JPH09127091A - Method for measuring saccarification hemoglobin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measurement method by the liquid chromatography for quickly measuring saccarification hemoglobin with further improved separation. SOLUTION: A sample containing saccarification hemoglobin is filled into a column for liquid chromatography where a filler (A) for cation exchange liquid chromatography (for example, acrylic) polymer with COOH group) and a filler for cation exchange liquid chromatography (B) (for example, styrenic polymer with SO3 H group) for improving chromatogram for saccarification hemoglobin are filled into one column, thus performing liquid chromatography.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring glycated hemoglobin by a liquid chromatography method.

[0002]

Glycated hemoglobin is formed by the non-enzymatic reaction of hemoglobin in red blood cells with glucose in blood. Since the average concentration of glucose in blood can be determined by measuring glycated hemoglobin, the measurement of glycated hemoglobin is widely used for the diagnosis of diabetes. This glycated hemoglobin is mainly measured by HPLC (high performance liquid chromatography). This HP
In LC, a weak cation ion-exchange liquid chromatography packing material is used. For example, JP-A-58-58
No. 221164 discloses a filler made of a copolymer of acrylic acid or methacrylic acid ester and acrylic acid or methacrylic acid, and in JP-A-2-309253, the surface portion of a hydrophobic crosslinked polymer particle is A filler made of coated polymer particles having a layer of acrylic acid and / or methacrylic acid (co) polymer formed thereon is disclosed.

When a sample obtained by hemolyzing blood is introduced into the above chromatography, glycated hemoglobin A _1a (HbA _1a ) and hemoglobin A _1b (Hb) are introduced.
A _1b ), hemoglobin F (HbF) and hemoglobin A
Peaks such as _1c (HbA _1c ) and hemoglobin A ₀ (HbA ₀ ) which are non-glycated hemoglobin appear. Among these peaks, it is effective in the diagnosis value diabetes HbA _1c, measurement of HbA _1c is to the sum of the areas of all peaks above, expressed as a percentage of the area of the peaks of HbA _1c, in healthy individuals It is set to about 4 to 6%.

Therefore, when the filler as disclosed above is used, glycated hemoglobin can be satisfactorily measured, and the result can be widely used for the diagnosis of diabetes. In particular, further improvement of the chromatogram such as the separability of the HbA _1c peak has been desired.

In HPLC, when it is desired to improve the chromatogram such as the separability of each peak and the reduction of the retention time of each peak, there are various methods such as changing the kind of eluent, measuring temperature or column size. The improvement of the filler is most effective. However, it is complicated and not easy to newly prepare a packing material suitable for the purpose and improve the chromatogram by using the packing material.

Further, as one method for simply improving the analysis method of liquid chromatography, a method using a column in which two kinds of packing materials are packed in one column is disclosed. For example, in JP-A-6-273401, a column having a packing material having a cation exchange group and a packing material having an anion exchange group is packed in one column, and an alkaline solution and an acidic solution are used as a mobile phase. , A method of analyzing cations and anions by ion exchange liquid chromatography, in which both of these solutions are switched and passed through a column and each ionic species is separately eluted for analysis, which is disclosed. It aims at simplifying the method by analyzing both ion species without exchanging the column, the chromatogram of the sample to be measured has not been improved, and even if it is applied to the chromatographic measurement of glycated hemoglobin. Not in.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and an object of the present invention is to provide a measuring method by a liquid chromatography method capable of measuring glycated hemoglobin with better separability in a short time. To provide.

[0008]

The method for measuring glycated hemoglobin according to claim 1 (hereinafter, the invention according to claim 1 is referred to as the present invention 1) comprises:
Cation exchange liquid chromatography packing material (A)
And a packing material (B) for cation exchange liquid chromatography, which is different from the packing material (A) and can improve the chromatogram of the packing material (A) with respect to the glycated hemoglobin, are packed in one column. It is characterized in that it is introduced into a column for liquid chromatography as described above to perform liquid chromatography.

The improvement of the chromatogram referred to in the present invention 1 means, for example, the separability of each peak, the shortening of the retention time of each peak, and the sharpness of the peak.

The packing material (A) used in the present invention 1 is a packing material which mainly acts on the separation of glycated hemoglobin, and is limited to the packing material for cation exchange liquid chromatography.

The filler (A) is not particularly limited as long as it is a filler for cation exchange liquid chromatography, and any known filler can be used. The packing material for cation exchange liquid chromatography is a packing material used for the above-mentioned cation exchange liquid chromatography, and, for example, as a functional group, —COOH, —SO ₃ H
And the like. The material of the skeleton of the filler (A) is not particularly limited, either organic type or inorganic type can be used. As organic type, synthetic type,
Any of natural type may be used. As the synthetic material,
For example, acrylic-based and styrene-based polymers are listed.
Examples of the natural material include cellulose-based materials, chitosan-based materials, polyamino acids, and the like, and inorganic-based materials include silica, zirconia, and the like.

The packing material (B) used in the present invention 1 is different from the packing material (A) described above and for cation exchange liquid chromatography capable of improving the chromatogram of the packing material (A) against glycated hemoglobin. Limited to fillers. As the selection range of the above-mentioned packing material (B), any packing material of the known packing materials for cation exchange liquid chromatography can be used as long as it is within the limited range as described above. Include those exemplified as the above-mentioned filler (A). Further, the filler (B) is 2
You may mix and use 1 or more types.

A combination of the filler (A) and the filler (B)
The functional groups of the filler (A) and the filler (B) are different.
There are combinations, specifically, fillers having COOH groups
SO _ThreeCombination of fillers with H groups, also filler (A)
There is a combination in which the basic skeleton of the filler and the filler (B) is different.
Physically, COOH group-containing polymeric filler and COOH
Having a COOH group, in combination with a silica-based filler having a group
Acrylic polymer filler and styrene with COOH group
Combination of polymer filler, high acrylic resin with COOH group
In the combination of molecular fillers, acrylic polymer
Polymer derived from acrylic acid and polymer derived from methacrylic acid
And the like.

As a combination of the filler (A) and the filler (B), the filler (A) and the filler (B) have the same functional groups and the same basic skeleton. The case where A) and the filler (B) are not the same is also included. In such a case, for example, even if the same material is used, the amount used is different, or in the case of a polymer material and the polymerization conditions (polymerization temperature, time, etc.) are different, the formulation is different. If they are different, they are not the same, and a combination of such substances can improve the chromatogram of the packing material (A) with the packing material (B).

The ratio of the amount of the filler (A) to the amount of the filler (B) used is different depending on the kind of the filler used, but usually, the filler in the total amount of the filler (A) and the filler (B) is used. The content of (B) is preferably 50% by weight or less.

The column used in the present invention 1 is not particularly limited as long as it has been conventionally used as a column for liquid chromatography, but the diameter is 1 to 10 m.
A stainless tube having a length of m and a length of 20 to 100 mm is preferable.

In order to manufacture the column for liquid chromatography used in the present invention 1, one column may be packed with the packing material (A) and the packing material (B). As the packing order, the packing (A) and the packing (B) may be mixed and then packed, or packed separately (in this case, the packing is present in the column in a multilayer state). You may. A wet filling method is preferable as the filling method. Specifically, in the eluent used for analysis, etc., disperse an amount of packing material suitable for the column size and transfer to a packer, connect a pump to the inlet side of the packer and a column to the outlet side, and set a constant flow rate or constant flow rate. A method of filling by pressure-feeding the liquid may be mentioned.

Examples of the sample containing glycated hemoglobin measured in the present invention 1 include a sample obtained by hemolyzing blood. Examples of the method for collecting a sample include a method in which blood is collected using an anticoagulant and then hemolyzed with a hemolytic reagent containing a surfactant. Also, usually, the sample hemolyzed blood, as hemoglobin A _1c, but are other unstable hemoglobin A _1c stable hemoglobin A _1c, in order to utilize the hemoglobin A _1c as an indicator of diabetes, anxiety Since the inclusion of the fixed form hemoglobin A _1c is not preferable in terms of accuracy, it can be removed. The unstable hemoglobin A _1c can be efficiently removed in a short time by adding an unstable hemoglobin A _1c removing reagent such as a polyphosphoric acid aqueous solution to the sample and heating.

The method for measuring glycated hemoglobin of the present invention 1 is the same as the method for measuring glycated hemoglobin by the conventional liquid chromatography method except that the above-mentioned column for liquid chromatography is used as the column used for the measurement. Is. Hereinafter, the method for measuring glycated hemoglobin of the first invention will be described in detail.

FIG. 1 shows an example of the construction of an apparatus for analyzing glycated hemoglobin using the above-mentioned liquid chromatography column. While supplying the mobile phase 2 to the liquid chromatography column 1 by the liquid feed pump 3, the sample containing the glycated hemoglobin is introduced from the sampler 4. The sample separated by the liquid chromatography column 1 is detected by the detector 5, and the result is recorded in the recorder 6.

As the above mobile phase, a buffer having a pH of 5 to 8 is preferable, and a buffer containing an inorganic acid (such as phosphoric acid), an organic acid (such as citric acid) and / or a salt thereof is used. The best chromatogram can be obtained by appropriately adjusting the pH and ionic strength of. The speed (flow rate) of passing the mobile phase is generally 0.5 to 5 ml / min.
The degree is chosen.

For the above separation, a mobile phase having an appropriate concentration is used.
Causes glycated hemoglobin to become HbA _1a, HbA_1b, Hb
F, HbA_1cIn that order, and further non-glycated hemoglobin
HbA₀Is eluted, but HbA_1cThe later eluate is
Not necessary for diabetes diagnosis, so high concentration or high pH
Alternatively, the mobile phase may be switched to the mobile phase to elute at once.

For the above detection, usually 415 nm
The absorbance measurement is performed.

The method for measuring glycated hemoglobin according to claim 2 (hereinafter, the invention according to claim 2 is referred to as the present invention 2),
Instead of the column for liquid chromatography used in the present invention 1, a cation exchange liquid chromatography packing material (A) and an anion exchange liquid chromatography capable of improving the chromatogram of the packing material (A) for glycated hemoglobin. A packing material for chromatography, a packing material for reversed-phase partition liquid chromatography, a packing material for normal-phase partition liquid chromatography, and a packing material for liquid chromatography (C) which is at least one selected from the group consisting of hydroxyapatite; In addition to the fact that the column for liquid chromatography packed in the column of is used,
Same as the first invention.

The above-mentioned anion-exchange liquid chromatography packing material is not particularly limited as long as it is a known anion-exchange liquid chromatography packing material.
-NR ₂ as a functional group, -NR _3, -NR ₄ (R is a hydrogen atom or an alkyl group) filler with the like.

The packing material for reversed phase partitioning liquid chromatography is not particularly limited as long as it is a known packing material for reversed phase partitioning liquid chromatography, and for example, a direct functional group such as an octyl group or an octadecyl group. Examples thereof include those having a chain alkyl group and fillers having a phenyl group and the like.

The packing material for normal phase partitioning liquid chromatography is not particularly limited as long as it is a known packing material for normal phase partitioning liquid chromatography, and for example, —NH ₂ , —CN, —OH as a functional group. And the like.

The hydroxyapatite is not particularly limited as long as it is a known hydroxyapatite used for liquid chromatography.

As a material for the skeleton of the filler (C),
The material is not particularly limited, and examples thereof include those exemplified as the material of the skeleton of the filler (A). Further, the filler (C) may be used as a mixture of two or more kinds.

In the present invention 2, it is possible to use two or more kinds of packings for cation exchange liquid chromatography as the packing (A), and the combination of the packing (A) and the packing (C) is used. As a specific example, it is possible to combine a filler of an acrylic polymer having a COOH group, a filler of a styrene polymer having a COOH group, and a hydroxyapatite filler.

The ratio of the amount of the filler (A) to the amount of the filler (C) used varies depending on the kind of the filler used, but usually, the filler in the total amount of the filler (A) and the filler (C) is used. The content of (C) is preferably 50% by weight or less.

The column used in Invention 2 is the same as that in Invention 1 described above.

In order to produce the column for liquid chromatography used in the second aspect of the present invention, the production of the column for liquid chromatography used in the first aspect of the present invention described above
It may be carried out in the same manner as in the present invention 1 except that the filler (C) is used instead of the filler (B).

The sample containing glycated hemoglobin measured in the second aspect of the present invention is the same as that of the first aspect of the present invention.

The method for measuring glycated hemoglobin according to the second aspect of the present invention is the same as the column for liquid chromatography used in the second aspect of the present invention, instead of the column for liquid chromatography used in the first aspect of the present invention. It is the same as the first embodiment except that a column is used.

The mobile phase and the detection method used in the present invention 2 are the same as in the present invention 1.

[0037]

Embodiments of the present invention will be described below. The measuring apparatus in the examples was constructed as shown in FIG. The following devices were used for each device. Liquid sending pump 3: LC-9A manufactured by Shimadzu Corporation. Sampler 4: Auto sampler. Sekisui Chemical Co., Ltd.
ASU-420. Detector 5: UV-visible spectrophotometer. Shimadzu Corp. SP
D-6AV.

The fillers used in the examples are as follows. Filler Cation-1 A cation exchange filler was produced by the method described in JP-A-2-309253 as follows. Benzoyl peroxide (polymerization initiator, Wako Pure Chemical Industries, Ltd.) was added to 200 g of diethylene glycol dimethacrylate (Shin Nakamura Chemical Co., Ltd.).
1 g was dissolved and added to a 3 wt% polyvinyl alcohol (Nippon Gosei) aqueous solution. Nitrogen was introduced with stirring, and polymerization was carried out at 80 ° C. for 2 hours. After cooling to room temperature, 40 g of acrylic acid was added, and the mixture was heated again to 80 ° C. and polymerized for 2 hours. The obtained particles are washed successively with water and acetone, dried and then classified by an air classifier (manufactured by Nittetsu Mining Co., Ltd.) to collect particles of 6 to 8 μm, which are used as filler cation-
It was set to 1. The filler cation-1 has --CO as a functional group.
Has OH.

Filler Cation-2 Commercially available cation exchange filler (Fractogel EM).
D, manufactured by Merck). As a scaffold material, styrenic polymers are used, it has a -SO ₃ H as a functional group.

Filler Anion-1 Commercially available anion exchange type filler (ES-502N, Asahi Kasei)
Company). As a material for the skeleton, vinyl alcohol polymer
-Is used as a functional group, -N (C_TwoH_Five) _TwoHas
ing.

Filler Reverse Phase-1 Commercially available reverse phase filler (Micronex RP-30,
Sekisui Chemical Co., Ltd.). A styrene-based polymer is used as a material for the skeleton and has a phenyl group as a functional group.

Filler Apatite-1 Commercially available hydroxyapatite-based filler (HCA-A-4
007, manufactured by Mitsui Toatsu Chemicals, Inc.).

Examples 1 to 4 and Comparative Example 1 Production of Liquid Chromatography Column Table 1 shows the packing material (A) and the packing material (B) or the packing material (C) shown in Table 1. The used amount (g) was dispersed in 30 ml of a phosphate buffer (pH 6) (Comparative Example 1 uses only the filler (A)). The dispersion was added to a packer (capacity 30 ml: Umeya Seiki Co., Ltd.) connected to a stainless steel column having an inner diameter of 6 mm and a length of 50 mm, and a liquid feed pump (Shimadzu Corporation LC-6AD) was connected to the packer. A column was manufactured by performing constant pressure packing.

[0044]

[Table 1]

Separation of glycated hemoglobin After collecting sodium fluoride from a healthy person, an aqueous solution containing a surfactant and polyphosphoric acid was added to remove hemolysin and unstable hemoglobin. The obtained sample was introduced into an apparatus in which the above liquid chromatography column was attached to the above apparatus to measure glycated hemoglobin. Elution is pH
Phosphate buffers of 5-6 were used and the pH and ionic strength were adjusted to obtain the best chromatogram for each example and comparative example. The eluate after HbA _1c was eluted at once by switching the mobile phase to a phosphate buffer solution of pH 7 and 500 mM. The chromatogram obtained in Example 1 is shown in FIG. 2, and the chromatogram obtained in Comparative Example 1 is shown in FIG. The chromatograms of Examples 2 to 4 were almost the same as the chromatogram of Example 1. In addition,
The peaks in FIGS. 2 and 3 are 11HbA _1a and Hb, respectively.
A _1b , 12HbF, 13HbA _1c and 14HbA ₀ .

[0046]

The structure of the method for measuring glycated hemoglobin of the present invention 1 is as described above, the chromatogram of glycated hemoglobin can be improved, and glycated hemoglobin can be measured with better separability in a short time. Further, the column for liquid chromatography used in the method for measuring glycated hemoglobin of the present invention 1 is easy to manufacture. Therefore, the method for measuring glycated hemoglobin of the present invention 1 is a method that can analyze glycated hemoglobin with high accuracy by a simple method.

The constitution of the method for measuring glycated hemoglobin of the present invention 2 is as described above, the chromatogram of glycated hemoglobin can be improved, and glycated hemoglobin can be measured in a shorter time with better separability. In addition, the column for liquid chromatography used in the method for measuring glycated hemoglobin of the present invention 2 is easy to manufacture. Therefore, the method for measuring glycated hemoglobin according to the second aspect of the present invention is a method capable of analyzing glycated hemoglobin with high accuracy by a simple method.

[0048]

[Brief description of the drawings]

FIG. 1 is an example of the configuration of an apparatus for measuring glycated hemoglobin of the present invention.

FIG. 2 is a chromatogram of Example 1.

FIG. 3 is a chromatogram of Comparative Example 1.

[Explanation of symbols]

 1 Column for liquid chromatography 2 Mobile phase 3 Liquid feed pump 4 Sampler 5 Detector 6 Recorder

Claims (2)

[Claims] 1. A sample containing glycated hemoglobin, and a packing material (A) for cation exchange liquid chromatography,
A single column is packed with a cation exchange liquid chromatography packing material (B), which is different from the packing material (A) and can improve the chromatogram of the packing material (A) with respect to the glycated hemoglobin. A method for measuring glycated hemoglobin, which comprises introducing into a column for liquid chromatography and performing liquid chromatography. 2. A sample containing glycated hemoglobin, and a packing material (A) for cation exchange liquid chromatography,
From the packing material for anion exchange liquid chromatography, packing material for reversed phase partitioning liquid chromatography, packing material for normal phase partitioning liquid chromatography and hydroxyapatite, which can improve the chromatogram of the packing material (A) with respect to the glycated hemoglobin Glycated hemoglobin, characterized in that liquid chromatography is carried out by introducing into a column for liquid chromatography in which one column is packed with at least one packing material for liquid chromatography (C) selected from the group consisting of Measuring method.