JP7496657B1 - Analytical Method for Agro-oligosaccharides - Google Patents

Abstract

[Problem] To provide a reliable analytical method capable of separating and detecting agarooligosaccharides easily and sensitively without labeling. [Solution] 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, and further characterized in that an acid is added to the mobile phase in the liquid chromatography to make the mobile phase acidic, and the agarooligosaccharide-containing substance contains carbohydrates other than agarooligosaccharides and their constituent sugars. [Selected Figure] Figure 3

Description

The present invention relates to a method for analyzing agarooligosaccharides.

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, with a β (1,4) bond between the 1st position of D-galactose and the 4th position of 3,6-anhydro-L-galactose, and an α (1,3) bond between the 1st position of 3,6-anhydro-L-galactose and the 3rd position of D-galactose.

Agarose is hydrolyzed at the α(1,3) bond by the action of an acid or an enzyme (α-agarase) to produce agarooligosaccharides such as agarobiose (disaccharide), agarotetraose (tetrasaccharide), agarohexaose (hexasaccharide), and agarooctaose (octasaccharide), which are oligosaccharides with 3,6-anhydro-L-galactose at the reducing end. These agarooligosaccharides have been reported to have various physiological activities such as prebiotic effects, apoptosis-inducing activity, anticancer activity, activity to inhibit active oxygen production, and immunomodulatory activity (Patent Document 1: Japanese Patent No. 4007760), and are therefore highly applicable industrially as functional ingredients in foods and medicinal ingredients in medicines.

In order to blend such agarooligosaccharides into products or formulations and ensure their quality, it is necessary to standardize a reliable analytical method for agarooligosaccharides. In particular, there is a demand for an analytical method that can separate and detect agarooligosaccharides contained in low concentrations in foods, medicines, etc. that contain contaminating ingredients such as other carbohydrates that have structures or properties similar to the target ingredient, agarooligosaccharide.

Patent No. 4007760

Patent Document 1 describes how an agar decomposition product (solution) was separated by liquid chromatography (LC) to collect a substance with a specific peak, which was then analyzed by mass spectrometry (MS) using fast atom bombardment (FAB) as the ionization method (FAB/MS) and nuclear magnetic resonance (NMR) to identify the collected substance as agarobiose (Examples 2 and 3). However, in this document, only agar, which is the source of agaro-oligosaccharides, is used without containing impurities, and a relatively high concentration agar solution, calculated to be about 10 mg/mL (Example 2) or 50 mg/mL (Example 3), is subjected to separation in a not-so-small amount of 2 mL, which is a decomposition product, and the separated material is then subjected to a separate qualitative analysis under these relatively mild conditions. Therefore, from these conventional documents, for example, it is not clear what analytical method and conditions are available for separating agaro-oligosaccharides contained in low concentrations from foods, medicines, etc. that contain impurities such as carbohydrates other than agaro-oligosaccharides, and detecting each sugar, and it is desirable to establish a highly sensitive and reliable analytical method for agaro-oligosaccharides.

On the other hand, in the past, in sugar analysis, sugar chains were sometimes labeled with fluorescent labels or the like to improve the sensitivity. However, even if the sensitivity is improved by labeling, such labeling is troublesome, and the analytical process tends to become complicated and the analytical equipment tends to become expensive.

The present invention has been made in consideration of the above circumstances, and aims to provide a reliable analytical method that can separate and detect agaro-oligosaccharides easily and with high sensitivity without labeling.

The present invention solves the above problems by the solution described below as one embodiment.

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, and further characterized in that an acid is added to the mobile phase in the liquid chromatography to make the mobile phase acidic, and the agarooligosaccharide-containing substance contains carbohydrates other than agarooligosaccharides and their constituent sugars.

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

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

The mobile phase can be an aqueous solution of 50 vol% or more of an organic solvent to which the acid has been added.

In addition, the agaro-oligosaccharide-containing material can be pre-treated with an enzyme before performing the liquid chromatography on the agaro-oligosaccharide-containing material.

The present invention can be directed to an agarooligosaccharide-containing product that specifically contains one or more types of monosaccharides, oligosaccharides, polysaccharides, and sugar alcohols other than the agarooligosaccharides and their constituent sugars.

The present invention provides a reliable method for analyzing agarooligosaccharides, which can be easily and sensitively separated and detected without labeling.

FIG. 1 is an explanatory diagram showing an example of an LC-MS (liquid chromatograph-mass spectrometer) according to an embodiment of the present invention. FIG. 2 is a diagram (chromatogram) explaining the evaluation criteria of Test 2. Fig. 3A is a diagram (chromatogram) showing the results of Example 7 of Test 3. Fig. 3B is a diagram (chromatogram) showing the results of Comparative Example 1 of Test 3. Fig. 4A is a diagram (chromatogram) showing the results of Example 8 of Test 4. Fig. 4B is a diagram (chromatogram) showing the results of Reference Example 1 of Test 4. FIG. 5 is a diagram (chromatogram) showing the results of Example 9 and Reference Example 2 in Test 5. FIG. 6 is a diagram (chromatogram) showing the results of Example 10 of Test 6. 7A is a diagram (chromatogram) showing the results of the control in Example 11 of Test 7. FIG. 7B is a diagram (chromatogram) showing the results of Sample 8 in Example 11 of Test 7. FIG. 8 is a diagram (chromatogram) showing the results of Sample 9 in Example 11 of Test 7. 9A is a diagram (chromatogram) showing the results of the control in Example 12 of Test 8. FIG. 9B is a diagram (chromatogram) showing the results of Sample 10 in Example 12 of Test 8. FIG. 10 is a chromatogram showing the results of Sample 11 in Example 12 of Test 8. Fig. 11A is a diagram (chromatogram) showing the results of the control in Example 13 of Test 9. Fig. 11B is a diagram (chromatogram) showing the results of Sample 12 in Example 13 of Test 9. FIG. 12 is a chromatogram showing the results of Sample 13 in Example 13 of Test 9.

Agaro-oligosaccharides (AOS) in this application consist of the disaccharide agarobiose (Abi), the tetrasaccharide agarotetraose (Ate), the hexasaccharide agarohexaose (Ahe), and the octasaccharide agarooctaose (Aoc), as well as modified versions of these. Abi, Ate, Ahe, and Aoc are produced by hydrolysis of agarose, and in practice, are produced by hydrolyzing agarose-containing substances such as agar with 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, a carboxy group or the like (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 a substance containing agaropectin, such as agar, with an acid or an enzyme (α-agarase). However, in terms of social implementation, it is not required to separate and detect Abi, Ate, Ahe and Aoc from these modified forms. In reality, when agaropectin is hydrolyzed with an acid or an enzyme (α-agarase), many of the above-mentioned substituents are eliminated, and those that are not eliminated are not decomposed sufficiently and remain as medium- or long-chains, so it is believed that the agaro-oligosaccharide-containing substance to be analyzed (sample) basically contains almost no modified forms. Therefore, the coexistence of Abi, Ate, Ahe, and Aoc with these modified forms is not 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 each separated and detected based on the difference in the degree of polymerization, each of these sugars may contain the respective modified forms.

The method for analyzing agarooligosaccharides of the present invention is a qualitative analysis method capable of separating, detecting and identifying 2-8 sugars of agarooligosaccharides. However, in the practice of the present invention, it is sufficient to qualitatively determine the desired sugar according to the purpose among the detectable agarooligosaccharide sugars. Therefore, for example, it is one embodiment of the present invention to qualitatively determine only the Abi of the disaccharide, or only the Abi of the disaccharide and the Ate of the tetrasaccharide, among the 2-8 sugars of agarooligosaccharides, as necessary. In addition, it is one embodiment of the present invention or an embodiment utilizing the present invention to obtain a quantitative analysis result by applying, for example, a known calibration curve based on the separation and detection of each sugar of agarooligosaccharide according to the present invention. Furthermore, it is one embodiment of the present invention or an embodiment utilizing the present invention to analyze (qualitatively and/or quantitatively) the 10 sugars together with any of the 2-8 sugars, when the present invention is carried out and can be separated and detected.

In this embodiment, agaro-oligosaccharides are separated from an agaro-oligosaccharide-containing material by liquid chromatography and detected by mass spectrometry. That is, this embodiment employs liquid chromatography mass spectrometry (LC/MS), which combines liquid chromatography (LC) and mass spectrometry (MS). As shown in FIG. 1, the LC/MS of this embodiment is basically an LC/MS (liquid chromatography mass spectrometry) that performs LC (liquid chromatography) and MS (mass spectrometry) continuously using an LC-MS (01) (liquid chromatography-mass spectrometer) that connects LC (02) (liquid chromatograph) and MS (03) (mass spectrometer). Therefore, unlike a method in which LC and MS are performed independently and separately, such as a method in which a substance of a specific peak separated by LC (liquid chromatography) using LC (02) (liquid chromatograph) is collected and then the collected substance is analyzed separately by MS (mass spectrometry) using MS (03) (mass spectrometer), the present invention includes constituent elements (processing or steps) that are unique to the method of analyzing agaro-oligosaccharides that applies LC/MS (liquid chromatography mass spectrometry) using LC-MS (01) (liquid chromatography-mass spectrometer).

According to this embodiment, agarooligosaccharides can be separated and detected by analyzing samples (various foods, medicines, quasi-drugs, cosmetics, etc.) containing agarooligosaccharides and carbohydrates other than agarooligosaccharides (monosaccharides, oligosaccharides, polysaccharides, other sugar alcohols). That is, according to this embodiment, agarooligosaccharides can be separated and detected by analyzing hydrolysates of agar containing agarose and agaropectin, but one of the advantageous features of this embodiment is that agarooligosaccharides can be separated and detected by analyzing various foods, medicines, quasi-drugs, cosmetics, etc. containing agarooligosaccharides and carbohydrates other than agarooligosaccharides. Here, although the content varies depending on the hydrolysis conditions and the subsequent purification degree, agarooligosaccharide-containing substances as hydrolysates of agar containing agarose and agaropectin may contain galactose and anhydrogalactose, which are the constituent sugars of these. In the example (Example 11) of this embodiment described later, glucose and agarooligosaccharides could be separated, so that according to this embodiment, galactose and the like, which are structurally very similar to glucose, can also be separated from agarooligosaccharides, but here, the advantageous feature of this embodiment described above can be expressed as being that the agarooligosaccharide-containing product can contain carbohydrates other than agarooligosaccharides and their constituent sugars. Therefore, in the present invention, as described above, "the agarooligosaccharide-containing product contains carbohydrates other than agarooligosaccharides and their constituent sugars." In the examples of this embodiment described below, agarooligosaccharides containing agarooligosaccharides and other sugars other than their constituent sugars, such as the polysaccharide dextrin, the monosaccharide glucose, the oligosaccharide sucrose, and the sugar alcohol sorbitol, which have been shown to be particularly difficult to separate in preliminary tests using LC/MS, were provided in a small amount of 1 μL of agarooligosaccharide solution containing agarooligosaccharides at a low concentration of 400 ppm (in the present invention, mg/L is defined as ppm. Therefore, 400 ppm is 0.4 mg/mL) or an extremely low concentration of 100 ppm (0.1 mg/mL), and the agarooligosaccharides were easily and sensitively separated without labeling, and each sugar (2-8 sugars) was detected.

Here, the liquid chromatography of the present invention includes not only the oldest medium-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 uses a high-pressure pump to deliver the mobile phase at a higher speed than medium-low pressure LC, and shortens the analysis time compared to medium-low pressure LC. HPLC is the most popular method at present, and LC (02) shown in Figure 1 shows an example of HPLC (High Performance Liquid Chromatograph), and HPLC is also used in the examples described below. UHPLC is a method developed recently that uses a high-pressure pump like HPLC, and further increases the flow rate of the mobile phase by finely dispersing the base material of the stationary phase to moderately increase the column pressure. In this way, improved methods such as HPLC and UHPLC are basically physical improvements to medium- and low-pressure LC (liquid chromatography), and do not essentially negate the effects based on the chemical characteristics (addition of acid, etc.) of the analysis method of the present invention, which will be described later. 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. Below, this embodiment will be explained using an example using LC-MS (01) (liquid chromatograph-mass spectrometer) shown in Figure 1.

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

The mobile phase container (04) is a container capable of holding liquid, such as a vial or bottle, and holds the mobile phase (eluent). The degassing device (05) is installed as desired, and may be, for example, a degassing unit that incorporates a resin membrane tube as a gas-liquid separation membrane into the flow path (A) and reduces the pressure on the outside of the tube, thereby utilizing the permeability of the membrane to expel small molecule gases from the mobile phase. Alternatively, it may be configured not to be incorporated into the flow path (A), for example by attaching an aspirator to the mobile phase container (04) and vibrating the mobile phase container (04) with an ultrasonic cleaner or by suctioning and degassing the mobile phase while stirring it.

The liquid delivery pump (06) delivers the mobile phase at a high pressure through the flow path (A) to a predetermined flow rate. The sample introduction section (07) introduces the sample to be analyzed, which has been appropriately pretreated by an autosampler or the like, into the mobile phase. The mobile phase to which the sample has been introduced passes through an optionally installed guard column and is introduced into the column (08) whose temperature has been adjusted to a predetermined temperature by the column thermostatic bath (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 in the stationary phase for different periods of time in the column (08) due to differences in affinity for the mobile phase and the stationary phase, and is eluted from the column (08) (packing material). This separates the agaro-oligosaccharides. It is also possible to prepare a solution in which the sample has been dissolved in the eluent in advance and introduce this into the column (08) via the sample introduction section (07) or the mobile phase container (04).

[LC: Sample and pretreatment]
Here, the agarooligosaccharide-containing substances that can be subjected to the analysis are, as described above, samples containing agarooligosaccharides and carbohydrates other than agarooligosaccharides (monosaccharides, oligosaccharides, polysaccharides, other sugar alcohols), and examples of such samples include various foods, medicines, quasi-drugs, cosmetics, etc. For example, foods are not limited in type or form, and examples of such foods include staple foods and side dishes such as rice, bread, noodles, and the like, confectioneries such as fresh confectioneries and frozen desserts, fermented foods such as yogurt, seasonings such as sauces and dressings, tablets such as supplements, jelly-like foods, foods for swallowing, beverages, powdered beverages, etc.

Before performing LC (liquid chromatography) on these agaro-oligosaccharide-containing materials, the agaro-oligosaccharide-containing materials may be subjected to a predetermined pretreatment. Pretreatment is particularly effective for foods that are likely to contain various impurities. As an example, the following crude extraction procedure can be performed on a crude sample (a sample that has not been treated in any way). That is, if the crude sample is a solid, it can be crushed in a mixer or the like, suspended in water, and the solids removed by centrifugation, filtration, or the like. Also, if the crude sample is a liquid, it can be diluted with water.

Next, the crude sample and/or the crude extract that has been subjected to the above-mentioned crude extraction operation can be subjected to the following crude purification operation. That is, for example, purification with an organic solvent can be performed by adding an aqueous solution of an organic solvent to the crude sample or crude extract, stirring with a mixer or the like, and then centrifuging to obtain a supernatant. Also, for example, purification with a solid-phase extraction column can be performed by selecting an appropriate column and performing conditioning, and then adding the crude sample or crude extract to elute and recover the target component, agaro-oligosaccharides, or by adsorbing the agaro-oligosaccharides and then eluting and recovering them.

Furthermore, the following enzyme treatment can be carried out on the crude sample/or the above-mentioned crude extract/or the crude product obtained by the above-mentioned crude purification procedure. This enzyme treatment is particularly effective for samples that are expected to contain carbohydrates other than agaro-oligosaccharides. That is, for example, a buffer such as ammonium acetate buffer (acetic acid/ammonium acetate buffer) (for example, adjusted to pH 4.5 within the buffer action range: pH 3.5 to 5.5) is added to the crude sample, crude extract, or crude product. Next, carbohydrate-degrading enzymes such as glucoamylase (α(1,4) glucoside bond hydrolase), invertase (sucrose hydrolase, also known as saccharase or β-D-fructofuranosidase), and amyloglucosidase (α(1,6) glucoside bond hydrolase, also known as amylo α-1,6-glucosidase or dextrin 6-α-D-glucosidase) are added, and the mixture is reacted at an optimal temperature (e.g., 37°C, within a range of, for example, 35°C to 40°C) for an optimal time (e.g., 60 minutes, within a range of, for example, 10 minutes to 120 minutes). A series of enzyme treatments can then be carried out, such as by boiling (e.g., 10 minutes, within a range of, for example, 5 minutes to 30 minutes) to inactivate the enzymes.

Whether or not to use a buffer solution is selected depending on the sample and enzyme. If a buffer solution is used, a volatile buffer solution such as the ammonium acetate buffer solution exemplified above is preferable, but is not limited to this. The type of buffer solution may be selected depending on the sample and enzyme, and the buffer solution may be prepared to have an optimal pH, molar concentration, etc. The enzyme is not limited to the carbohydrate-degrading enzymes exemplified above, and an appropriate enzyme may be selected depending on the anticipated impurity components. Therefore, proteolytic enzymes and lipid-degrading enzymes may be used. In addition, carbohydrate-degrading enzymes other than those exemplified above, such as amylases other than glucoamylase, maltase, lactase, etc. may be used. The temperature and time of enzyme treatment may also be appropriately set to the optimum.

As described above, the enzyme treatment is particularly effective for samples that are expected to contain carbohydrates other than agarooligosaccharides, and contributes to enabling separation of agarooligosaccharides with high sensitivity by subjecting a small amount of agarooligosaccharides contained in low concentrations to LC (liquid chromatography). In the examples described below, agarooligosaccharides containing dextrin, a polysaccharide that is particularly difficult to separate, and other impurity components were subjected to enzyme treatment, and agarooligosaccharides were detected with higher sensitivity through MS (mass spectrometry) as described below, compared to those not subjected to enzyme treatment. According to this embodiment, agarooligosaccharides can be detected to a degree that achieves the object of the present invention without labeling the sample (agarooligosaccharides) even without enzyme treatment, but as a means of further improving the separation or detection ability of agarooligosaccharides, for example, enzyme treatment can be performed instead of labeling, which tends to complicate the analysis process and require expensive analytical equipment. However, since both enzyme treatment and labeling are optional in the present invention, applying labeling rather than enzyme treatment, or applying labeling together with enzyme treatment, is not prohibited and is permitted.

[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 the polarity of the mobile phase. For example, the stationary phase is relatively high polarity unmodified silica gel or alumina, and the mobile phase is relatively low polarity 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 such as acetonitrile, methanol, or ethanol having a certain polarity, and the stationary phase with a higher polarity than that is silica gel or polymer gel modified with a polar group (e.g., amide group, cyano group, amino group, etc.). In the NPC mode and the HILIC mode, the more polar the molecule, the longer the retention time in the stationary phase, and the less polar the molecule, the earlier it elutes and the shorter the retention time. In the separation mode, the type of base material, polar group, size (particle size) and amount of the packing material packed in the stationary phase, i.e., the column (08), as well as the size of the column (08) associated therewith, the type, flow rate and flow rate of the mobile phase (eluent) 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 impart polarity to the stationary phase by modifying the amide group.

Compared to SEC (Size Exclusion Chromatography) mode, which is more suitable for the analysis of agaro-oligosaccharides with a high water content in the mobile phase, NPC mode and HILIC mode can use a relatively high concentration of a highly volatile organic solvent in the mobile phase, which increases the ionization efficiency in MS (mass spectrometry) and improves detection sensitivity, making them well suited to the separation mode of this embodiment. Furthermore, HILIC mode is more suitable for separating agaro-oligosaccharides, which are hydrophilic and highly polar, and is therefore more suited to the separation mode of this embodiment.

[LC: Mobile phase]
In the HILIC mode, the concentration of the aqueous solution of the organic solvent (mixed solvent of organic solvent and water) used in the mobile phase is preferably set to 50 vol% or more, 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 preferable. That is, in the HILIC mode, the mobile phase is preferably an aqueous solution of acetonitrile of 50 vol% or more, more preferably an aqueous solution of acetonitrile of 60 vol% or more, as an eluent as a solvent (dissolving solution) for dissolving the sample, or as a solution in which the sample is dissolved in advance in the eluent. Note that the water used may be pure water such as ion-exchanged water or distilled water, or ultrapure water, and the purity standard may be appropriately set according to the type of sample, etc.

Moreover, 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 types of acid, that is, a substance that exhibits acidity in the mobile phase, are added to the eluent of the mobile phase (a solvent (dissolving solution) that dissolves the sample, for example, a 60 vol% acetonitrile aqueous solution) or a solution in which the sample is dissolved in advance in the eluent (for example, a 60 vol% acetonitrile aqueous solution in which the sample is dissolved). The type of acid is not limited. Examples include formic acid (HCOOH), acetic acid (CH ₃ COOH), trifluoroacetic acid (TFA) (CF ₃ COOH), hydrochloric acid (HCl), sulfuric acid (H ₂ SO ₄ ), nitric acid (HNO ₃ ), propionic acid (C ₃ H ₆ O ₂ ), butyric acid (C ₄ H ₈ O ₂ ), and lactic acid (C ₃ H ₆ O ₃ ). If the purpose of making the mobile phase acidic can be achieved, an acid salt, which is a derivative of the specific acid, may be added. Among acids, volatile acids having a relatively low boiling point (so-called volatile acids), 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 having a relatively high boiling point, such as sulfuric acid, etc. Furthermore, one or more acids selected from formic acid, acetic acid, and trifluoroacetic acid can be particularly preferably applied.

In general, in LC/MS sugar analysis, the mobile phase is either neutral or alkaline by adding a base such as ammonia to the eluent to prevent anomeric separation and promote ionization of sugars. On the other hand, agaro-oligosaccharides have low base resistance and change structure and turn brown when they come into contact with a base, and it has been found that making the mobile phase alkaline makes it more likely to cause problems such as not detecting the target peak or detecting an unknown peak. Therefore, while a neutral range could be selected as the common knowledge of those skilled in the art, the inventor further studied the pH of the mobile phase and found that when an acid such as the above-mentioned examples is added, the molecules of each agaro-oligosaccharide can be detected with high sensitivity in MS (mass spectrometry) as specific ion-added molecules and/or deprotonated molecules. This makes it possible to easily and sensitively separate agaro-oligosaccharides without labeling the sample and detect each of the sugars (2-8 sugars).

The amount of acid added (addition ratio) can be adjusted according to the acidity of each acid. If an acid with a relatively strong acidity is added in excess, it may damage the inside of the LC (02) (liquid chromatograph) such as the column (08), so an acid with a relatively low acidity is generally more suitable as the acid. However, in the examples described below, trifluoroacetic acid, which has a relatively strong acidity among the acids tested, is relatively difficult to handle due to the limited amount of addition compared to formic acid and acetic acid, which have lower acidity, and the types of ion molecules capable of detecting 2-8 sugars of agaro-oligosaccharides with high sensitivity were relatively small, but with specific ion molecules, each saccharide of agaro-oligosaccharides could be reliably detected to achieve the object of the present invention. According to the examples, the amount of acid added was such that at least 0.01 vol% of formic acid was added to the mobile phase, or at least 0.01 vol% to 0.1 vol% of formic acid was added to the mobile phase, and agaro-oligosaccharides could be detected with a specific ion molecule (Examples 1 and 4). In addition, by adding at least 0.1 vol% acetic acid to the mobile phase, or by adding acetic acid in the range of 0.1 vol% to 1 vol% to the mobile phase, it was possible to detect agaro-oligosaccharides with a specific ion molecule (Examples 2 and 5). In addition, by adding at least 0.01 vol% trifluoroacetic acid to the mobile phase, it was possible to detect agaro-oligosaccharides with a specific ion molecule (Examples 3 and 6). Note that the acid concentration (unit: vol%) referred to here is a commonly used technique in the technical field to which the present invention belongs, regarding the pH adjustment of the mobile phase and its notation, for example, an acid-added eluent of x vol% represents an eluent prepared by adding an acid of x volume (e.g., x mL) to an eluent of 100x volume (e.g., 100x mL) (e.g., 60 vol% acetonitrile aqueous solution), and the present invention follows this.

[LC: Stationary phase]
As described above, in the LC (liquid chromatograph) (02), the stationary phase is configured as a column (08) packed with a packing material. The polar group of the base material in the packing material of the HILIC column is preferably an amide group compared to an amino group or a cyano group, which are easily basic even partially, from the viewpoint of the incompatibility of agaro-oligosaccharides with the basicity described above. In addition, as described above, the base material for bonding the polar group can be silica gel or a polymer gel such as polyvinyl alcohol. That is, in the HILIC mode, an amide column modified with an amide group (carbamoyl group) can be preferably used. This makes it possible to more reliably suppress problems caused by the incompatibility of agaro-oligosaccharides with the basicity, such as the tendency of the agaro-oligosaccharides to undergo structural changes, the difficulty of detecting a target peak, or the tendency of detecting an unknown peak. As a result, the agaro-oligosaccharides can be separated and detected with higher sensitivity. In addition, the temperature of the stationary phase (column temperature) is not limited and may be adjusted as appropriate, but a high temperature is preferable in order to prevent anomer separation. In particular, when an amide column is used, it is preferable to set the temperature at 60° C. or higher, more preferably at 80° C. or higher. On the other hand, amino columns and the like are easily basic, and therefore anomer separation is difficult to occur, so they can be preferably used at, for example, about 45° C.

[MS: Mass spectrometer]
Next, in this embodiment, MS (mass spectrometry: mass analysis) is performed on the sample separated by LC (liquid chromatography). MS (mass analysis) is performed in MS (03) (mass analyzer) in LC-MS (01) (liquid chromatograph-mass analyzer). As shown in FIG. 1, the MS (03) (mass analyzer) is configured with an ionization section (11), a mass separation section (12), and a detection section (13). Each of these components is operated under the control of a control section (14). The control section (14) is configured with 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 section (not shown). Note that the control section (10) in the LC (02) (liquid chromatograph) and the control section (14) in the MS (03) (mass analyzer) may be configured as one unit.

[MS: Ionization section/Ionization method]
The eluted sample containing agaro-oligosaccharides eluted from the column (08) of the LC (liquid chromatograph) is then introduced into the ionization section (11) of the MS (03) (mass spectrometer) connected to the LC (02) (liquid chromatograph). The ionization section (11) ionizes the introduced eluted sample. The ionization method can be any known ionization method in MS (mass spectrometry), and the ionization section (11) is configured according to the ionization method to be applied. Specific ionization methods include electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo ionization (APPI). ESI ionizes liquid phase sample molecules by applying a high voltage under atmospheric pressure. APCI heats and vaporizes the sample, and reacts the vaporized sample molecules with ions generated from the atmosphere by corona discharge to ionize the sample molecules. In APPI, a sample is heated and vaporized, and the vaporized sample molecules are ionized by UV light irradiated from a krypton lamp. Note that, although all of the above ionization methods ionize the sample under atmospheric pressure, thermospray ionization (TSI, TSPI), which ionizes the sample under vacuum, may also be used.

[MS: Mass separation unit/Mass separation method]
The mass separation section (12) separates the ionized ions (ion molecules) by m/z (the ratio of the mass (m) of the ion to the charge number (z): the so-called mass-to-charge ratio). As the separation method, a known mass separation method in MS (mass spectrometry) can be appropriately used, and the mass separation section (12) is configured according to the mass separation method to be applied. Specific mass separation methods include quadrupole, magnetic sector, time of flight, ion trap, and the like. In the quadrupole type, four electrodes are arranged in parallel to each other at equal distances from the central axis in a vacuum, and voltages of the same polarity are applied to the electrodes facing each other across the central axis, and voltages of opposite polarity are applied to adjacent electrodes. When a DC voltage and a high-frequency AC voltage are applied to each electrode in a superimposed manner, an electric field whose phase changes rapidly is generated in the quadrupole. Of the ions introduced therein, only those in a specific range of m/z can oscillate stably and pass through the quadrupole. Using this, the voltage is changed to perform mass separation. In the magnetic field type, when ions accelerated by voltage are introduced into a sector-shaped magnetic field, the ions are accelerated perpendicular to their speed and the direction of the magnetic field according to Fleming's left-hand rule, and their trajectories are bent. Mass separation is performed by utilizing the fact that the smaller the mass of the ion, the more its trajectory is bent. In the time-of-flight type, mass separation is performed by utilizing the fact that when ions are accelerated by the same voltage, ions with smaller masses fly faster and ions with larger masses fly slower. The ion trap type applies the principle of the quadrupole type and there are various types, but for example, the inlet and outlet of the quadrupole are connected to form a ring, and ions are trapped within the system, and mass separation is performed by gradually changing the radio frequency voltage to sequentially eject ions whose vibrations become unstable from the system.

The ionization method and mass separation method are not limited, and tandem methods such as MS/MS and MS ⁿ are also acceptable within the scope of the object of the present invention. Thus, the mass spectrometry of the present invention includes MS/MS and MS ⁿ as tandem types, in addition to MS as a single mass.

[LC: detector, detected ion molecules, mass spectrum, chromatogram]
The detection unit (13) is composed of a secondary electron multiplier, a photomultiplier, a channeltron, a microchannel plate, etc., and amplifies and detects the signals of ions (ion molecules) that have been separated and selected for each m/z. As a result, each agarooligosaccharide molecule M can be detected as an ion adduct molecule and/or a deprotonated molecule, such as a formate ion adduct molecule ([M+HCOO] ⁻ ), a deprotonated molecule ([M−H] ⁻ ), an acetate ion adduct molecule ([M+CH ₃ COO] ⁻ ), an ammonium ion adduct molecule ([M+NH ₄ ] ⁺ ), a sodium ion adduct molecule ([M+Na] ⁺ ), and a trifluoroacetate ion adduct molecule ([M+CF ₃ COO] ⁻ ). The detection signal detected by the detector (13) is converted by an appropriate converter and input to the controller (14), which analyzes the signal and performs a predetermined calculation, and outputs, for example, a mass spectrum with m/z on the horizontal axis and a signal value (ion intensity, etc.) related to the applied mass separation method on the vertical axis. And/or, the controller (14) outputs, for ions (ion molecules) separated and selected for each m/z, a chromatogram with the passage 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.

Here, the m/z of the ionic molecule of agarooligosaccharide can be calculated from its molecular formula. As an example, the formate ion adduct molecule ([ _C12H20O10 + _HCOO ] ^- ) of agarobiose ( _C12H20O10 ) can be calculated from its molecular formula to have _a molecular weight of about 369 (=agarobiose ₃₂₄ +formate ion 45) and _an m/z of about -369, assuming that the valence is -1. When the m/z 369(-) is detected by MS (mass spectrometry), for example, if a peak associated with the m/z 369(-) is observed in a chromatogram at the retention time of [C ₁₂ H ₂₀ O ₁₀ +HCOO] ^- determined by a standard sample, this can be taken as the peak of [C ₁₂ H ₂₀ O ₁₀ +HCOO] ^- and it can be determined that agarobiose is contained in the sample. In this way, agarobiose, agarotetraose, agarohexaose and agarooctaose can be qualitatively identified.

The display format of the mass spectrum, chromatogram, and the analysis results thereof is not limited, and they can be displayed on paper media such as chart paper, a display screen, or the like. Such a display unit (not shown) may be provided as part of the LC-MS (01), or may be provided in a separate device connected to the LC-MS (01) by wire and/or wirelessly. Alternatively, the data may be recorded as electronic data on a recording medium that is detachable from the LC-MS (01), and the user may take the electronic data from the LC-MS (01) via the recording medium and perform the necessary analysis on a separate device as appropriate to display it.

Thus, according to the method for analyzing agarooligosaccharides of this embodiment, agarooligosaccharides can be easily and sensitively separated and detected without labeling. When samples (various foods, medicines, quasi-drugs, cosmetics, etc.) containing agarooligosaccharides and carbohydrates other than agarooligosaccharides (monosaccharides, oligosaccharides, polysaccharides, other sugar alcohols) are analyzed, even if agarooligosaccharides are contained in extremely low concentrations, agarooligosaccharides can be separated and detected with high sensitivity by providing a minute amount.

[Production of agarooligosaccharides]
Agar-oligosaccharides were produced from agar by a conventional method. 50 g of agar (Ultra Agar AX-30: Ina Food Industry Co., Ltd.) was dissolved in 1000 g of distilled water by heating, and then 2 g of concentrated sulfuric acid was added and stirred at 90°C for 3 hours. The pH was then adjusted to 3.5 with sodium hydroxide, and the solution was treated with activated charcoal, filtered through filter paper, and further filtered through a 1 μm filter to obtain an agaro-oligosaccharide solution. This solution was vacuum freeze-dried to obtain powdered agaro-oligosaccharides. This was used in all subsequent tests.

[Test 1]
Agarooligosaccharides were dissolved in distilled water, and the final concentration of acetonitrile was adjusted to 60 vol%. The final concentration of agarooligosaccharides was adjusted to 400 ppm (0.4 mg/mL), and this was used as sample (1). Sample (1) was analyzed by LC-MS (HPLC-MS) in which HPLC (high performance liquid chromatograph) and MS (mass spectrometer) were connected under the following conditions (Examples 1 to 3).

In this test, sample (1) was not subjected to enzyme treatment or other pretreatment, and the prepared sample (1) was directly subjected to (injected into) HPLC.

<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 x length 25 cm)
Column temperature: 80℃
Mobile phase: 60 vol% acetonitrile aqueous solution
A specified acid was added to the mixture so that the amount was 0.01 vol% to 1 vol%.
0.01 vol% to 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

<MS conditions>
Ionization: ESI (parallel measurement using a negative and positive parallel measurement device)
Mass separation: quadrupole type Nebulizer gas flow rate: 1.5L/min
Drying gas flow rate: 15L/min
Interface temperature: 350℃
Desolvation Line (DL) temperature: 250°C
Heat block temperature: 200℃

The evaluation of detection sensitivity was made as follows: in the chromatogram of agaro-oligosaccharide (the m/z of its ion-adduct molecule or deprotonated molecule) obtained by HPLC/MS (high performance liquid chromatography mass spectrometry), if the agaro-oligosaccharide was detected as a symmetric normal peak or a similarly symmetric peak that could be distinguished from the baseline and impurity peaks, etc., it was marked as "++". If the agaro-oligosaccharide was not symmetric but was detected as a peak that could be distinguished from the baseline and impurity peaks, etc., it was marked as "+". If the agaro-oligosaccharide was not detected as a peak that could be distinguished from the baseline and impurity peaks, etc., it was marked as "-". The evaluation was made for each agaro-oligosaccharide, and the overall evaluation was made as "++" if all 2-8 sugars were "++", "+" if at least one was "+", and "-" if at least one was "-". An overall evaluation of "+" or higher means that each agaro-oligosaccharide sugar was detected at a qualitative level, and it can be evaluated that the object of the present invention is achieved. And the overall rating of "++" means that the detection was particularly sensitive.

[Example 1]
In Example 1, formic acid (FUJIFILM Wako Pure Chemical Industries, Ltd.; the same applies below) was selected as an example of an 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 1. In the table, formic acid adduct ions represent formic acid ion adduct molecules, deprotonated ions represent deprotonated molecules, acetate adduct ions represent acetate ion adduct molecules, ammonium adduct ions ( _NH4 adduct ions) represent ammonium ion adduct molecules, and sodium adduct ions (Na adduct ions) represent sodium ion adduct molecules (the same applies below).

As shown in Table 1, when the amount of formic acid added was 0.01 vol%, the sensitivity of 8 sugars was relatively low with acetate ion adduct molecules, but agaro-oligosaccharides could be detected. Furthermore, formate ion adduct molecules, deprotonated molecules, ammonium ion adduct molecules, and sodium ion adduct molecules could detect all 2-8 sugars with particularly high sensitivity. When the amount of formic acid added was 0.1 vol%, all five types of ion molecules tested could detect all 2-8 sugars with particularly high sensitivity. Example 1 showed that by adding at least 0.01 vol% formic acid to the mobile phase, or by adding at least 0.01 vol% to the mobile phase with formic acid in the range of 0.01 vol% to 0.1 vol%, agaro-oligosaccharides could be detected with five types of ion molecules, formate ion adduct molecules, deprotonated molecules, acetate ion adduct molecules, ammonium ion adduct molecules, and sodium ion adduct molecules.

[Example 2]
In Example 2, acetic acid (FUJIFILM Wako Pure Chemical Industries, Ltd.; the same applies below) was selected as an example of an 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 2.

As shown in Table 2, for either 0.1 vol% or 1 vol% acetic acid, no identifiable peaks of 2-8 sugars were detected in the formate ion adduct molecule. On the other hand, the deprotonated molecule, acetate ion adduct molecule, ammonium ion adduct molecule, and sodium ion adduct molecule were able to detect all 2-8 sugars with particularly high sensitivity in the deprotonated molecule, acetate ion adduct molecule, ammonium ion adduct molecule, and sodium ion adduct molecule. Example 2 showed that by adding at least 0.1 vol% acetic acid to the mobile phase, or at least by adding acetic acid in the range of 0.1 vol% to 1 vol% to the mobile phase, agaro-oligosaccharides could be detected with particularly high sensitivity in the four types of ion molecules, deprotonated molecule, acetate ion adduct molecule, ammonium ion adduct molecule, and sodium ion adduct molecule.

[Example 3]
In Example 3, trifluoroacetic acid (TFA) (FUJIFILM Wako Pure Chemical Industries, Ltd.; the same applies below) was selected as an example of an 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 3. In the table, trifluoroacetic acid adduct ion (TFA adduct ion) represents a trifluoroacetic acid ion adduct molecule (the same applies below).

As shown in Table 3, when the amount of trifluoroacetic acid added was 0.01 vol%, no identifiable peaks of 2-8 sugars were detected for the deprotonated molecules and acetate ion adduct molecules, and no peaks of 4-8 sugars were detected for the formate ion adduct molecules. On the other hand, agaro-oligosaccharides could be detected with particularly high sensitivity for all 2-8 sugars for the three types of ions, ammonium ion adduct molecules, sodium ion adduct molecules, and trifluoroacetate ion adduct molecules. Example 3 showed that by adding at least 0.01 vol% trifluoroacetic acid to the mobile phase, agaro-oligosaccharides could be detected with particularly high sensitivity for the three types of ion molecules, ammonium ion adduct molecules, sodium ion adduct molecules, and trifluoroacetate ion adduct molecules.

[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 molecule in Example 4 was selected, and samples (2), (3), and the control related to the 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 prepared by the agency, in the same manner as for other ion-added molecules.

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.

From the viewpoint of resolution, the evaluation "++" was given when the peak of sample (2) (AOS) and the peak of the control (blank) were clearly separated as shown in Figure 2A (peak of ammonium ion adduct molecules of disaccharides at 0.1 vol% added formic acid), and one peak of sample (3) (AOS + blank) overlaps with the peak of sample (2) (AOS) with almost no deviation, making it possible to clearly identify the peak as agaro-oligosaccharide (ion adduct molecule or deprotonated molecule), and from the viewpoint of detection, each peak compared between sample (2) and sample (3) is a symmetric normal peak or a similarly symmetric peak that can be distinguished from the baseline and impurity peaks, etc.

From the viewpoint of separation ability, the evaluation "+" was given when one peak of sample (3) (AOS + blank) is slightly shifted from the peak of sample (2) (AOS) as shown in Figure 2B (peak of ammonium ion adduct molecule of octasaccharide with 0.01 vol% formic acid added), but both peaks can be distinguished as the same substance (agaro-oligosaccharide). Alternatively, from the viewpoint of detection ability, a "+" was also given when any peak used for comparison in sample (2) and sample (3) is not symmetrical but can be recognized as distinct from the baseline and impurity peaks. In the example of Figure 2B, although there is some overlap between the peak of sample (2) (AOS) and the peak of the control (blank), the two peaks are distinctly separated. Furthermore, since the peaks of sample (3) (AOS + blank) are slightly shifted from the peaks of sample (2) (AOS) and the control (blank), but overlap and show the same tendency, it is possible to determine that one peak of sample (3) (AOS + blank) and one peak of sample (2) (AOS) are the same substance (agaro-oligosaccharides). Note that the peak shape itself in Figure 2B is symmetrical or similarly symmetrical.

The evaluation "-" was given when, from the viewpoint of separation ability, one peak of sample (3) (AOS + blank) cannot be distinguished as agaro-oligosaccharide, as shown in Figure 2C (peak of acetate ion adduct molecule of disaccharide at 0.01 vol% added formic acid). Alternatively, from the viewpoint of detection ability, a "-" was given when any peak used for comparison in sample (2) and sample (3) is not recognizable as distinguishable from the baseline and impurity peaks. In the example of Figure 2C, one peak of sample (3) (AOS + blank) overlaps with both peaks of sample (2) (AOS) and the control (blank) with almost no deviation, and it is impossible to distinguish whether the peak of sample (3) is the same substance as the peak of sample (2) or the control, i.e., whether it is agaro-oligosaccharide or impurity component. Note that the peak shape itself in Figure 2C is symmetrical or has a similar symmetry.

An overall rating of "+" or higher means that each agarooligosaccharide was detected at a qualitative level, and the objective of the present invention can be evaluated as being achieved. And an overall rating of "++" means that the sugars were detected with particularly high sensitivity.

[Example 4]
In Example 4, formic acid was selected as an example of an 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.

As shown in Table 4, when 0.01 vol% formic acid was added, disaccharides and octasaccharides could not be distinguished with the acetate ion-added molecules. On the other hand, formate ion-added molecules, ammonium ion-added molecules, and sodium ion-added molecules had relatively low sensitivity for octasaccharides, but were able to detect agaro-oligosaccharides. Furthermore, deprotonated molecules were able to detect all 2-8 sugars with particularly high sensitivity. When 0.1 vol% formic acid was added, acetate ion-added molecules, formate ion-added molecules, sodium ion-added molecules, and deprotonated molecules showed the same detection sensitivity as when 0.01 vol% formic acid was added. Furthermore, ammonium ion-added molecules showed even higher detection sensitivity, and were able to detect all 2-8 sugars with particularly high sensitivity. Example 4 shows that by adding at least 0.01 vol% formic acid to the mobile phase, or by adding at least 0.01 vol% to 0.1 vol% formic acid to the mobile phase, agaro-oligosaccharides can be detected with high sensitivity using four types of ion molecules, formate ion adduct molecules, deprotonated molecules, ammonium ion adduct molecules, and sodium ion adduct molecules, in samples containing impurities such as dextrins that are particularly difficult to separate as carbohydrates other than agaro-oligosaccharides.

[Example 5]
In Example 5, acetic acid was selected as an example of an 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.

As shown in Table 5, for the formate-ion adduct, no identifiable peaks of 2-8 sugars were detected at either 0.1 or 1 vol% acetic acid added. For the acetate-ion adduct and sodium-ion adduct, the sensitivity of 8 sugars was relatively low for both molecules at 0.1 vol% acetic acid added, and the sensitivity of 2-6 sugars was also relatively low for the acetate-ion adduct at 1 vol% acetic acid added, but both acetate-ion adduct and sodium-ion adduct molecules were able to detect agaro-oligosaccharides at either added amount. For the deprotonated and ammonium-ion adduct molecules, the agaro-oligosaccharides were able to be detected with particularly high sensitivity for all 2-8 sugars at either 0.1 or 1 vol% acetic acid added. Example 5 shows that by adding at least 0.1 vol% acetic acid to the mobile phase, or by adding at least 0.1 vol% to 1 vol% acetic acid to the mobile phase, agaro-oligosaccharides can be detected with high sensitivity using four types of ion molecules, namely deprotonated molecules, acetate ion-adduct molecules, ammonium ion-adduct molecules, and sodium ion-adduct molecules, in samples containing impurities such as dextrins that are particularly difficult to separate as carbohydrates other than agaro-oligosaccharides.

[Example 6]
In Example 6, trifluoroacetic acid (TFA) was selected as an example of an 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.

As shown in Table 6, when the amount of trifluoroacetic acid added was 0.01 vol%, the deprotonated molecule and the acetate ion adduct molecule could not detect a distinguishable peak of 2-8 sugars, and the formate ion adduct molecule could not detect a distinguishable peak of 4-8 sugars. In addition, the trifluoroacetate ion adduct molecule could not detect an octasaccharide. On the other hand, the ammonium ion adduct molecule had a relatively low sensitivity to octasaccharides, but was able to detect agaro-oligosaccharides. In addition, the sodium ion adduct molecule had a relatively low sensitivity to 2-8 sugars, but was able to detect agaro-oligosaccharides. Example 6 showed that by adding at least 0.01 vol% trifluoroacetic acid to the mobile phase, agaro-oligosaccharides could be detected with high sensitivity using two types of ion molecules, ammonium ion adduct molecules and sodium ion adduct molecules, for samples containing impurities such as dextrin, which is particularly difficult to separate as a carbohydrate other than agaro-oligosaccharides.

[Test 3]
Sample (4) was prepared from an agarooligosaccharide-containing material containing agarooligosaccharides and dextrin, a carbohydrate (polysaccharide) other than agarooligosaccharides. Sample (4) contained agarooligosaccharides and dextrin in a mass ratio of 10/50, 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) after the enzyme treatment described below. Sample (4) was analyzed by LC-MS (HPLC-MS) in which HPLC (high performance liquid chromatograph) and MS (mass spectrometer) were connected under the following conditions (Example 7, Comparative Example 1).

In this test, sample (4) was subjected to the following enzyme treatment beforehand and then subjected (injected) to LC. That is, 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 Industries, Ltd.), and 0.1 mL of amyloglucosidase (amyloglucosidase solution from Aspergillus niger, Sigma-Aldrich Japan Co., Ltd.) were added to 50 mL of ammonium acetate buffer (pH 4.5) containing agarooligosaccharides and dextrin in a mass ratio of 10/50, and the mixture was reacted at 37°C for 60 minutes, after which the reaction solution was boiled for 10 minutes to inactivate the enzymes. After the volume was adjusted to 100 mL with distilled water, acetonitrile was added to a final concentration of 60 vol% to prepare sample (4).

<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 x length 25 cm)
Column temperature: 80℃
Mobile phase:
Example 7: For a 60 vol% acetonitrile aqueous solution,
Formic acid was added to make it 0.1 vol%.
0.1 vol% formic acid - 60 vol% acetonitrile aqueous solution Comparative Example 1: 60 vol% acetonitrile aqueous solution Flow rate: 0.2 mL/min
Sample (4) pretreatment: performed Sample (4) injection volume: 1 μL

<MS conditions>
Ionization: ESI (using a negative and positive parallel measurement device)
Mass separation: quadrupole type Nebulizer gas flow rate: 1.5L/min
Drying gas flow rate: 15L/min
Interface temperature: 350℃
Desolvation Line (DL) temperature: 250°C
Heat block temperature: 200℃

Based on the chromatograms obtained by HPLC/MS (High Performance Liquid Chromatography Mass Spectrometry) under the above conditions, the detection sensitivity of each agarooligosaccharide sugar as a deprotonated molecule ([M-H] ^- ) was 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. Note that the horizontal axis of the chromatogram represents retention time, and the vertical axis represents ion intensity, which is the strength of the signal value, and the horizontal axis is aligned with 2-8 sugars for ease of visual recognition and comparison (the same applies below).

As shown in Figure 3A, in Example 7, in which formic acid was added to the mobile phase, a symmetric normal peak or a similar symmetric peak that could be distinguished from the baseline and impurity peaks was detected at the m/z of the deprotonated molecules for all 2-8 sugars. On the other hand, as shown in Figure 3B, in Comparative Example 1, in which formic acid was not added to the mobile phase, slight peaks that could be distinguished from the baseline were detected for 4 sugars, 6 sugars, and 8 sugars, but for disaccharides, no recognizable peaks were detected that could be distinguished from the baseline and impurity peaks. Thus, comparing Example 7 and Comparative Example 1, the addition of formic acid to the mobile phase clearly improved the detection sensitivity of agaro-oligosaccharides. As a result, in Example 7, by making the mobile phase acidic (here, adding 0.1 vol% formic acid), agarooligosaccharides could be detected with high sensitivity by providing only a small amount of 1 μL of an agarooligosaccharide solution containing polysaccharides (here, dextrin) as impurity components, and further containing an agarooligosaccharide at a low concentration of 400 ppm (0.4 mg/mL).

[Test 4]
Sample (5) was prepared from an agarooligosaccharide-containing material containing agarooligosaccharides and dextrin, which is a carbohydrate other than agarooligosaccharides, and other impurities. Sample (5) contained agarooligosaccharides, black tea extract, and dextrin 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 after enzyme treatment was 400 ppm (0.4 mg/mL). Sample (5) was analyzed under the following conditions by LC-MS (HPLC-MS) in which HPLC (high performance liquid chromatograph) and MS (mass spectrometer) were connected (Example 8, Reference Example 1). Sample (5) was subjected to enzyme treatment in advance as in Test 3 and then subjected (injected) to LC.

<LC conditions>
LC: HPLC
Separation mode: HILIC
column:
Example 8: Amide column: TSKgel Amide-80 5 μm (registered trademark) (Tosoh Corporation)
(Particle size: 5 μm Column size: inner diameter 2.0 mm x length 25 cm)
Reference Example 1: Amino column: Asahipak NH2P-40 2E (registered trademark) (Resonac Co., Ltd.)
(Particle size: 5 μm Column size: inner diameter 2.0 mm x length 25 cm)
Column temperature: Example 8; 80°C
Reference Example 1: 45°C
Mobile phase: 60 vol% acetonitrile aqueous solution
Formic acid was added to make it 0.1 vol%.
0.1vol% formic acid - 60vol% acetonitrile aqueous solution Flow rate: 0.2mL/min
Sample (5) pretreatment: performed Sample (5) injection volume: 1 μL

<MS conditions>
Ionization: ESI (using a negative and positive parallel measurement device)
Mass separation: quadrupole type Nebulizer gas flow rate: 1.5L/min
Drying gas flow rate: 15L/min
Interface temperature: 350℃
Desolvation Line (DL) temperature: 250°C
Heat block temperature: 200℃

In this test, as described above, the column type and column temperature were set to different values in Example 8 and Reference Example 1, but the column temperature was set to the optimum temperature for each column, and no unfavorable conditions were set for each example. Based on the chromatograms obtained by HPLC/MS (high performance liquid chromatography mass spectrometry) under the above conditions, the detection sensitivity of each agarooligosaccharide sugar as a formate ion adduct molecule ([M+HCOO] ⁻ ) was 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.

As shown in FIG. 4A, in Example 8, in which an amide column was used as the stationary phase, a symmetric normal peak or a similar symmetric peak that could be distinguished from the baseline and impurity peaks was detected at the m/z of the formate ion adduct molecules for all 2-8 sugars. On the other hand, as shown in FIG. 4B, in Reference Example 1, in which an amino column was used as the stationary phase, a peak that could be distinguished from the baseline and impurity peaks was detected for 2-6 sugars, but the intensity was significantly weak. Furthermore, for 8 sugars, no peak that could be recognized as being distinct from the baseline and impurity peaks was detected. Thus, comparing Example 8 with Reference Example 1, the detection sensitivity of agaro-oligosaccharides was further improved by using an amide column as the stationary phase.

[Test 5]
As in Test 4, sample (6) was prepared using distilled water and acetonitrile (final concentration of acetonitrile was 60 vol%) so that the sample contained agaro-oligosaccharides, black tea extract, and dextrin in a mass ratio of 10/40/50 and the final concentration of agaro-oligosaccharides was 400 ppm (0.4 mg/mL). Sample (6) was analyzed by LC-MS (HPLC-MS) in which HPLC (high performance liquid chromatograph) and MS (mass spectrometer) were connected under the following conditions (Example 9, Reference Example 2).

As for sample (6), in Example 9, the sample was pre-treated with an enzyme, as in Test 3, and then subjected (injected) to LC. The final concentration of agaro-oligosaccharides was adjusted to 400 ppm (0.4 mg/mL) after enzyme treatment. In Reference Example 2, the sample (6) was directly subjected (injected) to LC without enzyme treatment or other pretreatment.

<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 x length 25 cm)
Column temperature: 80℃
Mobile phase: 60 vol% acetonitrile aqueous solution
Formic acid was added to make it 0.1 vol%.
0.1 vol% formic acid, 60 vol% acetonitrile aqueous solution Flow rate: 0.2 mL/min
Sample (6) Pretreatment:
Example 9: Implemented Reference Example 2: Not implemented Sample (6) injection amount: 1 μL

<MS conditions>
Ionization: ESI (using a negative and positive parallel measurement device)
Mass separation: quadrupole type Nebulizer gas flow rate: 1.5L/min
Drying gas flow rate: 15L/min
Interface temperature: 350℃
Desolvation Line (DL) temperature: 250°C
Heat block temperature: 200℃

Based on the chromatograms obtained by HPLC/MS (high performance liquid chromatography mass spectrometry) under the above conditions, the detection sensitivity of each agarooligosaccharide as a formate ion adduct molecule ([M+HCOO] ⁻ ) was compared between Example 9 and Reference Example 2. The results are shown in FIG.

As shown in Fig. 5, in Example 9 in which the enzymatic treatment was performed on the sample (6), a symmetric normal peak or a similar symmetric peak that can be distinguished from the baseline and impurity peaks was detected in the m/z of the formate ion adduct molecules for all 2-8 sugars. On the other hand, in Reference Example 2 in which the enzymatic treatment was not performed, a symmetric normal peak or a similar symmetric peak that can be distinguished from the baseline and impurity peaks was detected for all 2-8 sugars, but the intensity of each peak was slightly weaker than that in Example 9, and an impurity peak was also detected for the 8 sugar. Thus, in Example 9, the enzymatic treatment was performed on the agaro-oligosaccharide-containing material containing dextrin and other impurity components, thereby improving the intensity of the peaks related to the agaro-oligosaccharide molecules and suppressing the appearance of impurity peaks, and as a result, a detection result of higher quality with higher sensitivity and reliability could be obtained.
[Test 6]
As in Test 4, sample (7) was prepared using distilled water and acetonitrile (final concentration of acetonitrile was 60 vol%) so that the sample contained agaro-oligosaccharides, black tea extract, and dextrin in a mass ratio of 10/40/50, and the final concentration of agaro-oligosaccharides was 100 ppm (0.1 mg/mL). Sample (7) was analyzed under the following conditions using LC-MS (HPLC-MS) in which HPLC (high performance liquid chromatograph) and MS (mass spectrometer) were connected (Example 10). Sample (7) was not subjected to enzyme treatment or other pretreatment, and the prepared sample (7) was directly subjected to (injected into) LC.

<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 x length 25 cm)
Column temperature: 80℃
Mobile phase: 60 vol% acetonitrile aqueous solution
Formic acid was added to make it 0.1 vol%.
0.1 vol% formic acid, 60 vol% acetonitrile aqueous solution Flow rate: 0.2 mL/min
Sample (7) pretreatment: not performed Sample (7) injection volume: 1 μL

<MS conditions>
Ionization: ESI (using a negative and positive parallel measurement device)
Mass separation: quadrupole type Nebulizer gas flow rate: 1.5L/min
Drying gas flow rate: 15L/min
Interface temperature: 350℃
Desolvation Line (DL) temperature: 250°C
Heat block temperature: 200℃

As shown in FIG. 6, in Example 10, a symmetric normal peak or a similar symmetric peak that could be distinguished from the baseline and impurity peaks was detected in the m/z of the formate ion adduct molecules for all 2-8 sugars. Since the amount of agaro-oligosaccharides tested was extremely small, a certain amount of noise appeared for the 8 sugars, but the peak shape was similar to a normal peak and could be distinguished from the baseline and impurity peaks. Thus, in Example 10, by adding formic acid to the mobile phase, agaro-oligosaccharides could be detected with high sensitivity even when only a small amount of 1 μL of an agaro-oligosaccharide solution containing dextrin and other impurity components, and containing an agaro-oligosaccharide at an extremely low concentration of 100 ppm (0.1 mg/mL), was provided.

[Test 7]
Sample (8) (AOS) consisting of agarooligosaccharides, sample (9) (AOS + glucose) consisting of agarooligosaccharide-containing material containing agarooligosaccharides and glucose, a carbohydrate (monosaccharide) other than agarooligosaccharides, and a control consisting of glucose (blank: glucose) were prepared. Sample (8) was prepared in the same manner as in Test 1, using distilled water and acetonitrile (final concentration of acetonitrile: 60 vol%) so that the final concentration of agarooligosaccharides was 400 ppm (0.4 mg/mL). Sample (9) was prepared using distilled water and acetonitrile (final concentration of acetonitrile: 60 vol%) so that the final concentration of agarooligosaccharides was 400 ppm (0.4 mg/mL) and contained agarooligosaccharides and glucose (FUJIFILM Wako Pure Chemical Industries, Ltd.) in a mass ratio of 10/100. A control (glucose) was prepared with distilled water and acetonitrile (final concentration of acetonitrile: 60%) so that the final concentration of glucose was the same as that of sample (9). Samples (8), (9), and the control were analyzed under the following conditions using LC-MS (HPLC-MS) in which HPLC (high performance liquid chromatograph) and MS (mass spectrometer) were connected (Example 11). Each sample was directly subjected (injected) to LC without enzyme treatment or other pretreatment.

<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 x length 25 cm)
Column temperature: 80℃
Mobile phase: 60 vol% acetonitrile aqueous solution
Formic acid was added to make it 0.1 vol%.
0.1 vol% formic acid, 60 vol% acetonitrile aqueous solution Flow rate: 0.2 mL/min
Samples (8) (9) and control Pretreatment: Not performed Samples (8) (9) and control Injection volume: 1 μL

<MS conditions>
Ionization: ESI (using a negative and positive parallel measurement device)
Mass separation: quadrupole type Nebulizer gas flow rate: 1.5L/min
Drying gas flow rate: 15L/min
Interface temperature: 350℃
Desolvation Line (DL) temperature: 250°C
Heat block temperature: 200℃

The results (chromatograms) of detection of glucose and agarooligosaccharides as formate ion adduct molecules ([M+HCOO] ⁻ ) for sample (8), sample (9), and the control are shown in the drawings. That is, the results of the control are shown in FIG. 7A, the results of sample (8) in FIG. 7B, and the results of sample (9) in FIG. 8.

As shown in FIG. 7A, in the control glucose, a peak was detected at the m/z of the formate ion adduct molecule for glucose, while no peak was detected for the agaro-oligosaccharide 2-8 sugars. Also, as shown in FIG. 7B, in sample (8) agaro-oligosaccharide, a symmetric normal peak or a similar symmetric peak that can be distinguished from the baseline and impurity peaks was detected at the m/z of the formate ion adduct molecule for all 2-8 sugars, while no peak for glucose was detected.

Furthermore, as shown in FIG. 8, in the sample (9) containing agarooligosaccharides and glucose, a symmetric normal peak or a similar symmetric peak that can be distinguished from the baseline and impurity peaks was detected in the m/z of the formate ion adduct molecules for all agarooligosaccharides 2-8 sugars, and a peak for glucose was also detected. When the horizontal axes (retention times) of the chromatograms in FIG. 7A and FIG. 8 are superimposed, the peaks for glucose in both figures overlap with almost no deviation at the retention times that are the basis for the qualitative analysis in the chromatograms. Therefore, it can be confirmed that the peak in FIG. 8 is indeed glucose. When the horizontal axes (retention times) of the chromatograms in FIG. 7B and FIG. 8 are superimposed, the peaks for each of the agarooligosaccharides 2-8 sugars in both figures overlap with almost no deviation at the retention times that are the basis for the qualitative analysis in the chromatograms. Therefore, it can be confirmed that the peaks in FIG. 8 are indeed agarooligosaccharides 2-8 sugars. In this way, it was possible to clearly separate each agarooligosaccharide sugar from glucose. That is, in Example 11, by making the mobile phase acidic (here, adding 0.1 vol% formic acid), agarooligosaccharides could be detected with high sensitivity by providing only a small amount of 1 μL of an agarooligosaccharide solution containing monosaccharides (here, glucose) as impurity components, and further containing agarooligosaccharides at a low concentration of 400 ppm (0.4 mg/mL). As a secondary finding, in this Test 7, it was also shown that this LC/MS (HPLC/MS) with an acidic mobile phase can also analyze monosaccharides (here, glucose) in addition to agarooligosaccharides.

[Test 8]
Sample (10) (AOS) consisting of agarooligosaccharides, sample (11) (AOS + sucrose) consisting of agarooligosaccharide-containing material containing agarooligosaccharides and sucrose, which is a carbohydrate (oligosaccharide) other than agarooligosaccharides, and a control consisting of sucrose (blank: sucrose) were prepared. Sample (10) was prepared in the same manner as in Test 1, using 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). Sample (11) contained agarooligosaccharides and sucrose (FUJIFILM Wako Pure Chemical Industries, Ltd.) in a mass ratio of 10/100, and was prepared using 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 (sucrose) was prepared with distilled water and acetonitrile (final concentration of acetonitrile: 60%) so that the final concentration of sucrose was the same as that of sample (11). These samples (10), (11), and the control were analyzed under the same conditions as in Test 7 (Example 12) (no enzyme treatment or other pretreatment was performed on any of the samples).

The results (chromatograms) of detection of sucrose and agarooligosaccharides as formate ion adduct molecules ([M+HCOO] ⁻ ) for sample (10), sample (11), and the control are shown in the drawings. That is, the results for the control are shown in FIG. 9A, the results for sample (10) in FIG. 9B, and the results for sample (11) in FIG. 10.

As shown in FIG. 9A, in the sucrose control, a peak was detected at the m/z of the formate ion adduct molecule for sucrose, while no peak was detected for the agaro-oligosaccharide 2-8 sugars. Also, as shown in FIG. 9B, in the agaro-oligosaccharide sample (10), a symmetric normal peak or a similar symmetric peak that can be distinguished from the baseline and impurity peaks was detected at the m/z of the formate ion adduct molecule for all 2-8 sugars, while no peak was detected for sucrose.

Furthermore, as shown in FIG. 10, in the sample (11) containing agarooligosaccharides and sucrose, a symmetric normal peak or a similar symmetric peak that can be distinguished from the baseline and impurity peaks was detected in the m/z of the formate ion adduct molecules for all agarooligosaccharides 2-8 sugars, and a peak for sucrose was also detected. When the horizontal axes (retention times) of the chromatograms in FIG. 9A and FIG. 10 are superimposed, the peaks for sucrose in both figures overlap with almost no deviation at the retention times that are the basis for the qualitative analysis in the chromatograms. Therefore, it can be confirmed that the peak in FIG. 10 is indeed sucrose. When the horizontal axes (retention times) of the chromatograms in FIG. 9B and FIG. 10 are superimposed, the peaks for agarooligosaccharides 2-8 sugars in both figures overlap with almost no deviation at the retention times that are the basis for the qualitative analysis in the chromatograms. Therefore, it can be confirmed that the peaks in FIG. 10 are indeed agarooligosaccharides 2-8 sugars. Thus, each agarooligosaccharide and sucrose could be clearly separated. That is, in Example 12, by making the mobile phase acidic (here, adding 0.1 vol% formic acid), agarooligosaccharides could be detected with high sensitivity by providing only a small amount of 1 μL of an agarooligosaccharide solution containing oligosaccharides (here, disaccharide sucrose) as impurity components, and further containing agarooligosaccharides at a low concentration of 400 ppm (0.4 mg/mL). In addition, in this test 8, it was shown as a secondary finding that this LC/MS (HPLC/MS) with an acidic mobile phase can also analyze oligosaccharides (here, sucrose) in addition to agarooligosaccharides.

[Test 9]
Sample (12) (AOS) consisting of agarooligosaccharides, sample (13) (AOS + sorbitol) consisting of agarooligosaccharide-containing material containing agarooligosaccharides and sorbitol, a carbohydrate (sugar alcohol) other than agarooligosaccharides, and a control consisting of sorbitol (blank: sorbitol) were prepared. Sample (12) was prepared in the same manner as in Test 1, using distilled water and acetonitrile (final concentration of acetonitrile: 60 vol%) so that the final concentration of agarooligosaccharides was 400 ppm (0.4 mg/mL). Sample (13) was prepared using distilled water and acetonitrile (final concentration of acetonitrile: 60 vol%) so that the final concentration of agarooligosaccharides was 400 ppm (0.4 mg/mL) containing agarooligosaccharides and sorbitol (FUJIFILM Wako Pure Chemical Industries, Ltd.) in a mass ratio of 10/100. The control (sorbitol) was prepared with distilled water and acetonitrile (final concentration of acetonitrile: 60%) so that the final concentration of sorbitol was the same as that of sample (13). These samples (12), (13), and the control were analyzed under the same conditions as in Test 7 (Example 13) (no enzyme treatment or other pretreatment was performed on any of the samples).

The results (chromatograms) of detection of sorbitol and agarooligosaccharides as formate ion adduct molecules ([M+HCOO] ⁻ ) for sample (12), sample (13), and the control are shown in the drawings. That is, the results for the control are shown in FIG 11A, the results for sample (12) in FIG 11B, and the results for sample (13) in FIG 12.

As shown in FIG. 11A, in the control sorbitol, a peak was detected at the m/z of the formate ion adduct molecule for sorbitol, while no peak was detected for the agaro-oligosaccharide 2-8 sugars. Also, as shown in FIG. 11B, in sample (12) agaro-oligosaccharide, a symmetric normal peak or a similar symmetric peak that can be distinguished from the baseline and impurity peaks was detected at the m/z of the formate ion adduct molecule for all 2-8 sugars, while no peak for sorbitol was detected.

Furthermore, as shown in FIG. 12, in the sample (13) containing agaro-oligosaccharides and sorbitol, a symmetric normal peak or a similar symmetric peak that can be distinguished from the baseline and impurity peaks was detected in the m/z of the formate ion adduct molecules for all agaro-oligosaccharides 2-8 sugars, and a peak for sorbitol was also detected. When the horizontal axis (retention time) of the chromatograms in FIG. 11A and FIG. 12 is superimposed, the peaks for sorbitol in both figures overlap with almost no deviation at the retention time that is the basis of the qualitative analysis in the chromatogram. Therefore, it can be confirmed that the peak in FIG. 12 is indeed sorbitol. Furthermore, when the retention times (horizontal axis) related to the m/z of the formate ion adduct molecules for agaro-oligosaccharides 2-8 sugars in FIG. 11B and FIG. 12 are superimposed, the positions of the peaks related to the retention times (left and right positions of the peaks representing the retention times of the target ions themselves) overlap with almost no deviation, even though there is a difference in the heights (ion intensities) of the peaks. When the horizontal axis (retention time) of the chromatograms in FIG. 11B and FIG. 12 is superimposed, the peaks of agarooligosaccharides 2-8 saccharides in both figures overlap almost without any shift in the retention time that is the basis of the qualitative analysis in the chromatograms. Therefore, it can be confirmed that the peaks in FIG. 12 are indeed agarooligosaccharides 2-8 saccharides. Thus, each agarooligosaccharide and sorbitol could be clearly separated. That is, in Example 13, by making the mobile phase acidic (here, adding 0.1 vol% formic acid), agarooligosaccharides could be detected with high sensitivity by supplying only a small amount of 1 μL of an agarooligosaccharide solution that contains sugar alcohols (here, sorbitol) as impurity components and further contains 400 ppm (0.4 mg/mL) of the agarooligosaccharides at a low concentration. In addition, as a secondary finding, this study 9 also showed that this LC/MS (HPLC/MS) with an acidic mobile phase can also be used to analyze sugar alcohols (sorbitol in this case) in addition to agarooligosaccharides.

From the above, according to the present invention, LC/MS in which the mobile phase in liquid chromatography is acidic can detect agarooligosaccharides with high sensitivity from agarooligosaccharide-containing materials that contain monosaccharides, oligosaccharides, polysaccharides, or sugar alcohols as impurity components. Moreover, the results of each example show that agarooligosaccharides can be detected in the same way even if they contain two or more types of these carbohydrates. Note that it is not necessary to distinguish between these carbohydrates here, but for example, oligosaccharides can be distinguished as carbohydrates with 2 to 10 sugars, polysaccharides as carbohydrates with 11 or more sugars, etc.

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 thermostatic bath 10 Control section 11 Ionization section 12 Mass separation section 13 Detection section 14 Control section

Claims (7)

The method is characterized in that agaro-oligosaccharides are separated from the agaro-oligosaccharide-containing material by liquid chromatography in a hydrophilic interaction mode and detected by mass spectrometry; and
adding an acid to a mobile phase in the liquid chromatography to make the mobile phase acidic;
The method for analyzing agarooligosaccharides, wherein the agarooligosaccharide-containing substance contains carbohydrates other than agarooligosaccharides and their constituent sugars. 2. The method for analyzing agaro-oligosaccharides according to claim 1, wherein an amide column is used in the hydrophilic interaction mode. 3. The method for analyzing agaro-oligosaccharides according to claim 1, wherein a volatile acid is added as the acid. 4. The method for analyzing agarooligosaccharides according to claim 3, wherein the acid added is at least one acid selected from the group consisting of formic acid, acetic acid, and trifluoroacetic acid. 3. The method for analyzing agaro-oligosaccharides according to claim 1, wherein the mobile phase is an aqueous solution of an organic solvent having a concentration of 50 vol % or more to which the acid is added. 3. The method for analyzing agarooligosaccharides according to claim 1, further comprising the step of treating the agarooligosaccharide-containing material with an enzyme before subjecting the agarooligosaccharide-containing material to the liquid chromatography. 3. The method for analyzing agarooligosaccharides according to claim 1 or 2, characterized in that the carbohydrates other than agarooligosaccharides and their constituent sugars are any one or more of monosaccharides, oligosaccharides, polysaccharides, and sugar alcohols other than agarooligosaccharides and their constituent sugars.

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