# US 12,110,309 B2 · Method for preparing analytical sample, analysis method, and kit for preparing analytical sample > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,110,309 B2 - Document ID: US12110309B2 - Status: Granted - Kind: B2 - Application number: 16/590,676 - Filed: 2019-10-02 - Published: 2020-04-09 - Granted: 2024-10-08 - Earliest claimed: 2018-10-03 - Assignees: Shimadzu Corporation - Applicants: Shimadzu Corporation - Inventors: Takashi Nishikaze - Web page: https://patentgrep.com/patent/US12110309B2 ## CPC - C07H 1/00: Processes for the preparation of sugar derivatives - C08B 37/006: {Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof} - G01N 33/6848: {Methods of protein analysis involving mass spectrometry} - G01N 2400/00: Assays, e.g. immunoassays or enzyme assays, involving carbohydrates - G01N 2400/10: Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters - G01N 2440/38: addition of carbohydrates, e.g. glycosylation, glycation - G01N 2560/00: Chemical aspects of mass spectrometric analysis of biological material - C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13) - C08B: POLYSACCHARIDES; DERIVATIVES THEREOF (polysaccharides containing less than six saccharide radicals attached to each other by glycosidic linkages C07H; fermentation or enzyme-using processes C12P19/00; sugar industry C13; production of cellulose D21) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract A method for preparing an analytical sample for analysis of a glycan contained in a sample includes: performing an amidation reaction that amidates a lactone structure included in the glycan through contacting the sample with a reaction solution that is basic; adding an acidic solution to the reaction solution after the reaction solution is subjected to the amidation reaction; and purifying the sample contained in the reaction solution after the acidic solution is added to the reaction solution by using a carrier for hydrophilic interaction chromatography. ## Claims ### Claim 1 A method for preparing an analytical sample for analysis of a glycan contained in a sample, the method comprising: performing an amidation reaction that amidates a lactone structure included in the glycan through contacting the sample with a reaction solution that has a pH of higher than 10; adding an acidic solution to the reaction solution after the amidation reaction; and purifying the sample contained in the reaction solution by using a carrier for hydrophilic interaction chromatography after the acidic solution is added to the reaction solution, wherein: the amidation reaction amidates the lactone structure included in the glycan by aminolysis based on an interaction between an amino group and a lactone; the reaction solution comprising at least one selected from the group consisting of ammonia, a primary amine, and a salt thereof; wherein the concentration of ammonia, a primary amine, or a salt thereof in the reaction solution is 0.5 M or more; a time during which the sample is in contact with the reaction solution for the amidation reaction is shorter than 30 minutes; performing a lactonization reaction that lactonizes at least a part of sialic acids included in the glycans before the amidation reaction, wherein in the lactonization reaction, at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid among the sialic acids is lactonized; an operation that reacts the sample with a dehydration condensation agent is not performed after the addition of the reaction solution to the sample, wherein: the reaction solution does not include a dehydration condensation agent that is reacted with the lactones, and wherein only contacting the sample with the amidation reaction solution is performed for the amidation reaction; pH of the reaction solution after the acidic solution is added to the reaction solution is 10 or lower; and the amidation reaction is performed in a liquid phase. ### Claim 2 (depends on 1) The method for preparing an analytical sample according to claim 1, the method further comprising: performing a lactonization reaction that lactonizes at least a part of sialic acids included in the glycans before the amidation reaction. ### Claim 3 (depends on 2) The method for preparing an analytical sample according to claim 2, wherein the lactonization reaction is performed through contacting the sample with a lactonization reaction solution containing a dehydration condensation agent. ### Claim 4 (depends on 3) The method for preparing an analytical sample according to claim 3, wherein the reaction solution to be used for the amidation reaction contains ammonia or an amine, the lactonization reaction solution further contains a nucleophilic agent that is reacted with a sialic acid included in the glycan, the nucleophilic agent is different from ammonia or the amine contained in the reaction solution to be used for the amidation reaction, and in the lactonization reaction, a part of the sialic acids is lactonized based on a linkage type of the sialic acid, and at least a part of the nucleophilic agent is linked to another part of the sialic acids through addition of the lactonization reaction solution to the sample. ### Claim 5 (depends on 2) The method for preparing an analytical sample according to claim 2, wherein at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid among the sialic acids is lactonized in the lactonization reaction. ### Claim 6 (depends on 3) The method for preparing an analytical sample according to claim 3, wherein at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid among the sialic acids is lactonized in the lactonization reaction. ### Claim 7 (depends on 4) The method for preparing an analytical sample according to claim 4, wherein at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid among the sialic acids is lactonized in the lactonization reaction. ### Claim 8 (depends on 4) The method for preparing an analytical sample according to claim 4, wherein α2,3-sialic acid is lactonized and a part of the nucleophilic agent is linked to α2,6-sialic acid through addition of the lactonization reaction solution to the sample in the lactonization reaction. ### Claim 9 (depends on 1) The method for preparing an analytical sample according to claim 1, wherein the acidic solution is a solution containing 0.2% by weight or more of trifluoroacetic acid. ### Claim 10 (depends on 1) An analysis method comprising: preparing an analytical sample by using the method for preparing an analytical sample according to claim 1; and analyzing the prepared analytical sample. ### Claim 11 (depends on 10) The analysis method according to claim 10, wherein the prepared analytical sample is analyzed through at least one of mass spectrometry and chromatography. ### Claim 12 (depends on 1) The method for preparing an analytical sample according to claim 1, wherein the reaction solution that has a pH of 12 or higher. ### Claim 13 (depends on 1) The method for preparing an analytical sample according to claim 1, wherein the amidation reaction amidates the lactone structure included in the glycan by aminolysis and the reaction solution comprising at least one selected from the group consisting of ammonia, an amine, and a salt thereof. ### Claim 14 (depends on 1) The method for preparing an analytical sample according to claim 1, wherein the amine is a primary amine. ### Claim 15 (depends on 1) The method for preparing an analytical sample according to claim 1, further comprising: performing a lactonization reaction that lactonizes at least one of α2,8-sialic acid and α2,9-sialic acid included in the glycans.