Methods of analyzing and / or extracting anion molecules contained in biological sample

Abstract

To provide methods of analyzing and / or extracting anion molecules contained in a biological sample.SOLUTION: A method of analyzing and / or extracting anion molecules contained in a biological sample is provided, the method comprising a step of bringing a biological sample into contact with an oxoacid molecule having one or more functional groups selected from monophosphate groups, diphosphate groups, and triphosphate groups, and having a total of three or more phosphate- and carboxyl groups.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present application relates to a method for the analysis and / or extraction of anionic molecules contained in a biological sample. [Background technology]

[0002] Various techniques are known for extracting or analyzing anionic molecules such as phospholipids, nucleotides, and glycolipids present in biological samples, and are appropriately selected depending on the type of anionic molecule to be extracted or analyzed. For example, techniques such as centrifugation, sedimentation, ethanol precipitation, liquid-liquid extraction methods such as the Folch method, filtration, suction filtration, ultrafiltration, gel filtration, dialysis, solid-phase extraction, and chromatography (e.g., ion exchange chromatography, affinity chromatography, normal phase chromatography, reverse phase chromatography, size exclusion chromatography, gas chromatography, and thin layer chromatography) are known.

[0003] However, anionic molecules in biological samples are non-specifically adsorbed to other proteins and / or metal parts of the equipment used, resulting in problems such as reduced analytical sensitivity and recovery rate. Although a method of adding molecules such as EDTA to a sample to reduce such non-specific adsorption (Non-Patent Document 1, Non-Patent Document 2, Patent Document 1) is known, the effect of this method is not sufficient. For this reason, a method for efficiently extracting or analyzing anionic molecules in biological samples is needed. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2021 / 111418 [Non-patent literature]

[0005] [Non-Patent Document 1] Analytical Chemistry, Vol. 90, pp. 9457-9464, 2018 [Non-Patent Document 2] Journal of the American Chemical Society, Vol. 103, No. 20, pp. 6152-6157, 1981 Summary of the Invention [Problem to be solved by the invention]

[0006] The present application aims to provide a method for analyzing and / or extracting anionic molecules contained in a biological sample. [Means for solving the problem]

[0007] The present application provides a method for analyzing and / or extracting anionic molecules contained in a biological sample, the method comprising a step of contacting the biological sample with an oxoacid molecule having one or more functional groups selected from a monophosphate group, a diphosphate group, and a triphosphate group, and having a total number of phosphate groups and carboxyl groups of 3 or more.

[0008] The present application also provides a kit for analyzing and / or extracting anionic molecules contained in a biological sample, the kit comprising an oxoacid molecule having one or more functional groups selected from a monophosphate group, a diphosphate group, and a triphosphate group, and the sum of the phosphate groups and the carboxyl groups is three or more. Effect of the Invention

[0009] The method of the present application makes it possible to efficiently analyze and / or extract anionic molecules contained in a biological sample. [Brief description of the drawings]

[0010] [Figure 1] Chromatographic analysis of lipid molecules that are highly adsorbed in the analytical system when four compounds containing a phosphate group were added to HPLC elution solvent A. [Diagram 2] Seven compounds containing phosphate groups were each added to HPLC elution solvent A, and the suppression of adsorption in the analytical system was examined for chromatographic peak tailing and quantification feasibility, and a four-level evaluation was performed. [Diagram 3] Targeted measurement of acyl-CoA (CoAC2:0, CoAC16:0 or CoAC17:1) in samples spiked with various additives (EDTA2K, H3PO4, MDPA, IP3, IP6, or IP6+EDTA2K). [Figure 4] S1P target measurement for samples containing various additives. [Diagram 5] PCa38:4 target measurement for samples with various additives. [Figure 6] PSa38:4 target measurement on samples spiked with various additives. [Figure 7] PIa38:4 target measurement for samples spiked with various additives. [Figure 8] PIPa38:4 target measurement on samples with various additives. [Figure 9] PIP2a38:4 target measurement for samples spiked with various additives. [Figure 10] LPIPa20:4 target measurement for samples containing various additives. [Figure 11] LPIP2a20:4 target measurement for samples spiked with various additives. [Figure 12] CoAC2:0 target measurement for samples containing various additives. [Figure 13] CoAC18:0 target measurement for samples spiked with various additives. [Figure 14] GD1 (d36:1) target measurement for samples with various additives. [Figure 15] GT1 (d36:1) target measurement for samples with various additives. [Figure 16]Non-targeted UDP-Glc measurement of samples containing various additives. [Figure 17] Non-targeted ADP measurement for samples containing various additives. [Figure 18] Non-targeted measurement of ATP in samples containing various additives. [Figure 19] S1P target measurement for samples spiked with various additives that have two or more anionic substituents in the molecule. [Figure 20] PSa38:4 target measurement on samples spiked with various additives that have two or more anionic substituents in the molecule. [Figure 21] PIa38:4 target measurement for samples spiked with various additives that have two or more anionic substituents in the molecule. [Figure 22] PIPa38:4 target measurement on samples spiked with various additives that have two or more anionic substituents in the molecule. [Diagram 23] PIP2a38:4 target measurement for samples spiked with various additives that have two or more anionic substituents in the molecule. [Figure 24] CoAC18:0 target measurement for samples spiked with various additives that have two or more anionic substituents in the molecule. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In this disclosure, when a numerical value is accompanied by the term "about," it is intended to include a range of ±10% of that value. For example, "about 20" is intended to include "18-22." A range of numerical values ​​includes all values ​​between and at the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20-30" is intended to include "18-33."

[0012] The present application provides a method for analyzing and / or extracting anionic molecules contained in a biological sample, the method comprising a step of contacting the biological sample with an oxoacid molecule having one or more functional groups selected from a monophosphate group, a diphosphate group, and a triphosphate group, and having a total number of phosphate groups and carboxyl groups of 3 or more.

[0013] The biological sample is not particularly limited as long as it contains components collected from a living organism. The biological sample may be derived from, for example, an animal, a plant, a cultured cell, a bacterium, a fungus, or a virus. The biological sample may be dissolved or suspended in water, an acid, an alkali, an organic solvent, or a mixture thereof, and may be further treated as necessary. In an embodiment, the biological sample is contained in a solution. Examples of solutions containing a biological sample include ammonium acetate, chloroform, methanol, ethanol, acetic acid, an acetic acid / methanol mixture (e.g., a 1% acetic acid / methanol solution), a chloroform / methanol mixture (e.g., a chloroform / methanol=2 / 1 solution), a chloroform / methanol / ethanol mixture (e.g., a chloroform / methanol / ethanol=1 / 2 / 2 solution), and a chloroform / methanol / ethanol / acetic acid mixture (e.g., a chloroform / methanol / ethanol / acetic acid=2 / 1 / 1 / 1 solution).

[0014] Biological samples derived from animals include blood, serum, plasma, urine, feces, cerebrospinal fluid, other body fluids and biological tissues, etc. Examples of animals include mammals such as mice, rats, hamsters, guinea pigs, cows, horses, pigs, sheep, monkeys, orangutans, chimpanzees, dogs, cats, and humans.

[0015] The anionic molecule contained in the biological sample is not particularly limited as long as it is a molecule that is negatively charged in the biological sample. The anionic molecule contained in the biological sample has, for example, a phosphate group, a carboxyl group, and / or a hydroxyl group. Examples of the anionic molecule contained in the biological sample include phospholipids, nucleotides, glycolipids, polysialylated sugars, monosaccharide phosphates, oligosaccharide phosphates, glycopeptides, and phosphorylated peptides.

[0016] Phospholipid means a lipid having phosphorus in the form of phosphoric acid. Examples of phospholipids include glycerophospholipids and sphingophospholipids. Phospholipids in this specification include phospholipids in which the fatty acid group of the phospholipid is replaced with a hydroxyl group. Examples of glycerophospholipids include phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP1), phosphatidylinositol diphosphate (PIP2), phosphatidylinositol triphosphate (PIP3), phosphatidylcholine (lecithin), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), cardiolipin (CL), and phosphatidic acid (PA). Examples of sphingophospholipids include sphingomyelin and sphingoethanolamine. In addition, examples of phospholipids in which the fatty acid group of these phospholipids has been replaced with a hydroxyl group include glycerophospholipids in which one fatty acid group has been replaced with a hydroxyl group, resulting in a fatty acid group number of 1, and glycerophospholipids in which two fatty acid groups have been replaced with hydroxyl groups, resulting in a fatty acid group number of 0. Examples of glycerophospholipids in which one fatty acid group has been replaced with a hydroxyl group, resulting in a fatty acid group number of 1, include lysophosphatidylinositol, lysophosphatidylinositol monophosphate (LysoPIP1), lysophosphatidylinositol diphosphate (LysoPIP2), lysophosphatidylinositol triphosphate (LysoPIP3), lysophosphatidylcholine (lysolecithin), lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylglycerol, and lysophosphatidic acid (LPA). Examples of glycerophospholipids in which two fatty acid groups have been replaced with hydroxyl groups, resulting in a fatty acid group number of zero, include glycerophosphorylcholine, glycerophosphorylethanolamine, glycerophosphorylserine, glycerophosphorylinositol, glycerophosphorylglycerol, and glycerophosphonic acid.

[0017] The fatty acid group constituting the phospholipid is not particularly limited. For example, it may be a saturated or unsaturated fatty acid group having 8 to 38 carbon atoms. Examples of the saturated or unsaturated fatty acid group having 8 to 38 carbon atoms include myristoleic acid, myristic acid, palmitoleic acid, palmitic acid, linoleic acid, linoleic acid, oleic acid, and stearic acid.

[0018] The base constituting the phospholipid is not particularly limited, and examples thereof include inositol, choline, ethanolamine, serine, glycerol, and a hydroxyl group bound to a phosphate group.

[0019] In certain embodiments, the phospholipid is phosphatidylinositol monophosphate (PIP1), phosphatidylinositol diphosphate (PIP2), phosphatidylinositol triphosphate (PIP3), lysophosphatidylinositol monophosphate (LysoPIP1), lysophosphatidylinositol diphosphate (LysoPIP2), lysophosphatidylinositol triphosphate (LysoPIP3), cardiolipin (CL), or a combination thereof.

[0020] A nucleotide refers to a substance in which a phosphate group is bound to a nucleoside. In the present specification, a nucleotide includes an oligonucleotide and a polynucleotide in which two or more nucleotides are linked. In an embodiment, the nucleotide is coenzyme A (CoA), acyl CoA, ATP, ADP, UTP, UDP, CTP, CDP, GTP, GDP, NAD, NADH, an oligonucleotide, a polynucleotide, a monosugar nucleotide, an oligosaccharide nucleotide, a derivative thereof, or a combination thereof. A monosugar nucleotide refers to a nucleotide to which a monosaccharide is bound. An oligosaccharide nucleotide refers to a nucleotide to which an oligosaccharide is bound. The number of monosaccharides constituting an oligosaccharide is, for example, 2 to 50.

[0021] Glycolipids refer to lipids bound to sugar chains. Glycolipids include sphingoglycolipids containing a glycosidic bond between sugar and sphingosine, and glyceroglycolipids containing an ether bond between sugar and glycerin. Examples of sphingoglycolipids include cerebrosides having one hexose molecule as a sugar, gangliosides containing sialic acid, and sulfatides containing sulfated sugars. Examples of gangliosides include GD1, GD2, GD3, GT1, GT2, GT3, GQ1, GQ2, GQ3, GP1, GP2, GP3, GM1, GM2, GM3, GM3, GM4, and LM1. In one embodiment, the glycolipid is GD1, GD2, GD3, GT1, GT2, GT3, GQ1, GQ2, GQ3, GP1, GP2, GP3, derivatives thereof, or combinations thereof. Such derivatives include acylated glycolipids, dehydrated glycolipids, glycolylated glycolipids, oxidized glycolipids, and the like.

[0022] The fatty acid group constituting the glycolipid is not particularly limited. For example, it may be a saturated or unsaturated fatty acid group having 8 to 38 carbon atoms. Examples of the saturated or unsaturated fatty acid group having 8 to 38 carbon atoms include myristoleic acid, myristic acid, palmitoleic acid, palmitic acid, linoleic acid, linoleic acid, oleic acid, and stearic acid.

[0023] Polysialylated saccharides refer to saccharides having two or more sialic acids. Polysialylated saccharides have, for example, two, three, four, five or more sialic acids. Polysialylated saccharides include, for example, gangliosides.

[0024] Monosaccharide phosphate refers to a monosaccharide having at least one phosphate group. Monosaccharide phosphates have, for example, one, two, three or more phosphate groups. Oligosaccharide phosphate refers to an oligosaccharide having at least one phosphate group. Oligosaccharide phosphates have, for example, one to ten or more phosphate groups.

[0025] A glycopeptide is a peptide in which one or more sugar chains are bound to a portion of the amino acids that constitute the peptide. Glycopeptides include oligosaccharide peptides in which oligosaccharides are bound to a portion of the amino acids that constitute the peptide. In this specification, peptide means at least two covalently bound amino acids, and includes proteins, polypeptides, and oligopeptides. Typical amino acids to which sugar chains are bound include asparagine (N-type sugar chain), serine, and threonine (O-type sugar chain). Examples of the constituent monosaccharides of the sugar chains that constitute glycopeptides include galactose, glucose, mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, and xylose.

[0026] A phosphopeptide refers to a peptide having at least one phosphate group. A phosphopeptide can have, for example, one, two, three, four, five or more phosphate groups.

[0027] In some embodiments, the anionic molecule contained in the biological sample has two or more functional groups selected from a phosphate group, a carboxyl group, and a hydroxyl group. In some embodiments, the anionic molecule contained in the biological sample is a phospholipid, a nucleotide, and / or a glycolipid.

[0028] The oxoacid molecule of the present application is not particularly limited as long as it has one or more functional groups selected from monophosphate, diphosphate, and triphosphate groups in the molecule, and the total number of phosphate and carboxyl groups is 3 or more. In some embodiments, the oxoacid molecule of the present application contains one, two, three, four, five, six, or more monophosphate groups. In further embodiments, the oxoacid molecule of the present application contains three to six monophosphate groups. In another embodiment, the oxoacid molecule of the present application contains one monophosphate group and one diphosphate group. In another embodiment, the oxoacid molecule of the present application contains one triphosphate group.

[0029] The phosphate group of the present application may be a monophosphate group, or a plurality of phosphate groups may be linked together. For example, a diphosphate group has two phosphate groups linked together, and a triphosphate group has three phosphate groups linked together. The phosphate group of the present application may also be contained within the molecule as, for example, a phosphate diester or a phosphate triester.

[0030] The oxoacid molecules of the present application have at least one phosphate group, since they have one or more functional groups selected from monophosphate, diphosphate, and triphosphate groups. The oxoacid molecules of the present application may contain three or more phosphate groups, may contain two phosphate groups and one or more carboxyl groups, or may contain one phosphate group and two or more carboxyl groups. In some embodiments, the oxoacid molecules of the present application have a total of three to six phosphate and carboxyl groups. In some embodiments, the oxoacid molecules of the present application have one, two, three, four, five, six, or more phosphate groups. In further embodiments, the oxoacid molecules of the present application have three to six phosphate groups.

[0031] Examples of oxoacid molecules of the present application include uridine triphosphate (UTP), 5-phospho-D-ribose 1-diphosphate (PRDP), pentose triphosphate, pentose tetraphosphate, pentose pentaphosphate, inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5), inositol hexaphosphate (IP6), (2R,3R)-2,3-bis(phosphonatooxy)succinic acid (CAS No. 1610451-96-1), and salts thereof. In some embodiments, the oxoacid molecule is uridine triphosphate (UTP), 5-phospho-D-ribose 1-diphosphate (PRDP), inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5), inositol hexaphosphate (IP6), (2R,3R)-2,3-bis(phosphonatooxy)succinic acid (CAS No. 1610451-96-1), and salts thereof. In some embodiments, the oxoacid molecule is inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5) or inositol hexaphosphate (IP6). In some embodiments, the oxoacid molecule is inositol triphosphate (IP3), inositol tetraphosphate (IP4) or inositol hexaphosphate (IP6). In certain embodiments, the oxoacid molecule is inositol hexaphosphate (IP6).

[0032] The method of the present application may be a method for extracting anionic molecules contained in a biological sample, comprising the steps of: (i) adding the oxoacid molecule to a biological sample; and (ii) extracting the anionic molecules from the biological sample to which the oxoacid molecules have been added.

[0033] The step of extracting anionic molecules can be performed using known extraction means, and is not particularly limited. Those skilled in the art can appropriately select the extraction means according to the type of biological sample and anionic molecules. Examples of the extraction means include liquid-liquid extraction methods such as centrifugation, precipitation, ethanol precipitation, and Folch method, filtration, suction filtration, ultrafiltration, gel filtration, dialysis, solid-phase extraction, and chromatography (e.g., ion exchange chromatography, affinity chromatography, normal phase chromatography, reverse phase chromatography, size exclusion chromatography, gas chromatography, and thin layer chromatography). The liquid-liquid extraction method also includes, for example, a method in which an extraction solution is directly added to a container containing a biological sample and then collected. In one embodiment, when the anionic molecule to be extracted is a phospholipid, a nucleotide, and / or a glycolipid, the extraction means can be centrifugation, liquid-liquid extraction, solid-phase extraction, chromatography, or a combination thereof. In another embodiment, when the anionic molecule to be extracted is a phospholipid, a nucleotide, and / or a glycolipid, the extraction means can be centrifugation, liquid-liquid extraction, reverse phase chromatography, or a combination thereof.

[0034] In one embodiment, the extraction method of the present application is centrifugation. Specifically, a biological sample containing the oxoacid molecule is centrifuged, and a supernatant or precipitate containing the anionic molecule is collected. Those skilled in the art can appropriately set the conditions for centrifugation depending on the type of biological sample and anionic molecule.

[0035] In another embodiment, the extraction means of the present application is a liquid-liquid extraction method. Those skilled in the art can appropriately set the conditions of the liquid-liquid extraction method according to the types of biological samples and anionic molecules. When the anionic molecules are phospholipids, the liquid-liquid extraction method is, for example, the Folch method. For example, the biological sample containing the oxoacid molecules is added to a mixture of chloroform:methanol=2:1 and stirred, and then the chloroform layer containing the anionic molecules, which are phospholipids, is collected by centrifugation, whereby the anionic molecules can be extracted.

[0036] The concentration of the oxoacid molecule in the biological sample is not particularly limited and can be appropriately set depending on the type of the oxoacid molecule and the extraction means, etc. For example, when the oxoacid molecule is IP6, the concentration of the oxoacid molecule after addition to the biological sample can be 10 μM to 1 M, 100 μM to 500 mM, or 1 mM to 100 mM, for example, about 10 mM.

[0037] The oxoacid molecule is added to the biological sample, for example, as a solution containing the oxoacid molecule. The addition may be performed by directly adding the biological sample to the solution, or by adding the solution to a solution containing the biological sample. The solution is not particularly limited as long as it contains the oxoacid molecule, and can be appropriately prepared by a person skilled in the art. The solvent of the solution may be an organic solvent such as methanol, ethanol, or chloroform, water, or a mixture thereof. In some embodiments, the solvent is ammonium acetate, chloroform, methanol, ethanol, acetic acid, an acetic acid / methanol mixture (e.g., a 1% acetic acid / methanol solution), a chloroform / methanol mixture (e.g., a chloroform / methanol / ethanol=2 / 1 solution), a chloroform / methanol / ethanol mixture (e.g., a chloroform / methanol / ethanol=1 / 2 / 2 solution), or a chloroform / methanol / ethanol / acetic acid mixture (e.g., a chloroform / methanol / ethanol / acetic acid=2 / 1 / 1 / 1 solution). The concentration of the oxoacid molecule in the solution can be adjusted so that the concentration of the oxoacid molecule after addition to the biological sample is 10 μM to 1 M, 100 μM to 500 mM, 1 mM to 100 mM, or about 10 mM.

[0038] The method of the present application may be a method for analyzing anionic molecules contained in a biological sample, comprising the steps of: (i) adsorbing anionic molecules in a biological sample onto a column and / or a flow path; (ii) adding a separation solvent containing the oxoacid molecules to the column and / or flow path, and eluting the anion molecules from the column and / or flow path; and (iii) detecting the eluted anionic molecules.

[0039] A column for analyzing anionic molecules contained in a biological sample can be a known column and is not particularly limited. A person skilled in the art can appropriately select a column depending on the type of biological sample and anionic molecules. Examples of the column include an ion exchange chromatography column, an affinity chromatography column, a normal phase chromatography column, and a reverse phase chromatography column. In an embodiment, the column is a reverse phase chromatography column. In an embodiment, the biological sample is a sample obtained by the extraction method of the present application.

[0040] The column of the present application may also be a column for supercritical fluid chromatography (SFC) using the above-mentioned chromatography, a column for supercritical fluid chromatography mass spectrometry (SF / MS), a column for supercritical fluid chromatography tandem mass spectrometry (SFC / MS / MS), a column for high performance liquid chromatography (HPLC), a column for liquid chromatography mass spectrometry (LC / MS), or a column for liquid chromatography tandem mass spectrometry (LC / MS / MS). Mass spectrometry includes quadrupole mass spectrometry, tandem quadrupole mass spectrometry, time-of-flight mass spectrometry, and ion trap mass spectrometry. In an embodiment, the mass spectrometry is quadrupole mass spectrometry or tandem quadrupole mass spectrometry.

[0041] The flow path for analyzing anionic molecules contained in a biological sample is a path from a container containing the biological sample to an analytical device, such as a container containing the biological sample, an injection needle, an injector, a tube connecting the injector to a column, and a tube from the column to an analytical device such as a mass spectrometer.

[0042] When the anionic molecule is a phospholipid, a nucleotide, or a glycolipid, the column of the present application may be a column for chromatography. In an embodiment, the column of the present application is a column for reversed-phase chromatography. The column for reversed-phase chromatography is not particularly limited as long as it can adsorb anionic molecules, and may be, for example, a porous silica gel whose surface is modified with an octadecylsilyl group (ODS group). It is also possible to use a commercially available column, for example, Acquity UPLC BEH C18 (Waters).

[0043] The separation solvent used in the present application is not particularly limited as long as it can elute anionic molecules from the column, and can be appropriately selected depending on the type of column and anionic molecules. The separation solvent may be used in one liquid form, or in two or more liquid forms for gradient elution. In the case of gradient elution, for example, 100% of the first separation solvent (0% of the second separation solvent) is passed through, and then a gradient is applied to increase the proportion of the second separation solvent until the proportion of the second separation solvent reaches 100%. The magnitude of the gradient may be appropriately adjusted depending on the performance of the column, and is not particularly limited.

[0044] The separation solvent used in reversed-phase chromatography is not particularly limited as long as it can elute anionic molecules from the column. For example, separation solvent A containing the oxoacid molecule of the present application, 5 mmol / L ammonium acetate, and 0.1 mmol / L phosphoric acid, and separation solvent B containing 0.5 mmol / L ammonium acetate in acetonitrile:methanol=4:1 solution may be used.

[0045] The concentration of the oxoacid molecule in the separation solvent can be appropriately set depending on the type of the oxoacid molecule, etc. For example, when the oxoacid molecule is IP6, the concentration of the oxoacid molecule can be 20 nM to 20 mM, 200 nM to 2 mM, or 2 μM to 200 μM, for example, about 20 μM.

[0046] The means for detecting the eluted anionic molecules is not particularly limited and can be appropriately selected according to the type of the anionic molecules. For example, mass spectrometry, spectroscopic analysis (e.g., absorbance detection, fluorescence detection, evaporative light scattering detection, differential refractive index detection, optical rotation detection, circular dichroism detection), electrical conductivity detection, and electrochemical detection can be mentioned. Conditions for detection can be appropriately set by those skilled in the art. In an embodiment, the means for detecting the eluted anionic molecules is spectroscopic analysis or mass spectrometry. In an embodiment, the means for detecting the eluted anionic molecules is mass spectrometry.

[0047] The present application also provides a kit for analyzing and / or extracting anionic molecules contained in a biological sample, the kit comprising an oxoacid molecule having one or more functional groups selected from a monophosphate group, a diphosphate group, and a triphosphate group, and the sum of the phosphate groups and the carboxyl groups is three or more.

[0048] Examples of the biological sample, anionic molecule, and oxoacid molecule in this embodiment are as described above. The contents of such a kit are not particularly limited, but may include reagents and devices used for analyzing and / or detecting anionic molecules. The kit may also include buffer solutions, reaction vessels, and instructions. The present disclosure provides, for example, the following embodiments. [1] A method for analyzing and / or extracting anionic molecules contained in a biological sample, the method comprising a step of contacting the biological sample with an oxoacid molecule having one or more functional groups selected from a monophosphate group, a diphosphate group, and a triphosphate group, and having a total number of phosphate groups and carboxyl groups of 3 or more. [2] The method according to [1] above, wherein the anionic molecule is a molecule having two or more functional groups selected from a phosphate group, a carboxyl group, and a hydroxyl group. [3] The method according to [1] or [2] above, wherein the anionic molecule is a phospholipid, a nucleotide, a glycolipid, a polysialylated sugar, a monosaccharide phosphate, an oligosaccharide phosphate, a glycopeptide, and / or a phosphorylated peptide. [4] The method according to any one of [1] to [3] above, wherein the phospholipid is phosphatidylinositol monophosphate (PIP1), phosphatidylinositol diphosphate (PIP2), phosphatidylinositol triphosphate (PIP3), lysophosphatidylinositol monophosphate (LysoPIP1), lysophosphatidylinositol diphosphate (LysoPIP2), lysophosphatidylinositol triphosphate (LysoPIP3), cardiolipin (CL), or a combination thereof. [5] The method according to any one of [1] to [3] above, wherein the nucleotide is CoA, acyl-CoA, ATP, ADP, UTP, UDP, CTP, CDP, GTP, GDP, NAD, NADH, an oligonucleotide, a polynucleotide, a monosaccharide nucleotide, an oligosaccharide nucleotide, a derivative thereof, or a combination thereof. [6] The method according to any one of [1] to [3] above, wherein the glycolipid is GD1, GD2, GD3, GT1, GT2, GT3, GQ1, GQ2, GQ3, GP1, GP2, GP3, a derivative thereof, or a combination thereof. [7] The method according to any one of [1] to [6] above, wherein the oxoacid molecule is a molecule containing a total of 3 to 6 phosphate groups and carboxyl groups, or one triphosphate group. [8] The method according to any one of [1] to [7] above, wherein the oxoacid molecule is a molecule containing one triphosphate group. [9] The method according to any one of [1] to [8] above, wherein the oxoacid molecule is selected from the group consisting of uridine triphosphate (UTP), 5-phospho-D-ribose 1-diphosphate (PRDP), pentose triphosphate, pentose tetraphosphate, pentose pentaphosphate, inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5), inositol hexaphosphate (IP6), (2R,3R)-2,3-bis(phosphonatooxy)succinic acid, and salts thereof.

[10] The method according to any one of [1] to [9] above, wherein the oxoacid molecule is selected from the group consisting of uridine triphosphate (UTP), 5-phospho-D-ribose 1-diphosphate (PRDP), inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5), inositol hexaphosphate (IP6), (2R,3R)-2,3-bis(phosphonatooxy)succinic acid, and salts thereof.

[11] The method according to

[10] above, wherein the oxoacid molecule is inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5) or inositol hexaphosphate (IP6).

[12] A method for extracting anionic molecules contained in a biological sample according to any one of [1] to

[11] above, comprising the following steps: (i) adding the oxoacid molecule to a biological sample; and (ii) extracting the anionic molecules from the biological sample to which the oxoacid molecules have been added.

[13] The method according to

[12] above, wherein the step of extracting anionic molecules from a biological sample is carried out by centrifugation, liquid-liquid extraction, reverse phase chromatography, or a combination thereof.

[14] The method according to

[12] or

[13] above, wherein the concentration of the oxoacid molecule after the addition in step (i) is 10 μM to 1 M (preferably 1 mM to 100 mM (more preferably about 10 mM)).

[15] The method according to any one of

[12] to

[14] above, wherein the biological sample is contained in a solution.

[16] The method according to

[15] above, wherein the solution is ammonium acetate, chloroform, methanol, ethanol, acetic acid, an acetic acid / methanol mixture (preferably, a 1% acetic acid / methanol solution), a chloroform / methanol mixture (preferably, a chloroform / methanol=2 / 1 solution), a chloroform / methanol / ethanol mixture (preferably, a chloroform / methanol / ethanol=1 / 2 / 2 solution), or a chloroform / methanol / ethanol / acetic acid mixture (preferably, a chloroform / methanol / ethanol / acetic acid=2 / 1 / 1 / 1 solution).

[17] A method for extracting an anionic molecule (preferably a phospholipid, a nucleotide, a glycolipid, a polysialylated sugar, a monosaccharide phosphate, an oligosaccharide phosphate, a glycopeptide, and / or a phosphopeptide (more preferably a phospholipid, a nucleotide, and a glycolipid)) contained in a biological sample, comprising: (i) adding to a biological sample an oxoacid molecule having one or more functional groups selected from a monophosphate group, a diphosphate group, and a triphosphate group, and having a total number of phosphate groups and carboxyl groups of 3 or more; (a) the oxoacid molecule is a molecule selected from the group consisting of uridine triphosphate (UTP), 5-phospho-D-ribose 1-diphosphate (PRDP), pentose triphosphate, pentose tetraphosphate, pentose pentaphosphate, inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5), inositol hexaphosphate (IP6), (2R,3R)-2,3-bis(phosphonatooxy)succinic acid, and salts thereof (preferably inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5), inositol hexaphosphate (IP6) (more preferably inositol hexaphosphate (IP6))); (b) a concentration of the oxoacid molecule after the addition in the step (i) is 10 μM to 1 M (preferably 1 mM to 100 mM (more preferably about 10 mM)); and (ii) extracting anionic molecules from the biological sample to which the oxoacid molecules have been added, (a) wherein said extraction is by centrifugation, liquid-liquid extraction, reverse phase chromatography, or a combination thereof (preferably reverse phase chromatography).

[18] A method for analyzing anionic molecules contained in a biological sample according to any one of [1] to

[11] above, comprising the following steps: (i) adsorbing anionic molecules in a biological sample onto a column and / or a flow path; (ii) adding a separation solvent containing the oxoacid molecules to the column and / or flow path, and eluting the anion molecules from the column and / or flow path; and (iii) detecting the eluted anionic molecules.

[19] The method according to

[18] above, wherein the column is a chromatography column.

[20] The method according to

[19] above, wherein the chromatography column is a reverse phase chromatography column.

[21] The method according to

[19] above, wherein the chromatography column is a column for supercritical fluid chromatography (SFC), supercritical fluid chromatography mass spectrometry (SF / MS), supercritical fluid chromatography tandem mass spectrometry (SFC / MS / MS), high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LC / MS), or liquid chromatography tandem mass spectrometry (LC / MS / MS).

[22] The method according to any one of the above

[18] to

[21] , wherein the concentration of the oxoacid molecule in the separation solvent is 20 nM to 20 mM (preferably 2 μM to 200 μM (more preferably about 20 μM)).

[23] The method according to any one of

[18] to

[22] above, wherein the step of detecting the eluted anionic molecules is carried out by spectroscopic analysis or mass spectrometry.

[24] A method for analyzing anionic molecules contained in a biological sample, comprising the steps of: (i) a step of adsorbing anionic molecules in a biological sample onto a column, (a) the column is a chromatography column, preferably a reverse phase chromatography column (more preferably a high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LC / MS), or liquid chromatography tandem mass spectrometry (LC / MS / MS) column); (ii) adding to the column a separation solvent containing an oxoacid molecule having one or more functional groups selected from a monophosphate group, a diphosphate group, and a triphosphate group, and the total number of phosphate groups and carboxyl groups being 3 or more, and eluting anionic molecules from the column, (a) the oxoacid molecule is a molecule selected from the group consisting of uridine triphosphate (UTP), 5-phospho-D-ribose 1-diphosphate (PRDP), pentose triphosphate, pentose tetraphosphate, pentose pentaphosphate, inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5), inositol hexaphosphate (IP6), (2R,3R)-2,3-bis(phosphonatooxy)succinic acid, and salts thereof (preferably inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5), or inositol hexaphosphate (IP6) (more preferably inositol hexaphosphate (IP6))); (b) the concentration of the oxoacid molecule in the separation solvent is 20 nM to 20 mM (preferably 2 μM to 200 μM (more preferably about 20 μM)); and (iii) detecting the eluted anionic molecules, (a) said detecting step is carried out by spectroscopic analysis or mass spectrometry.

[25] A kit for analyzing and / or extracting anionic molecules contained in a biological sample, comprising an oxoacid molecule having one or more functional groups selected from a monophosphate group, a diphosphate group, and a triphosphate group, and the total number of phosphate groups and carboxyl groups being 3 or more.

[26] A kit for analyzing and / or extracting anionic molecules contained in a biological sample, comprising an oxoacid molecule having one or more functional groups selected from a monophosphate group, a diphosphate group, and a triphosphate group, and the total number of phosphate groups and carboxyl groups is 3 or more, wherein the oxoacid molecule is selected from uridine triphosphate (UTP), 5-phospho-D-ribose 1-diphosphate (PRDP), pentose triphosphate, pentose tetraphosphate, pentose pentaphosphate, inositol triphosphate (IP3), , inositol tetraphosphate (IP4), inositol pentaphosphate (IP5), inositol hexaphosphate (IP6), (2R,3R)-2,3-bis(phosphonatooxy)succinic acid, and salts thereof (preferably inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5), or inositol hexaphosphate (IP6) (more preferably inositol hexaphosphate (IP6))). EXAMPLES

[0049] The present invention will be described in more detail below by showing examples, but the present invention is not limited to these examples in any way. In this disclosure, the abbreviations used may be those shown in Table 1. If an abbreviation is not specified, it represents the generally accepted meaning. [Table 1]

[0050] Improvement of LC-MS analytical sensitivity by adding various polyanions How it's done Collection of mouse brain tissue and preparation of tissue homogenate The samples used in this lipid analysis were prepared as follows: 10 volumes of 1% acetic acid in methanol cooled at -80°C were added to frozen cerebellar samples taken from male C57BL / 6J mice, and the samples were crushed with an ultrasonic homogenizer under ice cooling to prepare sample solutions, which were then stored at -80°C until extraction.

[0051] Standards, internal standards and reagents PCa16:0D31 / 18:1, PSa16:0D31 / 18:1, PIa16:0D31 / 18:1, PIP2a8:0 / 8:0, GM1d18:1 / 18:0d5, CoAC17:1, and Sph(d17:1 / 0:0)P were used as internal standards (IS). Reagents were diluted to the specified concentrations with ultrapure water and used. Commercially available special grade methanol, acetonitrile, ammonium acetate, and phosphoric acid were used, as well as those equivalent to HPLC and LC / MS grades. Ultrapure water was purified using a Millipore water purification system (Japan Millipore Corporation).

[0052] Method for extracting molecules from biological samples Example 1: For various internal standard substances, 1 mg / mL standard solutions were appropriately diluted with CM to prepare internal standard-added extraction solutions.

[0053] 50μL of sample solution was dispensed into a sample tube, 10μL of ultrapure water was added as sample: Folch(1), and 5μL each of 200mM IP6 and 1.2M ammonium acetate were added as sample: Folch+IP6(1), then 1.0mL of CM-IS solution was added, stirred for 1 minute, and left at room temperature for 1 hour. After centrifugation at 2500rpm and 4℃ for 10 minutes, the supernatant was dispensed into a vial, and chromatographic evaluation of molecules in various biological samples was performed by non-target measurement.

[0054] Example 2: For various internal standard substances, 1 mg / mL standard solution was diluted with chloroform / methanol / ethanol: 1 / 2 / 2 solution to prepare internal standard solution. Also, chloroform / methanol / ethanol / acetic acid = 2 / 1 / 1 / 1 diluted solution was prepared.

[0055] 50 μL of sample solution was dispensed into a sample tube, and 5 μL of each of the solutions to be tested (none, milliQ water, EDTA2K (10 mM, 20 mM), phosphoric acid (10 mM, 20 mM), MDPA (10 mM, 20 mM), IP6 (10 mM, 20 mM), IP3 (10 mM, 20 mM), IP6 + EDTA2K (10 mM, 20 mM)) was added, followed by 2,000 μL of IS solution and 100 μL of dilution solution, and stirring for 1 minute.

[0056] The sample was centrifuged at 2500 rpm, 4°C for 10 minutes, and the upper organic layer was collected and then dried under reduced pressure in a centrifugal dryer (1410 rpm, 30°C, 120 minutes). The residue was redissolved in an organic solvent and centrifuged at 2500 rpm, 4°C for 10 minutes. The supernatant was dispensed into vials and acyl-CoA target measurement was performed.

[0057] Example 3: For various internal standard substances, 1 mg / mL standard solutions were appropriately diluted with methanol, CM, or BM to prepare internal standard-added extraction solutions (hereinafter, IS diluted with methanol (M-IS), IS diluted with CM (CM-IS), and IS diluted with BM (BM-IS)).

[0058] Dispense 50μL of sample solution into sample tubes, add 200mM IP6 to samples No.5-7, 25, CDA to samples No.19-20, MDPA to samples No.21-22, 1.2M ammonium acetate to samples No.3, 6, 7, 11, 12, 15, 16, 20, 22, 26, 1.2M triethylamine / 50% methanol aqueous solution to samples No.4, 7, 8, 20μL and 40μL of physiological saline to samples No.17, 18, 15μL and 30μL of 200mM Na2SO4 to samples No.13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 30 μL of 1M ammonium formate was added to sample No. 24, 5 μL to sample No. 25, and 6 μL to sample No. 26. In addition, ultrapure water was added as shown in Tables 2 and 3.

[0059] To sample No.1, 1.0 mL of M-IS solution was added, to samples No.2-21, 1.0 mL of CM-IS solution was added, and to samples No.22-25, 500 μL of BM-IS solution was added, and then the mixture was stirred for 1 minute. The mixture was left at room temperature for 1 hour and centrifuged at 2500 rpm and 4°C for 10 minutes. The supernatant was dispensed into vials and target and non-target measurements were performed. The main additives and their concentrations in the samples after addition are shown in Tables 2 and 3. [Table 2] [Table 3]

[0060] Example 4: For various internal standard substances, 1 mg / mL standard solutions were diluted with an appropriate amount of methanol or CM to prepare internal standard-added extraction solutions (IS diluted with methanol (M-IS), IS diluted with CM (CM-IS)).

[0061] Into a sample tube, 50 μL of sample solution was dispensed for Samples No. 1 to 14, and 25 μL of sample solution was dispensed for Samples No. 15 to 27, and the additive aqueous solution and ultrapure water were added in the volumes shown in Tables 4 and 5, respectively.

[0062] 1.0 mL of M-IS solution was added to No. 1, and 1.0 mL of CM-IS solution was added to samples No. 2 to 27, and the mixture was stirred for 1 minute. The mixture was left at room temperature for 3 hours and centrifuged at 2500 rpm and 4°C for 10 minutes. The supernatant was dispensed into vials and the target measurement was performed. [Table 4] [Table 5]

[0063] Here, the compound names of Compounds 1 to 10 are shown in Table 6. [Table 6]

[0064] Equipment and materials used For the measurements, an LC-MS / MS system was used, in which a triple quadrupole mass analyzer: API4000QTRAP (AB Sciex) was connected to an LC instrument: Vanquish UHPLC system (Thermo Scientific) for targeted measurements, and an LC-MS system was used, in which an Orbitrap type high resolution mass analyzer: Q-Exactive HF (Thermo Scientific) was connected for non-target measurements. The separation column used was an Acquity UPLC BEH C18 (Waters, product no. 186002346, 1.7 μm particle size, 100 mm × 1 mm).

[0065] LC-MS / MS system target measurement conditions LC conditions Mobile Phase A: 5 mmol / L ammonium acetate, 0.1 mmol / L phosphoric acid, aqueous solution (add various additives to 20 μM as necessary) Mobile Phase B: 0.5 mmol / L ammonium acetate, acetonitrile / methanol; 4 / 1 Column temperature: 50℃ Injection volume: 3μL The LC gradient conditions are shown in Table 7: [Table 7]

[0066] MS / MS conditions Scan type:sMRM (total scan time: 0.8 sec) Collision Gas:Nitrogen Source Gas:Nitrogen Desolvation Gas:Nitrogen Ion Source:ESI The MS / MS measurement conditions are shown in Tables 8 and 9: [Table 8] [Table 9]

[0067] LC-MS system non-target measurement conditions LC conditions Mobile Phase A: 5 mmol / L ammonium acetate, 0.1 mmol / L phosphoric acid aqueous solution (add various additives to 20 μM as necessary) Mobile Phase B: 0.5 mmol / L ammonium acetate, acetonitrile / methanol; 4 / 1 Column temperature: 50℃ Injection volume: 3μL The LC gradient conditions are shown in Table 10: [Table 10]

[0068] MS conditions Scan type: Full MS Desolvation Gas:Nitrogen Ion Source:ESI Scan Range: m / z 120.000~1800.000 The MS measurement conditions are shown in Tables 11 and 12: [Table 11] [Table 12]

[0069] result Example 1 Four compounds containing phosphate groups were added to HPLC elution solvent A, and the chromatographic peak tailing and detection of lipid molecules with high adsorption in the analytical system were examined (Figure 1). The peak tailing was reduced for all lipids, PA, PS, PI, PIP, and PIP2, when IP6 was added compared to when IP6 was not added. In particular, PIP2 molecules could be detected with the addition of IP6 alone, demonstrating a remarkable adsorption suppression effect.

[0070] Seven compounds containing a phosphate group were added to HPLC elution solvent A, and the suppression of adsorption in the analytical system was examined for chromatographic peak tailing and quantification potential, as shown in Figure 2. A four-point evaluation was performed, and it was confirmed that IP6 showed the highest detectability and quantification potential and reduced peak tailing.

[0071] Example 2 Although the same mouse brain homogenate sample contained a constant amount of acyl-CoA, the detection value (detection amount) differed depending on the additive (Figure 3). The same results were obtained when a constant amount of commercially available non-natural internal standard (IS) CoAC17:1 was added to all samples. In detecting IS contained at a constant concentration in biological samples, the addition of IP6 showed a significant improvement in detection sensitivity compared to Cont, indicating that the difference in detection sensitivity due to the difference in additives is due to the difference in acyl-CoA adsorption in the presence of additives during the extraction process. Furthermore, this effect of IP6 was similar in the extraction of natural acyl-CoA from biological samples, indicating that the difference in detection sensitivity is not due to artifacts such as ionization suppression.

[0072] Therefore, IP6 was the most effective adsorption inhibitor for the extraction of polyanions: acyl-CoA from biological samples.

[0073] In acyl-CoA extraction, the addition of IP3 also improved detection sensitivity compared to other additives, indicating that the addition of a compound having three or more anionic substituents, such as phosphate groups, in the same molecule is effective in extracting polyanions from biological samples.

[0074] Example 3 Although the same mouse brain homogenate sample contained a certain amount of the analyte molecule, the detection value (detection amount) for polyanion molecule extraction and detection varied depending on the additive (Figures 4 to 18).

[0075] Regarding the extraction and detection of S1P, PCa38:4, PSa38:4, and PIa38:4, which have only one anion substituent such as a phosphate group in the molecule, there was no difference due to the presence or absence of additives or the type of additive (Figures 4-7), but the extraction and detection of polyanions such as PIPa38:4, PIP2a38:4, LPIPa20:4, LPIP2a20:4, CoAC2:0, CoAC18:0, GD1(d36:1), GT1(d36:1), UDP-Glc, ADP, and ATP, which have two or more anion substituents such as phosphate groups in the molecule, differed depending on the type of additive (Figures 8-18). In the case of MDPA and CDA, which have two phosphate groups in the molecule, the adsorption suppression effect was limited and was not effective for all polyanions. In contrast, IP6 showed stable high detection values ​​(detection amounts) for all polyanion molecules examined, demonstrating that IP6 is the most excellent additive for the extraction and detection of polyanion molecules.

[0076] Example 4 As polyanion molecule extraction additives, various known compounds other than IP6 that have two or more anionic substituents such as phosphate groups in the molecule were also examined (Figures 19 to 23).

[0077] Although the same mouse brain homogenate sample contained a constant amount of the analyte molecule, the detection value (detection amount) of a specific polyanion molecule varied depending on the additive.

[0078] Regarding the extraction of S1P, PSa38:4, and PIa38:4, which have only one anion substituent such as a phosphate group in the molecule, no difference was observed due to the presence or absence of additives or the type of additive (Figures 19-21), but the extraction of PIPa38:4, PIP2a38:4, and CoAC18:0, which have two or more anion substituents such as phosphate groups in the molecule, differed depending on the type of additive (Figures 22-24). In particular, IP4 (compound 2), which has four or more phosphate groups in the molecule, and IP6 showed stable high detection values ​​(detection amounts) for all of these molecules, and this study also showed that IP6 is the most excellent additive for the extraction and detection of polyanion molecules.

Claims

1. A method for analyzing and / or extracting anionic molecules contained in a biological sample, the method comprising the step of contacting the biological sample with an oxoacid molecule having one or more functional groups selected from a monophosphate group, a diphosphate group, and a triphosphate group, and having a total number of phosphate groups and carboxyl groups of 3 or more.

2. 2. The method of claim 1, wherein the anionic molecule is a phospholipid, a nucleotide, a glycolipid, a polysialylated sugar, a monosaccharide phosphate, an oligosaccharide phosphate, a glycopeptide, and / or a phosphopeptide.

3. 3. The method of claim 2, wherein the phospholipid is phosphatidylinositol monophosphate (PIP1), phosphatidylinositol diphosphate (PIP2), phosphatidylinositol triphosphate (PIP3), lysophosphatidylinositol monophosphate (LysoPIP1), lysophosphatidylinositol diphosphate (LysoPIP2), lysophosphatidylinositol triphosphate (LysoPIP3), cardiolipin (CL), or a combination thereof.

4. 3. The method of claim 2, wherein the nucleotide is CoA, acyl-CoA, ATP, ADP, UTP, UDP, CTP, CDP, GTP, GDP, NAD, NADH, an oligonucleotide, a polynucleotide, a monosaccharide nucleotide, an oligosaccharide nucleotide, a derivative thereof, or a combination thereof.

5. The method of claim 2, wherein the glycolipid is GD1, GD2, GD3, GT1, GT2, GT3, GQ1, GQ2, GQ3, GP1, GP2, GP3, a derivative thereof, or a combination thereof.

6. 2. The method of claim 1, wherein the oxoacid molecule is selected from the group consisting of uridine triphosphate (UTP), 5-phospho-D-ribose 1-diphosphate (PRDP), pentose triphosphate, pentose tetraphosphate, pentose pentaphosphate, inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5), inositol hexaphosphate (IP6), (2R,3R)-2,3-bis(phosphonatooxy)succinic acid, and salts thereof.

7. 7. The method of claim 6, wherein the oxoacid molecule is inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5), or inositol hexaphosphate (IP6).

8. An anionic molecule phospholipids that are phosphatidylinositol monophosphate (PIP1), phosphatidylinositol diphosphate (PIP2), phosphatidylinositol triphosphate (PIP3), lysophosphatidylinositol monophosphate (LysoPIP1), lysophosphatidylinositol diphosphate (LysoPIP2), lysophosphatidylinositol triphosphate (LysoPIP3), cardiolipin (CL), or combinations thereof; Nucleotides that are CoA, acyl-CoA, ATP, ADP, UTP, UDP, CTP, CDP, GTP, GDP, NAD, NADH, oligonucleotides, polynucleotides, monosaccharide nucleotides, oligosaccharide nucleotides, derivatives thereof, or combinations thereof, and Glycolipids such as GD1, GD2, GD3, GT1, GT2, GT3, GQ1, GQ2, GQ3, GP1, GP2, GP3, derivatives thereof, or combinations thereof is selected from the group consisting of 2. The method of claim 1, wherein the oxoacid molecule is selected from the group consisting of inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5), and inositol hexaphosphate (IP6).

9. A method for extracting anionic molecules contained in a biological sample according to any one of claims 1 to 8, comprising the following steps: (i) adding the oxoacid molecule to a biological sample; and (ii) extracting anionic molecules from the biological sample to which the oxoacid molecules have been added;

10. 10. The method of claim 9, wherein the step of extracting the anionic molecules from the biological sample is carried out by centrifugation, liquid-liquid extraction, chromatography, or a combination thereof.

11. The method according to claim 9, wherein the concentration of the oxoacid molecule after addition in step (i) is 10 μM to 1 M.

12. A method for analyzing anionic molecules contained in a biological sample according to any one of claims 1 to 8, comprising the following steps: (i) adsorbing anionic molecules in a biological sample onto a column and / or a flow path; (ii) adding a separation solvent containing the oxoacid molecules to the column and / or flow path, and eluting the anion molecules from the column and / or flow path; and (iii) detecting the eluted anionic molecules.

13. 13. The method of claim 12, wherein the column is a chromatographic column.

14. 14. The method of claim 13, wherein the chromatography column is a column for supercritical fluid chromatography (SFC), supercritical fluid chromatography mass spectrometry (SF / MS), supercritical fluid chromatography tandem mass spectrometry (SFC / MS / MS), high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LC / MS), or liquid chromatography tandem mass spectrometry (LC / MS / MS).

15. 13. The method of claim 12, wherein the concentration of the oxoacid molecule in the separation solvent is 20 nM to 20 mM.

16. 13. The method of claim 12, wherein the step of detecting the eluted anionic molecules is performed by spectroscopic analysis or mass spectrometry.

17. A kit for analyzing and / or extracting anionic molecules contained in a biological sample, comprising an oxoacid molecule having one or more functional groups selected from a monophosphate group, a diphosphate group, and a triphosphate group, and having a total number of phosphate groups and carboxyl groups of 3 or more.

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