Hemoglobin analysis method

Abstract

Provided is a hemoglobin assay method that enables high-precision separation of the HbA0 fraction.　This hemoglobin assay method comprises: inducing elution of the fraction containing hemoglobin A0 by ion-exchange chromatography using an eluent containing a buffer at a concentration of less than 25 mM, the buffer being a phosphate or Good's buffer and the eluent pH being within ± 0.1 of the buffer pKa.

Description

Technical Field

[0001] The present invention relates to a method for analyzing hemoglobin.

Background Art

[0002] In the fields of organic chemistry, biochemistry, medicine, etc., liquid chromatography is widely used for analyzing components in a sample. For example, in the medical field, liquid chromatography is used for analyzing hemoglobin (Hb). Most of normal human Hb is HbA, and HbA mainly contains HbA0 and glycated HbA1c. HbA1c is a glycated hemoglobin in which blood sugar and hemoglobin are bound, and is used as a marker for diabetes and the like. The blood HbA1c concentration (%), that is, the ratio of HbA1c in total blood Hb, reflects the average blood sugar level in the past 1 to 2 months and is widely used as an index for diabetes diagnosis.

[0003] Patent Documents 1 and 2 describe a method for separating an HbA1c fraction and an HbA0 fraction by cation exchange chromatography, and the method uses the following eluents A and B: Eluent A: having a pH of 4.0 to 6.0 and containing (1) a chaotropic ion, (2) an acid or its salt selected from the group consisting of a monocarboxylic acid, a dicarboxylic acid, a tricarboxylic acid, and an amino acid having an acid dissociation constant in the range of 3.5 or more and less than 6.5, and (3) an acid or its salt which is at least one of an inorganic oxo acid and an amino acid having an acid dissociation constant in the range of 6.5 or more and 9.5 or less, Eluent B: having a pH 0.5 to 3.0 higher than that of Eluent A and a concentration 20 to 200 mmol / L lower than that of Eluent A, or having a pH of 7.0 to 10.0, using them in this order, and including setting the pressure value generated in the measurement system to be 9.8×10 3 Pa or more and 19.6×10 5 Pa or less. Patent Document 3 describes a method for separating various hemoglobins including abnormal hemoglobin and thalassemia markers from a blood sample by cation exchange chromatography using an eluent with a pH of 8.1 or higher and an osmotic pressure of 40 mOsm / kg or lower.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0005] For accurate calculation of hemoglobin (Hb) A1c concentration, it is required to separate HbA1c and other Hbs, particularly HbA0, with high precision in liquid chromatography analysis. When the separation accuracy of HbA0 decreases, not only can the HbA1c concentration not be accurately calculated, but also HbA2, HbS, HbC, etc. (which are used as indicators of abnormal Hb formation) having peaks near HbA0 cannot be detected.

[0006] However, in actual chromatography analysis, even under the same measurement conditions using the same sample, the elution time and separation ability of HbA0 may differ. From the research of the present inventor, it is presumed that this deviation in elution time and separation ability is caused by variations in the structure of the chromatography analysis apparatus (for example, component dimensions and assembly) and variations in the liquid feeding conditions of the eluent due to slight differences in operation. The present invention provides a hemoglobin analysis method capable of separating the HbA0 fraction with high precision without being affected by variations in the structure of the analysis apparatus and differences in operation.

Means for Solving the Problems

[0007] Therefore, the present invention provides the following. 〔1〕A hemoglobin analysis method, comprising eluting a fraction containing hemoglobin A0 by ion exchange chromatography using an eluent containing a buffer at a concentration of less than 25 mM, wherein the buffer is a Good buffer, and the pH of the eluent is within ±0.1 of the pKa of the buffer, method. 〔2〕The method according to 〔1〕, wherein the Good buffer is TES. 〔3〕The method according to 〔1〕 or 〔2〕, wherein the osmotic pressure of the eluent is 60 mOsm / kg or less. 〔4〕The method according to any one of 〔1〕 to 〔3〕, further comprising eluting a fraction containing hemoglobin A1c with eluent A before eluting the fraction containing hemoglobin A0, wherein the eluent A is a buffer solution having a pH of 4.0 to 6.8. 〔5〕The method according to 〔4〕, wherein the switching between the eluent A and the eluent containing the buffer at a concentration of less than 25 mM is performed by a stepwise gradient. 〔6〕The method according to any one of 〔1〕 to 〔5〕, further comprising passing eluent B through after the eluent containing the buffer at a concentration of less than 25 mM, wherein the eluent B is a buffer solution having a pH of 6.0 to 10.0. 〔7〕The method according to 〔6〕, wherein the switching between the eluent containing the buffer at a concentration of less than 25 mM and the eluent B is performed by a stepwise gradient. 〔8〕The method according to any one of 〔1〕 to 〔7〕, wherein the ion exchange chromatography is cation exchange chromatography. 〔9〕The method according to any one of 〔1〕 to 〔8〕, wherein the chromatography is high performance liquid chromatography. 〔10〕An eluent for separating hemoglobin A0 in ion exchange chromatography, which contains a buffer at a concentration of less than 25 mM, has a pH within ±0.1 of the pKa of the buffer, and the buffer is a Good buffer. The eluent according to

[10] , wherein the good buffer is TES. The eluent according to

[10] or

[11] , having an osmotic pressure of 60 mOsm / kg or less. The eluent according to any one of

[10] to

[12] , which is used after eluent A in ion exchange chromatography, and the eluent A is a buffer solution having a pH of 4.0 to 6.8. The eluent according to any one of

[10] to

[13] , which is used before eluent B in ion exchange chromatography, and the eluent B is a buffer solution having a pH of 6.0 to 10.0. [Advantages of the Invention]

[0008] According to the present invention, HbA0 can be separated from a sample with high precision without being affected by variations in the structure of the analyzer or differences in operation. The present invention is useful for highly sensitive detection of various hemoglobin species and accurate measurement of HbA1c concentration. [Brief Description of the Drawings]

[0009]

Figure 1

Figure 2

[0010] The present invention relates to a method for analyzing hemoglobin by ion exchange chromatography. As a sample used in the method for analyzing hemoglobin according to the present invention (hereinafter, also referred to as the method of the present invention), a blood sample containing hemoglobin, which is used in ordinary hemoglobin analysis, can be used. For example, a blood sample obtained by hemolyzing or diluting blood collected from a human can be used.

[0011] Preferably, the chromatography performed by the method of the present invention is liquid chromatography, more preferably high performance liquid chromatography (HPLC). The ion exchange chromatography in the present invention can be carried out using a known liquid chromatography system, that is, an ion exchange column connected to a system equipped with a pump for eluent delivery, a sampler, a detector, etc.

[0012] Preferably, the ion exchange chromatography performed by the method of the present invention is cation exchange chromatography using a stationary phase having a cation exchange group. Preferably, a cation exchange column filled with the stationary phase having the cation exchange group is used for the cation exchange chromatography. Examples of the cation exchange group include a carboxyl group, a phosphate group, a sulfonic acid group, etc., and among these, a sulfonic acid group is preferred.

[0013] Examples of the stationary phase include filler particles, porous bodies, etc., and filler particles are preferred. Examples of the filler particles include inorganic particles, organic particles, etc. Examples of the inorganic particles include particles composed of silica, zirconia, etc. Examples of the organic particles include natural polymer particles such as cellulose, polyamino acid, chitosan, etc., and synthetic polymer particles such as polystyrene, polyacrylate ester, etc. A preferred example of the stationary phase having a cation exchange group is a filler containing crosslinked polymer particles obtained by polymerizing a mixture of a non-crosslinkable hydrophilic acrylic monomer and polyglycidyl ethers disclosed in JP-A-2011-047858, and a layer of an acrylic monomer having a cation exchange group polymerized on the surface of the crosslinked polymer particles.

[0014] In the method of the present invention, similar to the hemoglobin analysis by conventional ion exchange chromatography, after adsorbing hemoglobin in a sample to a stationary phase (for example, an ion exchange column), an eluent is passed through the stationary phase to elute hemoglobin from the stationary phase, thereby separating hemoglobin in the sample. Therefore, the eluent used in the method of the present invention is the eluent used for hemoglobin separation by ion exchange chromatography. More preferably, the eluent is the eluent used for elution of hemoglobin from the stationary phase in cation exchange chromatography.

[0015] In the method of the present invention, as the eluent for HbA0 separation used for elution of HbA0 from the stationary phase, a buffer solution containing a low concentration of Good buffer is used. Preferred examples of the Good buffer include Bicine (N,N-bis(2-hydroxyethyl)glycine), Tricine (N-[tris(hydroxymethyl)methyl]glycine), and TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), among which TES is preferred.

[0016] The concentration of the above-mentioned buffer in the eluent for HbA0 separation is preferably less than 25 mM, more preferably 20 mM or less, still more preferably 17.5 mM or less, and still more preferably 15 mM or less. On the other hand, it is preferably 2 mM or more, more preferably 2.5 mM or more, still more preferably 5 mM or more, and still more preferably 10 mM or more. Examples of the range of the concentration of the buffer in the eluent for HbA0 separation include preferably 2 mM or more and less than 25 mM, more preferably 2.5 mM or more and 20 mM or less, still more preferably 5 mM or more and 17.5 mM or less, and still more preferably 10 mM or more and 15 mM or less.

[0017] The pH of the eluent for HbA0 separation can be adjusted based on the pKa of the buffer contained in the eluent. The pH of the eluent for HbA0 separation is preferably within ±0.1 of the pKa of the buffer, more preferably within ±0.08 of the pKa of the buffer, still more preferably within ±0.05 of the pKa of the buffer, and even more preferably within ±0.03 of the pKa of the buffer. Also, in this specification, unless otherwise specified, pH and pKa indicate the pH and pKa values at 25°C, respectively. The pKa of the buffer can be calculated from its composition. For example, the pKa of TES at 25°C is 7.55 (J. Phys. Chem. Ref. Data, Vol. 31, No. 2: 231 - 370, 2002).

[0018] The eluent for HbA0 separation may further contain an organic acid, an inorganic acid, or a salt thereof. Examples of the organic acid include citric acid, succinic acid, tartaric acid, malic acid, etc. Examples of the inorganic acid include hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boric acid, acetic acid, perchloric acid, etc. Examples of the salt include sodium salt, potassium salt, etc. The eluent for HbA0 separation can contain any one or a combination of two or more of these organic acids, inorganic acids, or salts thereof.

[0019] The osmotic pressure of the eluent for HbA0 separation is preferably 45 mOsm / kg or less, more preferably 40 mOsm / kg or less. On the other hand, it is preferably 20 mOsm / kg or more, more preferably 25 mOsm / kg or more. Examples of the range of the osmotic pressure of the eluent for HbA0 separation include preferably 20 - 45 mOsm / kg, and more preferably 25 - 40 mOsm / kg. The value of the osmotic pressure in this specification refers to the value of the osmotic pressure measured by the freezing point depression method. The osmotic pressure can be measured using a commercially available osmometer, such as the Osmometer 3250 (manufactured by Advanced Instruments), etc. Also, in this specification, the unit mOsm / kg of the osmotic pressure means mOsm / kgH2O, which may also be expressed as mOsm / L or mOsm.

[0020] In the method of the present invention, a fraction containing HbA0 is separated from a sample by ion exchange chromatography using the above-described eluent for HbA0 separation. Preferably, in the method of the present invention, similar to the hemoglobin analysis by conventional ion exchange chromatography, before separating the fraction containing HbA0, first, a fraction containing hemoglobin A1c (HbA1c) is separated.

[0021] The fraction containing HbA1c is eluted with an eluent A different from the above-described eluent for HbA0 separation. The eluent A may be a buffer solution containing a buffer agent. Preferably, the buffer agent is an acid having a buffering capacity at pH 4.0 to 6.8 or a salt thereof. Examples of the acid include organic acids and inorganic acids. Examples of the organic acid include citric acid, succinic acid, tartaric acid, malic acid, etc., and salts thereof. Examples of the inorganic acid include hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boric acid, acetic acid, perchloric acid, etc., and salts thereof. The buffer solution used for the eluent A can contain any one or a combination of two or more of these organic acids, inorganic acids, and salts thereof. Among these, a buffer solution containing a phosphate, succinate, or perchlorate is preferred, and a succinate buffer solution is more preferred. Examples of the phosphate include sodium salts and potassium salts, etc., and monosodium phosphate and disodium phosphate are preferred. Examples of the succinate include sodium salts and potassium salts, etc., and monosodium succinate and disodium succinate are preferred. An example of the perchlorate is sodium perchlorate. These phosphates, succinates, and perchlorates may be used alone or in combination of two or more. The concentration of the buffer agent in the eluent A may be 0.1 mM or more and 400 mM or less, preferably 1 mM or more and 300 mM or less.

[0022] The pH of the eluent A is preferably 4.0 or higher, more preferably 5.0 or higher, still more preferably 5.2 or higher, and preferably 6.8 or lower, more preferably 6.0 or lower, still more preferably 5.8 or lower. The pH range of the eluent A is preferably 4.0 to 6.8, more preferably 5.0 to 6.0, still more preferably 5.2 to 5.8. Also, the osmotic pressure of the eluent A is preferably 160 mOsm / kg or higher, more preferably 180 mOsm / kg or higher, and preferably 240 mOsm / kg or lower, more preferably 220 mOsm / kg or lower. The osmotic pressure range of the eluent A is preferably 160 to 240 mOsm / kg, more preferably 180 to 220 mOsm / kg. In a preferred embodiment, the eluent A has a pH of 5.0 to 6.0 and an osmotic pressure of 160 to 240 mOsm / kg, more preferably 180 to 220 mOsm / kg. In a more preferred embodiment, the eluent A has a pH of 5.2 to 5.8 and an osmotic pressure of 160 to 240 mOsm / kg, more preferably 180 to 220 mOsm / kg.

[0023] In the method of the present invention, following the passage of the above-described eluent for HbA0 separation, an eluent B may be further passed through. The eluent B is an eluent having a composition different from that of the above-described eluent for HbA0 separation and the eluent A. By the eluent B, components eluted later than the fraction containing HbA0, for example, fractions containing HbA2, HbS, HbC, etc. can be eluted.

[0024] Eluent B may be any eluent that has been conventionally used for the elution of HbA0, HbA2, HbS, HbC, etc. in hemoglobin analysis by conventional ion exchange chromatography. For example, eluent B is a buffer solution, and examples of the buffer solution used for eluent B include the same ones as those exemplified for eluent A. Among these, a buffer solution containing phosphate, succinate, or perchlorate is preferred, and a phosphate buffer solution is more preferred. The concentration of the buffer agent in eluent B may be 0.1 mM or more and 400 mM or less, preferably 1 mM or more and 300 mM or less. The pH of eluent B is preferably pH 6.0 or more, more preferably pH 7.0 or more, still more preferably pH 7.5 or more, and preferably pH 10.0 or less, more preferably pH 9.0 or less, still more preferably pH 8.5 or less. The pH range of eluent B is preferably pH 6.0 to 10.0, more preferably pH 7.0 to 9.0, still more preferably pH 7.5 to 8.5. Also, the osmotic pressure of eluent B is preferably 150 mOsm / kg or more, more preferably 200 mOsm / kg or more, still more preferably 250 mOsm / kg or more, and preferably 500 mOsm / kg or less, more preferably 400 mOsm / kg or less, still more preferably 350 mOsm / kg or less. The osmotic pressure range of eluent B is preferably 150 to 500 mOsm / kg, more preferably 200 to 400 mOsm / kg, still more preferably 250 to 350 mOsm / kg. In a preferred embodiment, eluent B has a pH of 6.0 to 10.0 and an osmotic pressure of 150 to 500 mOsm / kg. In a more preferred embodiment, eluent B has a pH of 7.0 to 9.0 and an osmotic pressure of 200 to 400 mOsm / kg. In a still more preferred embodiment, eluent B has a pH of 7.5 to 8.5 and an osmotic pressure of 250 to 350 mOsm / kg.

[0025] The eluents (the above-mentioned eluents A, B, and the eluent for HbA0 separation) used in the present invention may contain, in addition to the above-mentioned buffer agents, a solvent and additives such as a hemoglobin denaturant, a binder to trivalent heme iron, a pH adjuster, and a surfactant.

[0026] Preferably, the eluent used in the present invention contains a hemoglobin denaturant. The hemoglobin denaturant may be an oxidizing agent that can change the heme iron of hemoglobin from divalent to trivalent to produce methemoglobin. Examples of such oxidizing agents include nitrite, potassium ferricyanide, methylene blue, hydrogen peroxide, ascorbic acid, hydrogen sulfide, etc. Among these, nitrite is preferred, and sodium nitrite or potassium nitrite is more preferred. The concentration of the hemoglobin denaturant in the eluent is preferably 0.05 to 15 mmol / L, more preferably 1.0 to 10 mmol / L.

[0027] Preferably, the eluent used in the present invention contains a binder to trivalent heme iron. The binder to trivalent heme iron stabilizes the methemoglobin produced by the hemoglobin denaturant and stabilizes its elution behavior. Examples of the binder to trivalent heme iron include azide, cyanide, etc. Examples of azide include sodium azide, diphenylphosphoric acid azide, 4-dodecylbenzenesulfonyl azide, 4-acetylaminobenzenesulfonyl azide, potassium azide, lithium azide, iron azide, hydrogen azide, lead azide, mercury azide, copper azide, silver azide, etc. Examples of cyanide include potassium cyanide, hydrogen cyanide, sodium cyanide, silver cyanide, mercury cyanide, copper cyanide, lead cyanide, iron cyanide, lithium cyanide, ammonium cyanide, etc. Preferably, the binder to trivalent heme iron is azide, more preferably sodium azide. The concentration of the binder to trivalent heme iron in the eluent is preferably 0.05 to 15 mmol / L, more preferably 0.5 to 10 mmol / L.

[0028] Examples of the pH adjuster include known acids and bases. Examples of acids include hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, etc. Examples of bases include sodium hydroxide, potassium hydroxide, lithium hydroxide, magnesium hydroxide, barium hydroxide, calcium hydroxide, etc.

[0029] Examples of the solvent include water-soluble organic solvents such as methanol, ethanol, acetonitrile, and acetone. The concentration of the solvent is preferably such that salts and the like do not precipitate. For example, it is preferably 80% (v / v) or less, and a more preferable range is 0.1% (w / w) or more and 50% (w / w) or less.

[0030] Preferably, in the method of the present invention, while switching the above-described eluents A, B, and the eluent for HbA0 separation, fractions containing various hemoglobins are eluted. Preferably, in the method of the present invention, gradient elution is performed by switching the eluents in the order of eluent A → eluent for HbA0 separation. More preferably, in the method of the present invention, gradient elution is performed by switching the eluents in the order of eluent A → eluent for HbA0 separation → eluent B.

[0031] Preferably, in the gradient elution in the method of the present invention, first, the eluent A (hereinafter referred to as solution A or simply A) is passed through at 100% to elute the fraction containing HbA1c. Next, a gradient is applied to the eluent to increase the ratio of the eluent for HbA0 separation (hereinafter referred to as solution C or simply C) until the ratio of solution C reaches 100%. The fraction containing HbA0 is eluted with solution C. The gradient from solution A to solution C is carried out so that A:C = 100:0 → 0:100. The gradient from solution A to solution C may be a stepwise gradient or a linear gradient, and the mixing ratio of the stepwise gradient or the gradient of the linear gradient may be changed stepwise in the middle. Preferably, the gradient from solution A to solution C is stepwise.

[0032] Preferably, the solution C is then switched to the eluent B (hereinafter referred to as solution B or simply B). Preferably, the gradient from solution C to solution B is carried out such that C:B = 100:0 → 0:100. The gradient from solution C to solution B may be a stepwise gradient or a linear gradient, and the mixing ratio of the stepwise gradient or the gradient of the linear gradient may be changed stepwise in the middle. By solution B, Hb fractions eluting later than HbA0 (for example, fractions containing HbA2, HbS, HbC, etc.) are eluted.

[0033] If necessary, after solution B, solution A may be passed through again. A flow time with A:B = 100:0 may be provided. The gradient from solution B to solution A may be a stepwise gradient or a linear gradient, but preferably it is a stepwise type.

[0034] In the method of the present invention, by passing the above-described eluent for separating HbA0 following the eluent A, the HbA0 fraction can be separated with high precision. Therefore, according to the method of the present invention, without being affected by variations in the structure of the analyzer and differences in operations, the peak of HbA0 can be clearly detected in chromatographic analysis, and thus, highly sensitive detection of various hemoglobin species and accurate measurement of the HbA1c concentration become possible.

Example

[0035] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited to the following examples.

[0036] (Reference Example 1: Preparation of Hemoglobin Separation Column) 200 g of tetraethylene glycol monomethacrylate (manufactured by NOF Corporation), a non-crosslinkable hydrophilic acrylic monomer, and 200 g of polyethylene glycol diglycidyl ether (manufactured by NOF Corporation), a polyglycidyl ether, were mixed, and 1.0 g of benzoyl peroxide (manufactured by Nacalai Tesque, Inc.) as a polymerization initiator was dissolved therein. The resulting mixture was dispersed in 5 L of an aqueous solution of 4 wt% polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Industry Co., Ltd., "Gosenol GH-20"), heated to 80°C under a nitrogen atmosphere with stirring, and subjected to a polymerization reaction for 1 hour. After cooling the temperature to 30°C, 100 g of 2-methacrylamido-2-methylpropanesulfonic acid (manufactured by Toagosei Co., Ltd.), an acrylic monomer having a cation exchange group, was added to the reaction system, and the mixture was heated to 80°C again and subjected to a polymerization reaction for 1 hour. The resulting crosslinked polymer particles were washed with ion-exchanged water and acetone to obtain crosslinked polymer particles into which sulfonic acid groups were introduced. The obtained column packing material for hemoglobin separation was packed into a stainless steel column having an inner diameter of 4 mm and a length of 20 mm to prepare a cation exchange column for hemoglobin separation.

[0037] (Reference Example 2: Preparation of Sample) A sample was prepared by appropriately dissolving and diluting a glycated Hb control (manufactured by Sysmex Corporation) with a phosphate buffer solution (pH 7.00) containing 0.1% Triton X-100.

[0038] (Test 1) Hemoglobin analysis was performed on the sample prepared in Reference Example 2 by HPLC. Two HPLC devices of the same type were prepared (Device A and B), and analysis was performed using each of them under the following conditions. <Analysis Conditions> HPLC device: Prominence 20A series (manufactured by Shimadzu Corporation) Detector: SPD-M20A (manufactured by Shimadzu Corporation) Liquid delivery pump: LC-20AD (manufactured by Shimadzu Corporation) Degassing unit: DGU-20A5R (manufactured by Shimadzu Corporation) Column oven: CTO-20AC (manufactured by Shimadzu Corporation) Autosampler: SIL-20AC (manufactured by Shimadzu Corporation) Column: Cation exchange column for hemoglobin separation prepared in Reference Example 1 Flow rate: 2.1 mL / min Detection wavelength: 415 nm Sample injection volume: 5 μL Eluent Eluent A: Buffer solution containing 40 mmol / L sodium succinate, 55 mmol / L sodium perchlorate, and 1 mmol / L sodium azide (pH 5.3, osmotic pressure 200 mOsm / kg) Eluent B: Buffer solution containing 45 mmol / L sodium phosphate, 100 mmol / L sodium perchlorate, and 9 mmol / L sodium azide (pH 7.6, osmotic pressure 300 mOsm / kg) Eluent C: Prescription described in Table 1 Gradient: Stepwise gradient (0 - 21 seconds: Eluent A → over 21 seconds to 39 seconds: Eluent C → over 39 seconds to 47 seconds: Eluent B → over 47 seconds to 90 seconds: Eluent A) The pH (25 °C) and osmotic pressure of the eluent were measured using a pH meter: F - 52 (manufactured by Horiba, Ltd.) and an osmometer 3250 (manufactured by Advanced Instruments).

[0039]

Table 1

[0040] The analysis results of the samples are shown in Figures 1 - 2. Table 2 also shows the elution time (top peak time) and full width at half maximum of the HbA0 peak when each Eluent C was used. When an Eluent C with a TES concentration of less than 25 mM and a difference between the pH and the pKa of TES within 0.1 was used, clear peaks of HbA0 were obtained by chromatography for any model, and furthermore, the differences in the peak rise time, top peak time, and peak width due to the model became smaller.

[0041]

Table 2

Claims

1. A method for hemoglobin analysis, comprising: eluting a fraction containing hemoglobin A0 by ion exchange chromatography using an eluent containing a buffer at a concentration of less than 25 mM, wherein the buffer is TES, the pH of the eluent is within ±0.1 of the pKa of the buffer; before eluting the fraction containing hemoglobin A0, eluting a fraction containing hemoglobin A1c using eluent A, where eluent A is a buffer having a pH of 4.0 to 6.8; passing eluent B through after the eluent containing the buffer at a concentration of less than 25 mM, where eluent B is a buffer having a pH of 6.0 to 10.0; A method comprising the above.

2. The method according to claim 1, wherein the osmotic pressure of the eluent containing the buffer at a concentration of less than 25 mM is 60 mOsm / kg or less.

3. The method according to claim 1 or 2, wherein the switching between eluent A and the eluent containing the buffer at a concentration of less than 25 mM is performed by a stepwise gradient.

4. The method according to any one of claims 1 to 3, wherein the switching between the eluent containing the buffer at a concentration of less than 25 mM and eluent B is performed by a stepwise gradient.

5. The method according to any one of claims 1 to 4, wherein the ion exchange chromatography is cation exchange chromatography.

6. The method according to any one of claims 1 to 5, wherein the chromatography is high performance liquid chromatography.

7. An eluent for separating hemoglobin A0, which contains a buffer at a concentration of less than 25 mM, has a pH within ±0.1 of the pKa of the buffer, and the buffer is TES, and is used after eluent A and before eluent B in ion exchange chromatography. Eluent A is a buffer solution with a pH of 4.0 to 6.

8. Eluent B is a buffer solution with a pH of 6.0 to 10.

0. Eluent.

8. The eluent according to claim 7, having an osmotic pressure of 60 mOsm / kg or less.

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