JP2003207497A - Eluting solvent, hemolyzing reagent and method for measuring hemoglobin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide eluting solvent and a hemolyzing reagent capable of measuring a stable HbA1c without deteriorating the antibacterial activity and to provide a method for measuring hemoglobin by using them. <P>SOLUTION: The eluting solvent and/or the hemolyzing reagent contain at least one kind of preservatives (and preferably chaotropic ion added) being selected from a group including an amine family compound, an alcohol family compound, an aldehyde family compound, a carboxylic acid family compound, a phenol family compound, an amide family compound, an organo-halogen family compound, an isothiazolone family compound, an organo-metallic compound, an EDTA and a boric acid, which are used for measuring the hemoglobin by a liquid chromatography. The method for measuring hemoglobin uses the eluting solvent and the hemolyzing reagent. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an eluent suitable for a method for measuring hemoglobins by liquid chromatography, a hemolytic reagent, and a method for measuring hemoglobins using the same.

[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 in the blood in the past 1-2 months (glucose concentration in blood). ) 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.

HbA1c is a glycated hemoglobin (hereinafter referred to as GHb) produced by a reaction between glucose in the blood and hemoglobin A (hereinafter referred to as HbA), and a reversible reaction of this is unstable hemoglobin. A1c (unstableHbA1c: hereinafter referred to as unstable HbA1c), which is irreversibly reacted through the unstable HbA1c to obtain stable hemoglobin A1c
(Stable HbA1c: Hereinafter, stable HbA1c
Is called). And, as the index of the above-mentioned diabetes diagnosis, it is preferable to use stable HbA1c,
In the field of clinical testing, it has been desired to develop a method capable of measuring the ratio of stable HbA1c with high accuracy.

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). In addition, HbA1a, HbA1b and HbA1c are GHb in which HbA is glycated, hemoglobin F (hereinafter referred to as HbF) is fetal hemoglobin consisting of α chain and γ chain, and the HbF value is for diagnosis of hemolytic anemia. It is also used as an index.

HbA1c used as an index for diagnosis of diabetes has recently been stable HbA1c as described above, and is calculated as a ratio (%) of the area of stable HbA1c peak to the area of total hemoglobin peak. ing.

As the eluent used in the liquid chromatography method, sodium azide has been generally used as a preservative for the eluent.

[0008]

However, according to the studies made by the present inventors, when sodium azide is used as a preservative, the pH of the eluent is lowered, so that hydrogen azide is easily generated. However, there is a problem that the antibacterial property is poor. The object of the present invention, in view of the problems in the measurement of hemoglobins by the liquid chromatography,
An object of the present invention is to provide an eluent capable of favorably separating stable HbA1c and the like used as an index of diabetes without lowering antibacterial properties, a hemolytic reagent, and a method for measuring hemoglobins using the same.

[0009]

The eluent according to claim 1 is an eluent used in the measurement of hemoglobins by liquid chromatography, which is an amine compound, an alcohol compound, an aldehyde compound, a carboxylic acid compound. Compounds, phenolic compounds, amide compounds,
It is characterized by containing at least one preservative selected from the group consisting of organic halogen compounds, isothiazolone compounds, organometallic compounds, EDTAs and boric acid. The hemolytic reagent according to claim 2 is a hemolytic reagent used for hemolyzing a blood sample in the measurement of hemoglobins by liquid chromatography, which comprises an amine compound, an alcohol compound, an aldehyde compound,
Contain at least one preservative selected from the group consisting of carboxylic acid compounds, phenolic compounds, amide compounds, organic halogen compounds, isothiazolone compounds, organometallic compounds, EDTAs and boric acid. Is characterized by. An eluent or hemolytic reagent according to claim 3 is the eluent according to claim 1 or the hemolytic reagent according to claim 2, which is characterized by further containing chaotropic ions. The method for measuring hemoglobins according to claim 4 is characterized by using the eluent according to claim 1 or 3 and / or the hemolytic reagent according to claim 2 or 3.

The present invention will be described in more detail below. In the present invention, an amine compound, an alcohol compound, an aldehyde compound,
At least one preservative selected from the group consisting of carboxylic acid compounds, phenol compounds, amide compounds, organic halogen compounds, isothiazolone compounds, organometallic compounds, EDTAs and boric acid is used. Specific examples thereof are not particularly limited, and examples thereof include the following.

Examples of amine compounds include 2-
Examples include imidazole compounds such as (4-thiazolyl) -benzimidazole, pyridine compounds such as 2,3,5,6-tetrachloromethylsulfonylpyridine, (2-pyridylthio-1-oxide) sodium, trialkyltriamines, and the like. To be Examples of alcohol compounds include methanol, ethanol, n-propyl alcohol, isopropanol, 2-phenoxyethanol, 2-phenoxypropanol, 2-phenylethanol and the like. Examples of the aldehyde compound include formaldehyde and glutaraldehyde. Examples of the carboxylic acid compound include benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, propionic acid, potassium propionate, sodium propionate, calcium propionate, salicylic acid, sodium salicylate, sodium dehydroacetate,
10-undecylenic acid, zinc undecylenate, magnesium dihydrogen bismonoperoxyphthalate and the like can be mentioned. Examples of the phenolic compound include thymol,
Isopropylmethylphenol, orthophenylphenol, sodium orthophenylphenol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, m-cresol, p-chlorophenol, etc. Is mentioned. As the amide compound, for example,
2-chloroacetamide and the like can be mentioned. Examples of the organic halogen-based compound include 2,4,5,6-tetrachloroisophthalonitrile, N- (fluorodichloromethylthio) -phthalimide, N, N-dimethyl-N '(fluorodichloromethylthio) -N'-. Phenylsulfamide and the like can be mentioned. As isothiazolone compounds,
For example, 1,2-benzothiazolin-3-one and the like can be mentioned. Examples of the organometallic compound include 10,10
-Oxybisphenoxyarsine and the like. ED
As TAs, EDTA, sodium salt of EDTA, E
Examples thereof include potassium salt of DTA.

The content of the above-mentioned preservative is not particularly limited, but is usually 0.000 in the eluent or the hemolysis reagent.
It is set to 1% by weight to 10% by weight, and preferably 0.000
It is set to 5% by weight to 5% by weight. The above preservatives may be used alone or in combination of two or more. Also, 2
Even when more than one species are used in combination, the content is preferably within the above range. Content is 0.0001% by weight
If it does not meet the requirements, sufficient antibacterial properties may not be obtained, and the measurement system for hemoglobins may be adversely affected. On the other hand, if the content exceeds 10% by weight, the solubility of the preservative may be poor, or the measurement system for hemoglobin may be affected, and stable HbA1c may not be accurately measured.

In the present invention, it is preferable that the above preservative is sufficiently dissolved in the eluent, and when a preservative having poor solubility alone is used, the content is kept low to the extent that it does not adversely affect the measurement system for hemoglobins. Alternatively, it is preferable to adopt a method of using a plurality of preservatives in combination. In addition, in order to increase the solubility, methanol, ethanol,
A water-soluble organic solvent such as acetonitrile or acetone may be mixed and used. In this case, the concentration of these organic solvents does not adversely affect the measurement system for hemoglobins,
Moreover, it is preferable to adjust the concentration so that chaotropic ions, inorganic acids, organic acids and salts thereof do not precipitate.

The eluent used in the measurement of hemoglobins by liquid chromatography is usually pH.
A method of eluting a plurality of types of hemoglobins by sequentially using a plurality of types of eluents having different salt concentrations and the like (so-called stepwise elution method) is used. In the stepwise elution method, the preservative in the present invention may be used in at least one eluent among a plurality of eluents.

In the eluent of the present invention, the eluent used for eluting hemoglobins other than the HbA0 component is an inorganic acid, an organic acid and / or a salt thereof having a buffering capacity at pH 4.0 to 5.8. The one containing Specific examples of these include the following. Examples of the inorganic acid include phosphoric acid and carbonic acid. As the organic acid, for example, carboxylic acid,
Examples thereof include dicarboxylic acid, carboxylic acid derivative, hydroxycarboxylic acid, cacodylic acid and amino acid.

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. As the salt of the inorganic acid or the organic acid,
Conventionally known compounds are used, and examples thereof include sodium salts and potassium salts.

The above-mentioned inorganic acid, organic acid and / or salt thereof may be used alone or in combination of two or more kinds. For example, inorganic acid and organic acid may be mixed and used. Good.

The above-mentioned inorganic acid, organic acid and / or salt thereof have an acid dissociation constant (pKa) of 2.15 to 6.3.
It is also preferable to use the substance of No. 9 and the substance having pKa of 6.4 to 10.5 in combination. Also,
Instead of using two or more substances in combination,
Single substance with pKa of 2.15-6.39 and 6.4
What exists at least one each in the range of 10.5 may be used. The combination of the more preferable range of pKa is 2.61 to 6.39 and 6.40 to 10.5.
0, more preferably 2.80 to 6.35 and 6.80 to 10.00, and particularly preferably 3.5.
0 to 6.25 and 7.00 to 9.50.

The above-mentioned inorganic acid, organic acid and / or
Or, the concentration of these salts is such that the pH of the eluent is 4.0 to
From the viewpoint of easily obtaining a buffering effect in the range of 5.8, it is preferably 1 mM to 1000 mM, more preferably 10 mM.
It is mM-500 mM.

When the pH of the eluent is less than 4.0, the hemoglobins may be denatured, and when the pH exceeds 6.8, the positive charge of the hemoglobins decreases and the cation exchange groups become cation exchange groups. It may become difficult to retain and the separability may deteriorate. More preferably, the pH is 4.5 to 5.8.
Is.

In the eluent of the present invention, the eluent used for eluting the HbA0 component is preferably on the alkaline side of the isoelectric point of hemoglobins. this is,
Among hemoglobins separated by liquid chromatography, HbA0 is composed of HbA and the like, is eluted after HbA1c and is strongly retained in the packing material, which tends to increase the measurement time. is there. Specifically, the pH of the eluent is preferably 7.0 to 12. More preferably 7.5-11.0
And more preferably 8.0 to 9.5.

Examples of the buffering agent that can be used in the eluent used for eluting the HbA0 component are inorganic acids such as phosphoric acid, boric acid, carbonic acid or salts thereof; hydroxycarboxylic acids such as citric acid, β, Examples of the buffer solution include carboxylic acid derivatives such as β-dimethylglutaric acid, dicarboxylic acids such as maleic acid, organic acids such as cacodylic acid, and salts thereof. Others, 2- (N-Morihorino)
Ethanesulfonic acid (MES), N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPE
S), bis (2-hydroxyethyl) iminotris-
(Hydroxymethyl) methane (Bistris), Tr
is, ADA, PIPES, Bistrispropa
ne, ACEA, MOPS, BES, TES, HEPP
S, Tricine, Bicine, TAPS, CAP
What is generally called a Good's buffer solution such as S can also be used. Further, organic substances 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 salts thereof, and organic substances may be mixed and used, for example, inorganic acids and organic substances may be mixed and used.

It is preferable that the eluent and / or the hemolytic reagent in the present invention contain chaotropic ions. A chaotropic ion is a compound that dissociates into an aqueous solution to destroy the structure of water and suppresses a decrease in entropy of water that occurs when a hydrophobic substance comes into contact with water. Tribromoacetate ion, trichloroacetate ion, thiocyanate ion,
Examples thereof include iodide ion, perchlorate ion, dichloroacetate ion, nitrate ion, bromide ion, chloride ion, acetate ion and the like, and other examples include urea and the like.
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, anions such as tribromoacetate ion, trichloroacetate ion, thiocyanate ion, iodide ion, perchlorate ion and dichloroacetate ion are used. , Nitrate ion, bromide ion and the like are preferably used, and as the cation, barium ion, calcium ion, magnesium ion, lithium ion, cesium ion, guanidine ion and the like are preferably used. 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 used for eluting hemoglobins other than the HbA0 component or in the hemolytic reagent is 0.1 mM to
3000 mM is preferable and 1 mM to 1000 mM is more preferable. More preferably, it is 10 mM to 500 mM. If the concentration of the chaotropic ion is less than 0.1 mM, the separation effect will be reduced and the measurement accuracy will be poor in the measurement of hemoglobins. In addition, 3000m
If it exceeds M, the separation effect of hemoglobins will not be further improved, and improvement in measurement accuracy cannot be expected.

In the eluent used for eluting the HbA0 component, the concentration of the chaotropic ion is preferably 1 mM to 3000 mM, preferably 10 mM to
1000 mM is more preferable, more preferably 50 m
M to 500 mM.

In the present invention, these chaotropic ions may be used alone or in combination of two or more kinds. Furthermore, these chaotropic ions may be used by being contained in a liquid (for example, a sample diluting liquid) that comes into contact with the measurement sample.

The eluent and / or the hemolytic reagent in the present invention may contain the following substances (1) to (6) unless the effects of the present invention are impaired. (1) Inorganic salts; examples thereof include sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, and sodium phosphate. The concentration of these salts is not particularly limited, but is preferably 1 to 1500 mM. (2) pH adjuster; examples thereof include known acids and bases. Examples of the acid include hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid and the like, and as the base, sodium hydroxide,
Examples thereof include potassium hydroxide, lithium hydroxide, magnesium hydroxide, barium hydroxide, calcium hydroxide and the like. The concentrations of these acids and bases are not particularly limited,
Preferably, it is 0.001 to 500 mM. (3) Water-soluble organic solvent; examples thereof include methanol, ethanol, acetonitrile, and acetone. The concentration of these organic solvents is not particularly limited, but is preferably 80% by volume or less, and it is preferable to use a concentration at which chaotropic ions, inorganic acids, organic acids, salts thereof, etc. do not precipitate. (4) Stabilizer; a known stabilizer may be contained, for example, as a stabilizer for hemoglobin. The stabilizer may contain, for example, a reducing agent such as glutathione or an antioxidant. (5) Amines; In order to reduce non-specific adsorption of hemoglobin, amines may be contained. Known amines are used as the amines, and preferred examples include primary amines, secondary amines and tertiary amines having a molecular weight of 20 to 500. (6) Surfactant: A nonionic surfactant, a cationic surfactant, an anionic surfactant and the like may be contained. By using a surfactant, not only efficient hemolysis but also high performance liquid chromatography (H
When the measurement is performed by PLC) or the like, it has an effect of washing the flow path through which the hemolytic reagent passes. A nonionic surfactant is preferably used as the above-mentioned surfactant, and examples thereof include polyoxyethylenes (hereinafter, polyoxyethylene is represented by POE and the number of moles of ethylene oxide added is represented by (n)), POE (7) decyl. Ether, P
OE (n) dodecyl ether, POE (10) tridecyl ether, POE (11) tetradecyl ether, P
OE (n) cetyl ether, POE (n) stearyl ether, POE (n) oleyl ether, POE (1
7) Cetyl-stearyl ether, POE (n) octylphenyl ether, POE (n) nonylphenyl ether, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, POE (n) sorbitan monolaurate, POE (n) sorbitan monopalmitate, POE
Examples include (n) sorbitan monostearate and POE (n) sorbitan monooleate. These surfactants may be used alone or in combination of two or more. The content of these surfactants is preferably 0.0
It is 1 to 10% by weight.

The method for measuring hemoglobins of the present invention is not particularly limited as long as it uses the above-mentioned eluent and / or hemolytic reagent. For example, a liquid chromatograph used by packing a column with a packing material having an ion exchange group. The graphic method and the like are preferable. The filler is preferably composed of particles having at least one kind of cation exchange group, and can be obtained, for example, by introducing cation exchange groups into polymer particles.

As the above-mentioned cation exchange group, known ones can be used and are not particularly limited. For example, a cation exchange group such as a carboxyl group, a sulfonic acid group and a phosphoric acid group is preferably used. These cation exchange groups may be used alone or in combination of two or more.

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

Examples of the polymer particles include inorganic polymer particles such as silica and zirconia; cellulose,
Examples include natural polymer particles such as polyamino acid and chitosan; synthetic polymer particles such as polystyrene and polyacrylic acid ester. Also, known non-porous polymer particles can be used. The constituent components other than the ion-exchange groups introduced into the polymer particles are preferably 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 polymer particles by a known method. For example, after the polymer particles are prepared, functional groups (hydroxyl group, amino group, carboxyl group, etc.) possessed by the particles can be used. Epoxy group, etc.)
For example, a method of chemically reacting a cation exchange group is adopted.
Alternatively, a method of polymerizing a monomer having a cation exchange group to prepare polymer particles may be used. For example, a cation exchange group-containing monomer and a crosslinkable monomer are mixed to initiate polymerization. Examples include a method of polymerizing in the presence of an agent. Also,
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, and then the obtained particles are hydrolyzed. A method of treating to convert the ester into a cation exchange group may be used. Furthermore, Japanese Examined Patent Publication 8-7197
After preparing the crosslinked polymer particles, as described in Japanese Patent Publication No.
A method of adding a monomer having a cation exchange group and polymerizing the monomer near the surface of the polymer particles may be used.

The inner diameter of the column is 0.1
.About.50 mm and length 1 to 300 mm are preferable, and more preferably inner diameter 0.2 to 30 mm and length 5 to 200.
mm. Column size is 0.1mm inner diameter, 1m length
If it is less than m, the workability tends to be poor and the separation ability tends to be poor. Further, when the inner diameter exceeds 50 mm and the length exceeds 300 mm, not only the amount of the filler to be used increases but also the separability may deteriorate.

As a method of packing the packing material into the column, any known method can be used, but for example, a slurry packing method is preferable. Specific examples of the slurry filling method include a method in which a slurry in which filler particles are dispersed in a buffer solution such as an eluent is pressed into a column by a liquid feed pump or the like to be filled.

As the material of the column, for example, metal such as stainless steel, glass, resin such as PEEK (polyether ether ketone) is used. Further, the part where the packing material and the column main body are in contact with each other may be coated with an inert material, and as the coating material, for example,
Examples include PEEK, polyethylene, Teflon (registered trademark), titanium compounds, silicon compounds, and silicon.

The column filter and / or pre-filter used in the above column is not particularly limited, but a filter made of an inert material or a filter whose surface is covered with an inert material is used. preferable. 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 material, acrylic Examples thereof include polymers, oxide ceramics, carbide ceramics, nitride ceramics, silicide ceramics, boride ceramics, and titanium.
Further, these materials can be used in appropriate combination. On the other hand, when a non-inert material such as stainless steel is used, a method of coating with the above-mentioned inert material, a method of performing an inert treatment such as silicone treatment, or blocking of bovine serum albumin, gelatin, casein, globulin, hemoglobin, etc. It is preferable to appropriately use a method of blocking with a reagent.

The liquid chromatography device used in the method of the present invention may be a known one, for example,
A device including a liquid feed pump, a sample injection device (sampler), a column, a detector and the like is used. Further, other accessory devices (column constant temperature bath, eluent degassing device, 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.
The temperature is preferably 60 ° C., more preferably 35 to 35 ° C.
55 ° C.

Further, the column pressure for measuring hemoglobins is, in terms of column durability and device durability, 9.
8 × 10 ^{4 to} 3.9 × 10 ⁶ Pa (1 to 40 kgf / cm
² ), more preferably 2.0 ×
10 ^{5 to} 3.4 × 10 ⁶ Pa ( ^{2 to} 35 kgf / cm ² )
Is.

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. The measurement sample is preferably a sample hemolyzed by the above-mentioned hemolytic reagent. 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.

In the above measuring method, an example of the constitution of the apparatus in the case of performing the measurement by the stepwise dissolution method is shown in FIG. The eluents A, B, C, and D have different elution outputs (that is,
The eluent is different in either pH or salt concentration, or both), and is configured to be switched by the solenoid valve 1 to each eluent at preset time intervals. The eluent is introduced by the liquid feed pump 2 into the column 4 together with the sample introduced from the sample injection unit 3, and each component is detected by the detector 5. Each peak area, height, etc. are calculated by the integrator 6.

(Function) The eluent and hemolytic reagent of the present invention are
Since a specific compound is contained as a preservative, stable HbA1c and the like can be satisfactorily separated without decreasing the antibacterial property in the measurement of hemoglobins. When the eluent and / or the hemolytic reagent contains chaotropic ions, the above effect becomes more certain.

[0044]

The present invention will be described in detail below 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 again to 80 ° C. with stirring for 1 hour, and polymerization was performed for 1.3 hours. After polymerization, washing, drying and classification were carried out to obtain particles having a sulfonic acid group as a cation exchange group having an average particle size of 6.5 μm.

(Packing of column) The obtained packing material was packed in a column as follows. Particles 0.7g, 50m
The mixture was dispersed in 30 mL of M phosphate buffer (pH 5.8), sonicated for 5 minutes, and then thoroughly stirred. The entire amount was injected into a packer (manufactured by Umeya Seiki Co., Ltd.) connected to an empty stainless steel column (inner diameter 4.6 mm × length 35 mm). Connect the liquid delivery pump (made by Sanuki Industry Co., Ltd.) to the packer, and pressure about 3 × 1
It was filled at a constant pressure of 0 ⁷ Pa (300 kgf / cm ² ).

(Measurement of Hemoglobins) Using the obtained column, hemoglobins were measured by liquid chromatography by the following method. (Measurement conditions) System: -Liquid-sending 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: 50 mM phosphate buffer containing 45 mM perchloric acid and 0.01 wt% sodium benzoate (pH
5.3) Eluent B: containing 200 mM perchloric acid, sodium azide 0.05% by weight, EDTA 0.01% by weight 5
0 mM phosphate buffer (pH 8.4) Eluent C: 60 mM perchloric acid, 50 mM phosphate buffer containing 0.01% by weight of sodium benzoate (pH
5.3) Eluent delivery method: Eluent C is flowed for 0 to 1.5 minutes and eluent B is flowed for 1.5 to 1.6 minutes from the start of measurement.
Eluent A was flowed between 1.6 and 1.9 minutes. Flow rate: 2.0 mL / min Detection wavelength: 415 nm Sample injection amount: 10 μL

(Measurement Sample) Blood of a healthy person was collected, and sodium fluoride was added as an anticoagulant at 10 mg / mL. Add 150 volumes of hemolysis reagent (0.1% by weight polyethylene glycol mono-4 as a surfactant).
-Octyl phenyl ether (Triton X-100, manufactured by Tokyo Kasei), potassium sorbate 0.0 as a preservative
A phosphate buffer solution (pH 7) containing 5% by weight was added and hemolyzed to obtain a measurement sample.

(Cultivation of Pseudomonas aeruginosa) The above eluents (eluents A, B and C) and hemolytic reagents were used by culturing Pseudomonas aeruginosa by the following method in order to evaluate the storage stability. . As a culture method, first, after preparing the above-mentioned eluent, Pseudomonas aeruginosa (P
Seudomonas aeruginosa) was cultured in a soybean casein digest medium described on page B-408 of the 13th edition of the Japanese Pharmacopoeia to make Pseudomonas aeruginosa fresh. Next, after collecting Pseudomonas aeruginosa by centrifugation, the concentration of the bacteria is about 100,000 cells / mL with physiological saline.
A bacterial inoculum was prepared so that Next, 2.8 each of the above-mentioned eluent A, eluent B, eluent C and hemolytic reagent are respectively added.
Each L was placed in a polyethylene 3 L bottle, 100 µL of the above-mentioned bacterial inoculum was added, and the mixture was stirred well and then cultured at 30 ° C for 20 days.

(Measurement Results) The chromatogram obtained as a result of the measurement of hemoglobins by the above method 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. Figure 2
It was found that the peaks 8, 9 and 10 were well separated.

[Example 2] Hemoglobins were measured in the same manner as in Example 1 except that the eluent, hemolytic reagent, and eluent solution feeding method were as follows. Eluent: Eluent A: 25 mM succinic acid-2 containing 40 mM perchloric acid, 0.1% by weight of 2-phenoxyethanol.
0 mM phosphate buffer (pH 5.3) Eluent B: 20 mM succinic acid containing 250 mM perchloric acid and 0.1% by weight of 2-phenoxyethanol-
20 mM phosphate buffer (pH 8.4) Eluent C: 25 mM succinic acid-2 containing 60 mM perchloric acid and 0.1% by weight of 2-phenoxyethanol.
0 mM phosphate buffer (pH 5.3) Hemolysis reagent: phosphate buffer solution (pH 7) containing 0.05% by weight of sodium dehydroacetate instead of the preservative of Example 1 (potassium sorbate). Eluent feeding method: Eluent A is flowed for 0 to 0.7 minutes from the start of measurement, and eluent C is flowed for 0.7 to 1.4 minutes,
Eluent B is allowed to flow for 1.4 to 1.5 minutes, and 1.5 to 1.
Eluent A was run for 9 minutes. (Measurement Results) The obtained chromatogram was similar to that of Example 1, and it was found that the HbF, unstable HbA1c, and stable HbA1c peaks were well separated.

[Example 3] Hemoglobins were measured under the same conditions as in Example 1 except that the eluent had the following composition. Eluent: Eluent A: 25 mM succinic acid-20, containing 50 mM perchloric acid, 0.05% by weight sodium salicylate.
mM phosphate buffer (pH 5.3) Eluent B: 20 mM succinic acid-20 m containing 250 mM perchloric acid and 0.05% by weight sodium azide
M phosphate buffer (pH 8.4) Eluent C: 10 mM succinic acid-35 containing 35 mM perchloric acid and 0.05% by weight sodium salicylate
mM phosphate buffer (pH 5.3) (Measurement result) The obtained chromatogram was the same as in Example 1, and it was revealed that the HbF, unstable HbA1c, and stable HbA1c peaks were well separated.

[Example 4] Hemoglobins were measured under the same conditions as in Example 1 except that the eluent was as follows. Eluent: Eluent A: 60 mM perchloric acid, m-cresol 0.
25 mM succinic acid-20 mM phosphate buffer (pH 5.3) containing 01 wt% Eluent B: 250 mM perchloric acid, 4.6 mM (0.
20 mM succinic acid-20 mM phosphate buffer (pH 8.4) containing sodium azide. Eluent C: 40 mM perchloric acid, m-cresol.
10 mM succinic acid-35 mM phosphate buffer solution (pH 5.3) containing 01% by weight (measurement result) The obtained chromatogram is the same as that of Example 1, and each peak of HbF, unstable HbA1c, and stable HbA1c. Were found to be well separated.

[Example 5] Hemoglobins were measured under the same conditions as in Example 1 except that the eluent was changed as follows. Eluent: Eluent A: 60 mM perchloric acid, sodium salicylate 0.02% by weight, 2-phenoxyethanol 0.06
25 mM succinic acid-20 mM phosphate buffer (pH 5.3) containing wt% Eluent B: 250 mM perchloric acid, 4.6 mM (0.
Sodium azide, 0.02 wt% EDTA, 20 mM succinic acid-20 mM phosphate buffer (pH 8.4) Eluent C: 40 mM perchloric acid, 0.02 wt% sodium salicylate, 2-phenoxyethanol 0.06
10 mM succinic acid-35 mM phosphate buffer solution (pH 5.3) containing wt% (measurement results) The obtained chromatogram is the same as in Example 1, and each peak of HbF, unstable HbA1c, and stable HbA1c is shown. It turned out to be well separated.

[Example 6] Hemoglobins were measured under the same conditions as in Example 1 except that the eluent was as follows. Eluent: Eluent A: 60 mM perchloric acid, sodium salicylate 0.02% by weight, 2-phenoxyethanol 0.06
%, Containing 0.05% by weight EDTA, 25 mM
Succinic acid-20 mM phosphate buffer (pH 5.3) -Eluent B: 250 mM perchloric acid, 4.6 mM (0.
Sodium azide, 0.02 wt% EDTA, 60 mM phosphate buffer (pH 8.4) Eluent C: 40 mM perchloric acid, sodium salicylate 0.02 wt%, 2-phenoxyethanol 0 .06
%, Containing 0.05% by weight EDTA, 10 mM
Succinic acid-35 mM phosphate buffer solution (pH 5.3) (Measurement result) The obtained chromatogram is the same as that of Example 1, and each peak of HbF, unstable HbA1c, and stable HbA1c can be well separated. found.

[Comparative Example 1] Hemoglobins were measured in the same manner as in Example 1 except that the preservative in the present invention was not used and the eluent and hemolyzing reagent were changed as follows. Eluent: • Eluent A: 50 mM phosphate buffer (pH 5.3) containing 45 mM perchloric acid • Eluent B: 200 mM perchloric acid, 50 mM phosphorus containing 0.05% by weight sodium azide Acid buffer (pH
8.4) Eluent C: 50 mM phosphate buffer (pH 5.3) containing 60 mM perchloric acid Hemolysis reagent: 0.1% by weight polyethylene glycol mono-
4-octyl phenyl ether (Triton X-100,
Phosphate buffer solution (pH 7) containing (Tokyo Kasei Co., Ltd.) (Measurement result) The eluent A, the eluent C, the eluent B, and the hemolytic reagent have a high bacterial growth rate. Then, hemoglobins were measured, but the baseline variation was large, and stable HbA1c could not be separated.

[0057]

Since the eluent and hemolytic reagent of the present invention contain a specific compound as a preservative, they can be used as an index of diabetes without lowering the antibacterial property in the measurement of hemoglobins by liquid chromatography. It is possible to satisfactorily separate and measure the stable HbA1c or the like used. When the eluent and / or the hemolytic reagent contains chaotropic ions, the above effect becomes more certain. Since the method for measuring hemoglobins of the present invention is characterized by using the above-mentioned eluent and / or hemolytic reagent, stable HbA1c can be satisfactorily separated and measured without decreasing the antibacterial property as in the above. It will be possible.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a configuration example of a method for measuring hemoglobins of the present invention.

FIG. 2 is a diagram showing a chromatogram obtained by measurement of hemoglobins in 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 30/56 G01N 30/56 A

Claims (4)

[Claims] 1. An eluent used in the measurement of hemoglobins by liquid chromatography, which comprises an amine compound, an alcohol compound, an aldehyde compound, a carboxylic acid compound, a phenol compound, an amide compound, and an organic halogen compound. An eluent containing at least one preservative selected from the group consisting of a compound, an isothiazolone compound, an organometallic compound, EDTAs and boric acid. 2. A hemolytic reagent used for hemolyzing a blood sample in the measurement of hemoglobins by liquid chromatography, which comprises an amine compound, an alcohol compound, an aldehyde compound, a carboxylic acid compound, a phenol compound, Amide compounds, organic halogen compounds, isothiazolone compounds, organometallic compounds, ED
At least one selected from the group consisting of TAs and boric acid
A hemolytic reagent comprising a preservative of a species. 3. The eluent according to claim 1 or the hemolytic reagent according to claim 2, further comprising chaotropic ions. 4. A method for measuring hemoglobin, which comprises using the eluent according to claim 1 or 3 and / or the hemolytic reagent according to claim 2 or 3.