JP2003107069A - Method for measuring hemoglobins - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring hemoglobins that can quickly and accurately separate stabilized HbAlc more stably than in the conventional method. SOLUTION: In the method for measuring hemoglobins by cation exchange liquid chromatography, at least three types of eluates are used, namely eluate for allowing hemoglobins that flow before hemoglobin AO to flow out (eluate A), eluate for allowing the hemoglobin AO to flow out (eluate B), eluate for reducing the flow output of the eluate B (eluate C). In the method for measuring homoglobins, pH in the eluate B is higher than an isoelectric point in hemoglobins, pH in the eluate C is the same as or less than pH in the eluate A, the difference between pH in the eluate C and that in the eluate A is within a range of 0-1.0, and the concentration of salt in the eluate C is lower than that of the eluate A by 1-500 mM.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring hemoglobins by liquid chromatography.

[0002]

2. Description of the Related Art Hemoglobin A1c (hereinafter referred to as HbA1c
Is produced by irreversibly binding to hemoglobin after sugar in blood enters erythrocytes, and the average blood sugar level (glucose concentration in blood in the past 1-2 months). ) Is reflected. Therefore, HbA1c is widely used as an index for diabetes diagnosis, such as a screening test for diabetes and grasping the blood sugar control status of a diabetic patient.

More specifically, in HbA1c, glucose in blood and hemoglobin A (hereinafter referred to as HbA) are
Glycated hemoglobin (hereinafter referred to as GHb) produced by the reaction, which is reversibly reacted, is called unstable hemoglobin A1c (unstable HbA1c: hereinafter referred to as unstable HbA1c), and is irreversible via unstable HbA1c. Stable hemoglobin A1c
(Stable HbA1c: hereinafter referred to as stable HbA1c). Then, it is preferable to use stable HbA1c as an index for the above-mentioned diabetes diagnosis, and in the field of clinical examination, development of a method capable of measuring the ratio of stable HbA1c with high accuracy has been desired.

Conventionally, a liquid chromatography method or an immunization method has been generally used as a method for measuring HbA1c.

HbA1 by liquid chromatography
The measurement of c is mainly performed by a cation exchange liquid chromatography method (Japanese Patent Publication No. 8-7198, etc.). When a hemolyzed sample is separated by cation exchange liquid chromatography, hemoglobin A1a (hereinafter referred to as HbA1a), hemoglobin A1b (hereinafter referred to as HbA1b), hemoglobin F (hereinafter referred to as Hb)
Peaks such as unstable HbA1c, stable HbA1c and hemoglobin A0 (hereinafter referred to as HbA0).

Further, HbA1a, HbA1b and HbA
1c is GHb in which HbA is glycosylated, and HbF is
Fetal hemoglobin composed of α chain and γ chain, and the HbF value is also used as a diagnostic index for hemolytic anemia.

HbA1c, which is used as an index for the diagnosis of diabetes, has recently been stable HbA as described above.
1c, which is calculated as the ratio (%) of the area of the stable HbA1c peak to the area of all hemoglobin peaks.

However, since it is difficult to separate the stable HbA1c peak and the unstable HbA1c peak, it is difficult to separate the peaks.
No. 3-298063 discloses red blood cells and / or
A method is described in which a sample containing hemoglobin is brought into contact with a dissociation solvate containing a phosphoric acid condensate and / or a salt of a phosphoric acid condensate to dissociate unstable hemoglobin into hemoglobin and glucose. However, in this method, the separation of stable HbA1c is insufficient,
It had the drawback of poor measurement accuracy.

In addition, in the method for measuring hemoglobins by liquid chromatography, it is said that it is influenced by modified hemoglobin such as acetylated hemoglobin (hereinafter referred to as AHb) and carbamylated hemoglobin (hereinafter referred to as CHb), and AHb and CHb. Peak is stable type H
Since it appears near the bA1c peak, it is stable H in the past.
In order to improve the separation of bA1c, there was also a method of performing separation up to stable A1c by a gradient elution method using two kinds of eluents. However, in this method, stable HbA1c could not be stably and accurately separated due to the influence of the concentration of the eluent used over time, the difference between column lots, the deterioration of the column due to use, and the like.

[0010]

SUMMARY OF THE INVENTION In view of the problems of the method for measuring hemoglobins according to the conventional method described above, the object of the present invention is to stably separate the stable HbA1c from the conventional method in a short time and with high accuracy. It is to provide a method for measuring hemoglobins.

[0011]

The present invention provides a method for measuring hemoglobins by cation exchange liquid chromatography, wherein an eluent for eluting hemoglobins that elutes before hemoglobin A0 (hereinafter referred to as "eluent A"). ), At least three kinds of eluents for eluting hemoglobin A0 (hereinafter referred to as "eluent B") and eluents for decreasing the elution output of eluent B (hereinafter referred to as "eluent C"). Liquid, the pH of the eluent B is higher than the isoelectric point of hemoglobins, the pH of the eluent C is the same as or lower than the pH of the eluent A, and the p
The difference between H and the pH of the eluent A is in the range of 0 to 1.0,
The salt concentration of the eluent C is 1 mM to 50 than the salt concentration of the eluent A.
It is a method for measuring hemoglobins characterized by being 0 mM lower.

In the present invention, the eluent A further contains chaotropic ions and has a pH of 4.0 to 6.
It is preferable to contain an inorganic acid, an organic acid and / or a salt thereof having a buffering capacity at 8.

Further, in the present invention, the above eluent A,
More preferably, each of B and C contains an azide ion. The present invention will be described in detail below.

When hemoglobins are usually separated by cation exchange liquid chromatography, HbA1a and HbA1b, HbF, unstable HbA1c and stable HbAb.
Peaks appear in the order of A1c and HbA0. In the method for measuring hemoglobins according to the present invention, HbA0 is measured by the eluent A.
Hemoglobins that elute before HbA1
a to stable HbA1c, and then eluent B
Is used to elute HbA0, and then the eluent C is caused to flow to weaken the elution output of the eluent B.

As the eluent A used in the present invention, it is preferable to use a solution containing an inorganic acid, an organic acid and / or a salt thereof having a buffering capacity at a pH of 4.0 to 6.8.

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

Examples of the above-mentioned inorganic acid include phosphoric acid and the like. Examples of the organic acid include carboxylic acid, dicarboxylic acid, carboxylic acid derivative, hydroxycarboxylic acid, cacodylic acid, amino acid and the like.

Examples of the carboxylic acid include acetic acid,
Propionic acid and the like can be mentioned. 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 and β-
Examples thereof include dimethyl glutaric acid, barbituric acid and aminobutyric acid. Examples of the hydroxycarboxylic acid include citric acid, tartaric acid, lactic acid and the like. Examples of the amino acid include aspartic acid and histidine.

The salt of the above-mentioned inorganic acid or organic acid may be a known one, and examples thereof include sodium salt and potassium salt.

The above-mentioned inorganic acid, organic acid or salts thereof are
They may be used alone or as a mixture of two or more kinds.
You may mix and use an inorganic acid and an organic acid.

As the above-mentioned inorganic acid or organic acid, a substance having an acid dissociation constant (pKa) value of 2.15 to 6.39 and a substance having a pKa value of 6.4 to 10.5 are combined. It is also good to use. Instead of using two or more kinds of substances in combination, a single substance having pKa values of 2.15 to 6.39 and 6.4 to 10.5 may be used. The more preferable range of the pKa value is 2.61 to 6.39 and 6.40 to 10.50, and the more preferable range is 2.80 to 6.35 and 6.80 to 10.00. , A particularly preferred range is
3.50 to 6.25 and 7.00 to 9.50.

The concentration of these inorganic acids, organic acids, etc. in the eluent A should be such that it has a buffering action to bring the pH of the eluent A to 4.0 to 6.8 when dissolved in water. However, it is preferably 1 mM to 1000 mM, more preferably 10 mM to 500 mM.

The pH of the eluent A is pH 4.0 to 6.
8 is preferable, and more preferably pH is 4.5 to 5.8. If the pH is lower than 4.0, hemoglobin may be denatured, and if the pH is higher than 6.8, the positive charge of hemoglobins decreases, and it becomes difficult for the hemoglobins to be retained by the cation exchange group, resulting in poor resolution. Is.

It is preferable that the eluent A further contains chaotropic ions. The chaotropic ions, by the ions generated by dissociating in an aqueous solution, the structure of water is destroyed, which suppresses the entropy reduction of water, which occurs when a hydrophobic substance and water come into contact, and specifically, Examples of the anion include tribromoacetate ion, trichloroacetate ion, thiocyanate ion, iodide ion, perchlorate ion, dichloroacetate ion, nitrate ion, bromide ion, chloride ion, acetate ion, and the like. Urea etc. are mentioned. Examples of the cation include barium ion, calcium ion, magnesium ion, lithium ion, cesium ion, potassium ion and guanidine ion.

Among the above chaotropic ions, in order to improve the measurement accuracy of stable HbA1c, the anion is 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, more preferably tribromoacetate ion,
Trichloroacetate ion, thiocyanate ion, iodide ion, perchlorate ion, nitrate ion, guanidine ion and the like are used.

The concentration of the above chaotropic ions in the eluent A is preferably 0.1 mM to 3000 mM, and 1 mM.
-1000 mM is more preferable, and even more preferably 1
It is 0 mM-500 mM. If the concentration of chaotropic ions is lower than 0.1 mM, the separation effect will be reduced and the measurement accuracy will be poor in the measurement of hemoglobins. Also, 3
Even if it is higher than 000 mM, the separation effect of hemoglobins does not improve any more, and therefore the measurement accuracy does not improve. Further, these chaotropic ions may be used alone or in combination of two or more. Further, it can be used by adding it to a liquid that comes into contact with the measurement sample, for example, a sample diluent.

More preferably, the eluent A contains azide ion. The azide ion is not particularly limited as long as it dissociates in an aqueous solution to generate an azide ion, and for example, a known azide salt or the like is used, and preferably sodium azide, barium azide or the like. Is mentioned.

The concentration of the azide in the eluent A is 0.15 mM to 76.9 mM (0.001% by weight).
0.5% by weight), and more preferably 0.77 mM to 61.5 mM (0.005% by weight).
0.4% by weight). The above concentration is 0.15 mM
If the amount is less than (0.001% by weight), the separation of HbF becomes poor, and if the amount is more than 76.9 mM (0.5% by weight), the H
This is because the separation of bF does not improve.

Further, the following substances (1) to (6) may be added to the eluent A. (1) Examples of the inorganic salts include sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium phosphate and the like. The concentration of these salts is not particularly limited, but is preferably 1 to 1500 mM. (2) A known acid or base may be added as a pH adjusting agent. 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. Can be mentioned. The concentrations of these acids and bases are not particularly limited,
Preferably, it is 0.001 to 500 mM. (3) You may mix with water-soluble organic solvents, such as methanol, ethanol, acetonitrile, and acetone. The concentration of these organic solvents is not particularly limited, but is preferably 8
0% by volume or less, chaotropic ions, inorganic acids,
It is preferable to use the organic acid and salts thereof in such an extent that they do not precipitate. (4) A known stabilizer may be added as a stabilizer for hemoglobin. As the stabilizer, for example, a chelating agent such as ethylenediaminetetraacetic acid (EDTA), a reducing agent such as glutathione, an antioxidant and the like may be added. (5) In order to reduce non-specific adsorption of hemoglobin, amines may be added. As the amines,
A known one is used, and preferably has a molecular weight of 20 to 50.
0 primary amines, secondary amines and tertiary amines.

(6) As the surface active agent, a nonionic surface active agent, a cationic surface active agent, an anionic surface active agent or the like may be added. By using a surfactant,
Not only is hemolysis performed efficiently, but, for example, in the case of performing measurement by high performance liquid chromatography (HPLC) or the like, it has an effect of washing a flow path through which a hemolytic reagent passes. The surfactant is preferably a nonionic surfactant,
For example, polyoxyethylenes (hereinafter, polyoxyethylene is represented by POE, the number of moles of ethylene oxide added is represented 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) octyl phenyl ether, POE (n) nonyl phenyl ether,
Sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, POE (n) sorbitan monolaurate, PO
Examples thereof include sorbitan monopalmitate E (n), sorbitan monostearate POE (n), and sorbitan monooleate POE (n). These surfactants are
You may use individually or in mixture of multiple. Further, the addition amount of these surfactants is preferably 0.01 to 10% by weight.

Next, the eluent B will be described. The eluent B used in the present invention is characterized by having a pH higher than the isoelectric point of hemoglobins. Among hemoglobins separated by cation exchange liquid chromatography, HbA0 is composed of HbA and the like, and is eluted after HbA1c and strongly retained in the packing material, which tends to increase the measurement time. Therefore, as the eluent B,
A hemoglobin having a high elution output is used, and specifically, an eluent having a pH higher than the isoelectric point of hemoglobin is used. Specifically, a pH 7.0 to 12 is used, a pH 7.1 to 11.0 is more preferable, and a pH 8.0 to 9.5 is more preferable.

As the eluent B, for example, phosphoric acid,
Inorganic acids such as boric acid and carbonic acid, or salts thereof; hydroxycarboxylic acids such as citric acid, carboxylic acid derivatives such as β, β-dimethylglutaric acid, dicarboxylic acids such as maleic acid, organic acids such as cacodylic acid, or salts thereof. A buffer solution containing is used. In addition, 2- (N-morpholino) ethanesulfonic acid (MES), N-2-hydroxyethylpiperazine-N′-ethanesulfonic acid (HEPES),
Bis (2-hydroxyethyl) iminotris- (hydroxymethyl) methane (Bistris), Tris, A
DA, PIPES, Bistrisprane, A
CEA, MOPS, BES, TES, HEPPS, Tr
What is generally called Good's buffer solution such as icine, Bicine, TAPS, and CAPS can also be used. Further, a buffer solution containing an organic substance such as imidazoles such as imidazole; amines such as diethanolamine and triethanolamine; amino acids such as glycine; A plurality of inorganic acids, organic acids and / or their salts, and organic substances may be mixed and used, or inorganic acids and organic substances may be mixed and used.

Further, in order to more effectively elute the HbA0 component, it is preferable to add chaotropic ions to the eluent B. As the chaotropic ion, the same one as the eluent A described above can be used. The concentration of chaotropic ion in the eluent B is 1 mM to 3
000 mM is preferable, 10 mM to 1000 mM is more preferable, and 50 mM to 500 mM is particularly preferable.

Further, it is more preferable that the eluent B contains the above-mentioned azide ion. The concentration of azide ions contained in the eluent B should be approximately the same as the concentration of azide ions contained in the eluent A.

The eluent B contains various additives such as (1) inorganic salts (2) p added to the eluent A described above.
Substances such as H regulator (3) water-soluble organic solvent (4) stabilizer (5) amines (6) surfactant may be added.

Next, the eluent C will be described. The eluent C used in the present invention has a pH equal to that of the eluent A.
Is equal to or less than, and the difference from the pH of the eluent A [= (pH of eluent A)-(pH of eluent C)] is 0 to 1.
It is characterized by being in the range of 0. Further, the eluent C is characterized in that its salt concentration is lower than that of the eluent A by 1 mM to 500 mM. This is because when the pH of the eluent C becomes higher than the pH of the eluent A, the elution output of the eluent B cannot be sufficiently reduced, and the separation performance of the stable HbA1c becomes poor. Also, eluent C and eluent A
This is because if the difference in pH is more than 1.0, the pH of the eluent C becomes too low and denaturation of hemoglobins is likely to occur, degrading the separation performance of stable HbA1c. Still more preferably, the difference in pH between the eluent C and the eluent A is 0 to 0.5. The timing of flowing the eluent C is not particularly limited as long as it can weaken the elution power of the eluent B at least, but basically it is after the eluent B and before the eluent A. It is good to flush. The eluent C has a pH equal to or lower than that of the eluent A and has a salt concentration of 1 mM to 500 mM from the salt concentration of the eluent A.
The elution output of hemoglobins is lower than that of the eluent A because it is low. Therefore, by flowing the eluent C prior to flowing the eluent A after the eluent B having a high elution output, the liquid property in the column can be brought closer to the eluent A more efficiently in a shorter time. As a result, stable HbA1c can be separated accurately in a short time.

The salt concentration of the eluent C is set to be 1 mM to 500 mM lower than the salt concentration of the eluent A. The salt concentration as used herein means the total concentration of anions in the eluent. The difference between the salt concentration of the eluent C and the salt concentration of the eluent A is 1
If it is less than mM, the eluent output of the eluent B is weakened in a short time, the liquid property in the column cannot be brought close to that of the eluent A, and the stabilized HbA1c cannot be measured for a short time. Conversely, 500 mM
If it exceeds, the difference in salt concentration between the eluent B and the eluent A becomes large, the column pressure fluctuation becomes large, and the reproducibility of the stabilized HbA1c measurement deteriorates.

The eluent C has a pH of 4.0 to 6.
It is preferable to use a solution containing an inorganic acid, an organic acid and / or a salt thereof having a buffering capacity at 8 as the above-mentioned eluent A.
The same substances as those contained in can be used.

It is also preferable to add chaotropic ions to the eluent C. Chaotropic ion
The same eluent A as described above can be used.
The concentration of chaotropic ions in the eluent C is 0.1 m.
M to 400 mM is preferable, 0.2 mM to 300 mM is more preferable, and 0.5 mM to 200 mM is particularly preferable.
Is.

It is more preferable that the eluent C also contains the above-mentioned azide ion. The concentration of azide ions contained in the eluent C is preferably 0.15 mM to 50 mM.

Various additives, such as (1) inorganic salts (2) p, added to the above-mentioned eluent A are also included in the above-mentioned eluent C.
Substances such as H regulator (3) water-soluble organic solvent (4) stabilizer (5) amines (6) surfactant may be added.

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

The cation exchange group may be a known one and is not particularly limited. Examples thereof include cation exchange groups such as a carboxyl group, a sulfonic acid group, and a phosphoric acid group. In addition, plural kinds of cation exchange groups may be introduced.

The diameter of the above particles is preferably 0.5 to 2
It is 0 μm, more preferably 1 to 10 μm. The particle size distribution is preferably 40% or less, more preferably 30% or less as a coefficient of variation value (CV value) (standard deviation of particle size / average diameter x 100 (%)).

Examples of the polymer particles include inorganic particles such as silica and zirconia; natural polymer particles such as cellulose, polyamino acid and chitosan; synthetic polymer particles such as polystyrene and polyacrylic acid ester. . Also, known non-porous particles can be used. In the polymer particles, the constituent components other than the ion-exchange group to be introduced are preferably more hydrophilic.
Further, from the viewpoint of pressure resistance and swelling resistance, those having a high degree of crosslinking are preferable.

The cation exchange group can be introduced into the above-mentioned polymer particles by a known method. For example, after preparing the polymer particles, functional groups (hydroxyl group, amino group, carboxyl group, epoxy group) possessed by the particles are prepared. Group) and a cation exchange group is introduced into the particle by a chemical reaction. The cation exchange filler can also be prepared by a method of polymerizing a monomer having a cation exchange group to prepare polymer particles. For example, a method of mixing a cation exchange group-containing monomer and a crosslinkable monomer, and polymerizing in the presence of a polymerization initiator can be mentioned. In addition, a polymerizable cation exchange group-containing ester such as methyl (meth) acrylate or ethyl (meth) acrylate is mixed with a crosslinkable monomer and the like, and the obtained particles are polymerized in the presence of a polymerization initiator. The ester may be converted into a cation exchange group by a hydrolysis treatment. Furthermore, as described in Japanese Patent Publication No. 8-7197, after preparing crosslinked polymer particles, a monomer having a cation exchange group is added, and the monomer is added near the surface of the polymer particles. It may be polymerized.

The packing material is packed in a column and used for liquid chromatography measurement. The size of the column is
An inner diameter of 0.1 to 50 mm and a length of 1 to 300 mm are preferable, and an inner diameter of 0.2 to 30 mm and a length of 5 to 200 mm are more preferable. Column size is 0.1m inner diameter
If it is less than m and the length is less than 1 mm, the workability is poor and the separability is poor. Further, if the inner diameter is larger than 50 mm and the length is larger than 300 mm, not only the amount of the filler to be used increases but also the separability deteriorates.

As a method of packing the packing material into the column, any known method can be used, but the slurry packing method is more preferable. Specifically, for example, it is carried out by press-fitting a slurry in which filler particles are dispersed in a buffer solution such as an eluent into a column with a liquid feed pump or the like.

As the material of the column, known metals such as stainless steel, glass, resins such as PEEK (polyetheretherketone) and the like are used. In addition, the part where the packing material comes into contact with the column body may be covered with an inert material such as PEE.
Examples thereof include K, polyethylene, Teflon (registered trademark), titanium compounds, silicon compounds, and silicon films.

As the column filter and pre-filter in the chromatography used in the present invention, it is preferable to use a filter made of an inert material or a filter whose surface is covered with the inert material. Examples of inert materials include
Cellulose ester, cellulose acetate, cellulose triacetate, cellulose, cellulose nitrate, polytetrafluoroethylene, polyvinylidene difluoride, polysulfone, polyethylene, polyether ether ketone (PEEK), polyether sulfone, polypropylene, nylon, polyvinylidene fluoride , Glass materials, acrylic copolymers, oxide ceramics, carbide ceramics, nitride ceramics, silicide ceramics, boride ceramics, titanium and the like. Further, these materials can be used in appropriate combination. When using a non-inert material such as stainless steel, a method of coating with the above-mentioned inert material, a method of performing an inert treatment such as silicone treatment, a blocking reagent such as bovine serum albumin, gelatin, casein, globulin, hemoglobin, etc. It is preferable to appropriately use a method such as blocking with.

The liquid chromatography device used in the method of the present invention may be a known device, and is composed of, for example, a liquid feed pump, a sample injection device (sampler), a column, a detector and the like. Further, other accessory devices (column constant temperature bath, degassing device for the eluent, etc.) may be appropriately attached.

The column temperature at the time of measuring hemoglobins ranges from 25 to 25 in order to improve the separation of stable HbA1c.
60 degreeC is good, More preferably, it is 35-55 degreeC.

The column pressure for measuring hemoglobins is 9.8 × 10 ^{4 to} 3.9 × 10 ⁶ Pa (1 to ⁴⁰ k) in order to improve column durability and device durability.
gf / cm ² ) is good. More preferably, 2.0 × 10 ⁵
It is -3.4 * 10 < ⁶ > Pa (2-35kgf / cm < ² >).

Other measurement conditions in the method of the present invention may be known conditions, and the flow rate of the eluent is preferably 0.05 to 5 ml / min, more preferably 0.2 to 3 ml.
/ Min. It is preferable to use visible light of 415 nm for the detection of hemoglobins, but the detection is not limited to this. As the measurement sample, a sample hemolyzed by a known hemolytic reagent is used. The amount of the sample injected into the liquid chromatography device varies depending on the dilution ratio, but it is preferably about 0.1 to 100 μl.

FIG. 1 shows an example of the structure of an apparatus in the case of performing measurement by the stepwise elution method. The eluents A, B, C, and D are eluents having different elution outputs (that is, different pH, salt concentration, or both), and the eluents are switched to each eluent at every set time by the solenoid valve 1. It is configured to be. The eluent is supplied to the column 4 together with the sample introduced from the sample injection unit 3 by the liquid feed pump 2.
And each component is detected by the detector 5. Each peak area, height, etc. are calculated by the integrator 6.

[0056]

EXAMPLES Next, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples.

[Example 1] (Preparation of filler) 450 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and 2-hydroxy-1,3-dimethacryloxypropane (manufactured by Wako Pure Chemical Industries, Ltd.)
Benzoyl peroxide (manufactured by Wako Pure Chemical Industries) 2 in 50 g of mixture
g was dissolved. Disperse this in 2500 ml of 4 wt% polyvinyl alcohol (Nippon Gosei Kagaku) aqueous solution,
While stirring, the temperature was raised to 80 ° C. under a nitrogen atmosphere, and polymerization was performed for 1.5 hours. Then, after cooling the reaction system to 35 ° C., 2
-400 g of a 50% aqueous solution of acrylamido-2-methylpropanesulfonic acid (manufactured by Tokyo Kasei), methanol 40
0 ml was added, the temperature was raised to 80 ° C. again with stirring for 1 hour, and polymerization was performed for 1.3 hours. After the polymerization, the product was washed, dried and then classified to obtain particles having an average particle size of 6.5 μm.

(Packing of column) The obtained packing material was packed in a column as follows. 0.7 g of filler particles,
The mixture was dispersed in 30 ml of 50 mM phosphate buffer (pH 5.8), sonicated for 5 minutes, and well stirred. The whole amount was injected into a packer (manufactured by Umeya Seiki Co., Ltd.) to which an empty stainless steel column (inner diameter 4.6 × 35 mm) was connected. Connect a liquid-sending pump (made by Sanuki Industry Co., Ltd.) to the packer and pressure 300k
It was filled under constant pressure with gf / cm ² (about 3 × 10 ⁷ Pa).

(Measurement of Hemoglobins) Using the obtained column, hemoglobins were measured under the following measurement conditions. (Measurement conditions) System: Liquid delivery pump: LC-9A (manufactured by Shimadzu Corporation) Autosampler: ASU-420 (manufactured by Sekisui Chemical Co., Ltd.) Detector: SPD-6AV (manufactured by Shimadzu Corporation) Eluent: Eluent A : Containing 5OmM perchloric acid, 5
0 mM phosphate buffer (pH 5.3) Eluent B: 50 mM containing 200 mM perchloric acid
Phosphate buffer (pH 8.4) Eluent C: 50 mM phosphate buffer (pH 5.3) containing 45 mM perchloric acid Eluent A was flowed for 0 to 1.5 minutes from the start of measurement, 1.
Eluent B was run for 5 to 1.6 minutes, Eluent C was run for 1.6 to 1.9 minutes, and Eluent A was run for 1.9 to 2.0 minutes. Flow rate: 2.0 ml / min Detection wavelength: 415 nm Sample injection amount: 10 μl

(Measurement Sample A) Normal human blood was collected and sodium fluoride was added as an anticoagulant at 10 mg / ml. To this, 150 times the amount of hemolysis reagent (0.1% by weight polyethylene glycol mono-
4-octyl phenyl ether (Triton X-100,
(Tokyo Kasei Co., Ltd.) and a phosphate buffer solution (pH 7)) were added and hemolyzed to obtain a measurement sample A.

(Measurement Results) Under the above measurement conditions, sample A
The chromatogram obtained by measuring is shown in FIG. Peak 7 is HbA1a and b, peak 8 is HbF, peak 9 is unstable HbA1c, and peak 10 is stable HbA1.
c, peak 11 shows HbA0. From Figure 2, peak 8
It can be seen that and 9 and 10 are well separated.

Example 2 Hemoglobins were measured under the same conditions as in Example 1 except that the eluent had the following composition. Eluent: Eluent A: containing 50 mM perchloric acid, 2
5 mM succinic acid-20 mM phosphate buffer (pH 5.3) Eluent B: 20 mM containing 250 mM perchloric acid
Succinic acid-20 mM phosphate buffer (pH 8.4) Eluent C: 25 mM succinic acid-20 mM phosphate buffer (pH 5.3) containing 25 mM perchloric acid The obtained chromatogram is the same as in FIG. The separation between the peaks was good.

[Example 3] Hemoglobins were measured under the same conditions as in Example 1 except that the eluent had the following composition. Eluent: Eluent A: containing 50 mM perchloric acid, 2
5 mM succinic acid-20 mM phosphate buffer (pH 5.3) Eluent B: 20 mM containing 250 mM perchloric acid
Succinic acid-20 mM phosphate buffer (pH 8.4) Eluent C: 3 mM succinic acid-5 mM phosphate buffer (pH 5.3) containing 5 mM perchloric acid The obtained chromatogram is the same as in FIG. The separation between the peaks was good.

[Example 4] Hemoglobins were measured under the same conditions as in Example 1 except that the eluent and the measurement sample B were changed as follows. Eluent: Eluent A: 50 mM perchloric acid, 4.6 mM
25, containing (0.03% by weight) sodium azide
mM succinic acid-20 mM phosphate buffer (pH 5.3) Eluent B: 250 mM perchloric acid, 4.6 mM (0.0
Containing 3% by weight) containing sodium azide, 2
3 mM containing 0 mM succinic acid-20 mM phosphate buffer (pH 8.4) Eluent C: containing 5 mM perchloric acid, 4.6 mM (0.03 wt%) sodium azide
Succinic acid-5 mM phosphate buffer (pH 5.3)

(Measurement Sample B) A high HbF blood sample was collected and 10 mg / mL of sodium fluoride was used as an anticoagulant.
It was added to make up to ml. To this, 150 times the amount of hemolyzing reagent (0.1% by weight of polyethylene glycol mono-4-octyl phenyl ether as a surfactant (Triton X
-100, manufactured by Tokyo Kasei Co., Ltd. and phosphate buffer solution (pH)
7)) was added and hemolyzed to obtain measurement sample B.

(Measurement Results) Sample B was measured under the above measurement conditions.
The chromatogram obtained by measuring is shown in FIG. Peak 7 is HbA1a and b, peak 8 is HbF, peak 9 is unstable HbA1c, and peak 10 is stable HbA1.
c, peak 11 shows HbA0. From Figure 3, peak 8
It can be seen that and 9 and 10 are well separated.

[Example 5] Hemoglobins were measured under the same conditions as in Example 2 except that the eluent C was changed as follows. Eluent C: 25 mM succinic acid-20 mM phosphate buffer (pH 4.8) containing 25 mM perchloric acid (measurement result) The measurement results were almost the same as those in FIG. 2, and favorable results were obtained.

[Comparative Example 1] Hemoglobins were measured under the same conditions as in Example 1 except that the eluent had the following composition. Eluent: Eluent A: containing 50 mM perchloric acid, 2
5 mM succinic acid-20 mM phosphate buffer (pH 5.3) Eluent B: 20 mM containing 250 mM perchloric acid
Succinic acid-20 mM phosphate buffer (pH 8.4) Eluent A was flowed for 0 to 1.5 minutes from the start of measurement, and 1.
Eluent B was run for 5 to 1.6 minutes, and eluent A was run for 1.6 to 2 minutes.

(Measurement Results) The obtained chromatogram is shown in FIG. Peak 7 shows HbA1a and b, peak 8 shows HbF, peak 9 shows unstable HbA1c, peak 10 shows stable HbA1c, and peak 11 shows HbA0. In Comparative Example 1, peaks 7, 8, and 9 were broader than those in Examples, and the separation between the peaks was poor.

[Comparative Example 2] Hemoglobins were measured under the same conditions as in Example 2 except that the eluent C had the following composition. Eluent C: 25 mM succinic acid-20 mM phosphate buffer solution (pH 4.0) containing 25 mM perchloric acid (measurement result) The measurement result was measured because the pH of the eluent C was as low as 4.0. The hemoglobins in the sample were easily denatured, and the chromatogram was deteriorated, so that good results were not obtained.

[0071]

EFFECTS OF THE INVENTION Since the present invention has the above-mentioned constitution, the stable HbA1c peak is sharply separated, and even when the eluent is concentrated with time, the separation pattern is not easily affected, and stable and short. Stable HbA with good time accuracy
1c can be separated.

[Brief description of drawings]

FIG. 1 is a view showing an example of a cation exchange liquid chromatography apparatus used in the present invention.

FIG. 2 is a diagram showing a chromatogram obtained when measuring hemoglobins (Sample A) under the measurement conditions of Example 1.

FIG. 3 is a diagram showing a chromatogram obtained when measuring hemoglobins (Sample B) under the measurement conditions of Example 4.

FIG. 4 is a diagram showing a chromatogram obtained when measuring hemoglobins (Sample A) under the measurement conditions of Comparative Example 1.

[Explanation of symbols]

A, B, C, D Eluent 1 Solenoid valve 2 Liquid feed pump 3 Sample injection part 4 columns 5 detectors 6 Integrator 7 HbA1a and b peaks 8 HbF peak 9 Unstable HbA1c peak 10 Stable HbA1c peak 11 HbA0 peak

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 30/54 G01N 30/54 F 30/56 30/56 A 30/74 30/74 E

Claims (3)

[Claims] 1. In a method for measuring hemoglobins by cation exchange liquid chromatography, hemoglobin A0, which is an eluent for eluting hemoglobins that elute before hemoglobin A0 (hereinafter referred to as "eluent A"), is eluted. Of at least three kinds of eluents (hereinafter referred to as “eluent B”) and eluents (hereinafter referred to as “eluent C”) for decreasing the elution volume of the eluent B. The pH is higher than the isoelectric point of hemoglobins, the pH of the eluent C is the same as or lower than the pH of the eluent A, and the difference between the pH of the eluent C and the pH of the eluent A is 0.
A method for measuring hemoglobins, wherein the eluent C has a salt concentration in the range of 1.0 and the eluent C has a salt concentration lower than that of the eluent A by 1 mM to 500 mM. 2. The method for measuring hemoglobins according to claim 1, wherein the eluent A contains chaotropic ions and has an inorganic acid having a buffering capacity at a pH of 4.0 to 6.8.
A method for measuring hemoglobins, which comprises an organic acid and / or a salt thereof. 3. The method for measuring hemoglobins according to claim 2, wherein each of the eluents A, B and C contains an azide ion.