Method for purifying oxytocin, method for measuring the same, and kit

Abstract

The present invention addresses the problem of providing a simple oxytocin purifying method and measuring method and kit. The present invention pertains to an oxytocin purifying method, comprising treating a sample with an acid or a salt thereof and treating the sample treated with the acid or a salt thereof with a hydrophobic carrier, wherein the acid or a salt thereof is at least one acid or salt thereof selected from the group consisting of hydrochloric acid, acetic acid, sulfuric acid, sodium hydrogen sulfate, potassium hydrogen sulfate, lithium hydrogen sulfate, ammonium sulfate and mixtures thereof, and the hydrophobic carrier is a hydrophobic carrier having at least one functional group selected from the group consisting of C4-6 alkyl groups, alkylene glycol groups and phenyl group on the surface thereof, and a measuring method and kit comprising the purifying method.

Description

Technical Field

[0001] The present invention relates to a method for purifying oxytocin, a method for measuring the same, and a kit.

Background Art

[0002] Oxytocin is a peptide hormone composed of 9 amino acids synthesized in the hypothalamus and secreted from the posterior pituitary gland. As a method for measuring the oxytocin concentration in a biological sample, immunological measurement methods, HPLC (high performance liquid chromatography), LC / MS (liquid chromatography mass spectrometer method), etc. are commonly used. However, the oxytocin contained in the biological sample is trace, and since proteins such as albumin, peptides, and measurement interfering substances such as mucin contained in the biological sample bind to oxytocin, accurate quantification of oxytocin is difficult.

[0003] In the commercially available ELISA kit for oxytocin (Non-Patent Document 1) and the pretreatment method described in Non-Patent Document 2, a biological sample containing TFA (trifluoroacetic acid) and oxytocin is mixed, and a complex of oxytocin and a measurement interfering substance is adsorbed onto a gel (C18 column), and the measurement interfering substance is washed away from the gel with 0.1% TFA-H 2 O, and then the oxytocin fraction is eluted with a mixed solution of 95% acetonitrile and 5% of 0.1% TFA-H 2 O. Further, in order to prevent the adverse effect of TFA on the measurement of oxytocin, the oxytocin fraction needs to be concentrated by centrifugation, further dried under reduced pressure, and then restored with a measurement solvent. TFA in the pretreatment method described in Non-Patent Documents 1-2 is used as an ion pair reagent, and fractionation is performed using a C18 column while the complex of oxytocin and a measurement interfering substance is formed.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

[0005] The pretreatment methods described in Non-Patent Documents 1 - 2 have many steps and are complex to prevent the adverse effects of TFA on the measurement, and require a long time for pretreatment. Furthermore, in hospitals or the like without a centrifuge, it is difficult to implement the pretreatment methods of Non-Patent Documents 1 - 2. In view of the above situation, an object of the present invention is to provide a simple method for purifying oxytocin. [Means for Solving the Problems]

[0006] [1] A method for purifying oxytocin, comprising treating a sample with an acid or a salt thereof, and treating the sample treated with the acid or a salt thereof using a hydrophobic carrier, wherein the acid or a salt thereof is at least one acid or a salt thereof selected from the group consisting of hydrochloric acid, acetic acid, sulfuric acid, sodium bisulfate, potassium bisulfate, lithium bisulfate, ammonium sulfate, and mixtures thereof, and the hydrophobic carrier is a hydrophobic carrier having at least one functional group selected from the group consisting of an alkyl group having 4 to 6 carbon atoms, an alkylene glycol group, and a phenyl group on the surface. [2] The method according to [1], wherein the acid or a salt thereof is hydrochloric acid. [3] The method according to [1] or [2], wherein the hydrophobic carrier is a hydrophobic carrier having an alkyl group having 4 to 6 carbon atoms on the surface. [4] The method according to [1], wherein the acid or a salt thereof is hydrochloric acid, and the hydrophobic carrier is a hydrophobic carrier having an alkyl group having 4 to 6 carbon atoms on the surface. [5] The method according to any one selected from [1] to [4], wherein the sample is selected from the group consisting of a blood sample, urine, and saliva. [6] A method for measuring oxytocin, comprising the method for purifying oxytocin according to any one selected from [1] to [5]. [7] A kit for purifying oxytocin, comprising at least one or more acids or salts thereof selected from the group consisting of hydrochloric acid, acetic acid, sulfuric acid, sodium bisulfate, potassium bisulfate, lithium bisulfate, ammonium sulfate, and mixtures thereof, and a hydrophobic carrier having on its surface at least one or more functional groups selected from the group consisting of an alkyl group having 4 to 6 carbon atoms, an alkylene glycol group, and a phenyl group. [Effects of the Invention]

[0007] According to the present invention, oxytocin can be easily purified. [Modes for Carrying Out the Invention]

[0008] In this specification, when indicating the upper and lower limits of a range, unless otherwise specified, A to B indicates that it is A or more and B or less. Also, in this specification, "measurement" may include the meanings of quantification, semi - quantification, and qualitative determination. "Purification" in this specification means making the amount of oxytocin contained in a sample measurable, and may include the meanings of isolation, recovery, extraction, concentration, and pretreatment.

[0009] [The Method for Purifying Oxytocin of the Present Invention] The method for purifying oxytocin of the present invention (hereinafter referred to as the purification method of the present invention) comprises treating a sample with at least one or more acids selected from the group consisting of hydrochloric acid, acetic acid, sulfuric acid, sodium bisulfate, potassium bisulfate, lithium bisulfate, ammonium sulfate and mixtures thereof (hereinafter referred to as the acid treatment step), and treating the sample treated with the acid using a hydrophobic carrier having on its surface at least one or more functional groups selected from the group consisting of an alkyl group having 4 to 6 carbon atoms, an alkylene glycol group, and a phenyl group (hereinafter referred to as the hydrophobic carrier treatment step). According to the purification method of the present invention, oxytocin can be easily purified regardless of the equipment of the purification environment.

[0010] Oxytocin is a polypeptide consisting of 9 amino acids having the sequence of Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (SEQ ID NO: 1). The oxytocin in the present invention may be obtained from a biological sample (natural oxytocin) or produced by a genetic recombination method, and may also contain a precursor of oxytocin. Further, oxytocin may be a peptide (mutant type) into which a mutation functionally equivalent to natural oxytocin has been introduced. Such mutations include deletion or substitution of one or more amino acids, or addition of one or more amino acids. The amino acid sequence of the mutant type of oxytocin has at least 80% or more sequence identity with natural oxytocin, and may have 85% or more, 90% or more, 95% or more sequence identity.

[0011] Examples of the sample according to the present invention include biological samples obtained from a test animal, such as blood samples like serum, plasma, whole blood, buffy coat, cerebrospinal fluid, urine, saliva, semen, chest effusion, tear fluid, sputum, mucus, lymph fluid, ascites, pleural effusion, amniotic fluid, bladder washings, bronchoalveolar lavage fluid, etc. Blood samples, urine, and saliva are preferred. Further, since the present invention is rich in mucin which is a measurement interfering substance and binds to oxytocin, it is particularly useful for saliva. Examples of the test animal include mammals such as humans, monkeys, mice, rats, dogs, cats, pigs, rabbits, chimpanzees, etc. Humans, monkeys, mice, or rats are preferred, and humans are more preferred. The sample according to the present invention may be a sample derived from a medium (culture solution) in which cells or microorganisms are cultured. For example, the culture supernatant of a medium in which oxytocin is recombinantly expressed using animal cells, plant cells, or bacterial cells as hosts by genetic recombination methods, extracts obtained by lysing or disrupting cells, etc. may be mentioned. Since the medium may contain animal proteins such as albumin to which oxytocin binds, the present invention is also useful for the medium. The sample according to the present invention may be directly used as it is taken from the test animal or the medium, or may be subjected to pretreatment such as recovery, concentration, dilution with a buffer, etc., and filtration sterilization. These pretreatments may be appropriately performed according to conventional methods.

[0012] The acid treatment step according to the present invention is a step of treating a sample with at least one or more acids or salts thereof (hereinafter, the acid according to the present invention) selected from the group consisting of hydrochloric acid, acetic acid, sulfuric acid, sodium bisulfate, potassium bisulfate, lithium bisulfate, ammonium sulfate, and mixtures thereof. Specifically, in the acid treatment step according to the present invention, for example, by co-existing the sample according to the present invention and the acid according to the present invention, the pH of the solution after co-existence may be set to an acidic condition of less than 7, preferably pH 1.5 to 6.0, and more preferably pH 2.0 to 6.0. The amount of the acid according to the present invention co-existing with the sample according to the present invention is, for example, 1 to 50 times (v / v) with respect to the sample containing oxytocin, preferably 5 to 20 times (v / v), and more preferably 8 to 15 times (v / v). As the acid according to the present invention, hydrochloric acid is preferred because oxytocin can be obtained with a high recovery rate regardless of the type of the sample. The acid according to the present invention may be used alone or in combination of two or more. According to the acid treatment step according to the present invention, it is considered that oxytocin is separated from the complex formed by binding to measurement interfering substances such as proteins and peptides in the sample by treating the specimen with a specific acid.

[0013] The hydrophobic carrier treatment step according to the present invention is a step of treating the sample after the acid treatment step according to the present invention (hereinafter, the acid-treated sample) with a hydrophobic carrier having at least one or more functional groups selected from the group consisting of an alkyl group having 4 to 6 carbon atoms, an alkylene glycol group, and a phenyl group on the surface (hereinafter, the hydrophobic carrier according to the present invention). Specifically, it is performed by bringing the acid-treated sample into contact with the hydrophobic carrier according to the present invention and separating oxytocin from other components in the acid-treated sample. The hydrophobic carrier treatment step according to the present invention is, for example, adding the acid-treated sample to the hydrophobic carrier (suspension) filled in a column or the like and eluting it, or mixing and / or suspending the acid-treated sample in the hydrophobic carrier (suspension) in a container, and if necessary, recovering the supernatant after centrifugation.

[0014] In the hydrophobic carrier according to the present invention, the alkyl group having 4 to 6 carbon atoms may be linear, branched or cyclic. Specific examples of such alkyl groups include, for example, butyl groups such as n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, cyclobutyl group, etc., n-pentyl group, isopentyl group, sec-pentyl group, tert-pentyl group, neopentyl group, 2-methylbutyl group, 1,2-dimethylpropyl group, 1-ethylpropyl group, cyclopentyl group, etc. pentyl groups, n-hexyl group, isohexyl group, sec-hexyl group, tert-hexyl group, neohexyl group, 2-methylpentyl group, 1,2-dimethylbutyl group, 2,3-dimethylbutyl group, 1-ethylbutyl group, cyclohexyl group, etc. hexyl groups, a linear or branched alkyl group having 4 to 6 carbon atoms is preferred, and a butyl group is more preferred.

[0015] Examples of the alkylene glycol group in the hydrophobic carrier according to the present invention include, for example, oligoethylene glycol group, oligopropylene glycol group, oligo-1,3-propanediol group, oligo-1,4-butanediol group, polyethylene glycol group, polypropylene glycol group, poly-1,3-propanediol group, poly-1,4-butanediol group. Polyethylene glycol, polypropylene glycol group, poly-1,3-propanediol group, and poly-1,4-propanediol group may have, for example, 100 or more repeating units (alkylene glycol or alkanediol), and may have 100 to 500 units. Oligoethylene glycol group, oligopropylene glycol group, oligo-1,3-propanediol group, and oligo-1,4-butanediol group may have, for example, 2 to 99 repeating units (alkylene glycol or alkanediol), and may have 10 to 99 units.

[0016] The hydrophobic carrier according to the present invention has at least one or more functional groups selected from the group consisting of an alkyl group having 4 to 6 carbon atoms, an alkylene glycol group, and a phenyl group on the surface of a solid phase (base material). The solid phase is not particularly limited as long as it is insoluble. For example, polystyrene, carboxylated polystyrene, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylamide, polyglycidyl methacrylate, polypropylene, polyolefin, polyimide, polyurethane, polyester, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, silicone rubber, agarose, dextran, ethylene-maleic anhydride copolymer and other organic substances; glass, silicon oxide, diatomaceous earth, porous glass, sintered glass, alumina, silica gel, metal oxides and other inorganic substances; cellulose derivatives; porous polymers; iron, cobalt, nickel, magnetite, chromite and other magnetic substances; and those prepared using alloys of these magnetic substances as materials, etc. may be mentioned. Examples of the form of the solid phase include a gel form (polymer gel), microplate, tube, disk-shaped piece, particles (beads), etc., and a gel form (polymer gel) is preferred. The hydrophobic carrier according to the present invention may be used alone or in combination of two or more. The hydrophobic carrier according to the present invention may be filled in a column or accommodated in a container such as a microtube.

[0017] Examples of commercially available hydrophobic carriers according to the present invention include, for example, TOYOPEARL TM Ether-650 (Tosoh Corporation, oligoethylene glycol group), TOYOPEARL TM PPG-600 (Tosoh Corporation, oligopropylene glycol group), TOYOPEARL TM Hexyl-650 (Tosoh Corporation, hexyl group), TOYOPEARL TM Butyl-650 (Tosoh Corporation, butyl group), TOYOPEARL TM Phenyl-650 (Tosoh Corporation, phenyl group), etc. may be mentioned.

[0018] The functional groups on the surface of the hydrophobic carrier according to the present invention are preferably an alkyl group having 4 to 6 carbon atoms or an alkylene glycol group, more preferably a linear or branched alkyl group having 4 to 6 carbon atoms, an oligoethylene glycol group, a polyethylene glycol group, an oligopropylene glycol group, or a polypropylene glycol group, and even more preferably a butyl group. The functional groups on the surface of the hydrophobic carrier according to the present invention may be used alone or in combination of two or more.

[0019] The hydrophobic carrier treatment step according to the present invention may be carried out in the presence of a salt, and it is preferably carried out in the presence of a salt. Examples of the salt include sodium salts, potassium salts, magnesium salts, and ammonium salts. The salt concentration when the acid-treated sample is brought into contact with the hydrophobic carrier according to the present invention is, for example, 0.10 M to 5.0 M, and preferably 0.30 M to 1.0 M. By coexisting the salt, the solution (supernatant or eluate) obtained by the hydrophobic carrier treatment step according to the present invention becomes neutral, and the measurement of oxytocin becomes easy. Further, by adjusting the binding force of oxytocin and other components in the acid-treated sample to the hydrophobic carrier according to the present invention, the separation of oxytocin and other components becomes easy. Specifically, the hydrophobic carrier treatment step according to the present invention is, for example, contacting a suspension containing the hydrophobic carrier according to the present invention and an amount of salt such that the final concentration is in the above range with the acid-treated sample, or contacting the acid-treated sample containing an amount of salt such that the final concentration is in the above range with the hydrophobic carrier according to the present invention (if necessary, a suspension of the hydrophobic carrier according to the present invention).

[0020] The hydrophobic carrier treatment step according to the present invention specifically includes, for example, mixing or / and suspending a suspension of a polymer gel having an alkyl group or alkylene glycol group having 4 to 6 carbon atoms, which is the hydrophobic carrier according to the present invention, with the acid-treated sample in a container (if necessary, in the presence of 0.30 M to 1.0 M of the salt), and if necessary, recovering the supernatant after centrifugation. Further, the hydrophobic carrier treatment step according to the present invention includes, for example, adding the acid-treated sample to a suspension of a polymer gel having an alkyl group or alkylene glycol group having 4 to 6 carbon atoms, which is the hydrophobic carrier according to the present invention, filled in a column or the like (if necessary, in the presence of 0.30 M to 1.0 M of the salt), and recovering the solution eluted from the column.

[0021] <Method for Measuring Oxytocin> The method for measuring oxytocin of the present invention (hereinafter, the measurement method of the present invention) is a method for measuring oxytocin using a sample obtained by performing the purification method of the present invention. The measurement method of the present invention includes, for example, methods commonly used for measuring proteins or peptides such as immunoassay methods, HPLC (high performance liquid chromatography), and mass spectrometry. Examples of the principles of these measurement methods include sandwich methods, competitive methods, agglutination methods (immunoturbidimetry, immunonephelometry), immunochromatography, Luminescent Oxygen Channeling Immunoassay (LOCI method), Liquid-phase Binding Assay-ElectroKinetic Analyte Transport Assay (LBA-EATA method), capillary electrophoresis methods such as lectin electrophoresis, Western blot method, surface plasmon resonance method (SPR method), lectin column method, etc. Also, it may be a homogeneous measurement system or a heterogeneous measurement system. According to the present invention, since it is presumed that the binding to oxytocin by the anti-oxytocin antibody becomes easy, the immunoassay method is more useful. The label in the immunoassay method is not particularly limited, and examples include enzyme-linked immunosorbent assay (ELISA method), enzyme immunoassay (EIA method), radioimmunoassay (RIA method), fluorescence enzyme immunoassay (FEIA method), fluorescence immunoassay (FIA method), chemiluminescent enzyme immunoassay (CLEIA method), chemiluminescent immunoassay (CLIA method), electrochemiluminescent immunoassay (ECLIA method), etc.

[0022] <Kit for Purifying Oxytocin of the Present Invention> The kit for purifying oxytocin of the present invention (hereinafter, the kit of the present invention) includes (i) at least one or more acids or their salts (acids according to the present invention) selected from the group consisting of hydrochloric acid, acetic acid, sulfuric acid, sodium bisulfate, potassium bisulfate, lithium bisulfate, ammonium sulfate, and mixtures thereof, and (ii) a hydrophobic carrier having on its surface at least one or more functional groups selected from the group consisting of an alkyl group having 4 to 6 carbon atoms, an alkylene glycol group, and a phenyl group (hydrophobic carrier according to the present invention). By being used in the method of the present invention, the kit of the present invention can easily purify oxytocin.

[0023] The hydrophobic carrier having at least one or more acids or salts thereof selected from the group consisting of hydrochloric acid, acetic acid, sulfuric acid, sodium bisulfate, potassium bisulfate, lithium bisulfate, ammonium bisulfate, and mixtures thereof, and at least one or more functional groups selected from the group consisting of an alkyl group having 4 to 6 carbon atoms, an alkylene glycol group, and a phenyl group on the surface in the kit of the present invention is the same as that of the method for purifying oxytocin of the present invention, and the preferred ones and specific examples are also the same.

[0024] The acid according to the present invention in the kit of the present invention may be a composition containing other components in addition to the acid. Examples of the other components include preservatives (for example, sodium azide, salicylic acid, benzoic acid, etc.), reaction accelerators, excipients, buffers, and the like. According to the present invention, in addition to the kit of the present invention, a kit for measuring oxytocin containing any components used for measuring oxytocin by various measurement methods such as immunological measurement methods and mass spectrometry methods is also provided. Examples of the optional components include a sample diluent (a solution for diluting a sample), an antibody against oxytocin, a carrier, a labeling substance, a buffer solution, an enzyme solution, a substrate solution, and the like. Further, the kit of the present invention may include an instruction manual describing the purification method of the present invention.

Example

[0025] Hereinafter, the present invention will be specifically described based on Examples and Comparative Examples, but the present invention is not limited by these examples at all.

[0026] Experimental Example 1. Evaluation of the method for purifying oxytocin (1) Acid treatment In a 1.5 mL PP vial, oxytocin (Anygen) was added to a urine specimen (Fuji Film Wako Shibayagi Co., Ltd.) so as to have a known concentration (within the range of 4.0 to 1000 pg / mL). 20 μL of room-temperature 1M hydrochloric acid was added to each of 200 μL of the obtained urine specimen containing oxytocin and the same urine specimen without added oxytocin, stirred, and then allowed to stand for 10 minutes to obtain acid-treated samples. (2) Hydrophobic carrier treatment After that, 110 μL of a gel suspension [50% (w / v) TOYOPEARL TM Butyl-650M (Tosoh Corporation), phosphate buffer (0.1 M phosphate buffer, 1.5 M NaCl)] was added to 220 μL of the acid-treated sample, respectively, stirred, and then allowed to stand for 10 minutes. After stirring the gel, centrifugation (5000 g, 10 minutes, 4°C) was performed, and the supernatant was used as the sample for measurement. (3) Measurement by ELISA method Anti-Rabbit IgG Goat IgG (manufactured by Fujifilm Wako Shibayagi Co., Ltd.) purified by Protein G (manufactured by Cytiva) chromatography was immobilized on a 96-well microplate (manufactured by Thermo Fisher Scientific). 100 μL / well of an anti-oxytocin rabbit polyclonal antibody (manufactured by Air Plants Bio Co., Ltd.) solution was added and left standing at room temperature for 2 hours. After removing and washing the antibody solution, 50 μL / well of an oxytocin standard solution {an oxytocin standard solution prepared with Tris-HCl buffer [0.1 M Tris-HCl (manufactured by Fujifilm Wako Pure Chemical Corporation), 0.15 M NaCl (manufactured by Fujifilm Wako Pure Chemical Corporation), 0.1% BSA (manufactured by Merck), 0.01% Tween 20 (manufactured by Fujifilm Wako Pure Chemical Corporation), 1 mg / m EDTA (manufactured by Dojindo Laboratories)] so as to be in the range of 4.0 to 1000 pg / mL oxytocin}, or 50 μL / well of a specimen for measurement, and 50 μL / well of a 10 pg / mL biotin-labeled oxytocin solution [a solution prepared by binding biotin (manufactured by Dojindo Laboratories) to oxytocin (manufactured by Anygen) and dissolving it in the Tris-HCl buffer] were each added and stirred, and left standing at room temperature for 2 hours. The reaction solution was removed and washed, and then 100 μL / well of Avidin-HRP (manufactured by Thermo Fisher Scientific) prepared with the Tris-HCl buffer was added and left standing at room temperature for 30 minutes. The reaction solution was removed and washed, and then 100 μL / well of a luminescence reagent (ELISA-Star Chemiluminescent Peroxidase Substrate, manufactured by Fujifilm Wako Pure Chemical Corporation) was added to each, and the luminescence intensity was measured for each by the endpoint measurement method using Infinite M200 PRO (manufactured by Tecan). A calibration curve was created from the measured values of the oxytocin standard solution by the above ELISA method, and the oxytocin concentration was calculated based on the luminescence intensity of each specimen. The recovery rate (%) was calculated by multiplying the oxytocin concentration calculated from the calibration curve from the value obtained by subtracting the measured value of the "urine specimen without added oxytocin" from the measured value of the "urine specimen with added known-concentration oxytocin" by 100 and dividing by the oxytocin concentration (the value of the added known-concentration oxytocin) in the "urine specimen with added known-concentration oxytocin". The obtained recovery rates are shown in Table 1 below.

[0027]

Table 1

[0028] Example 2 - 15. Evaluation of Oxytocin Purification Method Except for following the conditions described in Table 1 above, after purifying oxytocin by the same method as in Example 1, measurement was performed by the ELISA method, and the recovery rate was calculated. The reagents in Table 1 are as follows. TOYOPEARL TM Ether - 650M (Tosoh Corporation) TOYOPEARL TM Phenyl - 650M (Tosoh Corporation) TOYOPEARL TM Hexyl - 650C (Tosoh Corporation) Serum sample (BioIVT), saliva sample (FUJIFILM Wako Shibayagi Co., Ltd.) The obtained recovery rates are shown in Table 1.

[0029] Comparative Example 1 - 5. Evaluation of Oxytocin Purification Method Except for using the serum sample itself as the measurement sample without performing the purification of oxytocin (acid treatment and hydrophobic carrier treatment), measurement was performed by the ELISA method in the same manner as in Example 1(3), and the recovery rate was calculated (Comparative Example 1). Also, except for following the conditions described in Table 1 above, after purifying oxytocin by the same method as in Example 1, measurement was performed by the ELISA method, and the recovery rate was calculated (Comparative Examples 2 - 5). In the table, "-" indicates that the corresponding treatment was not performed. The reagents in Table 1 are as follows. Sep - Pak C8 (Waters) The obtained recovery rates are shown in Table 1.

[0030] From Table 1, it was found that when oxytocin was measured without purification, the value was extremely high and measurement was impossible. Also, in the case of purification by only acid treatment with hydrochloric acid, only hydrophobic carrier treatment having a butyl group on the surface, or a combination of acid treatment with hydrochloric acid and hydrophobic carrier treatment with C8, the value was extremely high and measurement was impossible. On the other hand, it was found that by purification combining acid treatment with hydrochloric acid, sulfuric acid or acetic acid, and hydrophobic carrier treatment having a butyl group, a hexyl group or an oligoethylene glycol group on the surface, oxytocin can be purified with a high recovery rate and accurate measurement can be performed. In particular, when combining acid treatment with hydrochloric acid and hydrophobic carrier treatment having a butyl group on the surface, it was found to be particularly useful because oxytocin can be purified with a high recovery rate regardless of whether a serum specimen, a urine specimen or a saliva specimen is used as a sample.

Industrial Applicability

[0031] According to the oxytocin purification method, measurement method, and purification kit of the present invention, since it is possible to easily purify oxytocin, it is useful in the field of measuring oxytocin, particularly in the field of clinical tests.

Claims

**Claim 1** A method for purifying oxytocin, comprising treating a sample with an acid or a salt thereof and treating the sample treated with the acid or a salt thereof using a hydrophobic carrier, wherein the acid or a salt thereof is at least one acid or a salt thereof selected from the group consisting of hydrochloric acid, acetic acid, sulfuric acid, sodium bisulfate, potassium bisulfate, lithium bisulfate, ammonium sulfate, and mixtures thereof, the hydrophobic carrier is a hydrophobic carrier having on its surface at least one functional group selected from the group consisting of an alkyl group having 4 to 6 carbon atoms, an alkylene glycol group, and a phenyl group, and treating the sample treated with the acid or a salt thereof using a hydrophobic carrier is to mix the hydrophobic carrier and the sample treated with the acid or a salt thereof in the presence of 0.10 M to 5.0 M of a salt, stir, and recover the supernatant. **Claim 2** The method according to claim 1, wherein the acid or a salt thereof is hydrochloric acid. **Claim 3** The method according to claim 1, wherein the hydrophobic carrier is a hydrophobic carrier having on its surface an alkyl group having 4 to 6 carbon atoms. **Claim 4** The method according to claim 1, wherein the acid or a salt thereof is hydrochloric acid and the hydrophobic carrier is a hydrophobic carrier having on its surface an alkyl group having 4 to 6 carbon atoms. **Claim 5** The method according to claim 1, wherein the sample is selected from the group consisting of a blood sample, urine, and saliva. **Claim 6** A method for measuring oxytocin, comprising purifying oxytocin by the method according to any one of claims 1 to 5 and measuring oxytocin using the sample obtained by the above purification. ​

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