JPH03118466A - Assay of saccharified hemoglobin - Google Patents

Abstract

PURPOSE:To perform assay with a high accuracy and in a short time by forming a polymer layer having a carboxyl group on the surface part of a filler. CONSTITUTION:The mixed liquid of a hydrophobic monomer is added into a water phase wherein suspension stabilizer is dispersed. After the substitution with nitrogen, the liquid is agitated and heated, and suspension polymerization is performed. When diluent is added and the polymerization is performed, organic solvent is removed after the end of the polymerization, and porous spherical particles are obtained. Then, the hydrophobic cross link polymer is impregnated with a polymerization initiator. The particles are dispersed in a dispersion medium wherein a monomer having a hydrolytic group is dissolved. After the substitution with hydrogen, the material is agitated and heated, and polymerization is performed. Thus, the polymer particles having a double-layered structure are obtained. Then, the polymer particles are washed and undergo hydrolysis with acid catalyst or alkali catalyst. The functional group on the coating layer on the surface of the particle is made to be a carboxyl group. The particles are filtered, washed with water and dried. Thus a filler is obtained. A column is filled with the filler. A sample blood is hemolyzed and applied on the column. Thus, saccharified hemoglobin can be assayed by liquid chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for quantifying tli-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.

Quantification is mainly performed by high performance liquid chromatography (hereinafter referred to as HPLC). According to the HPLC method, conventional column chromatography method, electrophoresis method,
Rapid measurement is possible compared to colorimetric methods.

The filler 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 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. When the first liquid is passed, hemoglobin other than glycated hemoglobin in the sample is
It is relatively easier to be retained in the filler than glycated hemoglobin, and 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 a polymer filler in the above method, there are the following problems. Polymer-based fillers are prepared using hydrophobic and hydrophilic monomers, as shown in JP-A-58-221164, for example, so that ion exchange groups mainly due to hydrophilic heptads are filled. distributed throughout the particle. 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 a second 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, it is necessary to flow a considerable amount of the first liquid for equilibration in order to sufficiently return the ion-exchange groups present inside the filler to a free form, so that the measurement time becomes long. It has become possible to measure glycated hemoglobin at a relatively high speed using HPLC 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. Separation performance deteriorates. Alternatively, the pressure within the column increases, deforming the packing material and reducing the separation performance. Therefore, in order to perform highly accurate separation, it is necessary to reduce the elution rate, making it difficult to shorten the measurement time.

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

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

(Problems to be Solved by the Invention) The present invention solves the drawbacks of the above-mentioned conventional methods, and its purpose is to provide a method for separating and quantifying glycated hemoglobin in a short time and with high precision by HPLC. It is about providing. Another object of the present invention is to use 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. An object 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.

It consists of coated polymer particles in which a layer of a (co)polymer having carboxyl groups is formed on the surface portion of hydrophobic crosslinked polymer particles; the coated polymer particles can generate carboxyl groups through a hydrolysis reaction. A monomer having a functional group is polymerized on the surface portion of the hydrophobic cross-linked polymer particles, and the resulting polymer is coated on the hydrophobic cross-linked polymer particles; and the functional group is hydrolyzed. The above objectives are thereby achieved.

The material for the hydrophobic crosslinked polymer particles used in the method of the present invention is a (co)polymer obtained by (co)polymerizing a hydrophobic crosslinkable monomer or a hydrophobic crosslinkable monomer and a hydrophobic crosslinkable monomer. A copolymer with a non-crosslinkable monomer is used. These monomers are required to form a polymer that does not undergo a chemical reaction (for example, hydrolysis) under the hydrolysis reaction conditions described below. Therefore, a monomer that does not have a functional group capable of chemically reacting under the hydrolysis reaction conditions 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 crosslinking monomer include divinylbenzene.

Aromatic compounds having two or more vinyl groups, such as divinyltoluene, divinylxylene, and divinylnaphthalene, are used. Examples of the hydrophobic non-crosslinkable monomer include styrene, methylstyrene, and the like.

When the above-mentioned crosslinkable and non-crosslinkable monomers are mixed and used, the crosslinkable monomer is used in an amount of 1 part by weight per 100 parts by weight of the total monomers.
0 parts by weight or more.

It is preferably used in an amount of 20 parts by weight or more.

The polymerization initiator used when preparing the above-mentioned hydrophobic crosslinked polymer particles and the polymerization initiator to be impregnated with the obtained hydrophobic crosslinked polymer particles are catalysts that generate radicals, and especially if they are hydrophobic. Not limited. Any of the known radical generating catalysts can be used, such as organic peroxides such as benzoyl peroxide and cumene peroxide; and ab compounds such as azobisisobutyronitrile and azobisisobutyramide.

In the method of the present invention, the monomer used to coat the hydrophobic crosslinked polymer particles is a monomer having a functional group that can generate a carboxyl group by hydrolysis (hereinafter, a monomer having a hydrolyzable group). ). Such monomers include alkyl esters of acrylic acid or methacrylic acid (hereinafter referred to as (meth)acrylic acid) such as methyl acrylate and methyl methacrylate; (meth)acrylamide, (meth)acrylonitrile, etc. can be mentioned. The above-mentioned monomer having a hydrolyzable group is as follows.

Two or more types may be used as a mixture if necessary.

The amount of the monomer having a hydrolyzable group varies depending on the type of monomer, but is in the range of 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 polymerization initiator is dissolved in the hydrophobic monomer (hydrophobic crosslinkable monomer and, if necessary, hydrophobic non-crosslinkable monomer). Furthermore, a porous hydrophobic crosslinked polymer can be obtained by adding a diluent if necessary. As the diluent, any organic solvent that dissolves the above-mentioned monomers but does not dissolve the polymer can be used. For example, toluene, xylene, diethylbenzene,
Aromatic hydrocarbons such as dodecylhenzen; hexane,
Saturated hydrocarbons such as heptane, octane and decane 1; Alcohols such as isoamyl alcohol, hexyl alcohol and octyl alcohol. The amount used is not limited at all, but it is preferably up to 200 parts by weight per 100 parts by weight of the monomer. A mixture of the above monomers.

Suspension polymerization is carried out by adding a suspension stabilizer such as polyvinyl alcohol or calcium phosphate to the dispersed aqueous phase, purging with nitrogen, and heating to 40 to 100'C with stirring. When polymerization is performed with the addition of a diluent, the organic solvent as the diluent is dispersed in the resulting polymer particles, so by removing the organic solvent after the completion of the double polymerization, porous spherical particles can be formed. particles are obtained. 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 the 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 necessary, heat this at a temperature below the decomposition point of the polymerization initiator.

By distilling off the solvent, particles containing a polymerization initiator in the hydrophobic crosslinked polymer particles can be obtained. The polymerization initiator-containing particles are dispersed in a dispersion medium in which the monomer having a hydrolyzable group is dissolved, or the monomer having a hydrolyzable group is dissolved in a dispersion medium in which the particles are dispersed. The mixture is added and dissolved, and after purging with nitrogen, polymerization is carried out by heating while stirring. As the dispersion medium, water or an organic solvent that dissolves the monomer having a hydrolyzable group, or a mixture of the two is used.

Carboxymethylcellulose to stabilize the dispersibility of the hydrophobic crosslinked polymer in the dispersion medium.

Dispersion stabilizers such as polyvinyl alcohol may also be used. The temperature and time of polymerization vary depending on the type of monomer having a hydrolyzable group to be reacted and the type of polymerization initiator, but are approximately 0.5 to IO hours at 40 to 100°C.

In addition to the method in which hydrophobic crosslinked polymer particles impregnated with a polymerization initiator are subjected to a polymerization reaction of a monomer having a hydrolyzable group, following the preparation of hydrophobic crosslinked polymer particles, The above two-layer structure polymer particles can also be prepared by a continuous method in which monomers having the following are reacted. In this method, first.

A polymerization reaction for preparing the hydrophobic crosslinked polymer particles is started. When the polymerization has progressed to some extent and unreacted monomers remain, the monomer having the hydrolyzable group is added to the reaction system. In such a state, since a polymerization initiator is present in the oil phase in the system and inside the generated hydrophobic crosslinked polymer particles, polymerization of monomers having hydrolyzable groups occurs subsequently. A layer of a polymer having a hydrolyzable group 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 polymer particles are contained in one particle.

Alternatively, adhering suspension stabilizers, solvents, residual monomers, etc. are removed.

By hydrolyzing the obtained polymer particles with an acid catalyst or an alkali catalyst, the hydrolyzable functional groups present in the coating layer on the surfaces of the two particles are hydrolyzed to become carboxyl groups. For example, when methyl acrylate is used as a monomer having a hydrolyzable group, one polymer particle is dissolved in a 15 to 25 wt% methanol solution of sodium hydroxide for 60 min.
By reacting at a temperature of ~80°C for 4 to 7 hours, the C00CI+3 groups on the particle surface become carboxyl groups.

After the hydrolysis reaction, the polymer particles are collected by filtration, washed several times with water and dried, and if necessary, the particles are classified to obtain a filler.

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 a hydrophobic crosslinked polymer is used as a skeleton and the surface portion of the hydrophobic crosslinked polymer is coated with a polymer having a carboxyl group. 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. Since no hydrophilic groups are present in the skeleton of this filler, the degree of swelling and shrinkage of the filler by an aqueous eluent is extremely low. 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 polymer layer having carboxyl groups, the ion exchange ability of the filler is high, and the carboxyl groups are equilibrated very quickly. Therefore, the separation performance of glycated hemoglobin is excellent, and measurement can be performed 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 6nnn and a length of 75M, 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. The swelling property is
It 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. Furthermore, human blood hemoglobin was analyzed using Kyoto Dai-Kagaku's old AUTOA, c, and its separation ability was compared with conventional products. The measurement method is as follows. As human blood samples, samples from the same person (R
Immediately after the blood of a normal person was collected, heparin was added to it. 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 (p), which are exclusive reagents attached to this device.
2 phosphate buffer) and C solution (phosphate buffer pH 5, 9) were used.

100 g of styrene (hydrophobic non-crosslinking monomer), 200 g of divinylbenzene (hydrophobic cross-linking monomer) and 1 g of benzoyl peroxide (polymerization initiator) were dissolved in 200 g of toluene (diluent). Is this 4% polyvinyl alcohol water? The mixture was added to a volume of 2.542 ml for 7 nights, stirred, and then heated to 80°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 acetone IP in which 0.5 g of acetyl peroxide was dissolved to impregnate the particles with the polymerization initiator. The acetone was then distilled off under reduced pressure at 20°C. 50% methanol aqueous solution 2.
The above-mentioned impregnated hydrophobic crosslinked polymer was suspended in No. 52, 50 g of methyl acrylate (a monomer having a hydrolyzable group) was added with stirring, and the polymerization reaction was carried out at 70°C for 5 hours after purging with nitrogen. I did it.

The product was washed successively with hot water and acetone and dried.

150 g of this polymer particle was added to 500 d of a 20% by weight methanol solution of sodium hydroxide and heated at 75"C for 5 hours to hydrolyze the ester moiety of polymethyl acrylate. After cooling the reaction mixture to room temperature. 1 Polymer particles were collected by filtration and washed several times with water.

Dry. The obtained microscopic polymer gel was processed using an air classifier Turbo Classifier TC~1 manufactured by Nisshin Engineering ■.
Classify with 5N to collect particles with a particle size of 8 to 10 μm,
A filler was obtained. This has an inner diameter of 6rM1 and a length of 75Mn.
was packed into a stainless steel column. Fill with 35m of purified water
Add 2g of gel (filler) to 1 and stir for 5 minutes, then add 2.
This was done by constant flow filling at 0 d/min.

Pressure resistance and swelling properties were evaluated using the methods described above.

In the pressure resistance evaluation, the pressure loss was proportional to the flow rate up to 150 kg/cd. 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. When the carboxyl groups on the gel surface were quantified by titration, 0.07 mmol of carboxyl groups were present per 1 g of gel, and therefore, 1.2 g of methyl acrylate was present on the gel surface before the hydrolysis reaction. ■Human blood was analyzed using Kyoto Kagaku's old AUTOAIC. The resulting chromatogram is shown in FIG.

In Fig. 1 and Figs. 2 and 3 described below, 1 is hemoglobin (hereinafter referred to as Hb), AI- and AIb, 2
is fetal Hb (F), 3 is unstable type 1 (bA, C,
4 is stable HbA.

And 5 is a peak due to normal ub (Aa).

In practice, 100 g of styrene (hydrophobic non-crosslinking monomer), 200 g of divinylbenzene (hydrophobic cross-linking monomer), and 1 g of benzoyl peroxide (polymerization initiator) were combined with 200 g of isoamyl alcohol (diluent). It was dissolved in Hydrophobic crosslinked polymer particles were prepared in the same manner as in Example 1.

200 g of the hydrophobic crosslinked polymer particles were immersed in 1 ρ of acetone in which 0.5 g of benzoyl peroxide was dissolved to impregnate the particles with the polymerization initiator. Next, add acetone to 20℃
The residue was distilled off under reduced pressure. 50% methanol aqueous solution 2
．． The above impregnated hydrophobic crosslinked polymer was suspended in No. 51, 50 g of methyl methacrylate was added with stirring, and the polymerization reaction was carried out at 80"C for 5 hours after purging with nitrogen. The product was dissolved in hot water and acetone. The resulting dried gel was hydrolyzed and classified into 1st class in the same manner as in Example 1, and then filled and evaluated.

As a result, regarding the pressure resistance, the pressure loss was proportional to the flow rate up to 150 kg/c111. 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. When the carboxyl groups on the gel surface were quantified by titration, there were Q, 1 mmol of carboxyl groups per 1 g of gel, and therefore, 1.7 g of methyl methacrylate was present on the gel surface before the hydrolysis reaction. Human blood was analyzed in the same manner as in Example 1. The resulting chromatogram is shown in FIG.

Main Example In this example, a continuous polymerization method was employed in which hydrophobic crosslinked polymer particles were prepared and then a monomer having a hydrolyzable group was reacted.

100 g of styrene, 200 g of divinylbenzene, and 1 g of benzoyl peroxide were dissolved in 200 g of toluene, added to 2.5 g of a 4% polyvinyl alcohol aqueous solution, barbed with stirring, and heated at 80° under nitrogen substitution.
Suspension polymerization was carried out by heating to C. After polymerizing at 80°C for 2 hours, 50 g of acrylamide was added, followed by further polymerization at 80°C for 2 hours, and the product was hydrolyzed and classified into 2 parts, packed and evaluated in the same manner as in Example 1.

As a result, regarding pressure resistance, pressure loss was proportional to flow rate up to 150 kg/cd. When a swelling test was conducted,
When the eluent was changed from 40 mM phosphate buffer to 2001 phosphate buffer, no increase in column pressure was observed. When the carboxyl groups on the gel surface were quantified by titration, 0.4 mmol of carboxyl groups were present per 1 g of gel, and therefore, 5.7 g of acrylamide was present on the gel surface before the hydrolysis reaction. Example 1
Human blood was analyzed in the same manner. The resulting chromatogram was similar to that shown in FIG.

100 g of styrene, 200 g of divinylbenzene
, 70g of methyl acrylate, 1g of acetyl peroxide to 200g of toluene? Dissolve and add 4% polyvinyl alcohol aqueous solution 2.5! The mixture was added to the solution, stirred while stirring, and then heated to 70°C for suspension polymerization. 8 at 70'C
After polymerization for a period of time, the product was hydrolyzed into 1 fraction by the same procedure as in Example 1, and then packed and evaluated.

As a result, regarding pressure resistance, pressure loss was proportional to flow rate up to 80 kg/c+fl. When a swelling test was conducted,
When the eluent was changed from 40mM phosphate buffer to 200mM phosphate buffer, the column pressure was 20kg/cff.
l rose. Thus, it is clear that the pressure resistance and swelling resistance are inferior to the filler of Example 1. Furthermore, human blood was analyzed in the same manner as in Example 1.

The results are shown in FIG. Compared to Fig. 1, the retention power for glycated hemoglobins is clearly weak.

Separation power is poor.

(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 can be made quickly and accurately by measuring glycated hemoglobin in the blood.

4.J's i' day Figures 1 to 3 are the results of Example 1, respectively. Chromatograms obtained when glycated hemoglobin was separated by filling a column with the packing materials obtained in Example 2 and Comparative Example 1 are shown.

that's all 1st figure Figure 2

Claims (1)

[Claims] 1. A method for quantifying glycated hemoglobin in a sample by liquid chromatography, wherein the packing material used in the liquid chromatography is a layer of a (co)polymer having carboxyl groups on the surface portion of hydrophobic crosslinked polymer particles. The coated polymer particles are formed by polymerizing a monomer having a functional group capable of producing a carboxyl group through a hydrolysis reaction on the surface portion of the hydrophobic crosslinked polymer particles, A method for quantifying glycated hemoglobin obtained by coating the hydrophobic crosslinked polymer particles with the resulting polymer and hydrolyzing the functional groups.