JPH02309253A - Determination of saccharified hemoglobin - Google Patents

Abstract

PURPOSE:To determine saccharified hemoglobin within a short time with high accuracy by using coated polymer particles wherein an acrylic or methacrylic acid (co)polymer layer is formed to the surface parts of hydrophobic crosslinked polymer particles as a packing material. CONSTITUTION:A blood specimen is hemolized if necessary to be applied to a column packed with a packing material and the determination of saccharified hemoglobin is performed by a liquid chromatography technique. By selecting a proper buffer solution, hemoglobin is separated and eluted. As the packing material, a polymer particle having a two-layer structure wherein a hydrophobic crosslinked polymer forms a skeletal and the surface part thereof is coated with a (meth)acrylic acid polymer is used. Since the polymerization degree of the skeletal part is high, mechanical strength is high. Since only the surface part is coated with the (meth)acrylic acid polymer, the ion exchange capacity of the packing material is high and a carboxyl group becomes an equilibrium state rapidly. Therefore, the separation capacity of saccharified hemoglobin is excellent and saccharified hemoglobin can be measured within a short time.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for quantifying glycated hemoglobin, particularly to a method for quantifying glycated hemoglobin using liquid chromatography.

(Prior Art) Glycated hemoglobin is formed when hemoglobin in red blood cells non-enzymatically reacts with glucose in blood. Since the average concentration of glucose in blood can be determined by measuring glycated hemoglobin, measurement of glycated hemoglobin is widely used in the diagnosis of diabetes. Glycated hemoglobin is now.

Mainly high performance liquid chromatography (hereinafter 1 (PLC)
Quantification is carried out by According to the HPLC method, rapid measurement is possible compared to conventional column chromatography, electrophoresis, colorimetry, and the like.

The filler used in IPLc (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 fillers for HPLC, organic polymer fillers or inorganic fillers are mainly used.

Examples of organic polymer fillers include: 1;

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.

As inorganic fillers, Japanese Patent Application Laid-open No. 63-75558,
A filler in which a carboxyl group is introduced into a silica base material is disclosed.

Generally, glycated hemoglobin is quantified using a step or continuous gradient method using two types of eluents: a solution with a weak elution power (hereinafter referred to as the first solution) and a solution with a strong elution power (hereinafter referred to as the second solution). Implemented by The first liquid increases the free carboxyl groups in the filler. As a result, hemoglobin other than glycated hemoglobin in the sample is retained in the filler.

Glycated hemoglobin is separated and eluted. Since the second liquid has a high ionic strength, free carboxyl groups become salts.

Therefore, hemoglobin other than the retained glycated hemoglobin is rapidly eluted.

However, when using the above method with a polymer filler, there are the following problems. Polymer-based fillers are prepared using hydrophobic and hydrophilic monomers, as shown in 1) the above-mentioned JP-A No. 58421164, and 2) ion exchange groups mainly derived from hydrophilic monomers are distributed throughout the filler particles. to be distributed. When such a packing material is brought into contact with the second liquid, the packing material swells and the pressure within the column increases. To measure the 9th sample after measuring one sample.

It is necessary to flow the first liquid to return the carboxyl group to its free form. At this time, in order to sufficiently return the ion exchange groups present inside the filler to a free form, it is necessary to flow a considerable amount of the first liquid, resulting in a long measurement time. It has become possible to measure glycated hemoglobin at a relatively high speed using IPLC (using a polymer-based packing material), but when trying to further increase the measurement speed, as mentioned above, the ion exchange groups inside the packing material are not exchanged sufficiently. Alternatively, the separation performance decreases because the filler swells. To perform separation with high precision, it is necessary to reduce the elution rate.

On the other hand, inorganic fillers based on silica generally have excellent pressure resistance and swelling resistance.

Problems have been pointed out, such as the silanol groups remaining on the surface that tend to cause non-specific adsorption of proteins.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned drawbacks of the conventional method, and its purpose is to provide a method for separating and quantifying glycated hemoglobin in a short time with high precision using IIPLc. It is about providing. Another object of the present invention is to perform (IPL) using a packing material that does not increase column pressure during column operation, quickly equilibrates with an eluent, and does not non-specifically adsorb proteins, etc. The purpose of the present invention 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.

The packing material used in the liquid chromatography.

The coated polymer particles have a layer of acrylic acid and/or methacrylic acid (co)polymer formed on the surface portion of the hydrophobic crosslinked polymer particles, thereby achieving the above object.

As a material for the hydrophobic crosslinked polymer particles used to prepare the filler used in the method of the present invention.

(Co) obtained by (co)polymerizing hydrophobic crosslinking monomers
A polymer or a copolymer of a hydrophobic 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. Examples of the hydrophobic crosslinkable monomer include di(meth)acrylics such as ethylene glycol di(meth)acrylate, polyethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, and polypropylene glycol di(meth)acrylate. Acid esters; poly(meth)acrylic esters of polyhydric alcohols such as tetramethylolmethane tri(meth)acrylate and tetramethylolmethanetetra(meth)acrylate; divinylbenzene.

Aromatic compounds having two or more vinyl groups, such as divinyltoluene, divinylxylene, and divinylnaphthalene, are used. As the hydrophobic non-crosslinkable monomer, any non-crosslinkable polymerizable monomer having hydrophobic properties can be used. For example, methyl (meth)acrylate, engineered methyl (meth)acrylate, propyl (
meth)acrylate, isopropyl(meth)acrylate.

(Meth)acrylic acid esters such as butyl (meth)acrylate and t-butyl (meth)acrylate; vinyl acetate; and styrenic monomers such as styrene and methylstyrene are used. When using a mixture of the above-mentioned crosslinkable and non-crosslinkable monomers, the amount of the crosslinkable monomer is 10 parts by weight or more, preferably 20 parts by weight or more based on 100 parts by weight of the total monomers. used.

The polymerization initiator used when preparing the hydrophobic crosslinked polymer particles and the polymerization initiator (described later) with which the obtained hydrophobic crosslinked polymer particles are impregnated are as follows.

It is a catalyst that generates radicals, and is not particularly limited as long as it is hydrophobic. Any of the known radical generating catalysts can be used, such as organic peroxides such as benzoyl peroxide and cumene peroxide; and azo compounds such as azobisisobutyronitrile and azobisisobutyramide.

The acrylic acid and/or methacrylic acid (hereinafter referred to as (meth)acrylic acid) used to coat the hydrophobic crosslinked polymer particles has a carboxyl group, so it polymerizes on the surface of the hydrophobic crosslinked polymer particles. In this case, the carboxyl group functions as an ion exchange group.

It has carboxyl groups in addition to acrylic acid and methacrylic acid.

Other monomers can also be used as long as they are water-soluble monomers. What is the amount of (meth)acrylic acid used?

Although it varies depending on the type of monomer, it is usually 5 to 50 parts by weight per 100 parts by weight of the hydrophobic crosslinked polymer.

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, if necessary, the hydrophobic non-crosslinkable monomer and the polymerization initiator are dissolved in a diluent. As the diluent, any organic solvent that dissolves the above-mentioned monomers but does not dissolve the polymer can be used. For example, aromatic hydrocarbons such as toluene, xylene, diethylbenzene, and dodecylbenzene; saturated hydrocarbons such as hexane, heptane, octane, and decane;
Examples include alcohols such as isoamyl alcohol, hexyl alcohol, and octyl alcohol.

The amount used is not limited at all, but it is preferably 15 to 200 parts by weight per 100 parts by weight of the above monomer. The diluted solution of the above monomer is mixed with polyvinyl alcohol,
Suspension polymerization is carried out by adding a suspension stabilizer such as calcium phosphate to the dispersed aqueous phase, and heating to 50 to 100° C. with stirring after nitrogen substitution. Since an organic solvent as a diluent exists dispersedly in the obtained polymer particles, porous spherical particles can be obtained by removing the organic solvent after one polymerization is completed. By using various organic solvents having different compatibility with the 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. To impregnate with polymerization initiator.

The polymerization initiator is dissolved in a solvent that has a low boiling point and has good affinity with the hydrophobic crosslinked polymer, and the hydrophobic crosslinked polymer particles are immersed in this. This allows 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 2 containing the polymerization initiator in the hydrophobic crosslinked polymer particles can be obtained. These 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 an aqueous medium in which the particles are dispersed, and the mixture is heated under stirring after nitrogen substitution. to perform polymerization. In order to stabilize the dispersibility of the hydrophobic crosslinked polymer, it is desirable to use a dispersion stabilizer such as carboxymethyl cellulose or polyvinyl alcohol in the aqueous dispersion medium. The temperature and time of polymerization are
It varies depending on whether acrylic acid or methacrylic acid is used or the type of polymerization initiator, but the
It takes about 0.5 to 10 hours at 100°C.

Crosslinked polymer particles impregnated with the above polymerization initiator (meth)
In addition to the method of subjecting acrylic acid to a polymerization reaction.

The above two-layered polymer particles can also be prepared by a continuous method in which crosslinked polymer particles are prepared and then (meth)acrylic acid is reacted. In this method, first, a polymerization reaction for preparing the above-mentioned crosslinked polymer particles is started. Polymerization progresses to some extent.

(Meth)acrylic acid is added to the reaction system when unreacted monomers remain. In such a state, a polymerization initiator exists in the oil layer in the system and inside the generated hydrophobic crosslinked polymer particles.

Subsequently, polymerization of (meth)acrylic acid occurs.

Moreover, a layer of (co)polymer of (meth)acrylic acid is formed to cover the surface portion of the hydrophobic crosslinked polymer particles.

The polymer particles obtained by each of the above methods are thoroughly washed with hot water, an organic solvent, etc., and the suspension stabilizer, solvent, etc. attached to each particle are removed.
Residual monomers etc. are removed. Furthermore, if necessary, the particles are classified to obtain a packing material for chromatography.

To measure glycated hemoglobin by the method of the present invention, first, the blood sample is hemolyzed as necessary. This is applied to a column filled with the above-mentioned packing material, and glycated hemoglobin is quantified by a conventional liquid chromatography method. By selecting an appropriate buffer solution, glycated hemoglobin and then other hemoglobins in the sample 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 in which the skeleton is a hydrophobic cross-linked polymer and the surface portion of the hydrophobic cross-linked polymer is coated with a (meth)acrylic acid polymer. By using a polymer with a high degree of crosslinking as the skeleton part, it is possible to obtain a packing material for liquid chromatography that has extremely high mechanical strength and excellent pressure resistance.

This is because there is no hydrophilic group in the skeleton of this filler.

Extremely low degree of swelling and shrinkage. Therefore, in quantifying glycated hemoglobin, the pressure increase during passage of the second liquid is extremely small. Since only the surface portion is covered with a (meth)acrylic acid polymer layer having carboxyl groups, the ion exchange ability of the filler is high, and the carboxyl groups are equilibrated very quickly. Therefore, it has excellent separation performance for glycated hemoglobin.

Measurement can be carried out in a short time. Furthermore, no non-specific adsorption of proteins was observed.

(Example) The invention will be explained below 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 filler" The obtained filler was packed into a stainless steel column with an inner diameter of 6 mm and a length of 75 m+n, and its pressure resistance and swelling property with respect to water were examined. 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. Swellability 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 filler were quantified using an automatic potentiometric titrator AT-310 manufactured by Kyoto Electronics Industry Co., Ltd. In addition, we analyzed hemoglobin in human blood using Kyoto Dai-Kagaku Old-A (JTOA+c) and compared the separation ability with the conventional product.The measurement method was as follows.
Immediately after the blood of a healthy person was collected, heparin was added to the blood. Blood specimen.

The blood is automatically diluted 290 times and hemolyzed using the dedicated hemolysis 21L (phosphate buffer containing nonionic surfactant) attached to this device. The eluents are solution A (phosphate buffer with pH 5, 9) and solution B (pH 7, 2), which are exclusive reagents included with this device.
phosphate buffer) and C solution (phosphate buffer with pH 5, 9) 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 a 4% aqueous polyvinyl alcohol solution at 2.51° C., granulated with stirring, and then heated to 80.degree. C. under nitrogen substitution to carry out suspension polymerization. After polymerization at 80°C for 8 hours.

The product was sequentially washed 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 1 liter of acetone in which 0.5 g of benzoyl peroxide was dissolved to impregnate the particles with the polymerization initiator. next.

Acetone was distilled off under reduced pressure at 20°C. The above impregnated hydrophobic crosslinked polymer was suspended in 2.5 g of a 1% polyvinyl alcohol aqueous solution, 50 g of acrylic acid was added with stirring, and after purging 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 microscopic polymer gel was passed through an air classifier manufactured by Nisshin Engineering, Turbo Classifier TC-15.
The mixture was classified with N and particles having a particle size of 8 to 10 μm were collected to obtain a filler.

This was packed into a stainless steel column with an inner diameter of 6 mm and a length of T5+nm. For filling, add gel (>2g of filler) to purified water 35rd, stir for 5 minutes, and then add 2.Ord/
This was done by constant flow filling in minutes.

The pressure resistance (tJ) and swelling property were evaluated using the above method. In the pressure resistance evaluation, the pressure loss was proportional to the flow rate up to 150 kg/cut. 200mM from acid buffer
When changing to phosphate buffer, no increase in column pressure was observed. The amount of carboxyl groups on the gel surface was determined by titration to be 0.08 mmol per 1 g of gel, and therefore, the amount of reacted acrylic acid was 4.3 g. Furthermore, human blood was analyzed using the old AUTOArc manufactured by Kyoto Dai-Kagaku. The resulting taromadogram is shown in FIG. In FIG. 1 and later-described FIGS. 2 and 3,
1 is hemoglobin Ala and A+b, 2 is fetal hemoglobin (P), 3 is unstable hemoglobin A
, e, 4 is stable hemoglobin AIC+ and 5 is the peak due to normal hemoglobin.

Example 2 300 g of diethylene glycol dimethacrylate (diethylene glycol dimethacrylate) was used as a hydrophobic crosslinked polymer, and 200 g of isoamyl alcohol was used instead of toluene as a solvent.

Hydrophobic crosslinked polymer particles were obtained according to Example 1.

Further, a polymer gel (filler) was prepared in the same manner as in Example 1 by using 50 g of methacrylic acid instead of acrylic acid and reacting at 80° C. for 2 hours.

As a result of the same evaluation as in Example 1, the pressure loss was proportional to the flow rate up to 150 kg/cffl with respect to pressure resistance. When a swelling test was conducted, no increase in column pressure was observed when the eluent was changed from 40 mM phosphate buffer to 200 mM phosphate buffer.

Furthermore, human blood was analyzed using ``Kyoto Dai-Kagaku old-^UTOA,c''. The resulting chromatogram is shown in FIG.

Example 3 In this example, a sequential polymerization method was employed in which hydrophilic monomers were reacted following the preparation of hydrophobic crosslinked polymer particles.

100g of styrene, 200g of divinylbenzene, 1g of benzoyl peroxide and 200g of toluene.
After adding to 4% polyvinyl alcohol aqueous solution (2.51 g) and granulating it while stirring, it was 80 g.
Suspension polymerization was carried out by heating to ℃. After polymerizing at 80° C. for 2 hours, 50 g of acrylic acid was added, followed by further polymerization at 80° C. for 2 hours, and the product was sequentially washed with hot water and acetone, dried, and classified into 2 parts. The obtained microscopic polymer gel was prepared in Example 1.
It was evaluated using the same method.

Regarding pressure resistance, pressure loss and flow rate were proportional up to 150 kg/Crl. When a swelling test was performed, the eluent was mixed with 200ml of 40mM phosphate buffer. ! When changing to phosphate buffer, no increase in column pressure was observed. Furthermore, human blood was analyzed using HiJUT RoArc manufactured by Kyoto Dai-Kagaku. The resulting chromatogram was similar to that shown in FIG.

Comparative example 1 100 g of styrene, 200 g of divinylbenzene,
150g acrylic acid and 11g benzoyl peroxide
g was dissolved in 200 g of toluene, added to 4% polyvinyl alcohol aqueous solution*2,51, granulated with stirring, and heated to 80° C. for suspension polymerization. After polymerization at 80° C. for 8 hours, the product was classified and packed in the same manner as in Example 1 and evaluated.

As a result of the same evaluation as in Example 1, the pressure resistance was BQkg/a+! The pressure drop and flow rate were proportional until A swelling test showed that when the eluent was changed from 40mM phosphate buffer to 200mM phosphate buffer, the column pressure was 20kg/ca! Rose. Thus, it is clear that the filler of Example 1 is inferior in pressure resistance and swelling resistance. In addition ■ Kyoto Dai-Kagaku) 1i-A [IT
Human blood was analyzed at OArc. The resulting chromatogram is shown in FIG. Comparing with Figure 2,
Apparently, the retention power for glycated hemoglobins is weak and the separation ability is inferior.

(Effects of the Invention) According to the present invention, glycated hemoglobin can be quantified with high precision and in a short time.

Diagnosis of diabetes, etc. can be made quickly and accurately by measuring glycated hemoglobin in the blood using a secondary method.

[Brief explanation of drawings]

FIGS. 1 to 3 show Example 1, respectively. A chromatogram is shown when a column was filled with the packing material obtained by wiping in Example 2 and Comparative Example 1, and glycated hemoglobin was separated. that's all

Claims (1)

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