JP4507682B2 - Sugar chain separation method, sample analysis method, liquid chromatography apparatus, sugar chain analysis method and sugar chain analysis apparatus - Google Patents

Description

  The present invention relates to a method for separating sugar chains used for analysis of glycoproteins in disease diagnosis and the like, a method for analyzing samples using normal phase and reverse phase liquid chromatography, a liquid chromatography apparatus used therefor, and a sugar chain The present invention relates to an analysis method and an analysis apparatus.

The sugar chains of glycoproteins play an important role in the diagnostic field, particularly in cancer diagnosis. It has already been clarified that sugar chains such as sialyl Lewis X, sialyl Tn, CA50, and CA15-3 increase in blood concentration with canceration, and the presence or absence thereof is examined by an antibody that specifically binds to these. Can do.
However, antibodies do not always recognize only the target sugar chain, and some antibodies may recognize sugar chains other than the target. In recent years, analysis of sugar chains by a highly selective analytical chemistry method has attracted attention.

  As a method for analyzing a sugar chain of a glycoprotein, the sugar chain is cut out from the glycoprotein and then labeled with a fluorescent compound, which is then subjected to normal phase liquid chromatography (hereinafter referred to as “normal phase HPLC” as appropriate) and reverse phase liquid chromatography. An analysis method is generally used (hereinafter referred to as “reverse phase HPLC” as appropriate) (Non-patent Document 1).

  Methods for cleaving a sugar chain from a glycoprotein include a method using a sugar chain cleaving enzyme (Non-Patent Document 2) and a method using anhydrous hydrazine (Non-Patent Document 3). However, the method using an enzyme has a drawback that a sugar chain deeply incorporated into a protein cannot be completely cut out. Therefore, in many cases, a chemical method using anhydrous hydrazine is used.

  Many natural sugar chains include acetylated sugars (specifically, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, etc.) as constituent sugars. Is eliminated during hydrazine decomposition. For this reason, it is necessary to acetylate after hydrazine decomposition. Therefore, conventionally, a method of acetylating by adding a saturated aqueous carbonate solution and acetic anhydride after distilling off hydrazine has been common.

  Furthermore, in the process of separation by normal phase HPLC and reverse phase HPLC, normal phase HPLC and reverse phase HPLC have different resolutions, and depending on the type of sugar chain, separation by normal phase HPLC is difficult. However, it can often be easily separated by reverse phase HPLC. Therefore, conventionally, a method has been used in which tens or hundreds of sugar chains are finally separated into a single sugar chain by a combination of normal phase HPLC and reverse phase HPLC.

  By the way, for the purpose of labor saving and speedup, many examples of connecting an apparatus used for liquid chromatography (hereinafter referred to as “HPLC apparatus” as appropriate) online via a plurality of automatic valve switches have been reported. For example, Wolters et al. Succeeded in separating a large number of protein fragments (peptides) in a living body by connecting an ion exchange column and a reverse phase column (Non-patent Document 4).

Hase S. Ikenaka T .; , Matsushima Y .; J .; Biochem. , 90, 407-414 (1981) S. Takasaki, T .; Mizuchi, A.M. Kobata, “Methods in Enzymology”, ed. by V. Ginsburg, Vol. 83, p. 263, Academic Press, New York (1982) Takahashi N. et al. , Biochemical and Biophysical Research Communications, 76, 1194-1200 (1977). Wolters D.W. A. , Washburn M .; P. , YatesJ. R. Anal. Chem. 73 (23) 5683-5690 (2001)

  However, when sugar chains are obtained from glycoproteins by the conventional method using anhydrous hydrazine, (1) it takes time to remove hydrazine having a high boiling point, and (2) when a large amount of hydrazine is evaporated at once. However, it took a lot of time to process a large number of specimens. That is, the conventional method for obtaining sugar chains from glycoproteins with anhydrous hydrazine has problems in processing time and handling.

  In addition, when the acetyl group elimination site of a sugar chain cut out by a method using anhydrous hydrazine is acetylated again, the conventional method of acetylating by adding saturated aqueous carbonate and acetic anhydride after distilling off hydrazine is an acetylation method. In addition to the time required for the reaction, a lot of time was required for desalting a large amount of salt remaining in the system. In other words, sugar chains containing acetylated sugars as constituent sugars require a long treatment time in a series of treatments such as acetylation and desalting of sugar chains after hydrazine removal. It was difficult to get.

  Furthermore, when analysis was performed by combining normal phase HPLC and reverse phase HPLC, the eluate of normal phase HPLC could not be directly led to reverse phase HPLC by connecting the apparatuses. This is because the eluent of normal phase HPLC has a very strong elution effect for reverse phase HPLC. For example, when direct flow from normal phase HPLC to reverse phase HPLC, the sugar chain is completely retained in the reverse phase column. Because it is not done. The same applies to the case of deriving from reverse phase HPLC to normal phase HPLC. In order to cope with this, conventionally, after analysis by normal phase HPLC, solvent replacement was performed before reverse phase HPLC. Specifically, the solvent displacement is a process in which the eluate from normal phase HPLC is once taken into a test tube, the solvent is distilled off with a concentrating dryer or an evaporator, and then redissolved in water. To do. However, this method requires a great amount of labor and requires a lot of time when analyzing a large number of samples. That is, since normal phase HPLC and reverse phase HPLC could not be directly connected online, the eluate was once collected between the apparatuses used for normal phase HPLC and reverse phase HPLC, and the solvent was distilled off. A solvent replacement step of redissolving was necessary. In other words, there is a problem that much labor is required for analysis and it takes time to analyze a large number of samples.

  In addition, examples such as Non-Patent Document 4 can be combined only when one elution condition is the other retention condition, and one elution condition such as normal phase HPLC and reverse phase HPLC is the other. There have been no reports on-line connection of combinations that require so-called solvent replacement, which would result in elution conditions.

  The present invention was devised in view of the above-described problems, and efficiently separates glycoprotein sugar chains in a short time, and various samples represented by the separated sugar chains can be efficiently obtained in a short time. The purpose is to analyze it.

  As a result of studying to solve the above-mentioned problems, the present inventors have passed a hydrazine decomposition solution of glycoprotein through a column packed with an alkali-resistant packing, and further passed a washing solvent for eluting hydrazine. Thus, it was found that sugar chains can be separated from a hydrazine decomposition solution of glycoprotein. In addition, the first eluate containing the specimen separated by one of normal phase liquid chromatography and reverse phase liquid chromatography is passed through the ion exchange column to adsorb the specimen, and then the specimen is removed from the ion exchange column. It was found that the sample can be efficiently analyzed in a short time by eluting and separating by the other of normal phase liquid chromatography and reverse phase liquid chromatography.

  That is, the gist of the present invention is a method for separating a sugar chain from a hydrazine degradation solution of glycoprotein, wherein the hydrazine degradation solution of glycoprotein is passed through a column packed with an alkali-resistant packing capable of retaining the sugar chain. The present invention resides in a method for separating sugar chains, comprising: a sugar chain retaining step for liquefying, and a washing step for passing a washing solvent for eluting hydrazine through the column (claim 1). By this sugar chain separation method, sugar chains of glycoproteins can be efficiently separated in a short time.

  Another gist of the present invention is a method for separating a sugar chain from a hydrazine decomposition solution of glycoprotein, and passing the hydrazine decomposition solution of a glycoprotein through a column packed with a filler capable of retaining a sugar chain. A sugar chain holding step, a washing step of passing a washing solvent for eluting hydrazine through the column, an acetylation step of passing an acetylation reagent through the column, and an acetylation reagent through the column. And a reaction agent washing step of passing a desalting agent through which the salt is eluted. (Claim 2) By this sugar chain separation method, the sugar chain of the glycoprotein can be efficiently separated in a short time. When the sugar chain of the glycoprotein originally has an acetyl group, it is detached in the hydrazine decomposition solution. The acetyl group that had been added can be added in a short time.

  Another aspect of the present invention is a method for separating sugar chains from a hydrazine degradation solution of glycoprotein, wherein the hydrazine degradation solution of glycoprotein is passed through a column packed with a filler capable of retaining the sugar chains. A sugar chain retention step for liquid, a washing step for passing a washing solvent for eluting hydrazine through the column, and an acetylation elution step for passing an acetylation reagent capable of eluting sugar chains to the column. The present invention resides in a sugar chain separation method characterized by comprising (claim 3). This sugar chain separation method can also efficiently separate glycoprotein sugar chains in a short time, and if the glycoprotein sugar chain originally has an acetyl group, it can be removed in the hydrazine decomposition solution. The separated acetyl group can be added in a short time.

  Furthermore, the filler is preferably alkali-resistant (claim 4). Thereby, it can prevent that the filler decomposed | disassembled with the alkali elutes from a column and mixes with a sugar_chain | carbohydrate, and can prevent that a filler is decomposed | disassembled with an alkali and the retention strength of sugar_chain | carbohydrate falls.

Further, another aspect of the present invention is to provide a decomposition step in which glycoprotein is decomposed with hydrazine to prepare a hydrazine decomposition solution of glycoprotein, and the hydrazine decomposition solution prepared in the decomposition step can retain a sugar chain. A sugar chain holding step for passing through a column packed with a packing material, a washing step for passing a washing solvent for eluting hydrazine through the column, and an acetylation step for passing an acetylation reagent through the column. A reaction agent washing step of passing a desalting agent to elute the acetylation reagent through the column, and elution of the sugar chain from the column and subjecting the sample liquid containing the resulting sugar chain to normal phase liquid chromatography And a first separation step that is separated by one of reverse-phase liquid chromatography and a first eluate containing a sugar chain separated in the first separation step through an ion exchange column. A sugar chain adsorption step for adsorbing sugar chains, and elution of sugar chains from the ion exchange column, and a second eluate containing the obtained sugar chains is obtained by the other of normal phase liquid chromatography and reverse phase liquid chromatography. A sugar chain analysis method comprising a second separation step of separating (claim 5 ). By this analysis method, it is possible to analyze the sugar chain of glycoprotein efficiently in a short time.

Further, another gist of the present invention is a glycan analyzer used in the above glycan analysis method, wherein the glycan is analyzed by a column packed with a filler capable of retaining a glycan and by normal phase liquid chromatography. A normal phase liquid chromatography part for separating the glycan, a reverse phase liquid chromatography part for separating sugar chains by reverse phase liquid chromatography, and an ion exchange column, wherein the column is a detection solution that has flowed out of the column. Is connected to flow into one of the normal phase liquid chromatography part and the reverse phase liquid chromatography part, and the ion exchange column is connected to the normal phase liquid chromatography part and the reverse phase liquid chromatography part. The first eluate flowing out from one of them flows into the ion exchange column, and the second eluent flowing out from the ion exchange column becomes the normal phase liquid chroma A sugar chain analyzer characterized in that it is connected between the normal phase liquid chromatography unit and the reverse phase liquid chromatography unit so as to flow into the other of the chromatography unit and the reverse phase liquid chromatography unit (Claim 6 ). By using this analyzer, it is possible to efficiently analyze the sugar chains of glycoproteins in a short time.

According to the sugar chain separation method of the present invention, a sugar chain of a glycoprotein can be efficiently separated in a short time.
Moreover, according to the analysis method or liquid chromatography apparatus of the present invention, sugar chains and other specimens can be analyzed efficiently in a short time.
Furthermore, according to the sugar chain analysis method or sugar chain analyzer of the present invention, sugar chains can be efficiently analyzed in a short time.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. However, the present invention is not limited to the following embodiments, and can be arbitrarily modified and implemented without departing from the gist thereof.

I. Separation of glycochain of glycoprotein First, a method for separating a sugar chain from a glycoprotein will be described.
In this embodiment, first, a decomposition step of preparing a hydrazine decomposition solution for glycoprotein is performed to cut out sugar chains from the glycoprotein, and then the sugar chains are separated from the hydrazine decomposition solution by the sugar chain separation method of the present invention. Here, the sugar chain separation method of the present invention is a method for separating sugar chains from a hydrazine decomposition solution of glycoprotein, and a hydrazine decomposition solution of glycoprotein is applied to a column packed with a filler capable of retaining sugar chains. It has a sugar chain holding step for passing liquid and a washing step for passing a washing solvent for eluting hydrazine through the column. Furthermore, when the sugar chain of the glycoprotein has an acetyl group before the sugar chain is cleaved by hydrazine, an acetylation step in which the acetylation reagent is passed through the column after the washing step, and an acetyl group in the column. And a reactive agent washing step of passing a desalting agent that elutes the chemical reaction agent.

Details will be described below.
[1] Decomposition step In the decomposition step, glycoprotein is decomposed with hydrazine to prepare a hydrazine decomposition solution of glycoprotein.
There is no restriction | limiting in particular in the preparation method of a hydrazine decomposition solution, It can prepare by well-known arbitrary methods. However, it is usually prepared by adding hydrazine to a sample (organ, tissue, glycoprotein, etc.) to be analyzed and heating it. As a result, the protein is almost completely decomposed in the hydrazine decomposition solution, and the sugar chain bound to the protein is released from the protein. In addition, when preparing a hydrazine decomposition solution by said method, the temperature to heat is 60 degreeC or more normally, Preferably it is 90 degreeC or more, and is 150 degrees C or less normally, Preferably it is 110 degrees C or less.

  At this time, if the sugar chain in the sample has an acetyl group, the acetyl group is desorbed from the sugar chain by hydrazine, and the sugar chain in the hydrazine decomposition solution becomes a sugar chain having no acetyl group. In this manner, a sugar chain having an acetyl group in a state of being bound to a glycoprotein and having lost its acetyl group by hydrazine decomposition is hereinafter referred to as “acetyl group-eliminated sugar chain” as appropriate. In the present specification, the term “sugar chain” is used as a broad term without distinguishing between those having an acetyl group and those having no acetyl group. In addition, the sugar chain referred to here is glucose, galactose, mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid or the like as a constituent sugar, which are connected by a plurality of glycosidic bonds, or Monosaccharides are also included.

[2] Sugar chain holding step In the sugar chain holding step, the hydrazine decomposition solution prepared in the decomposition step is passed through a column (hereinafter referred to as “sugar chain holding column” as appropriate).
The packing material of the sugar chain holding column is not particularly limited as long as it has a performance capable of holding a sugar chain, and any substance can be used. Specific examples include usually silica-based fillers such as octadecylsilane, octylsilane, cyclohexylsilane, phenylsilane, cyanopropylsilane, silica gel; magnesium silicate-based fillers such as florisil; polystyrene-based fillers such as styrenedivinylbenzene Agents; normal phase fillers such as amino and amide; carbon fillers such as graphite carbon; high molecular sugar fillers such as cellulose and Sephadex. However, since the hydrazine solution is alkaline, an alkali-resistant substance is preferable from the viewpoint of degradation of the filler due to hydrolysis and prevention of debris outflow. Specifically, graphite carbon, styrene divinylbenzene, and the like are preferable. Here, the alkali resistance means that it does not decompose or denature when it comes into contact with a solution having pH = 7 or higher. In addition, these fillers may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio.
Further, the packing density of the filler is optional, usually 0.2 g / cm ² or more, preferably 0.3 g / cm ² or more, and usually 1.0 g / cm ² or less, preferably 0.8 g / cm ² or less.

By passing the hydrazine decomposition solution through the sugar chain holding column, the sugar chains in the hydrazine decomposition solution are held in the column filler.
The flow rate of the hydrazine decomposition solution is arbitrary, but is preferably set according to the amount of the sample such as glycoprotein and the volume of the column to be used. For example, it is usually 0.1 mL / min or more, preferably 0.5 mL / min or more, and usually 100 mL / min or less, preferably 5 mL / min or less.

[3] Washing step In the washing step, a washing solvent for eluting hydrazine is passed through the sugar chain holding column through which the hydrazine decomposition solution was passed in the sugar chain holding step. By passing the washing solvent through the sugar chain holding column, hydrazine can be completely removed while the sugar chain is held in the filler of the sugar chain holding column.

  Here, as the cleaning solution, any liquid can be used as long as it has a property of eluting hydrazine while the sugar chain is held in the sugar chain holding column. Therefore, the specific type of the washing solution to be used can be selected according to the sugar chain holding column to be used.

For example, for a reverse phase column in which the sugar chain retention column is a carbon-based filler such as graphite carbon or a polystyrene-based filler such as styrenedivinylbenzene, water or an aqueous acetate solution is used as a cleaning liquid. It is preferable to use an aqueous solution such as an aqueous solution of ammonium formate, an aqueous solution of triethylamine acetate, an aqueous solution of sodium acetate, an aqueous solution of sodium phosphate, etc., more preferably water, an aqueous solution of ammonium acetate, an aqueous solution of ammonium formate, etc. preferable.
For normal phase columns in which the packing material is cellulose or the like, it is preferable to use an organic solvent such as methanol, ethanol, butanol, acetonitrile, acetone, chloroform, but in particular, butanol, ethanol, or a mixed solvent thereof is used. More preferably, it is used.
In addition, said washing | cleaning solution may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and ratios.

The flow rate and flow rate of the washing solution are preferably set according to the amount of the sample such as glycoprotein, the volume of the sugar chain holding column to be used, and the like. For example, the flow rate is arbitrary, but it is usually 1 or more times, preferably 3 or more times the column volume, and usually 50 or less, preferably 20 or less. Further, although the liquid passing speed is also arbitrary, it is usually 0.1 mL / min or more, preferably 1.0 mL / min or more, and usually 100 mL / min or less, preferably 10 mL / min or less.
As described above, by performing the sugar chain holding step and the washing step, the sugar chains in the hydrazine decomposition solution can be separated and obtained as sugar chains held in the filler of the sugar chain holding column.

[4] Acetylation step Next, an acetylation step is performed. When the sugar chain originally has an acetyl group in the glycoprotein, the target sugar chain cannot be obtained immediately after the washing step. That is, in such a case, since the acetyl group is released from the sugar chain in the hydrazine decomposition solution, an acetyl group-removed sugar chain is obtained immediately after the washing step, from which the acetyl group is released from the target sugar chain. It is done. Therefore, in order to obtain a sugar chain having an acetyl group, it is necessary to acetylate the portion of the acetyl group-eliminated sugar chain from which the acetyl group is eliminated.

In the acetylation step, the acetylation reagent is passed through the sugar chain holding column after hydrazine is removed in the washing step. By passing the acetylation reagent through the sugar chain-holding column, the acetyl group-eliminated sugar chain held in the filler can be acetylated to obtain the target sugar chain.
The acetylation reagent is not limited as long as it is a fluid that can acetylate an acetyl group-eliminated sugar chain. However, usually, an acetylating reagent dissolved in water or a buffer is used.

  The acetylating reagent refers to one that can convert alcohol and amine into ester and amide, respectively, and any known acetylating reagent can be used. Specific examples include acetic anhydride, acetyl bromide, acetyl chloride, acetylimidazole and the like. Of these, acetic anhydride is preferred. In addition, an acetylation reagent may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio.

Also, any liquid can be used as a buffer or solvent for dissolving the acetylating reagent. Specific examples include ammonium acetate aqueous solution, ammonium formate aqueous solution, sodium acetate aqueous solution for reversed-phase columns using carbon-based fillers such as graphite carbon and polystyrene-based fillers such as styrenedivinylbenzene as fillers. , An aqueous trishydroxymethylaminomethane solution, an aqueous trimethylamine acetate solution and the like are used, and among them, an aqueous trishydroxymethylaminomethane solution is preferable. In particular, when an aqueous solution as described above is used as a buffer or a solvent, the concentration of the aqueous solution is such that the sugar chain does not flow out of the column due to the passage of the aqueous solution, and pH variation due to the acetylation reaction is suppressed. It is optional if possible. It is usually 0.1M or more, preferably 0.5M or more, and usually 2.0M or less, preferably 1.0M or less. If this concentration is too low, the pH may be lowered by the acid generated by the acetylation reaction and the reaction may be stopped, and if this altitude is too high, sugar chains may flow out of the column.
In addition, for normal phase columns in which the filler is cellulose or the like, alcohols such as ethanol and butanol; water-soluble solvents such as acetone and acetonitrile can be used. In addition, these may be used individually by 1 type and may use 2 or more types together by arbitrary combinations and ratios.

  The concentration of the acetylating reagent is also arbitrary, but is usually set according to the packing material of the sugar chain holding column to be used. For example, when a carbon-based filler such as graphite carbon or a polystyrene-based filler such as styrenedivinylbenzene is used as the filler for the sugar chain holding column, the concentration of the acetylating reagent is usually 0.1% by weight or more, preferably 0. .5% by weight or more, and usually 10% by weight or less, preferably 3% by weight or less. For example, when cellulose is used as the filler, the concentration of the acetylating reagent is usually 0.1% by weight or more, preferably 1% by weight or more, and usually 80% by weight or less, preferably 10% by weight or less. .

  Furthermore, it is desirable to adjust the pH by appropriately mixing a buffer with the acetylation reagent. For example, when a carbon-based filler such as graphite carbon or a polystyrene-based filler such as styrenedivinylbenzene is used as a filler for the sugar chain retention column, the pH of the acetylating reagent is usually 4 or more with a buffer. Preferably it is 5 or more, usually 10 or less, preferably 8 or less. If the pH is too high, the acetylating reagent may be hydrolyzed, and if the pH is too low, acetylation may not proceed.

  Further, the flow rate of the acetylation reagent is preferably set according to the amount of the sample such as glycoprotein, the volume of the sugar chain holding column to be used, and the like. The flow rate is usually 0.1 mL / min or more, preferably 0.2 mL / min or more, more preferably 0.5 mL / min or more, and usually 10 mL / min or less, preferably 5 mL / min or less, more preferably Although 2 mL / min or less is desirable, it is not particularly limited thereto.

[5] Reactant cleaning step In the reagent cleaning step, a desalting agent (reactant cleaning agent) is passed through the sugar chain-holding column after the acetylation reactant is passed through in the acetylation step. By passing a desalting agent through the sugar chain holding column, the acetylation reagent in the sugar chain holding column can be removed to obtain the target sugar chain.
The desalting agent is particularly suitable as long as it can hold the sugar chain in the column and can elute the acetylation reagent (if the buffer contains a salt as a buffering agent, the salt is included). There is no restriction | limiting and arbitrary things can be used. As a specific example, the same cleaning solution as that used in the cleaning step can be used. Moreover, a desalinization material may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio.
The amount of the desalting agent to be passed is usually 1 or more times, preferably 2 or more times, more preferably 3 or more times, and usually 10 or less times, preferably the volume of the sugar chain holding column used. It is 8 times or less, more preferably 6 times or less.

  As described above, the target sugar chain can be obtained by performing the reactant washing step. However, the acetylation step and the reactant cleaning step may be omitted. That is, a method for separating a sugar chain from a hydrazine degradation solution of glycoprotein, wherein the sugar chain retention step of passing a glycoprotein hydrazine degradation solution through a column packed with an alkali-resistant packing capable of retaining the sugar chain The target sugar chain can also be obtained by performing a washing step of passing a washing solvent for eluting hydrazine through the column. Specifically, this is the case when the sugar chain is a kind of sugar chain having no acetyl group even when it is bound to the glycoprotein.

[6] Others Sugar chains can be obtained in a state of being held in a filler. This sugar chain may be used as it is held in the packing material, but usually the elution solvent is passed through the sugar chain holding column to elute the sugar chain held in the packing material into the elution solvent. . The step of eluting sugar chains is called an elution step.
The elution solvent is not particularly limited as long as it can elute the sugar chain held in the packing material, but it is usually desirable to select it according to the packing material of the sugar chain holding column. As a specific example, when a carbon-based filler such as graphite carbon or a polystyrene-based filler such as styrenedivinylbenzene is used as a filler for the sugar chain holding column, alcohols such as methanol, ethanol, and propanol, acetone And water-soluble solvents such as acetonitrile. Further, these elution solvents may be passed as a solution containing the elution solvent in other liquids such as water or a mixed solution with a buffer solution. Moreover, when cellulose is used as the filler, a solution containing a buffer solution such as a buffer solution or a buffer solution / ethanol mixed solution may be used. In addition, these may be used individually by 1 type and may use 2 or more types together by arbitrary combinations and ratios.

Furthermore, the flow rate of the elution solvent is usually 1 time or more, preferably 2 times or more, more preferably 3 times or more, and usually 20 times or less, preferably 10 times or less, more preferably the volume of the sugar chain holding column. Is 5 times or less.
Here, if acetic anhydride is added to the elution solvent, the effect of acetylation can be further increased. The amount of acetic anhydride to be added is usually 0.1% by volume or more, preferably 0.5% by volume or more, more preferably 1.0% by volume or more, and usually 50% by volume or less, preferably with respect to the elution solvent. It is 10 volume% or less, More preferably, it is 5 volume% or less.
For the eluted liquid (specimen liquid), for example, the solvent is distilled off with an evaporator or a concentrating centrifuge to obtain a glycoprotein sugar chain.

  Moreover, it may replace with the acetylation process mentioned above, and may perform the acetylation elution process which passes the acetylation reaction agent which can elute a sugar chain through a sugar chain holding column. That is, after performing [1] separation step, [2] sugar chain retention step, and [3] washing step, [4 ′] acetylation elution step may be performed. Accordingly, acetylation of the acetyl group-eliminated sugar chain and elution of the sugar chain from the sugar chain holding column can be performed in one step.

  The acetylation reagent used in the acetylation elution step is not limited as long as it can acetylate an acetyl group-eliminated sugar chain and can elute the sugar chain held in the sugar chain holding column. There is no limit. Specific examples include those obtained by dissolving the above-described acetylating reagent in an elution solvent such as aqueous triethylamine acetate or acetonitrile.

  In particular, when acetic anhydride is used as the acetylating reagent, the advantage of performing the [4 ′] acetylation elution step is remarkable. That is, when performing the above-mentioned [4] acetylation step, when a substance that hydrolyzes with a change in pH such as acetic anhydride is used as an acetylation reagent, the pH is controlled by coexisting a buffer in the acetylation reaction solution. It becomes desirable. However, in the [4 ′] acetylation elution step, acetylation is carried out in the same step as the elution of sugar chains, so that the acetylating reagent can be dissolved in the elution solvent (acetonitrile). Since acetic anhydride is stable in acetonitrile, for example, even if acetic acid is generated by acetylation reaction and the pH changes, acetic anhydride is not easily hydrolyzed. Therefore, when acetic anhydride is used, acetylation can be reliably performed without using a buffering agent, and a reagent washing step is not required. Elution of sugar chains from the holding column can be performed in one step.

As described above, according to the sugar chain separation method of the present invention, the time required for heating becomes unnecessary, and a sugar chain can be obtained in a shorter time than before. Further, since the sugar chain is separated from the hydrazine decomposition solution using the sugar chain holding column, the operation becomes easy and the sugar chain can be efficiently separated.
Moreover, since acetylation of acetyl group-eliminated sugar chains is performed in a series of flows using a single sugar chain-holding column, the operation can be simplified as compared with the conventional method, and it is necessary for acetylation. Time can be shortened. Furthermore, the reactant cleaning step for removing the acetylation reactant can be performed in a shorter time than before.

II. Analysis by Liquid Chromatography Next, analysis of a sample using the sample analysis method (sugar chain analysis method) of the present invention will be described. Here, a case where a sugar chain of a glycoprotein obtained by the above-described separation method is analyzed as a sample will be described as an example. However, there is no limitation on the sample, and any simple substance or compound can be used as the sample.
The specimen is usually analyzed as a specimen liquid dissolved or dispersed in an appropriate solvent. Here, there is no limitation on the solvent in which the sample is dissolved or dispersed, and any solvent can be used depending on the type of the sample, but when the sample is a sugar chain, water, a buffer solution, and the like can be given. . In the present embodiment, it is assumed that water is used as the solvent, and therefore an aqueous solution of sugar chains is used as the sample liquid. Moreover, the specimen may be labeled according to the detectors 12 and 16 used.

1. Analysis Device FIG. 1 is a block diagram showing a main part of an analysis device (liquid chromatography device) used in a sample analysis method as one embodiment of the present invention. This chromatography apparatus comprises a normal phase liquid chromatography part (hereinafter referred to as “normal phase HPLC part” as appropriate) 1, a reverse phase liquid chromatography part (hereinafter referred to as “reverse phase HPLC part” as appropriate) 2, and an ion exchange column 3. A regenerative liquid supply unit 4, a replacement liquid supply unit 5, an eluate supply unit 6, a valve switching (mobile phase switching unit) 7, a valve switching 8, and a control unit 100.

The normal phase HPLC unit 1 is configured to separate samples by normal phase HPLC, and includes a mobile phase supply unit 9, an autoinjector 10, a normal phase column 11, and a detector 12.
The mobile phase supply unit 9 is a part that supplies the mobile phase toward the normal phase column 11, and includes a plurality (two in this case) of mobile phase storage units 9A and gradients connected to the respective mobile phase storage units 9A. And a pump 9B.

  The mobile phase storage unit 9A stores a liquid (hereinafter referred to as “normal phase eluent”) as a mobile phase of normal phase HPLC, and the normal phase eluent stored in each mobile phase storage unit 9A. The polarities are different. Here, there is no restriction | limiting in particular in the kind of normal phase eluent, A well-known thing can be used arbitrarily. Specific examples include acetonitrile, acetone, chloroform, toluene, benzene, hexane and the like. In addition, a normal phase eluent may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio. Furthermore, the normal phase eluent in each mobile phase storage unit 9A may be used by mixing or dissolving with any liquid, for example, as an aqueous solution. Here, description will be made assuming that acetonitrile aqueous solutions having different concentrations are used as the normal phase eluent.

  The gradient pump 9B sends the normal phase eluent in the mobile phase storage unit 9A toward the normal phase column 11, and is connected to the downstream of each mobile phase storage unit 9A. Further, the operation of each gradient pump 9B is controlled by the control unit 100. In FIG. 1, the connection between the gradient pump 9 </ b> B and the control unit 100 is omitted in the middle, and all the chain line arrows indicated by reference character c represent control by the control unit 100. In FIG. 1, the control of the control unit 100 for other components is also indicated by a chain line arrow indicated by a symbol c.

As described above, the gradient pump 9B is configured to change the delivery amount of the normal phase eluent according to the control of the control unit 100. Therefore, the mobile phase supply unit 9 adjusts the delivery ratio of the normal phase eluents having different polarities stored in the mobile phase storage unit 9A by changing the delivery amounts of the gradient pumps 9B. The polarity of the normal phase eluent flowing through the column 11 is controlled.
The gradient pump 9B and the gradient pump 13B described later may be either a high pressure type or a low pressure type, but the high pressure gradient pump is preferable because it can effectively mix the solvent.

  The autoinjector 10 is a device that automatically injects the sample liquid into the normal phase column 11, and is installed on the downstream side of the mobile phase supply unit 9 and the upstream side of the normal phase column 11. Therefore, the sample liquid injected from the autoinjector 10 is mixed with the normal phase eluent sent from the mobile phase supply unit 9 on the upstream side of the normal phase column 11 and flows into the normal phase column 11. Yes. Further, the autoinjector 10 is connected to the control unit 100, and the operation is controlled by the control unit 100.

  The normal phase column 11 is a column for separating a sample in an inflowing sample solution by normal phase HPLC, and a normal phase eluent sent from the mobile phase supply unit 9 and a sample solution injected by the auto injector 10. Are connected to the downstream side of the auto-injector 10. In the following description, the liquid flowing out from the normal phase column 11, that is, the eluate containing the sample after being separated by normal phase HPLC in the normal phase column 11 is referred to as a first eluate.

  The packing material packed in the normal phase column 11 is a packing material having a polarity higher than that of the normal phase eluent, which is usually a mobile phase, and the specific type thereof can be appropriately selected according to the purpose of analysis. it can. Examples of the normal phase column 11 include an amino group column, a cyano group column, a diol group column, and an amide group column. In particular, when performing analysis using a sugar chain as a specimen, an amide group column is preferable from the viewpoint of the ability to separate sugar chains. Specific examples of the normal phase column include Tosoh Amide-80 column and Showa Denko Shodex Asahipak NH2P-50.

  The detector 12 is a device that detects a sample in the first eluate flowing out from the normal phase column 11 by an arbitrary technique, and is connected to the downstream of the normal phase column 11. Although there is no restriction | limiting in particular in the kind, As a specific example suitable for normal phase HPLC, a ultraviolet absorption detector, a fluorescence detector, and a differential refractive index detector sugar are used. Of these, a fluorescence detector is preferred in order to detect sugar chains with high sensitivity. In this embodiment, a fluorescence detector is used as the detector 12.

  Next, the reverse phase HPLC unit 2 will be described. The reverse phase HPLC unit 2 is configured to separate the sample by reverse phase HPLC, and is connected downstream of the ion exchange column 3. Further, the reverse phase HPLC unit 2 includes a mobile phase supply unit 13, a valve switching 14, a reverse phase column 15, and a detector 16.

  Similarly to the mobile phase supply unit 9, the mobile phase supply unit 13 is a part that supplies the mobile phase toward the reverse phase column 15 while changing the polarity. The mobile phase supply unit 13 includes a plurality (two in this case) of mobile phase storage units 13A and 13A. , And a gradient pump 13B connected to each mobile phase storage unit 13A.

  The mobile phase storage unit 13A stores a liquid (hereinafter referred to as “reverse phase eluent”) as a mobile phase of reverse phase HPLC, and the reverse phase eluent stored in each mobile phase storage unit 13A. The polarities are different. Here, there is no restriction | limiting in particular in the kind of reverse phase eluent, A well-known thing can be used arbitrarily. Specific examples include a mixed solution of a buffer solution such as an ammonium acetate aqueous solution, a sodium acetate aqueous solution, or a sodium phosphate aqueous solution, and methanol, ethanol, propanol, butanol, acetonitrile, and the like. In addition, a reverse phase eluent may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio. Furthermore, the normal phase eluent in each mobile phase storage unit 13A may be used by mixing or dissolving with any liquid. Here, it demonstrates as what uses the aqueous solution of ammonium acetate and a butanol as a reverse phase eluent.

  The gradient pump 13B sends out the reverse phase eluent in the mobile phase storage unit 13A toward the reverse phase column 15, and is connected to the downstream of each mobile phase storage unit 13A. Further, each gradient pump 13B is connected to the control unit 100, and the operation is controlled by the control unit 100.

  As described above, the gradient pump 13B is configured to change the delivery amount of the reverse-phase eluent according to the control of the control unit 100. Therefore, the mobile phase supply unit 13 adjusts the delivery rate of the reverse phase eluent having different polarities by changing the delivery amount of each gradient pump 13B, and the polarity of the mobile phase passing through the reverse phase column 15 is adjusted. Is to control.

  The valve switching 14 is a liquid path switching device connected downstream of the ion exchange column 3 and downstream of the mobile phase supply unit 13. In addition, the valve switching 14 is connected to the control unit 100 and is configured to switch the flow destination of the liquid that flows in according to the control of the control unit 100. Furthermore, the outflow side of the valve switching 14 is connected to a reverse phase column 15 and a waste liquid discharge port (not shown) that leads to the outside of the apparatus. Therefore, the valve switching 14 is configured so that the liquid flowing in from the ion exchange column 3 and the reverse phase eluent flowing in from the mobile phase supply unit 13 are either the outside of the apparatus or the reverse phase column 15 according to the control of the control unit 100. It is configured so as to perform switching to be sent out.

  The valve switching 14 and the valve switching 7 and 8 are not particularly limited as long as they are devices for switching the solvent, eluate, and cleaning liquid feeding paths, respectively, but any one can be used as in this embodiment. In order to save labor and increase the speed, it is preferable that the liquid supply path is automatically switched by computer control.

  The reverse phase column 15 is a column for separating the sample in the eluate containing the sample flowing out from the ion exchange column 3 (hereinafter referred to as “second eluate” as appropriate) by reverse phase HPLC. Is connected downstream of the valve switching 14 so that the second eluate sent out from the ion exchange column 3 flows through.

The packing material packed in the reverse phase column 15 is a packing material having a polarity lower than that of the reverse phase eluent, which is usually a mobile phase, and the specific type thereof can be appropriately selected according to the purpose of analysis. it can. Examples of the reverse phase column 15 include an octadecyl group column, an octyl group column, a butyl group column, a phenyl group column, and a _C30 column. In particular, when performing analysis using a sugar chain as a specimen, an octadecyl group column or a _C30 column is preferable from the viewpoint of the ability to separate sugar chains. Specific examples of the reversed phase column include Shimadzu Shim-pack CLC-ODS and Nacalai Tesque Cosmosil 5C18-P.

The detector 16 is a device that detects a specimen in a liquid flowing out from the reverse phase column 15 (hereinafter, appropriately referred to as “third eluate”) by an arbitrary method, and is connected downstream of the reverse phase column 15. . Although there is no restriction | limiting in particular in the kind, As a specific example suitable for reverse phase HPLC, a ultraviolet absorption detector, a fluorescence detector, a differential refractive index detector, a mass spectrometer etc. are used. Among these, a fluorescence detector and a mass spectrometer are preferable for detecting sugar chains with high sensitivity. In this embodiment, a fluorescence detector is used as the detector 16.
Moreover, the downstream side of the detector 16 is connected to a waste liquid discharge port (not shown).

  The ion exchange column 3 allows the first eluate to pass through and adsorbs the specimen contained therein. That is, for the ion exchange column 3, the normal phase eluent that is the solvent of the first eluate is a mobile phase that does not elute the sample adsorbed on the ion exchange column 3. When the liquid is passed, the specimen is adsorbed, but the normal phase eluent flows out of the ion exchange column 3 as it is.

  Moreover, the ion exchange column 3 is installed so that the 1st eluate sent out from the normal phase HPLC part 1 may flow in. Specifically, it is connected to the downstream side of the normal phase HPLC unit 1, that is, the downstream side of the detector 12 via a valve switching 7 described later. Further, the valve switching 14 of the reverse phase HPLC unit 2 is connected to the downstream side of the ion exchange column 3 so that the second eluate flowing out from the ion exchange column 3 flows into the reverse phase HPLC unit 2. Has been. Here, in order to increase the analysis efficiency, a plurality of ion exchange columns 3 (five in this embodiment) are provided in parallel here. Note that the ion exchange column 3 may be formed by only one, or a plurality of ion exchange columns 3 may be connected in series.

The ion exchange column 3 may be either an anion exchange column or a cation exchange column, but is preferably selected according to the type of specimen and the purpose of analysis. Moreover, there is no restriction | limiting in the ion exchange resin used for the ion exchange column 3, Arbitrary ion exchange resins can be used. For example, examples of the ion exchange resin used in the anion exchange column include a strongly basic anion exchange resin having a quaternary ammonium group and the like, and a weakly basic anion exchange resin having a diethylaminoethyl group and the like. Examples of the ion exchange resin in the cation exchange column include a strong acid cation exchange resin having a sulfopropyl group and the like, and a weak acid cation exchange resin having a carboxymethyl group and the like. Among these, when a sugar chain is analyzed as a specimen, it is preferable to use a strongly acidic cation exchange resin. In this embodiment, the ion exchange column 3 is described as a cation exchange column.
When analyzing a sugar chain as a specimen, the exchange capacity of the ion exchange resin is usually preferably 0.1 meq / g (that is, 0.1 × 10 ⁻³ equivalent / g) or more.

  The valve switching 7 is a delivery path switch that functions as a mobile phase switching unit that switches a fluid (in this embodiment, a first eluate, a regeneration solution, a replacement solution, and an adsorbed sample eluate) that flows through the ion exchange column 3. Yes, and connected to the downstream side of the normal phase HPLC unit 1 and the upstream side of the ion exchange column 3 as described above. Further, the valve switching 7 is connected to the control unit 100 and is configured to switch the outflow destination of the liquid that flows in according to the control of the control unit 100.

On the inflow side of the valve switching 7, in addition to the normal phase HPLC unit 1 (specifically, the detector 12), a later-described valve switching 8 is connected. A regeneration solution supply unit 4, a replacement solution supply unit 5, and an eluate supply unit 6 are connected upstream of the valve switching 8.
In addition to the ion exchange column 3, the outflow side of the valve switching 7 is connected to a waste liquid discharge port (not shown) that leads to the outside of the apparatus. Therefore, the valve switching 7 includes the first eluate flowing from the normal phase HPLC unit 1 and the regenerating liquid and replacement liquid supplying unit from the regenerating liquid supplying unit 4 flowing through the valve switching 8 according to the control of the control unit 100. The replacement liquid from 5 and the adsorbed sample eluate from the eluate supply unit 6 are switched to be sent to either the outside of the apparatus or the ion exchange column 3.

  Specifically, the first eluate from the normal phase HPCL section 1, that is, the first eluate containing the normal phase eluent, the regenerant liquid from the regenerant liquid supply section 4, and the replacement from the replacement liquid supply section 5. The liquid and the adsorbed specimen eluate from the eluate supply unit 6 are switched. Here, the normal phase eluent in the first eluate is a mobile phase that does not elute the adsorbed specimen for the ion exchange column 3. Further, the adsorbed sample eluate from the eluate supply unit 6 is a mobile phase for the ion exchange column 3 to elute the adsorbed sample. Therefore, the valve switching 7 functions as a mobile phase switching unit that switches the mobile phase of the ion exchange column 3 between the mobile phase that elutes the sample adsorbed on the ion exchange column 3 and the mobile phase that does not elute the sample.

  As described above, the valve switching 8 is a liquid path switching device connected to the downstream side of the regeneration liquid supply unit 4, the replacement liquid supply unit 5, and the eluate supply unit 6 and to the upstream side of the valve switching 7. It is. Further, the valve switching 8 is connected to the control unit 100 and is configured to switch the liquid to be discharged according to the control of the control unit 100. Therefore, the valve switching 8 is configured so that any one of the regenerative liquid, the replacement liquid, and the adsorbed sample eluate flowing from the regenerative liquid supply section 4, the replacement liquid supply section 5, and the elution liquid supply section 6 according to the control of the control section 100. The liquid supply path is switched so as to be sent to the valve switching 7.

  The regenerating liquid supply unit 4 is a part that supplies a regenerating liquid for regenerating the ion exchange resin in the ion exchange column 3, and includes a regenerating liquid storage unit 4A and a pump 4B. Any regeneration solution can be used depending on the ion exchange column 3. For example, an acetic acid aqueous solution, a formic acid aqueous solution, a weak hydrochloric acid aqueous solution, or the like can be used. Here, it is assumed that an acetic acid aqueous solution is used as the regenerating solution.

The regenerated liquid storage unit 4A stores an acetic acid aqueous solution that is a regenerated liquid.
The pump 4B is formed so as to send the aqueous acetic acid solution in the regenerating liquid storage unit 4A to the valve switching 8, and is connected to the downstream side of the regenerating liquid storage unit 4A. Further, the pump 4B is connected to the control unit 100, and the operation is controlled by the control unit 100. In addition, as the pump 4B and pumps 5B and 6B described later, a double blanker type, a single blanker type, or the like is used. However, the double blanker type is preferable because it is excellent in obtaining a stable flow rate.

  The replacement liquid supply unit 5 regenerates the ion exchange resin in the ion exchange column 3 or passes the first eluate through the ion exchange column 3 and then the regenerated liquid remaining in the ion exchange column 3 and the order. It is configured to supply a replacement liquid for removing the phase eluent, and includes a replacement liquid storage unit 5A and a pump 5B. Any substitution liquid can be used according to the ion exchange column 3. For example, desalted water, pure water, distilled water and the like can be mentioned. When sugar chains are used as the specimen, pure water and distilled water are preferred from the viewpoint of preventing impurity mixing. Here, it is assumed that water is used as the replacement liquid.

In the replacement liquid storage unit 5A, water that is a replacement liquid is stored.
Further, the pump 5B sends water in the replacement liquid storage section 5A to the valve switching 8, and is connected downstream of the replacement liquid storage section 5A. Further, the pump 5B is connected to the control unit 100, and the operation is controlled by the control unit 100.

  The eluate supply unit 6 is a part for supplying an adsorbed sample eluate that is an eluate for eluting the sample adsorbed in the ion exchange column 3 as will be described later. The eluate supply unit 6A and the pump 6B are connected to each other. I have. Any adsorbed sample eluate can be used depending on the ion exchange column 3 or the sample. For example, a high-concentration salt solvent can be used as the adsorbed sample eluate when the sample is a sugar chain. Any known high-concentration salt solvent can be used as long as it can elute sugar chains. Specific examples of the salt dissolved in the high-concentration salt solvent include ammonium acetate salt, triethylamine acetate salt, sodium acetate salt, sodium phosphate salt, sodium hydrogen phosphate salt, ammonium phosphate salt, hydrogen phosphate salt. An ammonium salt etc. are mentioned. Furthermore, the concentration of the high-concentration salt solvent is usually 0.001M or more, preferably 0.1M or more, and usually 1M or less, preferably 0.5M or less. In the present embodiment, description will be made assuming that an aqueous ammonium acetate solution is used as the adsorbed specimen eluate.

The eluate storage unit 6A stores an aqueous ammonium acetate solution that is an adsorbed specimen eluate.
The pump 6B is for sending the ammonium acetate aqueous solution in the eluate reservoir 6A to the valve switching 8, and is connected downstream of the eluate reservoir 6A. Further, the pump 6B is connected to the control unit 100, and the operation is controlled by the control unit 100.

2. Analysis Method Next, an analysis method using the above-described analyzer will be described. In addition, each process other than the pre-processing process demonstrated below is automatically performed by control of the control part 100. FIG. Furthermore, in the following description, each step is described as being performed sequentially, but each step may be performed continuously as appropriate.

<Pretreatment process>
First, the sample in the sample solution is labeled as necessary. In this embodiment, the sugar chain of the glycoprotein is used as a specimen, and since a fluorescence detector is used as the detectors 12 and 16, labeling is performed with a fluorescent labeling reagent. Although there is no restriction | limiting in particular in the reagent to be used, For example, 2-aminopyridine, 2-aminobenzamide, 3-aminoquinone, butyl 4-aminobenzoate, 4-trimethylammonium aniline, 1-phenyl-3-methyl-5-pyrazolone, etc. The derivatizing reagent can be used as a fluorescent labeling reagent.

  An example of a specific labeling method is as follows. When 2-aminopyridine is used as a labeling reagent, an excess amount of a mixed solution of 2-aminopyridine and acetic acid is added to the sugar chain as a sample, and the mixture is heated at 90 ° C. for 1 hour. Heat. Thereafter, the reducing agent is added in an excess amount of acetic acid / aqueous solution with respect to the sugar chain, and further reacted by heating at 80 ° C. for 30 minutes. The reducing agent used at this time is preferably dimethylamine boron or sodium cyanotrihydroborate, but is not particularly limited. After the reaction, a phenol / chloroform (1: 1) mixed solution and water are added, and after stirring well, the aqueous phase is taken out and dried to obtain a labeled sugar chain.

<HPLC preparation process>
Subsequently, the ion exchange column 3 is regenerated.
First, the control unit 100 switches the valve switchings 7 and 8 to connect the regeneration solution supply unit 4 and the ion exchange column 3, and operates the pump 4B in the regeneration solution supply unit 4. As a result, an aqueous acetic acid solution is sent from the regenerating solution supply unit 4A to each ion exchange column 3. When an aqueous acetic acid solution is passed through the ion exchange column 3, the ion exchange column 3 is converted into a hydrogen type.
Moreover, the control part 100 switches the valve switching 14, and makes the ion exchange column 3 and the discharge port communicate. Thereby, the acetic acid aqueous solution flowing out from the ion exchange column 3 is discharged from the discharge port via the valve switching 14.

Next, the control unit 100 switches the valve switching 8 to cause the substitution liquid supply unit 5 and the ion exchange column 3 to communicate with each other and to operate the pump 5B in the substitution liquid supply unit 5. Further, the pump 4B is stopped along with the switching of the valve switching 8.
Thereby, water is sent to each ion exchange column 3 from 5 A of substitution liquid supply parts. When water passes through the ion exchange column 3, the inside of the ion exchange column 3 is replaced by water, and the aqueous acetic acid solution remaining in the ion exchange column 3 is removed from the ion exchange column 3. The aqueous acetic acid solution and water that have flowed out of the ion exchange column 3 are discharged from the discharge port via the valve switching 14.
When the regeneration is completed, the control unit 100 stops the pump 5B.

<First separation step>
Next, in the first separation step, the sample solution is passed through the normal phase column 1 as follows, and sugar chains are separated by normal phase HPLC to analyze the sugar chains.
First, the control unit 100 switches the valve switching 7 so that the normal phase HPLC unit 1 communicates with the waste liquid discharge port. Next, the autoinjector 10 is controlled to inject the sample liquid from the autoinjector 10. The injected sample liquid flows into the normal phase column 11, and sugar chains in the sample liquid remain at the entrance of the normal phase column 11. Water that is the solvent of the detection liquid is discharged from the discharge port through the valve switching 7.

When a predetermined time elapses after the injection of the sample liquid, the control unit 100 controls the valve switching 7 to cause the normal phase HPLC unit 1 and the ion exchange column 3 to communicate with each other, operates each of the gradient pumps 9B, and moves the mobile phase. The feeding of the normal phase eluent from the supply unit 9 to the normal phase column 11 is started. Further, the valve switching 14 is controlled so that the ion exchange column 3 communicates with the discharge port.
As a result, the sugar chain remaining at the inlet of the normal phase column 11 passes through the normal phase column 11 together with the normal phase eluent. At this time, the control unit 100 adjusts the amount of each gradient pump 9B to send the solution while changing the polarity of the normal phase eluent with a predetermined gradient.

The sugar chain is separated by normal phase HPLC in the normal phase column 11, flows out from the outlet of the normal phase column 11 together with the normal phase eluent according to the polarity, and passes through the detector 12. Here, the liquid containing a sugar chain in the normal phase eluent that has flowed out of the normal phase column 11 is the first eluent.
When the first eluate passes through the detector 12, the detector 12 detects the sugar chain by fluorescence, and thus analysis by normal phase HPLC is performed. The measured value by the detector 12 is, for example, a two-dimensional analysis in combination with the measured value by the detector 16, and the liquid chromatography retention time (LC retention time) of the standard sugar chain whose structure is determined in advance. Used to identify sugar chains by comparison.
After passing through the detector 12, the first eluate flows into the ion exchange column 3 through the valve switching 7.

<Specimen adsorption process>
Subsequently, a specimen adsorption process is performed. Usually, this specimen adsorption process is performed continuously with the first separation process.
The first eluate containing the sugar chain separated in the first separation step flows into the ion exchange column 3 as described above. In general, a specimen adsorbs on a cation exchange column when it is positively charged, and adsorbs on an anion exchange column when it is negatively charged. When the sugar chain is labeled with 2-aminopyridine as in this embodiment, since the sugar chain is positively charged, when the first eluate flows into the ion exchange column 3, the sugar chain in the first eluate Adsorbs to the ion exchange column 3 which is a cation exchange column. Further, the normal phase eluent of the first eluate flowing into the ion exchange column 3 is discharged from the discharge port via the valve switching 14.

<Ion exchange column washing process>
Next, the control unit 100 switches the valve switchings 7 and 8 to connect the replacement liquid supply unit 5 and the ion exchange column 3 and to operate the pump 5B. Moreover, with the switching of the valve switching 7, the control unit 100 stops the gradient pump 9B.
Thereby, water is sent to each ion exchange column 3 from 5 A of substitution liquid supply parts. When water passes through the ion exchange column 3, the inside of the ion exchange column 3 is replaced with water, and the normal phase eluent remaining in the ion exchange column 3 is removed from the ion exchange column 3. On the other hand, the sugar chain maintains the adsorbed state on the ion exchange column 3.
The aqueous acetic acid solution and water that have flowed out of the ion exchange column 3 are discharged from the discharge port via the valve switching 14.

<Second separation step>
Next, in the second separation step, sugar chains are eluted from the ion exchange column 3 as follows, and the obtained second eluate is passed through the reverse phase column 15 to separate the sugar chains by reverse phase HPLC. To analyze the sugar chain.

  First, the control unit 100 switches the valve switching 8 to connect the eluate supply unit 6 and the ion exchange column 3, and switches the valve switching 14 to connect the ion exchange column 3 and the reverse phase column 15. Further, the pump 5B is stopped with the switching of the valve switching 7. Next, the control unit 100 controls the pump 6B to send the aqueous ammonium acetate solution in the eluate storage unit 6A to the ion exchange column 3.

  Thereby, the sugar chain adsorbed on the ion exchange column 3 is eluted by the aqueous ammonium acetate solution. At this time, the liquid eluted from the ion exchange column 3 and containing sugar chains in the aqueous ammonium acetate solution is the second eluent. The second eluate flows into the reverse phase column 15 via the valve switching 14, the sugar chain in the second eluate remains at the entrance of the reverse phase column 15, and the aqueous ammonium acetate solution as the solvent is discharged from the waste liquid discharge port. The

Next, the control unit 100 switches the valve switching 14 to cause the mobile phase supply unit 13 and the reverse phase column 15 to communicate with each other, and to operate each of the gradient pumps 13B, so that the reverse flow from the mobile phase supply unit 13 to the reverse phase column 15 is performed. Start feeding phase eluent. Moreover, the pump 6B is stopped with the switching of the valve switching 14.
As a result, the sugar chain remaining at the inlet of the reverse phase column 15 passes through the reverse phase column 15 together with the reverse phase eluent. At this time, the controller 100 adjusts the amount of each gradient pump 13B to send the solution while changing the polarity of the reverse phase eluent with a predetermined gradient.

The sugar chain is separated by reverse-phase HPLC in the reverse-phase column 15, flows out from the outlet of the reverse-phase column 15 together with the reverse-phase eluent according to the polarity, and passes through the detector 16. At this time, the liquid containing sugar chains in the reverse phase eluent that has flowed out of the reverse phase column 15 is referred to as a third eluent.
When the third eluate passes through the detector 16, the detector 16 detects the sugar chain by fluorescence, and thereby analysis by reverse phase HPLC is performed. Further, as described in the first separation step, the measurement value obtained by the detector 16 is subjected to two-dimensional analysis together with the measurement value obtained from the detector 12, and liquid chromatography of a standard sugar chain whose structure has been determined in advance is performed. It is used to specify the sugar chain in comparison with the retention time (LC retention time).
In addition, the 3rd eluate which passed the detector 16 is discharged | emitted from a waste-liquid discharge port.

  As described above, according to the sample analysis method and the liquid chromatography apparatus of the present invention, it is possible to efficiently analyze sugar chains and other samples in a short time. That is, when attempting to separate a specimen using normal phase HPLC and reverse phase HPLC, conventionally, after performing normal phase HPLC, the specimen was once taken out from the first eluate and then reverse phase HPLC was performed. It took a lot of work. However, according to the present invention, since the specimen is operated in the flow of the liquid, it can be easily separated in a short time.

  Further, since the normal phase HPLC unit 1 and the reverse phase HPLC unit 2 are connected online via the ion exchange column 3, the steps required for the analysis can be automated using the control unit 100, and labor saving and high speed can be achieved. It becomes possible.

<Others>
As described above, the sugar chain separation method, the sample analysis method, the sugar chain analysis method, and the liquid chromatography apparatus as one embodiment of the present invention have been described, but the present invention is not limited to the above embodiment and can be arbitrarily modified. Can be implemented.
For example, the order of the normal phase HPLC unit 1 and the reverse phase HPLC unit 2 may be reversed. That is, the specimen may be configured to flow in the order of the reverse phase HPLC unit 2, the ion exchange column 3, and the normal phase HPLC unit 1. That is, first, the sample solution is passed through the reverse phase HPLC unit 2 and the sample is separated by reverse phase HPLC, and then the first eluate containing the sample separated by the reverse phase HPLC unit 2 is passed through the ion exchange column 3. Alternatively, the sample may be eluted from the ion exchange column 3, and the second eluate containing the obtained sample may be passed through the normal phase HPLC unit 1 and separated by normal phase HPLC. Also by this, the same effect as the above embodiment can be obtained.

  In addition, according to the liquid chromatography apparatus and the sample analysis method, it is possible to separate and analyze samples other than sugar chains. Samples to be analyzed can be separated by normal phase and reverse phase HPLC, and include simple substances and compounds having chargeability. Examples of ionic samples that are ionic include sugar chains containing sialic acid, amino sugars, proteins, peptides, amino acids, and the like.

  Further, even if the sample itself does not have charging properties, those having charging properties by reacting with or interacting with compounds that can be charged, such as the labeling reagent of the above-described embodiment, can be used as the samples. Here, examples of compounds that can be charged positively include 2-aminopyridine, 2-aminobenzamide, 3-aminoquinone, butyl 4-aminobenzoate, 4-trimethylammonium aniline, 1-phenyl-3-methyl-5- Examples include pyrazolone. Examples of the negatively charged compound include 8-aminonaphthalene-1,3,6-trisulfonic acid and 8-aminopyrene-1,3,6-trisulfonic acid. In particular, when a sugar chain is to be analyzed as a specimen, it is preferably used for a sugar chain derivatized with 2-aminopyridine or 2-aminobenzamide.

Further, the valve switching 7, 8, and 14 can be used in combination with each other for the purpose of distributing the liquid feeding paths (see valve switching 7, 17, and 18 in FIG. 3).
In the above embodiment, the sugar chain to be analyzed is described as being obtained by the sugar chain separation method of the present invention. However, the sugar chain may be obtained by another method such as an enzyme treatment method. Good. However, from the viewpoints of safety, labor saving, high speed, etc., it is preferable to obtain a sugar chain using the sugar chain separation method of the present invention.

  Further, in order to perform the sugar chain analysis method described above, a sugar chain holding column used for “I. Separation of sugar chains of glycoprotein” may be connected upstream of the autoinjector 10. The main configuration of the sugar chain analyzer used in that case is shown as a block diagram in FIG. In FIG. 2, the same reference numerals as those in FIG. 1 denote the same parts.

  In the configuration of FIG. 2, a sugar chain holding column 20 filled with a filler capable of holding a sugar chain is provided upstream of the autoinjector 10, and a labeling unit 21 provided between the sugar chain holding column 20 and the autoinjector 10. Are connected in the same manner as the analyzer described with reference to FIG.

The sugar chain holding column 20 is a column for separating sugar chains as described in “I. Separation of sugar chains of glycoproteins”, and has a discharge port with a switching valve (not shown) on the downstream side. . Therefore, the sample liquid containing the sugar chain eluted from the sugar chain holding column 20 flows into the autoinjector 10 through the labeling unit 21, and the liquid other than the sample liquid flowing out from the sugar chain holding column 20 is discharged from a discharge port (not shown). It is configured to be. Here, switching between the sample liquid and the liquid other than the sample liquid is performed by the switching valve.
The labeling unit 21 is a part for labeling the sample in the sample liquid. Here, it is the part where the labeling reagent for fluorescence is mixed with the sample liquid.

By using such a sugar chain analyzer, separation and analysis of sugar chains can be performed efficiently in a short time.
Furthermore, upstream of the sugar chain holding column 20, a hydrazine decomposition solution introduction unit that introduces a hydrazine decomposition solution into the sugar chain holding column 20 in the sugar chain holding step, and a cleaning solvent that introduces a washing solvent into the sugar chain holding column 20 in the washing step An introduction unit, an acetylation reagent introduction unit for introducing an acetylation reagent into the sugar chain holding column 20 in the acetylation step, a desalting agent introduction unit for introducing a desalting agent into the sugar chain holding column 20 in the reaction agent washing step, An elution solution introduction section for introducing an elution solvent to be eluted from the sugar chain holding column 20, an acetylated eluate introduction section for introducing an acetylation reagent capable of eluting sugar chains in the acetylation elution step into the sugar chain holding column 20, etc. The steps described in “I. Separation of sugar chains of glycoprotein” may be automated.

  EXAMPLES Next, although an Example demonstrates this invention still in detail, this invention is not limited to a following example, unless the summary is exceeded.

I. Separation of sugar chains from glycoprotein [Example 1]
As a decomposition step, 200 μL of hydrazine was added to 2 mg of a mouse kidney acetone precipitate as a sample and heated at 95 ° C. for 10 hours to obtain a hydrazine decomposition solution of glycoprotein.
Then, as a sugar chain retention step, 2 mL of 50 mM ammonium acetate aqueous solution (hereinafter α solution) is added as a washing solvent and mixed well, and the hydrazine decomposition solution mixed with α solution is passed through a graphite carbon column (300 mg manufactured by GL Science). did.
Further, as a washing step, 10 ml of α solution, which is a washing solvent, was passed through the column to completely elute hydrazine from the column.

Next, as an acetylation step, 20 mL of 0.5 M tris (hydroxymethyl) aminomethane buffer (pH = 7) containing 1% by volume of acetic anhydride as an acetylation reagent was passed through the column.
Thereafter, as a reactant washing step, 5 mL of α solution, which is a washing solvent, was further passed through the column to elute the reaction residual solution.

  Subsequently, 5 mL of an elution solution obtained by adding 1% by volume of acetic anhydride to a mixed solution of acetonitrile: α solution (volume ratio 6: 4) was passed through the column to completely acetylate the unreacted sugar chain and remove the sugar chain. Elute from the column.

  Next, after distilling off the solvent with an evaporator, 20 μL of a solution prepared by dissolving 552 mg of 2-aminopyridine in 200 μL of acetic acid was added, and heated at 90 ° C. for 60 minutes in a heat block. Next, 70 μL of a solution in which 200 mg of dimethylamine borane was dissolved in a mixed solution of 80 μL of acetic acid and 50 μL of water was added and heated at 80 ° C. for 35 minutes. After the reaction, 180 μL of water and 90 μL of a water saturated phenol / chloroform (weight ratio 1/1) solution were added, and after mixing well, the lower layer was removed. After repeating this operation three times in total, 90 μL of chloroform was added and mixed well in the same manner to remove the lower layer. This operation is called pyridylamination.

  The upper layer was lyophilized and then added with 25 μL of water, 3 μL of 0.5 M aqueous ammonium acetate solution (pH = 5), and 2 μL of neuraminidase aqueous solution (manufactured by Nacalai) and reacted at 37 ° C. for 1 hour to release sialic acid from the sugar chain. This operation is called sialidase treatment.

Next, after filtering with a cartridge filter, the filtrate was fractionated into 10 fractions by normal phase liquid chromatography, and each fraction was analyzed by reverse phase chromatography. The conditions for normal phase and reverse phase HPLC are shown in Tables 1 and 2, respectively. In addition, A liquid and B liquid of Tables 1 and 2 are liquids constituting mobile phases, respectively, and these liquid A and liquid B are mixed to adjust the polarity of the mobile phase. It corresponds to the normal phase eluent and the reverse phase eluent stored in the mobile phase storage units 9A and 13A. In Tables 1 and 2, the description “B: a% (T ₁ min) → B: b% (T ₂ min)” indicates that the concentration of the B solution is set to (T ₂ −T ₁ ) min. It means that it was changed from% to b%. However, T ₁ , T ₂ , a, and b each represent a real number. In Tables 1 and 2,% represents a percentage based on volume.

  When 10 specimens were carried out simultaneously, the time required from hydrazine decomposition to acetylation of the sugar chain (end of the elution process) was 30 minutes. Table 3 shows the acetylation rate of the sugar chain by this method.

[Example 2]
In the same manner as in Example 1, the separation step and the washing step were performed.
Subsequently, as an acetylation elution step, 5 mL of an elution solution obtained by adding 2% by weight of acetic anhydride to a mixed solution of acetonitrile: β solution (weight ratio 6: 4) is passed through the column to completely acetylate the sugar chain. At the same time, the sugar chain was eluted from the column. Here, the β solution means a 50 mM aqueous solution of triethylamine acetate.

Next, after distilling off the solvent with an evaporator, pyridylamination, sialidase treatment, analysis by normal phase and reverse phase HPLC were performed in the same manner as in Example 1, and the reaction rate of sugar chain acetylation was measured.
When 10 specimens were carried out simultaneously, the time required from the decomposition of hydrazine to the acetylation of the sugar chain (end of the elution process) was 20 minutes. Table 3 shows the acetylation rate of the sugar chain by this method.

[Comparative Example 1]
A hydrazine decomposition solution was prepared in the same manner as in the decomposition step of Example 1.
Next, hydrazine was distilled off from the hydrazine decomposition solution with a vacuum pump, and toluene having a high azeotropic effect was further added to completely remove hydrazine.
Next, 200 μL of saturated aqueous sodium hydrogen carbonate solution and 8 μL of acetic anhydride were added and reacted on ice for 5 minutes, and further 200 μL of saturated aqueous sodium bicarbonate solution and 8 μL of acetic anhydride were added and reacted on ice for 30 minutes.

Next, about 2 g of ion exchange resin (Dowex X8: proton activated) was added and stirred well, followed by filtration with a Pasteur pipette filled with glass wool. The filtrate was distilled off water with an evaporator. Pyridylamination was carried out in the same manner as in Example 1 and analyzed by normal phase liquid chromatography and reverse phase liquid chromatography.
About 10 specimens, it took 10 hours to obtain acetylated sugar chains after hydrazine degradation.

  Table 3 shows the acetylation reaction rates of Examples 1 and 2 when the reaction rate of Comparative Example 1 is 100%. According to Table 3, in Examples 1 and 2, it was confirmed that acetylated sugar chains can be separated and obtained in a very short time with a reaction rate almost the same as that of the comparative example.

II. Analysis of Glycoprotein Sugar Chain An outline of the analyzer used in this example is shown in FIG. In the analyzer shown in FIG. 3, two groups of five ion exchange columns 3 are used, and a total of ten ion exchange columns are used. Corresponding to each of the five groups of the ion exchange column 3, a valve switching 17 was connected on the upstream side, and a valve switching 18 was connected on the downstream side. The valve switchings 17 and 18 are controlled by the control unit 100, respectively. In addition to the above configuration, the analyzer shown in FIG. 3 has the same configuration as the analyzer described above with reference to FIG. In FIG. 3, the same reference numerals as those in FIG. 1 denote the same parts.

  That is, the analyzer used in the present example is connected to the valve switching 7 (Valvemate manufactured by Guilson) downstream of the normal phase HPLC unit 1 (gradient pump 9B, color 11 / detector 12), and further valve switching 17 The ion exchange columns 3 (Showa Denko's Shodex IEC SP-825 8 mm ID × 75 mm) were connected in parallel to each valve switching 17. The respective ion exchange columns 3 were aggregated again in the valve switching 18, further aggregated in the valve switching 14 in the reverse phase HPLC unit 2, and led to the reverse phase column 15. However, the concentration of the acetic acid aqueous solution as the regeneration solution was 0.5 M, and the concentration of the ammonium acetate aqueous solution as the adsorbed specimen eluate was 0.5 M.

First, 5 mL (1 mL / min × 5 min) of 0.5 M acetic acid aqueous solution in the regenerative liquid storage unit 4A is passed through all the ion exchange columns 3 by the pump 4B through the valve switchings 7 and 8, and ions are supplied. Exchange column 3 was converted to the hydrogen form. Moreover, the valve switching 14 and 18 was switched, and the liquid that passed through the ion exchange column 3 was discharged from the discharge port via the valve switching 14.
Thereafter, the valve switching 8 was switched, and 5 mL (1 mL / min × 5 min) of water in the replacement liquid storage unit 5A was passed through the pump 5B to replace the acetic acid solution in the column with water.

  Next, as a first separation step, the sample liquid was injected from the autoinjector 10 into the normal phase column 11, the sample was separated by normal phase HPLC under the conditions shown in Table 1 above, and detected by the detector 12. At the same time, as the sugar chain adsorption step, the valve switching 7 is switched according to a preset time, and the first eluate sent from the normal phase HPLC unit 1 is divided into time and passed through each of the ten ion exchange columns 3. Let it liquid. Hereinafter, the ten ion exchange columns 3 will be referred to as the first ion exchange column, the second ion exchange column,..., The tenth ion exchange column in the order in which the first eluate is passed. And

Thereafter, the valve switching 7 and 8 are switched, and water is passed from the replacement liquid supply unit 5 to all the ion exchange columns 3 at a flow rate of 1 mL / min for 5 minutes, so that the normal phase eluent in the ion exchange column 3 is completely discharged. The water was replaced with water.
Next, as the second separation step, the first ion exchange column 3 and the reverse phase column 15 were connected by switching the valve switching 8, 18, and 14. At the same time, 0.5 M ammonium acetate aqueous solution was passed through the first ion exchange column 3 from the eluate supply unit 6 for 5 minutes. The sugar chain adsorbed on the ion exchange column 3 was eluted with an aqueous ammonium acetate solution at this stage and led to the reverse phase column 15 as a second eluate. Subsequently, the reverse phase gradient pump 13 and the reverse phase column 15 were connected by switching the valve switching 14, and the reverse phase eluent was sent to the reverse phase column 15 to separate the sample.
Similarly, separation by reverse phase HPLC was also performed on the second to tenth ion exchange columns.

As a measurement sample, as a result of using a pyridylaminated glucose oligomer (manufactured by Takara) as a sugar chain, as shown in FIG. 4, glucose chain lengths of 6 to 15 fractionated into 10 ion exchange columns 3 by normal phase HPLC are shown. It was confirmed that each sugar chain was detected as a single component by reverse phase HPLC. In addition, in FIG. 4, the number attached | subjected to each peak of normal phase HPLC represents the glucose chain length of a sugar chain.
The peak area (recovery rate) was 99%.
From the above, it was confirmed that the sample analysis method of the present invention can reliably analyze the sample using normal phase and reverse phase HPLC.

  By using the present invention, labor saving and speeding up of two-dimensional analysis using normal phase liquid chromatography and reverse phase liquid chromatography are expected. Analysis and diagnosis of glycoprotein sugar chains, diagnosis of cancer, rheumatism, etc. It is used in fields such as pharmaceutical quality control and testing drug search.

It is a block diagram which shows the principal part of the liquid chromatography apparatus used for the sample analysis method as one Embodiment of this invention. It is a block diagram which shows the principal part of the sugar chain analyzer used for the sugar chain analysis method as one Embodiment of this invention. It is a block diagram which shows the principal part of the liquid chromatography apparatus used for the Example of this invention. It is the normal phase HPLC chromatogram and reverse phase HPLC chromatogram of the sugar chain which were detected in the Example of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Normal phase HPLC part 2 Reverse phase HPLC part 3 Ion exchange column 4 Regeneration liquid supply part 4A Regeneration liquid storage part 4B, 5B, 6B Pump 5 Replacement liquid supply part 5A Replacement liquid storage part 6 Eluate supply part 6A Eluate storage part 7 Valve switching (fluid switching part)
8, 14, 17, 18 Valve switching 9, 13 Mobile phase supply unit 9A, 13A Mobile phase storage unit 9B, 13B Gradient pump 10 Autoinjector 11 Normal phase column 12, 16 Detector 15 Reverse phase column 20 Sugar chain retention column 21 Marking unit 100 Control unit

Claims (6)

A method for separating sugar chains from a hydrazine degradation solution of glycoprotein,
A sugar chain holding step in which a hydrazine decomposition solution of glycoprotein is passed through a column packed with an alkali-resistant packing capable of holding sugar chains;
A method for separating sugar chains, comprising a washing step of passing a washing solvent for eluting hydrazine through the column. A method for separating sugar chains from a hydrazine degradation solution of glycoprotein,
A sugar chain holding step of passing a hydrazine decomposition solution of glycoprotein through a column packed with a packing capable of holding sugar chains;
A washing step of passing a washing solvent for eluting hydrazine through the column;
An acetylation step of passing an acetylation reagent through the column;
A method for separating sugar chains, wherein the column comprises a reagent washing step for passing a desalting agent for eluting the acetylation reagent. A method for separating sugar chains from a hydrazine degradation solution of glycoprotein,
A sugar chain holding step of passing a hydrazine decomposition solution of glycoprotein through a column packed with a packing capable of holding sugar chains;
A washing step of passing a washing solvent for eluting hydrazine through the column;
An acetylation elution step of passing through the column an acetylation reaction agent capable of eluting sugar chains, and a method for separating sugar chains. The sugar chain separation method according to claim 2 or 3, wherein the filler is alkali resistant . A degradation step of degrading glycoprotein with hydrazine to prepare a hydrazine degradation solution of glycoprotein;
A sugar chain holding step of passing the hydrazine decomposition solution prepared in the decomposition step through a column packed with a filler capable of holding sugar chains;
A washing step of passing a washing solvent for eluting hydrazine through the column;
An acetylation step of passing an acetylation reagent through the column;
A reactant washing step of passing a desalting agent to elute the acetylated reactant through the column;
A first separation step of eluting the sugar chain from the column and separating the sample liquid containing the obtained sugar chain by one of normal phase liquid chromatography and reverse phase liquid chromatography;
A sugar chain adsorption step of adsorbing sugar chains by passing the first eluate containing the sugar chains separated in the first separation step through an ion exchange column;
A second separation step of eluting sugar chains from the ion exchange column and separating the second eluate containing the obtained sugar chains by the other of normal phase liquid chromatography and reverse phase liquid chromatography. A method for analyzing sugar chains. A sugar chain analyzer used in the sugar chain analysis method according to claim 5 ,
A column packed with a filler capable of retaining sugar chains;
A normal phase liquid chromatography section for separating sugar chains by normal phase liquid chromatography;
A reverse phase liquid chromatography section for separating sugar chains by reverse phase liquid chromatography;
An ion exchange column,
The column is connected so that the detection liquid flowing out from the column flows into one of the normal phase liquid chromatography part and the reverse phase liquid chromatography part,
In the ion exchange column, the first eluate flowing out from one of the normal phase liquid chromatography part and the reverse phase liquid chromatography part flows into the ion exchange column and flows out of the ion exchange column. The eluent is connected between the normal phase liquid chromatography unit and the reverse phase liquid chromatography unit so that the eluate flows into the other of the normal phase liquid chromatography unit and the reverse phase liquid chromatography unit. A sugar chain analyzer characterized by the above.

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