Method for detecting presence or absence of sugar, sugar derivative, or nucleotide sugar

Abstract

The present invention is able to provide a method whereby the presence or absence of a sugar, a sugar derivative, or a nucleotide sugar can be detected using the same column and without contaminating the device. This is achieved by means of a method for detecting the presence or absence of a sugar, a sugar derivative, or a nucleotide sugar, the method comprising a washing step in which a washing solution is passed through a graphite carbon column, and an elution step in which the graphite carbon column which has undergone the washing step is used to separate a sugar and a sugar derivative by means of gradient elution or separate a nucleotide sugar by means of isocratic elution.

Description

Methods for detecting the presence or absence of sugars, sugar derivatives, or sugar nucleotides

[0001] The present invention relates to methods for detecting the presence or absence of a sugar, sugar derivative, or sugar nucleotide.

[0002] Sugars, sugar derivatives, and sugar nucleotides are all highly hydrophilic and therefore cannot be retained on conventional reversed-phase columns. For liquid chromatography-mass spectrometry (LC / MS), ion-pair chromatography, hydrophilic interaction liquid chromatography (HILIC), or graphite carbon columns are used. For example, Xuan Qin et al., "Quantification of nucleotides and their sugar conjugates in biological samples: Purposes, instruments and applications," Journal of Pharmaceutical and Biomedical Analysis 158 (2018) pp. 280-287 (Non-Patent Document 1) describes analysis using ion pair chromatography, Jun Ito et al., "Analysis of plant nucleotide sugars by hydrophilic interaction liquid chromatography and tandem mass spectrometry," Anal. Biochem. 448 (2014) pp. 14-22 (Non-Patent Document 2) describes analysis using HILIC, and Martin Pabst et al., "Nucleotide and Nucleotide Sugar Analysis by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry on Surface-Conditioned Porous Graphitic Carbon," Analytical Chemistry, Vol. 82, No. 23, 2010, pp. 9782-9788 (Non-Patent Document 3) describe analysis using a graphite carbon column.

[0003] However, ion pair chromatography has the problem that it cannot retain uncharged sugars and sugar derivatives, and the device is contaminated with the ion pair reagent. In addition, HILIC columns and graphite carbon columns generally have low durability and are not stable in retention.

[0004] Xuan Qin et al., "Quantification of nucleotides and their sugar conjugates in biological samples: Purposes, instruments and applications", Journal of Pharmaceutical and Biomedical Analysis 158(2018)p280-287Jun Ito et al., "Analysis of plant nucleotide sugars by hydrophilic interaction liquid chromatography and tandem mass spectrometry", Anal. Biochem. 448 (2014) p14-22Martin Pabst et al., "Nucleotide and Nucleotide Sugar Analysis by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry on Surface-Conditioned Porous Graphitic Carbon", Analytical Chemistry, Vol. 82, No. 23, 2010, p9782-9788Tashinga E. Bapiro et al., "Understanding the Complexity of Porous Graphic Carbon (PGC) Chromatography: "Modulation of Mobile-Stationary Phase Interactions Overcomes Loss of Retention and Reduces Variability", Anal. Chem. 2016, 88, 6190-6194

[0005] The present invention aims to solve the above problems and provide a method for detecting the presence or absence of sugars, sugar derivatives, or sugar nucleotides using the same column without contaminating the device.

[0006] A first aspect of the present invention relates to a method for detecting the presence or absence of a sugar, a sugar derivative, or a sugar nucleotide, the method comprising: a washing step of passing a washing solution through a graphite carbon column; and an elution step of separating sugars and sugar derivatives by gradient elution or separating sugar nucleotides by isocratic elution using the graphite carbon column that has undergone the washing step.

[0007] According to the method of the present invention, since an ion pair reagent is not used, the device is not contaminated, the presence or absence of sugars, sugar derivatives, or sugar nucleotides can be detected using the same column, and column retention and separation are stable.

[0008] 1(a) is a graph showing an example of gradient elution, and FIG. 1(b) is a graph showing an example of isocratic elution. It shows the results of analyzing fructose, glucose, and galactose in Experimental Example 1. It shows the results of analyzing N-acetylglucosamine (GlcNAC), N-acetyl-D-mannosamine (ManNAC), and N-acetyl-D-galactosamine (GalNAC) in Experimental Example 1. It shows the results of analyzing uridine diphosphate galactose (UDP-galactose) and uridine diphosphate glucose (UDP-glucose) in Experimental Example 1. It shows the results of analyzing uridine diphosphate N-acetylgalactosamine (UDP-GalNAC) and uridine diphosphate N-acetylglucosamine (UDP-GluNAC) in Experimental Example 1. FIGS. 6(a) and 6(b) show the results of analyzing fructose, glucose, and galactose using columns from different lots in Experimental Example 2. Figures 7(a) and 7(b) show the results of analyzing UDP-galactose and UDP-glucose using columns from different lots in Experimental Example 2. This graph shows the results of comparative analysis of UDP-glucose for different antibody-producing cell extracts in Experimental Example 3. Figures 9(a) and 9(b) show the results of analysis of fructose, glucose, and galactose using mobile phases of different compositions in Experimental Example 4. Figures 10(a) and 10(b) show the results of analysis of GlcNAC, ManNAC, and GalNAC using mobile phases of different compositions in Experimental Example 4. Figure 11 shows the results of analysis of UDP-galactose and UDP-glucose using mobile phases of different compositions in Experimental Example 4. Figures 12(a) and 12(b) show the results of analysis of UDP-galactose and UDP-glucose using mobile phases of different compositions in Experimental Example 4. Figure 13(a) shows the results of Experimental Example 5, in which sugar derivatives were analyzed with and without a washing step. Figure 14(a) shows the results of Experimental Example 2, in which sugar nucleotides were analyzed with and without a washing step.

[0009] A first aspect of the present invention relates to a method for detecting the presence or absence of a sugar, a sugar derivative, or a sugar nucleotide, the method comprising: a washing step of passing a washing solution through a graphitized carbon column; and a step of separating sugars and sugar derivatives by gradient elution or separating sugar nucleotides by isocratic elution using the graphitized carbon column that has undergone the washing step.

[0010] According to the first aspect of the present invention, since no ion pair reagent is used, the device is not contaminated, the presence or absence of sugars, sugar derivatives, or sugar nucleotides can be detected using the same column, and column retention and separation are stable.

[0011] In the first aspect of the present invention, the sugar to be detected may be any of monosaccharides, oligosaccharides, and polysaccharides, and examples thereof include glucose, galactose, mannose, fructose, fucose, trehalose, maltose, and glucose. １３ C6, but is not limited to these.

[0012] In the first aspect of the present invention, sugar derivatives to be detected include, but are not limited to, N-acetyl-D-galactosamine (GalNAC), N-acetyl-D-mannosamine (ManNAC), N-acetylglucosamine (GlcNAC), sialic acid, glucosamine, mannosamine, galactosamine, N-valeryl-D-glucosamine, glucosamine acid, glucosamine oxime, and N-trifluoroacetyl-D-glucosamine.

[0013] In the first aspect of the present invention, the sugar nucleotides to be detected include uridine diphosphate-N-acetylglucosamine (UDP-GluNAC), uridine diphosphate galactose (UTP-galactose / UTP-Gal), and cytidine. Examples of such phosphates include, but are not limited to, 5'-monophosphate-N-acetylneuraminic acid (CMP-Neu5AC), guanosine diphosphate mannose (GDP-mannose / GDP-Man), uridine diphosphate glucose (UDP-glucose / UDP-Glc), uridine diphosphate N-acetylgalactosamine (UDP-GalNAC), guanosine diphosphate fucose (GDP-fucose / GDP-Fuc), adenosine diphosphate glucose (ADP-glucose / ADP-Glc), adenosine diphosphate ribose (ADP-ribose / ADP-rib), uridine diphosphate glucuronic acid (UDP-glucuronic acid), and guanosine diphosphate glucose (GDP-glucose / GDP-Glc).

[0014] The first aspect of the present invention is premised on the use of a graphite carbon column. Specific examples of graphite carbon columns include "Supel （商標） Examples of suitable columns include, but are not limited to, porous graphite carbon LC "High performance liquid chromatography (HPLC) columns" (manufactured by Merck Life Sciences Co., Ltd.).

[0015] In a first aspect of the present invention, a washing step is first performed in which a washing solution is passed through a graphite carbon column. In the first aspect of the present invention, the separation ability of the graphite carbon column is regenerated by passing the washing solution through the graphite carbon column for a certain period of time, and the presence or absence of sugars, sugar derivatives, or sugar nucleotides can be detected using the same column. Washing the graphite carbon column itself is a known technique, as described, for example, in Tashinga E. Bapiro et al., "Understanding the Complexity of Porous Graphitic Carbon (PGC) Chromatography: Modulation of Mobile-Stationary Phase Interactions Overcomes Loss of Retention and Reduces Variability," Anal. Chem. 2016, 88, 6190-6194 (Non-Patent Document 4).

[0016] The cleaning solution is not particularly limited as long as it can be used as a cleaning solution for a graphite carbon column, but it is preferable that it contains water (in the experimental example described below, 95 (v / v)% methanol / water is used as the cleaning solution as mobile phase C).

[0017] In the first aspect, the graphite carbon column that has been subjected to the washing step is then used to perform an elution step in which sugars and sugar derivatives are separated by gradient elution, or sugar nucleotides are separated by isocratic elution. Figure 1(a) is a graph showing an example of gradient elution, and Figure 1(b) is a graph showing an example of isocratic elution.

[0018] Gradient elution is a method of elution in which the mobile phase composition is continuously changed. For example, elution is performed by changing the ratio of two mobile phases over time. In the example shown in Figure 1(a) (Experimental Example 1, described below), gradient elution was performed under the following conditions: oven temperature: 40°C; flow rate: 0.2 mL / min; 0-5 min: mobile phase A concentration: 100%, mobile phase B concentration: 0%; 5-15 min: mobile phase A concentration: 100% → 5%, mobile phase B concentration: 0% → 95%; 15-20 min: mobile phase A concentration: 5%, mobile phase B concentration: 95%; 20-30 min: mobile phase C concentration: 100% (wash); 30-40 min: mobile phase A concentration: 100%. However, the gradient elution conditions are not limited to these. By performing this gradient elution, sugars and sugar derivatives are separated, enabling the presence or absence of sugars and sugar derivatives to be detected. When gradient elution is performed, the sample to be detected should not contain sugar nucleotides.

[0019] Isocratic elution is a method of elution in which the mobile phase composition is maintained constant, for example, by maintaining a constant ratio between two mobile phases. In the example shown in Figure 1(b) (Experimental Example 1, described later), isocratic elution was performed under the following conditions: oven temperature: 60°C; flow rate: 0.2 mL / min; 0-15 min: mobile phase A concentration: 97%, mobile phase B concentration: 3%; 15-25 min: mobile phase C concentration: 100% (wash); 25-35 min: mobile phase A concentration: 97%, mobile phase B concentration: 3%. However, the conditions for isocratic elution are not limited to these. This isocratic elution separates sugar nucleotides, enabling the detection of their presence or absence. When performing isocratic elution, the sample to be detected should be free of sugars (although sugar derivatives may be present).

[0020] In the first embodiment, the mobile phases used for gradient elution and isocratic elution may have the same composition or different compositions. In Experimental Example 1 described later, the mobile phases used for gradient elution and isocratic elution were: Mobile phase A: 0.1 (w / w)% NH３ aq (pH 11.0) Mobile phase B: acetonitrile / isopropyl alcohol (1:1) was used, but the present invention is not limited to this.

[0021] In the first aspect of the present invention, the washing step and the elution step are preferably repeated in this order two or more times. The presence or absence of sugars, sugar derivatives, or sugar nucleotides can be detected multiple times using the same column. The number of repetitions is not particularly limited.

[0022] When the washing step and the elution step are repeated in this order, for example, twice, the following four patterns may be possible.

[0023] (Pattern 1: Gradient elution → Isocratic elution) This pattern includes a washing step in which a washing solution is passed through a graphite carbon column, an elution step in which sugars and sugar derivatives are separated by gradient elution using the graphite carbon column that has undergone the washing step, a washing step in which a washing solution is passed through the graphite carbon column after gradient elution, and an elution step in which sugar nucleotides are separated by isocratic elution using the graphite carbon column that has undergone the washing step after gradient elution. In this case, after detecting the presence or absence of sugars and sugar derivatives, the presence or absence of sugar nucleotides can be detected using the same column.

[0024] (Pattern 2: Isocratic elution → Gradient elution) This pattern includes a washing step in which a washing solution is passed through a graphite carbon column, an elution step in which sugar nucleotides are separated by isocratic elution using the graphite carbon column that has undergone the washing step, a washing step in which a washing solution is passed through the graphite carbon column after isocratic elution, and an elution step in which sugars and sugar derivatives are separated by gradient elution using the graphite carbon column that has undergone the washing step after isocratic elution. In this case, after detecting the presence or absence of sugar nucleotides, the presence or absence of sugars and sugar derivatives can be detected using the same column.

[0025] (Pattern 3: Gradient elution → Gradient elution) This pattern includes a washing step of passing a washing solution through a graphite carbon column, a step of separating sugars and sugar derivatives by gradient elution using the graphite carbon column that has undergone the washing step, a washing step of passing a washing solution through the graphite carbon column after gradient elution, and an elution step of separating sugars and sugar derivatives by gradient elution using the graphite carbon column that has undergone the washing step after gradient elution. In this case, after detecting the presence or absence of sugars and sugar derivatives, the presence or absence of sugars and sugar derivatives can be detected using the same column.

[0026] (Pattern 4: Isocratic elution → Isocratic elution) This pattern includes a washing step of passing a wash solution through a graphite carbon column, an elution step of isocratically eluting sugar nucleotides using the graphite carbon column that has undergone the washing step, a washing step of passing a wash solution through the graphite carbon column after isocratic elution, and an elution step of isocratically eluting sugar nucleotides using the graphite carbon column that has undergone the washing step after isocratic elution. In this case, after detecting the presence or absence of sugar nucleotides, the presence or absence of sugar nucleotides can be detected using the same column.

[0027] The graphite carbon column may be equilibrated before the first washing step.

[0028] The present invention will be explained in more detail below by way of experimental examples, but the present invention is not limited to these examples.

[0029] <Experimental Example 1> "Supel" was used as a graphite carbon column. （商標） A Carbon LC 2.7 μm HPLC column (150 mm × 2.1 mm) and a guard column were used. The column was first equilibrated, and then 95 (v / v)% methanol / water (mobile phase C) was passed through the column at a flow rate of 0.2 mL / min (washing step).

[0030] The following samples were analyzed six times using a graphite carbon column that had undergone a washing step: Sample 1: containing 20 μM each of fructose, glucose, and galactose; Sample 2: containing 2 μM each of GlcNAC, ManNAC, and GalNAC; Sample 3: containing 0.5 μM each of UDP-galactose and UDP-glucose; and Sample 4: containing 0.5 μM each of UDP-GalNAC and UDP-GluNAC. The average area value and relative standard deviation (RSD) were calculated to evaluate the area reproducibility.

[0031] Gradient elution was performed under the following conditions shown in Figure 1(a) for Sample 1 containing sugar and Sample 2 containing sugar derivative: oven temperature: 40°C, flow rate: 0.2 mL / min, 0 to 5 min: mobile phase A concentration: 100%, mobile phase B concentration: 0%, 5 to 15 min: mobile phase A concentration: 100% → 5%, mobile phase B concentration: 0% → 95%, 15 to 20 min: mobile phase A concentration: 5%, mobile phase B concentration: 95%, 20 to 30 min: mobile phase C concentration: 100% (washing), 30 to 40 min: mobile phase A concentration: 100%. Gradient elution was performed under the following conditions shown in Figure 1(b) for Samples 3 and 4 containing sugar nucleotide: oven temperature: 60°C, flow rate: 0.2 mL / min, 0 to 15 min: mobile phase A concentration: 97%, mobile phase B concentration: 3%, Isocratic elution was performed under the following conditions: 15-25 min: Mobile phase C concentration: 100% (washing), 25-35 min: Mobile phase A concentration: 97%, Mobile phase B concentration: 3%. In both cases, the mobile phases were: Mobile phase A: 0.1 (w / w)% NH ３ aq (pH 11.0) Mobile phase B: acetonitrile (ACN) / isopropyl alcohol (IPA) (1:1) was used.

[0032] Figure 2 shows the results of analyzing fructose, glucose, and galactose in Sample 1, Figure 3 shows the results of analyzing GlcNAC, ManNAC, and GalNAC in Sample 2, Figure 4 shows the results of analyzing UDP-galactose and UDP-glucose in Sample 3, and Figure 5 shows the results of analyzing UDP-GalNAC and UDP-GluNAC in Sample 4. The results of the area reproducibility evaluation are shown in Table 1.

[0033]

[0034] <Experimental Example 2> We investigated whether there were any lot differences in graphite carbon columns. （商標） Carbon LC" 2.7 μm HPLC columns (150 mm × 2.1 mm) were equilibrated and washed in the same manner as in Experimental Example 1 using two lots (BL10289 and BL10416). Samples 1 and 3 used in Experimental Example 1 were then analyzed under the same conditions as in Experimental Example 1.

[0035] Figures 6(a) and 6(b) show the results of analyzing fructose, glucose, and galactose using columns from different lots (Figure 6(a) is BL10289, Figure 6(b) is BL10416), and Figures 7(a) and 7(b) show the results of analyzing UDP-galactose and UDP-glucose using columns from different lots (Figure 7(a) is BL10289, Figure 7(b) is BL10416). It was confirmed that sugars and sugar nucleotides can be separated using multiple lots.

[0036] Experimental Example 3: A comparative analysis was performed on different antibody-producing cell extracts. Specifically, each sample containing 2 μl of H (antibody-producing cell extract) or B (antibody-producing cell extract), which produces UDP-glucose, was equilibrated in the same manner as in Experimental Example 1, and after a washing step, was subjected to isocratic elution in the same manner as in Experimental Example 1. Figure 8 is a graph showing the results of Experimental Example 3, with the vertical axis representing the area value and the horizontal axis representing the number of days the cells had been cultured. This demonstrated that analysis of real samples is also possible.

[0037] Experimental Example 4: A study was conducted on the case where mobile phase A was changed. Samples 1 and 2 used in Experimental Example 1 were equilibrated and washed in the same manner as in Experimental Example 1, and then the mobile phase A was changed to 0.1 (w / w)% NH ３ aq (pH 11.0) or 20 mM NH ４ CO ３ aq (pH 9.0) was used, and gradient elution was carried out in the same manner as in Experimental Example 1. After equilibration and a washing step in the same manner as in Experimental Example 1, Sample 3 used in Experimental Example 1 was eluted with 0.1 (w / w)% NH ３ aq (pH 11.0), 20mM NH ４ CO ３ aq (pH 9.0) or 0.02 (w / w)% NH ３ Isocratic elution was carried out in the same manner as in Experimental Example 1 using aq (pH 11.0).

[0038] 9(a) and 9(b) show the results of analyzing fructose, glucose, and galactose using mobile phases with different compositions (FIG. 9(a) shows the results of analyzing fructose, glucose, and galactose using mobile phases with 0.1 (w / w)% NH ３ aq (pH 11.0), and Figure 9(b) shows 20 mM NH ４ CO ３ aq (pH 9.0), and Figures 10(a) and 10(b) show the results of analyzing GlcNAC, ManNAC, and GalNAC using mobile phases of different compositions (Figure 10(a) shows the results of analyzing GlcNAC, ManNAC, and GalNAC using mobile phases of different compositions (0.1 (w / w)% NH ３ aq (pH 11.0), and Fig. 10(b) shows 20 mM NH ４ CO ３ aq (pH 9.0) was used. Figures 11, 12(a), and 12(b) show the results of analyzing UDP-galactose and UDP-glucose using mobile phases of different compositions (Figure 11 shows the results for 0.1 (w / w)% NH ３ aq (pH 11.0), Figure 12(a) shows 20 mM NH ４ CO ３ aq (pH 9.0), and Fig. 12(b) shows 0.02 (w / w)% NH ３ aq (pH 11.0) was used).

[0039] From the results shown in Figs. 9(a), 9(b), 10(a), and 10(b), NH ４ CO ３ When aq was used as mobile phase A, the separation of monosaccharides deteriorated slightly and the sensitivity decreased, but the retention time did not change significantly. Furthermore, from the results shown in Figures 11, 12(a), and 12(b), it was observed that the retention of sugar nucleotides weakened as the concentration of mobile phase A increased, while the retention tended to increase as the pH of the mobile phase decreased, suggesting that the main separation mechanism was anion exchange.

[0040] Experimental Example 5: The case where the washing step was not performed was investigated. Samples containing sugar derivatives or sugar nucleotides were equilibrated in the same manner as in Experimental Example 1, with and without the washing step. After the washing step, the graphite carbon column was subjected to gradient elution for samples containing sugar derivatives and isocratic elution for samples containing sugar nucleotides, in the same manner as in Experimental Example 1.

[0041] 13(a) and 13(b) show the results of Experimental Example 5, in which sugar derivatives were analyzed with and without a washing step, respectively, and Fig. 14(a) and 14(b) show the results of Experimental Example 2, in which sugar nucleotides were analyzed with and without a washing step. The results showed that, for samples containing sugar derivatives, separation deteriorated when the washing step was not performed, and, for samples containing sugar nucleotides, separation deteriorated and retention decreased when the washing step was not performed.

[0042] Aspects It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects.

[0043] (Item 1) A method according to one embodiment is a method for detecting the presence or absence of sugars, sugar derivatives, or sugar nucleotides, and includes a washing step in which a washing solution is passed through a graphite carbon column, and an elution step in which sugars and sugar derivatives are separated by gradient elution or sugar nucleotides are separated by isocratic elution using the graphite carbon column that has undergone the washing step.

[0044] According to the method described in paragraph 1, the presence or absence of sugars, sugar derivatives, or sugar nucleotides can be detected using the same column without contaminating the device.

[0045] (Item 2) In a method according to one embodiment, the washing step and the elution step are repeated in this order two or more times.

[0046] According to the method described in item 2, the presence or absence of sugars, sugar derivatives, or sugar nucleotides can be detected multiple times using the same column.

[0047] (Item 3) A method according to one embodiment includes a washing step of passing a washing solution through a graphite carbon column, an elution step of separating sugars and sugar derivatives by gradient elution using the graphite carbon column that has undergone the washing step, a washing step of passing a washing solution through the graphite carbon column after gradient elution, and an elution step of separating sugar nucleotides by isocratic elution using the graphite carbon column that has undergone the washing step after gradient elution.

[0048] According to the method described in item 3, after detecting the presence or absence of sugars and sugar derivatives, the presence or absence of sugar nucleotides can be detected using the same column.

[0049] (Item 4) A method according to one embodiment includes a washing step of passing a washing solution through a graphite carbon column, an elution step of separating sugar nucleotides by isocratic elution using the graphite carbon column that has undergone the washing step, a washing step of passing a washing solution through the graphite carbon column after the isocratic elution, and an elution step of separating sugars and sugar derivatives by gradient elution using the graphite carbon column that has undergone the washing step after the isocratic elution.

[0050] According to the method described in item 4, after detecting the presence or absence of sugar nucleotides, the presence or absence of sugars and sugar derivatives can be detected using the same column.

[0051] (Item 5) A method according to one aspect includes a washing step of passing a washing solution through a graphite carbon column, an elution step of separating sugars and sugar derivatives by gradient elution using the graphite carbon column that has undergone the washing step, a washing step of passing a washing solution through the graphite carbon column after gradient elution, and an elution step of separating sugars and sugar derivatives by gradient elution using the graphite carbon column that has undergone the washing step after gradient elution.

[0052] According to the method described in item 5, after detecting the presence or absence of sugars and sugar derivatives, the presence or absence of sugars and sugar derivatives can be detected using the same column.

[0053] (Item 6) A method according to one aspect includes a washing step of passing a washing solution through a graphite carbon column, an elution step of isocratically eluting sugar nucleotides using the graphite carbon column that has undergone the washing step, a washing step of passing a washing solution through the graphite carbon column after the isocratic elution, and an elution step of isocratically eluting sugar nucleotides using the graphite carbon column that has undergone the washing step after the isocratic elution.

[0054] According to the method described in item 6, after detecting the presence or absence of a sugar nucleotide, the presence or absence of a sugar nucleotide can be detected using the same column.

[0055] (Item 7) In the method according to one aspect, the cleaning liquid comprises water.

[0056] According to the method described in item 7, the separation ability of the graphite carbon column is restored, and the presence or absence of sugars, sugar derivatives, or sugar nucleotides can be detected using the same column.

Claims

1. A method for detecting the presence or absence of sugars, sugar derivatives, or sugar nucleotides, comprising: a washing step of passing a washing solution through a graphitized carbon column; and an elution step of separating sugars and sugar derivatives by gradient elution, or separating sugar nucleotides by isocratic elution, using the graphitized carbon column that has been subjected to the washing step.

2. The method of claim 1, wherein the washing step and the elution step are repeated in that order two or more times.

3. The method according to claim 1, comprising: a washing step of passing a washing solution through a graphite carbon column; an elution step of separating sugars and sugar derivatives by gradient elution using the graphite carbon column that has undergone the washing step; a washing step of passing a washing solution through the graphite carbon column after gradient elution; and an elution step of separating sugar nucleotides by isocratic elution using the graphite carbon column that has undergone the washing step after gradient elution.

4. The method according to claim 1, comprising: a washing step of passing a washing solution through a graphite carbon column; an elution step of separating sugar nucleotides by isocratic elution using the graphite carbon column that has undergone the washing step; a washing step of passing a washing solution through the graphite carbon column after isocratic elution; and an elution step of separating sugars and sugar derivatives by gradient elution using the graphite carbon column that has undergone the washing step after isocratic elution.

5. The method according to claim 1, comprising: a washing step of passing a washing solution through a graphite carbon column; a step of separating sugars and sugar derivatives by gradient elution using the graphite carbon column that has undergone the washing step; a washing step of passing a washing solution through the graphite carbon column after gradient elution; and an elution step of separating sugars and sugar derivatives by gradient elution using the graphite carbon column that has undergone the washing step after gradient elution.

6. The method according to claim 1, comprising: a washing step of passing a washing solution through a graphite carbon column; an elution step of separating sugar nucleotides by isocratic elution using the graphite carbon column that has undergone the washing step; a washing step of passing a washing solution through the graphite carbon column after isocratic elution; and an elution step of separating sugar nucleotides by isocratic elution using the graphite carbon column that has undergone the washing step after isocratic elution.

7. The method of any one of claims 1 to 6, wherein the cleaning liquid comprises water.

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