JP2001021555A - Hemolysis reagent and method for measuring hemoglobins using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a hemolytic reagent which improves the measurement accuracy of stable HbA1c as compared with a conventional method, and a method for measuring hemoglobins using the same. SOLUTION: In a method for measuring hemoglobins by cation exchange liquid chromatography, a hemolytic reagent used for hemolyzing a blood sample, wherein the hemolytic reagent contains chaotropic ions, and further contains a buffering agent, A hemolytic reagent having a pH of 5.0 to 10.0, and a method for measuring hemoglobins using these hemolytic reagents.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring hemoglobins by liquid chromatography, which relates to a hemolytic reagent used for hemolyzing a blood sample and a method for measuring hemoglobins using the same.

[0002]

BACKGROUND OF THE INVENTION Glycated hemoglobin, especially hemoglobin A
1c (hereinafter referred to as HbA1c) is widely used as an index for diagnosing diabetes. HbA1c is formed by irreversible binding of hemoglobin after blood sugar enters erythrocytes, and reflects the average sugar concentration in blood over the past one to two months.

As a method for measuring HbA1c, a liquid chromatography method and an immunoassay are generally used.

HbA1 by liquid chromatography
The measurement of c is mainly performed by the cation exchange liquid chromatography method (Japanese Patent Publication No. 8-7198). When a hemolyzed blood sample is separated by cation exchange liquid chromatography, hemoglobin A1a (hereinafter referred to as HbA1a), hemoglobin A1b (hereinafter referred to as HbA1b), and hemoglobin F (hereinafter referred to as Hb
F), unstable HbA1c, stable HbA1c, and hemoglobin A0 (hereinafter, referred to as HbA0). In addition, HbA1c used as an index for diagnosing diabetes is a stable HbA1c among the above, and is recently obtained as a ratio (%) of the area of the stable HbA1c peak to the area of all hemoglobin peaks. .

However, it is difficult to separate the stable HbA1c peak from the unstable HbA1c peak.
In Japanese Patent Publication No. 3-298063, red blood cells and / or
A method is described in which a sample containing hemoglobin is brought into contact with a dissociation solvent containing a phosphate condensate and / or a salt of a phosphate condensate to dissociate unstable hemoglobin into hemoglobin and glucose and to measure the hemoglobin. However, in this method, the separation of stable HbA1c is insufficient,
There was a disadvantage that the measurement accuracy was poor.

In the method for measuring hemoglobins by the liquid chromatography method, it is said that the method is affected by modified hemoglobin such as acetylated hemoglobin (hereinafter, referred to as AHb) and carbamylated hemoglobin (hereinafter, referred to as CHb). And CHb peaks are also stable H
Because of elution near the bA1c peak, separation in a short time was difficult depending on the conventional method.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for measuring hemoglobins in view of the above-mentioned problems. Thus, it is possible to separate stable HbA1c with higher precision than conventional methods and to measure hemolysis in a shorter time. An object of the present invention is to provide a reagent and a method for measuring hemoglobins using the same.

[0008]

In order to achieve the above object, according to the present invention, there is provided a method for measuring hemoglobins by cation exchange liquid chromatography, which comprises a hemolysis reagent used for hemolyzing a blood sample. In addition, the present invention provides a hemolytic reagent containing a chaotropic ion. The present invention according to claim 2 provides:
The hemolytic reagent according to claim 1, further comprising a buffer, and having a pH of 5.0 to 10.0. Further, the present invention according to claim 3 provides a method for measuring hemoglobins, characterized by using the hemolytic reagent according to claim 1 or 2. Hereinafter, the present invention will be described in detail.

The hemolytic reagent in the present invention may be any aqueous solution that is hypotonic compared to red blood cells. In addition, the hemolysis referred to here means that the erythrocyte membrane is broken and contents such as hemoglobin (for example, hemoglobins) go out of the erythrocytes.

The chaotropic ion in the present invention is an ion generated by dissociation into an aqueous solution, which destroys the structure of water and suppresses the decrease in entropy of water, which occurs when a hydrophobic substance comes into contact with water. 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. And urea and the like. Examples of the cation include barium ion, calcium ion, magnesium ion, lithium ion, cesium ion, potassium ion, and guanidine ion. Stable HbA
In order to improve the measurement accuracy of 1c, more preferably, as anions, tribromoacetate ion, trichloroacetate ion, thiocyanate ion, iodide ion,
Examples include perchlorate ion, dichloroacetate ion, and nitrate ion. Examples of the cation include guanidine ion.

The concentration of the chaotropic ion in the hemolysis reagent is preferably 0.1 mM to 3000 mM, and 1 mM
~ 1000 mM is more preferred, more preferably 10
mM to 500 mM. If the concentration is lower than 0.1 mM, the separation effect is reduced in the measurement of hemoglobins, and the measurement accuracy is deteriorated. Further, even if the concentration is higher than 3000 mM, the effect of separating hemoglobins is not further improved, so that the measurement accuracy is not improved. Further, these chaotropic ions may be used alone or in combination of two or more.

[0012] The hemolytic reagent of the present invention has a pH containing a buffer in addition to the chaotropic ion.
Preferably, the hemolytic reagent is 5.0 to 10.0. Examples of the buffer include inorganic acids, organic acids, salts thereof, and organic substances. As the inorganic acid, for example,
Phosphoric acid, boric acid, carbonic acid and the like. Examples of the organic acids and organic substances include carboxylic acids, dicarboxylic acids, carboxylic acid derivatives, hydroxycarboxylic acids, anilines or aniline derivatives, amino acids, amines, imidazoles,
Examples include pyridine, cacodylic acid, tris (hydroxymethyl) aminomethane, glycylglycine, and pyrophosphoric acid.

As the carboxylic acid, for example, acetic acid,
And propionic acid. Examples of the dicarboxylic acid include malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, fumaric acid, phthalic acid and the like. Examples of the carboxylic acid derivative include β, β-
Dimethyl glutaric acid, barbituric acid, aminobutyric acid and the like can be mentioned. Examples of the hydroxycarboxylic acid include citric acid, tartaric acid, lactic acid and the like. Examples of the aniline or the aniline derivative include dimethylaniline. Examples of the above amino acids include aspartic acid, asparagine, glycine and the like.
Examples of the amines include ethylenediamine, triethanolamine, and the like. Examples of the imidazoles include imidazole, 5 (4) -methylimidazole, and 2,5 (4) -dimethylimidazole.

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

As the buffer, 2- (N-morpholino) ethanesulfonic acid (MES), N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (H
EPES), bis (2-hydroxyethyl) iminotris- (hydroxymethyl) methane (Bistris),
Tris (hydroxymethyl) aminoethane (Tri
s), and the like, which make up what is commonly referred to as Good's buffer.

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

The concentration of the above buffer in the hemolysis reagent may be within the range of a buffering action when dissolved in water.
1 mM to 1000 mM, preferably 1 to 500 m
M, more preferably 2 to 200 mM.

The pH of the hemolytic reagent of the present invention according to claim 2
Has a pH of 5.0 to 10, preferably a pH of 5.5 to 10.
9.5, more preferably 6.0 to 9.0. If the pH is below 5.0 or above 10, the hemoglobin will denature.

The following substances may be added to the above hemolytic reagent. (1) Examples of the inorganic salts 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) Known acids and bases may be added as pH adjusters. Examples of the acid include hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, and the like, and examples of the base include sodium hydroxide, potassium hydroxide, lithium hydroxide, magnesium hydroxide, barium hydroxide, calcium hydroxide, and water. Examples include ammonium oxide, strontium hydroxide, cesium hydroxide, nickel hydroxide, aluminum hydroxide, and cadmium hydroxide. The concentration of these acids and bases is not particularly limited, but is preferably 0.001 to 500 mM. (3) It may be mixed with a water-soluble organic solvent such as methanol, ethanol, acetonitrile, and acetone. The concentration of these organic solvents is not particularly limited, but is preferably 0.
~ 80% by volume, chaotropic ions, inorganic acids,
It is preferable to use it to such an extent that an organic acid or a salt thereof is not precipitated.

(4) As the surfactant, a nonionic surfactant, a cationic surfactant, an anionic surfactant, or the like may be added. By using a surfactant,
In addition to efficiently performing hemolysis, for example, when measurement is performed by high performance liquid chromatography (HPLC) or the like, there is an effect of washing a flow path through which a hemolysis reagent passes. The surfactant is preferably a nonionic surfactant,
For example, polyoxyethylenes (hereinafter, polyoxyethylene is represented by POE and the number of moles of ethylene oxide added by (n)), POE (7) decyl ether, POE (n)
Dodecyl ether, POE (10) tridecyl ether, POE (11) tetradecyl ether, POE
(N) cetyl ether, POE (n) stearyl ether, POE (n) oleyl ether, POE (17) cetyl-stearyl ether, POE (n) octylphenyl ether, POE (n) nonylphenyl ether,
Sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, POE (n) sorbitan monolaurate, PO
E (n) sorbitan monopalmitate, POE (n) sorbitan monostearate, POE (n) sorbitan monooleate, and the like. These surfactants are
They may be used alone or in combination.

The addition amount of these surfactants is preferably 0.01 to 10% by weight.

A third aspect of the present invention is a method for measuring hemoglobins using the hemolytic reagent according to the first or second aspect.

The eluent used in the cation exchange liquid chromatography in the present invention is used by dissolving the same inorganic acid, organic acid or salt or organic substance as the above-mentioned buffer in an aqueous solution.

The above-mentioned inorganic acids include, for example, phosphoric acid, boric acid, carbonic acid and the like. Examples of the organic acid / organic substance include carboxylic acid, dicarboxylic acid, carboxylic acid derivative, hydroxycarboxylic acid, aniline or aniline derivative, amino acid, amine, imidazole, pyridine,
Examples include cacodylic acid, tris (hydroxymethyl) aminomethane, glycylglycine, and pyrophosphoric acid.

Examples of the carboxylic acid include acetic acid,
And propionic acid. Examples of the dicarboxylic acid include malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, fumaric acid, phthalic acid and the like. Examples of the carboxylic acid derivative include β, β-
Dimethyl glutaric acid, barbituric acid, aminobutyric acid and the like can be mentioned. Examples of the hydroxycarboxylic acid include citric acid, tartaric acid, lactic acid and the like. Examples of the aniline or the aniline derivative include dimethylaniline. Examples of the above amino acids include aspartic acid, asparagine, glycine and the like.
Examples of the amines include ethylenediamine, triethanolamine, and the like. Examples of the imidazoles include imidazole, 5 (4) -methylimidazole, and 2,5 (4) -dimethylimidazole.

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

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

The concentration of the above buffer in the eluent may be within a range that provides a buffering action when dissolved in water.
mM to 1000 mM, preferably 1 to 500 m
M, more preferably 2 to 200 mM.

The pH of the eluent used in the present invention is usually from pH 4.0 to 13.5, preferably from pH 4.5 to pH 4.5.
12.0, more preferably 5.0 to 10.0. The hemoglobin denatures at pH below 4.0 and above 13.5. As the buffer in the eluent, those having an acid dissociation constant (pKa) of 1.5 to 6.8 and 6.8 to 13 are preferably used. In this case, even when a plurality of substances are contained as the buffer solution composition, the pKa value of each substance may be in both of 1.5 to 6.8 and 6.8 to 13. Alternatively, a buffer having a pKa value of 1.5 to 6.8 and 6.8 to 13 with one kind of substance may be used.

In the measurement method of the present invention, the eluent may be changed during the measurement, or gradient elution or step-up gradient elution may be performed.

The filler used in the cation exchange liquid chromatography of the present invention is composed of particles having at least one kind of cation exchange group. For example, a cation exchange group is introduced into polymer particles. Obtained by:

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

The diameter of the particles is preferably 0.1 to 1
0 μm, more preferably 0.2 to 8 μm. Also,
The particle size distribution is represented by a coefficient of variation (CV value) (standard deviation of particle size 粒径
(Average diameter x 100 (%)) is preferably 20% or less, more preferably 15% or less.

Examples of the polymer particles include inorganic particles such as silica and zirconia; natural polymer particles such as cellulose, polyamino acid and chitosan; and synthetic polymer particles such as polystyrene and polyacrylate. . It is preferable that the component other than the ion exchange group to be introduced into the polymer particles is more hydrophilic. Further, those having a high degree of crosslinking are preferable from the viewpoint of pressure resistance and swelling resistance.

The cation exchange group can be introduced into the polymer particles by a known method. For example, after preparing the polymer particles, the functional groups (hydroxyl group, amino group, carboxyl group, epoxy group) And the like) into the particles by a chemical reaction. The cation exchange filler can also be prepared by a method of preparing polymer particles by polymerizing a monomer having a cation exchange group. For example, there is a method in which a cation exchange group-containing monomer and a crosslinkable monomer are mixed and polymerized in the presence of a polymerization initiator. Further, a polymerizable cation exchange group-containing ester such as methyl (meth) acrylate and ethyl (meth) acrylate is mixed with a crosslinkable monomer and polymerized in the presence of a polymerization initiator. The ester may be converted to a cation exchange group by a hydrolysis treatment. Further, as described in JP-B-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 above packing material is packed in a column and used for liquid chromatography measurement. The column size is
Preferably, the inner diameter is 0.1 to 4 mm and the length is 5 to 200 mm, more preferably, the inner diameter is 0.3 to 3 mm and the length is 7 to
18 mm. When the column size is smaller than 0.1 mm in inner diameter and 5 mm in length, workability is poor and the separation function is also poor. When the inner diameter is larger than 4 mm and the length is larger than 200 mm, not only the amount of the filler used is increased, but also the separating function is deteriorated.

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

As the material of the column, known metals such as stainless steel, glass, and resins such as PEEK are used. In addition, a portion where the column packing and the column main body are in contact with each other may be covered with an inert material. Examples of the material include PEEK, polyethylene, Teflon, a titanium compound, a silicon compound, and a silicon film.

The liquid chromatograph used for the above measurement may be a known one, and includes, for example, a liquid sending pump, a sample injection device (sampler), a column, a detector and the like. Further, other accessory devices (such as a column thermostat and an eluent deaerator) may be appropriately attached.

The other measuring conditions in the above measuring method 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. For the detection of hemoglobins, visible light of 415 nm is preferable, but not particularly limited thereto. As the measurement sample, a sample obtained by diluting hemolyzed blood with a solution containing a substance having hemolytic activity such as a surfactant is used. The amount of sample injected into the liquid chromatograph is
Although it depends on the dilution ratio, it is preferably 0.1 to 100 μm.
It is about l.

[0041]

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

(Example 1) [Preparation of filler 1: carboxyl group] 400 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.)
Then, 1.5 g of benzoyl peroxide (manufactured by Wako Pure Chemical Industries) was dissolved in a mixture of 150 g of methacrylic acid (manufactured by Wako Pure Chemical Industries). This was dispersed in 2500 ml of a 4% by weight aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Industry Co., Ltd.), and the temperature was raised to 75 ° C. in a nitrogen atmosphere with stirring, followed by polymerization for 8 hours. After polymerization, the polymer was washed, dried, and classified to obtain particles having an average particle size of 6 μm.

[Packing of Column] The obtained packing material 1 was packed in a column as follows. 0.7 g of particles is
Disperse in 30 ml of mM phosphate buffer (pH 5.8)
After sonication for minutes, the mixture was stirred well. The whole amount was injected into a packer (manufactured by Umetani Seiki Co., Ltd.) connected to a stainless steel empty column (4.6 φ × 30 mm). A liquid feed pump (manufactured by Sanuki Kogyo) is connected to the packer and the pressure is 300 kg / cm.
2 was filled at a constant pressure.

[Measurement of Hemoglobins] Using the obtained column, hemoglobins were measured under the following measurement conditions. (Measurement conditions) System: Liquid pump: LC-9A (manufactured by Shimadzu Corporation) Autosampler: ASU-420 (manufactured by Sekisui Chemical) Detector: SPD-6AV (manufactured by Shimadzu Corporation) Eluent: Eluent A: 70 mM phosphate buffer (manufactured by Wako Pure Chemical Industries; pH 5.7) Eluent B: 70 mM phosphate buffer (pH 8.5), 250 mM sodium nitrate For 0 to 2 minutes from the start of measurement, eluent A was used. The eluent B was allowed to flow for 2 to 2.1 minutes, and the eluent A was allowed to flow for 2.1 to 3 minutes. Flow rate: 2.0 ml / min Detection wavelength: 415 nm Sample injection volume: 10 μl

(Measurement sample: A) Blood of a healthy human was collected, and sodium fluoride was added as an anticoagulant to a concentration of 10 mg / ml. To this, 150 times the amount of a hemolytic reagent (0.1% by weight of polyethylene glycol mono-4-octylphenyl ether (Triton X-10) as a surfactant) was added.
0, manufactured by Tokyo Chemical Industry Co., Ltd.) and a hemolytic reagent (pH 7.5) composed of 100 mM guanidine and 10 mM phosphate buffer as a compound that generates chaotropic ions. (Measurement sample B; AHb) Sodium fluoride was added to 10 ml of healthy human blood (10 mg / ml), and 1 ml of a 0.3 wt% acetaldehyde physiological saline solution was further added.
The reaction was performed at 37 ° C. for 3 hours. To this was added a 150-fold amount of a hemolysis reagent (a hemolysis reagent (pH 7.5) composed of 0.1% by weight of Triton X-100, 100 mM guanidine, and 10 mM phosphate buffer solution), and hemolyzed to obtain a measurement sample.

(Measurement Results) FIGS. 1 and 2 show chromatograms obtained by measuring a sample under the above measurement conditions. FIG. 1 shows the result of measuring the sample A, and FIG. 2 shows the result of measuring the sample B. Peak 1 is HbA1a and b, peak 2
Is HbF, peak 3 is unstable HbA1c, peak 4 is stable HbA1c, peak 5 is HbA0, and peak 6 is A
Hb is shown. In FIG. 2, peaks 4 and 6 are well separated. Table 1 shows the results obtained by measuring five samples of the measurement samples A and B and measuring the measurement accuracy (SD; standard deviation, CV; coefficient of variation) of stable HbA1c.

[0047]

[Table 1]

Example 2 Hemoglobins were measured under the same conditions as in Example 1 except that the following reagents were used as the hemolysis reagent. Hemolysis reagent (pH 8.0) (0.1% by weight Triton X-100, 80 mM thiocyanic acid and 10% by weight)
mM phosphate buffer solution) to lyse the measurement samples A and B.
The sample was diluted by a factor of 0 to obtain a measurement sample. The obtained chromatogram was as good as in FIG.

Example 3 Hemoglobins were measured under the same conditions as in Example 1 except that the following reagents were used as the hemolysis reagent. Hemolysis reagent (pH 8.0) (0.1% by weight Triton X-100, 40 mM perchloric acid and 10 mM
The measurement samples A and B were hemolyzed with a phosphate buffer solution) and diluted 150-fold to obtain measurement samples. The obtained chromatogram was as good as in FIG.

(Example 4) Hemoglobins were measured under the same conditions as in Example 1 except that the following reagents were used as the hemolysis reagent. The measurement samples A and B were hemolyzed with a hemolysis reagent (pH 8.0) (0.1% by weight Triton X-100, 50 mM urea and 10 mM phosphate buffer solution), and diluted 150 times to obtain a measurement sample. The resulting chromatogram is shown in FIG.
As good as.

(Example 5) [Preparation and filling of filler 2: sulfonic acid group] Instead of methacrylic acid as a polymerizable monomer, 2-acrylamide-
A filler was obtained in the same manner as in Example 1 except that 150 g of 2-methylpropanesulfonic acid was used. Further, the column was packed in the same manner as in Example 1.

[Measurement of hemoglobins] Using the obtained column, hemoglobins were measured under the following measurement conditions. The same measurement system as in Example 1 was used. (Measurement conditions) Eluent: Eluent A: 70 mM phosphate buffer (manufactured by Wako Pure Chemical Industries, Ltd .; pH 5.7) Eluent B: 70 mM phosphate buffer (pH 8.5), 250 mM sodium nitrate From the start of measurement The eluent A was allowed to flow for 0 to 2 minutes, the eluent B was allowed to flow for 2 to 2.1 minutes, and the eluent A was allowed to flow for 2.1 to 3 minutes. Flow rate: 2.0 ml / min Detection wavelength: 415 nm Sample injection amount: 10 μl (Measurement sample) Hemoglobin was measured using the following reagents as hemolysis reagents. Hemolysis reagent (pH 8.0) (0.1% by weight Triton X-100, 60 mM guanidine and 10 m
M phosphate buffer solution) to lyse the measurement samples A and B,
It was diluted by a factor of 2 to obtain a measurement sample. The obtained chromatogram was as good as in FIG. Comparative Example 1 Hemoglobins were measured in the same manner as in Example 1 except that the following eluent was used as the eluent and the following reagent was used as the hemolytic reagent. Eluent: Eluent A: 70 mM phosphate buffer (pH 5.7) Eluent B: 70 mM phosphate buffer (pH 8.5) (Measurement sample) Hemolysis reagent (pH 8.0) (0.1% by weight Triton X) -100 and 0.1% by weight polyphosphoric acid and 10m
M phosphate buffer solution) to lyse the measurement samples A and B,
It was diluted by a factor of 2 to obtain a measurement sample. (Measurement Results) The chromatogram obtained by measuring Sample B is shown in FIG. 3, but the separation of peaks 4 and 6 was poor. Further, as shown in Table 2, in Examples 1 to 5, the measurement sample A was used.
And B have substantially the same CV values for HbA1c,
In Comparative Example 1, the separation of B was poor and the CV value was large compared to the measurement sample A, and the result was that the difference between the measurement samples A and B was large.

[0053]

According to the first aspect of the present invention, the hemolytic reagent of the present invention is a hemolytic reagent used for hemolyzing a blood sample in a method for measuring hemoglobins by cation exchange liquid chromatography, and contains a chaotropic ion. The separation of stable HbA1c was improved. The hemolytic reagent of the present invention according to claim 2 is the hemolytic reagent according to claim 1, further comprising a buffer, and having a pH of 5.0 to 1.
Since it is 0.0, it has become possible to separate hemoglobins in a short time and with high accuracy. According to the method for measuring hemoglobin of the present invention described in claim 3, since the hemolytic reagent according to claim 1 or 2 is used, separation of stable HbA1c is improved, or separation of hemoglobin is performed in a short time with high accuracy. It became possible.

[Brief description of the drawings]

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

FIG. 2 is a view showing a chromatogram obtained when measuring hemoglobins (sample B) 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 Comparative Example 1.

[Explanation of symbols]

 1 Peak of HbA1a and b 2 Peak of HbF 3 Peak of unstable HbA1c 4 Peak of stable HbA1c 5 Peak of HbA0 6 Peak of AHb

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G045 AA07 BB14 BB16 BB29 BB34 BB41 BB51 CA02 CA25 DA45 DA48 DA51 FB06

Claims (3)

[Claims] 1. A method for measuring hemoglobins by cation exchange liquid chromatography, which is a hemolytic reagent used for hemolyzing a blood sample, wherein the hemolytic reagent contains chaotropic ions. 2. The hemolytic reagent according to claim 1, further comprising a buffer, and having a pH of 5.0 to 10.0. 3. A method for measuring hemoglobins, comprising using the hemolytic reagent according to claim 1 or 2.