JP5238319B2 - Method for measuring hemoglobins - Google Patents

Description

The present invention relates to a method for measuring hemoglobin that can be easily and highly accurately measured.

Hemoglobin (also referred to as Hb), particularly hemoglobin A1c (hereinafter also referred to as HbA1c), which is a type of glycated hemoglobin, reflects the average sugar concentration in the blood during the past 1-2 months. Widely used as a screening item for diabetes and as a test item for grasping blood glucose control status of diabetic patients.

HbA1c has been measured by liquid chromatography (HPLC), immunization, electrophoresis, and the like. Of these, the liquid chromatography method is widely used in the clinical laboratory field. In the liquid chromatography method, measurement in 1 to 2 minutes per sample is possible, and measurement accuracy of about 1.0% CV value in the simultaneous reproducibility test is realized. As a measurement method for grasping the blood glucose control state of a diabetic patient, at least this level of performance is required.

In the method for measuring hemoglobin A1c by the HPLC method, measurement by cation exchange chromatography is usually used. However, the conventional method has a problem that the separation performance between the peak of hemoglobin A1c and the peak of other hemoglobins is not always sufficient, and high-precision measurement is difficult.

On the other hand, for example, Patent Document 1 discloses a method for improving a column filler. However, improvement of the column packing is technically very difficult and not easy to study. In addition, in order to make the measurement time per sample shorter than a few minutes, a column packing material in which the amount of cation exchange groups introduced is controlled so that the retention time of the hemoglobin A1c peak can be managed at a level of several seconds is required. Become. Therefore, it has been a big problem such as a large production loss of column filler.

On the other hand, as another approach, attempts have been made to improve reagents such as an eluent. For example, Patent Documents 2 and 3 propose methods for improving the separation performance of the hemoglobin A1c peak using a special buffer solution. However, even these techniques have not been able to meet the current measurement accuracy requirements for hemoglobin A1c.
Japanese Patent Publication No. 08-007197 JP-A-11-295286 JP 2000-055899 A

An object of the present invention is to provide a method for measuring hemoglobin capable of easily and highly accurately measuring in view of the above situation.

The present invention is a method for measuring hemoglobin by a liquid chromatography method, and is a method for measuring hemoglobin using a buffer containing a polysaccharide as a buffer.
The present invention is described in detail below.

As a result of intensive studies, the present inventors have included a polysaccharide as an eluent used for measurement, a hemolyzed or diluted solution used for preparing a sample, a pretreatment solution used for pretreatment, etc., if necessary. The present inventors have found that the measurement accuracy of hemoglobin can be remarkably improved when the analysis is performed by the liquid chromatography method using the buffer solution, and the present invention has been completed.

In the method for measuring hemoglobin of the present invention, a buffer solution containing polysaccharide (hereinafter, also referred to as “sugar buffer solution”) is used.
The sugar buffer is, for example, an eluent used for measurement, a hemolyzed or diluted solution used for preparing a sample, and a part or all of the normal HPLC method used as necessary, such as pretreatment. It can be used for reagents. That is, it may be applied to all reagents, or may be used only for some reagents. For example, when a plurality of types of eluents are used, they may be used for all eluents or only for some eluents. More preferably, it is used for a hemolysis reagent used for hemolyzing a blood sample, a dilution reagent used for diluting the sample, and an eluent used for measuring the sample. It is particularly preferable to use it as an eluent.

The buffer used as the base of the sugar buffer is not particularly limited, and examples thereof include organic acids such as citric acid, succinic acid, tartaric acid, malic acid and salts thereof; amino acids such as glycine, taurine, arginine; hydrochloric acid, nitric acid Conventionally known buffer solutions such as inorganic acids such as sulfuric acid, phosphoric acid, boric acid and acetic acid and salts thereof, and Good's buffer solutions can be used. Moreover, you may add well to well-known additives, such as surfactant, various polymers, and a hydrophilic low molecular weight compound, to the said buffer solution.

The preferable lower limit of the salt concentration of the buffer solution is 10 mM, and the preferable upper limit is 1000 mM. If the concentration is less than 10 mM, the ion exchange reaction may not be performed, and hemoglobins may not be separated. If the concentration exceeds 1000 mM, the salt may precipitate and adversely affect the system.
The preferable lower limit of the pH of the buffer solution is 4.0, and the preferable upper limit is 9.0. If the pH is outside this range, hemoglobins may be greatly denatured and measurement reproducibility may be reduced. A more preferred lower limit is 4.5, and a more preferred upper limit is 8.5.

The polysaccharide used in the sugar buffer can be used polysaccharides with cationic exchange groups.
The polysaccharide having an upper Symbol cationic exchange groups, for example, hyaluronic acid, muramic acid, pectic acid, a polysaccharide having a carboxyl group of alginic acid and the like; polysaccharides having teichoic acid, a phosphoric acid group such as phosphomannan; Chondroitin And polysaccharides having a sulfonic acid group such as sulfuric acid, dermatan sulfate, keratan sulfate, heparan sulfate, heparin and fucoidan; and cation exchange group-introduced derivatives of the above neutral polysaccharides.
This polysaccharide of these may be used alone, it may be used in combination of two or more thereof. Among them, polysaccharides having a scan sulfonic acid group is more preferable. Particularly preferred is chondroitin sulfate.

The minimum with the preferable molecular weight of the said polysaccharide is 1,000, and a preferable upper limit is 10,000,000. If it is less than 1,000, the effect of improving the measurement accuracy by adding a polysaccharide may not be obtained. If it exceeds 10,000,000, it may precipitate in a buffer solution and adversely affect the measurement. is there. A more preferred lower limit is 5,000, and a more preferred upper limit is 5,000,000.

The concentration of the polysaccharide in the sugar buffer solution varies depending on the target reagent and the polysaccharide used, but the preferred lower limit is 0.01% by weight and the preferred upper limit is 20% by weight. If it is less than 0.01% by weight, the effect of improving the measurement accuracy due to the addition of polysaccharide may not be obtained. If it exceeds 20% by weight, the viscosity of the sugar buffer solution will increase and adversely affect the measurement. There is. A more preferred lower limit is 0.1% by weight and a more preferred upper limit is 10% by weight.

The method for measuring hemoglobin of the present invention is measured by liquid chromatography using the sugar buffer.
The measurement is performed by connecting a column packed with a column packing material to a known liquid chromatography system, that is, a system including a pump, a sampler, a detector and the like for feeding an eluent.

As the column filler, a column filler having an ion exchange group is suitable. Examples of the ion exchange group of the column packing having an ion exchange group include cation exchange groups such as carboxyl group, phosphate group and sulfonate group; anion exchange groups such as primary to tertiary amino group and quaternary ammonium group. Groups. Among these, a cation exchange group is preferable, and a sulfonic acid group is more preferable.

When a buffer solution containing the polysaccharide having an ion exchange group is used as at least one of the eluents, a column filler having no ion exchange group can be used. Since it is not necessary to introduce ion exchange groups into the column packing material, it is very advantageous for the reproducibility of the column packing manufacturing.

As the base material for the column filler, a cross-linked polymer made of an organic polymer, more preferably a cross-linked (meth) acrylic acid ester cross-linked polymer is suitable.
The crosslinkable (meth) acrylic acid ester is not particularly limited. For example, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, tetraethylene glycol di (meth) Polyethylene glycol di (meth) acrylates such as acrylate and polyethylene glycol di (meth) acrylate; propylene glycol di (meth) acrylate, dipropylene glycol di (meth) acrylate, tripropylene glycol di (meth) acrylate, tetrapropylene glycol di Polypropylene glycol di (meth) acrylates such as (meth) acrylate and polypropylene glycol di (meth) acrylate; polytetramethylene glycol di ( Acrylate), poly (propylene glycol-tetramethylene glycol) -di (meth) acrylate, polyethylene glycol polypropylene glycol polyethylene glycol-di (meth) acrylate, 1,3-butylene glycol di (meth) acrylate, 1,4-butylene Alkylene glycol di (meth) acrylates such as glycol di (meth) acrylate, 1,6-hexaglycol di (meth) acrylate, 1,9-nonanediol di (meth) acrylate, neopentyl glycol di (meth) acrylate; 2-hydroxy-1,3-di (meth) acryloxypropane, 2-hydroxy-1- (meth) acryloxy-3- (meth) acryloxypropane, 2-hydroxy-3- (meth) acryloyloxypropi (Meth) acryl, glycerol di (meth) acrylate, glycerol acrylate methacrylate, urethane (meth) diacrylate, isocyanuric acid di (meth) acrylate, isocyanuric acid tri (meth) acrylate, 1,10-di (meth) acryloxy-4 , 7-Dioxadecane-2,9-diol, 1,10-di (meth) acryloxy-5-methyl-4,7-dioxadecane-2,9-diol, 1,11-di (meth) acryloxy-4,8 -Hydroxyalkyl di (meth) acrylates such as dioxaoundegan-2,6,10-triol; trimethylolpropane tri (meth) acrylate, tetramethylolpropane tri (meth) acrylate, ditrimethylolpropane tetra (meth) acrylate , Tetramethylo And xalkalol alkane (meth) acrylates such as methane methane tri (meth) acrylate, tetramethylol methane tetra (meth) acrylate, trimethylol ethane tri (meth) acrylate, and the like. These crosslinkable (meth) acrylic acid esters may be used alone or in combination of two or more.

The cross-linked polymer constituting the substrate may be a copolymer of the cross-linkable (meth) acrylic acid ester and a non-crosslinkable monomer.
Although it does not specifically limit as said non-crosslinkable monomer, Non-crosslinkable (meth) acrylic acid ester is suitable.
The non-crosslinkable (meth) acrylate esters are not particularly limited, and examples thereof include methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, and butyl (meth) acrylate. Examples include alkyl (meth) acrylates; acrylic acid derivatives such as (meth) acrylic acid-2-ethylhexyl and (meth) acrylic acid-2-ethylhexyl.
Ethylene glycol mono (meth) acrylate, diethylene glycol mono (meth) acrylate, triethylene glycol mono (meth) acrylate, tetraethylene glycol mono (meth) acrylate, polyethylene glycol mono (meth) acrylate, methoxytri (poly) ethylene glycol mono (meta) ) Acrylate, polyethylene glycol mono (meth) acrylates such as methoxypolyethylene glycol mono (meth) acrylate; propylene glycol mono (meth) acrylate, dipropylene glycol mono (meth) acrylate, tripropylene glycol mono (meth) acrylate, tetrapropylene Polypropylene such as glycol mono (meth) acrylate and polypropylene glycol mono (meth) acrylate Poly (ethylene glycol / propylene glycol) mono (meth) acrylate, poly (ethylene glycol / tetramethylene glycol) mono (meth) acrylate, poly (propylene glycol / tetramethylene glycol) mono (meth) Alkylene glycol mono (meth) acrylates such as acrylate, octoxy polyethylene glycol polypropylene glycol-mono (meth) acrylate; glycidyl (meth) acrylate, glycerin mono (meth) acrylate, 2-hydroxyethyl (meth) acrylate, 2-hydroxy Hydroxy acids such as propyl (meth) acrylate, 2,3-dihydroxylethyl (meth) acrylate, 2,3-dihydroxylpropyl (meth) acrylate Or a glycidyl group-containing (meth) acrylates. These non-crosslinkable (meth) acrylic acid esters may be used alone or in combination of two or more.

When the cross-linked polymer constituting the substrate is a copolymer of the cross-linkable (meth) acrylic ester and a non-cross-linkable monomer, 100 parts by weight of the cross-linkable (meth) acrylic ester The preferable upper limit of the blending amount of the non-crosslinkable monomer is 50 parts by weight. If the amount exceeds 50 parts by weight, the cross-linked polymer constituting the base material has a low degree of cross-linking and tends to swell and shrink. Therefore, it takes time to equilibrate when the buffer solution is switched in the measurement by liquid chromatography. May take longer measurement time. A more preferred upper limit is 20 parts by weight.

The cross-linked polymer constituting the substrate is a mixture of the cross-linkable (meth) acrylic acid ester and a non-cross-linkable monomer, and is an emulsion polymerization method, a soap-free polymerization method, a dispersion polymerization method, a suspension weight, It can be produced by copolymerization by a known polymerization method such as a combination method or a seed polymerization method.

As the column filler having no ion exchange group, the above-mentioned substrate can be used as it is.
On the other hand, the column filler having the ion exchange group includes, for example, the crosslinkable (meth) acrylic acid ester (and the non-crosslinkable monomer to be added as necessary) and an ion exchange monomer. Can be produced by copolymerization.

As a method for producing the column filler having an ion exchange group, specifically, for example, the crosslinkable (meth) acrylic acid ester (and the non-crosslinkable monomer to be added if necessary) and A polymerization initiator and various additives as necessary are added to a mixture with a monomer having an ion exchange group (hereinafter also referred to as “ion exchange monomer”), and the polymerization reaction is carried out by heating. A method is mentioned.
Also, a column filler having an ion exchange group can be produced by a two-stage polymerization method. That is, first, the crosslinkable (meth) acrylic acid ester is polymerized to obtain a crosslinked polymer. Next, the ion-exchangeable monomer is polymerized on the surface of the obtained crosslinked polymer. When the ion-exchangeable monomer is polymerized on the surface of the crosslinkable polymer, for example, after the polymerization of the crosslinkable monomer mixture, the obtained crosslinkable polymer is washed, and then the polymerization initiator is changed to the crosslinkable polymer. After impregnation, a method in which an ion-exchangeable monomer is added to the reaction system for polymerization; in the middle of the polymerization reaction of the crosslinkable monomer mixture, the ion-exchangeable monomer is added to the reaction system for polymerization. The method etc. are mentioned. In addition, when adding an ion exchange monomer, you may dilute and use with water or various aqueous solution, an organic solvent, etc.
As the column filler having an ion exchange group, those obtained by the two-stage polymerization method are particularly suitable.

Examples of the ion exchangeable monomer include a polymerizable monomer having a functional group having a cation exchange group and a polymerizable monomer having a functional group having an anion exchange group.
The cation exchange group is not particularly limited, and examples thereof include a carboxyl group, a phosphoric acid group, and a sulfonic acid group. Of these, sulfonic acid groups are preferred.
Moreover, as said ion exchange monomer, (meth) acrylic acid derivatives are preferable.

The cation exchange monomer having a carboxyl group is not particularly limited, and examples thereof include (meth) acrylic acid, 2- (meth) acryloyloxyethyl succinate, 2- (meth) acryloyloxyethylphthalic acid, and the like ( (Meth) acrylic acid derivatives; crotonic acid, itaconic acid, citraconic acid, mesaconic acid, maleic acid, fumaric acid and the like.
The cation exchange monomer having a phosphoric acid group is not particularly limited. For example, ((meth) acryloyloxyethyl) acid phosphate, (2- (meth) acryloyloxyethyl) acid phosphate, (3- (meth) And (meth) acrylic acid derivatives such as (acryloyloxypropyl) acid phosphate.
The cation exchange monomer having a sulfonic acid group is not particularly limited, and examples thereof include (meth) acrylic acid such as 2- (meth) acrylamido-2-methylpropanesulfonic acid and 3-sulfopropyl (meth) acrylic acid. Derivatives; (3-sulfopropyl) -itaconic acid, allylsulfonic acid and the like.

It does not specifically limit as a polymerizable monomer which has an anion exchange group, For example, amino group containing (meth) acrylates, such as 2-diethylaminoethyl (meth) acrylate which has an amino group, and 2-diethylaminopropyl (meth) acrylate Etc.

The ion exchange group may be one obtained by converting a functional group that can be converted into an ion exchange group of a monomer having a functional group that can be converted into an ion exchange group into an ion exchange group.
That is, in the production of the column filler having the ion exchange group, a monomer having a functional group that can be converted into an ion exchange group is added to the monomer mixture and copolymerized. A method of converting a convertible functional group into an ion exchange group by a known chemical reaction, or after forming the above crosslinked polymer, polymerizing an ion exchange monomer on the surface of the crosslinkable polymer, The functional group that can be converted into an ion exchange group may be obtained by a method of converting into a ion exchange group by a known chemical reaction.

The functional group that can be converted into the ion exchange group is not particularly limited, and examples thereof include monomers having ester groups such as carboxylic acid esters, phosphoric acid esters, and sulfonic acid esters; monomers having epoxy groups and glycidyl groups. Etc.
The ester group derived from the monomer having an ester group can be easily converted into a carboxyl group, a phosphoric acid group, and a sulfonic acid group, respectively, by a known method of hydrolysis in the presence of an acid or a base. .
In the monomer having an epoxy group or glycidyl group, a carboxyl group, a phosphate group, or a sulfonic acid group can be introduced by a known method after the ring opening of the epoxy group.
These monomers having a functional group that can be converted into a cation exchange group may be used alone or in combination of two or more.

As the blending amount of the ion-exchangeable monomer or the monomer having a functional group that can be converted into an ion-exchange group, the crosslinkable (meth) acrylic acid ester (when a non-crosslinkable monomer is added) Is preferably 10 parts by weight with respect to 100 parts by weight, and a preferred upper limit is 100 parts by weight. If the amount is less than 10 parts by weight, the amount of ion exchange groups introduced is too small to perform sufficient ion exchange reaction, and the peak to be measured may not be sufficiently separated. Aggregates may be generated and a polymer may not be obtained. A more preferred lower limit is 20 parts by weight, and a more preferred upper limit is 80 parts by weight.

According to the liquid chromatography method using the sugar buffer, in addition to the hemoglobin A1c, abnormal hemoglobins such as hemoglobin S and hemoglobin C, hemoglobin F (fetal hemoglobin), hemoglobin A2 and the like are measured with high accuracy. be able to.

ADVANTAGE OF THE INVENTION According to this invention, the measuring method of hemoglobin which can perform a highly accurate measurement easily can be provided.

Hereinafter, embodiments of the present invention will be described in more detail with reference to examples. However, the present invention is not limited only to these experimental examples.

(Preparation of column packing material having ion exchange group)
1.2 g of benzoyl peroxide was dissolved in a mixture of 200 g of triethylene glycol dimethacrylate, 100 g of tetramethylol methane triacrylate, 100 g of tetramethylol methane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of glycerol dimethacrylate. .

The obtained monomer mixture was dispersed in 5 L of a 4% polyvinyl alcohol aqueous solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and a polymerization reaction was performed for 1 hour. After cooling the temperature to 30 ° C., 140 g of 2-methacrylamideamido-2-methylpropane sulfonic acid (cation exchange sugar monomer) is added to the reaction system, and the mixture is heated again to 80 ° C. for 1 hour. I did it. The obtained polymer was washed with ion exchange water and acetone to obtain a crosslinked polymer (column filler) having a cation exchange group. The obtained packing material was packed in a stainless steel column having an inner diameter of 3 mm and a length of 30 mm to obtain a hemoglobin A1c separation column.

(Preparation of column packing without ion exchange groups)
1.2 g of benzoyl peroxide was dissolved in a mixture of 200 g of triethylene glycol dimethacrylate, 100 g of tetramethylol methane triacrylate, 100 g of tetramethylol methane tetraacrylate (crosslinkable monomer: manufactured by Shin-Nakamura Chemical) and 50 g of glycerol dimethacrylate. .

The obtained monomer mixture was dispersed in 5 L of a 4% aqueous polyvinyl alcohol solution, heated to 80 ° C. in a nitrogen atmosphere with stirring, and subjected to a polymerization reaction for 12 hours. The obtained polymer was washed with ion-exchanged water and acetone to obtain a crosslinked polymer (column filler) having no cation exchange group. The obtained packing material was packed in a stainless steel column having an inner diameter of 3 mm and a length of 30 mm to obtain a hemoglobin A1c separation column.

(Experimental Examples 1 to 11)
The obtained column was connected to an HPLC system (LC-10A manufactured by Shimadzu Corporation). As a measurement sample, blood of a healthy human blood sampled from sodium fluoride was diluted 200 times with a phosphate buffer solution (pH 6.8) containing 0.05% Triton X-100. Two types of eluents, eluent A: 200 mM phosphate buffer (pH 5.3) and eluent B: 400 mM phosphate buffer (pH 7.5), were sent stepwise with a flow rate of 1.0 mL. Separated by a gradient method, the absorbance at 415 nm was measured.

In the experimental examples, as shown in Table 1, the polysaccharide was added to the hemolytic diluent, eluent A and eluent B so as to have a concentration of 1.5% by weight, and the measurement was performed.

(Evaluation 1) Evaluation of simultaneous reproducibility:
Experimental examples 1 to 6 were performed as measurement examples when a column filler having an ion exchange group was used. FIG. 1 shows a chromatogram when normal human blood is measured under the conditions of Experimental Example 1 (experimental example not including polysaccharides). In the figure, peak 1 represents hemoglobin A1c, and peak 2 represents hemoglobin Ao. Similar good chromatograms were obtained in the measurement system (Experimental Examples 2 to 6) to which polysaccharide was added.
Table 1 shows the reproducibility (CV value) when the blood of the healthy human was measured 10 times repeatedly. In Experimental Example 1 in which no polysaccharide was added, the CV value was 3.5%.
When the polysaccharide was added to the hemolyzed dilution, the CV value was greatly improved to 2.5%. Moreover, in the example (Experimental Examples 3-6) which added the polysaccharide to the eluent, the CV value was very good at 1.0% or less.

Experimental examples 7 to 11 were performed as measurement examples when a column packing material having no ion exchange group was used. FIG. 2 shows a chromatogram when normal human blood is measured under the conditions of Experimental Example 7 (experimental example not including polysaccharides). As shown in FIG. 2, hemoglobin A1c could not be separated. The same chromatogram as in FIG. 2 was obtained in the system in which the polysaccharide was added only to the hemolytic dilution of Experimental Example 8.
On the other hand, in the measurement system (Experimental Examples 9 to 11) in which polysaccharide was added to the eluent, hemoglobin A1c could be separated, and the same chromatogram as in FIG. 1 was obtained.
Table 1 shows the reproducibility (CV value) when the blood of the healthy human was measured 10 times repeatedly. In the system containing no polysaccharide at all (Experimental Example 7) and the system in which the polysaccharide was added only to the hemolytic dilution (Experimental Example 8), hemoglobin A1c could not be separated. On the other hand, when a polysaccharide was added to the eluent, hemoglobin A1c could be separated, and the reproducibility CV value was as good as 1% or less.

(Evaluation 2: Evaluation of separation performance of modified hemoglobins)
Instead of healthy human blood used in the above experimental examples, a sample containing modified hemoglobins was artificially prepared and measured, and the separation performance from the hemoglobin A1c peak was evaluated.
As modified hemoglobins, three kinds of samples including a labile hemoglobin A1c-containing sample (sample L), an acetylated hemoglobin-containing sample (sample A), and a carbamylated hemoglobin-containing sample (sample C) were prepared by a known method.
That is, the sample L was prepared by adding glucose to a healthy human whole blood to 2000 mg / dL and heating at 37 ° C. for 3 hours. Sample A was prepared by adding acetaldehyde at 50 mg / dL to healthy human whole blood and heating at 37 ° C. for 2 hours. Sample C was prepared by adding sodium cyanate to 50 mg / dL and heating at 37 ° C. for 2 hours.
The above-mentioned modified hemoglobin sample (sample L, sample A, sample C) and healthy human blood (non-modified product) used for the preparation of modified hemoglobin were measured using the above column filler, and the measured value of hemoglobin A1c was compared. . Separation performance was performed by calculating and comparing a value (Δ value) obtained by subtracting the hemoglobin A1c value of the unmodified product from the hemoglobin A1c value of the modified hemoglobin sample. The obtained Δ values are shown in Table 1.

From Table 1, the Δ value of Experimental Example 1 using a reagent not containing a polysaccharide was about 0.3 to 0.4%, and the influence of modified hemoglobins was recognized. On the other hand, in the measurement system using the reagent containing polysaccharide, particularly the measurement system (Experimental Examples 3 to 6 and 9 to 11) in which the polysaccharide was added to the eluent, it was hardly affected by the modified hemoglobin.

(Evaluation 3: Evaluation of separation performance of abnormal hemoglobins)
Using the above column filler, a sample containing hemoglobin S and hemoglobin C as abnormal hemoglobin (AFSC hemocontrol: manufactured by Helena Laboratories) was measured.
The chromatogram obtained as a result of measurement using the measurement conditions of Experimental Example 3 is shown in FIG. In FIG. 3, peak 1 represents hemoglobin HbA1c, peak 2 represents hemoglobin Ao, peak 4 represents hemoglobin F (fetal Hb), peak 5 represents hemoglobin S, and peak 6 represents hemoglobin C. In the experimental examples using reagents containing other polysaccharides, almost the same separation performance was shown.
On the other hand, when measured using a reagent not containing polysaccharide (Experimental Examples 1, 7 and 9), hemoglobin S and hemoglobin C, which are abnormal hemoglobins, could not be separated.

(Evaluation 4: Evaluation of separation performance of hemoglobin A2)
A2 control (level 2) (manufactured by Bio-Rad) was measured as a sample containing hemoglobin A2 using the above column filler.
The chromatogram obtained as a result of measurement using the measurement conditions of Experimental Example 3 is shown in FIG. In FIG. 4, peak 1 represents hemoglobin HbA1c, peak 2 represents hemoglobin Ao, peak 4 represents hemoglobin F (fetal Hb), and peak 7 represents hemoglobin A2. In the experimental examples using reagents containing other polysaccharides, almost the same separation performance was shown.
On the other hand, when it measured using the reagent which does not contain polysaccharide (Experimental example 1, 7, and 9), hemoglobin A2 was not able to be isolate | separated.

ADVANTAGE OF THE INVENTION According to this invention, the measuring method of hemoglobin which can perform a highly accurate measurement easily can be provided.

It is the chromatogram obtained when measuring hemoglobin A1c using the measurement conditions of Experimental Example 1. It is the chromatogram obtained when measuring hemoglobin A1c using the measurement conditions of Experimental Example 7. It is the chromatogram obtained when measuring abnormal hemoglobin using the measurement conditions of Experimental example 3. FIG. It is the chromatogram obtained when measuring hemoglobin A2 using the measurement conditions of Experimental Example 3.

Claims (8)

A method for measuring hemoglobin by a liquid chromatography method, wherein a buffer containing a polysaccharide having a cationic exchange group is used as an eluent used for measuring a blood sample. Method. The method for measuring hemoglobin according to claim 1, wherein the polysaccharide having a cationic exchange group is a polysaccharide having a sulfonic acid group. The method for measuring hemoglobin according to claim 1, wherein the polysaccharide having a cationic exchange group is chondroitin sulfate or dextran sulfate. The method for measuring hemoglobin according to claim 1, 2 or 3, wherein a column packing material having no ion exchange group is used. A method for measuring hemoglobin by a liquid chromatography method, wherein a buffer solution containing chondroitin sulfate or dextran sulfate is used for hemolysis reagent used for hemolyzing a blood sample, or for dilution used for diluting the blood sample A method for measuring hemoglobin, which is used as a reagent. 6. The method for measuring hemoglobin according to claim 1, 2, 3, or 5, wherein a column filler having an ion exchange group is used. The method for measuring hemoglobin according to claim 1, 2, 3, 4, 5 or 6, wherein the hemoglobin to be measured is hemoglobin A1c. The method for measuring hemoglobin according to claim 1, 2, 3, 4, 5 or 6, wherein the hemoglobin to be measured is hemoglobin A1c and abnormal hemoglobin .

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