JP2007315816A - Method of measuring hemoglobin types - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of measuring hemoglobin types using cation exchange chromatography, especially a measuring method of hemoglobin types capable of measuring stable hemoglobin A1c with high accuracy. <P>SOLUTION: In the method of measuring hemoglobin types for measuring at least one kind of hemoglobin selected from among a group consisting of acetylated hemoglobin, carbamylated hemoglobin, unstable hemoglobin A1c, stable hemoglobin A1c, hemoglobin A, hemoglobin A2, hemoglobin S and hemoglobin C using the cation-exchange chromatography is used an elute with dissolved oxygen concentration of 2.5 mg/L or higher. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to a method for measuring hemoglobin using cation exchange liquid chromatography, and more particularly to a method for measuring hemoglobin capable of measuring stable hemoglobin A1c with high accuracy.

In the fields of organic chemistry, biochemistry, medicine, and the like, liquid chromatography is widely used as an instrument for performing operations such as separation and fractionation of components in a sample. The liquid chromatography is usually configured as shown in FIG. 32, and the eluent sent from the mobile phase tanks 31a and 31b is degassed by a degasser 32, and then the manifold. A pressure is applied by a liquid feed pump 35 through a (liquid switching device) 34, and a sample is introduced from a sample tank 38 via a sample introduction device 37, and then introduced into a separation column 40 via a filter 39. The Then, each component in the sample is separated by the separation column 40. Each separated component is detected by the detector 41, for example, by measuring the absorbance, and the result is processed by the data processing device 42 and expressed as a chromatogram. In addition, a damper 36 is installed as necessary for the purpose of constant flow rate and constant pressure of the mobile phase.

Such a measurement method using liquid chromatography is also used for measurement of glycated hemoglobin in blood, and in particular, hemoglobin A1c (hereinafter referred to as HbA1c) is an average in blood for the past 1 to 2 months. Therefore, it is widely used as a screening item for diabetes and as a test item for grasping the blood glucose control state of diabetic patients. HbA1c is glycated hemoglobin (hereinafter referred to as GHb) produced by the reaction of glucose in blood with hemoglobin A (hereinafter referred to as HbA), and the reversibly reacted product is unstable HbA1c (unstable). HbA1c), which reacts irreversibly via unstable HbA1c, is called stable HbA1c (stable HbA1c).

Usually, hemoglobin is a tetrameric protein composed of two types of subunits. The subunits of HbA are an α chain and a β chain, and HbA1c is obtained by binding glucose to the N-terminal amino acid of the β chain. Among these, stable HbA1c reflects well the average blood glucose level over the past 1 to 2 months, and in the clinical laboratory field, it is possible to obtain a stable HbA1c value (%) with high accuracy. The development of a law is desired.

Conventionally, as a main measurement method of HbA1c, for example, as disclosed in Patent Document 1, hemoglobins in a sample prepared by hemolyzing and diluting a blood sample are separated for each hemoglobin component by a cation exchange method. A method using liquid chromatography for separating by utilizing the difference between different positive charge states has been performed.

Usually, when hemoglobins in a hemolyzed sample are separated with sufficient time using cation exchange liquid chromatography, hemoglobin A1a (hereinafter referred to as HbA1a), hemoglobin A1b (hereinafter referred to as HbA1b), hemoglobin F (hereinafter referred to as “HbA1b”). HbF), unstable HbA1c, stable HbA1c, and hemoglobin A0 (hereinafter referred to as HbA0). Here, HbA1a, HbA1b and HbA1c are GHb in which HbA is glycated, HbF is fetal hemoglobin composed of α chain and γ chain, HbA0 is a group of hemoglobin components mainly composed of HbA, and HbA1c More strongly held in the column.

However, the conventional method using cation exchange liquid chromatography not only cannot sufficiently separate unstable HbA1c from stable HbA1c, but also acetylated hemoglobin (hereinafter referred to as acetylated Hb) and carbamylated hemoglobin. There was a problem that modified hemoglobin such as (hereinafter referred to as carbamylated Hb) was eluted with overlapping with stable HbA1c.
That is, for the purpose of measuring the stable HbA1c value (%) by cation exchange liquid chromatography, the stable HbA1c and the stable HbA1c are measured so as not to affect the measured value of the stable HbA1c. It was difficult to separate unstable HbA1c, acetylated Hb, and carbamylated Hb peaks with similar elution behavior from stable HbA1c peaks.

In contrast, in recent years, a technique has been developed that improves the separation performance of modified hemoglobin such as carbamylated Hb and acetylated Hb, and can measure stable HbA1c. For example, Patent Document 2 discloses a cation exchange liquid. A method for measuring hemoglobin by chromatography, comprising using an eluent containing an inorganic acid, an organic acid and / or a salt thereof containing chaotropic ions and having a buffer capacity at pH 4.0 to 6.8. Discloses a method for separating stable HbA1c.
However, such a method has a disadvantage that highly modified acetylated Hb and carbamylated Hb cannot be sufficiently separated.

Patent Document 3 defines the average particle diameter of the packing material, the column size, the flow rate of the mobile phase, and the like using an eluent containing urea, a surfactant and the like in the measurement of hemoglobins by liquid chromatography. Accordingly, a technique for improving the stable HbA1c separation performance as compared with the conventional method is disclosed. Patent Document 4 discloses a method for measuring hemoglobin by cation exchange liquid chromatography, which contains urea and the like in addition to chaotropic ions and has a buffering ability at pH 4.0 to 6.8. In addition, by using an eluent containing an organic acid and / or a salt thereof, and further defining the pH and salt concentration of the three types of eluent used for the separation of hemoglobins, more stable HbA1c separation performance is improved. Technology is disclosed.

However, the methods described in Patent Documents 3 and 4 significantly improve the separation performance of highly modified acetylated Hb and carbamylated Hb, but have the disadvantage that the stability of the eluent is significantly reduced. It was. Therefore, there has been a demand for a method for measuring hemoglobins which has high separation performance of stable HbA1c, can measure stable HbA1c with high accuracy, and has good storage stability of the eluent used for the measurement. .
Japanese Patent Publication No.8-7198 JP 2000-111539 A JP 2000-171454 A JP 2003-107069 A

In view of the above situation, the present invention provides a method for measuring hemoglobin using cation exchange liquid chromatography, and in particular, provides a method for measuring hemoglobin capable of measuring stable HbA1c with high accuracy. With the goal.

The present invention uses cation exchange liquid chromatography to acetylate Hb, carbamylated Hb, unstable HbA1c, stable HbA1c, HbA, hemoglobin A2 (hereinafter referred to as HbA2), hemoglobin S (hereinafter referred to as HbS) and A method for measuring hemoglobin for measuring at least one selected from the group consisting of hemoglobin C (hereinafter referred to as HbC), wherein an eluent having a dissolved oxygen concentration of 2.5 mg / L or more is used. This is a method for measuring hemoglobins.
The present invention is described in detail below.

As a result of intensive studies, the present inventors have conducted degassing of the eluent and reducing the amount of dissolved oxygen in conventional cation exchange liquid chromatography measurement, and modified hemoglobin such as acetylated Hb and carbamylated Hb. It was found that this is a major factor that degrades the separation performance. As a result of further earnest studies, the present inventors have made it possible to highly isolate the modified hemoglobin by setting the concentration of dissolved oxygen, which has been reduced as much as possible, to a predetermined value or higher, and this is a measurement target. The inventors have found that the measurement accuracy of hemoglobins is greatly improved, and have completed the present invention.

In the method for measuring hemoglobin of the present invention, an eluent having a dissolved oxygen concentration of 2.5 mg / L or more is used. In the present invention, unlike a conventional method, a special filler is not used, or a new substance is not added to the eluent, so that hemoglobins to be measured can be measured by a simple and simple method. A significant improvement in measurement accuracy can be realized. In the present invention, the dissolved oxygen concentration of the eluent at the time of measurement may be 2.5 mg / L or more, and the dissolved oxygen concentration of the eluent during storage is not particularly limited. It will not affect

In the present invention, when the dissolved oxygen concentration of the eluent is 2.5 mg / L or more, the reason why the separation performance is improved is as follows. Usually, hemoglobins contained in a specimen are mainly oxygen-bound hemoglobin (oxyhemoglobin). However, when a specimen containing hemoglobin is injected into the eluent that has been deaerated by the deaerator and the dissolved oxygen concentration has decreased, a portion of the oxygen-bound oxyhemoglobin is gradually deoxygenated. It is thought to change to (deoxyhemoglobin). Thereby, since the hemoglobin (for example, carbamylated Hb) of the same component is in a mixed state of oxyhemoglobin and deoxyhemoglobin, it is considered that the separation performance deteriorates.
On the other hand, when the dissolved oxygen concentration of the eluent is 2.5 mg / L or more as in the present invention, deoxygenation hardly occurs and the ratio of oxyhemoglobin to deoxyhemoglobin becomes very high, so that the separation performance is high. It is thought to improve.

If the dissolved oxygen concentration is less than 2.5 mg / L, highly modified acetylated Hb and carbamylated Hb cannot be sufficiently separated, and the measurement accuracy of hemoglobins to be measured is lowered. In addition, although the upper limit of dissolved oxygen concentration is not specifically limited, It is preferable to set it as below saturated oxygen concentration. If the saturated dissolved oxygen concentration is exceeded and the state is supersaturated, bubbles are likely to be generated in the eluent during measurement, and the amount of eluent delivered becomes unstable due to the bubbles entering the liquid feed pump. The accuracy of hemoglobin measurement may be reduced.
The preferred lower limit of the dissolved oxygen concentration is 3.5 mg / L, the preferred upper limit is saturated dissolved oxygen concentration -0.5 mg / L; the more preferred lower limit is 4.5 mg / L, and the more preferred upper limit is saturated dissolved oxygen concentration -1.0 mg. / L; A more preferred lower limit is 5.5 mg / L, and a more preferred upper limit is a saturated dissolved oxygen concentration of -1.5 mg / L.

Examples of a method for setting the dissolved oxygen concentration of the eluent to 2.5 mg / L or more include, for example, a method of adjusting the deaeration capability of the deaerator; a pipe having a known oxygen permeability from the deaerator to the column A method of adjusting the length and thickness of the pipe using a method; a method of increasing the saturated dissolved oxygen concentration in the eluent by lowering the temperature of the eluent; an eluent, a deaerator, a pipe having oxygen permeability, A method of increasing the saturated dissolved oxygen concentration by lowering the temperature of a part of the measurement system including the liquid pump, sample injector, column, and detector; a method of measuring hemoglobin without using a deaerator Can be used. In addition, these methods may be used independently and may use several methods together. Specifically, a method of adjusting the length of the tube 33 between the deaerator 32 and the liquid switching electromagnetic valve 34 shown in FIG. 32 can be used. In this method, by using a deaeration device, it is possible to increase the amount of dissolved oxygen while suppressing the generation of bubbles in the eluent.

The dissolved oxygen concentration can be measured by using a known dissolved oxygen meter, but in order to measure with higher accuracy, it is necessary to use a closed system so that oxygen in the atmosphere does not dissolve in the measurement target. is there. Therefore, as the dissolved oxygen meter, it is preferable to use a flow type dissolved oxygen meter capable of measuring the dissolved oxygen concentration while flowing the eluent. Moreover, it is preferable to use a pipe made of stainless steel or PEEK having a good oxygen barrier property as a pipe material for feeding the eluent.

An example of the flow type dissolved oxygen meter described above is shown in FIG. As shown in FIG. 30, the transferred mobile phase (eluent) is introduced into the flow type dissolved oxygen meter 10 through a pipe 7 made of polyetheretherketone (PEEK) or the like. Then, the dissolved oxygen concentration is measured by the dissolved oxygen electrode 8 for the mobile phase introduced into the flow type 9 and then led out of the dissolved oxygen meter 10 through the pipe. Although the position where the dissolved oxygen meter 10 is installed is not particularly limited, it is preferably installed after the liquid feed pump 35 shown in FIG. 32 and is preferably installed before the filter 39.

The eluent preferably contains a buffer having an acid dissociation constant (hereinafter referred to as pKa) in the range of 2.15 to 6.39 and 6.40 to 10.50.
If the pKa is outside the above range, the pH of the eluent cannot be adjusted to an appropriate range for separating the peak for measurement. As a result, hemoglobins are denatured or hemoglobins are separated. It may be difficult.
The buffer may be a single substance having at least one pKa in the range of 2.15 to 6.39 and 6.40 to 10.50, and may be 2.15 to 6.39. In combination, a substance having at least one pKa in the range of 1.40 and a substance having at least one pKa in the range of 6.40 to 10.50 may be used as a buffering agent. Moreover, you may use combining the said buffering agent in multiple numbers.

The pKa range of the above-mentioned buffering agent is 2.61 to 6.39 and 6.40 to be able to exhibit a better buffering capacity in the vicinity of the pH of the eluent appropriate for separating the peak for measurement. It is preferably in the range of 10.50, more preferably in the range of 2.80 to 6.35 and 6.80 to 10.00. More preferably, it exists in the range of 3.50-6.25 and 7.00-9.50.

Examples of the buffer include inorganic substances such as phosphoric acid, boric acid, and carbonic acid, carboxylic acid, dicarboxylic acid, carboxylic acid derivatives, hydroxycarboxylic acid, aniline or aniline derivatives, amino acids, amines, imidazoles, and alcohols. Organic substances such as In addition, ethylenediaminetetraacetic acid, pyrophosphoric acid, pyridine, cacodylic acid, glycerol phosphoric acid, 2,4,6-collidine, N-ethylmorpholine, morpholine, 4-aminopyridine, ammonia, ephedrine, hydroxyproline, peridine, tris (hydroxy Organic substances such as methyl) aminomethane and glycylglycine may be used.

Examples of the carboxylic acid include formic acid, acetic acid, propionic acid, benzoic acid and the like. Examples of the dicarboxylic acid include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, phthalic acid, and fumaric acid.

Examples of the carboxylic acid derivative include β, β′-dimethylglutaric acid, barbituric acid, 5,5-diethylbarbituric acid, γ-aminobutyric acid, pyruvic acid, furancarboxylic acid, and ε-aminocaproic acid. It is done.

Examples of the hydroxycarboxylic acid include tartaric acid, citric acid, lactic acid, malic acid and the like. Examples of the aniline or aniline derivative include aniline and dimethylaniline. Examples of the amino acid include aspartic acid, asparagine, glycine, α-alanine, β-alanine, histidine, serine, leucine and the like.

Examples of the amines include ethylenediamine, ethanolamine, trimethylamine, and diethanolamine. Examples of the imidazoles include imidazole, 5 (4) -hydroxyimidazole, 5 (4) -methylimidazole, and 2,5 (4) -dimethylimidazole.

Examples of the alcohols include 2-amino-2-methyl-1,3-propanediol, 2-amino-2-ethyl-1,3-propanediol, and 2-amino-2-methyl-1-propanol. Is mentioned.

Examples of the buffer include 2- (N-morpholino) ethanesulfonic acid (MES), bis (2-hydroxyethyl) iminotris- (hydroxymethyl) methane (Bistris), N- (2-acetamido) imidodiacetic acid ( ADA), piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), 1,3-bis (tris (hydroxymethyl) -methylamino) propane (Bistrispropane), N- (acetamido) -2- Aminoethanesulfonic acid (ACES), 3- (N-morpholine) propanesulfonic acid (MOPS), N, N′-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), N-tris ( Hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), N-2-hydroxy Tilpiperazine-N′-ethanesulfonic acid (HEPES), N-2-hydroxyethylpiperazine-N′-propanesulfonic acid (HEPPS), N-tris (hydroxymethyl) methylglycine (Tricine), tris (hydroxymethyl) aminoethane (Tris), N, N′-bis (2-hydroxyethyl) glycine (Bicine), glycylglycine, N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), glycine, cyclohexylaminopropanesulfone Substances that make up what is commonly referred to as Good's buffer, such as acids (CAPS), can also be used. Tables 1 and 2 show the pKa values of these substances. (Cited by Takeo Horio and Nihei Yamashita, basic experiment method for proteins and enzymes, Nanedo)

The content of the buffering agent in the eluent is not particularly limited as long as it has a buffering effect, and the preferred lower limit is 1 mM and the preferred upper limit is 1000 mM. A more preferable lower limit is 10 mM, and a more preferable upper limit is 500 mM. Moreover, the said buffer may be used individually or in mixture of multiple, for example, you may mix and use an organic substance and an inorganic substance.

The eluent may contain inorganic salts such as sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, and sodium phosphate for the purpose of optimizing the peak elution of hemoglobins. In addition, although the density | concentration of these inorganic salts is not specifically limited, A preferable minimum is 1 mM and a preferable upper limit is 1500 mM.

The eluent may contain a known acid or base as a pH adjuster. Examples of the acid include hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, and the like. Examples of the base include sodium hydroxide, potassium hydroxide, lithium hydroxide, magnesium hydroxide, barium hydroxide, calcium hydroxide. Etc. The contents of these acids and bases are not particularly limited, but the preferred lower limit is 0.001 mM and the preferred upper limit is 500 mM.

The eluent may contain a water-soluble organic solvent such as methanol, ethanol, acetonitrile, or acetone. The concentration of the organic solvent is preferably used so that salts and the like do not precipitate, and the preferable upper limit is 80% (v / v).

The eluent may contain a preservative such as sodium azide and thymol. As a stabilizer for hemoglobin, a known stabilizer such as a chelating agent such as ethylenediaminetetraacetic acid (EDTA), glutathione, You may contain reducing agents, antioxidants, etc., such as sodium azide.

The eluent used in the present invention preferably contains chaotropic ions. The chaotropic ion is an ion generated by dissociation when a compound is dissolved in an aqueous solution, which destroys the structure of water and suppresses the decrease in entropy of water that occurs when the hydrophobic substance and water come into contact with each other. .

The chaotropic ions include anionic and cationic chaotropic ions, and the anionic chaotropic ions include tribromoacetate ion, trichloroacetate ion, thiocyanate ion, iodide ion, perchlorate ion, dichloroacetate ion. Nitrate ions, bromide ions, chloride ions, acetate ions, and the like. Examples of cationic chaotropic ions include barium ions, calcium ions, lithium ions, cesium ions, potassium ions, magnesium ions, and guanidine ions. .

Among the chaotropic ions, it is preferable to use tribromoacetate ion, trichloroacetate ion, thiocyanate ion, iodide ion, perchlorate ion, dichloroacetate ion, nitrate ion, bromide ion, etc. As the cation chaotropic ions, barium ions, calcium ions, magnesium ions, lithium ions, cesium ions, guanidine ions and the like are preferably used. More preferably, thiocyanate ion, perchlorate ion, nitrate ion, guanidine ion or the like is used.

The preferable lower limit of the content of chaotropic ions in the eluent is 0.1 mM, and the preferable upper limit is 3000 mM. If it is less than 0.1 mM, the separation effect may be reduced in the measurement of hemoglobin, and even if it is added in excess of 3000 mM, the separation effect of hemoglobin is not further improved. A more preferred lower limit is 1 mM, a more preferred upper limit is 1000 mM, a still more preferred lower limit is 10 mM, and a still more preferred upper limit is 500 mM.

In the present invention, when measuring hemoglobin, it is preferable to use at least two kinds of eluents having different pHs. In that case, it is preferable to use an eluent containing the same buffer as the eluent used for separating the peak for measurement. However, the baseline fluctuation of the detector output when the eluent is switched becomes the measured value. It is not necessarily limited to this as long as it does not have an adverse effect. The pH of the eluent can be adjusted by, for example, the amount of pH adjuster added.
Further, in order to further reduce the baseline fluctuation, it is more preferable to use an eluent having the same buffer concentration as the eluent used for separating the peak for measurement.

In the present invention, the method for feeding the eluent is not particularly limited. However, when two or more types of eluents having different pH are used, at least three types of eluents having different elution powers are used to elute HbA0. For the purpose of separating each hemoglobin component eluted before HbA0 before sending the eluent for elution, a method for sending an eluent other than the eluent for eluting the HbA0 is performed. It is preferable.
In the measurement of normal hemoglobins, hemoglobins to be measured include HbA1a, HbA1b, HbF, unstable HbA1c, stable HbA1c, HbA0, etc., of which they are eluted before HbA0. Each hemoglobin component refers to HbA1a, HbA1b, HbF, unstable HbA1c, and stable HbA1c.

Further, when measuring a blood sample containing a hemoglobin component such as HbA2, HbS, HbC and the like that may adversely affect the measurement of stable HbA1c, HbA2 is mainly eluted as HbA0 component (peak), and then HbA2 It is preferable to elute HbS, HbC and the like. Thereby, since hemoglobin components other than HbA can be removed from the HbA0 peak, a more accurate stable HbA1c (%) can be calculated.
In addition, it is necessary to use an eluent having a stronger dissolution power as an eluent used for eluting HbA2, HbS, HbC, etc. after feeding an eluent for eluting HbA0.

Further, in the method for measuring hemoglobins of the present invention, it is preferable that the eluent is sent by a gradient elution method or a step elution method, and an eluent having a low dissolution power is sent in the middle.

In conventional hemoglobin measurement methods, a gradient elution method using a plurality of eluents is used to sharpen the peaks of components to be separated or to improve the resolution of two or more peaks that elute next to each other. The step elution method was used.

The gradient elution method is a method called “gradient elution”. For example, the method uses a plurality of liquid feeding pumps and continuously feeds the liquid by changing the liquid feeding ratio of a plurality of eluents having different melt outputs. It is. Thereby, as shown in FIG. 27, elution is performed so that the melting power continuously increases with time.

The step elution method is called “stepwise elution”. For example, one elution pump is connected to a plurality of eluents via a solenoid valve or the like, and the elution is performed by switching the solenoid valves. This is a method of switching from an eluent having a low force to an eluent having a high dissolution power and feeding it. Therefore, as shown in FIG. 28, the melting power increases stepwise.

However, in the conventional gradient elution method and step elution method, if the properties of each eluted component are similar or if it is required to elute in a short time, peaks overlap between components of similar properties, There was a risk that the degree of separation would decrease.

On the other hand, when the eluent is sent by the gradient elution method or the step elution method, in the middle, that is, while the plural eluents are sequentially switched and sent by the gradient elution method or the step elution method, By sending an eluent having a low melt output, the separation target peak or the separation state between peaks can be improved. Specifically, in the case of the step elution method, after switching from an eluent having a weak dissolution power to an eluent having a high dissolution power, switching to an eluent having a weak dissolution power, and after a while, eluting with a strong dissolution power. For example, a method of switching to a liquid and feeding it.

A configuration example of the apparatus when the method of the present invention is performed by the step elution method is shown in FIG. A, B, C, and D are eluents having different elution powers (for example, different in salt concentration, pH, polarity, etc.), and are configured to be switched to each eluent at a set time by the solenoid valve 1. Yes. The eluent is introduced to the column 4 by the liquid feed pump 2 together with the sample introduced from the sample injection unit 3, and each component is detected by the detector 5. The area, height, etc. of each peak are calculated by the integrator 6.

In the present invention, cation exchange liquid chromatography is used for measuring hemoglobins. The packing material of the cation exchange liquid chromatography is composed of particles having at least one kind of cation exchange group, and can be obtained, for example, by introducing a cation exchange group into polymer particles. .

A well-known thing can be used as said cation exchange group, For example, cation exchange groups, such as a carboxyl group, a sulfonic acid group, and a phosphoric acid group, etc. are mentioned. A plurality of the cation exchange groups may be introduced.

The preferable lower limit of the diameter of the particles used as the filler is 0.5 μm, the preferable upper limit is 20 μm, the more preferable lower limit is 1 μm, and the more preferable upper limit is 10 μm.
Further, the preferable upper limit of the coefficient of variation (CV value) indicating the particle size distribution of the particles is 40%, and the more preferable upper limit is 30%. The coefficient of variation value (CV value) can be obtained from the standard deviation of particle diameter ÷ average diameter × 100.

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.
In the polymer particles, the constituent components other than the cation exchange group to be introduced are preferably more hydrophilic. Further, those having a high degree of crosslinking are preferred from the viewpoint of pressure resistance and swelling resistance.

The introduction of the cation exchange group into the polymer particle can be carried out by a known method. For example, after preparing the polymer particle, the functional group of the particle (hydroxyl group, amino group, carboxyl group, epoxy group, etc.) Further, it can be carried out by a method of introducing a cation exchange group into the particles by a chemical reaction.

Further, filler particles having a cation exchange group can also be prepared by a method of preparing a polymer particle by polymerizing a monomer having a cation exchange group. For example, the method etc. which mix a cation exchange group containing monomer, a crosslinkable monomer, etc., and superpose | polymerize in presence of a polymerization initiator etc. are mentioned.

Furthermore, after mixing a polymerizable cation exchange group-containing ester such as methyl (meth) acrylate and ethyl (meth) acrylate with a crosslinkable monomer and the like and polymerizing in the presence of a polymerization initiator, You may prepare the filler particle | grains which have a cation exchange group by the method of hydrolyzing and converting ester into a cation exchange group.
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 placed near the surface of the polymer particles. Filler particles having a cation exchange group may be prepared by a polymerization method.

In the present invention, the above packing material is packed into a column and used. The material of the column is not particularly limited, and examples thereof include known stainless steel, glass, and resin.
The column size is preferably 0.1 to 50 mm in inner diameter and 1 to 300 mm in length, and more preferably 0.2 to 30 mm in inner diameter and 5 to 200 mm in length.

The method for filling the column into the column is not particularly limited, and a known method can be used, but the slurry filling method is particularly preferable. Specifically, for example, a method in which a slurry in which filler particles are dispersed in a buffer solution such as an eluent is press-fitted into a column by a liquid feed pump or the like can be used.

As the cation exchange liquid chromatography device used in the present invention, known devices can be used. For example, as shown in FIG. 32, an eluent tank 31, a degassing device 32, a liquid switching electromagnetic valve. 34, a liquid feed pump 35, a damper 36, a sample introduction device 37, a specimen tank 38, a filter 39, a column 40, a detector 41, and a data processing device 42 can be used. In addition, other attached devices such as a column thermostat may be added as appropriate. The liquid switching electromagnetic valve 34 may be a manifold system using an electromagnetic valve or a known liquid switching apparatus such as a rotary valve system.

As the degassing device 32, any known degassing device can be used as long as it can be set within a range of dissolved oxygen concentration capable of improving the separation performance of hemoglobins.
An example of the deaeration device used in the present invention is shown in FIG. The deaerator 20 has a configuration in which a gas-liquid separation film 22 is introduced into a vacuum chamber 23 that can be evacuated by a vacuum pump 21, and a mobile phase (eluent) that has entered the vacuum chamber 23. ) Is configured such that only the gas is degassed by the gas-liquid separation membrane 22.

The filter 39 is preferably a known filter material that does not affect the measurement values of the measurement components. Specifically, for example, a filter in which a nonwoven fabric and a filter paper are laminated, a filter in which a nonwoven fabric and a membrane filter are laminated, a filter in which a sintered filter is treated with a surface treatment agent (silicone treatment, blocking agent), or the like is used. it can.

As the material of the filter, a material with little adsorption of the measurement component is preferable. For example, as the material of the nonwoven fabric, polyethylene, polypropylene, polyester, polyethylene terephthalate, nylon, rayon, acrylic, coconut fiber, vinylidene chloride, cotton, wool, Examples include hemp and glass fiber.
Further, if necessary, in order to reduce the adsorption of biological sample components, the surface of the non-woven fabric is at least a cellulose resin, a fluorine resin, a sulfone resin, polyethylene, a polymer of an ethylene alkyl derivative, an acrylic resin. , Nylon, carbide ceramic, nitride ceramic, silicide ceramic, boride ceramic, at least one selected from the group consisting of silylated silicon dioxide, glass and titanium on the surface, or a combination of these May be treated with different materials.

The preferable lower limit of the thickness of the nonwoven fabric is 0.1 mm, and the preferable upper limit is 10 mm. The preferable lower limit of the porosity of the nonwoven fabric is 40%, and the preferable upper limit is 90%.

As the filter paper, known materials can be used, and the material is not particularly limited, but materials made of cellulose, glass fiber, fluororesin, silica fiber, and the like can be used. Moreover, in order to implement | achieve the intensity | strength improvement of a filter paper, adsorption | suction suppression of a biological sample, etc., what gave the special process to the well-known filter paper can be used. Specific examples include filter paper with improved wet strength (manufactured by Advantech Toyo Co., Ltd., wet strength filter paper). The retained particle diameter of the filter paper is preferably 3 μm or less in order to remove foreign substances in the biological sample.

In order to increase the effective filtration area of the nonwoven fabric and the filter paper, the filter is configured in the order of “support, nonwoven fabric, filter paper, support” or “nonwoven fabric, filter paper, support” from the side away from the column. be able to. Although the shape of the said support body will not be specifically limited if the effective filtration area of a nonwoven fabric and a filter paper can be enlarged, A mesh-shaped thing is preferable. Examples of the material for the support include polypropylene, polyethylene, nylon, polyethylene terephthalate, and metal mesh (stainless steel, titanium).

The filter may be silicone-coated as necessary, and is preferably subjected to a blocking treatment with a blocking reagent. Examples of the blocking reagent include proteins such as bovine serum albumin, casein, gelatin, hemoglobin, and myoglobin; polar lipids such as phospholipids; and SDS, polyethylene glycol mono-4-octylphenyl ether (Triton X-100). ) And the like.

Other measurement conditions in the measurement method of the present invention can be appropriately selected depending on the type of measurement sample, column, etc. to be used. The flow rate of the eluent is preferably 0.05 to 5 mL / min, more preferably 0.2. ~ 3 mL / min. The detection of hemoglobin is preferably visible light of 415 nm, but is not limited to this. As the measurement sample, a solution obtained by diluting hemolyzed blood with a solution containing a substance having hemolytic activity such as a surfactant is usually used. The sample injection amount varies depending on the dilution rate of the blood sample, but is preferably about 0.1 to 100 μL.

According to the present invention, modified hemoglobin and the like are highly separated by setting the concentration of dissolved oxygen to a predetermined value or higher in the eluent used for measuring hemoglobin using cation exchange liquid chromatography. Therefore, it is possible to provide a method for measuring hemoglobin capable of significantly improving the measurement accuracy of hemoglobin as a measurement target.

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

Example 1
(1) Preparation of filler 1.5 g of benzoyl peroxide (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in a mixture of 400 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and 150 g of 2-acrylamido-2-methylpropanesulfonic acid. did. This was dispersed in 2500 mL of a 4% by weight polyvinyl alcohol (manufactured by Nippon Synthetic Chemical), heated to 75 ° C. in a nitrogen atmosphere with stirring, and polymerized for 8 hours. After polymerization, washing and drying, classification was performed to obtain particles having an average particle diameter of 6 μm.

(2) Packing of packing material into column 0.7 g of the obtained particles were dispersed in 30 mL of 50 mM phosphate buffer (pH 5.8), subjected to ultrasonic treatment for 5 minutes, and then well stirred. The whole amount was injected into a packer (Umeya Seiki Co., Ltd.) connected with a stainless steel empty column (inner diameter 4.6 × 30 mm). Next, a liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer and filled at a constant pressure of 30 MPa.

(3) Preparation of eluents A and B Eluent A was prepared by adding 60 mM sodium perchlorate to a 50 mM phosphate buffer and adjusting the pH to 5.3. Further, 300 mM sodium perchlorate was added to 80 mM phosphate buffer, and the pH was adjusted to 8.0 to obtain Eluent B.

(4) Preparation of measurement samples The following samples were prepared from whole blood samples obtained by collecting sodium fluoride from healthy human blood. As a hemolysis reagent, a phosphate buffer solution (pH 7.0) containing 0.1 wt% polyethylene glycol mono-4-octylphenyl ether (Triton X-100) (manufactured by Tokyo Chemical Industry Co., Ltd.) was used.
(A) Glucose-loaded blood: A 20 g / L glucose aqueous solution was added to a whole blood sample, reacted at 37 ° C. for 3 hours, then hemolyzed with the above hemolytic reagent, and diluted 90 times to give sample a.
(B) Carbamylated Hb-containing sample: To 15 mL of a whole blood sample, 1 mL of a physiological saline solution of 7.5 g / L sodium cyanate was added, allowed to react at 37 ° C. for 2 hours, and then hemolyzed with the above hemolysis reagent. The sample was diluted to 2 times.
(C) Acetylated Hb-containing sample: To 15 mL of a whole blood sample, add 1 mL of a physiological saline solution of 7.5 g / L acetaldehyde, react at 37 ° C. for 2 hours, and then hemolyze with the above hemolysis reagent to 90 times Diluted to sample c.

(5) Measurement of hemoglobins Using the obtained columns, eluents A and B, and samples a to c, hemoglobins were measured under the following conditions. In addition, a whole blood sample obtained by collecting sodium fluoride from healthy human blood was hemolyzed with the above hemolysis reagent, and a sample diluted 90 times was measured in the same manner. The measurement was performed at room temperature (25 ° C.). The eluent was kept at 25 ° C., eluent A was sent for 0 to 2 minutes from the start of measurement, eluent B was sent for 2 to 3 minutes, and 3 to 5 Eluent A was fed for a minute.

As the measuring device, the one shown in FIG. 32 is used, the liquid feed pump is LC-9A (manufactured by Shimadzu Corporation), the autosampler is ASU-420 (manufactured by Sekisui Chemical Co., Ltd.), and the detector is SPD-6AV ( As a tube 33 for feeding between the deaerator 32 and the liquid switching solenoid valve 34, the length is 40 cm for both the eluent A and the eluent B, the outer diameter is 3 mm, the inner diameter is made by Shimadzu Corporation) A 2 mm fluororesin tube (manufactured by GL Sciences, Cat. No. 6010-35305) was used. 32, a flow type trace dissolved oxygen meter (DO-55G, manufactured by Toa DKK Corporation) as shown in FIG. 30 is installed immediately before the filter 39 after the liquid feed pump 35 in FIG. The dissolved oxygen concentration of was measured. The measurement conditions were as follows.
Measurement conditions: Flow rate: 2.0 mL / min
Detection wavelength: 415 nm
Sample injection volume: 10 μL

(Examples 2-5, Comparative Examples 1-3)
In Example 1 (5), hemoglobins were measured in the same manner as in Example 1 except that the fluororesin tube having the length shown in Table 3 was used. The fluororesin tubes were the same length for eluent A and eluent B.
The results of dissolved oxygen concentration measurement in Examples 1 to 5 and Comparative Examples 1 to 3 are shown in Table 3. As shown in Table 3, it can be seen that the length of the fluororesin tube and the magnitude of the dissolved oxygen concentration are substantially proportional, and the longer the length of the fluororesin tube, the greater the dissolved oxygen concentration. This is considered to be because the dissolved oxygen increases as the eluent feeding distance increases as the fluororesin tube has oxygen permeability.

(Measurement result)
The chromatogram obtained by measuring hemoglobin using the method of Example 1 is shown in FIGS. FIG. 1 shows a sample a, FIG. 2 shows a sample b, FIG. 3 shows a sample c, and FIG. 4 shows the results of measurement of healthy human blood. Peak 1 is unstable HbA1c, peak 2 is stable HbA1c, peak 3 is HbA0, peak 4 is carbamylated Hb, and peak 5 is acetylated Hb.
In FIG. 1, peaks 1 (unstable HbA1c) and 2 (stable HbA1c) are well separated. In FIG. 2, peak 4 (carbamylated Hb) is well separated from peak 2 (stable HbA1c) in FIG. 3 and peak 5 (acetylated Hb) is well separated.
Moreover, the chromatogram obtained by measuring hemoglobin using the method of Examples 2-5 is shown to FIGS. 5 to 8 are obtained by measurement by the method of Example 2, FIGS. 9 to 12 are obtained by the method of Example 3, FIGS. 13 to 16 are obtained by the method of Example 4, and FIGS. Is a chromatogram obtained. 5, 9, 13, and 17 are samples a; FIGS. 6, 10, 14, and 18 are samples b; FIGS. 7, 11, 15, and 19 are samples c; and FIGS. It is the result of having measured. As shown in FIGS. 5-20, also in Examples 2-5, it turns out that the unstable type HbA1c, the carbamylated Hb peak, and the acetylated Hb peak were able to be isolate | separated from the stable type HbA1c peak satisfactorily.

On the other hand, the chromatogram obtained by measuring hemoglobin using the method of the comparative example 1 is shown in FIGS. FIG. 21 shows a sample a, FIG. 22 shows a sample b, FIG. 23 shows a sample c, and FIG. In FIG. 21, peaks 1 and 2 were well separated. In FIG. 22, peak 4 (carbamylated Hb) was not separated from peak 2 (acetylated Hb) in FIG. 23 from peak 2 (stable HbA1c). Moreover, it was a result similar to FIGS. 21-24 also when the hemoglobin was measured using the method of the comparative examples 2 and 3. FIG.
Therefore, when the amount of dissolved oxygen was small, good separation performance was obtained with the glucose-loaded blood sample, but the carbamylated Hb peak and acetylated Hb peak could not be separated well from the stable HbA1c peak. I understand.

1 to 24, the separation performance of modified hemoglobin (acetylated Hb, carbamylated Hb, unstable HbA1c) when the methods of Examples 1 to 5 and Comparative Examples 1 to 3 were used was evaluated. Table 3 It was shown to. The evaluation criteria were as follows.
○: Each modified hemoglobin peak and the stable HbA1c peak are well separated.
Δ: Incomplete separation between each modified hemoglobin peak and stable HbA1c peak
X: Separation between each modified hemoglobin peak and stable HbA1c peak is poor.

Table 3 shows the measurement accuracy of the stable HbA1c evaluated by the deviation value. The divergence value is a numerical value obtained by subtracting the stable HbA1c value obtained from the chromatogram obtained by measuring the unmodified specimen from the stable HbA1c value obtained from the chromatogram obtained by measuring the samples a to c. The stable HbA1c value can be calculated by determining the ratio (%) of the area of the stable HbA1c peak to the area of the HbA0 peak.
Usually, when a blood sample of the same person is measured by a measurement method with good separation performance, the stable HbA1c value shows a constant value regardless of the presence or absence of modified hemoglobin or the like. Therefore, the divergence value is a standard indicating the separation performance of the stable HbA1c, and it can be said that the separation performance of the stable HbA1c is worse as the deviation value is larger.

(Example 6)
In Example 5 (5), the measurement temperature was 4 ° C., the eluent was kept at 4 ° C., and the same procedure as in Example 1 was used except that the deaerator was not turned on and used. Hemoglobins were measured.

(Example 7)
Hemoglobins were measured in the same manner as in Example 6 except that the measurement temperature was 20 ° C and the eluent was kept at 20 ° C.

(Example 8)
Hemoglobins were measured in the same manner as in Example 6 except that the measurement temperature was 40 ° C. and the eluent was kept at 40 ° C.

(Measurement result)
The chromatograms obtained by measuring hemoglobin using the methods of Examples 6 to 8 were the same as those shown in FIGS. In addition, Table 3 shows the separation performance and the deviation value of the modified hemoglobin. From these results, it can be seen that when the methods of Examples 6 to 8 are used, the unstable HbA1c, carbamylated Hb peak, and acetylated Hb peak can be well separated from the stable HbA1c peak.

Example 9
Hemoglobin was measured in the same manner as in Example 1 except that the following operation was performed.

(3) Preparation of eluents C to F Eluent C, 40 mM phosphate buffer (pH 7.5) prepared by adding 60 mM sodium perchlorate to 50 mM phosphate buffer and adjusting pH to 5.3 Eluent D, 90 mM sodium perchlorate added to 20 mM phosphate buffer, pH adjusted to 5.3, eluent E, 50 mM phosphate buffer, sodium perchlorate 300 mM Eluent F was prepared by adding and adjusting the pH to 8.5.

(4) Preparation of measurement sample AFSC control (manufactured by Helena) was hemolyzed with a hemolysis reagent and diluted 67 times to obtain sample d. As a hemolysis reagent, a phosphate buffer solution (pH 7.0) containing 0.1 wt% polyethylene glycol mono-4-octylphenyl ether (Triton X-100) (manufactured by Tokyo Chemical Industry Co., Ltd.) was used.

(5) Measurement of hemoglobins Using the obtained column, eluents C to F and sample d, hemoglobins were measured under the following conditions. The measurement was performed at room temperature (25 ° C.). The eluent was kept at 25 ° C., and eluent C was sent for 0 to 1.7 minutes from the start of measurement, and eluent D was sent for 1.7 to 3.0 minutes. The eluent E is sent for 3.0 to 3.5 minutes, the eluent F is sent for 3.5 to 3.6 minutes, and 3.6 to 4.0 minutes. In the meantime, the eluent C was fed.

As the measuring apparatus, an apparatus improved so that four types of eluents can be used with the configuration shown in FIG. 32 is used. The liquid feed pump is LC-9A (manufactured by Shimadzu Corporation), and the autosampler is ASU-420 (Sekisui Chemical Co., Ltd.). (Manufactured by Kogyo Co., Ltd.), detector SPD-6AV (manufactured by Shimadzu Corporation) is used, and the length of the tube 33 for feeding between the deaerator 32 and the liquid switching solenoid valve 34 is for the eluent C. For both eluent D, eluent E, and eluent F, a fluororesin tube (Catalog No. 6010-35305, manufactured by GL Science Inc.) having an outer diameter of 3 mm and an inner diameter of 2 mm was used. 32, a flow type trace dissolved oxygen meter (DO-55G, manufactured by Toa DKK Corporation) as shown in FIG. 30 is installed immediately before the filter 39 after the liquid feed pump 35 in FIG. The dissolved oxygen concentration of was measured. The measurement conditions were as follows.
Measurement conditions: Flow rate: 2.0 mL / min
Detection wavelength: 415 nm
Sample injection volume: 10 μL

(Example 10, comparative example 4)
In Example 9 (5), hemoglobins were measured in the same manner as in Example 9 except that the fluororesin tube having the length shown in Table 3 was used. The fluororesin tubes have the same length for eluent C, eluent D, eluent E, and eluent F. Table 3 shows the results of the dissolved oxygen concentration measurement in Example 10 and Comparative Example 4.

(Measurement result)
FIG. 25 shows a chromatogram obtained by measuring hemoglobin using the method of Example 9. FIG. 25 shows the result of measuring the sample d. Peak 11 is HbA1a and HbA1b, Peak 12 is HbF, Peak 13 is unstable HbA1c, Peak 14 is HbA0 (mainly HbA), Peak 15 is HbA2, Peak 16 is HbS, and Peak 17 is HbC.
In FIG. 25, peaks 11 to 17 are well separated. Moreover, when the hemoglobins were measured using the method of Example 10, the same results as in FIG. 25 were obtained.

On the other hand, the chromatogram obtained by measuring hemoglobin using the method of Comparative Example 4 is shown in FIG. FIG. 26 shows the result of measuring the sample d. Peak 11 is HbA1a and HbA1b, Peak 12 is HbF, Peak 13 is unstable HbA1c, Peak 14 is HbA0 (mainly HbA), Peak 15 is HbA2, Peak 16 is HbS, and Peak 17 is HbC. In FIG. 26, peak 11 and peak 12 are well separated, but peaks 14 to 17 are not well separated.

The present invention relates to a method for measuring hemoglobin using cation exchange liquid chromatography, and in particular, can provide a method for measuring hemoglobin capable of measuring stable HbA1c with high accuracy.

FIG. 4 is a diagram showing a chromatogram obtained when sample a is measured under the measurement conditions of Example 1. FIG. 3 is a diagram showing a chromatogram obtained when sample b is measured under the measurement conditions of Example 1. FIG. 3 is a diagram showing a chromatogram obtained when sample c is measured under the measurement conditions of Example 1. FIG. 2 is a diagram showing a chromatogram obtained when measurement of healthy human blood (unmodified specimen) is performed under the measurement conditions of Example 1. 6 is a diagram showing a chromatogram obtained when measurement of a sample a is performed under the measurement conditions of Example 2. FIG. FIG. 6 is a diagram showing a chromatogram obtained when measuring a sample b under the measurement conditions of Example 2. FIG. 4 is a diagram showing a chromatogram obtained when sample c is measured under the measurement conditions of Example 2. It is a figure which shows the chromatogram obtained when the healthy human blood (unmodified sample) was measured on the measurement conditions of Example 2. FIG. FIG. 6 is a diagram showing a chromatogram obtained when sample a is measured under the measurement conditions of Example 3. FIG. 6 is a diagram showing a chromatogram obtained when sample b is measured under the measurement conditions of Example 3. FIG. 6 is a diagram showing a chromatogram obtained when sample c is measured under the measurement conditions of Example 3. It is a figure which shows the chromatogram obtained when the healthy human blood (unmodified sample) was measured on the measurement conditions of Example 3. FIG. FIG. 10 is a diagram showing a chromatogram obtained when sample a is measured under the measurement conditions of Example 4. It is a figure which shows the chromatogram obtained when measuring the sample b on the measurement conditions of Example 4. FIG. It is a figure which shows the chromatogram obtained when the measurement of the sample c was performed on the measurement conditions of Example 4. FIG. It is a figure which shows the chromatogram obtained when the healthy human blood (unmodified specimen) was measured on the measurement conditions of Example 4. FIG. 6 is a diagram showing a chromatogram obtained when sample a is measured under the measurement conditions of Example 5. FIG. 10 is a diagram showing a chromatogram obtained when sample b is measured under the measurement conditions of Example 5. 6 is a diagram showing a chromatogram obtained when sample c is measured under the measurement conditions of Example 5. FIG. It is a figure which shows the chromatogram obtained when the healthy human blood (unmodified sample) was measured on the measurement conditions of Example 5. FIG. It is a figure which shows the chromatogram obtained when the sample a was measured on the measurement conditions of the comparative example 1. FIG. It is a figure which shows the chromatogram obtained when the sample b was measured on the measurement conditions of the comparative example 1. FIG. It is a figure which shows the chromatogram obtained when the measurement of the sample c was performed on the measurement conditions of the comparative example 1. FIG. It is a figure which shows the chromatogram obtained when the normal human blood (unmodified sample) was measured on the measurement conditions of the comparative example 1. FIG. 10 is a diagram showing a chromatogram obtained when measurement of a sample d is performed under the measurement conditions of Example 9. FIG. It is a figure which shows the chromatogram obtained when the sample d was measured on the measurement conditions of the comparative example 4. FIG. It is a graph which shows the relationship between the melt | dissolution power and time in gradient elution method. It is a graph which shows the relationship between the melt | dissolution power and time in a step elution method. It is a structural example of the apparatus in the case of performing a step elution method. It is a cross-sectional schematic diagram which shows an example of a dissolved oxygen meter. It is a cross-sectional schematic diagram which shows an example of a deaeration apparatus. It is the schematic of the liquid chromatography generally used.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Solenoid valve 2 Liquid feed pump 3 Sample injection part 4 Column 5 Detector 6 Integrator

Claims (1)

Using cation exchange liquid chromatography, at least one selected from the group consisting of acetylated hemoglobin, carbamylated hemoglobin, unstable hemoglobin A1c, stable hemoglobin A1c, hemoglobin A, hemoglobin A2, hemoglobin S and hemoglobin C is used. A method for measuring hemoglobin, which comprises using an eluent having a dissolved oxygen concentration of 2.5 mg / L or more.

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