JP2021081379A - Sample diluent for blood sample measurement by hplc method, and glycated hemoglobin measurement method - Google Patents

Abstract

To solve problems such as a reduction in column separability and an increase in device pressure that tend to occur during repeated measurement when analyzing a blood sample by the HPLC method.SOLUTION: Provided is a sample diluent for blood sample measurement by the high-performance liquid chromatography (HPLC) method that uses column filled with a filler consisting of non-porous organic polymer particles having an ion-exchange group, the sample diluent having a pH in a range of 9.0-10.0. A method of measuring glycated hemoglobin by the HPLC method using the sample diluent is also provided.SELECTED DRAWING: None

Description

The present invention relates to a sample diluent used when measuring a blood sample by a high performance liquid chromatography (HPLC) method, and a method for measuring glycated hemoglobin using the sample diluent.

When a blood sample is measured by an HPLC method, a sample diluent for preparing the blood sample in a state suitable for measurement is used in advance.
For example, Japanese Patent No. 40947776 (Patent Document 1) describes that a hemolytic reagent containing a chaotropic ion is used as a diluent for a blood sample when measuring glycated hemoglobin (HbA1c) used for diagnosing diabetes. Has been done. In Patent Document 1, a hemolytic reagent is an aqueous solution hypotonic than erythrocytes, and hemolysis means that erythrocytes are torn and contents such as hemoglobin (for example, hemoglobins) go out of erythrocytes, and chaotropic ions are used. It is disclosed that this improves the separation of HbA1c. As a column for measuring HbA1c, a column made of a filler having a cation exchange group is used in the examples.

WO2019 / 004440 (Patent Document 2) discloses a filler in which a sulfo group is bonded to particles of a copolymer having a monomer unit derived from (meth) acrylic acid ester and a monomer unit derived from divinylbenzene. .. It is described that the filler can have a small particle size, has a high separability, and has properties suitable for analysis of HbA1c.

Japanese Patent No. 40947776 WO2019 / 004440

When a blood sample is analyzed by an HPLC method, problems such as a decrease in the separability of the column and an increase in the device pressure at the time of measurement may occur due to repeated measurements.
In recent years, more efficient analysis by HPLC method for diagnosing diabetes has been required. However, in order to measure blood samples more efficiently with higher resolution, if a column filled with a packing material having a small particle size as described in Patent Document 2 is used, the above-mentioned problem due to repeated measurement Is more likely to occur than in previous columns.
Thus, there is a trade-off between efficient blood sample measurement and the problems that arise in repeated measurements. Therefore, there has been a strong demand for a means capable of reducing problems that occur in repeated measurements.

As a result of diligent studies, the present inventors have described the above by using a sample diluent having a pH of 9.0 to 10.0 as the sample diluent when measuring a blood sample by the HPLC method. We have found that the problem can be solved and completed the present invention.

The present invention relates to the following sample diluents [1] to [5] and methods for measuring glycated hemoglobin according to [6] to [8].
[1] A sample diluent for measuring a blood sample by a high performance liquid chromatography (HPLC) method using a column packed with a packing material composed of non-porous organic polymer particles having an ion exchange group, and having a pH of 9.0. A sample diluent characterized by being ~ 10.0.
[2] The sample diluent according to item 1 above, wherein the sample diluent contains a buffer containing at least one of N-cyclohexyl-2-aminoethanesulfonic acid and N-cyclohexyl-3-aminopropanesulfonic acid.
[3] The sample diluent according to item 2 above, wherein the buffer in the sample diluent is N-cyclohexyl-2-aminoethanesulfonic acid.
[4] The sample diluent according to any one of Items 1 to 3 above, wherein the volume average particle diameter of the non-porous organic polymer particles having an ion exchange group is 1.5 μm to 3.5 μm.
[5] The filler according to any one of the above items 1 to 4, wherein the filler is a filler in which a sulfo group is bonded to copolymer particles having a monomer unit derived from (meth) acrylic acid ester and a monomer unit derived from divinylbenzene. Specimen diluent.
[6] A method for measuring glycated hemoglobin by an HPLC method, which comprises using the sample diluent according to any one of the above items 1 to 5.
[7] Claim 6 that the buffer in the sample diluent is N-cyclohexyl-2-aminoethanesulfonic acid, and a zwitterionic compound having an amino group and a sulfo group is used as a buffer for the eluent used in the HPLC method. The method for measuring saccharified hemoglobin according to.
[8] The method for measuring saccharified hemoglobin according to claim 7, wherein the zwitterionic compound having an amino group and a sulfo group is at least one selected from 2-morpholinoetan sulfonic acid and 3-morpholinopropane sulfonic acid.

By using the sample diluent of the present invention, problems that occur in repeated measurements can be reduced, and blood samples can be measured efficiently.

The state of the change of the column pressure in the repeated measurement of HbA1c of Example 1 using the sample diluent of pH 9.6 is shown. Chromatogram obtained in the measurement of HbA1c of Example 1 using a sample diluent having a pH of 9.6. In the figure, 1 is the peak of unstable HbA1c and 2 is the peak of stable HbA1c. The measurement result of HbA1c of Example 1 using the sample diluent of pH 9.6 is shown. The state of the change of the column pressure in the repeated measurement of HbA1c of Comparative Example 1 using the sample diluent of pH 7.0 is shown. Chromatogram obtained by repeated measurement of HbA1c of Comparative Example 2 using a sample diluent having a pH of 10.5. In the figure, 1 is the peak of unstable HbA1c and 2 is the peak of stable HbA1c. The graph which shows the relationship between the pH and the particle size in the measurement sample for HPLC when the measurement sample for HPLC was measured by the dynamic light scattering method. The graph which shows the relationship between the pH and the particle size in the measurement sample for HPLC when the measurement sample for HPLC using the sample diluent prepared without adding CHES was measured by the dynamic light scattering method.

The present invention will be described in detail below with reference to embodiments.
The blood sample in the present embodiment refers to a liquid obtained from the body of a mammal, particularly a human, and refers to a liquid collected in a container such as a vial for a medical examination or the like.

In the present specification, a blood sample to which a sample diluent is added is referred to as a measurement sample for HPLC. The sample diluent is added at a volume ratio of 50 to 200 times the blood sample. The ratio is preferably 80 to 150 times, more preferably 90 to 110 times. The blood sample may be diluted with the sample diluent at the same time as the collection, or the sample diluent may be added after some operation.

The sample diluent may contain a buffer to keep its pH stable. Examples of the buffer include phosphoric acid, acetic acid, formic acid, carbonic acid, amine compounds, salts thereof, and combinations thereof. Phosphate-based buffers are most often used because they have a wide pH range that exhibits a buffering action. A buffer solution to which an amine compound has been added is also often preferably used. Examples of amine compounds include ethanolamine, dimethylaminoethanol, triethanolamine, diethanolamine, piperazin, pyridine, imidazole, tris (hydroxymethyl) aminomethane (Tris) and the like. In addition, 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazin-N, N'-bis (2-ethanesulfonic acid), which are called Good buffers, (2-morpholinoethanesulfonic acid) ( PIPES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 2-hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid ( HEPSO), 3- [4- (2-Hydroxyethyl) -1-piperazinyl] propanesulfonic acid (EPPS), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), N- (2-Acetamide) -2-aminoethanesulfonic acid (ACES), N- (2-acetamide) iminodiacetic acid (ADA), N- [tris (hydroxymethyl) methyl] -2-aminoethanesulfonic acid (TES) , N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), etc. Ionic compounds are also preferably used. Among these, CHES and CAPS are more preferably used because they have a buffering capacity in the pH range required by the sample diluent of the present invention. Even in the measurement by the HPLC method using a column using a packing material having a small particle size, CHES is most preferable in that an increase in column pressure due to repeated use can be suppressed.

In addition to these components, the sample diluent may contain a surfactant, a water-soluble organic solvent, a preservative, an anticoagulant, and other salts, if necessary. Examples of surfactants include Triton® X-100, Tween® 20, Tween80, sodium dodecylbenzenesulfonate and the like. As the surfactant to be added, Triton X-100 is more preferable. Examples of water-soluble organic solvents include acetone, methanol, ethanol, isopropanol, ethylene glycol, diethylene glycol, diethylene glycol monomethyl ether, diethylene glycol dimethyl ether, diethylene glycol monobutyl ether, ethylene glycol monopropyl ether, ethylene glycol monomethyl ether, propylene glycol monomethyl ether, Ethylene, N, N-dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dimethylsulfoxide (DMSO) and the like can be mentioned. Examples of the preservative include sodium azide (NaN ₃ ), paraben, Proclin 300 and the like. Anticoagulants include ethylenediaminetetraacetic acid / disodium (EDTA / 2Na), ethylenediaminetetraacetic acid / dipotassium (EDTA / 2K), ethylenediaminetetraacetic acid / tripotassium (EDTA / 3K), sodium fluoride, potassium fluoride, etc. Can be mentioned. Examples of other salts to be added include sodium chloride, potassium chloride, ammonium chloride, sodium bromide, potassium bromide, ammonium bromide, sodium sulfate, potassium sulfate, ammonium sulfate and the like.

The buffer, salt combination, concentration, etc. are appropriately adjusted according to the purpose. In the HPLC method, it is not preferable that the salt concentration of the measurement sample for HPLC to be injected is too high, so the concentration is usually adjusted to 0.1 mmol / L to 500 mmol / L. The pH of the buffer is adjusted by a known method. For example, it is adjusted by adding alkali or acid while measuring the pH of an aqueous solution containing the necessary components (buffers and other compounds).

The pH of the sample diluent in this embodiment is 9.0 to 10.0. The effect of the sample diluent of the present invention is manifested in repeated measurements. For example, even if the measurement sample for HPLC prepared from the blood sample using the sample diluent of the present invention is repeatedly measured 2000 to 3000 times, the measured value of HbA1c shows a stable value and the column pressure is also stable. There is. On the other hand, when the pH of the sample diluent is less than 9.0, the column pressure increases in repeated measurements. As a result, the device pressure rises, making it difficult to feed the liquid and making measurement impossible. The device pressure here is the pressure applied to the pump outlet when the flow rate is 1.5 ml / min measured by a pressure gauge provided in the device. The device pressure is the pressure generated when the eluent flows through pipes, filters, columns, detectors, and the like. In many cases, the increase in device pressure is due to the increase in column pressure. It is presumed that the increase in column pressure is due to clogging of fine particles in the column, that is, contamination of the filler, alteration of the filler, and the like.
When the pH is higher than 10.0, an unfavorable effect appears on the measurement result. That is, a correct value cannot be obtained as the measured value of HbA1c. It is presumed that this is because the protein-based components in the blood sample are denatured. The sample diluent preferably has a pH of 9.2 to 9.8, more preferably 9.4 to 9.7. The pH is a value measured at 25 ° C.

The details of the reason why the sample diluent according to this embodiment is effective in blood sample measurement using the HPLC method are unknown. When a sample for column injection obtained by adding a sample diluent to a blood sample was analyzed by a dynamic light scattering method, the average diameter of what was supposed to be some fine particles changed depending on the pH of the sample diluent. It turned out to be. When the pH is 9 or higher, the average diameter of the measured particles becomes smaller. This tendency was commonly observed when CHES was used as a buffer for the diluent and when phosphoric acid was used. It is considered that such a decrease in the average diameter of the fine particles in the measurement sample for HPLC is a factor for preventing inconveniences such as an increase in the device pressure during repeated measurement.

The sample diluent of the present embodiment can be suitably used in the measurement of blood samples by the HPLC method. In particular, its effect is exhibited in the measurement of glycated hemoglobin. In glycated hemoglobin measurement, hemoglobins in erythrocytes contained in a blood sample are measured. Measurement of glycated hemoglobin is generally widely used as an index for diagnosis of diabetes and the like. Therefore, it is necessary to measure a large number of blood samples quickly. In order to measure a large number of samples quickly, it is desirable that more repeated measurements are possible.
Under these circumstances, the sample diluent of the present invention is particularly useful in the measurement of glycated hemoglobin.

The blood sample is diluted with the sample diluent by leaving it for a while after injecting the sample diluent into the blood sample, or by forcibly shaking the vial. An appropriate amount of the obtained sample for column injection is injected into the column.
Non-porous organic polymer particles having an ion exchange group are used as the packing material for the column into which the measurement sample for HPLC is injected. When the analysis target exists as a cation, a filler having a cation exchange group is used, and when it exists as an anion, a filler having an anion exchange group is used. In the measurement of glycated hemoglobin, a filler having a cation exchange group is generally used. Non-porous organic polymer particles have few fine pores on their surface. Specifically, it is preferable that the total pore volume is 0.05 cm ³ / g or less with respect to the mass of the particles measured by a gas adsorption method, and more preferably less 0.02 cm ³ / g. Particles with few fine pores have improved strength. In addition, when HPLC analysis is performed using a filler using these particles as a base material, compounds such as proteins contained in the sample do not diffuse in the pores, so that rapid analysis becomes possible.
The total pore volume can be measured using Autosorb® iQ manufactured by Kantachrome Co., Ltd. as an apparatus.

In order to fully obtain the effect of the sample diluent of the present embodiment, the volume average particle size of the non-porous organic polymer particles having an ion exchange group as a filler shall be 1.5 μm to 3.5 μm. Is preferable. In the column filled with the filler in the particle size range, the components to be measured can be separated quickly and efficiently. On the other hand, in the column using the filler having a small particle size, inconveniences such as an increase in the device pressure may occur. However, such inconvenience can be avoided by using the sample diluent of the present invention. From the viewpoint of the balance between the efficiency of separation and the increase in device pressure, the average particle size of the particles is more preferably 2.5 μm to 3.2 μm.

The volume average particle size means the following particle size. That is, the packing material particles are imaged with a particle size distribution measuring device so as to have 2000 or more particles, and from the obtained two-dimensional particle image (preferably a still image), the equivalent circle diameter of each particle (projected area of the particle image) Get the diameter of a circle with the same area). The volume of each particle is calculated from the equivalent circle diameter, and the particle diameter is averaged based on the calculated volume. At this time, each particle is regarded as a sphere having the same diameter as the above-mentioned circle-equivalent diameter. As the particle size distribution measuring device, FPIA-3000 (manufactured by Sysmex Corporation) or the like can be used.

In the measurement of components in blood samples such as glycated hemoglobin, the polymer filler described in Patent Document 2 is more preferable as the filler. Specifically, a column in which particles of a copolymer having a monomer unit derived from (meth) acrylic acid ester and a monomer unit derived from divinylbenzene are filled with a filler in which a sulfo group, which is an ion exchange group, is bonded is preferable. Such a filler is less likely to cause inconveniences such as deformation of the particles themselves due to the column pressure during use even when the particle size is small, and gives a more suitable analysis result of glycated hemoglobin. The (meth) acrylic acid ester refers to an acrylic acid ester or a methacrylic acid ester.

The above column will be described in more detail.
Divinylbenzene used in the above-mentioned copolymer particles is a mixture of isomers at the meta and para positions. As the (meth) acrylic acid ester, glycidyl methacrylate is preferable. When glycidyl methacrylate is used, the amount of glycidyl methacrylate used is preferably 70% by mass to 90% by mass in proportion to the total amount of monomers. The copolymer particles can be produced by suspension polymerization or the like. In suspension polymerization, when producing porous organic polymer particles, a diluent is added for polymerization. In the case of producing a non-porous organic polymer, the desired product can be obtained by polymerizing without adding a diluent.

A sulfo group is bonded by reacting the divinylbenzene obtained by the polymerization with the glycidyl methacrylate copolymer particles with a sulfolating agent. Examples of the sulphonating agent include sodium 2-hydroxyethanesulfonate, sodium 3-hydroxypropanesulfonate, sodium 2-mercaptoethanesulfonate, sodium 3-mercaptopropanesulfonate, sodium 2-bromopropanesulfonate, and 3-bromopropane. Examples thereof include sodium sulfonate, 1,3-propane sulton, 2,4-butane sulton, 1,3-butan sulton, and 1,4-butan sulton. The sulfonate is bonded to the surface of the substrate by utilizing the reaction with the epoxy group existing on the surface of the copolymer particles obtained by the polymerization. Among these sulfonates, sodium 3-mercaptopropanate sulfonate and 1,3-propane sultone are preferable, and 1,3-propane sultone is more preferable from the viewpoint of easiness of reaction. The amount of the sulfo group bonded to the copolymer particles is preferably 20 μmol / g to 300 μmol / g.

The amount of sulfo groups in the filler can be measured by the following method. That is, 10 mL of 0.5 M hydrochloric acid is added to 1 g of vacuum-dried particles to disperse the particles, and the particles are filtered and then washed with water. As a result, the sulfo group becomes an acid type, and the particles in a washed state are obtained. Next, 10 mL of 0.5 M sodium hydroxide is added to the particles, and the particles are filtered with a container for collecting the filtrate installed, and then the particles are washed with 10 mL of water. The mixture of the filtrate and the washing solution is titrated with 0.1 M hydrochloric acid. The number of moles of hydrochloric acid required at this time is determined, and the value obtained by subtracting the number of moles of sodium hydroxide (5 mM in the above case) is taken as the amount of sulfo groups.

In the HPLC measurement of saccharified hemoglobin using the measurement sample for HPLC according to the present embodiment, the step gradient method is preferably used for rapid measurement. Typical gradient conditions include adjusting the first solution of the eluent to a weak acidity of pH 4.0 to 6.0 and adjusting the second solution of the eluate to a pH of 8.0 to 11.0. Be done. Under this condition, HbA1c measurement can be achieved in a short time.
After each gradient measurement, it is also widely practiced to flush a third eluent to clean the column and prepare for the next measurement.

As the first liquid, the second liquid, and the third liquid of the eluent used in the step gradient method, those suitable for the purpose are appropriately used. For example, the first liquid, the second liquid, and the third liquid may contain a buffer to keep the pH stable. Examples of the buffer include phosphoric acid, acetic acid, formic acid, carbonic acid, amine compounds, salts thereof, and combinations thereof. The phosphoric acid-based buffer has a wide pH range showing a buffering action, and a buffer solution to which the most commonly used amine compound is added is often preferably used. Examples of amine compounds include ethanolamine, dimethylaminoethanol, triethanolamine, diethanolamine, piperazin, pyridine, imidazole, tris (hydroxymethyl) aminomethane (Tris) and the like. In addition, 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazin-N, N'-bis (2-ethanesulfonic acid) (PIPES), which are called Good buffers. ), 2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 2-hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid (HEPSO) ), 3- [4- (2-Hydroxyethyl) -1-piperazinyl] propanesulfonic acid (EPPS), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), N-( 2-acetamido) -2-aminoethanesulfonic acid (ACES), N- (2-acetamido) iminodiacetic acid (ADA), N- [tris (hydroxymethyl) methyl] -2-aminoethanesulfonic acid (TES), Amphoteric ions such as N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) Compounds are also preferably used.

In addition to these components, the eluent may contain a surfactant, a water-soluble organic solvent, a preservative, an anticoagulant, and other salts, if necessary. Examples of surfactants include Triton X-100, Tween 20, Tween 80, sodium dodecylbenzene sulfonate and the like. Examples of water-soluble organic solvents include acetone, methanol, ethanol, isopropanol, ethylene glycol, diethylene glycol, diethylene glycol monomethyl ether, diethylene glycol dimethyl ether, diethylene glycol monobutyl ether, ethylene glycol monopropyl ether, ethylene glycol monomethyl ether, propylene glycol monomethyl ether, Ethylene, N, N-dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dimethylsulfoxide (DMSO) and the like can be mentioned. Examples of the preservative include sodium azide, paraben, Proclin 300 and the like. Examples of the anticoagulant include ethylenediaminetetraacetic acid / disodium (EDTA / 2Na), ethylenediaminetetraacetic acid / dipotassium (EDTA / 2K), sodium fluoride, potassium fluoride and the like. Examples of other salts to be added include sodium chloride, potassium chloride, ammonium chloride, sodium bromide, potassium bromide, ammonium bromide, sodium sulfate, potassium sulfate, ammonium sulfate and the like.

The buffer, salt combination, concentration, etc. are appropriately adjusted according to the purpose. In the HPLC method, it is not preferable that the salt concentration of the measurement sample for HPLC to be injected is too high, so the concentration is usually adjusted to 0.1 mmol / L to 500 mmol / L. The pH of the buffer is adjusted by a known method. For example, it is adjusted by adding alkali or acid while measuring the pH of an aqueous solution containing the necessary components (buffers and other compounds).

In the measurement of saccharified hemoglobin by the step gradient method using a measurement sample for HPLC, the first solution is usually adjusted to a weak acidity of pH 4.0 to 6.0, and the second solution is adjusted to pH 8.0 to 11.0. adjust.

In the repeated measurement of the HPLC method using a column filled with a filler having a small particle size, it is preferable that the chemical properties of the eluent combinations used do not differ significantly from the viewpoint that the influence on the column can be reduced. it is conceivable that. Based on these inferences, in the method for measuring glycated hemoglobin, which is one aspect of the present invention, when CHES or CAPS is used as a buffer for a sample diluent, amino groups and sulfos having similar chemical term structures to CHES and CAPS. Zwitterionic compounds with groups are preferably used as buffers for eluents in step gradients. It is more preferable to use MES as the first liquid and MOPS as the second liquid as a combination having a buffering ability at each pH. It is more preferable to use CHES as the sample diluent, MES as the first solution, and MOPS as the second solution.

The column for liquid chromatography of the present embodiment is obtained by filling the column with a cation exchange type filler obtained by the above method by a known packing method such as a slurry method.

The liquid chromatography column of the present embodiment preferably has a housing made of PEEK (polyetheretherketone). Generally, a column made of SUS is also used as a column for liquid chromatography, but in the present invention, a column made of PEEK is preferably used in order to suppress adsorption of proteins contained in blood such as hemoglobin to the housing. .. A frit is installed in the column to prevent the filler particles from flowing out, but the frit is also made of PEEK to suppress adsorption, or made of a material obtained by mixing PEEK and PTFE (polytetrafluoroethylene) and sintering it. It is desirable to use one.

The column size is not particularly limited, but it is desirable that the measurement be completed in a short time, so that the length is preferably short, and specifically, 7 mm or more and 50 mm or less is preferable. More preferably, it is 8 mm or more and 20 mm or less. If it is too short, sufficient separation cannot be achieved. On the other hand, if it is too long, the time required for analysis will be long. Further, the thickness is preferably in the range of the thickness in which the column pressure does not become too large when the required flow velocity is applied. On the other hand, if it is too thick, the amount of eluate consumed will increase, making it uneconomical. Specifically, the inner diameter is preferably 1 mm or more and 10 mm or less. More preferably, it is 2 mm or more and 6 mm or less.
The required flow rate is related to the thickness of the column, the amount of sulfo groups in the filler, the average particle size of the filler, and the like. For example, when the thickness of the column is 4.6 mm, the flow rate is preferably 0.2 mL / min to 5.0 mL / min, more preferably 1.0 mL / min to 2.0 mL / min.

In measuring the concentration of glycated hemoglobin such as HbA1c in blood by the column to which the filler produced in this embodiment is applied, a liquid chromatography apparatus equipped with a gradient function capable of sending a plurality of types of eluents should be used. Is preferable. Examples of such an apparatus include Infinity (registered trademark) 1260 manufactured by Agilent Technologies, Prominense manufactured by Shimadzu Corporation, and the like.
It is also particularly preferably used in devices designed specifically for HbA1c measurement. A device designed exclusively for HbA1c measurement is installed in hospitals and the like and is widely used for diabetes diagnosis. In the HbA1c measurement dedicated device, the combination of the column and the eluent and the gradient conditions are optimized so that one measurement can be completed in the shortest possible time.

Example 1:
[Polymerization process]
50 g of a monomer mixture consisting of glycidyl methacrylate (manufactured by Nichiyu Co., Ltd.) and divinylbenzene (manufactured by Nippon Steel & Sumikin Chemical Co., Ltd., purity 99%) (glycidyl methacrylate: divinylbenzene = 82: 17 (mass ratio)) as a polymerization initiator. 0.5 g (amount corresponding to 1% by mass of the monomer mixture) of lauroyl peroxide (manufactured by Nacalai Tesque Co., Ltd.) was added to obtain a mixture. The obtained mixture was prepared into oil droplets using a microchannel suspension device (manufactured by EP Tech Co., Ltd.) using 500 g of a 1% aqueous sodium alkyldiphenyl ether sulfonate solution as an aqueous phase, and the polymerization reaction was further carried out at 80 ° C. for 15 hours to carry out a copolymer Obtained particles.
The average particle size of the obtained particles was 2.7 μm.

[Modification process]
To 3 g of the above copolymer particles, 24 g of 2-propanol and 3 g of 1,3-propanesulton (manufactured by Tokyo Chemical Industry Co., Ltd.) were added and heated to 50 ° C. 1.2 g of an 8 M potassium hydroxide aqueous solution is added thereto, and the mixture is stirred for 6 hours, filtered, and washed successively with 0.5 M hydrochloric acid, water, 0.5 M sodium hydroxide aqueous solution, and water to obtain a non-cation exchange group. Porous organic polymer particles (filler particles 1) were obtained. The average particle size of the obtained particles was 2.8 μm.
1.0 g of the vacuum-dried filler particles 1 was dispersed by adding 10 mL of 0.5 M hydrochloric acid, filtered, and then washed with water to obtain particles having an acid type sulfo group. 10 mL of 0.5 M sodium hydroxide was added to the particles, and the particles were filtered with a container for collecting the filtrate installed, and then the particles were washed with 10 mL of water. The mixed solution of the filtrate and the washing solution collected in the installed container was titrated with 0.1 M hydrochloric acid. The amount of sulfo group was determined from the difference between the number of moles of sodium hydroxide used at the beginning (5 mM) and the number of moles of hydrochloric acid required for titration. As a result, the amount of sulfo groups with respect to the mass of the particles was 200 μmol / g.
As a result of pore measurement by gas adsorption (using Autosorb (registered trademark) iQ manufactured by Kantachrome Co., Ltd.), the pore volume was a very small value of ^{0.01 cm 3 / g.}
The filler particles 1 were packed in a PEEK column having an inner diameter of 4.6 mm and a length of 10 mm to obtain an analytical column.

[Quantification of HbA1c]
The column was connected to an HPLC apparatus, Infinity 1260 (Agilent Technologies). The sample diluent and eluate were prepared by dissolving each reagent in water at the concentrations shown in Table 1. The pH at 25 ° C. was adjusted to the values shown in Table 1 using a 0.1 M aqueous potassium hydroxide solution. Table 2 shows the gradient conditions in the analysis. The flow rate of the eluent was 1.5 mL / min and the temperature was 37 ° C. A measurement sample for HPLC was prepared by diluting a whole blood sample 100-fold with a sample diluent on a volume basis. Repeated measurement was performed by injecting 5 μL of this HPLC measurement sample. No increase in pressure was observed even after 3000 repeated measurements. In addition, there was no significant change in the measured value of HbA1c. The state of the change in the column pressure is shown in FIG. The chromatogram of the measurement result of the 2000th HbA1c is shown in FIG. 2, and the state of change in the measurement result of glycated hemoglobin is shown in FIG. Stable measurement was possible.

CHES：Ｎ−シクロヘキシル−２−アミノエタンスルホン酸（緩衝剤）
MES：２−モルホリノエタンスルホン酸（緩衝剤）
MOPS：３−モルホリノプロパンスルホン酸（緩衝剤）
EDTA・３K：エチレンジアミン四酢酸・三カリウム（抗凝固剤）
NaN₃：アジ化ナトリウム（防腐剤）
Triton（登録商標）X-100：（界面活性剤）
Proclin300：プロクリン３００（防腐剤）
Tween（登録商標）２０：（界面活性剤）

CHES: N-cyclohexyl-2-aminoethanesulfonic acid (buffer)
MES: 2-morpholinoetan sulfonic acid (buffer)
MOPS: 3-morpholinopropanesulfonic acid (buffer)
EDTA ・ 3K: Ethylenediaminetetraacetic acid ・ Tripotassium (anticoagulant)
NaN ₃ : Sodium azide (preservative)
Triton® X-100: (surfactant)
Proclin300: Proclin 300 (preservative)
Tween® 20: (surfactant)

Comparative Example 1:
Repeated analysis of HbA1c was performed in the same manner as in Example 1 except that the pH of the sample diluent was set to 7.0. The change in pressure at that time is shown in FIG. The pressure increased when the number of measurements exceeded 1000. This made further measurements impossible.

Comparative Example 2:
A phenomenon in which the chromatogram changed was observed by repeated measurement in which HbA1c was measured in the same manner as in Example 1 except that the pH of the diluted solution was set to 10.5. The 500th chromatogram is shown in FIG. The measured value of HbA1c changed, and the correct measured value could not be obtained. It is speculated that protein denaturation and the like occur when the pH reaches 10.5, which may be one of the causes.

Measurement of Molecular Size in Measurement Sample for HPLC Based on the composition of the sample diluent used in Example 1, sample diluents having different pH were prepared. With respect to the measurement sample for HPLC obtained by treating this sample diluent into a blood sample, the Z average particle size contained in the sample was measured by a dynamic light scattering method. A Zetasizer (registered trademark) nano ZSP manufactured by Malvern was used for the measurement. The measurement results are shown in FIG. It was found that when the pH was 9.0 or higher, the particle size averaged about 20 nm and showed a small value that was almost constant, but when the pH was lower than 9.0, the Z average particle size increased.
It is unknown what the particle size in the measurement sample for HPLC is derived from. Moreover, it is unclear what kind of relationship it has with the increase in column pressure in repeated measurements. However, it is presumed that this is one of the factors that cause the effects of the present invention to be exhibited.
A similar experiment was performed on the sample diluent obtained by removing only CHES from the composition of the sample diluent used in Example 1. The state of the contained particle size is shown in FIG. It was found that when CHES was not used, the size of the particle size contained in the measurement sample for HPLC was large.

Claims (8)

It is a sample diluent for blood sample measurement by a high performance liquid chromatography (HPLC) method using a column packed with a packing material composed of non-porous organic polymer particles having an ion exchange group, and has a pH of 9.0 to 10. A sample diluent characterized by being 0. The sample diluent according to claim 1, wherein the sample diluent contains a buffer containing at least one of N-cyclohexyl-2-aminoethanesulfonic acid and N-cyclohexyl-3-aminopropanesulfonic acid. The sample diluent according to claim 2, wherein the buffer in the sample diluent is N-cyclohexyl-2-aminoethanesulfonic acid. The sample diluent according to any one of claims 1 to 3, wherein the volume average particle diameter of the non-porous organic polymer particles having an ion exchange group is 1.5 μm to 3.5 μm. The sample dilution according to any one of claims 1 to 4, wherein the filler is a filler in which a sulfo group is bonded to copolymer particles having a monomer unit derived from (meth) acrylic acid ester and a monomer unit derived from divinylbenzene. liquid. A method for measuring glycated hemoglobin by an HPLC method, which comprises using the sample diluent according to any one of claims 1 to 5. The sixth aspect of claim 6, wherein the buffer in the sample diluent is N-cyclohexyl-2-aminoethanesulfonic acid, and a zwitterionic compound having an amino group and a sulfo group is used as the buffer for the eluent used in the HPLC method. Method for measuring saccharified hemoglobin. The method for measuring saccharified hemoglobin according to claim 7, wherein the zwitterionic compound having an amino group and a sulfo group is at least one selected from 2-morpholinoetan sulfonic acid and 3-morpholinopropane sulfonic acid.

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