JP2929456B2 - Separation and quantification of glycohemoglobin - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for separating and quantifying glycohemoglobin by high performance liquid chromatography.

(Prior Art) Diagnosis methods for diabetes include measurement of blood sugar level (concentration of glucose in blood) which has been widely used conventionally. However, the measurement of the blood sugar level merely reflects the diabetic state only at the time of blood collection, and there is a problem that the long-term blood sugar control state cannot be accurately grasped.

In recent years, glycated hemoglobin in blood has been recognized as an index that accurately reflects long-term average blood glucose levels, and glycated hemoglobin measurement has begun to be widely used in clinical settings as a diagnostic method for diabetes.

Glycohemoglobin (hereinafter abbreviated as GHb) is a generic term for a combination of sugar in blood and hemoglobin A (hereinafter abbreviated as HbA). In the case of human, HbA _1C1 , HbA _1C2 and Hb
A _1b and HbA _1c are known. These are the main component of the HbA other than HbA ₁ are usually referred to as HbA ₁ collectively are referred to as HbA _0. The most quantitatively greater for the HbA _1c in HbA _1, the concentration is generated by non-enzymatic binding to HbA,
It has been recognized that a diabetic patient shows a higher value than a healthy person by quantitatively reflecting the average blood glucose level in blood for 1 to 2 months corresponding to the half-life of the life span of erythrocytes. Therefore, the measurement of GHb is more reliable and superior than the conventional measurement of blood glucose as an index for primary screening of diabetes and long-term blood glucose control.

At present, GHb is most often measured by liquid chromatography. In the method by liquid chromatography, various GHb are separated in an ion exchange chromatography mode usually using a cation exchanger as a packing material. As the cation exchanger, a porous cation exchanger having an appropriately sized pore diameter into which a cation exchange group such as a carboxylmethyl group or a sulfopropyl group is introduced is used. When ion exchange chromatography is performed using these porous cation exchangers by gradient elution of salt concentration or pH, GHb becomes HbA _1C1 , Hb
It is eluted in the order of A _1C2 , HbA _1b and HbA _1c .

(Problems to be Solved by the Invention) At present, in most clinical cases of diabetes, GHb is measured using liquid chromatography as described above. In particular, in clinical sites where a large number of specimens are handled, dedicated systems for measuring GHb, which are systemized, are widely used. However, with a dedicated device that is automatically accelerated, the separation time of various GHb is insufficient because the measurement time is very short. In particular, it is impossible labile HbA _1c and accurately quantify the concentration of stable HbA _1c to reflect the most accurate average blood glucose without complete separation is obtained of stable HbA _1c included in HbA _1c. As a method for solving this problem, either lengthening the measurement time, or a method of removing unstable HbA _1c pretreatment, both sample processing ability is reduced. When fetal hemoglobin F (hereinafter, referred to as HbF) shows a high value, HbF is also HbF.
In addition to not a little influence on the separation of A _1c in recent years, HbF
And other diseases have been studied, and it has been found that the values tend to be high in leukemia and recurrent anemia, and the usefulness of separation and quantification of HbF is increasing.

At present, a method for measuring GHb capable of rapidly and precisely separating GHb and HbF that satisfies all of them has not been developed.

(Means for Solving the Problems) In view of the above situation, the present inventors have considered that GHb and H
As a result of repeated research on a method for measuring GHb capable of rapid and precise separation of bF, it was found that a non-porous cation exchanger having a small particle diameter enables rapid and precise separation of GHb and HbF. Reached.

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

(Operation) Hereinafter, the present invention will be described in detail.

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

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

The non-porous spherical crosslinked polymer particles are prepared by adding a corresponding monomer, a crosslinking agent and a polymerization initiator to an aqueous solution containing a suspension stabilizer with stirring, and performing a crosslinking polymerization reaction with a monomer and a crosslinking agent by a known suspension polymerization method. Synthesized.

As the corresponding monomer, hydrophilic methacrylic acid and acrylic esters such as hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxypropyl acrylate, glycerin methacrylate, glycerin acrylate, polyethylene glycol methacrylate and polyethylene glycol acrylate are used.

As the crosslinking agent, polyfunctional methacrylates such as ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, and glycerin dimethacrylate, and divinylbenzene are used. The amount of the crosslinking agent used is preferably in the range of 0.15 to 0.35 based on the weight of the monomer.

In addition to the monomers and the crosslinking agent, methacrylic acid and alkyl acrylates such as methyl methacrylate, methyl acrylate, ethyl methacrylate and ethyl acrylate can be used as copolymerizable monomers. 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, an ordinary radical initiator can be used. Particularly, benzoyl peroxide, azobisisobutylnitrile, tert-butylperpivalate and the like are preferable. The amount used is based on the total of the monomer and the crosslinking agent.
A range of 0.1 to 10% by weight, especially 0.5 to 5% by weight is preferred.

As the suspension stabilizer, a commonly used suspension stabilizer such as polyvinyl alcohol and polyvinyl pyrrolidone can be used. The amount used is in the range of 0.1 to 10% by weight, preferably 0.5 to 5% by weight, based on water.

The non-porous spherical crosslinked polymer thus obtained is
Since the monomer itself has hydrophilicity, it is sufficiently compatible with the eluent used for the measurement of GHb, and does not exhibit swelling or shrinking properties with respect to salt concentration or pH change. Further, since it is appropriately crosslinked, it has mechanical strength enough to withstand use under a high flow rate.

Next, a method for introducing a cation exchange group into the non-porous spherical crosslinked polymer particles thus synthesized will be described.

Examples of the cation exchange group to be introduced include a sulfopropyl group, a sulfoethyl group, and a carboxymethyl group. Further, the introduction amount is in the range of 0.02 to 0.4 meq / ml, preferably in the range of 0.04 to 0.15 meq / ml.

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

Non-porous spherical silica particles can be prepared, for example, by the method of Stober et al. (Jourmal of Collid Interface Science, Vol., 26, 62, 1).
As shown in 968), alkoxysilanes can be synthesized by a known hydrolysis polymerization reaction in alcohol with water and ammonia as catalysts. The particle size of the synthesized non-porous spherical silica particles can be adjusted by the type and amount of the alkoxysilane and the concentrations of water and ammonia as a catalyst. It can also be adjusted by repeating the hydrolysis polymerization reaction.

Next, a method for introducing a cation exchange group into the non-porous spherical silica particles thus synthesized will be described.

Examples of the cation exchange group to be introduced include a sulfopropyl group, a sulfoethyl group, and a carboxylmethyl group. Further, the introduction amount is preferably in the range of 0.04 to 0.2 meq / ml.

As an introduction method, for example, an epoxy group-containing silane coupling agent such as γ-glycidoxypropyltrimethoxysilane is reacted with the non-porous spherical silica particles to introduce an epoxy group, and then a surface epoxy group, for example,
The corresponding cation exchange group can be introduced by reacting a cation exchange group-containing reagent such as sodium chloroacetate, glycolic acid or sodium sulfate. Alternatively, it can be synthesized by introducing a reagent having an allyl group with 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 thus obtained as a filler will be described. As the measuring device, a high-pressure liquid chromatograph composed of a high-pressure liquid pump capable of gradient elution of two liquids and a detector capable of detecting visible absorption is used.

The sample is adjusted 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 can be used.
Surfactants such as X-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 pHs can be used. As the buffer used for these eluents, those usually 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 substrate concentration of 10 to 50 mM,
An aqueous solution having a pH of 5 to 6.5 is particularly preferred. (B) The liquid
(A) The aqueous solution which has a 0.1-0.3M sodium salt in a liquid is especially preferable. Examples of the sodium salt include sodium chloride and sodium nitrate.

On the other hand, the solution (A) used for the pH gradient elution method is particularly preferably an aqueous solution having a buffer substrate concentration of 10 to 50 mM and a pH of 6.0 to 6.5, and the solution (B) is preferably an aqueous solution having a pH of 7.0 to 7.5. Particularly preferred. In the elution operation, in both the salt concentration gradient elution method and the pH gradient elution method, elution is started with 100% of solution (A), and the flow rates of solution (A) and solution (B) are continuously changed, and after 10 to 15 minutes, A linear gradient elution method using 100% of solution B is preferred.

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

The flow rate of the eluent is 0.5 to 2.0 ml / min, especially 1.0 to 1.5 ml / m
in is preferred.

Detection can be performed by visible absorption at 415 nm.

As described above, when GHb is separated under elution conditions, the ionic interaction between the sample and the ion-exchange groups introduced into the filler is limited to the particle surface because the non-porous filler has no pores. Since there is no diffusion of data inside the pores caused by the porous filler, precise separation can be performed in a shorter time than the porous filler. In addition, in the non-porous filler, since the ion-exchange group is introduced only on the particle surface, there is no non-specific adsorption often observed in the porous filler, so that the sample recovery rate is high, It is characterized by excellent quantification and very small analysis error.

(Examples) Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto.

Example 1 350 g of hydroxyethyl methacrylate, 70 g of ethylene glycol dimethacrylate, 30 g of methyl methacrylate
And a mixed solution of 10 g of tert-butyl perpivalate and Na ₂ SO
₄ Water at 60 ° C in which 40 g and 200 g of polyvinyl alcohol are dissolved 4.
Add the oil droplets into 5 liters with stirring so that the particle size of the oil droplets is 0.5 to 6 μm
The cross-linking polymerization reaction was carried out at a temperature of 60 ° C. for 12 hours while adjusting the stirring speed so that 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 diameter of 2 to 4 μm. Next, 100 g of the particles were dispersed well in 100 ml of water in which 20 g of sodium hydroxide was dissolved, and reacted at room temperature for 4 hours while dropwise adding 40 g of epichlorohydrin. 100 g of the epoxidized polymer particles thus obtained and 20 g of sodium sulfate are mixed with 100 ml of water.
And reacted at a temperature of 80 ° C. for 16 hours. After the reaction, the well was washed well with warm water and the sulfonyl group was quantified.
A sulfonyl group of 06 meq / ml was introduced. This non-porous cation exchanger was packed in a filter column having an inner diameter of 4.6 mm and a length of 3.5 cm to prepare a column for GHb measurement. As the GHb measuring device, CCPM was used for the liquid sending pump, and UV-8000 (both manufactured by Tosoh) was used for the detector. The elution method was a pH gradient elution method using a two-liquid ejection gradient method, and the detection wavelength was 415 nm, which is usually used for measurement of GHb. As a sample, 1 ml of 0.1% Triton X-100 was added to 2 μl of blood of a diabetic patient.
In addition, 10 μl of hemolyzed blood was used. The eluent is
20 mM phosphate buffer (pH 6.5) for solution (A), 20 mM for solution (B)
GHb was separated from the solution (A) at a flow rate of 1.5 ml / min using the M phosphate buffer (pH 6.8) to the solution (B) for 10 minutes with a linear gradient. FIG. 1 shows the obtained chromatogram.
As can be seen from the figure, the separation time was about 10 minutes, and the unstable HbA _1c
(1 in the figure), stable HbA _1c (2 in the figure), HbF (3 in the figure) and HbA ₀ (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 perpivalate was mixed with 40 g of Na ₂ SO ₄ and polyvinyl alcohol.
200 g was dissolved in 4 l of water at 60 ° C. with stirring, and the stirring speed was adjusted so that the particle size of the oil droplets was 0.5 to 7 μm, and the crosslinking polymerization reaction was carried out at a temperature of 60 ° C. for 16 hours. Washing and classification were performed in the same manner as in Example 1 to obtain about 120 g of nonporous spherical crosslinked polymer particles having a particle size of 3 to 5 μm. Next, 100 g of these particles and 40 g of sodium bromoethanesulfonate were added to 100 parts of water.
Then, 50 m of water in which 20 g of caustic soda was dispersed and dissolved in 20 ml of sodium hydroxide was dropped in about 1 hour, and the mixture was reacted at a temperature of 40 ° C. for 16 hours.
After the reaction was washed with water and quantified for the sulfonyl group, 0.042 m
An eq / ml sulfonyl group had been introduced. This non-porous cation exchanger was packed in a stainless steel column having an inner diameter of 6 mm and a length of 3.5 cm, and GHb was separated. The same results as in Example 1 were obtained.

Example 3 100 g of the epoxidized polymer particles obtained in the same manner as in Example 1 was thoroughly washed with dioxane, and 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. In addition, the reaction was performed at a temperature of 50 ° C. for 4 hours. After the reaction, the mixture was thoroughly washed with warm water and quantified for carboxyl groups. A carboxyl group of 0.12 meq / ml was observed. The separation of GHb was almost the same as that obtained in Example 1.

Example 4 10 g of nonporous spherical silica particles having a particle diameter of 2.1 μm synthesized by the Stober method were placed in a 100-ml round-bottomed flask, 60 ml of toluene was added and dispersed well, and 1 g of allyldimethylchlorosilane was added. The reaction was performed for 4 hours. After the completion of the reaction, the resultant was thoroughly washed with toluene and acetone, and dried under reduced pressure at 50 ° C. for 16 hours. Next, take the whole dried thing, put it in a 100 ml round bottom flask, add 60 ml of benzene and disperse well, then add 100 mg of periodic acid and 10 mg of sodium permanganate and react at 50 ° C for 16 hours, After cooling, it was washed with benzene and methanol, and the amount of carboxyl groups introduced was quantified. As a result, 0.11 meq / ml of carboxyl groups was found. When GHb was measured in the same manner as in Example 1, GHb and HbF could be completely separated in a separation time of about 8 minutes.

As apparent from the above description (Effect of the Invention), short time GHb by using the filler nonporous cation exchanger in the measurement of GHb by liquid chromatography, especially labile HbA _1c and stable HbA _1c And precision separation of HbF
More Reliable GHb for Clinical Practice in Diabetes Diagnosis
Can be provided.

[Brief description of the 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 filler.

──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int.Cl. ⁶ , DB name) G01N 30/88

Claims (2)

(57) [Claims] 1. Stable glycated hemoglobin (stable HbA1c) is separated from unstable glycated hemoglobin (unstable HbA1c) and fetal hemoglobin F (HbF) by high performance liquid chromatography using a non-porous cation exchanger. How to determine. 2. The non-porous cation exchanger having an average particle size of 1
The method according to claim 1, wherein the thickness is from 10 to 10 µm.