JPH10227780A - Method and device for measuring saccharification hemoglobin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve measurement accuracy of a HbA1C peak of saccharogenic hemoglobin, by using affinity chromatography filler and cation exchange chromatography filler. SOLUTION: As affinity chromatography filler, material provided by fixing boric acid derivative as ligand in the polymer based or inorganic compound based base material is used. As cation exchange chromatography filler, material provided by adding functional group such as carboxylato group or sulfo group which has cation exchange ability to polymer based or inorganic compound based base material is used. Average size of each filler is about 1-20μm. Both fillers are sequentially respectively charged into their columns, and valves are installed in front and back sides of the column which is filled with cation exchange chromatography filler. Thus, HbA1C peak which can not separated by affinity chromatography is accurately measured without suffering from effect of denatured hemoglobin which causes a problem to the cation exchange chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method and an apparatus for measuring glycated hemoglobin by liquid chromatography, and more particularly to a method and an apparatus for measuring glycated hemoglobin A1c.

[0002]

2. Description of the Related Art Glycated hemoglobin is formed by sugar in blood, which enters red blood cells in proportion to its concentration and then binds to hemoglobin. It is said to reflect the average sugar concentration. Since the blood sugar level and the urine sugar level are less affected by physiological factors, the concentration of glycated hemoglobin in the blood is used as an index for diagnosing diabetes. In particular, among glycated hemoglobin, glycated hemoglobin A1c (hereinafter, referred to as HbA1c) is used. ) Fractionation is widely used as an optimal indicator for the diagnosis of diabetes.

[0003] The concentration of glycated hemoglobin in blood is mainly measured by a cation exchange liquid chromatography method (see Japanese Patent Publication No. 8-7198). The concentration of HbA1c in the blood is defined as the percentage (%) of the HbA1c peak area when the total hemoglobin peak (glycated hemoglobin peak + non-glycated hemoglobin peak) area in a chromatogram obtained by subjecting a blood sample to cation exchange liquid chromatography is 100. ). In recent years, this measurement method has been shortened in measurement time, and measurement has been performed in several minutes per sample.

[0004] A method of separating glycated hemoglobin and non-glycated hemoglobin by affinity chromatography is also known (see JP-A-3-152455). In this method, a specific boronic acid derivative is used as a ligand. By using this method, glycated hemoglobin and non-glycated hemoglobin are separated, for example,
There is an advantage that HbA1c can be measured without being affected by denatured hemoglobin such as carbamylated hemoglobin (hereinafter, referred to as CHb) and acetylated hemoglobin (hereinafter, referred to as AHb).

[0005]

However, the cation exchange liquid chromatography method has a problem that the measured value of HbA1c is easily affected by denatured hemoglobin such as CHb and AHb. In the method using affinity chromatography, HbA1
There is a problem that glycated hemoglobin other than c is also measured.

[0006] The present invention has been made to solve the above-mentioned problems, and its object is to eliminate the influence of denatured hemoglobin, which has been a problem in cation exchange chromatography, and to prevent the separation by affinity chromatography. Glycated hemoglobin, especially Hb
An object of the present invention is to provide a method and an apparatus for measuring glycated hemoglobin, which can accurately measure A1c.

[0007]

The method for measuring glycated hemoglobin according to claim 1 of the present invention is characterized by using a packing material for affinity chromatography and a packing material for cation exchange chromatography.

[0008] The method for measuring glycated hemoglobin according to claim 2 of the present invention comprises the steps of: using a filler for affinity chromatography and a filler for cation exchange chromatography;
It is characterized by being used in this order.

In the method for measuring glycated hemoglobin according to claim 3 of the present invention, the sample is separated by a packing for affinity chromatography, and the component adsorbed on the packing for affinity chromatography is filled with the packing for cation exchange chromatography. It is characterized in that it is separated by an agent.

The apparatus for measuring glycated hemoglobin according to the present invention comprises a column packed with a packing material for affinity chromatography and a column packed with a packing material for cation exchange chromatography. Characterized in that valves are provided before and after the column packed with.

The packing material for affinity chromatography used in the present invention is not particularly limited as long as it is a packing material capable of adsorbing a sugar component. For example, a ligand in which a boronic acid derivative (such as m-aminophenylboronic acid) is immobilized on a substrate can be used. As the base material, a polymer base material (for example, synthetic polymer fine particles such as polystyrene and polyacrylate; polyamino acid,
Natural polymer fine particles such as polysaccharides); and inorganic base materials (eg, silica particles). The average particle size of the filler is preferably 1 to 20 μm.

As the filler for cation exchange chromatography used in the present invention, a known cation exchange filler is used. For example, a filler in which a functional group having a cation exchange ability such as a carboxyl group or a sulfonic acid group is introduced into particles serving as a base material may be used. As the base material, a polymer base material (for example, synthetic polymer fine particles such as polystyrene and polyacrylate; polyamino acid,
Natural polymer fine particles such as polysaccharides); and inorganic base materials (eg, silica particles). Examples of a method for introducing the functional group include, for example, polymerizing a monomer having the functional group as a material; preparing particles serving as a base material, and bonding a low molecular compound having a functional group by a chemical reaction. There is a way. The average particle size of the filler is preferably 1 to 20 μm.

The liquid chromatograph system used in the present invention will be described later, but the flow rate of the liquid chromatographic measurement condition is 0.2 to 5 ml / min.
For the detection of glycated hemoglobin and non-glycated hemoglobin, the absorbance at 415 nm is appropriate. The measurement sample is hemolyzed blood, for example, blood is collected by a blood collection tube containing a blood anticoagulant (for example, sodium fluoride or the like) (or a blood anticoagulant may be added after blood collection). ) Whole blood was lysed with a hemolytic agent (Triton X-100).
Hemolyzed and diluted about 50 to 300 times with a buffer solution containing a surfactant such as a surfactant. The injection amount of the measurement sample into the liquid chromatograph varies depending on the detection sensitivity, but usually about 10 to 300 μl is appropriate.

As the liquid chromatography system used in the present invention, the following three systems are exemplified. The first system is the system shown in FIG. 1, in which an eluent 1 passes through a switching mechanism (not shown), is passed through a sample introduction device 3 by a liquid sending pump 2, and is supplied with a packing material for affinity chromatography (hereinafter referred to as an affinity chromatography material). , A column for AFC) (hereinafter, referred to as AFC column 4), and then a column 5 for cation exchange chromatography (hereinafter, referred to as CEC filler). Hereinafter, the column is referred to as CEC column 5).
Each component in the sample is separated. The separated components are detected by the detector 6, for example, by measuring the absorbance.

The above system is described in more detail. (1) First, an eluent A for adsorbing glycated hemoglobin is passed through the AFC column 4. (2) Next, a sample is injected from the sample introduction device 3. (3) The glycated hemoglobin component including HbA1c in the sample is AFC
Adsorbed on the column 4 for non-glycated hemoglobin
Immediately move to column 5 for C. (4) Switch to the eluent B to desorb the glycated hemoglobin component adsorbed on the AFC column 4. (5) The detached glycated hemoglobin component is led to the CEC column 5 and is sequentially eluted by a cation exchange reaction. (6) The eluted glycated hemoglobin component is guided to the detector 6 in the order of elution.

In the first system, the eluent A is generally a high performance liquid chromatography (hereinafter referred to as H
For example, a buffer used for PLC), for example, an inorganic salt buffer such as phosphate is used. The pH of the eluent A is preferably from 7 to 11.

The eluent B is composed of a buffer containing a component having a higher affinity for boronic acid than the sugar component adsorbed on the AFC filler. Such components include, for example, sorbitol and tris (hydroxymethyl) aminomethane. An appropriate pH of the eluent B is 4 to 7.

The second system is the system shown in FIG. 2, in which a three-way valve 7 and a three-way valve 8 are provided before and after the CEC column 5 in the first system. In FIG. 2, the ports indicated as a, b, and c on the three-way valve 7 and the three-way valve 8 indicate the ports of each valve.

The second system will be described in more detail. First, (1) an eluent C for adsorbing glycated hemoglobin is passed through the AFC column 4. (2)
Next, a sample is injected from the sample introduction device 3. At this time, the ports of the three-way valve 7 and the three-way valve 8
-7c; 8c-8b are connected. (3)
The glycated hemoglobin component including HbA1c in the sample is adsorbed to the AFC column 4, and the non-glycated hemoglobin component is guided to the detector 6 through another flow path 9 (without passing through the CEC column 5). (4) After the non-glycated hemoglobin component has passed through the three-way valve 7 and the three-way valve 8, the ports of the three-way valve 7 and the three-way valve 8 are switched, and 7a
-7b; 8a-8b are connected. (5) Switch to the eluent D to desorb the glycated hemoglobin component adsorbed on the AFC column 4. (6) The desorbed glycated hemoglobin component is led to the CEC column 5 through the three-way valve 7 and is sequentially eluted by a cation exchange reaction.
(7) The eluted glycated hemoglobin component is guided to the detector 6 in the order of elution.

The eluent C is the same as the eluent A, and the eluent D is the same as the eluent B.

The feature of the second system is that the three-way valve 7 is provided before and after the CEC column 5 in the first system.
By providing the three-way valve 8, the non-glycated hemoglobin component is detected without passing through the CEC column 5.
Thereby, the non-glycated hemoglobin component is prevented from staying in the CEC column 5, and a more rapid analysis becomes possible. The valve is not limited to the three-way valve as long as it has a function of changing the flow path of the eluent as described above.

The third system is the system shown in FIG. 3. In the first system and the second system, the AFC packing material and the CEC packing material are packed in different columns. , In the third system, the two packings are combined into one packing column 10.
It is characterized by being filled inside.

The third system will be described in more detail. First, (1) AFC in the filler mixing column 10
The eluent E to which glycated hemoglobin is adsorbed is flowed through the packing material. (2) Next, the sample is introduced into the sample introduction device 3
Inject more. (3) The glycated hemoglobin component including HbA1c in the sample is mixed with the AF in the filler mixing column 10
Adsorbed to C filler. (4) On the other hand, the non-glycated hemoglobin component passes through the filler mixture column 10 and is guided to the detector 6. (5) Switch to the eluent F to desorb the glycated hemoglobin component adsorbed on the AFC packing material in the packing material mixing column 10. (6) The desorbed glycated hemoglobin component elutes while interacting with the CEC packing material in the packing material mixing column 10, and is guided to the detector 6.

In the third system, as for the eluent E, the non-glycated hemoglobin component (mostly hemoglobin A0) is added to the AFC filler in the filler mixing column 10 as described in (4) above. Is not adsorbed. However, hemoglobin A0 (hereinafter referred to as HbA0) is most strongly retained by the CEC filler. On the other hand, the glycated hemoglobin component is hardly retained by the filler for CEC. That is, C
When a column containing only the EC packing material is used, the HbA0 elution order is reversed as compared with the case where only the AFC packing material is used. Therefore, as described in (4) above, in order for the non-glycated hemoglobin component to be rapidly eluted by the present method, it is necessary to introduce the sample into the column under conditions where the non-glycated hemoglobin component is not easily retained by the CEC packing material. is there. That is, it is necessary to use an eluent E such that the glycated hemoglobin component is retained in the filler for AFC and the non-glycated hemoglobin component is not retained in the filler for CEC. Therefore, the HbA0 component is eluted without being held by the CEC filler at a specific ionic strength and pH, and the eluent E is set to such conditions. The above-mentioned specific ionic strength and pH vary depending on the combination thereof, and also vary depending on the ability to retain the filler, but generally, the pH is preferably about 5 to 10.

The method for producing the above-mentioned filler mixture column 10 is as follows.
There is no particular limitation, for example, AF before filling the column.
Filling after mixing C filler and CEC filler, AFC filler at eluent inlet side, CEC at outlet side
And a method of dividing and filling so that the filler for use is arranged. The mixing ratio of the two fillers differs depending on each filler. The average particle size of both is preferably as close as possible. As can be said of the first system and the second system, particularly in the third system, since both are packed in one column, the mixing of fillers having significantly different average particle diameters is not possible. This is not preferable because it induces void volume and causes a decrease in separation ability.

[0026]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to examples and comparative examples of the present invention, but the present invention is not limited thereto.

Example 1 ASK packed column TSKgel Boronate-5PW (inner diameter 7.5
mm × length 75 mm, manufactured by Tosoh Corporation).

A column containing a CEC filler was prepared by the method described in JP-B-8-7197. That is, a mixture of 100 g of diethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and 200 g of divinylbenzene (manufactured by Kishida Chemical Co., Ltd.) was mixed with benzoyl peroxide (polymerization initiator, manufactured by Wako Pure Chemical Industries, Ltd.).
g was dissolved. This mixture was added to 2.5 liters of a 4% by weight aqueous solution of polyvinyl alcohol (manufactured by Nippon Gosei),
After sieving with stirring, the mixture was heated to 80 ° C. under nitrogen replacement to perform polymerization. After polymerization at 80 ° C. for 2 hours, 50 g of acrylic acid was added, and polymerization was further performed at 80 ° C. for 2 hours. The product was washed with hot water and acetone sequentially and dried. The obtained particles were classified by an air classifier (Turbo Classifier TC-15N, manufactured by Nittetsu Mining Co., Ltd.), and the average particle size was 10 μm.
Was obtained. Filler with inner diameter 6mm, length 75
A column with a packing material for CEC was manufactured by packing in a column made of stainless steel having a diameter of 2 mm.

Measurement Conditions for Liquid Chromatography The amount of HbA1c in the sample was measured by the second system shown in FIG. The liquid sending pump 2 is LC-9A manufactured by Shimadzu Corporation, and the sample introduction device 3 is ASU-420 manufactured by Sekisui Chemical Co., Ltd.
Was SPD-6AV manufactured by Shimadzu Corporation.

The measurement conditions of the liquid chromatography were as follows. Elution conditions: Eluent C [20 mM HEPES (N-2-
Hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer: pH 8) and eluent D (HEPES buffer containing sorbitol at a concentration of 100 mM: pH 6)
Step gradient elution was carried out using the two solutions. Flow rate: 1.2 ml / min Detection: 415 nm The following samples 1 and 2 were used as samples. Sample 1 (healthy human blood): Blood was collected from a healthy human using a blood collection tube containing sodium fluoride, and 0.1% Triton X-10 was added thereto.
Sample 1 was prepared by adding 100 mM phosphate buffer (pH 7) containing 0 and hemolyzing and diluting 150-fold. Sample 2 (CHb-containing sample): A sample containing CHb at a high concentration was prepared as a sample containing denatured hemoglobin. That is, 0.5 ml of a 0.2 mg / dl sodium cyanate physiological saline solution was added to 0.5 ml of healthy human blood collected as in Sample 1 above. This mixture is
Warmed at 2 ° C. for 2 hours. After cooling, the sample was hemolyzed and diluted in the same manner as in Sample 1 above to obtain Sample 2.

Measurement of HbA1c Samples 1 and 2 were measured to determine the HbA1c value. Calculation of HbA1c value: FIG. 4 shows a representative chromatogram when the sample 1 was analyzed under the above measurement conditions. In this chromatogram, peak 1 is glycated hemoglobin HbA1a and HbA1b, peak 2 is HbA1c, and peak 3 is HbA0. The HbA1c value is determined from each peak area by the following formula. HbA1c (%) = [(area of HbA1c) / (HbA
0 area + HbA1a and HbA1b area + HbA1
area of c)] × 100

As a result of measuring Sample 1 and Sample 2 under the above measurement conditions, the HbA1c value of Sample 1 was 4.8%,
The HbA1c value of Sample 2 was 4.7%. That is,
Since there was almost no difference between the measured values of Sample 1 and Sample 2, the HbA1c value of Sample 2 could be measured without being affected by CHb. The chromatogram shapes of Sample 1 and Sample 2 are as shown in FIG. 4, and it is considered that CHb in Sample 2 was eluted overlapping HbA0 peak 3.

Comparative Example 1 Measurement Conditions of Liquid Chromatography The same CEC as used in Example 1 was used as the filler mixing column 10 in the third system shown in FIG.
Column packed with only packing material (ie, AFC
The HbA1c value of Samples 1 and 2 was measured in the same manner as in Example 1 using a system (containing no filler for liquid) (the liquid sending pump 2, the sample introduction device 3 and the detector 6 were the same as in Example 1). Was.

The measurement conditions for liquid chromatography were as follows. Elution conditions: Eluent G (50 mM phosphate buffer: pH 6)
Step gradient elution was performed using two liquids, eluent H (300 mM phosphate buffer: pH 7). Flow rate: 1.2 ml / min Detection: 415 nm

Results of measurement of HbA1c Calculation of HbA1c value: FIG. 5 shows a typical chromatogram when the sample 1 was analyzed under the above measurement conditions. In this chromatogram, peak 1 is glycated hemoglobin HbA1a and HbA1b, peak 2 is HbA1c, peak 3 is HbA0, and peak 4 is fetal hemoglobin HbF. The HbA1c value was determined from each peak area by the following formula. HbA1c (%) = [(area of HbA1c) / (HbA
Area of 1a and HbA1b + area of HbF + HbA1c
Area + HbA0 area)] × 100

FIG. 6 shows a typical chromatogram when Sample 2 was analyzed under the above measurement conditions. In this chromatogram, peak 1 is glycated hemoglobin Hb
A1a and HbA1b, peak 2 is HbA1c, peak 3 is HbA0, peak 4 is HbF, and peak 5 is CH
b. The HbA1c value was determined from each peak area by the following formula. HbA1c (%) = [(area of HbA1c) / (HbA
1a and HbA1b area + HbF area + CHb area + HbA1c area + HbA0 area)] × 100

As a result of measuring Sample 1 and Sample 2 under the above measurement conditions, the HbA1c value of Sample 1 was 4.8%,
The HbA1c value of Sample 2 was 3.2%. In sample 2, CHb peak 5 overlaps HbA1c peak 2 as seen in FIG. Therefore, HbA1c of sample 2
It is considered that the value was greatly reduced.

[0038]

The structure of the method for measuring glycated hemoglobin according to claim 1 of the present invention is as described above. When this method is used, it is affected by denatured hemoglobin, which has been a problem in cation exchange chromatography. Glycated hemoglobin, particularly HbA1c, which could not be separated without affinity chromatography
Can be accurately measured.

The structure of the method for measuring glycated hemoglobin according to the second aspect of the present invention is as described above. With this method, it is possible to eliminate the influence of denatured hemoglobin which is a problem in cation exchange chromatography. Hemoglobin, especially HbA1c, which could not be separated by affinity chromatography
Can be measured more accurately.

The structure of the method for measuring glycated hemoglobin according to the third aspect of the present invention is as described above. When this method is used, the method is not affected by denatured hemoglobin, which is a problem in cation exchange chromatography. Hemoglobin, especially HbA1c, which could not be separated by affinity chromatography
Can be measured with higher accuracy.

The configuration of the apparatus for measuring glycated hemoglobin of the present invention is as described above. When this apparatus is used, the apparatus is not affected by denatured hemoglobin, which has been a problem in cation exchange chromatography, and has affinity chromatography. Glycated hemoglobin, particularly HbA1c, which could not be separated by chromatography, can be measured accurately and more quickly.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing an example of a liquid chromatograph system used in the present invention.

FIG. 2 is an explanatory diagram showing another example of the liquid chromatography system used in the present invention.

FIG. 3 is an explanatory view showing another example of the liquid chromatography system used in the present invention.

FIG. 4 is a chromatogram obtained using Sample 1 in Example 1.

FIG. 5 is a chromatogram obtained using Sample 1 in Comparative Example 1.

FIG. 6 is a chromatogram obtained using Sample 2 in Comparative Example 1.

[Explanation of symbols]

REFERENCE SIGNS LIST 1 eluent 2 liquid sending pump 3 sample introduction device 4 column filled with packing for affinity chromatography 5 column packed with packing for cation exchange chromatography 6 detector 7, 8 three-way valve 9 other flow path 10 Packing column

Claims (4)

[Claims] 1. A method for measuring glycated hemoglobin, which comprises using a packing material for affinity chromatography and a packing material for cation exchange chromatography. 2. A packing material for affinity chromatography and a packing material for cation exchange chromatography,
A method for measuring glycated hemoglobin, which is used in this order. 3. A method for measuring glycated hemoglobin, comprising separating a sample with a packing material for affinity chromatography, and then separating components adsorbed on the packing material for affinity chromatography with a packing material for cation exchange chromatography. Method. 4. A column filled with a packing material for affinity chromatography and a column packed with a packing material for cation exchange chromatography, wherein a valve is provided before and after the column filled with the packing material for cation exchange chromatography. A device for measuring glycated hemoglobin, comprising: