JP2001349894A - Measuring method of hemoglobin or the like - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measuring method of hemoglobin or the like which achieves higher endurance of a column, and moreover enables separation of a stable type HbA1c accurately and in a shorter time. SOLUTION: In the measuring method of hemoglobin or the like cation exchange liquid chromatography, the column pressure is set to 9.8×104Pa-3.9×106Pa, and measurement is performed using an eluate containing chaotropic ion and an inorganic acid, an organic acid each having buffer capacity with pH4.0-6.8 and/or these salts thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring hemoglobins by cation exchange liquid chromatography.

[0002]

2. Description of the Related Art Hemoglobin A1c (hereinafter referred to as HbA1c)
) Is formed by irreversibly binding to hemoglobin after the sugar in the blood enters the red blood cells, and the average blood sugar level (blood glucose concentration in the blood) for the past one to two months ) To reflect. For this reason, HbA1c is widely used as an index for diabetes diagnosis, such as a screening test for diabetes and grasping the blood sugar management status of diabetic patients.

[0003] HbA1c is glycated hemoglobin (hereinafter referred to as GHb) produced by the reaction between glucose in blood and hemoglobin A (hereinafter referred to as HbA), and the reversible reaction of glycated hemoglobin is referred to as unstable hemoglobin. A1c (unstable HbA1c: hereinafter, unstable HbA1c)
Irreversible reaction via unstable HbA1c is referred to as stable hemoglobin A1c (stable HbA1c).
1c: hereinafter referred to as stable HbA1c). As an indicator of the above-mentioned diabetes diagnosis, stable HbA1
It is preferable to use c, and in the field of clinical examination, it has been desired to develop a method capable of measuring the ratio of stable HbA1c with high accuracy and in a short time.

Conventionally, HbA1c is generally measured by liquid chromatography or immunization, and HbA1c is mainly measured by cation exchange liquid chromatography by liquid chromatography. Japanese Patent Publication No. 8-7198, etc.). When a hemolyzed blood sample is separated by cation exchange liquid chromatography, hemoglobin A1a (hereinafter, HbA1
a) and hemoglobin A1b (hereinafter, HbA1b)
), Hemoglobin F (hereinafter, referred to as HbF), unstable HbA1c, stable HbA1c, and hemoglobin A0 (hereinafter, referred to as HbA0).

Among these, recently, stable HbA1 has been used as an index for diagnosing diabetes as described above.
c, and the stable HbA1c value is determined as the ratio (%) of the area of the stable HbA1c peak to the area of all hemoglobin peaks.

Therefore, in order to accurately measure the value of the stable HbA1c, a technique for separating only the peak of the stable HbA1c with high accuracy is necessary. However, conventionally, the technique appears near the peak of the stable HbA1c. Unstable HbA
1c peak or acetylated hemoglobin (hereinafter AHb
Carbamylated hemoglobin (hereinafter referred to as CHb) and the peak of modified hemoglobin and stable HbA
It has been extremely difficult to separate the peak of 1c with high precision in a short time.

Therefore, as described in JP-A-63-298063, a sample containing erythrocytes and / or hemoglobin is mixed with a dissociation solvent containing a phosphate condensate and / or a salt of a phosphate condensate. There has been conventionally known a method in which unstable hemoglobin is dissociated into hemoglobin and glucose by being brought into contact with the hemoglobin and measured. However, this method has a drawback that the separation of stable HbA1c becomes insufficient and the measurement accuracy is poor.

On the other hand, a method capable of separating unstable HbA1c and modified hemoglobin from stable HbA1c in a short time of 2.2 minutes has been developed (basis of fully automatic glycohemoglobin analyzer HLC-723GHbV type). Examination: Journal of the Society of Clinical Automation in Japan 21 (199
6) 840-843).

In this method, a non-porous filler is used without using the dissociating agent in the above-mentioned conventional method, and the particle size of the filler is extremely small as 2.5 μm. Of stable HbA by reducing the diffusion of
1c can be separated. However, when such a column packed with a packing material having a very small particle size is used for the measurement of hemoglobins, the column pressure in the initial stage of measurement is extremely high (for example, about 9.8 × 10 ⁶ Pa). There is a drawback that the pressure resistance is required for the device and the cost is increased.

[0010] In addition, when continuously measured, there is a drawback that the column pressure is high due to large clogging and the column durability is poor. Further, in this method, when the column pressure is lowered by increasing the particle size of the filler, there is a problem that the measurement sample is diffused in the column and the separation becomes insufficient.

[0011]

SUMMARY OF THE INVENTION An object of the present invention is to provide a stable HbA1c having good column durability and a stable HbA1c in view of the above-mentioned problems of the conventional method for measuring hemoglobins.
To provide a method for measuring hemoglobins that can be separated accurately and in a short time.

[0012]

According to the first aspect of the present invention,
In a method for measuring hemoglobins by cation exchange liquid chromatography, an inorganic acid containing chaotropic ions and having a buffering capacity at pH 4.0 to 6.8,
Using an eluent containing an organic acid and / or a salt thereof, the column pressure was increased from 9.8 × 10 ⁴ Pa to 3.9 × 10 ⁶ P
This is a method for measuring hemoglobins, characterized in that measurement is performed by setting the value to a.

According to a second aspect of the present invention, there is provided the method for measuring hemoglobins according to the first aspect, wherein the eluent is sent using a gradient elution method. is there.

Hereinafter, the present invention will be described in detail.

In the present invention, a chaotropic ion and an inorganic acid having a buffering ability at pH 4.0 to 6.8,
An eluent containing an organic acid and / or a salt thereof is used. This eluent may be a single type of eluent,
Preferably, two or more eluents having different dissolution outputs, for example, different chaotropic ion concentrations and pHs are used.

The above-mentioned chaotropic ions suppress the decrease in entropy of water, which occurs when water is destroyed by the ions generated by dissociation into an aqueous solution and the hydrophobic substance comes into contact with water.

Specifically, the anionic chaotropic ion includes tribromoacetate ion, trichloroacetate ion, thiocyanate ion, iodide ion, perchlorate ion, dichloroacetate ion, nitrate ion, bromide ion, chloride ion. And acetic acid ions, and urea.

Examples of the cation chaotropic ion include barium ion, calcium ion, magnesium ion, lithium ion, cesium ion, potassium ion, guanidine ion and the like.

Among the above chaotropic ions, in order to further improve the measurement accuracy of stable HbA1c, the anions are preferably tribromoacetate ion, trichloroacetate ion, thiocyanate ion, iodide ion, perchlorate ion. , Dichloroacetate ion, nitrate ion, bromide ion and the like, and as the cation, barium ion, calcium ion, magnesium ion, lithium ion, cesium ion, guanidine ion and the like are used. More preferably, tribromoacetate ion, trichloroacetate ion, thiocyanate ion, perchlorate ion, nitrate ion, guanidine ion and the like are used.

These chaotropic ions may be used alone or as a mixture of two or more.

The concentration of the chaotropic ion in the eluent is preferably 0.1 mM to 3000 mM, more preferably 1 mM to 1000 mM, and particularly preferably 10 mM.
５００500 mM. This is because, when the concentration is less than 0.1 mM, the separation effect is reduced and the measurement accuracy is deteriorated in the measurement of hemoglobins.
This is because even if the concentration exceeds 00 mM, the effect of separating hemoglobins is not further improved.

The chaotropic ion may be added to a solution that comes into contact with a measurement sample other than the eluent, for example, a sample diluent, hemolyzed blood, or the like.

The inorganic acids, organic acids and / or salts thereof having a buffering ability at a pH of 4.0 to 6.8 in the present invention include the following.

The above-mentioned inorganic acids include, for example, phosphoric acid, carbonic acid and the like. Examples of the organic acids include carboxylic acids, dicarboxylic acids, carboxylic acid derivatives, hydroxycarboxylic acids, cacodylic acids, amino acids, and pyrophosphoric acids.

Examples of the carboxylic acid include acetic acid,
And propionic acid. Examples of the dicarboxylic acid include malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, fumaric acid, phthalic acid and the like. Examples of the carboxylic acid derivative include β, β-
Dimethyl glutaric acid, barbituric acid, aminobutyric acid and the like can be mentioned. Examples of the hydroxycarboxylic acid include citric acid, tartaric acid, lactic acid and the like. Examples of the above amino acids include aspartic acid and asparagine.

The inorganic or organic acid salt may be a known salt, such as a sodium salt or a potassium salt.

The above-mentioned inorganic acids, organic acids and / or salts thereof may be used alone or in combination of two or more.

The concentration of the above-mentioned inorganic acid, organic acid and / or salt thereof in the eluent is within a range that has a buffering action of adjusting the pH of the eluent to 4.0 to 6.8 when dissolved in water. The concentration is preferably 1 mM to 1000 mM, and more preferably 10 mM to 500 mM.

The pH of the eluent in the present invention is 4.0 to 4.0.
6.8, and more preferably 4.5 to 5.8.
If the pH is less than 4.0, hemoglobin may be denatured. If the pH exceeds 6.8, the positive charge of hemoglobins decreases, it is difficult to be retained by the cation exchange group, and the separation ability deteriorates. It is.

The eluent includes the following (1) inorganic salts, (2) pH adjuster, (3) organic solvent, (4) stabilizer, (5) amines, (6) surfactant, (7) Preservatives and the like may be added.

(1) Examples of the inorganic salts include sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, and sodium phosphate. The concentration of these salts in the eluent is not particularly limited, but is preferably 1 to 1500 mM.

(2) Known acids and bases may be added as pH adjusters. Examples of the acid include hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid and the like, and examples of the base include sodium hydroxide, potassium hydroxide, lithium hydroxide, magnesium hydroxide, barium hydroxide, calcium hydroxide and the like. No. The concentration of these acids and bases in the eluent is not particularly limited, but is preferably 0.001 to 5
00 mM.

(3) It may be mixed with a water-soluble organic solvent such as methanol, ethanol, acetonitrile and acetone. The concentration of these organic solvents is not particularly limited,
The content is preferably from 0 to 80% by volume, and is preferably used to such an extent that chaotropic ions, inorganic acids, organic acids, salts thereof and the like do not precipitate.

(4) As a hemoglobin stabilizer, a known stabilizer may be added. Examples of the stabilizer include chelating agents such as ethylenediaminetetraacetic acid (EDTA) and reducing agents and antioxidants such as glutathione and sodium azide.

(5) In order to reduce nonspecific adsorption of hemoglobin, amines may be added. As the amines, known ones are used, and preferred are primary amines, secondary amines and tertiary amines having a molecular weight of 20 to 500.

(6) As the surfactant, a nonionic surfactant, a cationic surfactant, an anionic surfactant, or the like may be added. By using a surfactant,
In addition to efficiently performing hemolysis, for example, when measurement is performed by high performance liquid chromatography (HPLC) or the like, there is an effect of washing a flow path through which a hemolysis reagent passes. The surfactant is preferably a nonionic surfactant,
For example, polyoxyethylenes (hereinafter, polyoxyethylene is represented by POE and the number of moles of ethylene oxide added by (n)), POE (7) decyl ether, POE (n)
Dodecyl ether, POE (10) tridecyl ether, POE (11) tetradecyl ether, POE
(N) Cetyl ether, POE (n) stearyl ether, POE (n) oleyl ether, POE (17) cetyl stearyl ether, POE (n) octylphenyl ether, POE (n) nonylphenyl ether, sorbitan monolaurate, monopalmitin Acid sorbitan, sorbitan monostearate, sorbitan monooleate, POE (n) sorbitan monolaurate, PO
E (n) sorbitan monopalmitate, POE (n) sorbitan monostearate, POE (n) sorbitan monooleate, and the like. These surfactants are
A single species or a mixture of two or more species may be used. The addition amount of these surfactants is preferably 0.01 to 10% by weight.

(7) As preservatives, sodium azide,
Thymol and the like may be added.

In the present invention, when two or more eluents having different dissolution powers are used, it is more preferable that these eluents are sent by a gradient elution method. The gradient elution method includes, for example, a step elution method (step-up gradient elution), a linear gradient elution method, or a combination thereof, and is not limited to any method.

FIG. 1 shows an example of the configuration of a chromatography apparatus when the measurement is performed by the above-mentioned step elution method. The eluents A, B, C, and D are eluents having different dissolution powers, and are configured to be switched to the respective eluents by the electromagnetic valve 1 every set time. The eluent is guided to the column 4 together with the sample introduced from the sample injection unit 3 by the liquid sending pump 2, and each component is detected by the detector 5. Each peak area, height, and the like are calculated by the integrator 6.

Among the hemoglobins separated by the measuring method of the present invention, HbA0 consisting of HbA or the like is HbA0.
Since it elutes after 1c and is strongly retained by the filler, the measurement time tends to be longer. For this reason, for elution of HbA0, an eluent having a higher elution strength than the above-mentioned eluent, that is, a pH of the hemoglobin isoelectric point (the isoelectric point of hemoglobin, is described in the Physical and Chemical Dictionary (4th edition, September 1987,
As described in Iwanami Shoten, Ryogo Kubo et al., P. 1178, the pH is 6.8-7, and it is preferable to use an eluent on the more alkaline side.

That is, when the pH of the eluent changes from the acidic side to the alkaline side from the isoelectric point of hemoglobin, the total charge on the surface of hemoglobin changes from plus to minus, thereby changing the cation exchange groups on the filler surface. , And the HbA0 component can be rapidly eluted.

As described above, the eluent for eluting the HbA0 component is such that the pH when flowing into the column is more alkaline than the isoelectric point of hemoglobin in order to improve column durability and shorten the measurement time. It is preferable to use at least one or more buffers whose pH is set to 6.8 or higher.

The pH of the above eluent is preferably 7 to 12
And more preferably 7.5 to 11, and even more preferably 8 to 9.5. This is because when the pH of the eluent is 6.8 or less, elution of the HbA0 component tends to be insufficient, and when the pH is higher than 12, the filler may be decomposed. Further, when the decomposition of the filler does not affect the measurement, the pH of the eluent is preferably adjusted to 12 or more, more preferably 10 to 13.5. However, the eluent p
When H is set to 12 or more, the decomposition of the filler can be minimized by shortening the flow time of the eluent even if the decomposition of the filler affects the measurement.

It is preferably used for elution of the HbA0 component.
Examples of the eluent having a buffer capacity at a pH of 6.8 or more include inorganic acids such as phosphoric acid, boric acid, and carbonic acid, and salts thereof;
A buffer solution comprising an organic acid such as a hydroxycarboxylic acid such as citric acid, a carboxylic acid derivative such as β, β-dimethylglutaric acid, a dicarboxylic acid such as maleic acid, cacodylic acid, or a salt thereof. In addition, 2- (N-morpholino) ethanesulfonic acid (MES), N-2-hydroxyethylpiperazine-N′-ethanesulfonic acid (HEPE
S), bis (2-hydroxyethyl) iminotris-
(Hydroxymethyl) methane (Bistris), Tr
is, ADA, PIPES, Bistrispropa
ne, ACES, MOPS, BES, TES, HEPE
What is generally called a Good's buffer such as S, HEPPS, Tricine, Bicine, glycylglycine, TAPS, and CAPS can also be used. Also, Britton and Robinson's buffer; GT
A buffer can also be used. Further, organic substances such as imidazoles such as imidazole; amines such as ethylenediamine, methylamine, ethylamine, triethylamine, diethanolamine and triethanolamine; and amino acids such as glycine, β-alanine, aspartic acid and asparagine can also be used.

The concentration of the above-mentioned inorganic acid / organic acid, or a salt or an organic substance thereof may be within a range having a buffering action for adjusting the pH of the eluent to 6.8 or more in a state of being dissolved in water. Is 1 mM to 1000 mM, more preferably 10 to 500 mM.

In order to elute the HbA0 component more effectively, it is preferable to add chaotropic ions to the eluent suitably used for elution of HbA0. The same chaotropic ions as those described above can be used. The concentration of the chaotropic ion in the eluent is preferably 1 mM to 3000 mM,
It is more preferably from 10 mM to 1000 mM, particularly preferably from 50 mM to 500 mM.

The above-mentioned eluent includes various additives similar to those added to the above-described eluent, for example, (1) inorganic salts,
(2) pH adjuster, (3) organic solvent, (4) stabilizer,
You may add (5) amines, (6) surfactant, (7) preservative, etc.

In the present invention, the column pressure is set to 9.8 ×
10 ⁴ Pa to 3.9 × 10 ⁶ Pa (1 kgf / cm ² to 4 Pa)
0 kgf / cm ² ) for measurement. The more preferable range of the column pressure is 2.0 × 10 ⁵ Pa to 3.4 ×.
A ^{10 6 Pa (2kgf / cm 2} ~35kgf / cm 2), still more preferably in the range of, 4.9 × 10 ⁵ Pa~2.
9 × 10 ⁶ Pa (5 kgf / cm ² -30 kgf / c
m ² ). This means that the column pressure is 9.8 × 10 ⁴ Pa
If it is smaller, the pressure fluctuation is large, so the stable HbA1c
This makes it impossible to measure with high accuracy. On the other hand, if the column pressure is higher than 3.9 × 10 ⁶ Pa, the column pressure rises greatly when continuous measurement is performed in a short time, and the column durability deteriorates.

The column pressure in the present invention means the liquid pressure of the eluent flowing into the separation column, and is provided on the flow path of the eluent flowing into the separation column in the liquid chromatography apparatus. It is measured by a pressure gauge. The pressure gauge for measuring the column pressure is not particularly limited as long as it is generally used in a liquid chromatography apparatus. For example, a pressure gauge specified in JIS B 7505-1980 may be used. A tube pressure gauge is preferably used.

The setting of the column pressure is performed by adjusting various conditions which affect the magnitude of the column pressure. Specifically, the flow rate of the eluent, the size of the particle size of the packing material packed in the column, the column inner diameter, the column length, and the like are appropriately adjusted so as to be within the above range. In addition, a filler having a small particle size (for example, a filler having a particle size of 3 μm or less: including a porous filler and a non-porous filler)
Is used, the flow rate of the eluent may be adjusted so as to be within the above-mentioned column pressure range, or the size of a column filled with a filler having a small particle size may be appropriately reduced (for example, (Inner diameter: 0.1 mm to 4 mm, column length: about 5 mm to 200 mm).

For example, when only the flow rate of the eluent is changed under the same other conditions, the flow rate of the eluent may be increased to increase the column pressure, and the eluent may be decreased to decrease the column pressure. What is necessary is just to reduce the flow velocity of. Also, when only the size of the particle size of the packing material is changed, the particle size of the packing material may be reduced in order to increase the column pressure, and the particle size of the packing material may be reduced in order to lower the column pressure. Should be increased.

Other measurement conditions in the present invention include:
Known conditions may be used, and the flow rate of the eluent is preferably 0.0
01-5 ml / min, more preferably 0.01-4 ml / min
Min, particularly preferably 0.1 to 3 ml / min.

The filler used in the method for measuring hemoglobin of the present invention comprises particles having at least one or more cation exchange groups.
It is obtained by introducing a cation exchange group into the polymer particles.

The cation exchange group may be a known one and is not particularly limited. For example, a cation exchange group such as a carboxyl group, a sulfonic acid group, and a phosphoric acid group is exemplified. Further, a plurality of cation exchange groups may be introduced.

The diameter of the above-mentioned filler particles is preferably set to 0.1.
It is 5-20 μm, more preferably 1-10 μm. Further, the particle size distribution is preferably 40 as a coefficient of variation (CV value) (standard deviation of particle size 粒径 average diameter × 100 (%)).
%, More preferably 30% or less.

As the filler particles, if necessary, a plurality of types of fillers in the above range may be mixed and used.

Examples of the polymer particles include inorganic particles such as silica and zirconia; natural polymer particles such as cellulose, polyamino acid and chitosan; and synthetic polymer particles such as polystyrene and polyacrylate. .

In the above-mentioned polymer particles, it is preferable that the constituent components other than the ion exchange groups to be introduced are more hydrophilic. Further, those having a high degree of crosslinking are preferable from the viewpoint of pressure resistance and swelling resistance.

The introduction of the cation exchange group into the polymer particles can be performed by a known method. For example, after preparing the polymer particles, the functional groups (hydroxyl group, amino group, carboxyl group, epoxy group) And the like, by introducing a cation exchange group into the particles by a chemical reaction.

A cation exchange filler can also be prepared by a method of preparing polymer particles by polymerizing a monomer having a cation exchange group. For example, there is a method in which a cation exchange group-containing monomer and a crosslinkable monomer are mixed and polymerized in the presence of a polymerization initiator.

Further, a polymerizable cation-exchange group-containing ester such as methyl (meth) acrylate or ethyl (meth) acrylate is mixed with a crosslinkable monomer and polymerized in the presence of a polymerization initiator. The resulting particles may be subjected to a hydrolysis treatment to convert the ester into a cation exchange group.

Further, as described in JP-B-8-7197, after preparing a crosslinked polymer particle, a monomer having a cation exchange group is added, and the vicinity of the surface of the polymer particle is added.
The monomer may be polymerized.

As a method for filling the above-mentioned filler into the column, any known method can be used, but it is more preferable to use a slurry filling method. Specifically, for example, it is performed by pressing a slurry in which filler particles are dispersed in a buffer such as an eluent into a column by a liquid sending pump or the like.

The liquid chromatography apparatus used to carry out the measurement method of the present invention may be a known one,
For example, it comprises a liquid sending pump, a sample injection device (sampler), a column, a detector, and the like. Further, other accessory devices (such as a column thermostat and an eluent deaerator) may be appropriately attached.

Further, a semi-micro type device may be used. One example is NANOSPA manufactured by Shiseido.
There is CE series, and liquid sending pump: inert pump 20
It consists of 01NANOSPACE SI-1, autosampler 2003, detector UV-VIS detector 2002, and the like.

The column may be made of a known material.
For example, resins such as glass, stainless steel, and PEEK (polyetheretherketone) can be used.

The material of the filter mounted on the column may be a known material. Filters provided at both ends of the column reduce the adsorption of hemoglobins,
Inert (inert) materials are preferred. For example,
Examples include polyetheretherketone, polyethylene, Teflon (registered trademark), and titanium. In addition, a filter having a large adsorption of hemoglobin, such as a filter made of stainless steel, may have its surface treated with silicone or blocked with a known blocking reagent (such as albumin, bovine serum albumin, casein, and gelatin). The above filter may be used as a pre-filter.

Further, the area where the column is in contact with the packing material of the column may be covered with an inert material, such as PEEK, polyethylene, Teflon, a titanium compound, a silicon compound, a silicon film and the like. Can be

The other measuring conditions in the above measuring method may be known conditions and are not particularly limited, but preferably the following conditions are used. For detection of hemoglobins, it is preferable to use 415 nm visible light. Usually, it is preferable to use a measurement sample obtained by diluting the hemolyzed blood with a solution containing a substance having hemolytic activity such as a surfactant. The amount of sample injected into the liquid chromatography apparatus varies depending on the dilution ratio, but is preferably 0.1 to
It is about 100 μL.

One embodiment of the method for measuring hemoglobins in the present invention is described below. When a lysed blood sample is separated by cation exchange liquid chromatography, Hb
A1a and HbA1b, HbF, unstable HbA1c,
Peaks such as stable HbA1c and HbA0 appear.

Here, three kinds of eluents A having different dissolution powers are used.
To C (eluent A <eluent B <eluent C, the output of which is set to increase in the order of eluent C).
HbA1a to HbF were eluted and unstable HbA1c
And stable HbA1c are strongly separated by a column. Next, the eluent B having a stronger dissolution output is flowed by the step gradient elution method, and the unstable HbA1c peak and the stable HbA1c peak separated by being strongly retained in the column are eluted sharply. Finally, the eluent C having the strongest elution is eluted by flowing the eluent C having the strongest elution by the step gradient elution method. The column is equilibrated with eluent A to such an extent that the system is not adversely affected.

[Operation] Conventionally, in a column packed with a packing material having a large particle size, unstable HbA1c and stable HbA1c could not be separated due to the large diffusion of a measurement sample in the column. By adding chaotropic ions to the eluent, the difference in interaction between the unstable HbA1c and the stable HbA1c columns, which have very close separation and elution behavior, is enlarged, and the stable HbA1
Even with a filler having a large particle size for which c could not be separated and measured, stable HbA1c can be separated and measured. In addition, a column filled with a packing material having a large particle size has a very low column pressure, so that the measurement sample is difficult to clog the column.
Therefore, the column pressure rise is small even in continuous measurement, and the column durability is excellent. Furthermore, by combining the gradient elution method, stable HbA
1c can be measured separately.

[0073]

Next, the present invention will be described in detail with reference to examples and comparative examples, but the present invention is not limited to these examples.

Example 1 (1) Preparation of Filler 450 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and 50 g of 2-hydroxy-1,3-dimethacryloxypropane (manufactured by Wako Pure Chemical Industries, Ltd.) 2 g of benzoyl peroxide (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in the mixture.
This was dispersed in 2500 mL of a 4% by weight aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Industry Co., Ltd.), and the temperature was raised to 80 ° C. under a nitrogen atmosphere with stirring to carry out polymerization for 1.5 hours. Next, after cooling the reaction system to 35 ° C., 2-acrylamido-2-methylpropanesulfonic acid (manufactured by Tokyo Chemical Industry Co., Ltd.)
Of a 50% aqueous solution and 400 mL of methanol were added, and the mixture was heated to 80 ° C. again with stirring for 1 hour and polymerized for 1.3 hours. After the polymerization, the polymer was washed, dried, and classified to obtain particles having an average particle size of 9 μm.

(2) Packing into Column The packing obtained in (1) was packed into a column as follows. 0.7 g of the filler particles were dispersed in 30 mL of 50 mM phosphate buffer (pH 5.8), sonicated for 5 minutes, and then stirred well. The whole amount was injected into a packer (manufactured by Umetani Seiki Co., Ltd.) connected to a stainless steel empty column (inner diameter 4.6 mm x length 35 mm). A liquid feed pump (manufactured by Sanuki Kogyo KK) was connected to the packer, and the pressure was 300 kg / cm ^2.
At constant pressure.

(3) Measurement of hemoglobins Using the column obtained in (2), hemoglobins were measured under the following measurement conditions. (Measurement conditions) System: Liquid pump: LC-9A (manufactured by Shimadzu Corporation) Autosampler: ASU-420 (manufactured by Sekisui Chemical Co., Ltd.) Detector: SPD-6AV (manufactured by Shimadzu Corporation) Pressure gauge: strain gauge type Pressure Transducer Eluent: Eluent A: 10 mM adipic acid-50 mM phosphate buffer (pH 5.3) containing 56 mM perchloric acid Eluent B: 10 mM adipic acid-50 mM phosphoric acid containing 275 mM perchloric acid Acid buffer (pH 8.40) Feeding pattern: Eluent A is sent for 0 to 1.2 minutes from the start of measurement, and eluent B is sent for 1.2 to 1.3 minutes. The eluent A was sent for 1.3 to 1.9 minutes. Flow rate: 2.2 mL / min Detection wavelength: 415 nm Sample injection volume: 10 μL Column pressure: 2.9 × 10 ⁶ Pa (30 kgf / cm ² )

(Measurement Samples) The following samples were prepared from whole blood samples obtained by collecting sodium fluoride from healthy human blood. As a hemolysis reagent, 0.1% by weight of polyethylene glycol mono-4-octylphenyl ether (Triton X-10) was used.
0) (manufactured by Tokyo Chemical Industry Co., Ltd.)
H7.0) was used.

(4) Measurement Results The chromatogram obtained under the above measurement conditions is shown in FIG. Peak 11 is HbA1a and b, peak 12 is HbF, peak 13 is unstable HbA1c, and peak 14
Indicates stable HbA1c, and peak 15 indicates HbA0. FIG. 2 shows that the stable A1c was able to be separated and measured in a measuring time of 1.9 minutes.

Example 2 (1) Preparation of Filler 450 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical) and 50 g of 2-hydroxy-1,3-dimethacryloxypropane (manufactured by Wako Pure Chemical Industries) were used. 2 g of benzoyl peroxide (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in the mixture.
This was dispersed in 2500 mL of a 4% by weight aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Industry Co., Ltd.), and the temperature was raised to 80 ° C. under a nitrogen atmosphere with stirring to carry out polymerization for 1.5 hours. Next, after the reaction system was cooled to 35 ° C, 400 g of a 50% aqueous solution of 2-acrylamido-2-methylpropanesulfonic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) and 400 mL of methanol were added, and the temperature was raised to 80 ° C again with stirring for 1 hour. And polymerized for 1.3 hours. After polymerization, wash, dry, classify and average particle size 11
μm particles were obtained.

(2) Packing into a Column The same procedure as in Example 1 was performed.

(3) Measurement of Hemoglobins The same procedures as in Example 1 were carried out except for the following items. Eluent: Eluent A: 10 mM adipic acid-50 mM phosphate buffer (pH 5.3) containing 56 mM perchloric acid Eluent B: 10 mM adipic acid-50 mM phosphate buffer containing 70 mM perchloric acid (PH 5.3) Eluent C: 10 mM adipic acid-50 mM phosphate buffer (pH 8.40) containing 275 mM perchloric acid Transmission pattern: Eluent A from 0 to 0.6 minutes from the start of measurement The eluent B is sent for 0.6 to 1.2 minutes, the eluent C is sent for 1.2 to 1.3 minutes, and 1.3 to 1.6 minutes. The eluent A was sent during the minute. Column pressure: 7.8 × 10 ⁵ Pa (8 kgf / cm ² )

(4) Measurement Results The chromatogram obtained under the above measurement conditions is shown in FIG. Peak 11 is HbA1a and b, peak 12 is HbF, peak 13 is unstable HbA1c, and peak 14
Indicates stable HbA1c, and peak 15 indicates HbA0. 3. From FIG. 3, the stable A1c was able to be separated and measured in a measurement time of 1.6 minutes.

Comparative Example 1 (1) Preparation of Filler 450 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical) and 50 g of 2-hydroxy-1,3-dimethacryloxypropane (manufactured by Wako Pure Chemical Industries) were used. 2 g of benzoyl peroxide (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in the mixture.
This was dispersed in 2500 mL of a 4% by weight aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Industry Co., Ltd.), and the temperature was raised to 80 ° C. under a nitrogen atmosphere with stirring to carry out polymerization for 1.5 hours. Next, after the reaction system was cooled to 35 ° C, 400 g of a 50% aqueous solution of 2-acrylamido-2-methylpropanesulfonic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) and 400 mL of methanol were added, and the temperature was raised to 80 ° C again with stirring for 1 hour. And polymerized for 1.3 hours. After the polymerization, the polymer was washed, dried, and classified to obtain particles having an average particle size of 3 μm.

(2) Packing into a Column The same procedure as in Example 1 was performed.

(3) Measurement of hemoglobins The same procedures as in Example 1 were carried out except for the following items. Column pressure: 7.8 × 10 ⁶ Pa (80 kgf / c
m ² )

(4) Measurement Results The chromatogram obtained in Comparative Example 1 was the same as in FIG.

Comparative Example 2 (1) Preparation of Filler 450 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical) and 50 g of 2-hydroxy-1,3-dimethacryloxypropane (manufactured by Wako Pure Chemical Industries) were used. 2 g of benzoyl peroxide (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in the mixture.
This was dispersed in 2500 mL of a 4% by weight aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Industry Co., Ltd.), and the temperature was raised to 80 ° C. under a nitrogen atmosphere with stirring to carry out polymerization for 1.5 hours. Next, after the reaction system was cooled to 35 ° C, 400 g of a 50% aqueous solution of 2-acrylamido-2-methylpropanesulfonic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) and 400 mL of methanol were added, and the temperature was raised to 80 ° C again with stirring for 1 hour. And polymerized for 1.3 hours. After polymerization, wash, dry, classify and average particle size 3
0 μm particles were obtained.

(2) Packing into a Column The same procedure as in Example 1 was performed.

(3) Measurement of Hemoglobins The same procedures as in Example 1 were carried out except for the following items. Column pressure: 4.9 × 10 ⁴ Pa (0.5 kgf / cm ² )

(4) Measurement Results Measurement of hemoglobins was attempted under the above measurement conditions.
Could not measure.

Example 3 (1) Preparation of Filler The same operation as in Comparative Example 1 was performed.

(2) Packing into a Column The same procedure as in Comparative Example 1 was performed.

(3) Measurement of hemoglobins Using the column obtained in (2), hemoglobins were measured under the following measurement conditions. (Measurement conditions) System: Liquid pump: LC-9A (manufactured by Shimadzu Corporation) Autosampler: ASU-420 (manufactured by Sekisui Chemical Co., Ltd.) Detector: SPD-6AV (manufactured by Shimadzu Corporation) Pressure gauge: strain gauge type Pressure Transducer Eluent: Eluent A: 10 mM adipic acid-50 mM phosphate buffer (pH 5.40) containing 62 mM perchloric acid Eluent B: 10 mM adipic acid-50 mM phosphoric acid containing 70 mM perchloric acid Acid buffer (pH 5.40) Eluent C: 10 mM adipic acid-50 mM phosphate buffer (pH 8.40) containing 275 mM perchloric acid Transmission pattern: 0 to 0.6 minutes from the start of measurement Eluent A is sent, eluent B is sent for 0.6 to 1.2 minutes, eluent C is sent for 1.2 to 1.3 minutes, and 1.3 to 1.3 minutes. The eluent A was sent for 1.6 minutes. Flow rate: 1.0 ml / min Detection wavelength: 415 nm Sample injection amount: 10 μl Column pressure: 3.8 × 10 ⁶ Pa (39 kgf / cm ² )

(4) Measurement Results The chromatogram obtained under the above measurement conditions was the same as in FIG. 3, and stable A1c could be separated and measured in 1.6 minutes.

Example 4 (1) Preparation of Filler The same operation as in Comparative Example 1 was performed.

(2) Packing in Column The packing obtained in (1) was packed in a column as follows. 0.7 g of the filler particles were dispersed in 30 ml of a 50 mM phosphate buffer (pH 5.8), sonicated for 5 minutes, and then stirred well. The whole amount was injected into a packer (manufactured by Umeya Seiki Co., Ltd.) to which a stainless steel empty column (inner diameter 40 mm x length 30 mm) was connected. A liquid pump (manufactured by Sanuki Kogyo KK) was connected to the packer, and the pressure was 300 kg / cm ^2.
At constant pressure. (3) Measurement of hemoglobin Using the column obtained in (2), measurement of hemoglobin was performed under the following measurement conditions. (Measurement conditions) System: Liquid pump: LC-9A (manufactured by Shimadzu Corporation) Autosampler: ASU-420 (manufactured by Sekisui Chemical Co., Ltd.) Detector: SPD-6AV (manufactured by Shimadzu Corporation) Pressure gauge: strain gauge type Pressure transducer Eluent: Eluent A: 10 mM adipic acid-50 mM phosphate buffer (pH 5.40) containing 58 mM perchloric acid Eluent B: 10 mM adipic acid-50 mM phosphoric acid containing 68 mM perchloric acid Acid buffer (pH 5.40) Eluent C: 10 mM adipic acid-50 mM phosphate buffer (pH 8.40) containing 275 mM perchloric acid Transmission pattern: 0 to 0.6 minutes from the start of measurement Eluent A is sent, eluent B is sent for 0.6 to 1.2 minutes, eluent C is sent for 1.2 to 1.3 minutes, and 1.3 to 1.3 minutes. The eluent A was sent for 1.6 minutes. 1 ml / min Detection Wavelength: 415 nm Sample injection amount: 10 [mu] l column ^{pressure: 3.5 × 10 6 Pa (36kgf} / cm 2)

(4) Measurement Results The chromatogram obtained under the above measurement conditions was the same as in FIG. 3, and stable A1c could be separated and measured in a measurement time of 1.6 minutes.

Example 5 (1) Preparation of Filler The same operation as in Comparative Example 1 was performed.

(2) Packing into Column The procedure was the same as in Example 4, except that the filler obtained in (1) was packed in an empty stainless steel column (1.5 mm in inner diameter × 35 mm in length). (3) Measurement of hemoglobin Using the column obtained in (2), measurement of hemoglobin was performed under the following measurement conditions. (Measurement conditions) System: Liquid pump: Inert pump 2001: NANOSPACE SI-1 (made by Shiseido) Autosampler: Autosampler 2003 (made by Shiseido) Detector: UV-VIS detector 2002 (made by Shiseido) Elution Liquid: Eluent A: 10 mM succinic acid-47 mM phosphate buffer containing 49 mM perchloric acid (pH 5.30) Eluent B: 10 mM succinic acid-47 mM phosphate buffer containing 60 mM perchloric acid (pH 5.30) 30) Eluent C: 10 mM succinic acid-47 mM phosphate buffer (pH 8.40) containing 250 mM perchloric acid Sending pattern: Sending the eluent A from 0 to 0.6 minutes from the start of measurement The eluent B is sent for 0.6 to 1.2 minutes, the eluent C is sent for 1.2 to 1.3 minutes, and the eluent C is sent for 1.3 to 1.6 minutes. Sent eluent A. Flow rate: 0.18 ml / min Detection wavelength: 415 nm Sample injection amount: 2 μl Column pressure: 2.2 × 10 ⁶ Pa (22 kgf / cm ² ) (4) Measurement results The chromatogram obtained under the above measurement conditions is shown in FIG. Similarly, stable A1c could be separated and measured in 1.6 minutes.

[Evaluation of Column Durability] With respect to the columns of Examples 1 to 4 and Comparative Examples 1 and 2, 500 samples were continuously measured under the above-described measurement conditions to evaluate the column durability. As a measurement sample, the following sample was prepared from a whole blood sample obtained by collecting normal human blood with sodium fluoride. As a hemolysis reagent, 0.1% by weight of polyethylene glycol mono-4-octylphenyl ether (Triton X-10) was used.
0) (manufactured by Tokyo Chemical Industry Co., Ltd.)
H7.0) was used.

[Results of Column Durability Evaluation] Stable HbA
The column durability was evaluated using the change in the measured value of 1c as an index, and is shown in FIG. As a result, in Examples 1-4, 50
Even when 0 samples are continuously measured, the fluctuation of the stable HbA1c value is as follows.
Almost never. However, in Comparative Example 1, the stable HbA1c value gradually increased after 100 samples were continuously measured. This is because when the column pressure is set high, a part of the measurement sample is clogged in the column, and the column washing becomes insufficient.
This was thought to be due to insufficient separation of bA1c. In Comparative Example 2, the stable HbA1c value could not be measured because the column pressure was too low.

[0102]

As described above, the method for measuring hemoglobins of the present invention has the above-mentioned structure, so that the column durability is excellent, and
Stable HbA1c can be accurately separated and measured in a short time.

[Brief description of the drawings]

FIG. 1 shows an example of a liquid chromatography apparatus.

FIG. 2 is a diagram showing a measurement result in Example 1.

FIG. 3 is a diagram showing a measurement result in Example 2.

FIG. 4 shows the results of column durability evaluation.

[Explanation of symbols]

 A, B, C, D: Eluents A to D 1: Solenoid valve 2: Liquid sending pump 3: Sample injection section 4: Column 5: Detector 6: Integrator 11: HbA1a and HbA1b 12: HbF13: Unstable HbA1c 14: Stable HbA1c 15: HbA0

Claims (2)

[Claims] 1. A method for measuring hemoglobins by cation exchange liquid chromatography, comprising an inorganic acid, an organic acid and / or a salt thereof containing a chaotropic ion and having a buffering ability at a pH of 4.0 to 6.8. Column pressure was set to 9.8 × 10 ⁴ Pa to 3.9.
A method for measuring hemoglobins, wherein the measurement is carried out at a pressure of 10 ⁶ Pa. 2. The method for measuring hemoglobin according to claim 1, wherein said eluent is sent using a gradient elution method.