JPH10227779A - Measuring method for saccharogenic hemoglobin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily separate HbA1c peak, by adding ion pair reagent to one of eluate, dilution solution of a sample, and washing solution. SOLUTION: Buffer solution for cation exchange liquid chromatography, such as phosphoric acid, hydrochloric acid, or acetic acid, is used as eluate, and aqueous solution containing surface active agent such as triton X-100 is used as dilution solution of a sample or washing solution. Onium ion salt such as tetra butyl ammonium chloride, tetra butyl ammonium bromide, or tetra butyl ammonium phosphate is used as ion pair reagent. Contents of added ion pair reagent to at least one of eluate, dilution solution of a sample, and washing solution is about 0.1-10mM. By containing ion pair reagent, cation exchange reaction of a sample with filler can be adjusted without changing kind of filler, and separating ability of HbA1c peak for saccharogenic hemoglobin is easily improved.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring glycated hemoglobin by cation exchange liquid chromatography.

[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. Also,
As an eluent for this measurement, a buffer for general cation exchange liquid chromatography or the like is used.

[0004]

The HbA1 in this blood
In the cation exchange liquid chromatography for measuring the c concentration, the HbA1c fraction is adjacent to other glycated hemoglobin peaks, and depending on the blood sample, other peaks may overlap with the HbA1c peak, making accurate measurement impossible. There was a problem. However, it is very difficult to solve this problem by improving the packing material, and it is also difficult to solve this problem by improving the measurement conditions of cation exchange liquid chromatography.

The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a method for measuring glycated hemoglobin, particularly, glycated hemoglobin in which the separation of the HbA1c peak is easily improved using a conventional filler. It is in.

[0006]

According to the method for measuring glycated hemoglobin of the present invention, a cation exchange liquid is characterized in that at least one selected from an eluent, a sample diluent and a washing liquid contains an ion pair reagent. This is a method for measuring glycated hemoglobin by chromatography.

As the filler 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. Examples of the base material include polymer base materials (for example, synthetic polymer fine particles such as polystyrene and polyacrylate; natural polymer fine particles such as polyamino acids and polysaccharides), and inorganic base materials (for example, silica particles). Is mentioned. 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. Method. The average particle size of the filler is preferably 1 to 20 μm.

[0008] As the liquid chromatograph system used in the present invention, a known liquid chromatograph system is used. That is, for example, a system as shown in FIG. The eluent 1 passes through a switching mechanism (not shown), enters a separation column 4 filled with a filler via a sample introduction device 3 by a liquid sending pump 2, and the separation column 4 causes each component in the sample to flow. Are separated. The separated components are detected by the detector 5, for example, by measuring the absorbance.

The specific conditions of the liquid chromatography measurement are as follows: the flow rate is about 0.2 to 5 ml / min;
An absorbance measurement in nm is appropriate. The measurement sample is hemolyzed blood. For example, whole blood collected from a blood collection tube containing a blood anticoagulant (for example, sodium fluoride) is converted into a hemolytic agent (for example, an interface such as Triton X-100). Hemolysis about 50-300 times with a buffer solution containing an activator
Diluted ones can be mentioned. The injection amount of the measurement sample into the liquid chromatograph is suitably about 10 to 300 μl.

In the present invention, as for the elution conditions of the liquid chromatography, a buffer for general cation exchange liquid chromatography or the like is used as the eluent.
Examples thereof include inorganic acids and inorganic bases such as phosphoric acid, hydrochloric acid, acetic acid, sodium hydroxide and potassium hydroxide or salts thereof; organic acids such as citric acid and phthalic acid and salts thereof; and mixtures thereof.

As the ion pair reagent used in the present invention, a known reagent capable of forming an ion pair in a solution is used. For example, tetrabutylammonium chloride,
Onium ion salts such as tetrabutylammonium bromide and tetrabutylammonium phosphate; alkylsulfonic acids such as 1-hexasulfonic acid, 1-octasulfonic acid, and 1-pentanesulfonic acid; and sodium salts thereof.

The method for measuring glycated hemoglobin of the present invention comprises:
At least one selected from an eluent, a sample diluent, and a washing solution contains an ion pair reagent. The sample diluent also includes a sample lysate. The above-mentioned cleaning liquid refers to a liquid for cleaning a channel or the like of a liquid chromatograph system. The ion pair reagent may be added to at least one selected from an eluent, a sample diluent, and a washing liquid, as well as any of the reagents used in analysis and measurement. In order to stabilize the interaction between the reagent including the ion pair reagent and the filler, it is most preferable to add the reagent to the eluent. When a plurality of eluents are used, it is necessary to add the eluent to elute HbA1c, but in this case, it may be added to another eluent.

As the above eluent, sample diluent and washing solution, there may be mentioned known ones conventionally used for liquid chromatography analysis. As the eluent,
For example, the buffer for cation exchange liquid chromatography described in the above description of the elution conditions may be used. As a sample diluent, for example, Triton X-1
An aqueous solution containing a surfactant such as 00; a buffer solution comprising a substance constituting the above eluent; As the above-mentioned washing liquid, the same as the above-mentioned diluting liquid can be mentioned.

The content of the ion pair reagent contained in at least one selected from an eluent, a sample diluent, and a washing solution varies depending on a filler and a solution composition.
0.1-10 mM is preferred.

In the present invention, the ion exchange reagent is contained in at least one selected from the eluent, the sample diluent and the washing solution, so that the cation exchange reaction between the sample and the filler can be performed without changing the type of the filler. Thus, the present invention provides a method for easily measuring glycated hemoglobin, in particular, glycated hemoglobin with improved HbA1c peak resolution, since it can be finely adjusted.

[0016]

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 Preparation of packed column A cation exchange liquid chromatography packing was prepared by the method described in Japanese Patent Publication No. 8-7197. That is, 100 g of diethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and 2 parts of divinylbenzene (manufactured by Kishida Chemical Co., Ltd.)
1 g of benzoyl peroxide (polymerization initiator, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in the mixture of 00 g. This mixture is mixed with 4% by weight polyvinyl alcohol (manufactured by Nippon Gohsei).
The solution was added to 2.5 liters of an aqueous solution, and the mixture was sized with stirring, and then heated to 80 ° C. under nitrogen replacement to carry out polymerization. 80 ℃
After polymerization 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 in that order and dried. The obtained particles are subjected to an air classifier (Turbo Classifier TC-15N, manufactured by Nippon Steel Mining Co., Ltd.).
To obtain filler particles having an average particle size of 10 μm.
The filler was packed in a stainless steel column having an inner diameter of 6 mm and a length of 75 mm.

FIG. 1 shows a liquid chromatograph system.
System. The liquid sending pump 2 is made of Shimadzu Corporation, LC-9A, the sample introduction device 3 is made of Sekisui Chemical Co., Ltd., ASU-420, and the detector 5 is made of Shimadzu Corporation, SPD-6AV. did.

Measurement conditions of liquid chromatography Elution conditions: eluent A (50 mM phosphate buffer containing 1-pentanesulfonic acid at a concentration of 5 mM: pH 6) and eluent B (300 mM phosphate buffer: pH 7) A step gradient elution using the liquid was performed. Flow rate: 2.0 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-100 was added thereto.
100 mM phosphate buffer (pH 7) containing
Sample 1 was hemolyzed and diluted 50-fold. Sample 2 (carbamylated hemoglobin (hereinafter, referred to as 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. The mixture was warmed at 40 ° 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. 2 shows a chromatogram when Sample 1 was analyzed under the above measurement conditions. In this chromatogram, peak 1 is glycated hemoglobin HbA1
a and HbA1b, peak 2 are fetal hemoglobin Hb
F, peak 3 is glycated hemoglobin HbA1c, peak 4
Is non-glycated (normal) hemoglobin HbA0. HbA
The 1c value is 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. 3 shows a chromatogram when Sample 2 was analyzed under the above measurement conditions. In this chromatogram, peak 1 is glycated hemoglobin HbA1a
And HbA1b, peak 2 is fetal hemoglobin Hb
F, peak 3 is glycated hemoglobin HbA1c, peak 4
Is non-glycated (normal) hemoglobin HbA0, peak 5 is C
Hb. The HbA1c value is 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

The results of measuring the HbA1c values of Sample 1 and Sample 2 were 5.2% for Sample 1 and 5.1% for Sample 2. That is, HbA is not affected by CHb.
It can be seen that the 1c value could be measured.

Comparative Example 1 In Example 1, instead of eluent A (50 mM phosphate buffer containing 1-pentanesulfonic acid at a concentration of 5 mM: pH 6) in the section of the measurement conditions of liquid chromatography in Example 1, 1- 50 mM without pentanesulfonic acid
HbA1c values of Sample 1 and Sample 2 were determined in the same manner as in Example 1 except that phosphate buffer: pH 6 was used.

Calculation of HbA1c value: FIG. 4 shows a chromatogram when sample 1 was analyzed, and FIG. 5 shows a chromatogram when sample 2 was analyzed. Each HbA1
The c value was 5.1% for sample 1 and 3.7% for sample 2. That is, in Sample 2, the HbA1c value was significantly reduced. As can be seen from the chromatogram shown in FIG. 5, in sample 2, CHb peak 5 was HbA1c peak 3
And the HbA1c value was reduced.

[0025]

The structure of the method for measuring glycated hemoglobin of the present invention is as described above. If this method is used, glycated hemoglobin, particularly Hb, can be easily prepared using a conventional filler.
A1c can be measured accurately.

[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 a chromatogram obtained using Sample 1 in Example 1.

FIG. 3 is a chromatogram obtained using Sample 2 in Example 1.

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

FIG. 5 is a chromatogram obtained using Sample 2 in Comparative Example 1.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Eluent 2 Liquid sending pump 3 Sample introduction device 4 Separation column 5 Detector

Claims (1)

[Claims] 1. A method for measuring glycated hemoglobin by cation exchange liquid chromatography, wherein at least one selected from an eluent, a sample diluent and a washing solution contains an ion pair reagent.