High-performance liquid chromatography elution time standardization using a reversed-phase scale.

Abstract

To provide a method for accurately analyzing sugar chains by standardizing an elution time when separating the sugar chains by using reverse phase high performance liquid chromatography.SOLUTION: An elution time standardization method when separating sugar chains by reversed-phase high-performance liquid chromatography, the elution time standardization method comprises: (i) using, as standard substances, nine types of sugar chains including #R-0 and four pairs of combinations of standard substances having the same structure except for the presence / absence of core fucose, #R-1 and #R-2, #R-3 and #R-4, #R-5 and #R-6, and #R-7 and #R-8, and (ii) standardizing the elution time by converting the standard curve such that a difference in R values between the sugar chains having core fucose and the sugar chains not having core fucose in the respective pairs of (i) becomes constant.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for analyzing sugar chain structures. [Background technology]

[0002] Conventionally, elution time standardization methods using isomaltooligosaccharides and the like have been used for analyzing glycans (see Non-Patent Document 1). This method uses isomaltooligosaccharides with different degrees of glucose polymerization as standard substances, and identifies glycans from their elution times. In this method, the standard curve converges for standard substances with high degrees of polymerization and long elution times, and analysis is performed by extrapolation. In addition, the standard substances have structures different from those of glycans such as glycoproteins. Therefore, accurate analysis has been difficult using conventional methods for analyzing glycans. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] N Tomiya et al., Analytical Biochemistry, 1988 May 15;171(1):73-90. doi: 10.1016 / 0003-2697(88)90126-1. Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a method for standardizing elution times and accurately analyzing sugar chains when separating and analyzing sugar chains using reversed-phase high performance liquid chromatography. [Means for solving the problem]

[0005] The present inventors have conducted extensive research into methods for accurately analyzing glycans in biological samples. Specifically, they investigated methods for standardizing the elution positions of reversed-phase high-performance liquid chromatography (RPLC) when analyzing glycans using this method. When identifying glycans by comparing HPLC elution times, reducing the variability between experiments increases the accuracy of analysis. However, elution times can vary due to various factors, such as slight differences in eluent composition and column deterioration. Therefore, they used nine types of glycans with specific structures as standard substances (reference substances). They also used four pairs of N-linked glycans, each with the same structure except for the presence or absence of core fucose. They found that standardization by converting the standard curve so that the difference in elution time between the standard substance with core fucose and the standard substance without core fucose in each pair is constant can extend the effective elution time and enable accurate analysis.

[0006] That is, the present invention is as follows. [1] A method for standardizing elution times when separating glycans by reversed-phase high-performance liquid chromatography, comprising: (i) Nine types of glycans having the structures shown below were used as standards, including #R-0 and four pairs of standard substances with the same structure except for the presence or absence of core fucose: #R-1 and #R-2, #R-3 and #R-4, #R-5 and #R-6, and #R-7 and #R-8. (ii) Elution time normalization method, in which the elution time is normalized by converting the standard curve so that the difference in R value between the glycan with core fucose and the glycan without core fucose in each pair in (i) is constant: TIFF0007779480000001.tif165133[2] The elution time standardization method according to [1], wherein the biological sample is selected from the group consisting of serum, plasma, urine, and biological tissue. [3] The elution time standardization method of [1] or [2], in which glycans are separated by reversed-phase high-performance liquid chromatography using a C18 column. [4] Any of the elution time standardization methods [1] to [3], in which a mobile phase containing water and acetonitrile is used as the mobile phase for reversed-phase chromatography. [5] The elution time standardization method of [3] or [4], in which the C18 column size is 1 to 3 mm in inner diameter and 10 to 20 cm in length, and the mobile phase flow rate is 0.1 to 0.5 mL / min. [6] One of the elution time standardization methods [3] to [5], using the following mobile phases: Solvent A: A mixture of "water" and "0.5 M aqueous acetic acid adjusted to pH 4.0 with triethylamine" in a 9:1 (v / v) ratio; Solvent B: A mixture of "water" and "acetonitrile" in a 7:2:1 (v / v) ratio of "0.5 M aqueous acetic acid adjusted to pH 4.0 with triethylamine." [7] A method for analyzing glycan structures, which involves determining the R value of the glycan whose structure is to be identified using a standardized standard curve created by standardizing the elution time using any of the elution time standardization methods [1] to [6], and comparing it with the R value of glycan structures whose structures are known. [8] A glycan structure analysis method according to [7], in which the R values ​​of multiple glycans with known structures are measured using a standardized standard curve created by standardizing elution times using one of the elution time standardization methods [1] to [6], and the structure is identified by comparing the R values ​​of glycans with known structures in a database of R values ​​of glycan structures with known structures in advance with the R value of the glycan whose structure is to be identified. [Effects of the Invention]

[0007] The high performance liquid chromatography elution time standardization method using the reversed phase scale of the present invention makes it possible to accurately identify the structure of sugar chains in biological samples based on their elution times. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the elution conditions for reversed-phase HPLC. [Figure 2] FIG. 1 shows the relationship between elution time and glucose polymerization degree when PA-labeled isomaltooligosaccharides were analyzed by reverse-phase HPLC. [Figure 3] FIG. 1 shows sugar chains used as standard substances in the high-performance liquid chromatography elution time standardization method of the present invention. [Figure 4-1]FIG. 1 shows details of the standard substances (#R-0, #R-1, and #R-2) used in the high-performance liquid chromatography elution time standardization method of the present invention. [Figure 4-2] FIG. 1 shows details of the standard substances (#R-3 and #R-4) used in the high-performance liquid chromatography elution time standardization method of the present invention. [Figure 4-3] FIG. 1 shows details of the standard substances (#R-5 and #R-6) used in the high-performance liquid chromatography elution time standardization method of the present invention. [Figure 4-4] FIG. 1 shows details of the standard substances (#R-7 and #R-8) used in the high-performance liquid chromatography elution time standardization method of the present invention. [Figure 5] FIG. 1 shows a standard curve obtained by standardizing the elution time of PA glycans in reversed-phase chromatography. [Figure 6] FIG. 1 shows a comparison of chromatograms obtained when PA-labeled isomaltooligosaccharides were used as a standard substance and when the PA-labeled sugar chains of the present invention were used. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention will be described in detail below. The present invention relates to a method for analyzing sugar chain structures.

[0010] In the method of the present invention, the elution time or elution position is standardized in the analysis of glycan structure using high performance liquid chromatography. Here, standardizing the elution time or elution position means making the elution time or elution position constant depending on the glycan structure. The elution time is also called retention time.

[0011] To standardize the elution time, nine types of glycans with specific structures are used as standard substances. Furthermore, four pairs of standard substances with the same structure except for the presence or absence of core fucose are used as standard substances. The standard curve is transformed so that the difference in R value between the standard substance with core fucose and the standard substance without core fucose in each pair is constant.

[0012] standard material In the method of the present invention, multiple sugar chains having specific structures are used as standards.

[0013] Specifically, nine types of N-linked glycans, #R-0, #R-1, #R-2, #R-3, #R-4, #R-5, #R-6, #R-7, and #R-8, shown in Figure 3 and Figures 4-1 to 4-4, are used as standards. These glycans are structurally similar to the target glycans to be measured and belong to the same series. Because the structures of the target and standard substances are similar, the elution patterns are also similar, reducing measurement error. These glycans are also called elution time standardization glycans. They are also sometimes called reversed-phase standards. #R-1 and #R-2, #R-3 and #R-4, #R-5 and #R-6, and #R-7 and #R-8 differ in whether they contain core fucose (#R-1, #R-3, #R-5, and #R-7) or not (#R-2, #R-4, #R-6, and #R-8). #R-0 is the point where the R value becomes "R=0", and galactose (#R-0, Gal-PA) is used. In the present invention, by using galactose (#R-0, Gal-PA) as the point where the R value becomes "R=0", it is expected that the difference in values ​​due to differences in piping length and other factors between analytical instruments will be reduced.

[0014] The hydrophobicity increases in the order of #R-0, #R-1, #R-2, #R-3, #R-4, #R-5, #R-6, #R-7 and #R-8, and the elution time increases in this order.

[0015] The standard substance is prepared as follows. Gal-PA (#R-0), GlcNAc-PA (#R-1) and Fucα1-6GlcNAc-PA (#R-2) They are prepared by pyridylamination of commercially available D-galactose, N-Acetyl-D-glucosamine, and Fucα(1-6)GlcNAc (purchased from Tokyo Chemical Industry Co., Ltd., etc.). AG124(#R-3) and AG124F6(#R-4) Each antibody is prepared from mouse serum (purchased from Fujifilm Wako Pure Chemical Industries, Sigma-Aldrich, etc.) by hydrazinolysis or Peptide:N-glycanase (PNGase) to release the glycans, followed by pyridylamination, digestion with β-galactosidase (derived from bovine testis, Streptococcus pneumoniae, and / or Xanthomonas manihotis), and purification. BIBs(#R-5) and BIBsF6(#R-6) Each oligosaccharide is prepared by releasing the glycan from commercially available human γ-globulin (purchased from Fujifilm Wako Pure Chemical Industries, Sigma-Aldrich, etc.) using hydrazinolysis or Peptide:N-glycanase (PNGase), followed by pyridylamination. 06N(EA)-BIBs(#R-7) and 06N(EA)-BIBsF6(#R-8) Each glycan is prepared by releasing the glycans from commercially available human γ-globulin (purchased from Fujifilm Wako Pure Chemical Industries, Sigma-Aldrich, etc.) using hydrazinolysis or Peptide:N-glycanase (PNGase), followed by pyridylamination and ethylamidation of the carboxyl groups on the sialic acid residues with ethylamine. Because it is difficult to prepare highly hydrophobic standard glycans using natural structures, existing glycans are modified to enhance their hydrophobicity. Modification can be performed by modifying the carboxyl group of a glycan containing one sialic acid with an amine. Modifications can be performed with ammonia, methylamine, ethylamine, or isopropylamine, but ethylamine is preferred. The reaction is carried out using a condensing agent (such as EDC-HOBt or DMT-MM), and the resulting mixture is purified using Amide-HILIC (such as Discovery DPA-6S).

[0016] Target glycans In the present invention, the target glycans to be measured and the structures to be identified are glycans in mammalian biological samples. Examples of glycans include free glycans and glycans of glycoproteins. Mammals include humans and non-human mammals, such as primates. Humans are preferred. Examples of biological samples include blood, serum, plasma, urine, and biological tissues. Examples of biological tissues include normal tissues and abnormal tissues at cancer or inflammatory sites.

[0017] Extraction and processing of test material Extraction of free glycans from biological samples can be performed, for example, by the following method. The biological sample is pretreated with Dowex 50W-X8 resin (H+ form, 200-400 mesh, Fujifilm Wako Pure Chemical Industries, Osaka, Japan), neutralized with sodium bicarbonate solution, and desalted with a graphite carbon cartridge (InertSepGC 300 mg; GL Science, Tokyo, Japan). The reducing ends of the glycans can be labeled with 2-aminopyridine.

[0018] The sugar chains of glycoproteins can be released from glycoproteins in a biological sample by, for example, hydrazine treatment or cleavage with the enzyme Peptide:N-glycanase F (PNGase F) in the case of N-linked sugar chains.

[0019] In the method of the present invention, when separating glycans using reversed-phase chromatography, the glycans are labeled and used. Labeling can be performed using 2-aminopyridine or tritium, but preferably, the reducing end of the glycan is reacted with 2-aminopyridine to perform reductive amination, resulting in fluorescent labeling (PA) and the resulting glycan is used as a PA glycan.

[0020] High-performance liquid chromatography The high performance liquid chromatography used for the analysis in the present invention is reversed phase high performance chromatography.

[0021] Columns used in reversed-phase high-performance liquid chromatography may be low-polarity columns, such as alkylated silica gel, phenyl columns, polystyrene-crosslinked divinylbenzene columns, PFP columns, and CN columns. Columns containing packing materials with groups such as octadecylsilyl (C18), octyl (C8), butyl (C4), trimethyl (C3), triacontyl (C30), phenyl (Ph), and butyl groups can be used. Among these, columns containing packing materials with octadecylsilyl (ODS) groups (C18) or octyl (C8) groups are preferred, and columns containing packing materials with octadecylsilyl (ODS) groups (C18) are particularly preferred. Columns containing packing materials with octadecylsilyl (C18) groups are called C18 columns.

[0022] The packing material to be packed into the column can be spherical or crushed. The particle diameter is 3 to 10 μm, and in the case of spheres, it is preferably 5 μm or less, and more preferably about 3 μm. The size of the column to be used depends on the volume of the sample to be analyzed, but it may be, for example, an inner diameter of 1 to 6 mm and a length of 5 to 30 cm, preferably an inner diameter of 1 to 3 mm and a length of 10 to 20 cm.

[0023] The mobile phase can be water or a buffer solution, an organic solvent such as methanol or acetonitrile, or a mixture thereof. The higher the organic solvent content, the shorter the elution time. Examples of organic solvents used in the mobile phase include acetonitrile (MeCN), methanol, ethanol, and tetrahydrofuran (THF), with acetonitrile being preferred. The pH of the mobile phase is 3.0 to 8.0, preferably 3.0 to 5.0, and more preferably 4.0. The pH can be adjusted by adding an acid or alkali to the mobile phase. Examples of acids used for adjustment include organic acids such as trifluoroacetic acid, formic acid, and acetic acid, and inorganic acids such as hydrochloric acid. In the method of the present invention, the mobile phase used is preferably a mobile phase containing water and acetonitrile, such as a water-acetonitrile-formic acid system or a water-acetonitrile-triethylamine-acetic acid system.

[0024] Water and organic solvent may be mixed at a volume ratio of 1:1 to 9:1, preferably 2:1 to 9:1. Separation may be performed by gradient separation or isocratic separation, but gradient separation is preferred. Gradient separation involves extracting glycans using a concentration gradient of organic solvent and water in the mobile phase, for example, a concentration gradient of acetonitrile / water of 6:4 to 9:1.

[0025] Liquid chromatography may be performed using a commercially available HPLC system. Column equilibration and flow rate can be appropriately set depending on the column size and sample volume. The sample injection volume during analysis may be appropriately determined depending on the type of sample, the size of the column used, and other factors, and is, for example, several tens of μL to several hundreds of μL, e.g., 10 μL to 200 μL, and preferably 10 μL to 100 μL. The flow rate of the mobile phase during liquid chromatography analysis can be appropriately determined depending on the required resolution, etc. It is usually 0.1 to 3.0 mL / min, preferably 0.1 to 1.5 mL / min, more preferably 0.1 to 0.5 mL / min, and particularly preferably 0.1 to 0.3 mL / min. The column temperature during analysis is usually 20 to 60°C, preferably 30 to 45°C.

[0026] For example, a Shim-pack Scepter C18-120 column (3 μm, 2.1 × 150 mm) was used as the column, and the following was performed at a flow rate of 0.2 mL / min at 35°C: Solvent A: "water" / "0.5 M acetic acid aqueous solution adjusted to pH 4.0 with triethylamine" mixed in a 9:1 (v / v) ratio (in this case, 0.5 M acetic acid-triethylamine aqueous solution pH 4.0 can be prepared as a 10-fold concentrated eluent and diluted to a final concentration of 0.05 M before use); Solvent B: "water" / "acetonitrile" / "0.5 M acetic acid aqueous solution adjusted to pH 4.0 with triethylamine" mixed in a 7:2:1 ratio. A mixture of the two phases in a (v / v) ratio is used as the mobile phase, and the ratio (A:B) of the mobile phases is changed linearly from 100:0 to 80:15-25 (V / V) over 40 to 120 minutes, preferably 60 to 100 minutes. Thereafter, the concentration of mobile phase B is increased to 100% and maintained for 1 to 20 minutes, preferably 2 to 10 minutes. Thereafter, re-equilibration with mobile phase A is performed and elution is carried out.

[0027] Conversion of standard curves and identification of target glycans using the standard curves The nine standards are separated by reversed-phase chromatography, and a standard curve is created with elution time plotted on the horizontal axis and R-values ​​(reverse-phase scale values: R-values) plotted on the vertical axis. In the present invention, the R-value refers to a standardized elution time obtained by the following method. Here, the R-value of standard substance #R-0 is set to 0, and the R-value of standard substance #R-8 is set to a value close to the elution time (minutes) of #R-8. For example, if #R-8 elutes around 70 minutes, the R-value of #R-8 should be set to 70. Corresponding to the elution time of #R-8, the R-value should be set to, for example, any value between 50 and 100, preferably any value between 60 and 80, and more preferably 70. For combinations of standards with the same structure except for the presence or absence of core fucose (#R-1 and #R-2, #R-3 and #R-4, #R-5 and #R-6, and #R-7 and #R-8), the difference in elution time of glycans differs depending on whether or not the core fucose residue is present. However, the R value is calculated so that the contribution to the difference in R value due to the presence or absence of the core fucose residue in the standards is constant. A standard curve is created by plotting the "elution time" and "R value" of the standards. In other words, the elution position of reversed-phase high-performance liquid chromatography is standardized by standardization using the reversed-phase scale to create a standard curve. The created standard curve is called the standardized standard curve. Standardization using the reversed-phase scale is called the reversed-phase scale method and is described in Kanta Yanagida et al., J. Chromatogr. A, 800 (1998) 187-198.

[0028] Specifically, the R value can be calculated by the method described in the Examples, and a standardized standard curve can be created.

[0029] The R value is calculated from the elution time of the target glycan using a standardized standard curve, and the glycan is identified.

[0030] In this case, a database of R values ​​for multiple glycans with known structures is separately created, and the R value of the target glycan is compared with the R values ​​of the glycans in the database to identify the structure of the target glycan. The number of glycans with known structures used in creating the database is 100 or more, preferably several hundred or more, for example, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, and more preferably 1000 or more.

[0031] Although the high-performance liquid chromatography separation conditions used to create the standard curve and the database must be the same, accurate R values ​​can be obtained even if the separation conditions are slightly different when analyzing the target substance. In particular, accurate R values ​​can be obtained even if the separation conditions are slightly different when using a C18 column as the reversed-phase column.

[0032] The present invention encompasses a method for analyzing sugar chain structures by reversed-phase liquid chromatography in which elution times are standardized using the elution time standardization method described above. Standardizing elution times enables highly accurate structural identification. [Example]

[0033] The present invention will be specifically explained by the following examples, but the present invention is not limited to these examples.

[0034] PA-glycans were fractionated using either a Shimadzu LC20A or Shimadzu LC10A HPLC system (Shimadzu Corporation). Separation of glycans was achieved similarly using either of the two HPLC systems. PA-glycans were detected using a fluorescence detector RF-10Axl (Shimadzu Corporation) or Waters 2475 (Waters) connected to the HPLC.

[0035] Reverse-phase (RP-) HPLC was performed using a Shim-pack Scepter C18-120 column (3 μm, 2.1 × 150 mm; Shimadzu Corporation) at a flow rate of 0.2 mL / min at 35 °C. Solvent A was a 9:1 (v / v) mixture of water and 0.5 M acetic acid (pH 4.0) adjusted with triethylamine. (In this case, the 0.5 M acetic acid-triethylamine solution, pH 4.0, was prepared as a 10-fold concentrated eluent and diluted to a final concentration of 0.05 M before use.) Solvent B was a 7:2:1 (v / v) mixture of water, acetonitrile, and 0.5 M acetic acid (pH 4.0) adjusted with triethylamine. Detailed HPLC settings, including column temperature, solvent, and gradient conditions, are shown in Figure 1. Specifically, the column was equilibrated with eluent A (eluent B, 0%) and maintained at this concentration for 3 min after sample injection and analysis (eluent B, 0%). The concentration of eluent B was then linearly increased to 22.5% over 81 min (until 84 min after analysis). The concentration of eluent B was then increased to 100% and maintained for 4 min (from 84 to 88 min), after which the column was re-equilibrated with eluent A for 15 min (eluent B, 0%). The elution time of each PA-glycan was converted to an R value using a modified reversed-phase scale, as well as to RP glucose units (RP-GU). The conversion of elution time to an R value was performed as follows. Eight (four sets) standard PA-N-glycans, #R-1 to #R-8, were used, as shown in Figure 3. The R values ​​were determined so that the shift in position due to core-α1,6-fucose contributed equally. The R value of the elution position of PA-Gal (#R-0) was set to 0, and the elution time of #R-0 was subtracted from the elution time of each glycan to calculate the R value in the same manner as above. Furthermore, the elution position of #R-8 was set.

[0036] The specific calculation method is shown below. 1. The elution times (E) of the reference materials #R-0, #R-1, #R-2, #R-3, #R-4, #R-5, #R-6, #R-7, and #R-8 are designated as E0, E1, E2, E3, E4, E5, E6, E7, and E8, respectively. The respective inverse phase scale values ​​(R) are R0, R1, R2, R3, R4, R5, R6, R7 and R8. 2. Take E on the horizontal axis and R on the vertical axis, and connect each point with a straight line. In this case, let the slope of the E0-E1-E2 section be a, the slope of the E3-E4 section be b, the slope of the E5-E6 section be c, and the slope of the E7-E8 section be d. a (E2-E1) = b (E4-E3) = c (E6-E5) = d (E8-E7) = P In other words, the partial elution time P of core fucose is assumed to be equal among the four pairs of PA glycans. 3. Furthermore, the slope of the E2~E3 section is set to 1 / 2(a+b), the slope of the E4~E5 section is set to 1 / 2(b+c), and the slope of the E6~E7 section is set to 1 / 2(c+d). In other words, the slope of each section is set to the average of the previous and next sections. 4. Also, set the R value of E0 to 0, R=0. 5. When E is as follows, define R as follows: When E0 to E2: R = P×E / (E2-E1) For E2 to E3: R = R2 + (E-E2) × 1 / 2 × P{1 / (E2-E1) + 1 / (E4-E3)} For E3 to E4: R = R3 + P × (E-E3) / (E4-E3) For E4 to E5: R = R4 + (E-E4) × 1 / 2 × P{1 / (E4-E3) + 1 / (E6-E5)} For E5 to E6: R = R5 + P × (E-E5) / (E6-E5) For E6~E7: R = R6 + (E-E6)×1 / 2×P{1 / (E6-E5)+1 / (E8-E7)} For E7~: R = R7 + P×(E-E7) / (E8-E7) 6. Furthermore, if we set R8 = 70, R will be the following value: R1 = P×E1 / (E2-E1) R2-R1 = P R3-R2 = 1 / 2×P×(E3-E2){1 / (E2-E1)+1 / (E4-E3)} R4-R3 = P R5-R4 = 1 / 2×P×(E5-E4){1 / (E4-E3)+1 / (E6-E5)} R6-R5 = P R7-R6 = 1 / 2×P×(E7-E6){1 / (E6-E5)+1 / (E8-E7)} R8-R7 = P The sum of the right-hand side of the above equation can be set to any value, 70 in this example, to find P, and by substituting this value into equation 5, the R value for any elution time E can be found.

[0037] FIG. 5 shows a standard curve obtained by standardizing the elution time of PA-glycans in reversed-phase chromatography, and also shows the method for converting R values. [Industrial Applicability]

[0038] The high performance liquid chromatography elution time standardization method using the reversed phase scale of the present invention can be used for the analysis of sugar chains in biological samples.

Claims

1. A method for standardizing elution times when separating sugar chains by reversed-phase high-performance liquid chromatography, comprising: (i) Nine types of glycans having the structures shown below were used as standards, including #R-0 and four pairs of standard substances with the same structure except for the presence or absence of core fucose: #R-1 and #R-2, #R-3 and #R-4, #R-5 and #R-6, and #R-7 and #R-8. (ii) Elution time normalization method, in which the elution time is normalized by converting the standard curve so that the difference in R value between the glycan with core fucose and the glycan without core fucose in each pair in (i) is constant:

2. 2. The method for normalizing elution time according to claim 1, wherein the biological sample is selected from the group consisting of serum, plasma, urine, and biological tissue.

3. 3. The elution time standardization method according to claim 1, wherein the sugar chains are separated by reversed-phase high performance liquid chromatography using a C18 column.

4. 4. The method for normalizing elution times according to claim 1, wherein a mobile phase containing water and acetonitrile is used as a mobile phase for reversed phase chromatography.

5. 5. The elution time standardization method according to claim 3, wherein the C18 column has an inner diameter of 1 to 3 mm and a length of 10 to 20 cm, and the flow rate of the mobile phase is 0.1 to 0.5 mL / min.

6. The method for normalizing elution times according to any one of claims 3 to 5, wherein a mobile phase is used in which Solvent A is a mixture of "water" and "0.5 M aqueous acetic acid adjusted to pH 4.0 with triethylamine" in a 9:1 (v / v) ratio, and Solvent B is a mixture of "water", "acetonitrile" and "0.5 M aqueous acetic acid adjusted to pH 4.0 with triethylamine" in a 7:2:1 (v / v) ratio.

7. A method for analyzing a sugar chain structure, comprising determining the R value of a sugar chain whose structure is to be identified using a standardized standard curve prepared by standardizing elution times using the elution time standardization method according to any one of claims 1 to 6, and comparing the R value with the R value of sugar chain structures whose structures are known.

8. The method for analyzing glycan structures according to claim 7, wherein the R values ​​of a plurality of glycans with known structures are measured using a standardized standard curve prepared by standardizing elution times using the elution time standardization method according to any one of claims 1 to 6, and the structure is identified by comparing the R values ​​of glycan structures of known structures in a database of R values ​​of glycan structures of known structures prepared in advance with the R value of the glycan whose structure is to be identified.

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