 g of tetramethylol methane triacrylate, 100 g of tetramethylol methane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of glycerol dimethacrylate. Dissolved.
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., 140 g of 2-methacrylamide-2-methylpropanesulfonic acid (cation exchange monomer) and 2,3-dihydroxyprol methacrylate (monomer having a hydroxyl group other than sugar structure) (Manufactured by Wako Pure Chemical Industries, Ltd.) 60 g of the mixture was added to the reaction system, and the mixture was heated again to 80 ° C. to conduct a polymerization reaction for 1 hour. The obtained polymer was washed with ion-exchanged water and acetone to obtain a crosslinked polymer (column filler) having a cation exchange group and a sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 2 was obtained.

(Comparative Example 8)
A column filler was prepared in the same manner as in Example 15 except that a monomer having a non-sugar polyhydroxyl group was used in place of the sugar monomer.
That is, 1.2 g of benzoyl peroxide was added to a mixture of 200 g of triethylene glycol dimethacrylate, 100 g of tetramethylol methane triacrylate, 100 g of tetramethylol methane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of glycerol dimethacrylate. Dissolved.
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., 150 g of 2-methacrylamideamido-2-methylpropanesulfonic acid (cation exchange monomer) was added to the reaction system, and the temperature was again raised to 80 ° C. to conduct a polymerization reaction. The obtained polymer was washed with ion-exchanged water and acetone, and then the polymer was added to an aqueous solution of 2% by weight of polyethylene glycol (average molecular weight of about 20,000: compound having a polyhydroxyl group other than sugar structure: manufactured by Wako Pure Chemical Industries, Ltd.). Was heated at 40 ° C. for 24 hours to obtain a crosslinked polymer (column filler) having a cation exchange group and a sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 2 was obtained.

(Comparative Example 9)
A column filler was prepared in the same manner as in Example 19 except that a cation-exchange polysaccharide having a non-sugar structure was used instead of the cation-exchange polysaccharide.
That is, 1.2 g of benzoyl peroxide was added to a mixture of 200 g of triethylene glycol dimethacrylate, 100 g of tetramethylol methane triacrylate, 100 g of tetramethylol methane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of glycerol dimethacrylate. Dissolved.
The obtained monomer mixture was dispersed in 5 L of a 4% by weight aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was carried out for 1 hour. After cooling the temperature to 30 ° C., 150 g of 2-methacrylamideamido-2-methylpropane sulfonic acid (cation exchange monomer) is added to the reaction system, and the mixture is heated again to 80 ° C. to conduct a polymerization reaction for 1 hour. It was. The obtained polymer was washed with ion-exchanged water and acetone, and then the polymer was added to 1 L of an aqueous solution of 5% by weight of polymethacrylic acid (molecular weight: about 100,000: cation-exchangeable compound having no sugar structure: manufactured by Wako Pure Chemical Industries). Was heated at 40 ° C. for 24 hours to obtain a crosslinked polymer (column filler) having a cation exchange group and a sugar structure.
When human blood was measured by the same method as in Example 1, the same chromatogram as in FIG. 2 was obtained.

(Evaluation of column packing material 1: recovery rate test)
A recovery rate test was performed using the obtained column filler. In the recovery test, a PEEK pipe having an inner diameter of 0.25 mm and a length of 1000 mm is attached to the HPLC instead of the column, and the peak total area when the sample is measured is 100%. It calculated | required by the ratio with respect to the said PEEK piping time of a total area. The obtained results are shown in Tables 1 and 2.

(Evaluation of column packing 2: Evaluation of separation performance of modified hemoglobins)
Instead of healthy human blood used in the above examples, a sample containing modified hemoglobins was artificially prepared and measured, and the separation performance from the hemoglobin A1c peak was evaluated.
Three types of modified hemoglobins were prepared by a known method: a labile hemoglobin A1c-containing sample (sample L), an acetylated hemoglobin-containing sample (sample A), and a carbamylated hemoglobin-containing sample (sample C).
That is, the sample L was prepared by adding glucose to healthy whole blood so as to be 2000 mg / dL and heating at 37 ° C. for 3 hours. Sample A was prepared by adding acetaldehyde at 50 mg / dL to healthy human whole blood and heating at 37 ° C. for 2 hours. Sample C was prepared by adding sodium cyanate to 50 mg / dL and heating at 37 ° C. for 2 hours.

The above-mentioned modified hemoglobin sample (sample L, sample A, sample C) and healthy human blood (unmodified product) used for the preparation of modified hemoglobin were measured using the obtained column filler, and the measured value of hemoglobin A1c was measured. Compared.
Separation performance was performed by calculating and comparing a value (Δ value) obtained by subtracting the hemoglobin A1c value of the unmodified product from the hemoglobin A1c value of the modified hemoglobin sample. The obtained Δ values are shown in Tables 1 and 2.

From Tables 1 and 2, when the column fillers of the examples are used, the recovery rate is 82% or more, and the column fillers manufactured by the two-stage polymerization method which is a particularly preferred embodiment are used (Examples 4 to 4). 10) In the case of using a column filler produced by a three-stage polymerization method (Examples 11 and 12) and in the case of using a column filler produced by a polysaccharide coating method (Examples 14 to 19) A recovery rate of almost 100% was obtained.
On the other hand, the column fillers (Comparative Examples 1, 2, 4, and 9) having no sugar structure had a low recovery rate of 70% or less. This is presumed to be because hemoglobin as a measurement target did not elute due to nonspecific adsorption to the column packing material. Further, when polymerized or coated with a polyhydroxy compound other than sugar (Comparative Examples 7 and 8), 75-76%, a slight improvement was seen when compared with the case without a sugar structure, but when compared with Examples. Significantly lower. Comparative Examples 3, 5, and 6 showed high recoveries, but these column fillers did not have a cation exchange group, and thus hemoglobin A1c could not be separated.

From Tables 1 and 2, it was found that the Δ value of the example is about 0.3% or less, and that hemoglobin A1c can be measured accurately even in the sample containing the modified hemoglobin. In particular, Examples 4 to 19 were hardly affected by the modified hemoglobin.
On the other hand, when the column filler of the comparative example was used, the hemoglobin A1c value fluctuated greatly when the modified hemoglobin was present, and the hemoglobin A1c value could not be measured accurately.

(Evaluation of column packing 3: Evaluation of separation performance of abnormal hemoglobins)
Using the column filler thus obtained, a sample containing hemoglobin S and hemoglobin C as abnormal hemoglobin (AFSC hemocontrol: manufactured by Helena Laboratories) was measured.
FIG. 4 shows the chromatogram obtained as a result of measurement using the column packing material of Example 6. In FIG. 4, peak 1 represents hemoglobin HbA1c, peak 2 represents hemoglobin Ao, peak 4 represents hemoglobin F (fetal Hb), peak 5 represents hemoglobin S, and peak 6 represents hemoglobin C. Even when the column fillers of other examples were used, almost the same separation performance was exhibited.
On the other hand, when measured using the column filler of the comparative example, hemoglobin S and hemoglobin C, which are abnormal hemoglobins, could not be separated.

(Evaluation of column packing 4: Evaluation of separation performance of hemoglobin A2)
Using the obtained column filler, A2 control (level 2) (manufactured by Bio-Rad) was measured as a sample containing hemoglobin A2.
As a result of measurement using the column packing material of Example 6, the obtained chromatogram is shown in FIG. In FIG. 5, peak 1 represents hemoglobin HbA1c, peak 2 represents hemoglobin Ao, peak 4 represents hemoglobin F (fetal Hb), and peak 7 represents hemoglobin A2. Even when the column fillers of other examples were used, almost the same separation performance was exhibited.
On the other hand, when measured using the column filler of the comparative example, hemoglobin A2 could not be separated.

ADVANTAGE OF THE INVENTION According to this invention, the nonspecific adsorption | suction to the column filler of hemoglobin can be reduced and the measuring method of hemoglobin A1c which can measure with high precision can be provided.

Claims (8)

A method for measuring hemoglobin A1c by a cation exchange chromatography method,
A method for measuring hemoglobin A1c, comprising using a methacrylic acid ester crosslinked polymer having a cation exchange group and a sugar structure introduced at least on the surface as a column filler. The cation exchange group is derived from a polymer obtained by polymerizing a monomer having a cation exchange group and / or a functional group that can be converted into a cation exchange group. Method for measuring hemoglobin A1c. The method for measuring hemoglobin A1c according to claim 1 or 2, wherein the sugar structure is derived from a polymer obtained by polymerizing saccharified (meth) acrylates. The method for measuring hemoglobin A1c according to claim 1 or 2, wherein the sugar structure is derived from a polysaccharide adsorbed or bonded to the surface of a crosslinked polymer of (meth) acrylic acid ester. The method for measuring hemoglobin A1c according to claim 4, wherein the polysaccharide is a compound having a cation exchange group and / or a functional group convertible to a cation exchange group. The cation exchange group and the sugar structure are derived from a polymer obtained by polymerizing a monomer having a sugar structure and having a functional group that can be converted into a cation exchange group and / or a cation exchange group. The method for measuring hemoglobin A1c according to claim 1, wherein the method is used. The column filler is a monomer having a cation exchange group and / or a functional group convertible to a cation exchange group on the surface of a cross-linked polymer of (meth) acrylic acid ester having a sugar structure on the surface. The method for measuring hemoglobin A1c according to claim 1, 2, 3, 4, or 5, wherein the hemoglobin A1c is produced by a method of polymerizing sucrose. A column filler used for measuring hemoglobin A1c by a cation exchange chromatography method, wherein a cation exchange group and a sugar structure are introduced on at least the surface of a crosslinked polymer of (meth) acrylate. Column filler for measuring hemoglobin A1c.

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