JP2007121240A - Method and device for analyzing sugars - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple analyzing method of high sensitivity constituted not only to accurately separate and analyze sugars but also to quantify them; and to provide an analyzer performed by the analyzing method. <P>SOLUTION: The analyzing method of sugars includes: a process for separating a sample to be inspected containing sugars by liquid chromatography using a ligand replacing column; a process for adding ammonia to the separated sample to be inspected; and a process for ionizing the sample to be inspected, to which ammonia is added, by an electrospray ionization process to perform mass analysis. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for analyzing saccharides and an apparatus for analyzing saccharides.

  Saccharides are important not only as sweeteners but also as biological energy sources and constituents, or physiologically active substances, and have been studied for a long time. In particular, in recent years, it has been clarified that sugar chains present on the cell surface are involved in cell recognition and the like, and the use of sugars in the diagnosis and treatment of diseases is expected. For example, since sugar chains on the surface of cancer cells are different from normal cells, cancer can be diagnosed by analyzing the composition of this sugar chain, or sugar chains specific to cancer cells can be recognized and therapeutic agents can be transferred to cancer tissues. It is conceivable to deliver selectively. Therefore, a technique for analyzing the saccharide itself contained in the sample and all or a part of the hydrolyzate thereof is very important.

  However, many sugars have similar structures, and there is a problem that analysis is difficult. In view of this, a technique has been developed that facilitates analysis by chemically modifying a saccharide contained in an analysis sample or by causing an enzyme to act. For example, Patent Document 1 describes a method for analyzing monosaccharides, which includes releasing sialic acid by allowing sialidase to act on a test sample. In addition, there is a fluorescence detection method in which saccharides are modified with 2-aminopyridine (pre-label method) or arginine (post-label method). However, the analysis method including the step of modifying the saccharide is inefficient.

On the other hand, a ligand exchange column is known as a column for separating and analyzing saccharides. This column has metal ions as counter ions of the functional groups of the packed gel, and the metal ions interact with the hydroxyl groups of the sugars. Since this interaction differs depending not only on the number of hydroxyl groups in the saccharide but also on the position, saccharides containing only a hydroxyl group as a functional group can be separated.
JP 2000-333698 A (Claims)

  As described above, various methods for analyzing saccharides have been known. However, there are few techniques that can easily separate and analyze saccharides having similar structures to each other, and many techniques are laborious and costly. On the other hand, a simple analysis method has little information to be obtained, and in particular, a sample containing a large number of saccharides may not be sufficiently analyzed.

  Therefore, the problem to be solved by the present invention is to provide a method that is simple and has high sensitivity and enables more accurate separation and analysis of saccharides as well as quantification, and an apparatus capable of performing such a method. There is.

  In order to solve the above-mentioned problems, the present inventors firstly prepared a liquid chromatography / mass spectrometry method (hereinafter referred to as “LC / MS method”) using a combination of liquid chromatography using a ligand exchange column and electrospray ionization mass spectrometry. In some cases).

  When the ligand exchange column alone is used, for example, when a sample containing a large number of saccharides is analyzed, if the peaks overlap, identification becomes difficult. However, by combining mass spectrometry, the molecular weight of each peak of the chart obtained by liquid chromatography can be obtained, so that saccharides can be easily identified. The electrospray ionization method is suitable for ionization of saccharides which are hardly volatile substances.

  However, the above method sometimes has insufficient sensitivity for mass spectrometry. Therefore, the present inventors have further conducted diligent research to find that the addition of an amine compound to a sample significantly improves the sensitivity of mass spectrometry and enables the quantification of each saccharide in the sample. Completed the invention.

That is, the method for analyzing saccharides according to the present invention comprises:
Separating a test sample containing saccharides by liquid chromatography using a ligand exchange column;
A step of adding an amine compound to the separated test sample, and a step of ionizing the test sample to which the amine compound has been added by electrospray ionization and performing mass spectrometry,
It is characterized by including.

  Ammonia is preferred as the amine compound. Further, the concentration of the amine compound in the sample to be subjected to mass spectrometry is preferably 1.5% by mass or more. This is because the sensitivity of mass spectrometry is about 10 times that in the case where no amine compound is added.

  The average particle size of the gel packed in the ligand exchange column is preferably 1 to 10 μm. This is because by making the gel particle size fine, good results can be obtained even in the separation of saccharides having similar structures.

In addition, the saccharide analyzer according to the present invention includes:
A liquid chromatography system having a ligand exchange column,
A pump for adding amine compounds to the eluate from the liquid chromatography system, and an electrospray ionization mass spectrometry system;
It is characterized by having.

  As the above apparatus, an apparatus having a means for controlling the concentration of the amine compound added to the eluate from the liquid chromatography system is preferable. By this means, it is possible to adjust the amine compound concentration to be optimal for improving the sensitivity of mass spectrometry. Furthermore, it is preferable to have a means for quantifying each saccharide based on the intensity of the mass spectrum obtained by mass spectrometry. Since the method of the present invention has high sensitivity, each saccharide can be quantified by the intensity of the mass spectrum. Therefore, for example, the saccharides can be quantified by comparing the molecular ion peak intensities of the respective concentrations by the means.

  According to the method of the present invention, saccharides contained in a sample can be easily and accurately separated and analyzed. Moreover, since the mass analysis can be performed with extremely high sensitivity, the saccharides can be quantified from the peak intensity of the mass spectrum. Moreover, the device of the present invention is useful as a device capable of carrying out the method.

  Therefore, the present invention is very useful industrially as it can carry out saccharide analysis that can be applied to diagnosis and treatment of diseases more simply and more accurately than the prior art.

The method for analyzing saccharides according to the present invention comprises:
Separating a test sample containing saccharides by liquid chromatography using a ligand exchange column;
A step of adding an amine compound to the separated test sample, and a step of ionizing the test sample to which the amine compound has been added by electrospray ionization and performing mass spectrometry,
It is characterized by including.

  The method of the present invention is for separating and analyzing saccharides. Therefore, the test sample to be analyzed is not particularly limited as long as it contains saccharides. For example, environmental samples and biological samples can be analyzed. It can also be applied to food analysis.

  In the present invention, first, a test sample containing saccharides is separated by liquid chromatography using a ligand exchange column.

  The ligand exchange column uses a gel having a group having a salt of an acidic group and a metal ion as a side chain. As the main chain of the gel, polystyrene or the like, sulfonic acid group or carboxyl group as acid group, and calcium ion, lead ion, zinc ion, sodium ion or the like as metal ion can be used. By using such a gel, when a saccharide is introduced into the column, the metal ion on the gel side chain and the hydroxyl group of the saccharide interact, so that the saccharide can be separated according to the number and position of the hydroxyl group.

  The average particle size of the gel packed in the ligand exchange column is preferably 1 to 10 μm. This is because by making the gel particle size fine, good results can be obtained even in the separation of saccharides having similar structures. A conventional method may be used as a method for producing the gel filled in the ligand exchange column. For example, in the case of a sulfonic acid type polystyrene styrene resin, styrene and divinylbenzene are mixed, an appropriate polymerization initiator and a dispersant are added, and the mixture is stirred in water to obtain a polymer, followed by sulfonation. Moreover, the method for refining the gel may be a conventional method. For example, in producing the gel, there is a method of rapidly increasing the stirring speed during suspension polymerization. The obtained gel is classified by the wet and dry methods before filling to make the particle diameter uniform.

In the present invention, the “average particle size” refers to a particle size distribution of 50% of the integrated value from the small particle size side obtained from the result obtained by measuring the particle size distribution of gel particles with a general particle size distribution meter. ₅₀ ). Such a particle size distribution can be measured by a diffraction or scattering intensity pattern generated by applying light to the particles. This is because the intensity pattern depends on the particle size.

  The implementation conditions such as the flow rate of the mobile phase and the column temperature in liquid chromatography may be determined by preliminary experiments, but water is usually used as the mobile phase. Moreover, when using what has a calcium base in a side chain as column gel, it is preferable that column temperature shall be 65-75 degreeC. According to the knowledge of the present inventors, the separation ability of saccharides in such a case is good in the temperature range, and about 70 ° C. is particularly preferable.

  In the present invention, an amine compound is added to the eluate of liquid chromatography before mass spectrometry. Addition of an amine compound facilitates ionization of saccharides that are difficult to ionize, and can significantly improve the sensitivity of mass spectrometry. The amine compound is added to the eluent of liquid chromatography because the mobile phase that can be used in the ligand exchange column is usually only water.

  Amine compounds include ammonia; lower alkyl primary amines such as methylamine, ethylamine, and isopropylamine; secondary amines such as dimethylamine, diethylamine, ethylmethylamine, pyrrolidine, and piperidine; tertiary amines such as trimethylamine, triethylamine, and pyridine. Preferably, ammonia is used.

  The addition amount of the amine compound may be appropriately adjusted according to the concentration of saccharides in the eluate, but is preferably added at 1.5% by mass or more with respect to the eluate. Even if the amount is less than 1.5% by mass, the sensitivity of the subsequent mass spectrometry is improved. However, the addition of 1.5% by mass or more particularly increases the sensitivity. However, the effect is saturated even if added excessively, so the upper limit is about 10% by mass.

  The test sample to which the amine compound has been added is then ionized by electrospray ionization (hereinafter sometimes referred to as “ESI”) and subjected to mass spectrometry. The reason for using ESI as an ionization method is that it is suitable for ionization of sugars that are difficult to ionize.

  In ESI, a liquid sample is introduced into a capillary and a voltage is applied to its tip. As a result, the liquid sample is sprayed as charged droplets by forming a strong unequal electric field. Further, the droplets break up due to the Coulomb repulsive force of the ions in the droplets, and the saccharide is finally ionized. In the present invention, it is considered that the ionization proceeds favorably and the sensitivity of mass spectrometry is improved by the action of the added amine compound.

  The ionized saccharide is introduced into the mass spectrometer under high vacuum. A DC voltage called a cone voltage is applied to the pores (cones) for this introduction. The cone voltage may be adjusted as appropriate so as to improve the sensitivity of mass spectrometry, but 10 to 30 V is preferable, and about 20 V is particularly preferable.

  By the method of the present invention described above, saccharides contained in the test sample are separated, and molecular weight data of each saccharide is obtained. In addition, chemical structure data other than molecular weight can be obtained by mass spectrum. In addition, it is not necessary to modify the saccharide with an expensive reagent such as 2-aminopyridine, it can be carried out inexpensively and easily, and the sensitivity of mass spectrometry is high. That is, the method of the present invention can efficiently perform separation analysis of a test sample containing saccharides.

  More specifically, first, a standard saccharide sample is prepared and analyzed by the method of the present invention, and a retention time, a mass spectrum, and a mass chromatogram of a standard saccharide are acquired. Next, the test sample is analyzed under the same conditions, and by comparing the obtained data with the data of the standard sample, the type of saccharide contained in the test sample can be analyzed, and each saccharide is quantified. It is also possible to do.

The saccharide analyzer of the present invention capable of carrying out the above method comprises:
A liquid chromatography system having a ligand exchange column,
A pump for adding an amine compound to the eluate from the liquid chromatography system, and an electrospray ionization mass spectrometry system;
It is characterized by having. The apparatus of the present invention will be described below with reference to FIG.

  The liquid chromatography system of the present invention is a normal liquid chromatography system having at least a pump 1 for feeding a mobile phase, an injector 2 for inserting a test sample, a column 3, a column oven 4, and control devices thereof. If it is. However, the ligand exchange column described above is used as the column.

  The apparatus of the present invention further includes a pump 5 for adding an amine compound to the eluate from the liquid chromatography system, and an electrospray ionization mass spectrometry system 6.

  The pump 5 is for adding a specified amount of the amine compound solution to the eluate from the liquid chromatography system. Preferably, means are provided for controlling the concentration of the amine compound added to the eluate from the liquid chromatography system. As this control means, for example, the flow rate (flow rate) of the eluate from the liquid chromatography system and the input of the concentration of the amine compound solution to be added are received, and the amine compound concentration in the mixture of the eluate and the amine compound solution is further received. The thing which can adjust the flow volume (flow velocity) of an amine compound solution can be mentioned by receiving input.

  As the ESI mass spectrometry system 6, a normal one can be used. In other words, the housing has a capillary through which the mobile phase sent from the liquid chromatography system passes, and the tip of the capillary has a needle shape so that the mobile phase is ejected. Furthermore, it has a discharge electrode for applying a voltage to the tip of the capillary.

  The ionized saccharide is detected by total ion chromatogram (TIC) monitoring and amplified as an electrical signal. A mass spectrum is obtained by analyzing the electrical signal with a computer or the like.

  In the method of the present invention, the sensitivity of mass spectrometry is remarkably improved. As a result, the inventors have clarified that the intensity of the mass spectrum is proportional to the concentration of each saccharide in the test sample. Therefore, the apparatus of the present invention preferably has a means for quantifying each saccharide based on the intensity of the mass spectrum obtained by mass spectrometry. Specifically, for example, a calibration curve regarding sample concentration and mass spectrum intensity (for example, molecular ion peak intensity) is prepared in advance for a saccharide to be quantified, and each measured by a computer that analyzes the mass spectrum. Quantification is performed by comparing the mass spectral intensity of saccharides with calibration curve data. According to the findings by the present inventors, it is 5 to 500 μM for raffinose, which is a trisaccharide, 5 to 1000 μM for monosaccharide and maltotriose, and 10 to 1000 μM for disaccharide. A calibration curve with high linearity is obtained.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. It is also possible to implement, and they are all included in the technical scope of the present invention.

Example 1
Samples each containing 1 mmol / L of the saccharides shown in Table 1 were prepared and analyzed with the LC / MS apparatus shown in FIG. PU-980 manufactured by JASCO Corporation was used as the liquid chromatography device, and VG Quattro II manufactured by micromass was used as the mass spectrometer. Further, a 12% by mass aqueous ammonia solution is used as the amine compound solution, the flow rate ratio between the eluate from liquid chromatography and the aqueous ammonia solution is 5: 1, and the ammonia concentration in the sample solution introduced into the mass spectrometer is 2% by mass. It was. The LC / MS performance conditions are as follows.
Column: manufactured by Shinwa Kako Co., Ltd., ULTRON PS-80C / 5S
Filled with calcium sulfonate polystyrene cation exchange resin
Inner diameter 2.0mm, length 250mm
Mobile phase: Water Flow rate: 0.05 mL / min Column temperature: 70 ° C
Ionization mode: ESI-negative mode Cone voltage: 20V
Capillary voltage: 3.5 kV
Ion source temperature: 90 ° C
Detection mode: TIC (m / z: 50 to 700)

  The results are shown in Table 1.

  As shown in the above results, molecular ion peaks were obtained for all saccharides. Further, trisaccharides were decomposed into monosaccharides and disaccharides, and disaccharides were decomposed into monosaccharides. In addition, a peak that was considered to be a dimer was obtained, and no peak was observed, and a simple mass spectrum was obtained for each monosaccharide. As described above, according to the method of the present invention, it was demonstrated that saccharides having similar structures can be separated and analyzed well.

Example 2 Optimization of Amine Compound Concentration A 1 mmol / L glucose aqueous solution was analyzed in the same manner as in Example 1 except that the ammonia concentration in the sample solution introduced into the mass spectrometer was 0 to 5%. It was. About the obtained result, the intensity ratio of the molecular ion peak is relatively represented and shown in FIG.

  From the results, when the ammonia concentration was 1% by mass, the mass spectrometry sensitivity was improved, but the effect was not sufficient, but the sensitivity at 2% by mass was very good, and the effect was saturated at 5% by mass. Therefore, it was found that the concentration of the amine compound in the sample subjected to mass spectrometry is preferably 1.5% by mass or more.

Example 3 Optimization of column temperature In Example 1 described above, saccharides were analyzed in the same manner except that 10 types of saccharides were used and the column temperature was 65, 70, or 75 ° C. About the obtained result, the intensity ratio of the molecular ion peak is relatively represented and shown in FIG.

  From the results, each molecular ion peak could be detected even when the column temperature was 65 ° C., but the results at 70 ° C. or 75 ° C. were good, and the best results were obtained at 70 ° C. Therefore, it was found that the column temperature for liquid chromatography is preferably 65 to 75 ° C., and 70 ° C. is optimal under the execution conditions of Example 1.

Example 4 Optimization of Cone Voltage Sugars were analyzed in the same manner as in Example 1 except that the cone voltage was changed to 10 to 80V. About the obtained result, the intensity ratio of molecular ion peak is relatively represented and shown in FIG.

  From the results, the sensitivity was relatively good when the cone voltage was 10 to 30V, and the best result was obtained at 20V. Therefore, it was found that the cone voltage in the execution conditions of Example 1 is 10 to 30V, and 20V is optimal.

Example 5 Examination of detection limit In the liquid chromatography of Example 1 above, saccharides were analyzed in the same manner except that the test sample was a single solution of each saccharide and the concentration was variously changed. Table 2 shows detection limits of molecular ion peaks in liquid chromatography of each sugar.

  As the result, according to the liquid chromatography using the ligand exchange column in which the gel particles are semi-micronized, the detection limit of any sugar was ng level. Therefore, it was demonstrated that liquid chromatography using a ligand exchange column in which gel particles are semi-micronized exhibits high sugar separation ability.

It is a schematic diagram of the apparatus for implementing this invention method. It is a figure which shows the intensity | strength ratio of the molecular ion peak at the time of changing the density | concentration of the amine compound which occupies for the sample which performs mass spectrometry. It is a figure which shows the intensity ratio of the molecular ion peak at the time of changing column temperature. It is a figure which shows the intensity | strength ratio of the molecular ion peak at the time of changing cone voltage.

Explanation of symbols

  1: mobile phase feed pump, 2: injector, 3: ligand exchange column, 4: column oven, 5: amine compound solution feed pump, 6: electrospray ionization mass spectrometry system

Claims (7)

Separating a test sample containing saccharides by liquid chromatography using a ligand exchange column;
A step of adding an amine compound to the separated test sample, and a step of ionizing the test sample to which the amine compound has been added by electrospray ionization and performing mass spectrometry,
A method for analyzing saccharides, comprising:   The method for analyzing saccharides according to claim 1, wherein ammonia is used as the amine compound.   The method for analyzing saccharides according to claim 1 or 2, wherein the concentration of the amine compound in the sample subjected to mass spectrometry is 1.5% by mass or more.   The method for analyzing a saccharide according to any one of claims 1 to 3, wherein an average particle size of the gel packed in the ligand exchange column is 1 to 10 µm. A liquid chromatography system having a ligand exchange column,
A pump for adding amine compounds to the eluate from the liquid chromatography system, and an electrospray ionization mass spectrometry system;
An apparatus for analyzing saccharides, comprising:   The saccharide analyzer according to claim 5, further comprising means for controlling the concentration of the amine compound added to the eluate from the liquid chromatography system.   Furthermore, the saccharide | sugar analyzer of Claim 5 or 6 which has a means for quantifying each saccharide | sugar by the intensity | strength of the mass spectrum obtained by mass spectrometry.

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