JPH087198B2 - Quantitative method for glycated hemoglobin - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for quantifying glycated hemoglobin, and more particularly to a method for quantifying glycated hemoglobin using liquid chromatography.

(Prior Art) Glycated hemoglobin is formed by non-enzymatic reaction of hemoglobin in red blood cells with glucose in blood. Since the average concentration of glucose in blood can be known by measuring glycated hemoglobin, the measurement of glycated hemoglobin is widely used for the diagnosis of diabetes. Glycated hemoglobin is currently quantified mainly by high performance liquid chromatography (hereinafter referred to as HPLC). HPLC
According to the method, it is possible to perform quick measurement as compared with the conventional column chromatography method, electrophoresis method, colorimetric method, and the like.

The packing material used in HPLC when measuring glycated hemoglobin is generally a weakly cationic ion exchange resin, and usually an ion exchange resin having a carboxyl group as an ion exchange group is used. As such a HPLC packing material, an organic polymer packing material or an inorganic packing material is mainly used. As the organic polymer-based filler, for example, JP-A-58-221164 discloses a filler made of a copolymer of an ester of acrylic acid or methacrylic acid such as tetramethylolmethane triacrylate and acrylic acid or methacrylic acid. Has been done. As an inorganic filler, JP-A-63-75558 discloses a filler in which a carboxyl group is introduced into a silica base material.

Generally, glycated hemoglobin is quantified by a step or continuous gradient method using two kinds of eluents, a liquid having a weak elution power (hereinafter referred to as the first liquid) and a liquid having a strong elution power (hereinafter referred to as the second liquid). Be implemented. The first liquid increases the free carboxyl groups in the filler. As a result, hemoglobin other than glycated hemoglobin in the sample is retained by the packing material, and glycated hemoglobin is separated and eluted. Second
Since the liquid has high ionic strength, the free carboxyl groups become salts. Therefore, hemiglobin other than the retained glycated hemoglobin is rapidly eluted.

However, when the above method uses a polymer-based filler, there are the following problems. Since the polymer-based filler is prepared by using a hydrophobic and hydrophilic monomer as disclosed in, for example, JP-A-58-221164, the ion-exchange group mainly derived from the hydrophilic monomer is used as the filler particle. Distributed throughout. When the second liquid is brought into contact with such a packing material, the packing material swells and the pressure in the column increases. In order to measure the next sample after measuring one sample, it is necessary to flow the first liquid to restore the carboxyl groups to the free form. At this time, since it is necessary to flow a considerable amount of the first liquid in order to sufficiently return the ion exchange groups existing inside the filler to the free form, the measurement time becomes long. It has become possible to measure glycated hemoglobin at a relatively high speed by HPLC using a polymer-based packing material. However, as the measurement speed is further increased, as described above, the ion-exchange groups inside the packing material do not sufficiently exchange, Alternatively, the separation performance decreases because the filler swells. In order to perform separation with high precision, it is necessary to reduce the elution rate.

On the other hand, silica-based inorganic fillers are generally excellent in pressure resistance and swelling resistance, but it has been pointed out that non-specific adsorption of proteins is likely to occur due to silanol groups remaining on the surface.

(Problems to be Solved by the Invention) The present invention is intended to solve the above-mentioned drawbacks of the conventional method, and an object thereof is to provide a method for highly accurately separating and quantifying glycated hemoglobin by HPLC in a short time. To provide. Another object of the present invention is to analyze by HPLC using a packing material that does not increase the column pressure during column operation, equilibrates quickly with the eluent, and does not nonspecifically adsorb proteins and the like. It is to provide a method for effectively quantifying glycated hemoglobin.

(Means for Solving the Problems) The present invention is a method for quantifying glycated hemoglobin in a sample by liquid chromatography, wherein the filler used in the liquid chromatography is a surface portion of hydrophobic cross-linked polymer particles. And coated polymer particles having a layer of acrylic acid and / or methacrylic acid (co) polymer formed thereon.

The material of the hydrophobic crosslinked polymer particles used for preparing the filler used in the method of the present invention is a (co) polymer or a hydrophobic polymer obtained by (co) polymerizing a hydrophobic crosslinkable monomer. A copolymer of a hydrophilic crosslinkable monomer and a hydrophobic non-crosslinkable monomer is used. The above-mentioned hydrophobic crosslinkable monomer and hydrophobic non-crosslinkable monomer may be used alone or in combination of two or more kinds. Examples of the hydrophobic crosslinkable monomer include di (meth) acryl such as ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate, propylene glycol di (meth) acrylate and polypropylene glycol di (meth) acrylate. Acid ester; poly (meth) acrylic acid ester of polyhydric alcohol such as tetramethylolmethane tri (meth) acrylate, tetramethylolmethane tetra (meth) acrylate; two or more such as divinylbenzene, divinyltoluene, divinylxylene, divinylnaphthalene An aromatic compound having a vinyl group is used. As the hydrophobic non-crosslinkable monomer, any non-crosslinkable polymerizable monomer having a hydrophobic property can be used. For example, (meth) acrylic acid esters such as methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, isopropyl (meth) acrylate, butyl (meth) acrylate, t-butyl (meth) acrylate;
Vinyl acetate; and styrene-based monomers such as styrene and methylstyrene are used. When the crosslinkable and non-crosslinkable monomers are mixed and used, the amount of the crosslinkable monomer is 10 parts by weight or more, preferably 20 parts by weight or more, relative to 100 parts by weight of the total monomers.
It is used so as to be more than weight part.

The polymerization initiator used when preparing the above hydrophobic crosslinked polymer particles and the polymerization initiator (described later) to be impregnated in the obtained hydrophobic crosslinked polymer particles are radical generating catalysts, and are hydrophobic. There is no particular limitation as long as it exists. For example, any of known radical generating catalysts such as organic peroxides such as benzoyl peroxide and cumene peroxide; azo compounds such as azobisisobutyronitrile and azobisisobutyroamide can be used.

Acrylic acid and / or methacrylic acid (hereinafter referred to as (meth) acrylic acid) used for coating the above hydrophobic crosslinked polymer particles have a carboxyl group, and therefore, are polymerized on the surface of the hydrophobic crosslinked polymer particles. Then, the carboxyl group functions as an ion exchange group.
Other than acrylic acid and methacrylic acid, other monomers can be used as long as they have a carboxyl group and are water-soluble. The amount of (meth) acrylic acid used depends on the type of monomer, but it is usually a hydrophobic cross-linked polymer.
It is 5 to 50 parts by weight with respect to 100 parts by weight.

In order to prepare the packing material for liquid chromatography used in the method of the present invention, first, hydrophobic crosslinked polymer particles are prepared. The hydrophobic crosslinked polymer particles can be prepared by any known aqueous suspension polymerization method. First, the hydrophobic crosslinkable monomer, and optionally the hydrophobic non-crosslinkable monomer and the polymerization initiator are dissolved in a diluent. As the diluent, any organic solvent that dissolves the above monomer and does not dissolve the polymer can be used. Examples thereof include aromatic hydrocarbons such as toluene, xylene, diethylbenzene and dodecylbenzene; saturated hydrocarbons such as hexane, heptane, octane and decane; alcohols such as isoamyl alcohol, hexyl alcohol and octyl alcohol. The amount used is not particularly limited, but it is preferably 15 to 200 parts by weight with respect to 100 parts by weight of the above monomer. The above diluted monomer solution was added to an aqueous phase in which a suspension stabilizer such as polyvinyl alcohol and calcium phosphate was dispersed, and the mixture was replaced with nitrogen and stirred for 50 to 10 minutes.
Suspension polymerization is carried out by heating to 0 ° C. Since the organic solvent that is a diluent is dispersed and present in the obtained polymer particles, by removing the organic solvent after completion of the polymerization,
Porous spherical particles are obtained. By using various organic solvents having different compatibility with the above-mentioned hydrophobic monomer mixture as a diluent, it is possible to arbitrarily change the size of the pores of the porous polymer.

Next, the obtained hydrophobic crosslinked polymer particles are impregnated with a polymerization initiator. In order to impregnate the polymerization initiator, the polymerization initiator is dissolved in a solvent having a low boiling point and having a good affinity for the hydrophobic crosslinked polymer, and the hydrophobic crosslinked polymer particles are immersed in the solvent. This causes the polymerization initiator to penetrate into the particles. If the solvent is distilled off while heating at a temperature below the decomposition point of the polymerization initiator, if necessary, particles containing the polymerization initiator in the hydrophobic crosslinked polymer particles can be obtained. The polymerization initiator-containing particles are dispersed in an aqueous dispersion medium in which (meth) acrylic acid is dissolved, or (meth) acrylic acid is added to the aqueous medium in which the particles are dispersed, and the mixture is heated under stirring after nitrogen substitution. And polymerize. In order to stabilize the dispersibility of the hydrophobic cross-linked polymer in the aqueous dispersion medium, it is desirable to use a dispersion stabilizer such as carboxymethyl cellulose or polyvinyl alcohol. The temperature and time of polymerization vary depending on whether acrylic acid or methacrylic acid is used or the type of polymerization initiator, but at 50 to 100 ° C 0.5
~ 10 hours.

In addition to the method of subjecting the crosslinked polymer particles impregnated with the above-mentioned polymerization initiator to the polymerization reaction of (meth) acrylic acid, a continuous method of reacting (meth) acrylic acid subsequent to the preparation of the crosslinked polymer particles may also be used. Layered polymer particles can be prepared. In this method, first, a polymerization reaction for preparing the crosslinked polymer particles is started. (Meth) acrylic acid is added to the reaction system when the polymerization proceeds to some extent and unreacted monomers remain. In such a state, since the polymerization initiator is present in the oil layer in the system and inside the formed hydrophobic crosslinked polymer particles, the polymerization of (meth) acrylic acid continues, and the hydrophobic crosslinked polymer particles A (co) polymer layer of (meth) acrylic acid is formed so as to cover the surface portion of the polymer.

The polymer particles obtained by each of the above methods are thoroughly washed with hot water, an organic solvent or the like to remove the suspension stabilizer, solvent, residual monomers and the like adhering to the particles. Further, if necessary, the particles are classified to obtain a packing material for chromatography.

In order to measure glycated hemoglobin by the method of the present invention, first, the blood of the sample is hemolyzed if necessary. This is applied to a column packed with the above-mentioned packing material, and glycated hemoglobin is quantified by an ordinary liquid chromatography method. By selecting an appropriate buffer solution, glycated hemoglobin in the sample and then other hemoglobin are separated and sequentially eluted.

The filler used in the method of the present invention is composed of polymer particles having a two-layer structure having a hydrophobic cross-linked polymer as a skeleton and a surface portion of the hydrophobic cross-linked polymer covered with a (meth) acrylic acid polymer. By using a polymer having a high degree of cross-linking as the skeleton, a packing material for liquid chromatography having extremely high mechanical strength and excellent pressure resistance can be obtained.
Since there is no hydrophilic group in the skeleton of this filler, the degree of swelling and shrinkage is extremely low. Therefore, in the quantification of glycated hemoglobin, the pressure rise during the passage of the second liquid is extremely small. Since only the surface part is covered with a (meth) acrylic acid polymer layer having a carboxyl group,
The ion exchange capacity of the packing material is high, and the equilibration of carboxyl groups is very fast. Therefore, the separation performance of glycated hemoglobin is excellent and the measurement can be performed in a short time. Furthermore, non-specific adsorption of protein is not observed at all.

(Example) Hereinafter, the present invention will be described with reference to Examples.

The methods for measuring the physical properties and evaluating the performance of the fillers obtained in the following examples and comparative examples are as follows.

"Evaluation method of packing material" The obtained packing material was filled in a stainless steel column having an inner diameter of 6 mm and a length of 75 mm, and the pressure resistance and the swelling property in water were investigated. The pressure resistance was measured by flowing purified water through the column, changing the flow rate, and measuring the relationship between flow rate and pressure loss. The swelling property was determined from the change in column pressure when liquids with different ionic strengths were passed.

The ion exchange groups on the surface of the packing material were quantified by an automatic potentiometric titrator AT-310 manufactured by Kyoto Electronics Manufacturing Co., Ltd. Furthermore, the hemoglobin of human blood was analyzed with Hi-AUTO A _1C manufactured by Kyoto Daiichi Kagaku Co., Ltd., and the separation ability was compared with that of the conventional product. The measuring method is as follows. As a human blood sample, heparin was added immediately after collecting blood of the same person (healthy person). The blood sample is the dedicated blood 21L attached to this device.
(Phosphate buffer containing nonionic surfactant) automatically dilutes and hemolyzes 290 times. The eluent is liquid A (phosphate buffer of pH 5.9), which is a dedicated reagent attached to this device,
Solution B (pH 7.2 phosphate buffer) and solution C (pH 5.9 phosphate buffer) were used.

Example 1 100 g of styrene (hydrophobic non-crosslinkable monomer), 200 g of divinylbenzene (hydrophobic crosslinkable monomer) and 1 g of benzoyl peroxide (polymerization initiator) were dissolved in 200 g of toluene. This was added to 2.5 l of a 4% aqueous polyvinyl alcohol solution, and the particles were sized with stirring, and then heated to 80 ° C. under nitrogen substitution to carry out suspension polymerization. After polymerizing at 80 ° C. for 8 hours, the product was washed successively with hot water and acetone and dried to obtain fine hydrophobic crosslinked polymer particles.

200 g of the hydrophobic crosslinked polymer particles were immersed in acetone 1 in which 0.5 g of benzoyl peroxide was dissolved to impregnate the polymerization initiator. Next, acetone was distilled off under reduced pressure at 20 ° C. 1% polyvinyl alcohol aqueous solution 2.
The impregnated hydrophobic cross-linked polymer was suspended in 5 liters, 50 g of acrylic acid was added with stirring, and after substitution with nitrogen, a polymerization reaction was carried out at 80 ° C. for 2 hours. The product was washed successively with hot water and acetone and dried. The obtained fine polymer gel was classified by an air classifier Turbo Classifier TC-15N manufactured by Nisshin Engineering Co., Ltd. to collect particles having a particle size of 8 to 10 μm to obtain a filler. It has an inner diameter of 6 mm and a length of 7
It was packed in a 5 mm stainless steel column. Filled with purified water 35m
2 g of gel (filler) was added to 1 l, stirred for 5 minutes, and then filled at a constant flow rate of 2.0 ml / min.

The pressure resistance and the swelling property were evaluated by the above methods. In the pressure resistance evaluation, the pressure loss up to 150 kg / cm ² was proportional to the flow velocity. A swelling test showed that the eluent was 40
No increase in column pressure was observed when the mM phosphate buffer was changed to 200 mM phosphate buffer. When the carboxyl groups on the gel surface were quantified by titration, it was 0.08 nmol / g gel, and the reacted acrylic acid was 4.3 g. Further, human blood was analyzed with Hi-AUTO A _1C manufactured by Kyoto Daiichi Kagaku Co., Ltd. The chromatogram obtained as a result is shown in FIG. In Fig. 1 and Figs. 2 and 3 described later, 1 is hemoglobin A _1a and A _1b , 2 is fetal hemoglobin (F), 3 is unstable hemoglobin A _1C , 4 is stable hemoglobin A _1C , and 5 is a peak resulting from normal hemoglobin.

Example 2 According to Example 1, hydrophobic crosslinked polymer particles were obtained by using 300 g of diethylene glycol dimethacrylate as the hydrophobic crosslinked polymer and 200 g of isoamyl alcohol in place of toluene as the solvent. Further, 50 g of methacrylic acid was used instead of acrylic acid, and the mixture was reacted at 80 ° C. for 2 hours to prepare a polymer gel (filler) by the same operation method as in Example 1.

As a result of performing the same evaluation as in Example 1, the pressure loss was proportional to the pressure loss up to 150 kg / cm ² and the flow velocity. A swelling test was performed and the eluent was changed from 40 mM phosphate buffer.
No increase in column pressure was observed when the buffer was changed to 200 mM phosphate buffer. Furthermore, Hi-manufactured by Kyoto Daiichi Kagaku Co., Ltd.
Human blood was analyzed using AUTO A _1C . The chromatogram obtained as a result is shown in FIG.

Example 3 In this example, a continuous polymerization method was employed in which the hydrophobic cross-linked polymer particles were prepared and subsequently reacted with a hydrophilic monomer.

Styrene 100 g, divinylbenzene 200 g and benzoyl peroxide 1 g were dissolved in toluene 200 g, added to 2.5% of a 4% polyvinyl alcohol aqueous solution, sized with stirring, and heated to 80 ° C. under nitrogen substitution to carry out suspension polymerization. I went. After polymerizing at 80 ° C. for 2 hours, 50 g of acrylic acid was added and further polymerized at 80 ° C. for 2 hours, and the product was washed successively with hot water and acetone, dried and classified. The obtained minute polymer gel was evaluated in the same manner as in Example 1.

Regarding pressure resistance, the pressure loss was proportional to the flow velocity up to 150 kg / cm ² . A swelling test showed that when the eluent was changed from 40 mM phosphate buffer to 200 mM phosphate buffer,
No increase in column pressure was observed. Further, human blood was analyzed with Hi-AUTO A _1C manufactured by Kyoto Daiichi Kagaku Co., Ltd.
The resulting chromatograph was similar to that shown in FIG.

Comparative Example 1 Styrene 100g, divinylbenzene 200g, acrylic acid 150g
And 1 g of benzoyl peroxide was dissolved in 200 g of toluene and added to 2.5 l of 4% polyvinyl alcohol aqueous solution,
After granulating with stirring, the suspension was polymerized by heating to 80 ° C. 8
After polymerizing at 0 ° C. for 8 hours, the product was classified, filled and evaluated in the same manner as in Example 1.

As a result of performing the same evaluation as in Example 1, the pressure loss was proportional to the pressure loss up to 80 kg / cm ² and the flow velocity. The swelling test showed that the eluent was 20 mM from 40 mM phosphate buffer.
When changing to 0 mM phosphate buffer, the column pressure is 20 kg / cm
² rose. Thus, it is clear that the pressure resistance and swelling resistance are inferior to those of the filler of Example 1. Furthermore, Inc.
Human blood was analyzed using Hi-AUTO A _1C manufactured by Kyoto Daiichi Kagaku. The chromatogram obtained as a result is shown in FIG.
Compared to Fig. 2, the retention of glycated hemoglobins is clearly weak and the separation ability is poor.

(Effects of the Invention) According to the present invention, as described above, glycated hemoglobin can be quantified with high accuracy and in a short time. By measuring glycated hemoglobin in blood using this method,
Diabetes can be diagnosed quickly and accurately.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 to FIG. 3 are chromatographs obtained when the packing materials obtained in Example 1, Example 2 and Comparative Example 1 were packed in a column and glycated hemoglobin was separated. Indicates gram.

Claims (1)

[Claims] 1. A method for quantifying glycated hemoglobin in a sample by liquid chromatography, wherein the filler used in the liquid chromatography is acrylic acid and / or methacrylic acid on the surface of hydrophobic crosslinked polymer particles. A method for quantifying glycated hemoglobin, which comprises coated polymer particles on which a layer of acid (co) polymer is formed.