JPH03255360A - Separation and determination of glycohemoglobin - Google Patents

Abstract

PURPOSE:To make it possible to perform precise separation in a short time and to obtain a highly reliable separating determination method by using a non-porous cation exchange material as a filler in measurement of glycohemoglobin by liquid chromatography. CONSTITUTION:In measurement of glycohemoglobin CHb by liquid chromatogrpahy, a non-porous cation exchange material is used as a filler. When the non-porous filler is used in the separation of the CHb, mutual ionic action between a sample and an ion-exchange group which is introduced into the filler is performed only at the surface of the particle because there is no small hole. Since the sample is not diffused in the inside of the small hole, precise separation can be achieved in a short time. Since the ion-exchange group is introduced only into the surface of the particle in the non-porous filler, there is no nonspecific absorption. Therefore, the recovery of the sample is high, quantitative performance is excellent and the analyzing errors can be reduced.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for separating and quantifying glycated hemoglobin using high performance liquid chromatography.

(Prior Art) Diagnostic methods for diabetes include measuring blood sugar levels (glucose concentration in the blood), which has been widely used in the past. but,
Measuring blood sugar levels only reflects the diabetic state at the time of blood collection, and there is a problem in that the long-term blood sugar control state cannot be accurately determined.

In recent years, glycated hemoglobin in the blood has come to be recognized as an indicator that accurately reflects average blood sugar levels over a long period of time, and measurement of glycated hemoglobin has begun to be widely used in clinical practice as a diagnostic method for diabetes.

Glycated hemoglobin (hereinafter referred to as GHb) is a general term for the combination of sugar in the blood and hemoglobin A (hereinafter referred to as HbA).
bA, HbAlb and Hb A 1°191
192 etc. are known. These are usually collectively called HbA1, and the main component of HbA other than HbA1 is HbA1.
It's called Ao. Among HbAt, HbAi is the most abundant in quantity, and it is produced by non-oxygenically binding to HbA, and its concentration lasts for 1 to 2 months, which corresponds to the half-life of red blood cells. It quantitatively reflects the average blood sugar level in the blood of people with diabetes, and it is recognized that diabetic patients show higher values than healthy people.

Therefore, GHb measurement is more reliable and superior to conventional blood sugar level measurements as an index for temporary screening for diabetes or long-term blood sugar level control.

Currently, liquid chromatography is the most commonly used method for measuring GHb. In liquid chromatography methods, various GHb species are usually separated in an ion exchange chromatography mode using a cation exchanger as a packing material. As the cation exchanger, a porous cation exchanger having an appropriate pore size and into which a cation exchange group such as a carboxylmethyl group or a sulfopropyl group is introduced is used. When ion exchange chromatography using these porous cation exchangers is performed using a salt concentration or pH gradient elution method, GHb is converted to HbA.
, HbA , HbA,b and 191 19
2HbA and o are eluted in this order.

(Problems to be Solved by the Invention) Currently, in most clinical settings for diabetes, GHb is measured using liquid chromatography as described above. Particularly in clinical sites where a large number of specimens are handled, dedicated devices for measuring GHb that are systemized are widespread and in use. However, with a dedicated device that is automatically accelerated, the measurement time is very short, so that the separation of GHb is insufficient. In particular, unstable HbA and stable HbA contained in HbA, o. There is a problem in that complete separation of C cannot be obtained and the concentration of stable Hb A lc that most accurately reflects the average blood sugar level cannot be accurately quantified. To solve this problem, either lengthen the measurement time or pre-process the unstable HbA.
There are methods for removing lc, but all of them reduce sample throughput. In addition, fetal hemoglobin F (hereinafter referred to as HbF
) shows a high value, HbF also has a high value.
In addition to having a considerable influence on the separation of Hb
Research has been conducted on F and other diseases, and it has been found that leukemia and male anemia tend to show high values1 (b
The usefulness of separating and quantifying F is increasing.

Currently, no method for measuring GHb that satisfies all of these requirements and is capable of rapid and precise separation of GHb and HbF has been developed.

(Means for Solving the Problems) In view of the above-mentioned current situation, the present inventors have conducted repeated research on a method for measuring GHb that enables rapid and precise separation of GHb and HbF, and as a result, they have found that The present invention was achieved by discovering that a porous cation exchanger enables rapid and precise separation of GHb and HbF.

That is, the present invention provides a method for separating and quantifying glycated hemoglobin by high performance liquid chromatography using a non-porous cation exchanger.

(effect) The present invention will be explained in detail below.

First, a method for synthesizing the non-porous cation exchanger used in the present invention will be explained.

The non-porous cation exchanger of the present invention can be synthesized by introducing a cation exchange group into pre-synthesized non-porous spherical crosslinked polymer particles or non-porous spherical silica particles.

Non-porous spherical cross-linked polymer particles have the corresponding 7-mer 1
A cross-linking agent and a polymerization initiator are added to an aqueous solution containing a suspension stabilizer to carry out a cross-linking polymerization reaction.The seven polymers and the cross-linking agent are synthesized by a known suspension polymerization method.

Corresponding seven polymers include hydroxyethyl methacrylate and hydroxyethyl acrylate.

Hydrophilic methacrylic acids and acrylic esters such as hydroxypropyl acrylate, glycerin methacrylate, glycerin acrylate, polyethylene glycol methacrylate, and polyethylene glycol acrylate are used.

As a crosslinking agent, ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate.

Polyethylene glycol dimethacrylate 1 Polyfunctional methacrylic acid esters such as glycerin dimethacrylate, divinylbenzene, etc. are used. The amount of crosslinking agent used is 0.15 to 0.35 based on the weight of the monomer.
A range of is preferred.

In addition to the above-mentioned monomers and overmaterials, copolymerization monomers such as methyl methacrylate, methyl acrylate,
Methyacrylic acid and acrylic acid alkyl esters such as ethyl methacrylate and ethyl acrylate can also be used. These copolymerizable monomers can be used alone or in combination of two or more. The amount used is preferably in the range of 0.01 to 0.1 based on the weight of the monomer.

As the polymerization initiator, ordinary radical initiators can be used. Especially benzoyl peroxide.

Azobisisobutylnitrile, tert-butyl perpivalate and the like are preferred. The amount used is 0.1 to 10% by weight, especially 0.5 to 5% by weight, based on the total of monomer and crosslinking agent.
% weight ranges are preferred.

As the suspension stabilizer, commonly used ones such as polyvinyl alcohol and polyvinylpyrrolidone can be used. The amount used ranges from 1 to 10% by weight of O2, preferably from 0.5 to 5% by weight, based on water.

The non-porous spherical cross-linked polymer obtained in this way is fully compatible with the eluent used for GHb measurement because the monomer itself is hydrophilic, and it swells and contracts in response to changes in salt concentration and pH. Does not show gender. In addition, since it is appropriately crosslinked, it has sufficient mechanical strength to withstand use under high flow rates.

Next, a method for introducing cation exchange groups into the nonporous spherical crosslinked polymer particles synthesized in this manner will be described.

Examples of the cation exchange group to be introduced include a sulfopropyl group, a sulfoethyl group, and a carboxymethyl group. In addition, the amount introduced is 0.02~0゜4 m eq /
XEII range, preferably 0.04 to 0°15 m
eq/ml range.

The introduction method is to introduce a corresponding cation exchange group via the hydroxyl group on the surface of the non-porous spherical cross-linked polymer particles, or to poxidize the surface of the non-porous spherical cross-linked polymer particles and then introduce the corresponding cation exchange group via the epoxy group. Examples include a method of introducing a corresponding cation exchange group. For example, in the former method, a sulfonyl group can be introduced by reacting nonporous spherical crosslinked polymer particles with a halogenated ethanesulfonic acid such as sodium bromoethanesulfonate in an aqueous solution of caustic soda. Furthermore, a carboxymethyl group can be introduced by reacting a halogenated acetic acid such as sodium chloroacetate in a similar manner. In the latter method, nonporous spherical crosslinked polymer particles are first reacted with an epoxy compound such as epichlorohydrin or glycerol triglycidyl ether in an aqueous solution of caustic soda to epoxidize the surface of the polymer particles, and then sodium sulfate, taurine, or glycolic acid is added to the surface of the polymer particles. A sulfonyl group or carboxyl group can be introduced by reacting with a corresponding cation exchange group-containing reagent such as.

Non-porous spherical silica particles can be prepared, for example, by the method of 5tober et al.
interface 5cLence, Vol., 26
．． 62.1968), alkoxysilanes can be synthesized by a known hydrolytic polymerization reaction in alcohol using water and ammonia as catalysts. The particle size of the non-porous spherical silica particles to be synthesized can be adjusted by the type and amount of alfukidisilane and the concentration of water and ammonia as catalysts. It can also be adjusted by repeating the hydrolysis polymerization reaction.

Next, a method for introducing cation exchange groups into the non-porous spherical silica particles synthesized in this manner will be described.

Examples of the cation exchange group to be introduced include a sulfopropyl group, a sulfoethyl group, and a carboxylmethyl group. In addition, the amount introduced is 0.04 to 0.2rr+eq/
A range of ml is preferred.

The introduction method is, for example, to introduce an epoxy group by reacting an epoxy group-containing silane coupling agent such as γ-glycidoxypropyltrimethoxysilane with non-porous spherical silica particles, and then to react with the epoxy group on the surface. for example,
Sodium chloroacetate.

A corresponding cation exchange group can be introduced by reacting with a cation exchange group-containing reagent such as glycolic acid or sodium sulfate. Alternatively, it can be synthesized by introducing a reagent having an allyl group using a silane coupling agent and then generating a carboxyl group by oxidative cleavage.

Next, a method for separating and quantifying GHb by high performance liquid chromatography using various non-porous cation exchangers obtained in this way as a packing material will be described. The measuring device used is a high-performance liquid chromatograph that is comprised of a high-pressure liquid pump capable of two-liquid gradient elution and a detector capable of detecting visible absorption.

The sample is prepared by adding a hemolytic agent to whole blood collected from a diabetic patient or the like. As a hemolytic agent, any commonly used hemolytic agent may be used, but for example, Trf
Surfactants such as tonX-100 can be used.

As the elution method, a salt concentration gradient elution method or a pH gradient elution method in which separation is performed using eluents (A) and (B) having different salt concentrations or pH can be used. Further, as the buffer used for these eluents, those commonly used in ion exchange chromatography using a cation exchanger can be used. The solution (A) used in the salt concentration gradient elution method has a buffer base material concentration of 10 to 50.
An aqueous solution having m M s pH of 5 to 6.5 is particularly preferred.

The liquid (B) is particularly preferably an aqueous solution containing a sodium salt of 0.1 to 0.3M in the liquid (A). Examples of sodium salts include sodium chloride and sodium nitrate.

On the other hand, the solution (A) used in the pH gradient elution method is particularly preferably a vinegar nutrient solution with a buffer base concentration of 10 to 50 mM and a pH of 6.0 to 6.5, and the solution (B) is a vinegar nutrient solution with a pH of 7. .0-7.5
The same aqueous solution of is particularly preferred.

For both the salt concentration gradient elution method and the pH gradient elution method, elution is started with 100% solution (A), and the flow rates of solution (A) and (B) are continuously changed, and after 10 to 15 minutes, B liquid 10
A linear gradient elution method using 0% is preferred.

Although the case where two eluents are used has been described above, the present invention may be applied to other combinations such as three eluents.

The flow rate of the eluent is 0.5 to 2.0 ml/min.

Particularly preferred is 1.0 to 1.5 ml/min.

Detection can be done by visible absorption at 415 nm.

When GHb is separated under the elution conditions described above, the ionic interaction between the sample and the ion-exchange groups introduced into the packing material occurs on the surface of the particles, since there are no pores in the non-porous photopacking material. Since this method is carried out using only a porous packing material, there is no diffusion of the sample inside the pores that occurs with porous packing materials, so precise separation can be performed in a shorter time than with porous packing materials. In addition, since non-porous optical agents have ion exchange groups introduced only on the particle surface, there is no non-specific adsorption that is often observed with porous packing materials, resulting in a high sample recovery rate. It is characterized by excellent quantitative performance and very small analytical error.

(Examples) Hereinafter, the present invention will be specifically explained using Examples, but the present invention is not limited to these.

Example 1 350 g of hydroxyethyl methacrylate, 70 g of ethylene glycol dimethacrylate, 30 g of methyl methacrylate and 10 g of tert-butyl pervivalate
A mixed solution of 440 g of Na2S0 and 200 g of polyvinyl alcohol was dissolved in 4.5 liters of water at 60°C while stirring, and the stirring speed was adjusted so that the particle size of the oil droplets was 0.5 to 6 μm, and the temperature was raised to 60°C. A crosslinking polymerization reaction was carried out for 12 hours. The obtained particles were washed with hot water on a glass filter and then classified to obtain about 110 g of non-porous spherical crosslinked polymer particles having a particle size of 2 to 4 μm.

Next, 10g of these particles were well dispersed in 100ml of water in which 20g of caustic soda was dissolved, and then reacted at room temperature for 4 hours while dropping 40g of epichlorohydrin. 100 g of epoxidized polymer particles thus obtained
and 20 g of sodium sulfate were well dispersed in 100 ml of water and reacted at a temperature of 80° C. for 16 hours. After the reaction, the sulfonyl group was thoroughly washed with warm water and the sulfonyl group was determined to be 0.06me.
q/ml of sulfonyl groups were introduced. This non-porous cation exchanger was packed into a filtration column with an inner diameter of 4.6 mm and a length of 3.5 cm to create a column for GHb measurement.

As a GHb measuring device, a CCPM was used as a liquid pump, and a Niha UV-8000 detector (Ifi was also manufactured by Tosoh) was used. Further, the elution method was performed by a pH gradient elution method using a two-liquid ejection gradient method, and the detection wavelength was 415 nm, which is normally used for measuring GHb. The sample was 0.1% TritonX-1 in 2 μl of diabetic patient blood.
Add 1 ml of 00 and lyse 10μ of hemolysate! used. The eluent was 20mM phosphate buffer (pH 6) added to solution (A).
, 5).

Using 20mM phosphate buffer (pH 6, 8) as solution (B), flow rate 1-, 5ml/min (from solution A) to (B)
Separation of GHb was performed with a 10 minute linear gradient to the liquid. Figure 1 shows the obtained chromatogram. As shown in the figure, unstable HbA can be obtained in about 10 minutes.
(1 in the figure), stable HbA1゜c (2 in the figure), HbF (3 in the figure), and HbA. (
4) in the figure could be completely separated.

Example 2 A mixed solution of 400 g of hydroxyethyl methacrylate, 100 g of ethylene glycol dimethacrylate, and 10 g of tert-butyl pervivalate was dissolved in NaSO.
40 g of a and 200 g of polyvinyl alcohol dissolved in water 41 at 60° C. are added with stirring, and the stirring speed is adjusted so that the particle size of the oil droplets is 0.5 to 7 μm.
A crosslinking polymerization reaction was carried out at a temperature of 60° C. for 16 hours. Example 1
Wash as well.

After classification, about 120 g of non-porous spherical crosslinked polymer particles having a particle size of 3 to 5 μm were obtained. Next, 100g of these particles and 40g of sodium bromoethanesulfonate were added to 100g of water.
After about 1 hour, 50 ml of water in which 20 g of caustic soda was well dispersed and dissolved was added dropwise over a period of about 1 hour, and the mixture was reacted at a temperature of 40° C. for 16 hours. After the reaction, the mixture was washed with water and the sulfonyl groups were quantified. As a result, 0.042 meq/ml of sulfonyl groups had been introduced.

This non-porous cation exchanger has an inner diameter of 6 mm and a length of 3°5.
When a CII+ stainless steel column was packed and GHb was separated, the same results as in Example 1 were obtained.

Example 3 100 g of epoxidized polymer particles obtained in the same manner as in Example 1 were thoroughly washed with dioxane, then the epoxidized polymer particles and 20 g of glycolic acid were well dispersed in 100 ml of dioxane, and 5 ml of boron trifluoride etherate was added. The mixture was added and reacted at a temperature of 50°C for 4 hours. After the reaction, the mixture was thoroughly washed with warm water and the carboxyl groups were quantified. 0.
12 meq/ml of carboxyl groups were observed. G
The results of separation of Hb were almost the same as those obtained in Example 1.

Example 4 10 g of non-porous spherical silica particles with a particle diameter of 2.1 μm synthesized by the Stober method were placed in a 100 ml round bottom flask, and 60 ml of toluene was added to disperse them well. Then, 1 g of allyldimethylchlorosilane was added and the mixture was heated at a temperature of 80 ml. The reaction was carried out at ℃ for 4 hours. After the reaction was completed, it was thoroughly washed with toluene and acetone and dried under reduced pressure at 50°C for 16 hours. Next, take the entire amount of this dried material, put it in a 1.00 ml round bottom flask, add 60 ml of benzene and disperse it well, then add 100 mg of periodic acid and 10 μ of sodium permanganate and keep it at 50℃ for 16 hours. After reaction and cooling, the mixture was washed with benzene and methanol, and the amount of carboxyl groups introduced was determined. As a result, 0.11 meq/ml of carboxyl groups was observed. When GHb was measured in the same manner as in Example 1, GHb and HbF could be completely separated in about 8 minutes.

(Effects of the Invention) As is clear from the above description, by using a non-porous cation exchanger as a filler in measuring GHb by liquid chromatography, GHb, especially unstable HbA, can be measured in a short time. This method enables precise separation of stable HbAlc and HbF, and provides a more reliable method for separating and quantifying GHb in clinical settings for diabetes diagnosis.

[Brief explanation of drawings]

FIG. 1 is a chromatogram obtained by measuring GHb by liquid chromatography using the non-porous cation exchanger obtained in Example 1 as a packing material.

Claims (2)

[Claims] (1) A method for separating and quantifying glycated hemoglobin by high performance liquid chromatography using a non-porous cation exchanger. (2) Non-porous cation exchanger has an average particle size of 1 to 10
The separation and quantitative method according to claim (1), wherein the measurement is μm.