JPH0743362B2 - Device for analysis of glycated albumin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention is for the analysis of saccharified arabamine in which the separation of albumin in a sample and the separation of glycated albumin and non-glycated albumin are performed in the same channel of chromatography. Regarding the device.

In diseases such as diabetes, where the blood sugar level of the long organs is high, various proteins in the living body undergo glycation reaction nonenzymatically. Serum albumin is one of those proteins.

Glycated albumin has a half-life of 17 days, and when compared with glycated hemoglobin, which is said to be an index that correlates well with the fasting blood glucose level three months ago, it responds quickly to blood glucose status, and As described above, it does not fluctuate significantly under the influence of temporary physiological conditions. Since information on the blood glucose status of several weeks to a month or so before is useful in the diagnosis of diabetes, glycated albumin has recently attracted attention from various fields as an index that can best meet this demand.

For example, glycated albumin has been suggested to be useful, for example, it is effective in determining whether or not the current or newly modified dietary therapy is suitable for the patient at an early stage. [Kay F. McFarland, Diabets
farland et al, DIA-BETS), 28,1011,1979, C.R. Gaslow, Prok Natal Acad Sai, You.
S.A. (C. Earl Guthrow etal, Proc.Natl.Acad.Sc
i, USA), 76, No. 9,4258 , 1979] (Prior Art) Conventionally, ion exchange chromatography using carboxymethyl cellulose as a stationary phase [James J. F. Day etal, JB
io.Chem.), 254, No. 3,595 , 1979], introducing a dihydroxyboronyl group into a soft gel such as cellulose and utilizing the interaction between this functional group and glucose [A.K.Maria]. , Analytical Letters (AKMallia etal, Analy−tical Lette
rs), 14 (B8), 649-661, 1981] and the like have been tried.

The above-mentioned conventional technology is capable of separating glycated albumin and non-glycated albumin, but other components in body fluid such as serum and plasma, for example, immunoglobulins (IgG, IgM, etc.),
Since it cannot be separated from haptoglobin, transferrin, etc., it is practically impossible to quantify glycated albumin and non-glycated albumin in body fluid.

Therefore, conventionally, a method has been employed in which albumin is isolated from a sample, then once taken out of the chromatography system, concentrated, and then introduced again into a column capable of separating glycated albumin and non-glycated albumin. That is, the operation of separating albumin from the sample and the operation of separating albumin into glycated albumin and non-glycated albumin are performed as at least two separate operations, which is complicated and requires a long time.

These are the biggest causes for the fact that glycated albumin is not measured at present and there is no analyzer for it in the clinical laboratory where rapidity, operability, reproducibility and automation are required.

Here, as a method for analyzing glycated albumin that overcomes these problems, there is a method described in JP-A-62-226999. That is, the method is characterized in that albumin is separated from the sample in the first column, and is guided to the second column without being discharged out of the flow channel system of chromatography, where albumin is separated into glycated albumin and non-glycated albumin. Is a method for separating glycated albumin by chromatography.

(Problems to be Solved by the Invention) According to the examples described in JP-A-62-226999, non-glycated albumin and glycated albumin are separated within 30 minutes. However, since it takes time to regenerate the first column and the second column between the end of analysis and the start of the next analysis, the analysis cycle becomes long, which makes it difficult to use in clinical laboratories. Still, there was still a problem, especially in terms of speed.

(Means for Solving the Problems) As a result of earnest research for overcoming the above-mentioned problems, the inventors of the present invention have excellent rapidity, operability, reproducibility, and automated glycated albumin and non-glycated albumin. The present invention has been completed and the present invention has been completed.

That is, the present invention, by liquid chromatography,
In a device for separating and analyzing glycated albumin and non-glycated albumin in a sample, a container for a mobile phase A solution used for separating albumin from the sample in a first column, a first mobile phase liquid sending section, and a sample injection section , And a first flow path in which a first column for separating albumin from the data is connected in this order, a container for mobile phase A solution used for eluting non-glycated arabamine in the second column, and glycated albumin are eluted. A container for the mobile phase B solution used for the above is connected in parallel to the second mobile phase liquid feeding section including the mobile phase switching means, and further a second column for separating albumin into glycated albumin and non-glycated albumin, and The second flow path, in which the detectors are connected in this order, is connected via the flow path switching means existing between the downstream side of the second mobile phase liquid sending section and the upstream side of the second column to the first color channel. It provides an apparatus for glycated albumin analysis, characterized in that it is attached in a downstream portion of the.

In the present invention, glycated albumin means albumin covalently bound to glucose, and non-glycated albumin means albumin not bound to glucose.

The albumin referred to in the present invention refers to both glycated albumin and non-glycated albumin.

The sample referred to in the present invention contains at least either glycated albumin or non-glycated albumin, or both, and examples thereof include body fluids such as serum, plasma, hemolyzed blood, and urine.

The first column used in the present invention is not particularly limited as long as it is a column capable of separating albumin and other components in body fluid. As a preferred example, a column packed with a gel to which a dye having high affinity with albumin such as Cibacron Blue F3G-A is packed, IgG (molecular weight: about 150,000), which is a large component in serum, and albumin (molecular weight: about 6.6). Examples thereof include a packed column for gel filtration capable of separating the above) and an ion-exchange gel packed column having an amino group.

However, as a carrier for introducing a substance having a high affinity for albumin such as cibacron blue and a stationary phase carrier for gel filtration, it has excellent mechanical strength and chemical stability, and because non-specific adsorption of proteins is small, A hard hydrophilic cross-linked copolymer having an alcoholic hydroxyl group of 1.0 to 14.0 meq / g, a specific surface area of 5 to 1000 m ² / g, and an amount of water that can be retained is 0.5 to 6.0 g / g is preferable per cross-linked copolymer weight. . The most preferable example is a cross-linked copolymer having an alcoholic hydroxyl group derived from a vinyl alcohol unit described in JP-A-57-30945. Alternatively, the cross-linked copolymers described in JP-A-56-64657 and JP-A-59-145036 are also preferable.

Examples of the method for introducing an amino group into the hydrophilic crosslinked copolymer include the following methods. That is, by reacting the above hydrophilic cross-linked copolymer with epihalohydrin bisepoxide and the like to obtain an epoxy group-containing cross-linked copolymer, and then reacting ammonia, a primary amine such as ethylamine, and a secondary amine such as diethylamine. Obtainable.

Examples of the amino group include an amino group having no substituent, a monosubstituted amino group such as an ethylamino group, and a disubstituted amino group such as a diethylamino group. The amount of amino groups is
0.02 to 5.0 meq / g, preferably 0.05 to 2.0 meq / g, more preferably 0.2 to 1.0 per weight of the cross-linked copolymer.
It is meq / g.

Examples of the method for introducing Cibacron Blue F3G-A include a method in which bromocyan is reacted with a hydroxyl group of the hydrophilic cross-linked copolymer, and then Cibacron Blue F3G-A is reacted.

The amount of Cibacron Blue F3G-A is 0.002 to 1.0 meq / g, preferably 0.005 to 0.05 me, per weight of the cross-linked copolymer.
q / g.

Among these gels, a gel obtained by introducing an amino group into a hydrophilic crosslinked copolymer is particularly preferable.

The most preferable example used in the present invention is JP-A-60-1508.
The cross-linked copolymer having an alcoholic hydroxyl group derived from a vinyl alcohol unit and a diethylamino group described in JP-A-39 can be mentioned. This crosslinked copolymer is disclosed in Japanese Patent Application No. 60-12.
By the method described in No. 22, albumin and other components in body fluid can be rapidly separated.

Examples of the second column used in the present invention include a column packed with an ion-exchange gel or a gel having a dihydroxyboronyl group, but are not particularly limited as long as at least glycated albumin and non-glycated albumin can be separated.

When using an ion exchange gel, needless to say, the slight difference in isoelectric point between glycated albumin and non-glycated albumin is used, but the cation exchange group is preferable as the ion exchange group, and among them, a carboxyl group and a sulfone group are preferable. Acid groups and the like are particularly preferable.

In addition, since the dihydroxyboronyl group specifically binds to a compound having 1,2-cisdiol, affinity chromatography using a gel having this functional group as a stationary phase can also separate glycated albumin and non-glycated albumin. It is valid.

As the stationary phase carrier for introducing these functional groups, cellulose, which is generally used as a stationary phase carrier for conventional liquid chromatography, a soft gel of a polysaccharide such as agarose,
Examples include silica gel and styrene-divinylbenzene copolymers, which have excellent mechanical strength and chemical stability.
In terms of low non-specific adsorption of proteins, alcoholic hydroxyl group is 1.0 to 14.0 meq / g, specific surface area is 5 to 1000 m ² / g, and the amount of water that can be retained is 0.5 to 6.0 g / g per weight of the cross-linked copolymer.
The soft hydrophilic cross-linked copolymer is preferred. The most preferable examples include crosslinked copolymers described in JP-A-57-30945, JP-A-56-64657 and JP-A-54-145036.

The following method can be mentioned as a method of introducing a dihydroxyboronyl group into the hydrophilic cross-linked copolymer. That is, it can be obtained by reacting the above hydrophilic crosslinked copolymer with epihalohydrin, bisepoxide or the like to obtain an epoxy group-containing crosslinked copolymer, and then reacting with metaaminophenylboronic acid.

Examples of the method of introducing a carboxyl group include a method of reacting a halogenated acetic acid such as monochloroacetic acid or monobromoacetic acid with a hydroxyl group of the hydrophilic crosslinked copolymer.

Further, as a method of introducing a sulfonic acid group, for example, a method of reacting propane sultone with a hydroxyl group of the hydrophilic crosslinked copolymer can be mentioned.

The amount of dihydroxyboronyl groups is 0.05 to 5.0 meq / g, preferably 0.05 to 3.0 meq / g, based on the weight of the cross-linked copolymer.
More preferably, it is 0.1 to 2.0 meq / g.

The amount of the carboxyl group or the sulfonic acid group is 0.02 to 5.0 meq / g per weight of the crosslinked copolymer, preferably 0.05 to
It is 2.0 meq / g, and more preferably 0.2 to 1.0 meq / g.

Among these gels, a gel obtained by introducing a dihydroxyboronyl group into a hydrophilic crosslinked copolymer is particularly preferable. The most preferable example used in the present invention is a boron-containing cross-linked copolymer having an alcoholic hydroxyl group and a dihydroxyboronyl group described in JP-A-62-192405.

The functional group-immobilized gel used in the present invention may have various shapes such as a spherical shape and a crushed shape, but a spherical shape is preferable. In that case, the weight average particle diameter is 1 to 500.
μm, preferably 1 to 20 μm, more preferably 1 to 10 μm.

A flow diagram of an example of the apparatus of the present invention is shown in FIG. 1 to explain the relationship between the first column, the second column, the first flow path and the second flow path.

In FIG. 1, the liquid A of the mobile phase is sent to the first flow path by the first mobile phase liquid sending section and is sent to the second flow path by the second mobile phase sending section. The sample injected from the sample injection unit is sent to the first column together with the transfer phase liquid A, and albumin and other components in the sample are separated. Then, the flow passage is switched by the flow passage switching means, and only the albumin separated in the first column is fed into the second column through the second flow passage. After the albumin has been fed into the second column, the flow channel is switched by the flow channel switching means, and the mobile phase A liquid fed from the second mobile phase liquid feeding section passes through the second flow channel. It is fed into the second column via the flow path switching means. By switching the mobile phase from solution A to solution B, glycated albumin and non-glycated albumin are separated in the second column and sent to the detector. After that, the mobile phase sent from the second mobile phase sending part is switched from the solution B to the solution A, passes through the second flow path, and is sent to the second column through the flow path switching means, Play the column. When the regeneration of the second column is completed, the next analysis becomes possible. In order to confirm the separation of albumin in the first column, a detector for monitoring is provided between the outlet of the first column and the flow path switching means or in the downstream portion of the flow path switching means of the first flow path. Can be prepared.

Next, the mobile phase used in the present invention will be described.

For separating glycated albumin and non-glycated albumin, or for separating glycated protein and non-glycated protein, a gel having a dihydroxyboronyl group immobilized thereon is used to separate albumin and other components. When using a gel having amino groups immobilized as a packing material, at least two types of mobile phases (liquid A and liquid B)
A preferred method is to prepare the solution and switch the solution A to the solution B on the way. The solution A can be switched to the solution B stepwise or by a so-called gradient method in which the ratio is continuously changed.

The solution A mentioned here has a buffering base of 10 to 500 mM,
-Aqueous solutions of pH 7.5 to 9.5 free of substances with disdiol are preferred. As the buffering base, a base having a buffering capacity at pH 7.5 to 9.5 is preferable, and particularly preferable bases are ammonium salts such as ammonium acetate, morpholine, N-2-hydroxyethylpiperazine N '.
2-ethanesulfonic acid and the like can be mentioned. Since these buffering bases can strengthen the bond between the dihydroxyboronyl group and the sugar moiety of the glycated protein such as glycated albumin, they are effective for highly separating the glycated protein such as glycated albumin from the non-glycated protein. Is. If the pH is lower than 7.5, the bond between the dihydroxyboronyl group and the sugar becomes weak, which is not preferable. On the other hand, if the pH is higher than 9.5, proteins such as albumin are denatured and precipitation may occur, which is not preferable. Further, the liquid A can contain a salt other than the buffer base. In particular, by including a salt of a divalent metal ion, such as magnesium chloride,
It is preferable because the bond between the sugar chain and the dihydroxyboronyl group can be strengthened. The concentration of the salt of the divalent metal ion is preferably
It is 5 mM to 100 mM. A salt of a monovalent metal ion, such as sodium chloride, may be included, and its concentration is preferably 500 mM or less.

On the other hand, solution B has a pH of 2 to 6.5 with a buffering base of 10 to 500 mM.
The above aqueous solution is preferable, and it is sufficient that it contains a substance having 1,2-disdiol even at a pM of 7.5 or more. Also in that case, pH 9.5 or less is preferable. Using this condition, the bond between the dihydroxyboronyl group and the sugar chain can be weakened, and the glycated protein such as glycated albumin bound to the dihydroxyboronyl group can be eluted.

As the buffering base, a buffering base having a buffering capacity at the pH used is preferable, but particularly preferable examples include tris (hydroxymethyl) aminomethane, triethanolamine and the like. These buffering bases are
The bond between the dihydroxyboronyl group and the sugar chain can be weakened, which facilitates the elution of the glycated protein bonded to the dihydroxyboronyl group.

Examples of the substance having 1,2-cisol include saccharides such as sorbitol and mannitol. The concentration of the substance having 1,2-cisdiol is 50 to 200.
0 mM is preferred.

Furthermore, when the liquid A contains a salt of a divalent metal ion,
It is preferable to add a metal chelating agent such as ethylenediaminetetraacetic acid (EDTA) to the solution B. In that case, the concentration of the metal chelating agent is preferably 5 to 100 mM. It may be preferable that both the A and B solutions contain a small amount of a water-soluble organic solvent such as ethanol, methanol, n-propyl alcohol, iso-propyl alcohol, acetonitrile or ethylene glycol.

The shape of the column used in the present invention is not particularly limited, but preferably has an inner diameter of 10 mm or less and a length of 30 cm or less, particularly preferably an inner diameter of 2 to 8 mm and a length of 1 to 10 cm. The flow rate of the mobile phase is not particularly limited, but is preferably 0.1 to 10 ml / min, particularly preferably 0.3 to 5 ml / min.

The flow channel switching means in the present invention is not particularly limited as long as it can switch the first flow channel and the second flow channel, but a flow channel switching means such as a four-way switching valve or a six-way switching valve is preferable. Here, for example, when a flow path switching means having a sample loop is used, albumin eluted from the first column is once introduced into the sample loop, and then the flow path is switched to transfer the albumin to the second column. It becomes possible to introduce.

In the present invention, glycated albumin and non-glycated albumin can be detected and quantified respectively by connecting an ultraviolet absorption detector, a fluorescence detector, etc. to the outlet of the second column.

(Effect of the Invention) The glycated albumin analyzer of the present invention has been conventionally used for a long time when performing a method for separating and quantifying glycated albumin and non-glycated albumin in a sample by only one sample injection operation. The time required for one analysis was greatly reduced.

In the present invention, since the mobile phase having a constant composition always flows in the first column, the column regeneration time required for switching the mobile phase is unnecessary. Until the albumin is introduced into the second column, the second column is regenerated by the mobile phase A solution sent by the second mobile phase sending section. Also, the second
The mobile phase for the analysis after albumin is introduced into the column and the regeneration of the second column is sent to the second column from the second mobile phase liquid sending section through a short flow path. For the above reasons, the time from the start of the analysis to the start of the water analysis was significantly reduced.

The device of the present invention can be automated, and as a result, glycated albumin and non-glycated albumin can be analyzed quickly, easily and with good reproducibility.

(Embodiment) An embodiment of the present invention will be described based on the configuration explanatory view shown in FIG.

In FIG. 2, (1) is a container for mobile phase A liquid, and in the first flow path from the container, a first liquid feed pump (4), a sample injection part (5), a first column (6). , The fluorescence detector A (7) and the flow path switching valve (8) are connected. Also,
(2) is a container for mobile phase A liquid, (3) is a container for mobile phase B liquid, the flow path of which is the mobile phase switching valve (9) and the second
Forming a second flow path connected to the flow path switching valve (8) via the liquid sending pump (10) of the second column (2) from the flow path switching valve (8). 11) and the fluorescence detector B (12) are connected. (13) is a data processor,
(14) is a sample cooler, (15) is a thermostat for columns, (16)
Is an automatic control system.

The first column was the gel described in Example 1 of JP-A-60-150839 (weight average particle size 9.0 μm, hydroxyl group density 4.9 meq /
g, the amount of water that can be retained is 1.9 g / g, and the diethylamino group is 0.5
Meq / g) was used by filling a stainless steel column having an inner diameter of 7.6 mm and a length of 100 mm.

The second column was prepared as follows.

Vinyl alcohol copolymer gel described in Example 1 of JP-A-57-30945 (X; 0.3, amount of hydroxyl group; 7.8 meq / g, amount of water that can be retained; 1.78 g / g, specific surface area; 78 m ² / g, particle size; 9.0
μm) 25 g, epichlorohydrin 116.8 g, dimethyl sulfoxide 250 ml, 10N aqueous sodium hydroxide solution 12.5 ml
Was added and reacted at 30 ° C. for 20 hours. The introduction amount of the epoxy group was 1.0 meq / g. The epoxy group was quantified by the method described in JP-A-57-190003.

Then, 13 g of m-aminophenylboronic acid hemisulfate was added to 250 g.
20 g of the above-mentioned epoxy group-introduced polymer was added to the one dissolved in water (ml) and adjusted to pH 11, and the reaction was carried out at 60 ° C. for 20 hours. Next, 25 mM tris (hydroxymethyl) aminomethane was added to protect the residual epoxy group, and the reaction was carried out at 50 ° C. for 5 hours. The amount of boronic acid introduced was 0.28 meq / g. The quantification of boronic acid was performed by the following method.

Polymer in which the above m-aminophenylboronic acid is introduced
Boron-carbon bond was cleaved by adding 10 ml of 30% hydrogen peroxide solution to 1 g and reacting for 5 hours. Next, the polymer was spun down and the supernatant was collected. After decarboxylation,
Boric acid ester was formed by adding sugar, strongly oxidized, and boric acid was quantified by titrating sodium hydroxide.

The instruments used for this analysis are all made of quartz.

The copolymer introduced with m-aminophenylboron had a hydroxyl group density of 9.2 meq / g (after cleavage of dihydroxyboronyl group) and an amount of water that could be retained was 1.70 g / g. The particle size and specific surface area were the same as before introduction.

The amount of water that can be retained and the specific surface area are described in JP-A-57-
It was determined by the method described in Japanese Patent No. 30945.

The above aminophenylboronic acid-introduced copolymer has an inner diameter of 6 m
It was packed in a stainless steel column having a length of m and a length of 100 mm to obtain a second column.

The operation of this analyzer will be described. The operation is divided into the following steps by switching the flow path.

Step I (from sample injection to albumin separation in the first column) First, the column thermostat (15) was used to remove the first column (6).
And the second column (11) is kept at 20-60 ° C. The sample is kept at 0 to 10 ° C by the sample cooler (14).
The mobile phase A liquid in the mobile phase A liquid container (1) is sent by the first liquid sending pump (4), and the sample is injected from the sample injection part (5). The injected sample is introduced into the first column (6), separated, and then sent to the fluorescence detector A (7).
Until the separated albumin comes out of the fluorescence detector A (7), the eluate from the first column (6) is discharged to the outside of the flow channel system via the flow channel switching valve (8).

During this period, the second liquid feed pump (10) has the mobile phase switching valve (9) in the solid line flow path and the mobile phase A liquid container (2).
The mobile phase liquid A therein is sent to the column (11) through the flow path switching valve (8).

Step II (Introduction of Albumin in Sample to Second Column) After the albumin in the sample separated by the first column (6) has passed through the fluorescence detector A (7), the flow path switching valve (8) is The flow path is switched to the broken line, and as a result, the eluate from the fluorescence detector A (7) is sent to the second column (10) via the flow path switching valve A (8).

Step III (change of flow path for separation of glycated albumin and non-glycated albumin in the second flow path) After the albumin in the sample is introduced into the second column (10), the flow path switching valve (8) is turned into a solid flow. As a result, the liquid is sent from the second liquid sending pump (10) to the second column (11) via the flow path switching valve (8).

Step IV (step of separating glycated albumin and non-glycated albumin) Next, the mobile phase switching valve (9) is switched to the broken line flow path, and the mobile phase B liquid in the mobile phase B liquid container (3) is changed to the second phase. The liquid is sent to the second column (11) by the liquid sending pump (10) through the flow path of step III. The glycated albumin and the non-glycated albumin separated in the second column (11) are sent to the fluorescence detector B (12), and the detection result is detected together with the detection result of the fluorescence detector A (7) in the data processor (13). ) Is processed.

Step V (regeneration of the second column) The mobile phase switching valve (9) is switched to the solid line flow path, and the mobile phase A liquid in the mobile phase A liquid container (2) is driven by the second liquid transfer pump (10). Then, it is sent to the second column (11) through the flow path of the above step III to regenerate the second column (11).

The above operation is controlled by the automatic control system (16).

An example of analysis of a sample containing glycated albumin and non-glycated albumin using the above-mentioned device will be shown. The analysis conditions are as follows.

Sample: Serum of healthy subjects (5μ) Mobile phase: (Solution A) Aqueous solution containing 250 mM ammonium acetate, 50 mM magnesium chloride and 200 mM sodium chloride (p
H8.5) / Ethanol = 95/5 (Solution B) 100 mM tris (hydroxymethyl) aminomethane, 100 mM sorbitol and 50 mM EDTA 2Na in water (pH 8.5) Flow rate: 1.0 ml / min Temperature: 30 ° C Source: Excitation wavelength: 285 nm, fluorescence wavelength: 340 nm for both fluorescence detector A and fluorescence detector B. The results are shown in FIGS. 3 and 4. FIG. 3 shows the result of separation of albumin in the sample by the first column, and FIG. 4 shows the result of separation of glycated albumin and non-glycated albumin by the second column.

In addition, in FIG. 5 showing a configuration diagram of an embodiment using a flow path switching means having a sample loop, in FIG. 5, albumin in the sample eluted from the fluorescence detector A (7) is After being introduced into the sample loop (17), it is introduced into the second column (11).

[Brief description of drawings]

FIG. 1 is a flow diagram showing an example of the glycated albumin analyzer of the present invention, FIG. 2 is a structural explanatory view showing an embodiment of the device of the present invention, and FIGS. 3 and 4 use the device of the present invention. Chromatogram obtained by analyzing the sample with
FIG. 5 is a flow diagram showing another example of the glycated albumin analyzer of the present invention. 1 ... container for mobile phase A liquid, 2 ... container for mobile phase A liquid, 3
...... Container for mobile phase B liquid, 4 ... First liquid feed pump, 5 ...
... Sample injection part, 6 ... First column, 7 ... Fluorescence detector A, 8 ... Flow path switching valve, 9 ... Mobile phase switching valve,
10 …… Second liquid feed pump, 11 …… Second column, 12 …… Fluorescence detector B, 13 …… Data processor, 14 …… Sample cooler,
15 …… Column thermostat, 16 …… Automatic control system, 17…
… Sample loop

Claims (1)

[Claims] 1. A device for separating and analyzing glycated albumin and non-glycated albumin in a sample by liquid chromatography, a container for mobile phase A liquid used for separating albumin from the sample in the first column, A mobile phase liquid feeding part, a sample injecting part, and a first column for separating albumin from the sample, which are connected in this order.
The flow path, the container for the mobile phase solution A used for eluting the non-glycated arabamine in the second column and the container for the mobile phase solution B used for eluting the glycated albumin are
A second flow path, which is connected in parallel to a second mobile phase liquid-feeding section including mobile phase switching means, and which is further connected with a second column for separating albumin into glycated albumin and non-glycated albumin, and a detector in this order. And glycated albumin are connected in the downstream part of the first column via a flow path switching means existing between the downstream of the second mobile phase liquid-feeding part and the upstream of the second column. Equipment for analysis.