Eluent for hemoglobin f separation and diagnostic method

Abstract

An object of the present invention is to provide an eluent for suppressing bimodality of hemoglobin F which is a β - thalassemia disease marker in hemoglobin analysis in cation exchange chromatography and stably separating hemoglobin F without depending on the type of diluent, and a method for separating hemoglobin F and a method for diagnosing hemoglobin F using the eluent.SOLUTION: In the analysis of a hemoglobin sample using cation exchange chromatography, the problem is solved by an eluent having a pH of 5.0 to 6.0, which is passed when separating and quantifying hemoglobin F from the hemoglobin sample, wherein the eluent contains at least one or more pH buffers, and a relationship between the pH of the eluent and a pKa value in a range of 5.0 to 7.0 among pKa values indicated by the pH buffers is a relationship of pH ≤ pKa ≤ pH + 1.0.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an eluent for separating hemoglobin F from a sample containing hemoglobins by liquid chromatography with cation exchange chromatography, and a diagnostic method using the eluent. [Background technology]

[0002] Various hemoglobins in blood are used as diagnostic markers. For example, hemoglobin A1c, which is glycated hemoglobin, is an effective diabetes marker. Quantitation of hemoglobin A1c and diagnosis of diabetes using liquid chromatography with a cation exchange column (hereinafter referred to as cation exchange chromatography) is widely used due to its rapidity and high reliability. It is also used to diagnose hemoglobinopathies (identification of hemoglobin E, D, S, and C) in which part of the hemoglobin is mutated, and β-thalassemia (quantitation of hemoglobin F and hemoglobin A2). In β-thalassemia, the production of β-chains that make up hemoglobin decreases. In this case, the proportion of hemoglobin A0, which is composed of β-chains, decreases in the total amount of hemoglobin in the blood, while the proportions of hemoglobin F and hemoglobin A2 in the blood increase relatively. Therefore, the proportion of hemoglobin F and hemoglobin A2 in total hemoglobin is used as a diagnostic indicator (Non-Patent Document 1).

[0003] When using a diluent in a typical cation exchange chromatography analysis, it is desirable to adjust the composition of the diluted measurement sample to match the composition of the initial eluent for cation exchange chromatography to minimize the impact of the diluent on the measurement results. However, analysis of blood samples requires a hemolysis process to elute hemoglobins from red blood cells in the blood. Therefore, the diluent is usually a surfactant-containing hemolyzing agent, which is not used in the initial eluent composition for liquid chromatography, or water. Specifically, when a surfactant-containing hemolyzing agent is used as a diluent, the hemolysis process proceeds rapidly, improving the speed of diagnosis, but also promoting the degradation of hemoglobins, reducing diagnostic reliability. When water is used as a diluent, the hemolysis rate of blood is slower, but the degradation of hemoglobins is less likely to occur and the diluent is more stable than when a surfactant-containing hemolyzing agent is used, making it easier to handle. These hemolysis methods have a trade-off between their advantages and disadvantages, and are therefore used appropriately depending on the application in clinical settings.

[0004] Hemoglobin F is eluted early among hemoglobin species in cation exchange chromatography analysis, and therefore its quantitative results may be particularly affected by the diluent used to dilute the sample. Patent documents 1 to 4 report the effects of the buffer type, buffer concentration, pH, etc. of the eluent used on hemoglobin separation in cation exchange chromatography analysis of hemoglobin A1c and hemoglobin A0. Patent documents 5 and 6 also report the pH and buffer concentration of the eluent that provide good separation for hemoglobin A2 and various abnormal hemoglobins E, D, S, and C. However, there are no reports on the reagent composition of the eluent that provides good separation for hemoglobin F.

[0005] Accurate quantification of hemoglobin F requires (1) the separation and elution of hemoglobin F from hemoglobin A1b or hemoglobin A1c, which have similar ionic properties, (2) the elution of hemoglobin F so that it can be detected as a single-peak, and (3) the elution without being affected by the diluent. However, under analytical conditions with high separation performance to achieve (1), the hemoglobin F peak may separate into hemoglobin F2 and hemoglobin F1, resulting in a bimodal peak. This does not satisfy requirement (2), making accurate quantification of hemoglobin F difficult. Furthermore, hemoglobin F, which elutes early among the hemoglobin species detected by cation exchange chromatography, is particularly susceptible to the effects of diluents. Therefore, a technology to reduce the effects of diluents on measurement results is desired. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2000-055899 [Patent Document 2] Patent No. 5322736 [Patent Document 3] Patent No. 4420539 [Patent Document 4] Patent No. 2014-095638 [Patent Document 5] Patent No. 5013560 [Patent Document 6] Patent No. 6600123 [Non-patent literature]

[0007] [Non-Patent Document 1] ADStepehens et.al.,Int.Jnl.Lab.Hem.,34,14-20 (2012) [Non-patent document 2] Takekazu Horio, Nihei Yamashita, Basic Experimental Methods for Proteins and Enzymes, Nankodo (1985) Summary of the Invention [Problem to be solved by the invention]

[0008] The inventors of the present application have found that in a method for separating and quantifying hemoglobin F using cation exchange chromatography, the eluent used in conventional hemoglobin F analysis using cation exchange chromatography may cause the hemoglobin F peak to separate into hemoglobin F1 and hemoglobin F2, resulting in a bimodal peak shape, and that the method is susceptible to the influence of the type of diluent.

[0009] Therefore, the present invention provides an eluent that can elute hemoglobin F with a unimodal peak shape and can stably separate hemoglobin F regardless of the type of diluent in hemoglobin analysis using cation exchange chromatography, as well as a method for assisting in the diagnosis of β-thalassemia and diabetes by separating and quantifying hemoglobin F with higher accuracy. [Means for solving the problem]

[0010] As a result of extensive investigations, the present inventors have found that in the analysis of a sample containing hemoglobins using liquid chromatography equipped with a cation exchange column, the use of an eluent containing a buffer having an acid dissociation constant (pKa) within a certain range from the pH of the eluent enables hemoglobin F to be eluted with a unimodal peak shape and enables stable separation of hemoglobin F regardless of the type of diluent, leading to the completion of the present invention. That is, the present invention relates to the following embodiments of an eluent. [1] In the analysis of a sample containing hemoglobin using liquid chromatography equipped with a cation exchange column, an eluent having a pH of 5.0 to 6.0 that is passed through a cation exchange column when separating and quantifying hemoglobin F from a sample containing hemoglobins, the eluent contains at least one pH buffer; The pH buffer has an acid dissociation constant (pKa value) in the range of 5.0 to 7.0, The pH value of the eluent has a relationship with a pKa value of the pH buffer agent that is in the range of 5.0 to 7.0, pH≦pKa≦pH+1.0 An eluent characterized by: [2] The eluent according to [1], wherein the pH of the eluent is 5.00 to 5.64, and at least one of the pH buffering agents is succinic acid. [3] The eluent according to [2], wherein the pH of the eluent is 5.40 to 6.0, and at least one of the pH buffering agents is citric acid. [4] The eluent according to [3], wherein the pH of the eluent is 5.23 to 6.0, and at least one of the pH buffering agents is maleic acid. [5] The eluent according to any one of [4] to [4], characterized in that the eluent contains perchloric acid. [6] The eluent according to any one of [5] to [5], wherein the concentration of the pH buffer is 10 mmol / L or more and 200 mmol / L or less. [7] A method for diagnosing β-thalassemia by separating and quantifying hemoglobin F, which uses the eluent according to any one of [6] to [6]. [8] A method for diagnosing diabetes by separating and quantifying hemoglobin F, which uses the eluent according to any one of [7] to [6]. [9] A liquid chromatography device capable of separating and analyzing hemoglobin F from a sample containing hemoglobins by the method according to any one of [8] to [8]. [Effects of the Invention]

[0011] When the eluent of the present invention is used in hemoglobin analysis using cation exchange chromatography, hemoglobin F can be eluted with a unimodal peak shape, and hemoglobin F can be stably separated regardless of the type of diluent. Furthermore, hemoglobin F can be separated and quantified with higher accuracy, and information necessary for diagnosing β-thalassemia and diabetes can be provided, enabling more accurate diagnosis. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows a chromatogram in Example 1. [Figure 2] FIG. 1 shows a chromatogram in Example 2. [Figure 3] FIG. 1 shows a chromatogram in Comparative Example 1. [Figure 4] FIG. 1 shows a chromatogram in Comparative Example 2. [Figure 5] FIG. 10 is a diagram showing a chromatogram in Comparative Example 3. [Figure 6] FIG. 1 shows a chromatogram in Example 3. [Figure 7] FIG. 1 shows a chromatogram in Example 4. [Figure 8] FIG. 1 shows a chromatogram in Example 5. [Figure 9] FIG. 1 shows a chromatogram in Example 6. [Figure 10] FIG. 10 shows a chromatogram in Example 7. [Figure 11] FIG. 10 is a diagram showing a chromatogram in Comparative Example 4. [Figure 12] FIG. 10 is a diagram showing a chromatogram in Comparative Example 5. [Figure 13] FIG. 10 is a diagram showing a chromatogram in Comparative Example 6. [Figure 14] FIG. 10 is a diagram showing a chromatogram in Comparative Example 7. [Figure 15] FIG. 10 is a diagram showing a chromatogram in Comparative Example 8. [Figure 16] FIG. 10 is a diagram showing a chromatogram in Comparative Example 9. [Figure 17] FIG. 1 shows a chromatogram in Example 8. [Figure 18] FIG. 10 shows a chromatogram in Example 9. [Figure 19] FIG. 1 shows a chromatogram in Example 10. [Figure 20] FIG. 1 shows the overall chromatogram in Example 11. [Figure 21] FIG. 10 shows the overall chromatogram in Comparative Example 10. [Figure 22] FIG. 1 shows the overall chromatogram in Comparative Example 11. [Figure 23] 1 is a conceptual diagram of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below. However, the present invention can be embodied in various forms and is not limited to the following embodiments and examples.

[0014] According to one embodiment of the present disclosure, an eluent used in the analysis of a sample containing hemoglobins using liquid chromatography (cation exchange chromatography) equipped with a cation exchange column has a pH of 5.0 to 6.0 and is passed through a cation exchange column when separating hemoglobin F from a hemoglobin sample. The eluent contains at least one pH buffer, which has an acid dissociation constant (pKa value) in the range of 5.0 to 7.0, and the pH of the eluent satisfies the relationship pH≦pKa≦pH+1.0 with respect to the pKa values ​​of the pH buffer in the range of 5.0 to 7.0. From another perspective, the relationship between the pH of the eluent and the pKa values ​​of the pH buffer in the range of 5.0 to 7.0 is such that the pH of the eluent is equal to or lower than the pKa value of the pH buffer, and at least one of the pKa values ​​of the pH buffer falls within the buffer capacity range defined by the pKa values ​​in the range of 5.0 to 7.0. Those skilled in the art believe that the range of buffer capacity of a pH buffer is within 1.0 around the pKa value, and the relationship between the pH value of the eluent in the present disclosure and the pKa value of the pH buffer in the range of 5.0 to 7.0 can also be expressed as pKa-1≦pH≦pKa.

[0015] The pH buffer used in the present disclosure is not particularly limited as long as at least one of the pKa values ​​of the pH buffer is in the range of 5.0 to 7.0. A pH buffer with a pKa of 5.0 to 6.8 is preferably used, and a pH buffer with a pKa of 5.5 to 6.5 is more preferably used. In this case, there are no limitations on the type of salt used as the buffer, but carboxylic acids are preferred. Examples include formic acid, malic acid, lactic acid, tartaric acid, citric acid, acetic acid, fumaric acid, maleic acid, and succinic acid. Divalent or higher carboxylic acids are preferred, with succinic acid, citric acid, and maleic acid being particularly preferred.

[0016] When the pH buffer used in the present disclosure is succinic acid (pKa=4.21, 5.64), the pH of the eluent may be 5.00 to 5.64. More preferably, the pH of the eluent is in the range of 5.30 to 5.64, and particularly preferably, the pH of the eluent is 5.30.

[0017] When the pH buffer used in the present disclosure is citric acid (pKa=3.13, 4.76, 6.40), the pH of the eluent may be 5.40 to 6.0, more preferably 5.40 to 5.80, and particularly preferably 5.55.

[0018] When the pH buffer used in the present disclosure is maleic acid (pKa=1.92, 6.23), the pH of the eluent may be 5.23 to 6.0, more preferably 5.30 to 5.80, and particularly preferably 5.30 or 5.55.

[0019] The concentration of the pH buffer used in the eluent of the present disclosure is not particularly limited as long as it is within a range that allows hemoglobin separation. However, if the pH buffer concentration exceeds 200 mmol / L, components other than hemoglobin F, which elutes early, will also be eluted, affecting the separation of hemoglobin F, so it is preferably 200 mmol / L or less. Furthermore, if the pH buffer concentration is less than 10 mmol / L, the effect of suppressing the variation in the retention time of the hemoglobin F peak cannot be obtained, so the pH buffer concentration is preferably 10 to 200 mmol / L, more preferably 10 to 30 mmol / L.

[0020] The eluent used in the present disclosure may contain a chaotropic salt to improve separation performance. Chaotropic salts are defined as salts that have a stronger salting-in effect than chloride anions in the Hofmeister series, which lists salt species according to their ease of salting out proteins. In other words, chaotropic salts reduce water-water interactions and promote protein denaturation. Examples of chaotropic salts include chlorides such as nitrate ions, chloride ions, perchlorate ions, bromide ions, iodide ions, and thiocyanate ions.

[0021] The eluent used in the present disclosure may contain a preservative, such as sodium azide or lithium azide.

[0022] In the present disclosure, liquid chromatography equipped with a cation exchange column (cation exchange chromatography) is not particularly limited as long as it is performed using a liquid chromatography system equipped with a cation exchange column commonly used for analyzing samples containing hemoglobins and a detector capable of detecting hemoglobins. Cation exchange chromatography uses a column packed with a separating agent having at least one type of cation exchange group. For example, a separating agent can be prepared by using polymer particles as a base and introducing cation exchange groups onto their surfaces. The cation exchange groups introduced onto the surface are not particularly limited, but examples include carboxy groups, sulfo groups, and phosphate groups. In the present disclosure, sulfo groups or carboxy groups are preferred as the cation exchange groups.

[0023] In the present disclosure, there is no limitation on the size of the column packed with the separating agent, and a column of an appropriate size can be selected depending on the separation conditions. In the present disclosure, there is no limitation on the material of the column, and columns made of metals such as stainless steel, glass, or resins such as PEEK can be used.

[0024] In the present disclosure, the sample containing hemoglobin may be a quality control sample or a sample for creating a calibration curve obtained by redissolving freeze-dried hemoglobin, or a blood sample directly collected from a human.

[0025] The diluent used in the present disclosure is typically water or a hemolytic agent, and may be selected depending on the purpose. The hemolytic agent may contain a surfactant, preservative, or hemoglobin stabilizer. While the type of surfactant is not particularly limited, cationic, anionic, amphoteric, or nonionic surfactants are preferred, with nonionic surfactants being preferred. Nonionic surfactants include ester, ether, and ester-ether types, with ether types such as polyoxyethylene octylphenyl ether being particularly preferred. While the type of hemoglobin stabilizer is not particularly limited in the present disclosure, a chelating agent is preferred, and sodium or potassium EDTA is particularly preferred. The type of preservative is not particularly limited. Furthermore, the surfactant is preferably used at a concentration of 0.01 to 1.00% by mass, and the pH of the hemoglobin diluent is preferably in the range of 5.5 to 9.5.

[0026] In the present disclosure, hemoglobins are a general term for hemoglobins derived from human red blood cells, and include normal hemoglobin (hemoglobin A, hemoglobin A2, hemoglobin F), abnormal hemoglobin (hemoglobin S, hemoglobin E, hemoglobin C, hemoglobin D), glycosylated hemoglobin A1c, and the like. By analyzing hemoglobins and measuring the abundance ratio of abnormal hemoglobin, the abundance ratio of glycosylated hemoglobin, and the like, specific diseases can be detected. That is, in the present disclosure, one embodiment of a sample containing hemoglobins is a sample containing hemoglobins derived from blood collected from a patient with a specific disease. Another embodiment includes a quality control sample or a sample for preparing a calibration curve, which is a mixture of various hemoglobins and is used in hemoglobin analysis.

[0027] In the present disclosure, hemoglobin F refers to one of the normal hemoglobins. Hemoglobin F exists as hemoglobin F1 and hemoglobin F2, and when separated by cation exchange chromatography, the peaks of hemoglobin F1 and hemoglobin F2 may separate, resulting in a bimodal peak shape. In current clinical medicine, it is considered that there is no clinical significance in quantifying hemoglobin F1 and hemoglobin F2 individually, and it is desirable to quantify them as hemoglobin F. In the present disclosure, one aspect of evaluating the peak shape of hemoglobin F may be to output a chromatogram and confirm the peak shape to determine whether the peaks of hemoglobin F1 and hemoglobin F2 have separated and resulted in a bimodal peak shape.

[0028] The method of the present disclosure can assist in the diagnosis of β-thalassemia or diabetes by separating and quantifying hemoglobin F by cation exchange chromatography. Thus, one aspect of the present disclosure is a method for assisting in the diagnosis of β-thalassemia, using an eluent capable of separating and quantifying hemoglobin F in the analysis of a sample containing hemoglobins using cation exchange chromatography. Another aspect of the present disclosure is a method for assisting in the diagnosis of diabetes, using an eluent capable of separating and quantifying hemoglobin F in the analysis of a sample containing hemoglobins using cation exchange chromatography.

[0029] In the present disclosure, "being able to stably separate hemoglobin F regardless of the diluent" may be evaluated as the variability (RT variability) of the elution time of hemoglobin F when a sample hemolyzed with a surfactant-containing hemolyzing agent is analyzed compared with the elution time of hemoglobin F when a sample hemolyzed with water is analyzed. The RT variability can be calculated as follows. When the RT variability is small, it can be evaluated that hemoglobin F can be stably separated regardless of the diluent. RT fluctuation rate = (1 - RT after hemolysis with hemolytic agent / RT after hemolysis with water) x 100 [Example]

[0030] The present invention will be specifically explained with reference to examples and comparative examples, but the present invention is not limited to these.

[0031] The filler prepared as follows was used in the investigations shown in Table 1. A mixture of 350 g of glycidyl methacrylate, 70 g of ethylene glycol dimethacrylate, 30 g of methyl methacrylate, and 10 g of t-butyl perpivalate was added to a solution of 40 g of sodium sulfate and 200 g of polyvinyl alcohol, and the mixture was stirred. A crosslinking polymerization reaction was carried out at a reaction temperature of 60°C for 12 hours to obtain non-porous crosslinked polymer particles with a particle diameter of 3.2 μm.

[0032] 20 g of crosslinked polymer particles and 20 g of 1,4-cyclohexanedimethanol were dispersed in 200 g of 1,4-dioxane, and then 1 mL of boron trifluoride diethyl etherate was added and reacted for 16 hours at 70° C. After the reaction, the gel was washed with 1,4-dioxane and water and air-dried.

[0033] 15 g of the dried gel was dispersed in a 42% aqueous solution of sodium hydroxide. Allyl glycidyl ether was added dropwise over 1 hour while maintaining the liquid temperature at 40°C. After stirring for 16 hours after the addition, the resulting polymer particles were washed again with water, dispersed in an aqueous solution of sodium sulfite, and reacted for 16 hours while blowing in oxygen gas.

[0034] The resulting liquid chromatography packing material was packed into a column having an inner diameter of 4.6 cm and a length of 2.0 cm to prepare a column for hemoglobin measurement. Flow rate: 2.2mL / min Monitor wavelength: 415nm Device: HLC-723 G11 (Tosoh Corporation) Eluent: Buffers listed in Table 1 Blood samples containing hemoglobin F were measured under the above conditions.

[0035] The eluent systems used are as shown in Table 1 below. A 100 mmol / L aqueous solution of Triton X-100 (Kishida Chemical) was used as the hemolyzing agent. In each Comparative Example and each Example, the buffer concentration was based on 20 mmol / L, and the perchloric acid concentration of the eluent was adjusted so that the retention time of the hemoglobin F peak was within 1 minute. When the pH was high, as in Comparative Example 2, the hemoglobin elution power of the eluent was too strong, so the buffer concentration was reduced to 5 mmol / L. In Comparative Example 3 and Examples 3 to 5, the pH was fixed at 5.30 and the succinic acid concentration was investigated. The retention times (RT) and peak shapes of the hemoglobin F peaks are shown in Table 2. The RT variation was calculated as follows: RT fluctuation rate = (1 - RT after hemolysis with hemolytic agent / RT after hemolysis with water) x 100

[0036] [Table 1]

[0037] [Table 2]

[0038] The solid lines in Figures 1 to 22 represent chromatograms obtained using water as the diluent, and the dotted lines represent chromatograms obtained using hemolyzed lecithin as the diluent. Figure 23 shows the relationship between the pH of the eluent and the pKa of the buffer on separation. When a buffer with a pKa lower than the pH by 1 or more is used, as in Region 1, the shape of the hemoglobin F peak deteriorates and the diluent significantly influences the retention time of the hemoglobin F peak. When a buffer with a pKa within pH -1 is used, as in Region 2, the peak shape is good, but the retention time fluctuation of the hemoglobin F peak cannot be suppressed. The buffer in Region 3 discovered in this invention was able to suppress the influence of the diluent type and achieve elution with a good peak shape. When a buffer in Region 4, with a pKa higher than the pH by 1 or more, is used, the hemoglobin F peak shape is fine, but the influence of the diluent type cannot be eliminated. The classification in Figure 23 was confirmed by the following experiment.

[0039] In the case of Examples 1 to 7, in which a buffer with a pKa higher than the pH of the eluent was used and falling within region 3 of Figure 23, both the separation and shape of the hemoglobin F peak were good. However, in the case of Comparative Examples 1 to 9, in which the eluent or buffer concentration outside region 3 of Figure 23 was too low, the retention time of the hemoglobin F peak was sometimes affected by the diluent, and the hemoglobin F peak sometimes became bimodal. Details are explained below.

[0040] In Examples 1 and 2, succinic acid, which is commonly used to separate glycated hemoglobin HbA1c, was used as a buffer. Because an eluent with a pH lower than the pKa of succinic acid was used, the retention time of the hemoglobin F peak did not change even when the diluent was changed, and the peak shape was extremely good. In contrast, in Comparative Example 1, the pH of the elution system was higher than the pKa of succinic acid, so the effects of the present invention were not achieved. Although the retention time variation of the hemoglobin F peak due to the diluent could not be suppressed, the hemoglobin F peak was eluted as a single peak because it fell within region 2 of Figure 23. In the eluent of Comparative Example 2, where the pKa of the buffer is 1.0 or more lower than the pH, which falls within region 1 of Figure 23, the retention time variation of the hemoglobin F peak due to the diluent was large, and the hemoglobin F peak was eluted as two broadened peaks.

[0041] In the present invention, it is necessary to use an appropriate buffer concentration. Even when a buffer having a pKa in Region 3 relative to the pH of the eluent is used, if the buffer concentration is too low as in Comparative Example 3, the effect of suppressing the variation in the retention time of the hemoglobin F peak cannot be obtained. However, if a buffer with a concentration of more than 10 mmol / L is used as in Examples 3 to 5, the effects of the present invention can be obtained.

[0042] In the present invention, buffers other than succinic acid can be used as long as they are in region 3. As in Examples 6 and 7, good results were also obtained when buffers such as citric acid and maleic acid, which have pKa values ​​that fall within region 3 in Figure 23 relative to the pH of the eluent used, were used.

[0043] Even when a typical buffer other than succinic acid was used as in Comparative Examples 4 to 9, the same results as in Comparative Examples 1 and 2 were obtained when an eluent outside of Region 3 was used. Comparative Examples 4, 6, and 8, which used a buffer having a pKa within −1.0 of the pH of the eluent, such as Region 2 in FIG. 23, produced a good shape of the hemoglobin F peak, but were unable to suppress the retention time variation of the hemoglobin F peak due to differences in the diluent type. Comparative Example 5, which used phosphoric acid, which does not have a pKa that falls within Region 3 in FIG. 23, was also unable to suppress the retention time variation of the hemoglobin F peak. The eluents of Comparative Examples 7 and 9, which fall within Region 1 in FIG. 23, were unable to suppress the retention time variation of the hemoglobin F peak, resulting in poor peak shape.

[0044] The present invention can be applied regardless of the type of packing material or apparatus. Table 3 lists the packing material and apparatus conditions, and Table 4 lists the results of actual measurements. As in Example 8, good separation characteristics were observed even when the column used was changed to a TSKgel SP-NPR (inner diameter 4.6 cm, length 2.0 cm) manufactured by Tosoh Corporation, under the same conditions as in Example 2, using the same apparatus and eluent. Even when a different apparatus was used as in Examples 10 and 11, the hemoglobin F peak could be separated and analyzed without fluctuations in retention time or abnormal peak shape, provided the same eluent as in Example 2 was used.

[0045] [Table 3]

[0046] [Table 4]

[0047] The use of the eluent of the present invention is useful for diagnosing β-thalassemia. Blood samples from patients with β-thalassemia were measured using different eluents and gradient conditions as shown in Table 1. Step gradient: Eluent A: Eluent listed in Table 5 (0.0-1.0 min) Eluent B: Eluent listed in Table 5 (20mmol / L, pH5.6) (1.0-3.0min) Eluent C: Phosphate buffer (200 mmol / L, pH 6.0) (3.0-5.0 min) The measurement conditions are as described above.

[0048] As in Example 11, the use of the eluent of the present invention enabled simultaneous analysis of hemoglobin F and hemoglobin A2. When a buffer with a pKa outside the claimed range was used as in Comparative Example 10, there was no problem with the separation of hemoglobin A2, but stable measurement of hemoglobin F was not possible. When solution A with a low buffer concentration was used as in Comparative Example 11, the separation of not only hemoglobin F but also hemoglobin A2 was affected, and the separation of hemoglobin A2 and hemoglobin A0 was insufficient.

[0049] [Table 5]

Claims

1. In the analysis of a sample containing hemoglobins using liquid chromatography equipped with a cation exchange column, an eluent having a pH of 5.0 to 6.0 which is passed through a cation exchange column when separating and quantifying hemoglobin F from a sample containing hemoglobins, the eluent contains at least one pH buffer; The pH buffer is a pH buffer having an acid dissociation constant (pKa value) in the range of 5.0 to 7.0, The relationship between the pH value of the eluent and the pKa value of the pH buffer agent, which is in the range of 5.0 to 7.0, is pH≦pKa≦pH+1.0 An eluent characterized by:

2. 2. The eluent according to claim 1, wherein the pH of the eluent is 5.00 to 5.64, and at least one of the pH buffering agents is succinic acid.

3. 2. The eluent according to claim 1, wherein the eluent has a pH of 5.40 to 6.00, and at least one of the pH buffering agents is citric acid.

4. 2. The eluent according to claim 1, wherein the pH of the eluent is 5.23 to 6.00, and at least one of the pH buffering agents is maleic acid.

5. 5. The eluent according to claim 1, further comprising perchloric acid.

6. 5. The eluent according to claim 1, wherein the concentration of the pH buffer is 10 mmol / L or more and 200 mmol / L or less.

7. A method for assisting in the diagnosis of β-thalassemia by separating and quantifying hemoglobin F, using the eluent according to any one of claims 1 to 4.

8. A method for assisting in the diagnosis of diabetes by separating and quantifying hemoglobin F, using the eluent according to any one of claims 1 to 4.

9. A liquid chromatography apparatus equipped with a cation exchange chromatography column and a hemoglobin detector, capable of separating and analyzing hemoglobin F from a sample containing hemoglobins by a method using the eluent according to claim 1.

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