JPH0296657A - Apparatus for analyzing saccharified albumin - Google Patents

Abstract

PURPOSE:To shorten a time for analysis by connecting a second channel to the downstream part of a first column through the intermediary of a channel switching means which is provided between the downstream of a second moving- phase liquid supply element and the upstream of a second column. CONSTITUTION:A liquid A of a moving phase is sent to a first channel by a first moving-phase liquid supply element and to a second channel by a second moving-phase liquid supply element. Next, a sample injected from a sample injection element sent into a first column, together with the liquid A, and albumin in the sample is separated from other components. Subsequently, the channel is switched over by a channel switching means and only the albumin separated in the first column is sent into a second column through the second channel. This albumin is sent into the second column, the channel is switched over by the channel switching means, and the liquid A from the second moving-phase liquid supply element passes through the second channel and is sent into the second column through the channel switching means. By switching the moving phase from the liquid A over to a liquid B in this way, saccharified albumin and non-saccharified albumin are separated in the second column and sent into a detector.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is an analysis method for glycated albumin in which separation of albumin in a sample and separation of glycated albumin and non-glycated albumin are performed in the same flow path of chromatography. Relating to equipment for use.

In diseases such as diabetes, where blood sugar levels are high for a long period of time, various proteins in living organisms undergo non-enzymatic glycation reactions. Serum albumin is one of those proteins.

Glycated albumin has a half-life of 17 days, and responds rapidly to blood sugar conditions compared to glycated hemoglobin, which is said to be a correlated indicator with fasting blood sugar levels three months ago. In the diagnosis of urinary disease, it is useful to know the blood sugar status from several weeks to a month ago. Recently, it has been attracting attention from various quarters as the index that can best meet the demands.

For example, the usefulness of glycated albumin has been suggested as being effective in determining at an early stage whether a current or newly changed dietary therapy is appropriate for the patient. [K.F. McFarland, Diapets
F, Mcfarland et al, DIA OTS)
, 28. Roll, 1979, C.R. Gaslow.

Brok Natal Akad Sai, Ni Nis Ni (
C, Earl Guthrow etal, Proe
, Natl, Aead, Sei.

USA), Yu, 6 No. 9, 4258. 1979
) (Prior Art) Conventionally, as a method for separating and detecting glycated albumin, ion exchange chromatography (James F.D., GA Bio-Chem. D
ay etal, J, Bio, Chem,) + 25
4 No.

3, 595.1979), affinity cloning 7 which introduces a 2-xyboronyl group into a soft gel such as cellulose by dihydration and utilizes the interaction between this functional group and glucose.
Analytical Letters (A), Mallia etal, Ana
lyrical I, etters), 14 (B8)
, 649-661.1981), etc. have been attempted.

Although it is possible to separate glycated albumin and non-glycated albumin with the above conventional technology, it is possible to separate them from other components in body fluids such as serum and plasma, such as immunoglobulins (IgG, IgM).
etc.), haptoglobin, transferrin, etc., and it is virtually impossible to standardize glycated albumin and non-glycated albumin in body fluids.

Therefore, the conventional method has been to isolate albumin from a sample, then take it out of the chromatography system, concentrate it, and then introduce it to a column that can separate glycated albumin and non-glycated albumin. That is, the operation of separating albumin from a sample and the operation of separating albumin into glycated albumin and non-glycated albumin are performed as at least two separate operations, which are complicated and take a long time.

These factors are the main reasons why glycated albumin is not currently measured in clinical tests that require speed, operability, reproducibility, and automation, and there are no analytical devices for this purpose.

Here, as a method for analyzing glycated albumin that overcomes these problems, there is a method described in JP-A-62-226999. That is, albumin is separated from the sample in the first column and guided to the second column without leaving the chromatography flow path system, where the albumin is separated into glycated albumin and non-glycated albumin. This is a method for separating zero-carbonized alpha-mine by chromatography.

(Problems to be Solved by the Invention) According to the example described in JP-A-62-226999, non-glycated albumin and glycated albumin are separated within 30 minutes. However, it takes time to regenerate the first and second columns between the end of an analysis and the start of the next analysis, which lengthens the analysis cycle, making it unsuitable for use in clinical testing. However, there were still problems, especially in terms of speed.

(Means for Solving the Problems) In order to overcome the above-mentioned problems, the present inventors, as a result of intensive research, have developed an automated method for glycated albumin that has excellent speed, operability, and reproducibility. We developed an analyzer for non-zero J9-albumin and completed the present invention.

That is, the present invention enables liquid chromatography to
In an apparatus that separates and analyzes glycated albumin and non-glycated albumin in a sample, there is a mobile phase, a first mobile phase liquid feeding section, a sample injection section, and a first mobile phase that separates albumin from the sample.
A first flow path in which the columns are connected in this order, a mobile phase, a second mobile phase feeding section, a second column for separating albumin into glycated albumin and non-glycated albumin, and a detector are connected in this order. The second flow path formed by A device for analyzing glycated albumin with characteristics is provided.

In the present invention, glycated albumin refers to albumin covalently bonded to glucose, and non-glycated albumin refers to albumin covalently bonded to glucose.
- Refers to albumin that is not bound to gas.

Furthermore, albumin as used in the present invention refers to both glycated albumin and non-glycated albumin.

The sample referred to in the present invention is one containing at least either glycated albumin or non-glycated albumin, or both, and includes, for example, body fluids such as serum, plasma, hemolysate, and urine.

The first column used in the present invention is not particularly limited as long as it can separate albumin from other components in body fluids. Preferred examples include columns filled with a gel bound with a dye such as Cibacron Blue F3G-A, which has a high affinity for albumin, and IgG, which is a large component in serum.
(molecular weight: approx. 150,000) and albumin (molecular weight: approx. 6.6)
Examples include a gel filtration packed column that can separate 1,000 yen) and an ion exchange gel packed column having amino groups.

However, as a carrier for introducing substances with high affinity for albumin such as Cibacron Blue, and as a stationary phase carrier for gel filtration, it is recommended because it has excellent mechanical strength, chemical stability, and less nonspecific adsorption of proteins. Alcoholic hydroxyl group per weight of crosslinked copolymer 1. 0-14. Om
A hard hydrophilic crosslinked copolymer having a specific surface area of 5 to 1000 g/g and a water retention capacity of 0.5 to 6.0 g/g is preferred. The most preferred example is a crosslinked copolymer having an alcoholic hydroxyl group derived from a vinyl alcohol unit, which is described in JP-A-57-30945. Alternatively, crosslinked copolymers described in JP-A-56-64657 and JP-A-59-145036 are also preferred.

Examples of methods for introducing amino groups into the hydrophilic crosslinked copolymer include the following method. That is, the above hydrophilic crosslinked copolymer is reacted with epihalohydrin bisepoxide or the like to obtain an epoxy group-containing crosslinked copolymer, and then ammonia, a primary amine such as ethylamine, or a secondary amine such as diethylamine are reacted. can be obtained by

Examples of the amino group include an unsubstituted amino group, 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 per weight of crosslinked copolymer.
．． On+eq/g, preferably 0.05 to 2,
Omeq/g, more preferably 0.2 to 1
, Omeq/g.

As a method for introducing Cibacron Blue F 3 GA, for example, the hydroxyl groups of the hydrophilic crosslinked copolymer are reacted with bromic cyanide, and then Cibacron Blue F 3 (
, -A may be reacted.

The amount of Cibacron Blue F3G-A was 0.0% per weight of crosslinked copolymer. 002-1. Os+eq/g,
Preferably O, OO5-0. 05meq/g.

Among these gels, gels in which amino groups are introduced into hydrophilic crosslinked copolymers are particularly preferred.

As the most preferable example used in the present invention, JP-A-60-1
A crosslinked copolymer having an alcoholic hydroxyl group derived from a vinyl alcohol unit and a diethylamino group described in Japanese Patent No. 50839 can be mentioned.

This crosslinked copolymer allows albumin to be rapidly separated from other components in body fluids by the method described in Japanese Patent Application No. 1222/1982.

The second column used in the present invention may be a column filled with an ion exchange gel or a gel having a dihydroxyboronyl group, but is not particularly limited as long as it can separate at least glycated albumin and non-glycated albumin.

When using an ion exchange gel, it goes without saying that the slight difference in isoelectric point between glycated albumin and non-glycated albumin is utilized, but cation exchange groups are preferable for ion exchange JAM, especially carboxyl groups and sulfone groups. Acid groups and the like are particularly preferred9.Also, dihydroxyboronyl groups are
Since it specifically binds to compounds having 1,2-cis diol, affinity chromatography using a gel having this functional group as a stationary phase is also effective in separating glycated albumin and non-glycated albumin.

Examples of stationary phase carriers into which these functional groups are introduced include soft gels of polysaccharides such as cellulose and agarose, silica gel, and styrene-divinylbenzene copolymers, which are commonly used as stationary phase carriers for liquid chromatography. The alcoholic hydroxyl groups per weight of the crosslinked copolymer are 1.0 to 14.
A hard hydrophilic crosslinked copolymer having a specific surface area of 5 to 1000 po/g and a water retention capacity of 0.5 to 6.0 g/g is preferred. Most preferred examples include crosslinked copolymers described in JP-A-57-30945, JP-A-56-64657 and JP-A-54-145036.

As a method for introducing dihydroxybonyl groups into the above-mentioned hydrophilic crosslinked copolymer, the following method can be mentioned. That is, it can be obtained by reacting the above-mentioned hydrophilic crosslinked copolymer with shrimp halohydrin, bisepoxide, etc. to obtain an epoxy group-containing crosslinked copolymer, and then reacting it with meta-aminophenylboronic acid.

Further, as a method for introducing a carboxyl group, for example, a method of reacting a halogenated acetic acid such as monochloroacetic acid or monobromoacetic acid with a hydroxyl group of the hydrophilic crosslinked copolymer can be mentioned.

Further, as a method for 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 group is 0.05 to 5.0% per weight of crosslinked copolymer. Omeq/g, preferably 0.
05-3. Omeq/g, more preferably 0
．． 1-2. Omeq/g.

The amount of carboxyl group or sulfonic acid group is 0.02 to 5.0% per weight of crosslinked copolymer. Omeq/g, preferably 0.05 to 2.0+++eq/g, more preferably 0. 2-1. Omeq/g.

Among these gels, gels in which dihydroxyboronyl groups are introduced into hydrophilic crosslinked copolymers are particularly preferred. As the most preferable example used in the present invention, JP-A-62-19240
The boron-containing crosslinked copolymer having an alcoholic hydroxyl group and a dihydroxyboronyl group described in No. 5 can be mentioned.

The gel having immobilized functional groups used in the present invention may have various shapes such as spherical and crushed shapes, but is preferably spherical. In that case, the weight average particle size is 1 to 50
0 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, and the relationship between the first column, the second column, the first flow path, and the second flow path will be explained.

In FIG. 1, a mobile phase liquid A is sent to a first channel by a first mobile phase feeding section, and is sent to a second channel by a second mobile phase feeding section. The sample injected from the sample injection section is sent to the first column together with the mobile phase A solution, and albumin and other components in the sample are separated. Next, the flow path is switched by the flow path switching means, and only the albumin separated in the first column is sent to the second column through the second flow path. After the albumin is sent to the second column, the flow path is switched by the flow path switching means, and the mobile phase A solution sent from the second mobile phase liquid feeding section passes through the second flow path. It is sent to the second column via the flow path switching means. By switching the mobile phase from liquid A to liquid 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 section is B
The liquid is switched to liquid A, passes through the second flow path, and is sent to the second column via the flow path switching means to regenerate the second column. Once the regeneration of the second column is complete, the next analysis is possible. In order to confirm the separation of albumin in the first column, a monitoring detector is installed between the outlet of the first column and the flow path switching means, or in the downstream part of the flow path switching means of the first flow path. You can prepare.

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

Gel with immobilized dihydroxyboronyl groups is used as a packing material to separate glycated albumin and non-glycated albumin or glycated protein and non-glycated protein, and to separate albumin and other components. When using a gel with immobilized amino groups as a packing material, at least two types of mobile phases (liquid A and liquid B) are used.
A preferred method is to prepare a liquid and switch from liquid A to liquid B midway through. Switching from liquid A to liquid B can be performed stepwise or by a so-called gradient method in which the ratio is continuously changed.

The A solution mentioned here is preferably an aqueous solution with a pH of 7.5 to 9.5, which has a buffering base of 10 to 500 a+M and does not contain a substance containing 1,2-cis diol. As the buffering base, a base having a buffering capacity at pH 7.5 to 9.5 is preferred, and particularly preferred bases include ammonium salts such as ammonium acetate, morpholine, N-2-
Examples include hydroxyethylpiperazine N°-2-ethanesulfonic acid. These buffering bases are
Because it can strengthen the bond between the dihydroxyboronyl group and the sugar part of glycated proteins such as glycated albumin,
It is effective in highly separating glycated proteins such as glycated albumin from non-glycated proteins. If the pH is lower than 7.5, the bond between the dihydroxyboronyl group and the sugar becomes weak, which is not preferable.

Furthermore, if the pH is higher than 9.5, proteins such as albumin may be denatured and precipitate may be produced, which is not preferable. Moreover, the A solution can contain salts other than the buffer base. In particular, it is preferable to include a salt of a divalent metal ion, such as magnesium chloride, because it can strengthen the bond between the sugar chain and the dihydroxybonyl group. The concentration of the divalent metal ion salt is preferably between 5IIIM and 11001n.
It is. Salts of monovalent metal ions, such as sodium chloride, may also be included, preferably at a concentration of 500+
It is less than nM.

On the other hand, solution B has a buffer base of 10 to 500 mM.
An aqueous solution having a pH of 2 to 6.5 is preferable, and even if the pH is 7.5 or higher, it is sufficient as long as it contains a substance having 1.2-cis diol. In that case as well, the pH is preferably 9.5 or less. Using this condition, the bond between the dihydroxyboronyl group and the #M chain can be weakened, and glycated proteins such as glycated albumin bonded to the dihydroxyboronyl group can be expelled.

As the buffering base, a buffering base having a buffering capacity at the pH used is preferred, and particularly preferred examples include tris(hydroxymethyl)aminomethane, triethanolamine, and the like. These buffering bases can weaken the bond between the dihydroxyboronyl group and the sugar chain, and facilitate the elution of the glycated kungbang substance bonded to the dihydroxyboronyl group.

Examples of substances having 1,2-cis diol include:
Examples include HM such as sorbitol and mannitol. The concentration of the substance containing 1,2-cis diol is 5
0-2000mM is preferred.

Furthermore, if the A solution contains a salt of a divalent metal ion,
It is preferable to add a metal chelating agent such as ethylenediaminetetraacetic acid (EDTA) to the B solution. In that case, the concentration of the metal chelating agent is preferably 5 to 100 IR1'I. A
, For both liquids B, for example, ethanol, methanol, n
It may be preferable to contain a small amount of a water-soluble organic solvent such as ~propyl alcohol, 1so-brobylalniol, 7cetonitrile, or ethylene glycol.

Although the shape of the column used in the present invention is not particularly limited,
Preferably, the inner diameter is 10 mm or less and the length is 30 cm or less, and the inner diameter is 2 to 8 mm and the length is particularly preferably 1 to 10 CII11. The flow rate of the mobile phase is also not particularly limited, but is 0.1~
10111/win is preferred, particularly preferably 0,
3 to 5 results/win.

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

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

(Effects of the Invention) The glycated albumin analyzer of the present invention is capable of separating and quantifying glycated albumin and non-glycated albumin in a sample with only one sample injection operation, which conventionally requires a long time. The time required for one analysis was significantly reduced.

In the present invention, since a mobile phase of a constant composition is always flowing through the first column, there is no need for column regeneration time associated with switching the mobile phase. Until albumin is introduced into the second column, the second column is regenerated by the mobile phase A liquid sent by the second mobile phase liquid sending section. Also, the second
After albumin has been introduced into the column, the mobile phase for analysis and second column regeneration is sent from the second mobile phase feeding section to the second column through a short channel. For the above reasons, the time from starting one analysis to starting the next analysis was significantly shortened.

The apparatus 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.

(Example) An example of the present invention will be described based on a configuration explanatory diagram shown in FIG.

In Fig. 2, (1) is a container for mobile phase A liquid, and the first flow path from there includes a first liquid pump (4), a sample injection part (5), and a first column (6). ), a fluorescence detector A (7), and a flow path switching pulp (8) are connected. Also,(
2) is a container for mobile phase A, and (3) is a container for mobile phase B, the flow path of which is connected to the mobile phase switching valve (9) and the second liquid feeding pump (00). A second flow path connected to the flow path switching pulp (8) is formed, and a second column (11) and a fluorescence detector BO are connected to the second flow path from the flow path switching pulp (8). 0 hot water is a data processing machine, 04
) is the sample cooler, 0ω is the constant temperature bath for the column, and 00 is the automatic control system.

The first column was a gel described in Example 1 of JP-A No. 60-150839 (weight average particle size 9.0 μm, hydroxyl group density 4.9 meq/g, amount of water that can be held t, 9 g/g).
g, diethylamino group is 0.5 meq/g) with an inner diameter of 7
．． A column made of stainless steel and having a length of 100+ nm was used.

The second column was prepared as follows.

Vinyl alcohol copolymer gel described in Example 1 of JP-A-57-30945 (X; 0°3, hydroxyl group content;
7. 8 meq/g, amount of water that can be held; 1,78
g/g, specific surface area: 78ryf/g, particle size: 9,
Qμm) 25g, epichlorohydrin 116.8g
, 250 g of dimethyl sulfoxide, and 12.5 d of 1ON aqueous sodium hydroxide solution were added, and the mixture was reacted at 30°C for 200 hours. Amount of epoxy group introduced: Omeq/
It was g. The method for quantifying epoxy groups is described in Japanese Patent Application Laid-open No. 57
The method described in Japanese Patent No. 190003 was used.

Next, 13 g of m-aminophenylboronic acid hemisulfate was dissolved in 250-degree water, and the pH was adjusted to 11. To this solution, 20 g of the epoxy group-introduced polymer was added, and the reaction was carried out at 60° C. for 200 hours. Next, 2511IM tris(hydroxymethyl)aminomethane was added to protect the remaining epoxy groups, and the reaction was carried out at 50°C for 5 hours. The amount of boronic acid introduced was 0.28 meq/g. The boronic acid was determined by the following method.

10 d of 30% hydrogen peroxide solution was added to 1 g of the polymer into which m-aminophenylboronic acid was introduced, and the reaction was allowed to proceed for 5 hours to cleave the boron-carbon bond. Next, the polymer was spun down and the supernatant was collected. After decarboxylation, a boric acid ester was formed by adding sugar, strongly oxidized, and boric acid was determined by titration with sodium hydroxide.

All instruments used in this analysis were made of quartz.

The hydroxyl group density of the copolymer into which m-aminophenylboronic acid has been introduced is 9. 2meq7g (after dihydroxyboronyl group cleavage), the amount of water that could be retained was 1.70g/g. Furthermore, 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 determined according to Japanese Patent Application Laid-open No. 57
It was determined by the method described in Publication No.-30945.

The above aminophenylboronic acid-introduced copolymer was mixed with an inner diameter of 6 cm.
A stainless steel column having a length of 100 m was filled with the mixture to form a second column.

To explain the operation of this analyzer, it operates in the following steps by switching channels.

Step 1 (From sample injection to albumin separation in the first column) First, the first column (6) and the second column (11) are maintained at 20 to 60°C using the column constant temperature bath 09. The sample is maintained at 0-10"C by a sample cooler 04). The mobile phase A liquid in the mobile phase A method container (1) is fed by the first liquid feeding pump (4). Sample injection part (5
). The injected sample is introduced into the first column (6), separated, and then subjected to fluorescence detection HA (7).
sent to. The separated albumin is detected by fluorescence detector A (7
), the eluate from the first column (6) is discharged to the outside of the flow path system via the flow path switching valve (8).

During this period, the second liquid feeding pump 0Φ transfers the mobile phase A liquid in the mobile phase A method container (2) to the flow path switching valve (8) with the mobile phase switching valve (9) in the solid line flow path state. The liquid is sent to the second column 01) via the column 01).

Step ■ (Introduction of albumin in the sample into the second column) After the albumin in the sample separated by the first column (6) passes through the fluorescence detector A (7), the flow path switching valve (
8) is switched to the broken line flow path, and as a result, the eluate from the fluorescence detector A (7) is sent to the second column 00 via the flow path switching valve A (8).

Step ■ (Changing the flow path to separate glycated albumin and non-glycated albumin in the second flow path) After the albumin in the sample is introduced into the second column (11), the flow path switching valve (8) is switched to the solid line flow. As a result, the liquid sent from the second liquid sending pump (+(+1) is sent to the second column (11) via the flow path switching valve (8).

Step IV (Step of separating tl-conjugated 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 solution in the mobile phase B method container (3) is transferred to the second The liquid is sent to the second column (11) through the flow path of step (2) by the liquid sending pump (10). Glycated albumin and non-glycated albumin separated in the second column G+) are sent to the fluorescence detector BO2), and the detection results are processed by a data processing device along with the detection results of the fluorescence detector A (7). .

Step ■ (Regeneration of the second column) The mobile phase switching valve (9) is switched to the solid line flow path, and the mobile phase A solution in the mobile phase A method container (2) is transferred by the second liquid sending pump 00). It is sent to the second column (II) through the flow path of step (2) above, and regenerates the second column (11).

Note that the above operations are controlled by an automatic control system 0ω.

The analysis conditions for an example of analysis of a sample containing glycated albumin and non-glycated albumin using the above apparatus are as follows.

Sample: Healthy subject serum (5 μl) Mobile phase: (Solution A) 250 mM ammonium acetate, 5 μl
Aqueous solution (pH 8,5) containing 0mM magnesium chloride and 200mM sodium chloride/ethanol = 9515 (Solution B) Aqueous solution (pH 8,5) containing 100fiM tris(hydroxymethyl)aminomethane, 100mM sorbitol and 50mM HDT^2Na Flow rate :1. Od/min Temperature: 230"C Detection: Excitation wavelength for both fluorescence detector A and fluorescence detector B: 285 nm, fluorescence wavelength: 340 ni The results are shown in Figs. 3 and 4. Figure 4 shows the results of separation of albumin in a sample using one column.
This figure shows the results of separation of glycated albumin and non-glycated albumin using a column.

Further, FIG. 5 shows a configuration diagram of an embodiment using a flow path switching means equipped with a sample loop. In FIG. 5, albumin in the sample eluted from fluorescence detector A (7) is
- first, it is introduced into the sample loop 07) and then into the second column (11).

[Brief explanation of the drawing]

FIG. 1 is a flow diagram showing an example of the glycated albumin analyzer of the present invention, FIG. 2 is a configuration explanatory diagram showing an example of the device of the present invention, and FIGS. 3 and 4 are diagrams showing the use of the apparatus of the present invention. A chromatogram obtained by analyzing a sample using
FIG. 5 is a flow diagram showing another example of the glycated albumin analyzer of the present invention. 1 Column 7...Fluorescence detector A 8...Flow path switching valve 9...Mobile phase switching valve 10...Second liquid pump 11...Second column 12...Fluorescence detector B
13. Data processor 14. Sample cooler 15.
・Thermostatic chamber for columns 16・・Automatic control system 17・
・Sample loop (1 other person)

Claims (1)

[Scope of Claims] An apparatus for separating and analyzing glycated albumin and non-glycated albumin in a sample by liquid chromatography, which includes: (1) a mobile phase, a first mobile phase liquid feeding section, a sample injection section, and a sample from a document; a first flow path in which a first column that separates albumin is connected in this order; (2) a mobile phase, a second mobile phase feeding section, and a second column that separates albumin into glycated albumin and non-glycated albumin;
and a detector are connected in this order, and a second flow path is connected downstream of the first column via a flow path switching means that exists between the downstream of the second mobile phase liquid feeding section and the upstream of the second column. A device for analyzing glycated albumin, characterized in that the parts are connected.