Method of analyzing agaro-oligosaccharides

Abstract

To provide a reliable analysis method which enables simple and high-sensitivity isolation and detection of agaro-oligosaccharides without labeling.SOLUTION: An agaro-oligosaccharide analysis method according to the present invention involves isolating an agaro-oligosaccharide from an agaro-oligosaccharide-containing substance using liquid chromatography in a hydrophilic interaction mode to mass-spectrometrically detect the agaro-oligosaccharide, where an acid is added to a mobile phase in the liquid chromatography.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to a method for analyzing agarooligosaccharides.

Background Art

[0002] Agarose, known as the main component of agar, is a polysaccharide in which D-galactose and 3,6-anhydro-L-galactose are alternately linked by glycosidic bonds. The 1-position of D-galactose and the 4-position of 3,6-anhydro-L-galactose are linked by a β(1,4) bond, and the 1-position of 3,6-anhydro-L-galactose and the 3-position of D-galactose are linked by an α(1,3) bond.

[0003] Agarose is hydrolyzed by the action of an acid or an enzyme (α-agarase) at the α(1,3) bond to produce agarooligosaccharides such as agarobiose (disaccharide), agaro-tetraose (tetrasaccharide), agaro-hexaose (hexasaccharide), and agaro-octaose (octasaccharide), which are oligosaccharides having 3,6-anhydro-L-galactose at the reducing end. It has been reported that these agarooligosaccharides have various physiological activities such as prebiotic effects, apoptosis-inducing activity, anti-cancer activity, active oxygen production inhibitory activity, and immunomodulatory activity (Patent Document 1: Japanese Patent No. 4007760), and have great industrial applicability as functional components of foods and medicinal ingredients of pharmaceuticals, etc.

[0004] In order to formulate and commercialize products containing such agarooligosaccharides and ensure their quality, it is necessary to standardize a reliable method for analyzing agarooligosaccharides. In particular, an analytical method capable of separating and detecting agarooligosaccharides contained at low concentrations from foods, pharmaceuticals, etc. that contain contaminating components such as other carbohydrates having a structure or properties similar to those of the target component, agarooligosaccharides, is required.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] Patent Document 1 describes that the degradation product (solution) of agar is separated by liquid chromatography (LC), the substance of a predetermined peak is fractionated, and then the fractionated substance is subjected to mass spectrometry (mass spectrometry, MS) using fast atom bombardment (FAB) as an ionization method; FAB / MS and nuclear magnetic resonance spectrometry (NMR) for analysis, and it is described that the fractionated substance was identified as agarobiose (Examples 2 and 3). However, in this document, basically, only agar, which is the source of agarooligosaccharide, is used as a test sample without containing impurities, and a relatively high-concentration agar solution of about 10 mg / mL (Example 2) or 50 mg / mL (Example 3) is used for separation of 2 mL, which is not a small amount, as a degradation product. Under such relatively mild conditions, the substance is fractionated, and then the fractionated substance is separately subjected to qualitative analysis. Therefore, from such conventional documents, for example, for foods and pharmaceuticals containing impurities such as saccharides other than agarooligosaccharides, even if they contain agarooligosaccharides at a low concentration, the analytical method and its conditions for separating and detecting each sugar are not clear, and it is desired to establish a highly sensitive and reliable analytical method for agarooligosaccharides.

[0007] On the other hand, conventionally, in sugar analysis, in order to improve its sensitivity, a label such as a fluorescent label may be applied to the sugar chain. However, even if the sensitivity is improved by the label, such a label is troublesome, the analysis process is likely to become complicated, and the analysis equipment is likely to be expensive.

Means for Solving the Problems

[0008] The present invention has been made in view of the above circumstances, and an object thereof is to provide a reliable analysis method capable of simply and highly sensitively separating and detecting agarooligosaccharides without applying a label.

[0009] In one embodiment of the present invention, the above problems are solved by the solution means described below.

[0010] The method for analyzing agarooligosaccharides according to the present invention is characterized in that agarooligosaccharides are separated from an agarooligosaccharide-containing substance in a hydrophilic interaction mode of liquid chromatography and detected by mass spectrometry. Further, an acid is added to the mobile phase in the liquid chromatography.

[0011] In the hydrophilic interaction mode, it is preferable to use an amide column.

[0012] The acid added to the mobile phase is not limited. In one example, a volatile acid can be added as the acid. Further, as the acid (the volatile acid), one or more acids selected from formic acid, acetic acid, trifluoroacetic acid, and derivatives thereof can be added.

[0013] Also, for the mobile phase, the acid can be added to an aqueous solution of an organic solvent of 50 vol% or more.

[0014] Also, before performing the liquid chromatography on the agarooligosaccharide-containing substance, the agarooligosaccharide-containing substance can be pretreated with an enzyme.

[0015] And the present invention can be directed to an agarooligosaccharide-containing substance containing saccharides other than agarooligosaccharides. More specifically, the present invention can be directed to, for example, an agarooligosaccharide-containing substance containing dextrin.

Advantages of the Invention

[0016] According to the present invention, agarooligosaccharides can be simply and highly sensitively separated and detected without labeling, and a reliable method for analyzing agarooligosaccharides can be provided.

Brief Description of the Drawings

[0017]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0018] As used in this application, the agaro-oligosaccharide (AOS) refers to disaccharide agarobiose (Abi), tetrasaccharide agarotetraose (Ate), hexasaccharide agarolhexaose (Ahe), octasaccharide agarooctaose (Aoc), and their modified forms. Abi, Ate, Ahe, and Aoc are produced by hydrolysis of agarose, and in practical use, they are produced by hydrolyzing substances containing agarose such as agar with an acid or an enzyme (α-agarase). On the other hand, the modified forms of Abi, Ate, Ahe, and Aoc are compounds in which at least one of the hydroxyl groups of Abi, Ate, Ahe, or Aoc is modified with a substituent, and the hydroxyl group is modified with a substituent such as a methoxy group, a sulfate group, a pyruvic acid group, or a carboxy group (more specifically, the hydrogen atom of the hydroxyl group is substituted with these substituents). These modified forms are produced by hydrolysis of agaropectin, and in practical use, they are produced by hydrolyzing substances containing agaropectin such as agar with an acid or an enzyme (α-agarase). However, in social practice, it is not required to separate and detect Abi, Ate, Ahe, and Aoc and their modified forms. In fact, as agaropectin is hydrolyzed with an acid or an enzyme (α-agarase), many of the above substituents are eliminated, or those that are not eliminated do not undergo sufficient decomposition and remain as medium-chain or long-chain residues. Therefore, it is considered that the analytical target (sample) of agaro-oligosaccharide content basically contains almost no modified forms. Therefore, there is a situation where the coexistence of Abi, Ate, Ahe, and Aoc and their modified forms does not pose a problem. Therefore, in the analysis of agaro-oligosaccharides in the present invention, when the disaccharide Abi, the tetrasaccharide Ate, the hexasaccharide Ahe, and the octasaccharide Aoc are separated and detected based on the difference in the degree of polymerization, each of these sugars may contain its respective modified form.

[0019] The method for analyzing agarooligosaccharides of the present invention is a qualitative analysis method capable of separating and detecting 2- to 8-saccharide agarooligosaccharides and identifying them. However, in the practice of the present invention, among the sugars of detectable agarooligosaccharides, it is only necessary to qualitatively analyze the desired sugar according to the purpose. Therefore, for example, among the 2- to 8-saccharide agarooligosaccharides, if necessary, only the Abi of the disaccharide can be qualitatively analyzed, or only the Abi of the disaccharide and the Ate of the tetrasaccharide can be qualitatively analyzed, which is also an embodiment of the present invention. Further, based on the separation and detection of each sugar of the agarooligosaccharides according to the present invention, for example, applying a known calibration curve or the like to obtain a quantitative analysis result is also an embodiment of the present invention or an embodiment using the present invention. Furthermore, by implementing the present invention, for agarodecaose with 10 sugars that is not included in the object of the present invention, when it can be separated and detected, it is also an embodiment of the present invention or an embodiment using the present invention to analyze (qualitatively and / or quantitatively) the 10 sugars together with any one of the 2- to 8-saccharide sugars.

[0020] In this embodiment, agarooligosaccharides are separated from an agarooligosaccharide-containing substance by liquid chromatography and detected by mass spectrometry (mass spectrometry). That is, this embodiment employs liquid chromatography-mass spectrometry (LC / MS) that combines liquid chromatography (LC) and mass spectrometry (MS). As shown in FIG. 1, the LC / MS according to this embodiment is basically an LC-MS (01) (liquid chromatograph-mass spectrometer) that connects an LC (02) (liquid chromatograph) and an MS (03) (mass spectrometer), and continuously performs LC (liquid chromatography) and MS (mass spectrometry). Therefore, it is different from methods in which LC and MS are performed independently and separately, for example, a method in which a substance of a predetermined peak separated by LC (liquid chromatography) using an LC (02) (liquid chromatograph) is fractionated and then the fractionated substance is analyzed separately by MS (mass spectrometry) using an MS (03) (mass spectrometer). It includes constituent elements (processes or steps) characteristic of the present invention unique to the method for analyzing agarooligosaccharides to which LC / MS using an LC-MS (01) (liquid chromatograph-mass spectrometer) is applied. According to this embodiment, a sample (various foods, pharmaceuticals, quasi-drugs, cosmetics, etc.) containing agarooligosaccharides and carbohydrates other than agarooligosaccharides (monosaccharides, oligosaccharides, polysaccharides, other sugar alcohols) can be analyzed to separate and detect agarooligosaccharides. In the examples of this embodiment described later, an agarooligosaccharide-containing substance containing dextrin, a polysaccharide, which was shown to be particularly difficult to separate by LC / MS in a preliminary test, was used. An agarooligosaccharide solution containing agarooligosaccharides at a low concentration of 400 ppm (in the present invention, mg / L is used as ppm. Therefore, 400 ppm is 0.4 mg / mL) to an extremely low concentration of 100 ppm (0.1 mg / mL) was subjected to only 1 μL of a small amount, and the agarooligosaccharides were easily and highly sensitively separated without labeling, and each sugar (2-8 sugars) could be detected.

[0021] Here, the liquid chromatography of the present invention includes not only the oldest medium- and low-pressure LC (Liquid Chromatography), but also improved methods such as high-pressure HPLC (High Performance Liquid Chromatography) and UHPLC (Ultra High Performance Liquid Chromatography). HPLC is a method that uses a high-pressure liquid delivery pump to deliver the mobile phase at a higher speed than medium- and low-pressure LC, and it shortens the analysis time compared to medium- and low-pressure LC. HPLC is the most widespread method at present. The LC (02) shown in FIG. 1 is an example of HPLC (High Performance Liquid Chromatography), and the following examples also use HPLC. UHPLC is a method developed in recent years. It uses a high-pressure liquid delivery pump similar to HPLC, and further increases the column pressure moderately by micronizing the base material of the stationary phase to increase the flow rate of the mobile phase at a higher speed. Thus, improved methods such as HPLC and UHPLC are basically physical improvements to medium- and low-pressure LC (Liquid Chromatography), and do not basically cancel out the effects based on the chemical characteristics (such as acid addition) described later in the analysis method of the present invention. Therefore, there is no problem in including medium- and low-pressure LC, high-pressure HPLC, UHPLC, etc. as subordinate concepts in the concept of LC (Liquid Chromatography) of the present invention. Hereinafter, this embodiment will be described by taking an example using the LC-MS (01) (Liquid Chromatograph-Mass Spectrometer) shown in FIG. 1.

[0022] [LC: Liquid Chromatograph] In this embodiment, first, LC (liquid chromatography) is performed on an agarooligosaccharide-containing substance, which is a sample to be analyzed. LC (liquid chromatography) is performed in the LC (02) (liquid chromatograph) in the LC-MS (01) (liquid chromatograph-mass spectrometer). The configuration of the LC (02) (liquid chromatograph) includes a mobile phase container (04), a degassing device (05), a liquid delivery pump (06), a sample introduction unit (07), a column (08), and a column thermostat (09), as shown in FIG. 1 as an example of an HPLC (high performance liquid chromatograph). Each of these components is operated under the control of a control unit (10). The control unit (10) is composed of a CPU and a memory, and performs a predetermined operation based on a preset operation program and / or a setting signal input from an operation unit (not shown).

[0023] The mobile phase container (04) is a container capable of accommodating a liquid such as a vial or a bottle, and accommodates the mobile phase (eluent). The degassing device (05) is optionally installed. For example, a degassing unit that discharges small molecule gases from the mobile phase by utilizing the permeability of the membrane by incorporating a resin membrane tube as a gas-liquid separation membrane into the flow path (A) and reducing the pressure outside the tube. Alternatively, for example, an aspirator may be attached to the mobile phase container (04), and the mobile phase container (04) may be vibrated with an ultrasonic cleaner or aspirated while stirring the mobile phase, without being incorporated into the flow path (A).

[0024] The liquid delivery pump (06) delivers the mobile phase in the flow path (A) at a high pressure so as to achieve a predetermined flow rate. The sample introduction unit (07) introduces a sample, which is an analysis target appropriately pretreated by an autosampler or the like, into the mobile phase. The mobile phase into which the sample has been introduced passes through an optionally installed guard column and is introduced into a column (08) appropriately adjusted to a predetermined temperature by a column thermostat (09). The column (08) is configured as a column (08) filled with a packing material and forms a stationary phase in LC (liquid chromatography). The sample to be analyzed is retained by the stationary phase at different times for each of the contained components (molecules) in the column (08) due to the difference in affinity for the mobile phase and the stationary phase, and elutes from the column (08) (packing material). Thereby, the separation of agarooligosaccharides is achieved. Note that a solution in which the sample is dissolved in advance in the eluent may be prepared and introduced into the column (08) via the sample introduction unit (07) or the mobile phase container (04).

[0025] [LC: Sample · Pretreatment] Here, as the agarooligosaccharide-containing substance that can be used for analysis, as described above, it is a sample containing agarooligosaccharides and saccharides other than agarooligosaccharides (monosaccharides, oligosaccharides, polysaccharides, other sugar alcohols), and various foods, pharmaceuticals, quasi-drugs, cosmetics, etc. can be cited. For example, as for foods, neither the type nor the form is limited, but for example, staple foods such as rice, bread, noodles, side dishes, confectionery such as fresh confectionery and frozen confectionery, fermented foods such as yogurt, seasonings such as sauce and dressing, and further, tablets such as supplements, jelly-like foods, swallowable foods, beverages, powdered beverages, etc. can be cited.

[0026] Before performing LC (liquid chromatography) on these agarooligosaccharide-containing substances, a predetermined pretreatment may be carried out on the agarooligosaccharide-containing substances in advance. In particular, the pretreatment for foods that are likely to contain various contaminants is effective. As an example, for a crude sample (a sample that has not been treated at all), the following crude extraction operation can be first carried out. That is, when the crude sample is a solid, it can be pulverized with a mixer or the like, then suspended in water, and operations such as removing the solid content by centrifugation or filtration can be carried out. Also, when the crude sample is a liquid substance, an operation such as dilution with water can be carried out.

[0027] Next, for the crude sample or for the crude extract obtained through the above crude extraction operation, the following crude purification operation can be carried out. That is, for example, as purification with an organic solvent, an operation such as adding an aqueous solution of an organic solvent to the crude sample or the crude extract, stirring with a mixer or the like, and then performing centrifugation to obtain the supernatant can be carried out. Also, for example, as purification with a solid-phase extraction column, after selecting an appropriate column and conditioning it, the crude sample or the crude extract is added, and the agarooligosaccharide, which is the target component, is eluted and recovered, or an operation such as adsorbing the agarooligosaccharide and then eluting and recovering it can be carried out.

[0028] Furthermore, the following enzymatic treatment can be performed on the crude sample and / or the above-mentioned crude extract and / or the crude purified product obtained through the above-mentioned crude purification operation. This enzymatic treatment is effective particularly for samples that are assumed to contain carbohydrates other than agarooligosaccharides. That is, for example, a buffer solution such as an ammonium acetate buffer (acetic acid / ammonium acetate buffer) (for example, adjusted to pH 4.5 within a buffering range of about pH 3.5 to 5.5) is added to the crude sample, or the crude extract, or the crude purified product. Subsequently, carbohydrases such as glucoamylase (α(1,4)-glucosidic bond hydrolase), invertase (sucrose hydrolase, also known as saccharase or β-D-fructofuranosidase), and amyloglucosidase (α(1,6)-glucosidic bond hydrolase, also known as amyl α-1,6-glucosidase or dextrin 6-α-D-glucosidase) are further added, and the reaction is carried out at the optimum temperature (for example, within a range of about 35°C to 40°C, such as 37°C) for the optimum time (for example, within a range of about 10 minutes to 120 minutes, such as 60 minutes). Then, a series of enzymatic treatments can be performed, such as boiling treatment (for example, within a range of about 5 minutes to 30 minutes, such as 10 minutes) to inactivate the enzyme.

[0029] The necessity of using the buffer solution is selected according to the sample and the enzyme. When used, a volatile buffer solution such as the ammonium acetate buffer exemplified above is preferred, but it is not limited thereto. The type can be selected according to the sample and the enzyme, and it may be adjusted to the optimum pH, molar concentration, etc. The enzyme is not limited to the carbohydrases exemplified above, and an appropriate enzyme can be selected according to the assumed contaminants. Therefore, proteases or lipases may be used. Also, for carbohydrases, those other than the ones exemplified above, for example, amylases other than glucoamylase, and other maltase, lactase, etc. may be used. The treatment temperature and treatment time by the enzyme may be appropriately set optimally.

[0030] As described above, the enzymatic treatment is effective for a sample that is assumed to contain carbohydrates other than agarooligosaccharides in particular, and even for agarooligosaccharides contained at a low concentration, it contributes to enabling the separation of trace amounts of agarooligosaccharides with high sensitivity by subjecting them to LC (liquid chromatography). In the examples described later, in the case where enzymatic treatment was carried out on an agarooligosaccharide-containing substance containing dextrin of polysaccharide and other impurity components, which are particularly difficult to separate, agarooligosaccharides could be detected with higher sensitivity through the MS (mass spectrometry) described later as compared with the case where it was not carried out. According to the present embodiment, even if the enzymatic treatment is not carried out, agarooligosaccharides can be detected to the extent that the object of the present invention can be achieved without labeling the sample (agarooligosaccharides). However, as a means for further improving the separation ability or detection ability of agarooligosaccharides, for example, enzymatic treatment can be carried out instead of labeling, which tends to make the analysis process complicated and the analysis equipment expensive. However, since both the enzymatic treatment and the labeling are optional treatments in the present invention, applying labeling instead of the enzymatic treatment or applying labeling together with the enzymatic treatment itself is not prohibited and is allowed.

[0031] [LC: Separation mode] The separation mode of LC (Liquid Chromatography) according to this embodiment is preferably Normal Phase Chromatography (NPC), and Hydrophilic Interaction Chromatography (HILIC) is applied. Normal Phase Chromatography (Normal Phase Mode, NPC Mode) is a separation mode in which the polarity of the stationary phase is set higher than that of the mobile phase. For example, the stationary phase is relatively highly polar, such as unmodified silica gel or alumina, and the mobile phase is relatively low polar, such as an organic solvent such as hexane or chloroform. Hydrophilic Interaction Chromatography (Hydrophilic Interaction Mode, HILIC Mode) is one of the NPC modes. For example, the mobile phase is an aqueous solution of an organic solvent having a certain polarity, such as acetonitrile, methanol, or ethanol, and the stationary phase, which is more polar than that, is silica gel or polymer gel modified with a polar group (for example, an amide group, a cyano group, an amino group, etc.). In the NPC mode and the HILIC mode, the higher the polarity of the molecule, the longer the retention time in the stationary phase, and the lower the polarity of the molecule, the earlier it elutes and the shorter the retention time. In the separation mode, the type of substrate, polar group, size (particle size) and amount of the packing material filled in the stationary phase, that is, the column (08), and the size of the column (08) associated therewith, the type, flow rate and flow velocity of the mobile phase (eluent), etc. can be freely selected. However, among them, the polar group that imparts polarity to the stationary phase is preferably an amide group, and it is preferable to modify the stationary phase with an amide group to impart polarity.

[0032] In the NPC mode and the HILIC mode, in the analysis of agarooligosaccharides, compared with the SEC mode (Size Exclusion Chromatography) and the like where it is more suitable to increase the water content rate of the mobile phase, conversely, a highly volatile organic solvent can be used in the mobile phase at a relatively high concentration. In this regard, the ionization efficiency in MS (mass spectrometry) can be increased to improve the detection sensitivity, which is preferably adapted to the separation mode according to this embodiment. Further, the HILIC mode is more suitable for the separation of hydrophilic and highly polar agarooligosaccharides, and is more preferably adapted to the separation mode according to this embodiment.

[0033] [LC: Mobile phase] In the HILIC mode, the aqueous solution of the organic solvent used in the mobile phase (mixed solvent of organic solvent and water) is preferably set at a concentration of 50 vol% or more, and more preferably 60 vol% or more, in order to increase the ionization efficiency in MS (mass spectrometry) as described above. As the organic solvent, acetonitrile, which is an aprotic solvent, is preferred. That is, in the HILIC mode, in the mobile phase, as the eluent as the solvent (dissolution solution) for dissolving the sample, or as the solution in which the sample is previously dissolved in the eluent, an acetonitrile aqueous solution of 50 vol% or more is preferred, and an acetonitrile aqueous solution of 60 vol% or more is more preferred. Note that for water, pure water or ultrapure water such as ion-exchanged water or distilled water may be used, and the purity standard may be appropriately set according to the type of sample and the like.

[0034] In addition, this embodiment is characterized in that a specific acid is added to the mobile phase to make the mobile phase acidic. Specifically, one or more acids, that is, substances that exhibit acidity in the mobile phase, are added to the eluent of the mobile phase (a solvent (dissolving solution) for dissolving the sample, for example, a 60 vol% aqueous acetonitrile solution) or a solution in which the sample is previously dissolved in the eluent (for example, a 60 vol% aqueous acetonitrile solution in which the sample is dissolved). The type of acid is not limited. As an example, formic acid (HCOOH), acetic acid (CH3COOH), trifluoroacetic acid (CF3COOH), hydrochloric acid (HCl), sulfuric acid (H2SO4), nitric acid (HNO3), propionic acid (C3H6O2), butyric acid (C4H8O2), lactic acid (C3H6O3), etc., and derivatives thereof are exemplified. As long as the purpose of making the mobile phase acidic can be achieved, an acid salt may be added as a specific acid. Among the acids, volatile acids (so-called volatile acids) with relatively low boiling points, such as formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, propionic acid, butyric acid, lactic acid, etc., can be preferably applied rather than non-volatile acids with relatively high boiling points, such as sulfuric acid. Furthermore, one or more acids selected from formic acid, acetic acid, and trifluoroacetic acid, and derivatives thereof can be particularly preferably applied.

[0035] Generally, in sugar analysis by LC / MS, a base such as ammonia is added to the eluent to make it alkaline for the purpose of keeping the mobile phase in the neutral range, preventing anomeric separation, or promoting the ionization of sugars. On the other hand, it has been found that agarooligosaccharides have low base resistance, and when they come into contact with a base, their structure changes and browning occurs, and by making the mobile phase alkaline, problems such as the target peak not being detected or an unknown peak being detected are likely to occur. Therefore, although those skilled in the art could select the neutral range as ordinary knowledge, the inventor further studied the pH of the mobile phase and found that when an acid as exemplified above is added, each sugar molecule of agarooligosaccharides can be detected with high sensitivity as a specific ion-added molecule and / or deprotonated molecule in MS (mass spectrometry). As a result, agarooligosaccharides can be simply and highly sensitively separated without labeling the sample, and each sugar (2-8 sugars) can be detected.

[0036] The addition amount (addition ratio) of the acid can be adjusted according to the acidity of each acid. Among the acids, if an acid with a relatively strong acidity is added excessively, it may damage the LC (02) (liquid chromatograph) such as the column (08). Therefore, generally, an acid with a relatively low acidity is more suitable as an acid. However, in the examples described later, among the tested acids, trifluoroacetic acid, which has a relatively strong acidity, has a limited addition amount and is relatively difficult to handle compared to formic acid and acetic acid, which have lower acidity. Also, the types of ion molecules capable of detecting 2-8 saccharides of agarooligosaccharides with high sensitivity were relatively few. However, for specific ion molecules, reliable detection of each saccharide of agarooligosaccharides for achieving the object of the present invention was possible. According to the examples, the addition amount of the acid enabled detection of agarooligosaccharides with a predetermined ion molecule by adding at least 0.01 vol% of formic acid to the mobile phase, or by adding formic acid to the mobile phase in the range of at least 0.01 vol% to 0.1 vol% (Examples 1 and 4). Also, detection of agarooligosaccharides with a predetermined ion molecule was possible by adding at least 0.1 vol% of acetic acid to the mobile phase, or by adding acetic acid to the mobile phase in the range of at least 0.1 vol% to 1 vol% (Examples 2 and 5). Also, detection of agarooligosaccharides with a predetermined ion molecule was possible by adding at least 0.01 vol% of trifluoroacetic acid to the mobile phase (Examples 3 and 6). Regarding the acid concentration (unit: vol%) mentioned here, as a conventional technique in the technical field to which the present invention belongs, regarding the pH adjustment of the mobile phase and its notation, for example, if it is an acid-added eluent of x vol%, it represents an eluent prepared by adding x volume amount (for example, x mL) of an acid to an eluent of 100x volume amount (for example, 100x mL) (for example, 60 vol% aqueous acetonitrile solution), and the present invention also follows this.

[0037] [LC: stationary phase] As described above, in the LC (liquid chromatograph) (02), the stationary phase is configured as a column (08) filled with a packing material. From the perspective of the incompatibility of the agarooligosaccharide with basicity as described above, the polar group of the base material in the packing material of the HILIC (used) column is preferably an amide group compared to an amino group or a cyano group that is partially basic and easily becomes basic. Further, as described above, as the base material to which the polar group is bonded, silica gel, a polymer gel such as polyvinyl alcohol, or the like can be used. That is, in the HILIC mode, an amide column modified with an amide group (carbamoyl group) can be preferably used. Thereby, it becomes possible to more surely suppress problems caused by the incompatibility of agarooligosaccharide with basicity, such as the agarooligosaccharide being easily structurally changed, the target peak being difficult to detect, or an unknown peak being easily detected. As a result, agarooligosaccharide can be separated and detected with higher sensitivity. Further, the temperature of the stationary phase (column temperature) is not limited and may be adjusted as appropriate, but a higher temperature is preferable to prevent anomer separation. In particular, when using an amide column, it is preferably set to 60°C or higher, more preferably 80°C or higher. On the other hand, in the case of an amino column or the like, since anomer separation that easily becomes basic hardly occurs, it can be preferably used, for example, at about 45°C.

[0038] [MS: Mass spectrometer] Subsequently, in this embodiment, MS (mass spectrometry) is performed on the sample separated by LC (liquid chromatography). The MS (mass spectrometry) is performed by the MS (03) (mass spectrometer) in the LC-MS (01) (liquid chromatograph-mass spectrometer). As shown in FIG. 1, the configuration of the MS (03) (mass spectrometer) includes an ionization unit (11), a mass separation unit (12), and a detection unit (13). Each of these components is operated under the control of the control unit (14). The control unit (14) is composed of a CPU and a memory, and performs a predetermined operation based on a preset operation program and / or a setting signal input from an operation unit (not shown). Note that the control unit (10) in the LC (02) (liquid chromatograph) and the control unit (14) in the MS (03) (mass spectrometer) may be integrally configured.

[0039] [MS: Ionization Unit · Ionization Method] The elution sample containing agarooligosaccharides eluted from the column (08) of the LC (liquid chromatograph) is subsequently introduced into the ionization section (11) of the MS (mass spectrometer) connected to the LC (02) (liquid chromatograph). The ionization section (11) ionizes the introduced elution sample. As the ionization method, a known ionization method in MS (mass spectrometry) can be appropriately used, and the ionization section (11) is configured according to the applied ionization method. Examples of specific ionization methods include Electrospray Ionization (ESI), Atmospheric Pressure Chemical Ionization (APCI), Atmospheric Pressure Photo Ionization (APPI), etc. ESI ionizes sample molecules in the liquid phase by applying a high voltage under atmospheric pressure. APCI heats and vaporizes the sample, and reacts the ions generated from the atmosphere by corona discharge with the vaporized sample molecules to ionize the sample molecules. APPI heats and vaporizes the sample, and ionizes the vaporized sample molecules by UV irradiated from a krypton lamp. Although the above ionization methods all ionize the sample under atmospheric pressure, Thermospray Ionization (TSI, TSPI), etc., which ionize the sample under vacuum, may also be applied.

[0040] [MS: Mass separation section · Mass separation method] The mass separation unit (12) separates the ionized ions (ion molecules) for each m / z (the ratio of the mass (m) to the charge number (z) of the ion: so-called mass-to-charge ratio). As the separation method, known mass separation methods in MS (mass spectrometry) can be appropriately used, and the mass separation unit (12) is configured in accordance with the applied mass separation method. Specific mass separation methods include, for example, the quadrupole type, the magnetic sector type, the time-of-flight type, the ion trap type, etc. In the quadrupole type, four electrodes are arranged in a vacuum at an equal distance from the central axis and parallel to each other. Electrodes facing each other across the central axis are applied with voltages of the same polarity, and electrodes adjacent to each other are applied with voltages of opposite polarities. Then, when a DC voltage and a high-frequency AC voltage are superimposed and applied to each electrode, an electric field with a rapidly changing phase is generated in the quadrupole. Only ions with a specific range of m / z can vibrate stably and pass through the quadrupole when introduced into this field. By utilizing this, the voltage is changed to perform mass separation. In the magnetic sector type, when ions accelerated by a voltage are introduced into a sector-shaped magnetic field, the ions receive an acceleration perpendicular to the velocity and the magnetic field direction according to Fleming's left-hand rule, and the trajectory is bent. Mass separation is performed by utilizing the fact that the orbit of ions with a smaller mass is bent more greatly. In the time-of-flight type, when ions are accelerated by the same voltage, mass separation is performed by utilizing the fact that ions with a smaller mass fly faster and ions with a larger mass fly slower. The ion trap type applies the principle of the quadrupole type and there are various methods. For example, it is a ring-shaped structure connecting the entrance and exit of the quadrupole. Ions are trapped in the system, and the high-frequency voltage is gradually changed to sequentially discharge ions with unstable vibrations out of the system to perform mass separation.

[0041] Also, the ionization method and the mass separation method are not limited, and tandem type applications such as MS / MS and MS n are also allowed within the scope of achieving the object of the present invention. Therefore, the mass spectrometry (mass spectrometry) of the present invention includes, in addition to MS as a single mass, MS / MS and MS n as tandem types.

[0042] [LC: Detection unit, detected ion molecule, mass spectrum, chromatogram] The detection unit (13) is composed of a secondary electron multiplier tube, a photomultiplier tube, a channeltron, a microchannel plate, etc., and amplifies and detects the signals of ions (ion molecules) separated and selected for each m / z. As a result, the molecule M of each sugar of the agarooligosaccharide is converted into a formic acid ion-added molecule ([M+HCOO] - ), deprotonated molecule ([M-H] - ), acetic acid ion-added molecule ([M+CH3COO] - ), ammonium ion-added molecule ([M+NH4] + ), sodium ion-added molecule ([M+Na] + ), and trifluoroacetic acid ion-added molecule ([M+CF3COO] - ), etc., and can be detected as ion-added molecules and / or deprotonated molecules exemplified above. When the detection signal detected by the detection unit (13) is appropriately converted by a converter and input to the control unit (14), it is analyzed by the control unit (14) and a predetermined calculation is performed. For example, it is output as a mass spectrum with m / z on the horizontal axis and the signal value (ion intensity, etc.) related to the applied mass separation method on the vertical axis. And / or, by the control unit (14), for the ions (ion molecules) separated and selected for each m / z, a chromatogram is output with the passing time (retention time, etc.) in LC(02) on the horizontal axis and the signal value (ion intensity, etc.) related to the applied separation mode on the vertical axis.

[0043] Here, the m / z of the ion molecule of the agarooligosaccharide can be calculated from its molecular formula. As an example, for the formic acid ion-added molecule of agarobiose (C 12 H 20 O 10 ), ([C 12 H 20 O 10 +HCOO] -) From the molecular formula, its molecular weight can be calculated to be approximately 369 (= 324 for agarobiose + 45 for formate ion), and its m / z can be calculated to be approximately -369 with a valence of -1. When the m / z 369(-) is detected by MS (mass spectrometry), for example, in a chromatogram, [C 12 H 20 O 10 +HCOO] - If a peak corresponding to the m / z 369(-) is observed in the retention time, etc. of, for example, the retention time of [C 12 H 20 O 10 +HCOO] - it can be determined that the sample contains agarobiose. In this way, agarobiose, agarotetraose, agaropentaose, and agarohexaose can be qualitatively analyzed.

[0044] Note that the mass spectrum and chromatogram, as well as the display form of their analysis results, are not limited and can be displayed on a paper medium such as chart paper or a display screen. Such a display unit (not shown) may be provided as part of the LC-MS(01), or may be provided in another device connected to the LC-MS(01) by wire and / or wirelessly. Alternatively, it may be recorded as electronic data on a recording medium detachable from the LC-MS(01), and the user may take out the electronic data from the LC-MS(01) via the recording medium and perform the necessary analysis and display on another device as appropriate.

[0045] Thus, according to the method for analyzing agarooligosaccharides according to this embodiment, agarooligosaccharides can be easily and highly sensitively separated and detected without labeling. Even for a sample (various foods, pharmaceuticals, quasi-drugs, cosmetics, etc.) containing agarooligosaccharides and carbohydrates other than agarooligosaccharides (monosaccharides, oligosaccharides, polysaccharides, other sugar alcohols), agarooligosaccharides contained at extremely low concentrations can be separated and detected with high sensitivity using a trace amount.

Examples

[0046] [Production of agarooligosaccharide] Agarooligosaccharide was produced from agar by a conventional method. After dissolving 50 g of agar (Ultra Agar AX-30: Ina Food Industries, Ltd.) in 1000 g of distilled water by heating, 2 g of concentrated sulfuric acid was added, and the mixture was stirred at 90 °C for 3 hours. Then, after adjusting the pH to 3.5 with sodium hydroxide, it was treated with activated carbon, filtered through filter paper, and further filtered through a 1-μm filter to obtain an agarooligosaccharide solution. This solution was freeze-dried under vacuum to obtain powdered agarooligosaccharide. This was used in all the following tests.

[0047] [Test 1] After dissolving agarooligosaccharide in distilled water, it was prepared so that the final concentration of acetonitrile became 60 vol%. The final concentration of agarooligosaccharide was prepared to be 400 ppm (0.4 mg / mL), and this was designated as sample (1). Sample (1) was analyzed under the following conditions by LC-MS (HPLC-MS) connected with HPLC (high performance liquid chromatography) and MS (mass spectrometer) (Examples 1 - 3).

[0048] In this test, without performing enzyme treatment or other pretreatment on sample (1), the prepared sample (1) was directly subjected to (injected into) HPLC.

[0049] [LC conditions] LC: HPLC Separation mode: HILIC Column: Amide column; TSKgel Amide-80 5 μm (registered trademark) (Tosoh Corporation) (Particle size; 5 μm Column size; Inner diameter 2.0 mm × Length 25 cm) Column temperature: 80 °C Mobile phase: An aqueous solution of 60 vol% acetonitrile with a predetermined acid added to be 0.01 vol% to 1 vol%, 0.01 vol% - 1 vol% acid - 60 vol% acetonitrile aqueous solution Flow rate: 0.2 mL / min Sample (1) pretreatment: Not performed Sample (1) injection volume: 1 μL

[0050] [MS conditions] Ionization: ESI (parallel measurement possible with equipment capable of negative and positive parallel measurement) Mass separation: Quadrupole type Nebulizer gas flow rate: 1.5 L / min Drying gas flow rate: 15 L / min Interface temperature: 350 °C Desolvation Line (DL) temperature: 250 °C Heat block temperature: 200 °C

[0051] The evaluation of the detection sensitivity was carried out in the chromatogram related to the agarooligosaccharide (m / z of its ion-added molecule or deprotonated molecule) obtained by HPLC / MS (High Performance Liquid Chromatography Mass Spectrometry). When it was detected as a symmetric normal peak or a symmetric peak equivalent thereto that could be distinguished from the baseline and interfering peaks, etc., it was designated as "++". Also, when it was detected as a peak that was not symmetric but could be recognized separately from the baseline and interfering peaks, etc., it was designated as "+". Further, when it was not detected as a peak that could be recognized separately from the baseline and interfering peaks, etc., it was designated as "-". The evaluation was performed for each sugar of the agarooligosaccharide. As a comprehensive evaluation, when all of the 2-8 sugars were "++", it was designated as "++"; when even one was "+", it was designated as "+"; and when even one was "-", it was designated as "-". A comprehensive evaluation of "+" or higher means that each sugar of the agarooligosaccharide was detected at a level where it could be qualitatively determined, and it can be evaluated that the object of the present invention was achieved. And a comprehensive evaluation of "++" means that it was detected with particularly high sensitivity.

[0052] [Example 1] In Example 1, as an example of the acid added to the mobile phase, formic acid (FUJIFILM Wako Pure Chemical Corporation. The same shall apply hereinafter) was selected. That is, the mobile phase was a 0.01 vol% or 0.1 vol% formic acid - 60 vol% acetonitrile aqueous solution. The results are shown in Table 1. In the table, the formic acid - added ion represents a formic acid - ion - added molecule, the proton - desorbed ion represents a de - protonated molecule, the acetic acid - added ion represents an acetic acid - ion - added molecule, the ammonium - added ion (NH4 - added ion) represents an ammonium - ion - added molecule, and the sodium - added ion (Na - added ion) represents a sodium - ion - added molecule (the same shall apply hereinafter).

[0053] [Table 1]

[0054] As shown in Table 1, at a formic acid addition amount of 0.01 vol%, the sensitivity of the octasaccharide with acetic acid - ion - added molecules was relatively low, but agarooligosaccharides could be detected. Furthermore, for formic acid - ion - added molecules, de - protonated molecules, ammonium - ion - added molecules, and sodium - ion - added molecules, all of the disaccharides to octasaccharides could be detected with particularly high sensitivity. At a formic acid addition amount of 0.1 vol%, for all five types of ion molecules tested, agarooligosaccharides could be detected with particularly high sensitivity for all of the disaccharides to octasaccharides. From Example 1, it was shown that by adding at least 0.01 vol% of formic acid to the mobile phase, or by adding formic acid to the mobile phase in the range of at least 0.01 vol% to 0.1 vol%, agarooligosaccharides could be detected with five types of ion molecules: formic acid - ion - added molecules, de - protonated molecules, acetic acid - ion - added molecules, ammonium - ion - added molecules, and sodium - ion - added molecules.

[0055] [Example 2] In Example 2, as an example of the acid added to the mobile phase, acetic acid (FUJIFILM Wako Pure Chemical Corporation. The same shall apply hereinafter) was selected. That is, the mobile phase was a 0.1 vol% or 1 vol% acetic acid - 60 vol% acetonitrile aqueous solution. The results are shown in Table 2.

[0056] [Table 2]

[0057] As shown in Table 2, in both cases where the acetic acid addition amount was 0.1 vol% and 1 vol%, no distinguishable peaks of 2-8 sugars could be detected in the formate ion-added molecules. On the other hand, in the deprotonated molecules, acetate ion-added molecules, ammonium ion-added molecules, and sodium ion-added molecules, agarooligosaccharides could be detected with particularly high sensitivity for all of the 2-8 sugars. From Example 2, by adding at least 0.1 vol% of acetic acid to the mobile phase, or by adding acetic acid to the mobile phase in the range of at least 0.1 vol% to 1 vol%, it was shown that agarooligosaccharides could be detected with particularly high sensitivity in four types of ionic molecules, namely, deprotonated molecules, acetate ion-added molecules, ammonium ion-added molecules, and sodium ion-added molecules.

[0058] [Example 3] In Example 3, as an example of the acid added to the mobile phase, trifluoroacetic acid (TFA) (FUJIFILM Wako Pure Chemical Corporation. The same shall apply hereinafter) was selected. That is, the mobile phase was a 0.01 vol% TFA - 60 vol% acetonitrile aqueous solution. The results are shown in Table 3. Note that the trifluoroacetic acid-added ions (TFA-added ions) in the table represent trifluoroacetic acid ion-added molecules (the same shall apply hereinafter).

[0059]

Table 3

[0060] As shown in Table 3, at a trifluoroacetic acid addition amount of 0.01 vol%, distinguishable peaks of 2-8 sugars could not be detected in the deprotonated molecules and acetic acid ion-added molecules, and peaks of 4-8 sugars could not be detected in the formic acid ion-added molecules. On the other hand, for the three types of ions, namely ammonium ion-added molecules, sodium ion-added molecules, and trifluoroacetic acid ion-added molecules, agarooligosaccharides could be detected with particularly high sensitivity for all of 2-8 sugars. From Example 3, it was shown that by adding at least 0.01 vol% of trifluoroacetic acid to the mobile phase, agarooligosaccharides could be detected with particularly high sensitivity for the three types of ion molecules, namely ammonium ion-added molecules, sodium ion-added molecules, and trifluoroacetic acid ion-added molecules.

[0061] [Test 2] Sample (2) (AOS) consisting of agarooligosaccharides, sample (3) (AOS+blank) consisting of agarooligosaccharide-containing material including agarooligosaccharides and dextrins (sugars other than agarooligosaccharides) and other impurities, and a control (blank) consisting of impurities were prepared. Sample (2) was prepared with distilled water and acetonitrile (final concentration of acetonitrile was 60 vol%) in the same manner as in Test 1 so that the final concentration of agarooligosaccharides was 400 ppm (0.4 mg / mL). Sample (3) contained agarooligosaccharides, black tea extract (Sato Foods Co., Ltd., the same below) and dextrin (Sanei Sugar Co., Ltd., the same below) in a mass ratio of 10 / 40 / 50, and was prepared with distilled water and acetonitrile (final concentration of acetonitrile was 60 vol%) so that the final concentration of agarooligosaccharides was 400 ppm (0.4 mg / mL). The control (blank) was prepared with distilled water and acetonitrile (final concentration of acetonitrile: 60%) so that the final concentration of dextrin was the same as that of sample (3) and contained black tea extract and dextrin in a mass ratio of 40 / 50. These samples (2), (3), and the control were analyzed under the same conditions as those of Test 1 (Examples 4 to 6) (no enzyme treatment or other pretreatment was performed on any of the samples). However, in order to further improve the reliability of the present invention and to improve the objectivity of the test results, the deprotonated molecules in Example 4 were selected, and samples (2), (3), and the control related to their detection were analyzed by an external analysis agency under conditions similar to those of Test 1. Then, the detection sensitivity was evaluated based on the following evaluation criteria of this test for the chromatograms created by the agency, in the same way as for other ion-added molecules.

[0062] The detection sensitivity of this test was evaluated from the viewpoints of both detection ability, which was evaluated by evaluating the shape of the peaks based on the chromatograms obtained by HPLC / MS (high performance liquid chromatography mass spectrometry) using the same criteria as in Test 1, and resolution ability, which was evaluated by comparing the peaks of sample (3) consisting of agarooligosaccharides and agarooligosaccharide-containing substances containing impurity components with the peaks of sample (2) consisting of agarooligosaccharides and the control consisting of impurity components.

[0063] The evaluation of "++" is as follows: From the perspective of separation ability, as shown in Fig. 2A (the peak of the ammonium ion-added molecule of disaccharide at a formic acid addition amount of 0.1 vol%), the peak of sample (2) (AOS) and the peak of the control (blank) are clearly separated, and one peak of sample (3) (AOS + blank) overlaps with the peak of sample (2) (AOS) with almost no deviation, and it can be clearly determined that the peak is an agarooligosaccharide (ion-added molecule or deprotonated molecule). From the perspective of detectability, when each peak used for comparison between sample (2) and sample (3) is a normal symmetric peak that can be distinguished from the baseline and interfering peaks, etc., or a symmetric peak similar thereto, it is rated as "++".

[0064] The evaluation of "+" is as follows: From the perspective of separation ability, as shown in Fig. 2B (the peak of the ammonium ion-added molecule of octasaccharide at a formic acid addition amount of 0.01 vol%), although one peak of sample (3) (AOS + blank) is slightly deviated from the peak of sample (2) (AOS), it is rated as "+" when it can be determined that both peaks are the same substance (agarooligosaccharide). Alternatively, from the perspective of detectability, when any peak used for comparison between sample (2) and sample (3) is a peak that is not symmetric but can be recognized separately from the baseline and interfering peaks, etc., it is also rated as "+". In the example of Fig. 2B, although there is some overlap between the peak of sample (2) (AOS) and the peak of the control (blank), both peaks are clearly separated. Furthermore, each peak of sample (3) (AOS + blank) is slightly deviated from the peaks of sample (2) (AOS) and the control (blank) respectively but overlaps, and shows the same trend, so it can be determined that one peak of sample (3) (AOS + blank) and the peak of sample (2) (AOS) are the same substance (agarooligosaccharide). Note that the peak shape in Fig. 2B itself is symmetric or has a symmetry similar thereto.

[0065] The evaluation of "-" was determined as "-" when, from the perspective of separation ability, as shown in Fig. 2C (the peak of the acetic acid ion-added molecule with disaccharide at a formic acid addition amount of 0.01 vol%), it was impossible to discriminate that one peak of the sample (3) (AOS + blank) was agarooligosaccharide. Or, from the perspective of detectability, when any peak for comparison between the sample (2) and the sample (3) was not a peak that could be recognized separately from the baseline and interfering peaks, etc., it was also determined as "-". In the example of Fig. 2C, one peak of the sample (3) (AOS + blank) almost overlapped without deviation with the peaks of both the sample (2) (AOS) and the control (blank), and it was impossible to discriminate whether the peak of the sample (3) was the same substance as the peaks of either the sample (2) or the control, that is, whether it was agarooligosaccharide or an interfering component. Note that the peak shape itself in Fig. 2C is symmetric or has symmetry similar thereto.

[0066] A comprehensive evaluation of "+" or higher means that each sugar of agarooligosaccharide was detected at a level where it could be qualitatively determined, and it can be evaluated that the object of the present invention was achieved. And a comprehensive evaluation of "++" means that it was detected with particularly high sensitivity.

[0067] [Example 4] In Example 4, formic acid was selected as an example of the acid added to the mobile phase. That is, the mobile phase was a 0.01 vol% or 0.1 vol% formic acid - 60 vol% acetonitrile aqueous solution. The results are shown in Table 4.

[0068]

Table 4

[0069] As shown in Table 4, at a formic acid addition amount of 0.01 vol%, disaccharides and octasaccharides could not be detected discriminably in acetate ion-added molecules. On the other hand, in formate ion-added molecules, ammonium ion-added molecules, and sodium ion-added molecules, the sensitivity of octasaccharides was relatively low, but agarooligosaccharides could be detected. Furthermore, in deprotonated molecules, all of the disaccharide to octasaccharide could be detected with particularly high sensitivity. At a formic acid addition amount of 0.1 vol%, acetate ion-added molecules, formate ion-added molecules, sodium ion-added molecules, and deprotonated molecules showed the same detection sensitivity as that at 0.01 vol%. In addition, in ammonium ion-added molecules, the detection sensitivity was further improved, and agarooligosaccharides could be detected with particularly high sensitivity for all of the disaccharide to octasaccharide. From Example 4, by adding at least 0.01 vol% of formic acid to the mobile phase, or by adding at least 0.01 vol% to 0.1 vol% of formic acid to the mobile phase, it was shown that agarooligosaccharides could be detected with high sensitivity by four types of ionic molecules, namely, formate ion-added molecules, deprotonated molecules, ammonium ion-added molecules, and sodium ion-added molecules, for samples containing interfering components such as dextrin, which are particularly difficult to separate as saccharides other than agarooligosaccharides.

[0070] [Example 5] In Example 5, acetic acid was selected as an example of the acid added to the mobile phase. That is, the mobile phase was a 0.1 vol% or 1 vol% acetic acid - 60 vol% acetonitrile aqueous solution. The results are shown in Table 5.

[0071]

Table 5

[0072] As shown in Table 5, for formate ion-added molecules, no distinguishable peaks of 2-8 sugars could be detected at either 0.1 vol% or 1 vol% of acetic acid addition. For acetate ion-added molecules and sodium ion-added molecules, the sensitivity for 8 sugars was relatively low for both molecules at 0.1 vol% of acetic acid addition, and for acetate ion-added molecules, the sensitivity for 2-6 sugars also became relatively low at 1 vol% of acetic acid addition. However, agarooligosaccharides could be detected for both acetate ion-added molecules and sodium ion-added molecules at any addition amount. For deprotonated molecules and ammonium ion-added molecules, agarooligosaccharides could be detected with particularly high sensitivity for all of 2-8 sugars at both 0.1 vol% and 1 vol% of acetic acid addition. From Example 5, by adding at least 0.1 vol% of acetic acid to the mobile phase, or by adding at least 0.1 vol% - 1 vol% of acetic acid to the mobile phase, it was shown that agarooligosaccharides could be detected with high sensitivity using four types of ionic molecules, namely deprotonated molecules, acetate ion-added molecules, ammonium ion-added molecules, and sodium ion-added molecules, for samples containing interfering components such as dextrin, which are particularly difficult to separate as carbohydrates other than agarooligosaccharides.

[0073] [Example 6] In Example 6, trifluoroacetic acid (TFA) was selected as an example of the acid added to the mobile phase. That is, the mobile phase was a 0.01 vol% TFA - 60 vol% acetonitrile aqueous solution. The results are shown in Table 6.

[0074] [Table 6]

[0075] As shown in Table 6, at a trifluoroacetic acid addition amount of 0.01 vol%, distinguishable peaks of 2 - 8 sugars could not be detected for deprotonated molecules and acetic acid ion - added molecules, and peaks of 4 - 8 sugars could not be detected for formic acid ion - added molecules. Also, for trifluoroacetic acid ion - added molecules, octasaccharides could not be detected distinguishably. On the other hand, for ammonium ion - added molecules, the sensitivity for octasaccharides was relatively low, but agarooligosaccharides could be detected. Also, for sodium ion - added molecules, the sensitivity for 2 - 8 sugars was relatively low, but agarooligosaccharides could be detected. From Example 6, by adding at least 0.01 vol% of trifluoroacetic acid to the mobile phase, it was shown that agarooligosaccharides can be detected with high sensitivity by two types of ion molecules, ammonium ion - added molecules and sodium ion - added molecules, for samples containing contaminants such as dextrin, which are particularly difficult to separate as saccharides other than agarooligosaccharides.

[0076] [Test 3] A sample (4) consisting of an agarooligosaccharide - containing substance containing agarooligosaccharides and dextrin, which is a saccharide other than agarooligosaccharides, was prepared. Sample (4) contained agarooligosaccharides and dextrin at a mass ratio of 10 / 50, and was prepared with distilled water and acetonitrile (the final concentration of acetonitrile was 60 vol%) so that the agarooligosaccharides had a final concentration of 400 ppm (0.4 mg / mL) after undergoing the following enzymatic treatment. Sample (4) was analyzed under the following conditions by LC - MS (HPLC - MS) connected with HPLC (high - performance liquid chromatograph) and MS (mass spectrometer) (Example 7, Comparative Example 1).

[0077] In this test, the sample (4) that had been previously subjected to the following enzymatic treatment was subjected to (injected into) LC. That is, to 50 mL of an ammonium acetate buffer (pH 4.5) containing agarooligosaccharide and dextrin at a mass ratio of 10 / 50, 23 mg of glucoamylase (Glucoamylase from Rhizopus (contains 50% Diatomaceous earth), Tokyo Chemical Industry Co., Ltd.), 0.25 mL of invertase (invertase solution, yeast-derived, Fujifilm Wako Pure Chemical Corporation), and 0.1 mL of amyloglucosidase (amyloglucosidase solution from Aspergillus niger, Sigma-Aldrich Japan) were added, and after reacting at 37 °C for 60 minutes, the reaction solution was boiled for 10 minutes to inactivate the enzyme. After making up the volume to 100 mL with distilled water, acetonitrile was added so that the final concentration became 60 vol%, and the sample (4) was obtained.

[0078] <LC Conditions> LC: HPLC Separation Mode: HILIC Column: Amide Column; TSKgel Amide-80 5μm (registered trademark) (Tosoh Corporation) (Particle size; 5μm Column size; Inner diameter 2.0 mm × Length 25 cm) Column Temperature: 80 °C Mobile Phase: Example 7; 0.1 vol% formic acid - 60 vol% acetonitrile aqueous solution prepared by adding formic acid to 60 vol% acetonitrile aqueous solution so that it becomes 0.1 vol% Comparative Example 1; 60 vol% acetonitrile aqueous solution Flow Rate: 0.2 mL / min Sample (4) Pretreatment: Performed Sample (4) Injection Volume: 1 μL

[0079] Ionization: ESI (using an instrument capable of parallel measurement of negative and positive) Mass Separation: Quadrupole type Nebulizer Gas Flow Rate: 1.5 L / min Drying Gas Flow Rate: 15 L / min Interface temperature: 350 °C Temperature of the desolvation line (DL): 250 °C Heat block temperature: 200 °C

[0080] Based on the chromatograms obtained by HPLC / MS (high performance liquid chromatography-mass spectrometry) under the above conditions, the detection sensitivities of the deprotonated molecules ([M-H] - ) of each sugar of the agarooligosaccharides were compared between Example 7 and Comparative Example 1. The results of Example 7 are shown in Fig. 3A. The results of Comparative Example 1 are shown in Fig. 3B. The horizontal axis of the chromatogram represents the retention time, and the vertical axis represents the ion intensity which is the intensity of the signal value. For easy visual recognition or comparison, the horizontal axes of 2-8 sugars were aligned (the same hereinafter).

[0081] As shown in Fig. 3A, in Example 7 where formic acid was added to the mobile phase, symmetric normal peaks or symmetric peaks similar thereto that could be distinguished from the baseline and interfering peaks were detected at the m / z of the deprotonated molecules for all of the 2-8 sugars. On the other hand, as shown in Fig. 3B, in Comparative Example 1 where formic acid was not added to the mobile phase, for the 4-sugar, 6-sugar, and 8-sugar, slightly detectable peaks that could be distinguished from the baseline were detected, but for the 2-sugar, no peaks that could be recognized as distinguishable from the baseline and interfering peaks were detected. Thus, when comparing Example 7 and Comparative Example 1, by adding formic acid to the mobile phase, the detection sensitivity of the agarooligosaccharides was clearly improved. As a result, in Example 7, by adding formic acid to the mobile phase, it was possible to highly sensitively detect an agarooligosaccharide-containing substance containing dextrin as an interfering component, and further an agarooligosaccharide solution containing it at a low concentration of 400 ppm (0.4 mg / mL) with only a trace amount of 1 μL.

[0082] [Test 4] A sample (5) consisting of an agarooligosaccharide-containing substance containing agarooligosaccharides and dextrin and other contaminating components that are carbohydrates other than agarooligosaccharides was prepared. Sample (5) contains agarooligosaccharides, black tea extract, and dextrin in a mass ratio of 10 / 40 / 50, and was prepared with distilled water and acetonitrile (the final concentration of acetonitrile is 60 vol%) so that the agarooligosaccharides are in a state after enzymatic treatment and the final concentration is 400 ppm (0.4 mg / mL). Sample (5) was analyzed under the following conditions by LC-MS (HPLC-MS) connected with HPLC (high performance liquid chromatography) and MS (mass spectrometer) (Example 8, Reference Example 1). For sample (5), as in Test 3, the one that had been enzymatically treated in advance was subjected to LC (injected).

[0083] <LC Conditions> LC: HPLC Separation Mode: HILIC Column: Example 8; Amide Column; TSKgel Amide-80 5μm (registered trademark) (Tosoh Corporation) (Particle Size; 5μm Column Size; Inner Diameter 2.0 mm × Length 25 cm) Reference Example 1; Amino Column; Asahipak NH2P-40 2E (registered trademark) (Resonac Corporation) (Particle Size; 5μm Column Size; Inner Diameter 2.0 mm × Length 25 cm) Column Temperature: Example 8; 80°C Reference Example 1; 45°C Mobile Phase: 0.1 vol% formic acid - 60 vol% acetonitrile aqueous solution prepared by adding formic acid to 60 vol% acetonitrile aqueous solution so that it becomes 0.1 vol% Flow Rate: 0.2 mL / min Sample (5) Pretreatment: Performed Sample (5) Injection Volume: 1 μL

[0084] Ionization: ESI (using an instrument capable of parallel measurement of negative and positive) Mass Separation: Quadrupole type Nebulizer Gas Flow Rate: 1.5 L / min Driving gas flow rate: 15 L / min Interface temperature: 350 °C Desolvation Line (DL) temperature: 250 °C Heat block temperature: 200 °C

[0085] In this test, as described above, the column type and column temperature were set differently between Example 8 and Reference Example 1. However, for the column temperature, the optimum temperature for each column was applied, and no conditions unfavorable to each example were set. Based on the chromatograms obtained by HPLC / MS (High Performance Liquid Chromatography-Mass Spectrometry) under the above conditions, the detection sensitivities of the formic acid ion-added molecules ([M+HCOO] - ) of each sugar of agarooligosaccharide were compared between Example 8 and Reference Example 1. The results of Example 8 are shown in Fig. 4A. The results of Reference Example 1 are shown in Fig. 4B.

[0086] As shown in Fig. 4A, in Example 8 using an amide column as the stationary phase, for the m / z of the formic acid ion-added molecules of all 2-8 sugars, symmetric normal peaks or symmetric peaks similar thereto that could be distinguished from the baseline and interfering peaks were detected. On the other hand, as shown in Fig. 4B, in Reference Example 1 using an amino column as the stationary phase, for 2-6 sugars, peaks that could be distinguished from the baseline and interfering peaks were detected, but their intensities were significantly weak. Also, for 8 sugars, no peaks that could be recognized as distinguishable from the baseline and interfering peaks were detected. Thus, when comparing Example 8 and Reference Example 1, the detection sensitivity of agarooligosaccharide was improved by using an amide column as the stationary phase.

[0087] [Test 5] Similar to Test 4, a sample (6) was prepared with distilled water and acetonitrile (final concentration of acetonitrile: 60 vol%) to contain agarooligosaccharide, black tea extract, and dextrin at a mass ratio of 10 / 40 / 50, such that the final concentration of agarooligosaccharide was 400 ppm (0.4 mg / mL). The sample (6) was analyzed under the following conditions by LC-MS (HPLC-MS) connected with HPLC (high performance liquid chromatography) and MS (mass spectrometer) (Example 9, Reference Example 2).

[0088] For the sample (6), in Example 9, similar to Test 3, the sample that had been pre-treated with an enzyme in advance was subjected to (injected into) LC. The final concentration of agarooligosaccharide was adjusted to 400 ppm (0.4 mg / mL) after the enzyme treatment. In Reference Example 2, the prepared sample (6) was directly subjected to (injected into) LC without performing enzyme treatment or other pre-treatments.

[0089] <LC Conditions> LC: HPLC Separation Mode: HILIC Column: Amide Column; TSKgel Amide-80 5μm (registered trademark) (Tosoh Corporation) (Particle Size; 5μm Column Size; Inner Diameter 2.0 mm × Length 25 cm) Column Temperature: 80°C Mobile Phase: 0.1 vol% formic acid 60 vol% acetonitrile aqueous solution with 0.1 vol% formic acid added to 60 vol% acetonitrile aqueous solution Flow Rate: 0.2 mL / min Sample (6) Pretreatment: Example 9; Performed Reference Example 2; Not Performed Sample (6) Injection Volume: 1 μL

[0090] Ionization: ESI (using an instrument capable of parallel measurement of negative and positive) Mass Separation: Quadrupole type Nebulizer Gas Flow Rate: 1.5 L / min Drying Gas Flow Rate: 15 L / min Interface temperature: 350 °C Temperature of the desolvation line (DL): 250 °C Heat block temperature: 200 °C

[0091] Based on the chromatogram obtained by HPLC / MS (high performance liquid chromatography mass spectrometry) under the above conditions, the detection sensitivity of each sugar of agarooligosaccharide as the formic acid ion-added molecule ([M+HCOO] - ) was compared between Example 9 and Reference Example 2. The results are shown in Fig. 5.

[0092] As shown in Fig. 5, in Example 9 where the sample (6) was subjected to enzymatic treatment, for the m / z of the formic acid ion-added molecule of all 2-8 sugars, a symmetric normal peak distinguishable from the baseline and interfering peaks or a symmetric peak similar thereto was detected. On the other hand, in Reference Example 2 where enzymatic treatment was not performed, for all 2-8 sugars, a symmetric normal peak distinguishable from the baseline and interfering peaks or a symmetric peak similar thereto was detected, but the intensity of any peak was slightly weaker compared to Example 9, and interfering peaks were also detected for the 8-sugar. Thus, in Example 9, by subjecting the agarooligosaccharide-containing material containing dextrin and other interfering components to enzymatic treatment, the intensity of the peak related to the agarooligosaccharide molecule was improved, and the appearance of interfering peaks was further suppressed. As a result, a higher-sensitivity and highly reliable high-quality detection result could be obtained. [Test 6] Similar to Test 4, a sample (7) was prepared with distilled water and acetonitrile (the final concentration of acetonitrile was 60 vol%) so as to contain agarooligosaccharide, black tea extract, and dextrin at a mass ratio of 10 / 40 / 50 and the final concentration of agarooligosaccharide was 100 ppm (0.1 mg / mL). The sample (7) was analyzed under the following conditions by LC-MS (HPLC-MS) connected with HPLC (high performance liquid chromatograph) and MS (mass spectrometer) (Example 10). For the sample (7), no enzymatic treatment or other pretreatment was performed, and the prepared sample (7) was directly subjected to LC (injected).

[0093] <LC conditions> LC: HPLC Separation mode: HILIC Column: Amide column; TSKgel Amide-80 5μm (registered trademark) (Tosoh Corporation) (Particle size; 5μm Column size; Inner diameter 2.0 mm × Length 25 cm) Column temperature: 80 °C Mobile phase: 0.1 vol% formic acid 60 vol% acetonitrile aqueous solution prepared by adding formic acid to 60 vol% acetonitrile aqueous solution to a concentration of 0.1 vol% Flow rate: 0.2 mL / min Sample (7) pretreatment: Not performed Sample (7) injection volume: 1 μL

[0094] Ionization: ESI (Using an instrument capable of parallel measurement in negative and positive modes) Mass separation: Quadrupole type Nebulizer gas flow rate: 1.5 L / min Drying gas flow rate: 15 L / min Interface temperature: 350 °C Desolvation line (DL) temperature: 250 °C Heat block temperature: 200 °C

[0095] As shown in Fig. 6, in Example 10, for all of the 2-8 saccharides, at the m / z of the formate ion-added molecules, symmetrical normal peaks or symmetrical peaks similar thereto that could be distinguished from the baseline and interfering peaks were detected. Since the amount of the tested agarooligosaccharide was extremely small, some noise appeared for the octasaccharide, but as the peak shape, a form that could be distinguished from the baseline and interfering peaks as a peak similar to the normal peak was maintained. Thus, in Example 10, by adding formic acid to the mobile phase, even when a trace amount of 1 μL of an agarooligosaccharide solution containing dextrin and other interfering components and further containing agarooligosaccharide at an extremely low concentration of 100 ppm (0.1 mg / mL) was used, the agarooligosaccharide could be detected with high sensitivity.

Explanation of Reference Signs

[0096] 01 LC-MS (Liquid Chromatograph-Mass Spectrometer) 02 LC (Liquid Chromatograph) 03 MS (Mass Spectrometer) 04 Mobile Phase Container 05 Degassing Device 06 Liquid Delivery Pump 07 Sample Introduction Section 08 Column 09 Column Thermostat 10 Control Section 11 Ionization Section 12 Mass Separation Section 13 Detection Section 14 Control Section

Claims

1. A method for analyzing agarooligosaccharides, comprising separating agarooligosaccharides from an agarooligosaccharide-containing substance by hydrophilic interaction mode of liquid chromatography and detecting them by mass spectrometry. Further, an acid is added to the mobile phase in the liquid chromatography. The method for analyzing agarooligosaccharides is characterized by the above.

2. In the hydrophilic interaction mode, an amide column is used. The method for analyzing agarooligosaccharides according to Claim 1, characterized by the above.

3. A volatile acid is added as the acid. The method for analyzing agarooligosaccharides according to Claim 1 or Claim 2, characterized by the above.

4. One or more acids selected from formic acid, acetic acid, trifluoroacetic acid, and their derivatives are added as the acid. The method for analyzing agarooligosaccharides according to Claim 3, characterized by the above.

5. The acid is added to an aqueous solution of an organic solvent at 50 vol% or more as the mobile phase. The method for analyzing agarooligosaccharides according to Claim 1 or Claim 2, characterized by the above.

6. Before performing the liquid chromatography on the agarooligosaccharide-containing substance, the agarooligosaccharide-containing substance is pretreated with an enzyme in advance. The method for analyzing agarooligosaccharides according to Claim 1 or Claim 2, characterized by the above.

7. The agarooligosaccharide-containing substance contains saccharides other than agarooligosaccharides. The method for analyzing agarooligosaccharides according to Claim 1 or Claim 2, characterized by the above.

8. The saccharide other than the agarooligosaccharide is dextrin. The method for analyzing agarooligosaccharides according to Claim 7, characterized by the above.

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