Method for analyzing samples containing organic substances and reagent for analyzing organic substances

Abstract

The present invention provides a novel method for producing a derivative of an organic substance that contains one or more groups selected from the group consisting of a sulfanyl group, a selanyl group and a sulfino group. A method for producing a derivative of an organic substance, said method comprising a process for obtaining a derivative of an organic substance that contains one or more groups selected from the group consisting of a sulfanyl group, a selanyl group and a sulfino group by reacting the organic substance with an olefin compound under acidic conditions. With respect to this method for producing a derivative of an organic substance, the olefin compound contains an ethylene structure that has at least two electron-withdrawing groups (excluding halogen atoms).

Description

[Technical field]

[0001] The present invention relates to a method for producing a derivative of an organic substance, and a method for analyzing a sample containing an organic substance. [Background technology]

[0002] Thiols play important roles in living organisms, such as maintaining the redox state in the body, controlling protein function, and detoxifying xenobiotics such as heavy metals. Therefore, attempts have been made to analyze thiols in samples such as biological samples. For example, there is a technique for analyzing thiols with an electrochemical detector by utilizing an oxidation-reduction reaction on the electrode surface (Non-Patent Documents 1 and 2). In addition, attempts have been made to convert thiols into derivatives and analyze them (Non-Patent Documents 3 to 8). Known reactions of thiols include reactions with carbon-carbon double bonds (Non-Patent Documents 9 and 10). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Anal.Biochem.,407(2010)151-159 [Non-Patent Document 2] Talanta,84(2011)789-801 [Non-Patent Document 3] Anal. Biochem., 27(1969)502-522 [Non-Patent Document 4] Biomed.Chromatogr.,20(2006)656-661 [Non-Patent Document 5] Chromatographia (1996) 42:515 [Non-Patent Document 6] Journal of Chromatography A,Volume844,Issues1-2,4 June1999,Pages 361-369 [Non-Patent Document 7] Sci Rep,6(2016)21433 [Non-Patent Document 8] J Chromatogr B Analyt Technol Biomed Life Sci,1083(2018)12-19 [Non-Patent Document 9] European Journal of Organic Chemistry,Volume722, Issue 1,14.May 1969,Pages 222-224 [Non-Patent Document 10] ARKIVOC 2009(viii)187-198 Summary of the Invention [Problem to be solved by the invention]

[0004] However, in the analytical methods of Non-Patent Documents 1 and 2, thiols are analyzed without being derivatized, so that thiols are easily oxidized by dissolved oxygen in the sample or the eluent used in the analysis, making it difficult to obtain stable analytical results. In addition, electrochemical detectors do not have sufficient selectivity for thiols, and when measuring samples containing amino acids, sugars, and other complex impurities, the measurement may be hindered by the impurities. Furthermore, electrochemical detectors require maintenance such as cleaning of the electrodes, and are difficult to handle because the detection sensitivity easily fluctuates and becomes unstable with use.

[0005] In the techniques of Non-Patent Documents 3 to 6, thiol is derivatized under neutral to basic conditions. However, the present inventors found that, under neutral to basic conditions, the analysis of thiols is difficult because the oxidation reaction of thiols and the exchange reaction between thiols and disulfide compounds easily proceed. Therefore, the techniques of Non-Patent Documents 3 to 6, which perform the derivatization of thiols under neutral to basic conditions, may make it difficult to accurately analyze thiols.

[0006] On the other hand, in the techniques of Non-Patent Documents 7 and 8, thiols are derivatized under acidic conditions (pH 3.5), but the reactivity is poor and special conditions such as a long reaction time or application of microwaves are required. [Means for solving the problem]

[0007] As a result of intensive research, the present inventors have found that a derivative of an organic substance obtained by reacting a specific olefin compound with an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino under acidic conditions is useful for the analysis of an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino, and have completed the present invention. The above prior art does not teach or suggest either (a) reacting a specific olefin compound with an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino under acidic conditions, or (b) that a derivative of an organic substance obtained by such a reaction is useful for the analysis of an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino.

[0008] That is, the present invention provides the following.

[0009] [1] A method for producing a derivative of an organic substance comprising reacting an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino with an olefin compound under acidic conditions, wherein the olefin compound contains an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms), [2] The method for producing the derivative according to [1], wherein the ethylene structure has two electron-withdrawing groups. [3] A method for producing the derivative according to [1] or [2], wherein the two electron-withdrawing groups are bonded to the same carbon atom constituting the ethylene structure. [4] The method for producing a derivative according to any one of [1] to [3], wherein at least two electron-withdrawing groups are the same group. [5] The method for producing a derivative according to any one of [1] to [4], wherein the olefin compound is a compound represented by the following formula (I): [ka] (However, EWG 1 and E.W.G. 2 Each of the groups independently represents an electron-withdrawing group, and may form a ring together with the carbon atom to which they are attached. [6] The electron withdrawing group, or EWG 1 and E.W.G. 2 each independently represents -C(=O)-OR 1 , -S(=O)2-R 2 , -P(=O)(-OR 3 )2, cyano, alkyl substituted with halogen atoms, carboxy, nitro, -S(=O)-R 4 , -C(=O)-R 5 or -C(=O)-NR 6 R 7 and Where: R 1 , R 2 , R 3 , and R 4 each independently represents a monovalent hydrocarbon group or a monovalent heterocyclic group, which may have a substituent; R 5 , R 6 , and R 7 each independently represents a hydrogen atom, a monovalent hydrocarbon group, or a monovalent heterocyclic group, which may have a substituent. The method for producing a derivative according to any one of [1] to [5]. [7] The method for producing a derivative according to any one of [1] to [6], wherein the acidic condition is a condition of less than pH 6.0. [8] The method for producing a derivative according to any one of [1] to [7], wherein the organic substance is one or more selected from the group consisting of cysteine, reduced glutathione, γ-glutamylcysteine, cysteinylglycine, homocysteine, N-acetylcysteine, cysteine ​​persulfide, hypotaurine, glutathione persulfide, and peptidic compounds containing a cysteine ​​residue. [9] The organic substance further contains an amino group; The method for producing the derivative according to any one of [1] to [8], comprising reacting the organic substance with the olefin compound under acidic conditions, and then further reacting them under neutral or basic conditions to obtain a derivative of the organic substance.

[10] (1) A sample containing an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino; An olefin compound containing an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms), mixing under acidic conditions to obtain a treated sample containing a derivative of said organic material; and (2) A method for analyzing a sample containing an organic substance, comprising: analyzing a derivative of the organic substance in the treated sample.

[11] The analysis in step (2), (2a) isolating a derivative of the organic material from the treated sample; (2b) detecting a derivative of the separated organic material; A method for analyzing a sample containing an organic substance according to

[10] , comprising:

[12] The method for analyzing a sample containing an organic substance according to

[10] or

[11] , wherein the sample further contains a disulfide compound.

[13] The method for analyzing a sample containing an organic substance according to

[12] , wherein the disulfide compound is one or more compounds selected from the group consisting of oxidized glutathione and cystine.

[14] The organic substance further contains an amino group; Step (1) is (1') A method for analyzing a sample containing an organic substance according to any one of

[10] to

[13] , comprising mixing the sample with the olefin compound under acidic conditions, and then further mixing them under neutral or basic conditions to obtain a treated sample containing a derivative of the organic substance.

[15] A derivatization agent under acidic conditions for organic substances containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino, including olefinic compounds containing an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms).

[16] A reagent for analyzing an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino, comprising the derivatizing agent according to

[15] . Effect of the Invention

[0010] According to the present invention, there are provided a novel method for producing derivatives of organic substances containing one or more groups selected from the group consisting of sulfanyl (-SH), selanyl (-SeH), and sulfino (-S(=O)-OH); and a novel method for analyzing samples containing such organic substances. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is an example of a chromatogram showing the cysteine ​​peak. [Diagram 2] FIG. 2 is an example of a chromatogram showing the peak of NEM-Cys. [Diagram 3] FIG. 3 is an example of a chromatogram showing the EMM-Cys peak. [Figure 4] FIG. 4 is an example of a chromatogram showing a peak of hypotaurine derivatized with EMM. [Diagram 5] FIG. 5 is an example of a chromatogram showing peaks of cysteine ​​and persulfated cysteine ​​derivatized with EMM. [Figure 6] FIG. 6 is an example of a chromatogram showing the results of simultaneous analysis of Cys, Cys2, and amino acids using an amino acid analyzer. [Figure 7] FIG. 7 is an example of a chromatogram of a thiol derivative by BPSE. [Figure 8] FIG. 8 is an example of a chromatogram of a derivatized chiral thiol. [Figure 9] FIG. 9 is a graph showing the changes over time in the peak area values ​​of Cys and GSH at pH 8.0. [Figure 10] FIG. 10 is a graph showing the changes over time in the peak area values ​​of Cys and GSH at pH 7.0. [Figure 11] FIG. 11 is a graph showing the changes over time in the peak area values ​​of Cys and GSH at pH 6.0. [Figure 12] FIG. 12 is a graph showing the changes over time in the peak area values ​​of Cys and GSH at pH 2.5. [Figure 13] FIG. 13 is a graph showing the change over time in the peak area value of Cys at pH 8.0, pH 7.0, pH 6.0, and pH 2.5. [Figure 14] FIG. 14 is an example of a chromatogram showing a peak of derivatized cysteine ​​or a peak of derivatized cystine by EMM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described in detail below with reference to embodiments and examples. However, the present invention is not limited to the embodiments and examples shown below. In addition, the "agent" may be a single substance or a composition composed of two or more substances.

[0013] [1. Method for producing organic derivatives] A method for producing a derivative of an organic substance according to one embodiment of the present invention comprises reacting an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino with an olefinic compound under acidic conditions to obtain a derivative of the organic substance. Here, the olefin compound contains an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms).

[0014] [1.1.Organic substances] The organic material comprises one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino, preferably one or more groups selected from the group consisting of sulfanyl and sulfino, and more preferably sulfanyl, where sulfanyl is -(S) n It is a group represented by -SH, selanyl is a group represented by -SeH, and sulfino is a group represented by -(S=O)-OH. Hereinafter, "one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino" may be referred to as "group A".

[0015] In the present invention, sulfanyl is -(S) n It is a group represented by -SH (n is an integer of 0 or more). According to the method for producing a derivative according to the present embodiment, -(S) such as -S-SH and -SS-SH can be used. n Organic substances (hereinafter also referred to as persulfides) having a group represented by -SH (where n is an integer of 1 or more) can also be derivatized with olefin compounds. Such persulfides (e.g., cysteine ​​persulfide) play an important role in redox in vivo. According to the method for producing a derivative according to this embodiment, persulfides can be derivatized while maintaining their redox state, so that the redox state in the living body can be understood. From the viewpoint of the number of types and abundance of sulfanyl-containing organic substances (particularly natural organic substances in biological samples such as blood, saliva, urine, and feces), -(S) n In the group represented by -SH, n is preferably 0, 1, 2, or 3, more preferably 0, 1, or 2, even more preferably 0 or 1, and particularly preferably 0.

[0016] In the present invention, selanyl is a group represented by -SeH. Selenium atom is an atom belonging to group 16 in the periodic table, the same as sulfur atom, and shows the same reactivity as sulfur atom. Therefore, the reactivity of selenium atom in organic material to olefin compound is the same as the reactivity of sulfur atom in organic material to olefin compound, so that the olefin compound described below having ethylene structure that can react well with sulfur atom in organic material can also react well with selenium atom in organic material.

[0017] One or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino (-S(=O)-OH) may be contained in a single molecule of the organic substance. The organic substance may also have one or more (eg, two, three, four) functional groups in addition to one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino. Examples of functional groups include, but are not limited to, hydroxy, carboxy, amino, alkylamino, dialkylamino, alkyloxy, alkyloxycarbonyl, alkylcarbonyl, and alkylcarbonyloxy.

[0018] In one embodiment, the organic substance may contain an amino group in addition to the group A. Here, the amino group that the organic substance may contain in addition to the group A includes unsubstituted amino groups (-NH2), mono-substituted amino groups and di-substituted amino groups. The amino group that the organic substance may contain in addition to the group A is preferably one or more selected from the group consisting of unsubstituted amino groups and mono-substituted amino groups, and more preferably an unsubstituted amino group. The organic substance containing the group A may contain only one amino group or may contain multiple amino groups in one molecule. The number of amino groups contained in one molecule of the organic substance may be 1, 2, or 3, preferably 1 or 2, and more preferably 1.

[0019] The organic material may be derived from a sample as described below. The organic substance may be a polymeric compound or a low molecular weight compound, and is preferably a low molecular weight compound. A low molecular weight compound refers to a compound with a molecular weight of 1500 or less. The molecular weight of a low molecular weight compound may be 1200 or less, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, or 300 or less. The molecular weight of a low molecular weight compound may also be 30 or more, 40 or more, or 50 or more. The low molecular weight compound may be an amino acid (eg, cysteine, selenocysteine), a peptide compound, or a salt thereof. Here, a peptidic compound is a compound having a structure obtained by condensing two or more amino acid molecules. The organic substance may be a peptidic compound, which is a polymeric compound. Examples of the peptidic compound, which is a polymeric compound, include proteins such as albumin.

[0020] The organic substances may be natural or synthetic compounds.

[0021] Specific examples of organic substances include cysteine, reduced glutathione, γ-glutamylcysteine, cysteinylglycine, homocysteine, N-acetylcysteine, persulfated cysteine ​​(e.g., S-mercaptocysteine, S-disulfanylcysteine, S-trisulfanylcysteine), hypotaurine, persulfated glutathione, peptide compounds containing a cysteine ​​residue, allyl mercaptan, 2-furfurylthiol, 3-mercapto-3-methylbutylformate, 3-sulfanyl-1-hexanol, thiophene-2-ylmethanethiol, 1,6-hexanedithiol, 4-methyl-4-sulfanylpentan-2-one, 3-sulfanylpentan-2-one, thioterpineol, and 4-methoxy-2-methylbutane-2-thiol. The organic substance may be one kind alone or two or more kinds in combination. The organic substance is preferably one or more selected from the group consisting of cysteine, reduced glutathione, γ-glutamylcysteine, cysteinylglycine, homocysteine, N-acetylcysteine, persulfated cysteine ​​(e.g., S-mercaptocysteine, S-disulfanylcysteine, S-trisulfanylcysteine), hypotaurine, persulfated glutathione, and peptidic compounds containing a cysteine ​​residue. Here, the peptidic compound containing a cysteine ​​residue may be a low molecular weight compound (eg, an oligopeptide) or a high molecular weight compound (eg, a protein).

[0022] [1.2. Olefin compounds] An olefin compound is a compound that contains an ethylene structure (ethene structure) having at least two electron-withdrawing groups and can react with sulfanyl, selanyl, or sulfino in an organic substance via the ethylene structure, although in this specification, the electron-withdrawing group does not include a halogen atom.

[0023] The number of electron-withdrawing groups in the ethylene structure is usually 2 or more and usually 4 or less, preferably 2 or more and 3 or less, more preferably 2. When the number of electron-withdrawing groups is within the above range, the reactivity of the olefin compound with the organic substance is improved.

[0024] The electron-withdrawing group may be monovalent or divalent. The number of electron-withdrawing groups in an ethylene structure means the number of bonds between the ethylene structure and the electron-withdrawing groups. Therefore, when a certain electron-withdrawing group is a divalent group and the electron-withdrawing group is bonded to one ethylene structure via two bonds, the number of electron-withdrawing groups in the ethylene structure can be two.

[0025] In the olefin compound, it is preferable that two electron-withdrawing groups are bonded to the same carbon atom constituting the ethylene structure, which can improve the reactivity of the carbon atom other than the one to which the two electron-withdrawing groups are bonded in the ethylene structure.

[0026] When two electron-withdrawing groups are bonded to the same carbon atom in the ethylene structure, and when two electron-withdrawing groups are bonded to adjacent carbon atoms in the ethylene structure, the two electron-withdrawing groups may form a ring together with the carbon atoms to which they are bonded.

[0027] In the olefin compound, it is preferable that at least two of the electron-withdrawing groups in the ethylene structure are the same group.

[0028] In the olefin compound, at least two of the electron-withdrawing groups in the ethylene structure are the same group, and it is more preferable that the two identical electron-withdrawing groups are bonded to the same carbon atom constituting the ethylene structure.

[0029] It is more preferable that the olefin compound has an ethylene structure having two identical electron-withdrawing groups, the two identical electron-withdrawing groups being bonded to the same carbon atom constituting the ethylene structure, and that two identical groups (which may be hydrogen atoms) are bonded to the carbon atom other than the carbon atom to which the two electron-withdrawing groups are bonded. This can prevent the organic substance from being converted into two diastereomeric derivatives due to the reaction between the olefin compound and the organic substance.

[0030] The ethylene structure may have groups other than the electron-withdrawing group. Examples of groups other than the electron-withdrawing group that the ethylene structure may have include monovalent hydrocarbon groups, preferably one or more types selected from the group consisting of monovalent linear hydrocarbons and monovalent aromatic hydrocarbon groups, and more preferably one or more types selected from the group consisting of alkyls and aryls. Preferably, the ethylenic structure has no groups other than electron-withdrawing groups.

[0031] Examples of electron withdrawing groups include, but are not limited to, -C(=O)-OR 1 , -S(=O)2-R 2 , -P(=O)(-OR3 )2, cyano, alkyl substituted with halogen atoms (e.g., perfluoroalkyl such as trifluoromethyl, perchloroalkyl such as trichloromethyl), carboxy, nitro, -S(=O)-R 4 , -C(=O)-R 5 and -C(=O)-NR 6 R 7 Examples include: Here, R 1 , R 2 , R 3 , and R 4 each independently represents a monovalent hydrocarbon group or a monovalent heterocyclic group, which may or may not have a substituent. R 5 , R 6 , and R 7 each independently represents a hydrogen atom, a monovalent hydrocarbon group, or a monovalent heterocyclic group, which may or may not have a substituent.

[0032] Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

[0033] The monovalent chain hydrocarbon group means a hydrocarbon group that is composed only of a chain structure and does not include a cyclic structure in the main chain. However, the chain structure may be linear or branched. Examples of the monovalent chain hydrocarbon group include alkyl, alkenyl, and alkynyl. The alkyl, alkenyl, and alkynyl may be linear or branched.

[0034] The alkyl group is preferably an alkyl group having 1 to 12 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms, and even more preferably an alkyl group having 1 to 4 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of the substituent. Examples of the alkyl group having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0035] The alkenyl is preferably an alkenyl having 2 to 12 carbon atoms, more preferably an alkenyl having 2 to 6 carbon atoms, and even more preferably an alkenyl having 2 to 4 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of the alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0036] The alkynyl is preferably an alkynyl having 2 to 12 carbon atoms, more preferably an alkynyl having 2 to 6 carbon atoms, and even more preferably an alkynyl having 2 to 4 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of the alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0037] The monovalent chain hydrocarbon group is preferably an alkyl group.

[0038] The monovalent alicyclic hydrocarbon group means a hydrocarbon group that contains only alicyclic hydrocarbons as a ring structure and does not contain an aromatic ring, and the alicyclic hydrocarbon may be either monocyclic or polycyclic. However, it is not necessary to be composed only of alicyclic hydrocarbons, and it may contain a chain structure as a part of it. Examples of the monovalent alicyclic hydrocarbon group include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be either monocyclic or polycyclic.

[0039] The cycloalkyl is preferably a cycloalkyl having 3 to 12 carbon atoms, more preferably a cycloalkyl having 3 to 6 carbon atoms, and even more preferably a cycloalkyl having 5 to 6 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of the substituent. Examples of the cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0040] The cycloalkenyl is preferably a cycloalkenyl having 3 to 12 carbon atoms, more preferably a cycloalkenyl having 3 to 6 carbon atoms, and even more preferably a cycloalkenyl having 5 to 6 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of the cycloalkenyl having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0041] The cycloalkynyl is preferably a cycloalkynyl having 3 to 12 carbon atoms, more preferably a cycloalkynyl having 3 to 6 carbon atoms, and even more preferably a cycloalkynyl having 5 to 6 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of the substituent. Examples of the cycloalkynyl having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0042] The monovalent alicyclic hydrocarbon group is preferably a cycloalkyl group.

[0043] The monovalent aromatic hydrocarbon group means a hydrocarbon group containing an aromatic ring structure. However, it is not necessary that the aromatic ring is composed only of the aromatic ring, and it may contain a chain structure, an alicyclic hydrocarbon structure, or the like in part. Therefore, the monovalent aromatic hydrocarbon group may be an aralkyl. The aromatic ring may be either a monocyclic or polycyclic ring. As the monovalent aromatic hydrocarbon group, an aryl having 6 to 12 carbon atoms is preferable, an aryl having 6 to 10 carbon atoms is more preferable, and an aryl having 6 carbon atoms is even more preferable. The number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of the aryl having 6 to 12 carbon atoms include phenyl and naphthyl.

[0044] The monovalent aromatic hydrocarbon group is preferably phenyl.

[0045] Among these, the monovalent hydrocarbon group is preferably alkyl, cycloalkyl, or aryl, and more preferably alkyl or aryl.

[0046] A monovalent heterocyclic group refers to a group obtained by removing one hydrogen atom from a cyclic compound containing a heterocycle. As the heteroatom constituting the heterocyclic group, it is preferable to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom, and a silicon atom, and it is more preferable to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, and a nitrogen atom. The monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group.

[0047] The monovalent aromatic heterocyclic group refers to a heterocyclic group containing a ring having aromaticity. As the monovalent aromatic heterocyclic group, an aromatic heterocyclic group having 1 to 15 carbon atoms is preferable, an aromatic heterocyclic group having 1 to 9 carbon atoms is more preferable, and an aromatic heterocyclic group having 1 to 6 carbon atoms is even more preferable. The number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the monovalent aromatic heterocyclic group include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, purinyl, anthraquinolyl, carbazonyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, acridinyl, coumarinyl, xanthenyl, and phthalazinyl.

[0048] The monovalent non-aromatic heterocyclic group refers to a heterocyclic group that does not contain a ring having aromaticity. As the monovalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 2 to 15 carbon atoms is preferable, a non-aromatic heterocyclic group having 2 to 9 carbon atoms is more preferable, and a non-aromatic heterocyclic group having 2 to 6 carbon atoms is even more preferable. The number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the monovalent non-aromatic heterocyclic group include oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, pyrrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

[0049] Among these, the monovalent heterocyclic group is preferably a 5- or 6-membered heterocyclic group.

[0050] Examples of the substituent include: a monovalent hydrocarbon group (which may be further substituted with a halogen atom); a monovalent heterocyclic group (which may be further substituted with a halogen atom); -OR s1 (where R s1 represents a hydrogen atom or a monovalent hydrocarbon group; -(C=O)-R s2 (where R s2 represents a hydrogen atom or a monovalent hydrocarbon group; -(C=O)-OR s3 (where R s3 represents a hydrogen atom or a monovalent hydrocarbon group; -O-(C=O)-R s4 (where R s4 represents a hydrogen atom or a monovalent hydrocarbon group; -N(R s5 )2(where R s5 each independently represents a hydrogen atom or a monovalent hydrocarbon group; -(C=O)-N(R s6 )2(where R s6 each independently represents a hydrogen atom or a monovalent hydrocarbon group; -N(R s7 )-(C=O)-R s8 (where R s7 R represents a hydrogen atom or a monovalent hydrocarbon group. s8 represents a monovalent hydrocarbon group; -SO2-R s9 (where R s9 represents a hydroxyl or monovalent hydrocarbon group; -S(=O)-R s10 (where R s10 represents a hydroxyl or monovalent hydrocarbon group; Nitro, Cyano, and Halogen atoms Examples include: Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and preferably a fluorine atom or a chlorine atom.

[0051] The substituent is preferably at least one selected from the group consisting of a monovalent hydrocarbon group (which may be further substituted with a halogen atom), nitro, cyano, and a halogen atom.

[0052] The electron withdrawing group is Preferably, -C(=O)-OR 1 , -S(=O)2-R 2 , -P(=O)(-OR 3 )2, cyano, alkyl substituted with halogen atoms, carboxy, nitro, -S(=O)-R 4 , -C(=O)-R 5 and -C(=O)-NR 6 R 7 One or more selected from the group consisting of More preferably, -C(=O)-OR 1 , -S(=O)2-R 2 , -P(=O)(-OR 32) one or more selected from the group consisting of cyano, alkyl substituted with a halogen atom, and carboxy; More preferably, -C(=O)-OR 1 , -S(=O)2-R 2 , -P(=O)(-OR 3 ) 2, and cyano. Here, R 1 , R 2 , R 3 , R 4 , R 5 , R 6、 and R 7 is as defined above.

[0053] R 1 is preferably a monovalent hydrocarbon group, more preferably a monovalent chain hydrocarbon group, even more preferably an alkyl group, particularly preferably an alkyl group having 1 to 4 carbon atoms. R 2 is preferably a monovalent hydrocarbon group, more preferably a monovalent aromatic hydrocarbon group, even more preferably aryl, and particularly preferably phenyl. R 3 is preferably a monovalent hydrocarbon group, more preferably a monovalent chain hydrocarbon group, and even more preferably an alkyl group. R 4 is preferably a monovalent hydrocarbon group, more preferably a monovalent aromatic hydrocarbon group, and even more preferably aryl. R 5 is preferably a monovalent hydrocarbon group, more preferably a monovalent aromatic hydrocarbon group, and even more preferably aryl. R 6 is preferably a hydrogen atom or a monovalent hydrocarbon group, and more preferably a hydrogen atom. R 7 is preferably a hydrogen atom or a monovalent hydrocarbon group, more preferably a monovalent hydrocarbon group, even more preferably a monovalent linear hydrocarbon group or a monovalent aromatic hydrocarbon group, and particularly preferably an alkyl or aryl.

[0054] From the viewpoint of improving reactivity with organic substances, the olefin compound is preferably a compound represented by the following formula (I).

[0055] [ka]

[0056] In formula (I), EWG 1 and E.W.G. 2 each independently represents an electron-withdrawing group, and may form a ring together with the carbon atom to which they are attached.

[0057] Preferred examples of the electron-withdrawing group are the same as those mentioned above. EWG 1 and E.W.G. 2 are preferably the same group. This can prevent the derivative of the organic substance produced by the reaction of the olefin compound with the organic substance from being two diastereomeric derivatives.

[0058] The olefin compound can be produced by a conventionally known method. In addition, a commercially available product can be used as the olefin compound.

[0059] [1.3. Reaction conditions] In the production method of this embodiment, an organic substance is reacted with an olefin compound under acidic conditions. This can suppress the oxidation reaction of group A of the organic substance. Furthermore, when an oxide such as a disulfide compound coexists in the reaction system, the reaction between group A and the oxide can be suppressed. As a result, the organic substance can be efficiently converted into a derivative.

[0060] The term "under acidic conditions" means that the reaction of the organic substance and the olefin compound is carried out in an acidic solution. The organic substance and the olefin compound do not have to be completely dissolved in the acidic solution, and the reaction may be carried out in a state where the organic substance and the olefin compound are dispersed in the acidic solution. The acidic solution usually has a pH of less than 7, preferably less than 6.5, more preferably less than 6.0, even more preferably less than 6.0, particularly preferably less than 5.5, 5.25, 5.0, 4.75, 4.5, or 4.25, and is preferably at least 0.0, more preferably at least 2.0. By setting the pH of the acidic solution within the above range, it is possible to effectively suppress side reactions such as oxidation of organic substances and exchange reactions with disulfide compounds while suppressing decomposition of the derivative. Here, the pH of the acidic solution and the pH of the neutral or basic solution described below can be measured at a temperature of 25° C. using a pH meter that employs the glass electrode method.

[0061] The acidic solution is preferably a solution containing water. The acidic solution may contain an organic solvent in addition to water. The organic solvent is preferably a solvent that can be mixed with water at any ratio. Examples of such organic solvents include alcohol solvents such as methanol and ethanol; nitrile solvents such as acetonitrile; and aprotic polar solvents such as dimethylsulfoxide, dimethylformamide, dioxane, and tetrahydrofuran. When the acidic solution is a solution containing water, the weight ratio of the organic solvent in the solution to the water is preferably 50% by weight or less, more preferably 10% by weight or less, even more preferably 5% by weight or less, and usually 0% by weight or more. More preferably, the acidic solution is an aqueous solution.

[0062] The acidic solution usually contains an acid. Examples of the acid include inorganic acids and organic acids. Examples of the inorganic acids include phosphoric acid, hydrochloric acid, sulfuric acid, and perchloric acid. Examples of the organic acids include formic acid, oxalic acid, acetic acid, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, toluenesulfonic acid, and cyanoacetic acid. As the acidic solution, a buffer solution whose pH has been adjusted to a desired range may be used.

[0063] The reaction temperature under acidic conditions is not particularly limited, but is preferably 50° C. or lower, more preferably 45° C. or lower, even more preferably 40° C. or lower, and is preferably 5° C. or higher, more preferably 10° C. or higher, even more preferably 15° C. or higher.

[0064] The reaction time under acidic conditions is not particularly limited, but is preferably 60 minutes or less, more preferably 30 minutes or less, further preferably 15 minutes or less, and is usually 0 minutes or more.

[0065] Reactions under acidic conditions are usually carried out by mixing the organic substance and the olefinic compound in an acidic solution. There is no particular limitation on the method for mixing an organic substance and an olefin compound in an acidic solution, and examples thereof include: 1) a method in which an acidic solution containing an organic substance is prepared, and an olefin compound is added thereto and mixed; and 2) a method in which a solution containing an organic substance is prepared, and a buffer solution or the like is added thereto to adjust the pH to a desired range, and then the olefin compound is added thereto and mixed. From the viewpoint of suppressing the oxidation reaction of the organic substance, it is preferable to prepare an acidic solution containing the organic substance, and add the olefin compound to the obtained solution and mix it in. The olefin compound may be added in its original form or in the form of a solution dissolved in a solvent (water, organic solvent).

[0066] The concentration of the organic substance in the acidic solution is not particularly limited, but may be, for example, 0.1 μmol / L to 100 mmol / L. The amount of the olefin compound relative to the organic substance is not particularly limited, but may be, for example, 1 to 100 in molar ratio.

[0067] When the organic substance contains an amino group in addition to group A, the organic substance may be reacted with the olefinic compound under acidic conditions and then further reacted under neutral or basic conditions (preferably basic conditions) to obtain a derivative of the organic substance. As a result, group A contained in the organic substance reacts with the olefin compound under acidic conditions to be converted into a substituent that is stable against side reactions such as oxidation. Next, under neutral or basic conditions, an amino group (which may be an unsubstituted amino group, a monosubstituted amino group, or a disubstituted amino group) contained in the organic substance reacts with the olefin compound to be converted into a substituted amino group having a substituent derived from the olefin compound (if the amino group contained in the organic substance is a disubstituted amino group, a substituted ammonio group). According to this production method, both the group A contained in the organic substance and the amino group that the organic substance may contain react with the olefin compound, thereby obtaining a derivative in which both the group A and the amino group are converted.

[0068] Derivatives containing more substituted amino groups have advantages in production and analysis compared to derivatives containing amino groups before substitution. Usually, more substituted amino groups have improved hydrophobicity compared to amino groups before substitution. Therefore, in liquid chromatography, separation conditions for organic derivatives can often be easily set. On the other hand, derivatives containing less substituted amino groups (e.g., derivatives containing unsubstituted amino groups such as cysteine ​​and reduced glutathione) are highly hydrophilic, so that retention is weak in reversed-phase liquid chromatography and retention is extremely strong in hydrophilic interaction chromatography (HILIC), and separation conditions may not be easily set. In addition, derivatives containing more substituted amino groups often have higher detection sensitivity in mass spectrometry compared to derivatives containing amino groups before substitution.

[0069] "Under neutral or basic conditions" means that an organic substance reacted under acidic conditions (hereinafter also referred to as an acidic derivative) is reacted with an olefin compound in a neutral or basic solution. The acidic derivative and the olefin compound do not have to be completely dissolved in the solution, and the reaction may be carried out in a state where the acidic derivative and the olefin compound are dispersed in the solution.

[0070] The neutral or basic solution usually has a pH of 7 or more, preferably a pH of 7.5 or more, more preferably a pH of 8 or more, even more preferably a pH of 8.5 or more, and is preferably a pH of 11 or less, more preferably a pH of 10 or less, even more preferably a pH of 9.5 or less. By setting the pH of the neutral or basic solution within the above range, the reaction between the amino group that the organic substance may contain and the olefin compound can be promoted, and decomposition of the derivative can be suppressed.

[0071] The neutral or basic solution is preferably a solution containing water. The neutral or basic solution may contain an organic solvent in addition to water. The organic solvent is preferably a solvent that can be mixed with water in any ratio. Examples of such organic solvents include the organic solvents exemplified in the description of the acidic solution. When the neutral or basic solution is a solution containing water, the weight range of the organic solvent in the solution relative to the water may be the same as the preferred range mentioned in the description of the acidic solution. More preferably, the neutral or basic solution is an aqueous solution.

[0072] The basic solution usually contains a base. Examples of the base include inorganic bases and organic bases. Examples of the inorganic base include metal hydroxides (e.g., sodium hydroxide, potassium hydroxide, calcium hydroxide), metal carbonates (e.g., sodium carbonate, sodium bicarbonate, potassium carbonate), sodium phosphate, potassium phosphate, ammonia, sodium borate, and potassium borate. Examples of the organic base include amines (e.g., trimethylamine, triethylamine). As the neutral or basic solution, a buffer solution having a pH adjusted to a desired range may be used.

[0073] The reaction temperature under neutral or basic conditions is not particularly limited, but is preferably 50° C. or lower, more preferably 45° C. or lower, even more preferably 40° C. or lower, and is preferably 5° C. or higher, more preferably 10° C. or higher, even more preferably 15° C. or higher.

[0074] The reaction time under neutral or basic conditions is not particularly limited, but is preferably 60 minutes or less, more preferably 30 minutes or less, further preferably 15 minutes or less, and usually 0 minute or more.

[0075] The reaction under neutral or basic conditions is usually carried out by mixing the derivative and the olefin compound under acidic conditions in a neutral or basic solution.

[0076] The derivative under acidic conditions may be isolated from the acidic solution and then mixed with the olefin compound in a neutral or basic solution, or the pH of the acidic solution may be adjusted to a desired neutral or basic pH without isolating the derivative under acidic conditions from the acidic solution, and then mixed with the olefin compound in a neutral or basic solution.

[0077] When the derivative is mixed with the olefin compound in a solution without isolating it under acidic conditions as described above, the olefin compound may be newly added to the solution, or an excess amount of the olefin compound may be added during the reaction under acidic conditions, and the excess olefin compound may be used for the reaction under neutral or basic conditions.

[0078] The production of the organic derivative can be confirmed by, for example, liquid chromatography, mass spectrometry, or the like.

[0079] The method for producing a derivative according to the present embodiment may include any step other than the reaction step of reacting an organic substance with an olefin compound. Examples of the optional step include a step of diluting the reaction solution containing the derivative after the reaction step, and a step of isolating the derivative from the reaction solution.

[0080] In the derivatives of organic substances produced by the above-mentioned method, the group A is converted to a group that is more stable against oxidation, and therefore the derivatives of organic substances can be analyzed more accurately than when the organic substances are analyzed as they are.

[0081] [2. Analytical methods for samples containing organic substances] According to one embodiment of the present invention, there is provided a method for analyzing a sample containing an organic substance, comprising: (1) a sample containing an organic substance that includes one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino; An olefin compound containing an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms), mixing under acidic conditions to obtain a treated sample containing a derivative of said organic material; and (2) analyzing the derivatives of the organic material in the treated sample.

[0082] [2.1. Process (1)] In step (1), a sample containing an organic substance containing one or more groups (group A) selected from the group consisting of sulfanyl, selanyl, and sulfino is mixed with an olefin compound containing an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms) under acidic conditions to obtain a treated sample containing a derivative of the organic substance.

[0083] Examples and preferred examples of the organic substance include those mentioned above. A plurality of types of organic substances may be contained in the sample. Examples of samples containing organic substances include, but are not limited to, biological samples derived from living organisms, synthetic samples obtained by organic synthesis reactions, and non-biological samples such as environmental samples present in the natural environment. Examples of organisms from which biological samples are derived include animals such as mammals (e.g., humans, monkeys, mice, rats, rabbits, cows, pigs, horses, goats, and sheep), birds, insects, mollusks, microorganisms, and plants, with mammals being preferred. Types of biological samples include, for example, blood samples (e.g., whole blood, serum, and plasma), saliva, urine, feces, bile, sweat, tears, cerebrospinal fluid, culture medium used for culturing cells or microorganisms, and tissue and cell extracts. Examples of synthetic samples include, for example, reaction products obtained by organic synthesis reactions that produce organic substances, and culture media used for culturing cells or microorganisms. Examples of environmental samples include samples derived from soil, seawater, and freshwater.

[0084] The concentrations of the organic substance and the olefin compound in the sample are not particularly limited as long as they react to produce a derivative of the organic substance and the product can be analyzed. The concentrations of the organic substance and the olefin compound in the sample may be, for example, 0.001 μmol / L to 1000 mmol / L, preferably 0.01 μmol / L to 100 mmol / L, more preferably 0.1 μmol / L to 10 mmol / L, and even more preferably 1 μmol / L to 1 mmol / L.

[0085] The sample may be a sample that has been pretreated, for example by deproteinization. In the analysis method of the present embodiment, since the sample is treated under acidic conditions, it is not necessary to adjust the liquid property of the sample to neutral to basic after performing an acidic treatment such as a deproteinization treatment as a pretreatment, and the sample can be mixed with an olefin compound while keeping the liquid property of the sample acidic. Under acidic conditions, side reactions such as the oxidation reaction of group A and the exchange reaction between group A and disulfide are unlikely to proceed. Therefore, the side reactions can be suppressed from proceeding between the pretreatment and the analysis. As a result, the organic substance having group A can be analyzed more accurately.

[0086] The sample may contain a disulfide compound. A disulfide compound is a compound containing a group represented by -SS-. A disulfide compound may be generated by oxidation of a thiol. For example, information on the ratio of thiols (eg, cysteine, reduced glutathione) to disulfide compounds (eg, cystine, oxidized glutathione) in a biological sample is useful for knowing the redox state of the biological sample. In the analysis method of this embodiment, a group A such as sulfanyl, which is easily oxidized in an organic substance, is reacted with an olefin compound to convert the organic substance into a derivative, and then the analysis is performed. In addition, in the analysis method of this embodiment, the sample is treated under acidic conditions. As described above, under acidic conditions, the exchange reaction between thiol and disulfide compounds is unlikely to occur. Therefore, the amount of organic substance present in the sample can be accurately determined.

[0087] Examples of disulfide compounds that the sample may contain include compounds in which sulfanyl that the organic substance may have is converted to a disulfide through an intermolecular reaction, and more specifically, examples of such compounds include dimethyl disulfide, diethyl disulfide, oxidized glutathione, and cystine. The disulfide compound that the sample may contain may contain an amino group. Here, the amino group that the disulfide compound may contain includes, in addition to an unsubstituted amino group (-NH2), a mono-substituted amino group and a di-substituted amino group. The amino group that the disulfide compound may contain is preferably one or more selected from the group consisting of an unsubstituted amino group and a mono-substituted amino group, and more preferably an unsubstituted amino group. The disulfide compound may contain only one amino group or may contain multiple amino groups in one molecule. The number of amino groups that may be contained in one molecule of the disulfide compound may be 1, 2, 3, or 4, preferably 1, 2, or 3, and more preferably 1 or 2. In one embodiment, the sample comprises one or more selected from the group consisting of oxidized glutathione and cystine.

[0088] Examples and preferred examples of the olefin compound are the same as those mentioned above.

[0089] A sample containing an organic substance is mixed with an olefin compound under acidic conditions to obtain a treated sample containing a derivative of the organic substance. Preferably, the sample containing the organic substance is mixed with the olefin compound in an acidic solution. The type and pH of the acidic solution may be the same as those described in the preferred examples in [1.3. Reaction conditions].

[0090] The mixing method is not particularly limited, and examples thereof include: 1) a method in which a solution containing a sample containing an organic substance and an acidic solution having a desired pH value is prepared, and an olefin compound is added to the solution and mixed; and 2) a method in which a sample containing an organic substance is diluted with dilute hydrochloric acid, a solvent, etc. to make a solution, a buffer solution, etc. is added to the solution to adjust the pH to a desired range, and then an olefin compound is added and mixed.

[0091] The treatment temperature and treatment time are not particularly limited, but may be the same as the preferred examples of the reaction temperature and reaction time described in [1.3. Reaction conditions].

[0092] When the organic substance further contains an amino group in group A, step (1) preferably includes step (1'). In step (1'), the sample and the olefin compound are mixed under acidic conditions, and then further mixed under neutral or basic conditions to obtain a treated sample containing a derivative of the organic substance. As described above, the group A contained in the organic substance in the sample reacts with the olefin compound under acidic conditions and is converted into a substituent that is stable against side reactions such as oxidation. Next, under neutral or basic conditions, the amino group (which may be an unsubstituted amino group, a monosubstituted amino group, or a disubstituted amino group) contained in the organic substance reacts with the olefin compound and is converted into a substituted amino group (if the amino group contained in the organic substance is a disubstituted amino group, a substituted ammonio group) to obtain a derivative of the organic substance. As described above, the hydrophobicity of the more substituted amino group is improved, so that the derivative in which the less substituted amino group is converted into the more substituted amino group can be easily separated by easily setting the separation conditions in normal liquid chromatography. In addition, the derivative in which the less substituted amino group is converted into the more substituted amino group usually has an increased detection sensitivity in a mass spectrometer.

[0093] When a sample contains a compound containing a lightly substituted amino group (e.g., a disulfide compound containing an unsubstituted amino group), the compound is highly hydrophilic and therefore is weakly retained in reversed-phase liquid chromatography, but is extremely retained in hydrophilic interaction chromatography (HILIC), making it difficult to set separation conditions. Even if a sample contains a compound containing a low-substitution amino group, the compound containing a low-substitution amino group, such as a disulfide compound containing an unsubstituted amino group, reacts with the olefin compound under neutral or basic conditions to convert the amino group to a more substituted amino group (or to a substituted ammonio group if the amino group is a disubstituted amino group). As with derivatives of organic substances, compounds such as disulfide compounds in which the amino group has been converted to a more substituted amino group can be easily separated by easily setting separation conditions in normal liquid chromatography. In addition, the detection sensitivity in normal mass spectrometers is increased.

[0094] For example, an analytical method will be described for the case where the organic substance that may be contained in the sample is cysteine ​​and the disulfide compound that may be contained in the sample is cystine, which is a dimer of cysteine. In the treated sample under acidic conditions obtained by mixing the olefin compound with the sample under acidic conditions, it is considered that a derivative of cysteine ​​(first derivative) and cystine obtained by reaction of the sulfanyl group of cysteine ​​with the olefin compound are present.

[0095] A cysteine ​​derivative has one unsubstituted amino group, and cystine has two unsubstituted amino groups. This causes a large difference in hydrophobicity between a cysteine ​​derivative and a cystine derivative. Therefore, when analyzing by liquid chromatography, it may not be easy to set conditions for simultaneously analyzing a cysteine ​​derivative and cystine.

[0096] On the other hand, when a sample and the olefin compound are mixed under acidic conditions and then further mixed under neutral or basic conditions to obtain a treated sample under neutral or basic conditions, the treated sample is considered to contain a cysteine ​​derivative (second derivative) obtained by reaction of the sulfanyl group and amino group with the olefin compound, and a cystine derivative obtained by reaction of the amino group with the olefin compound.

[0097] Since the amino groups of cysteine ​​and cystine react with the olefin compound, the difference in hydrophobicity between the cysteine ​​derivative (second derivative) and the cystine derivative becomes small, and it becomes relatively easy to set conditions for simultaneously analyzing the cysteine ​​derivative and cystine during analysis by liquid chromatography.

[0098] Therefore, by including step (1') in step (1), organic substances containing an amino group in addition to group A in a sample can be analyzed accurately and easily.

[0099] In step (1'), the sample containing the organic substance and the olefin compound are preferably mixed in an acidic solution, and then further mixed in a neutral or basic solution. The type and pH of the neutral or basic solution may be the same as the preferred examples described in [1.3. Reaction conditions].

[0100] The method of mixing in a neutral or basic solution is not particularly limited. For example, a method of mixing a sample and the olefin compound in an acidic solution, then adding a basic solution to the resulting mixed solution (sample treated under acidic conditions) to adjust the pH of the mixed solution to a desired neutral or basic pH, and further mixing the sample and the olefin compound in the neutral or basic solution; a method of adding the mixed solution to a prepared neutral or basic solution and mixing the mixture can be mentioned.

[0101] The treatment temperature and treatment time under neutral or basic conditions are not particularly limited, but can be the same as the preferred examples of reaction temperature and reaction time under neutral or basic conditions described in [1.3. Reaction conditions].

[0102] A sample containing an organic substance and the olefin compound are mixed in an acidic solution, and then further mixed in a neutral or basic solution, whereby a treated sample containing a derivative of the organic substance can be obtained.

[0103] [2.2. Process (2)] In step (2), the derivatives of the organic substances in the treated sample are analyzed. Step (2) may include step (2a) and step (2b) in this order. [2.2.1. Process (2a)] In step (2a), a derivative of the organic substance is separated from the sample treated in step (1). The separation can be carried out by any method capable of separating the derivative from substances other than the derivative (eg, disulfide compounds) contained in the treated sample. Examples of the separation method include, but are not limited to, liquid chromatography, supercritical fluid chromatography (SFC), capillary electrophoresis (CE), and gas chromatography (GC). The separation method may be appropriately selected depending on the type and properties of impurities contained in the sample and the type and properties of the derivative of the organic substance. As the separation method, liquid chromatography is preferable, and high performance liquid chromatography (HPLC) is more preferable. These separation methods can be performed by conventionally known methods. These separation methods may be used alone or in combination.

[0104] Examples of liquid chromatography include reversed-phase liquid chromatography, normal-phase liquid chromatography, hydrophilic interaction chromatography (HILIC), ion-exchange chromatography, and size-exclusion chromatography.

[0105] Examples of stationary phases for reversed-phase liquid chromatography include, but are not limited to, silica gel modified with a hydrophobic compound such as octadecylsilane. Examples of mobile phases include, but are not limited to, organic solvents, aqueous solutions, and mixed solvents thereof. Preferred examples of organic solvents include acetonitrile, methanol, ethanol, isopropanol, and the like. Preferred examples of aqueous solutions include water, an aqueous formic acid solution, an aqueous ammonium formate solution, an aqueous trifluoroacetic acid solution, an aqueous acetic acid solution, an aqueous ammonium acetate solution, an aqueous ammonium bicarbonate solution, and a buffer solution.

[0106] Examples of stationary phases for normal phase liquid chromatography include, but are not limited to, silica gel and alumina, and examples of mobile phases include, but are not limited to, hexane, ethyl acetate, methylene chloride, isopropanol, ethanol, methanol, tetrahydrofuran, and mixtures thereof.

[0107] Examples of stationary phases for hydrophilic interaction chromatography include, but are not limited to, silica gel and silica gel modified with aminopropyl, amide, diol, or cyano. Examples of mobile phases include, but are not limited to, organic solvents, aqueous solutions, and mixed solvents thereof. Preferred examples of organic solvents include acetonitrile, methanol, ethanol, isopropanol, and the like. Preferred examples of aqueous solutions include water, aqueous formic acid, aqueous ammonium formate, aqueous trifluoroacetic acid, aqueous acetic acid, aqueous ammonium acetate, aqueous ammonium bicarbonate, and buffer solutions.

[0108] Hydrophilic interaction chromatography is preferably used to separate highly hydrophilic objects that have poor retention in the stationary phase of reversed-phase chromatography. For example, disulfide compounds such as cystine and oxidized glutathione that are not derivatized with olefin compounds, and thiol derivatives such as cysteine ​​derivatives and reduced glutathione derivatives can be simultaneously analyzed by hydrophilic interaction chromatography.

[0109] In any liquid chromatography, when an organic substance is a mixture of chiral compounds, the chiral compounds can be separated by using a column packed with a chiral packing material (chiral column). For example, derivatives of D-cysteine ​​(which exists in a small proportion in nature) and L-cysteine ​​can be separated using a chiral column.

[0110] [2.2.2. Process (2b)] In step (2b), the derivatives of the separated organic substances are detected. Examples of detection methods include, but are not limited to, mass spectrometry using a mass spectrometer, ultraviolet absorption detection (UV), photodiode array detection (PDA), fluorescence detection (FL), visible light absorption detection, inductively coupled plasma optical emission spectrometry (ICP), corona charged particle detection, refractive index detection (RI), and evaporative light scattering detection (ELSD). An appropriate detection method may be selected depending on the type and properties of impurities contained in the sample and the type and properties of derivatives of organic substances. As the detection method, mass spectrometry, ultraviolet absorption detection, fluorescence detection, visible light absorption detection, and inductively coupled plasma optical emission spectrometry are preferred, and mass spectrometry and ultraviolet absorption detection are more preferred. These detection methods may be used alone or in combination. These separation methods can be carried out by known methods.

[0111] The mass spectrometry is not particularly limited, and a mass spectrometry method that combines various ionization methods, various ion separation methods, and various detectors may be used. Specific examples of mass spectrometers used in mass spectrometry include triple quadrupole mass spectrometers, time-of-flight mass spectrometers (TOF-MS), and ion trap mass spectrometers (eg, Orbitrap (registered trademark) mass spectrometers).

[0112] [2.3. Optional process] The analysis method of the present embodiment may include any optional step in addition to the steps (1) and (2). An example of the optional step is, when a sample containing amino acids is used as a sample, a step of derivatizing the amino acids with an amino acid derivatizing reagent such as ninhydrin, o-phthalaldehyde, etc. The step of derivatizing the amino acids is preferably carried out after the step (1) and before the step (2). By including such an optional step, the following advantages can be obtained, for example. For example, it is usually difficult to simultaneously analyze cysteine ​​and cystine using an amino acid analyzer. This is because when cysteine ​​and cystine are derivatized with an amino acid derivatizing agent, their retention times are usually similar. However, by carrying out step (1), of cysteine ​​and cystine, only cysteine ​​reacts with an olefin compound and is converted into a derivative, so that the retention time of cysteine ​​after being derivatized with an amino acid derivatizing agent is usually different from the retention time of cystine. Therefore, the analysis method of this embodiment allows cysteine ​​and cystine to be simultaneously analyzed using an amino acid analyzer.

[0113] [3. Derivatization agents for organic substances under acidic conditions] The agent according to one embodiment of the present invention is a derivatizing agent under acidic conditions for an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino, and includes an olefin compound containing an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms).

[0114] Examples and preferred examples of olefin compounds are the same as those mentioned above. Examples and preferred examples of organic substances are the same as those mentioned above. The conditions for producing a derivative using a derivatizing agent are acidic. Derivatization of an organic substance is preferably carried out in an acidic solution containing a derivatizing agent. The type and pH of the acidic solution may be the same as the preferred examples described in [1.3. Reaction conditions].

[0115] The concentration of the olefin compound in the derivatizing agent may vary depending on the dilution rate of the derivatizing agent, but may be, for example, 0.001 μmol / L to 1000 mol / L, preferably 0.01 μmol / L to 100 mol / L, more preferably 0.1 μmol / L to 10 mol / L, and even more preferably 1 μmol / L to 1 mol / L.

[0116] [4. Analytical Reagents for Organic Substances] A reagent according to one embodiment of the present invention is a reagent for analyzing an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino, and includes the derivatizing agent.

[0117] Examples and preferred examples of the olefin compound include the same as those mentioned above, and examples and preferred examples of the organic substance include the same as those mentioned above.

[0118] The concentration of the olefin compound in the analytical reagent may vary depending on the dilution rate of the analytical reagent, but may be, for example, 0.001 μm / L to 1000 mol / L, preferably 0.01 μmol / L to 100 mol / L, more preferably 0.1 μmol / L to 10 mol / L, and even more preferably 1 μmol / L to 1 mol / L.

[0119] The analytical reagent is preferably used in a method for analyzing a sample, which includes the above-mentioned steps (1) and (2). EXAMPLES

[0120] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples. The following operations were carried out at atmospheric pressure and room temperature unless otherwise specified.

[0121] [Explanation of abbreviations] In the following, the abbreviations have the following meanings: (Olefin compounds (derivatizing agents)) EMM:Diethyl methylenemalonate BPSE:1,1-Bis(phenylsulfonyl)ethylene BEPE:1,1-Bis(diethoxyphosphoryl)ethylene NEM: N-Ethylmaleimide

[0122] [ka]

[0123] In the above chemical formula, "Ph" stands for phenyl and "Et" stands for ethyl.

[0124] (Organic substances (chalcogen-containing compounds)) Thiols Cys: Cysteine Cys2: Cystine GSH: Glutathione, reduced form GSSG: oxidized glutathione gEC: γ-glutamylcysteine CG: Cysteinylglycine Hcy: homocysteine NAC: N-acetylcysteine

[0125] ·Cysteine ​​persulfides Cys-SH:S-mercaptocysteine Cys-SSH:S-disulfanylcysteine

[0126] [ka]

[0127] (Equipment, analysis method, etc.) LC-MS / MS: Liquid chromatograph-tandem mass spectrometer HILIC: Hydrophilic Interaction Chromatography

[0128] [Example A: Example using EMM and NEM] [Example A1] Reaction of EMM, NEM with cysteine ​​(Cys) under acidic conditions In this example, the reactivity of EMM and NEM under acidic conditions was estimated from the amount of Cys remaining after the reaction. In addition, the EMM-derivatized Cys peak was confirmed by LC-MS / MS.

[0129] [ka]

[0130] (A1-1. Production of Derivatives) L-cysteine ​​hydrochloride (Sigma) was dissolved in 0.1% formic acid solution (Fujifilm Wako Pure Chemical, prepared by diluting with pure water) to 10 mmol / L. The derivatization agent EMM (Asta Tech) was dissolved in acetonitrile (Fujifilm Wako Pure Chemical) to 10 μL / mL, and the derivatization agent NEM (Wako Pure Chemical) was dissolved in acetonitrile to 10 mg / mL. 10 μL of the derivatization agent solution (EMM or NEM) or 10 μL of acetonitrile was added to 10 μL of the cysteine ​​solution, and the mixture was left at room temperature for 10 minutes. After the reaction, 980 μL of 0.1% formic acid was added and thoroughly stirred to obtain a sample solution.

[0131] (A1-2. Analysis of Derivatives) The sample solution was analyzed by LC-MS / MS under the following conditions: Agilent 1200 series (Agilent) was used for LC, and 3200QTRAP system (Sciex) was used as the tandem mass spectrometer.

[0132] (LC conditions) Mobile phase A: 0.1% formic acid, Mobile phase B: acetonitrile Gradient conditions: 5-80%B (0-8min), 80%B (8-10min), 80-5%B (10-10.5min), 5%B (10.5-15min) Column: XBridge C18 3.5 μm, 2.1 × 100 mm (Waters) Column temperature: 40℃ ·Flow rate: 0.4mL / min Injection volume: 1μL

[0133] (MS / MS conditions) The experiment was carried out under the conditions in the table below. In the table below, "NEM-Cys" and "EMM-Cys" represent Cys derivatized with NEM and Cys derivatized with EMM, respectively.

[0134] [Table 1]

[0135] (Analysis results) The underivatized Cys peak was observed at 1.36 minutes, the NEM-Cys peaks at 2.00 and 2.14 minutes (two peaks were observed due to the generation of diastereomers), and the EMM-Cys peak at 8.23 ​​minutes. Each peak was integrated. The measurement was performed twice, and the results were the average of the two measurements. The derivatization rate was calculated according to the following formula (1).

[0136]

number

[0137] The analysis results are shown in the table below, and examples of chromatograms are shown in Figures 1 to 3. FIG. 1 is an example of a chromatogram showing the cysteine ​​peak. FIG. 2 is an example of a chromatogram showing the peak of NEM-Cys. FIG. 3 is an example of a chromatogram showing the EMM-Cys peak.

[0138] [Table 2]

[0139] These results show that Cys can be derivatized with NEM or EMM at a high reaction rate under acidic conditions and mild temperature conditions (room temperature). In particular, when EMM was used, a higher reaction rate was observed compared to NEM. Furthermore, when Cys, a chiral compound, was derivatized with NEM, diastereomers were generated and the NEM-Cys peak was split into two. On the other hand, when Cys was derivatized with EMM, no diastereomers were generated and no peak split occurred.

[0140] [Example A2] Simultaneous detection and quantification of EMM-derivatized thiols (separation by reversed-phase LC) In this example, six types of thiol compounds derivatized with EMM under acidic conditions were simultaneously separated and detected using LC-MS / MS.

[0141] (A2-1. Production of Derivatives) A 0.01 mol / L hydrochloric acid standard thiol mixture solution (Fujifilm Wako Pure Chemicals diluted with pure water) containing 25 μmol / L each of L-cysteine ​​hydrochloride, glutathione reduced (GSH) (Fujifilm Wako Pure Chemicals), D,L-homocysteine ​​(Hcy) (Sigma), cysteinylglycine (CG) (Bachem), gamma-glutamylcysteine ​​(gEC) (Sigma), and N-acetylcysteine ​​(NAC) (Junsei Kagaku) ​​was prepared. 10 μL of the above standard thiol solution was added to 60 μL of 20 mmol / L sodium phosphate buffer (pH 2.5) (Nacalai Tesque diluted with pure water), and 10 μL of EMM acetonitrile solution (10 μL / mL) was added, stirred, and left at room temperature for 10 minutes. 220 μL of 0.1% formic acid solution was added and stirred well to prepare the sample solution.

[0142] (A2-2. Analysis of Derivatives) The sample solution was analyzed by LC-MS / MS. The LC was a Nexera (Shimadzu Corporation) and the tandem mass spectrometer was a Triple Quad 6500 system (Sciex).

[0143] (LC conditions) Mobile phase A: 0.1% formic acid, Mobile phase B: acetonitrile Gradient conditions: 15-20%B (0-2.5min), 20-90%B (2.5-4.0min), 90%B (4.0-6.0min), 90-15%B (6.0-6.2min), 15%B (6.2-8.0min) Column: L-Column ODS, 3 μm, 2.1 × 50 mm (Chemicals Evaluation and Research Institute, Japan) with Inertsil ODS-3, 3 μm, 1.5 × 10 mm Guard column for UHPLC (GL-Science) attached as a guard column Column temperature: 40℃ ·Flow rate: 0.4mL / min Injection volume: 1μL

[0144] (MS / MS conditions) The test was carried out under the conditions shown in the table below.

[0145] [Table 3]

[0146] (Analysis results) The analysis results are shown in the table below.

[0147] [Table 4]

[0148] The above results show that various thiols can be derivatized using EMM, the derivatized thiols can be simultaneously separated by reversed-phase LC, and detected by MS / MS.

[0149] [Example A3] Analysis of sulfinic acid In this example, it was confirmed that not only thiols (-SH) but also sulfinic acids (-SO2H) are nucleophilic and can be derivatized with an EMM agent and analyzed.

[0150] [ka]

[0151] (A3-1. Production of Derivatives) Hypotaurine (Sigma) was dissolved in 0.01 mol / L hydrochloric acid to a concentration of 1 mmol / L. 10 μL of the above standard hypotaurine solution was added to 60 μL of 20 mmol / L sodium phosphate buffer (pH 2.5), and 10 μL of EMM acetonitrile solution (10 μL / mL) was added, stirred, and left at room temperature for 10 minutes. 920 μL of 0.1% formic acid solution was added and stirred well to prepare the sample solution.

[0152] (A3-2. Analysis of Derivatives) The sample solution was analyzed by LC-MS / MS under the following conditions: Agilent 1200 series (Agilent) was used for LC, and 3200QTRAP system (Sciex) was used as the tandem mass spectrometer.

[0153] (LC conditions) Mobile phase A: 0.1% formic acid, Mobile phase B: acetonitrile Gradient conditions: 15-40%B (0-3.0min), 40-90%B (3.0-3.2min), 90%B (3.2-5.5min), 90-15%B (5.5-5.8min), 15%B (5.8-9.5min) Column: L-Column ODS, 3 μm, 2.1 × 50 mm (Chemicals Evaluation and Research Institute, Japan) with Inertsil ODS-3, 3 μm, 1.5 × 10 mm Guard column for UHPLC (GL-Science) attached as a guard column Column temperature: 40℃ ·Flow rate: 0.4mL / min Injection volume: 10μL

[0154] (MS / MS conditions) The test was carried out under the conditions shown in the table below.

[0155] [Table 5]

[0156] (Analysis results) The analysis results are shown in the table below, and an example of a chromatogram is shown in Figure 4. FIG. 4 is an example of a chromatogram showing a peak of hypotaurine derivatized with EMM.

[0157] [Table 6]

[0158] The above results demonstrate that sulfinic acids such as hypotaurine can be derivatized and analyzed by EMM under acidic conditions.

[0159] [Example A4] Analysis of persulfides In this example, it was confirmed that persulfides can be derivatized and analyzed. Cysteine ​​persulfides (Cys-SH and Cys-SSH) were used as persulfides. Cysteine ​​persulfides play an important role in redox reactions in living organisms, and therefore, if it is possible to derivatize the persulfides while maintaining their redox state in the present invention, this would be useful for understanding the redox state in living organisms.

[0160] (A4-1. Production of derivatives) 70μL of pH6.7 ammonium formate solution (Fujifilm Wako Pure Chemical) was mixed with 10μL of 100mmol / L-cysteine ​​solution, 10μL of 100mmol / L sodium sulfide (Fujifilm Wako Pure Chemical diluted with pure water) aqueous solution, and 10μL of 0.1% hydrogen peroxide aqueous solution (Kanto Chemical diluted with pure water), and reacted at 30℃ for 30 minutes to generate cysteine ​​persulfide in the system. 60μL of 20mmol / L sodium phosphate buffer (pH2.5) and 10μL of EMM acetonitrile solution (10μL / mL) were added to 10μL of the above solution, stirred, and left at room temperature for 10 minutes. 420μL of 0.1% formic acid solution was added and stirred well to prepare the sample solution.

[0161] (A4-2. Analysis of Derivatives) The sample solution was analyzed by LC-MS / MS under the following conditions: Agilent 1200 series (Agilent) was used for LC, and 3200QTRAP system (Sciex) was used as the tandem mass spectrometer.

[0162] (LC conditions) Mobile phase A: 0.1% formic acid, Mobile phase B: acetonitrile Gradient conditions: 15-40%B (0-3.0min), 40-90%B (3.0-3.2min), 90%B (3.2-5.5min), 90-15%B (5.5-5.8min), 15%B (5.8-9.5min) Column: L-Column ODS, 3 μm, 2.1 × 50 mm (Chemicals Evaluation and Research Institute, Japan) with Inertsil ODS-3, 3 μm, 1.5 × 10 mm Guard column for UHPLC (GL-Science) attached as a guard column Column temperature: 40℃ ·Flow rate: 0.4mL / min Injection volume: 10μL

[0163] (MS / MS conditions) The test was carried out under the conditions shown in the table below.

[0164] [Table 7]

[0165] (Analysis results) The analytical results are shown in the table below, and an example of a chromatogram is shown in Figure 5. FIG. 5 is an example of a chromatogram showing peaks of cysteine ​​and persulfated cysteine ​​derivatized with EMM.

[0166] [Table 8]

[0167] The above results demonstrate that persulfides such as persulfated cysteine ​​can be derivatized and analyzed by EMM under acidic conditions.

[0168] [Example A5] Simultaneous analysis of Cys, Cys2, and amino acids using an amino acid analyzer In this example, it was confirmed that Cys, Cys2, and amino acids can be analyzed simultaneously using an amino acid analyzer. (Outline of analysis method) The samples were treated with EMM to derivatize Cys and Cys2, and then the amino acids were separated from the samples using an ion exchange column. The amino acids were then derivatized with ninhydrin by post-column derivatization, and Cys, Cys2, and amino acids were analyzed simultaneously. In addition, in an amino acid analyzer, Cys and Cys2 are usually eluted from the column at the same time, so it is usually difficult to analyze Cys and Cys2 simultaneously.

[0169] (A5-1. Production of derivatives) To 100μL of amino acid mixed standard solution, H type (manufactured by Fujifilm Wako Pure Chemical Industries) (a solution containing 2.5mmol / L each of L-aspartic acid, L-threonine, L-serine, L-glutamic acid, L-proline, glycine, L-alanine, L-cystine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-lysine, L-histidine, ammonium chloride, and L-arginine), 25μL of 10mmol / L cysteine ​​hydrochloride solution and 125μL of pure water were added to prepare a mixed standard solution. To 100μL of this product, 200μL of 20mmol / L sodium phosphate buffer (pH2.5) and 50μL of EMM acetonitrile solution (5μL / mL) were added, stirred, and left at room temperature for 10 minutes. After the reaction, 650 μL of 0.01 mol / L hydrochloric acid was added to prepare a sample solution.

[0170] (A5-2. Analysis of Derivatives) The sample solutions were analyzed using the following analytical equipment. Analytical equipment: L-8900 high-speed amino acid analyzer (Hitachi) -Analysis method: The "Biofluid Analysis Method" from the method package with an analysis time of "148 minutes" was used.

[0171] (Analysis results) Cysteine ​​derivatives eluted at a retention time of 40.3 minutes, and Cys2 eluted at a retention time of 48.8 minutes. Other amino acids eluted at the retention times of the standard "Biofluid Analysis" analysis time of "148 minutes." An example of the chromatogram is shown in Figure 6. Figure 6 is an example of a chromatogram showing the results of simultaneous analysis of Cys, Cys2, and amino acids using an amino acid analyzer. From the above results, it is evident that by treating a sample containing Cys, Cys2, and various amino acids under acidic conditions and analyzing it with a conventional amino acid analyzer, Cys, Cys2, and various amino acids can be analyzed simultaneously.

[0172] [Example B: Example using BPSE] [Example B1] Derivatization with BPSE and analysis In this example, it was confirmed that thiols can be derivatized and analyzed by BPSE. An example of the reaction of BPSE with a thiol is shown below.

[0173] [ka]

[0174] (B1-1. Production of Derivatives) Six types of standard thiol solutions were prepared by preparing 0.01 mol / L hydrochloric acid standard thiol solutions containing 10 mmol / L L-Cys, GSH, Hcy, CG, gEC, and NAC for each compound. 10 μL of the above standard thiol solution was added to 60 μL of 20 mmol / L sodium phosphate buffer (pH 2.5), and 10 μL of BPSE acetonitrile solution (10 mg / mL) was added, stirred, and left at room temperature for 10 minutes. 420 μL of 0.1% formic acid aqueous solution was added and stirred well to prepare the sample solution.

[0175] (B1-2. Analysis of Derivatives) The sample solution was analyzed by LC under the following conditions. Peaks were detected by an HPLC-UV detector. (LC conditions) Mobile phase A: 20mmol / L sodium phosphate buffer (pH 2.5) Mobile phase B: Acetonitrile Gradient conditions: 30%B (0-10min), 30-90%B (10-11min), 90%B (11-16min), 90-30%B (16-16.5min), 30%B (16.5-20min) Column: Triart C18, 3 μm, 150 × 4.6 mm (YMC) Column temperature: 40℃ ·Flow rate: 0.8mL / min Injection volume: 5μL (Detection conditions) Detection wavelength: 260nm (Analysis results) The analysis results are shown in the table below. An example of a chromatogram is shown in Figure 7. Figure 7 is an example of a chromatogram of a thiol derivative by BPSE.

[0176] [Table 9]

[0177] From the above results, it is evident that thiols can be derivatized by BPSE under acidic conditions, separated by column chromatography, and detected by a UV detector. It is expected that the amino acids having sulfanyl groups are converted to highly hydrophobic amino acids by derivatization, and can be easily separated from other less hydrophobic amino acids and detected by column chromatography using an ODS (C18) column.

[0178] [Example B2] Separation and detection of chiral thiols In this example, it is confirmed that chiral thiols can be derivatized with BPSE and separated and detected by column chromatography using a chiral column.

[0179] (B2-1. Production of Derivatives) A standard D,L-cysteine ​​solution containing 5 mmol / L each of L-Cys and D-Cys (Sigma) was prepared using 0.1 mol / L hydrochloric acid. 10 μL of the above standard thiol solution was added to 60 μL of 20 mmol / L sodium phosphate buffer (pH 2.5), 10 μL of BPSE acetonitrile solution (10 mg / mL) was added, stirred, and left at room temperature for 10 minutes. 420 μL of 20 mmol / L sodium phosphate buffer (pH 2.5) was added and stirred well. This solution was further diluted 10 times with the mobile phase to prepare the sample solution.

[0180] (B2-2. Analysis of Derivatives) The sample solution was analyzed by LC under the following conditions. Peaks were detected by an HPLC-UV detector. (LC conditions) Mobile phase: Methanol / acetonitrile / water (49 / 49 / 2 (v / v / v)) with formic acid and diethylamine at concentrations of 50mmol / L and 25mmol / L, respectively, used under isocratic conditions. Column: CHIRAL PAK (registered trademark) ZWIX, 3 μm, 150 × 3 mm (manufactured by Daicel) Column temperature: 25℃ ·Flow rate: 0.4mL / min Injection volume: 10μL (Detection conditions) Detection wavelength: 260nm

[0181] (Analysis results) The analytical results are shown in the table below. An example of a chromatogram is shown in Figure 8. Figure 8 is an example of a chromatogram of a derivatized chiral thiol.

[0182] [Table 10]

[0183] From the above results, it is found that chiral thiols can be derivatized with BPSE under acidic conditions, separated by a chiral column, and detected. Therefore, it is found that the method of the present invention can efficiently detect D-Cys, which is present in small amounts in nature, by separating it from L-Cys.

[0184] [Examples and Reference Example C: Evaluation of reactivity of olefin compounds] In this Example and Reference Example, the reactivity of olefin compounds (derivatizing agents) including EMM and BPSE with Cys was evaluated. The olefin compounds and their abbreviations used in the Examples and Reference Examples are shown below.

[0185] [ka]

[0186] (C-1. Sample preparation and derivative production) L-Cys hydrochloride monohydrate was dissolved in 0.01 mol / L hydrochloric acid to a concentration of 1 mmol / L. 60 μL of buffer solutions of various pH values ​​and 10 μL of derivatizing agent solution (or acetonitrile) were added to 10 μL of this Cys solution, and the mixture was left to stand at room temperature for 10 minutes. After the reaction, 920 μL of 0.1% formic acid aqueous solution was added to prepare a sample solution. Acetonitrile solutions of the derivatizing agents were prepared at a concentration of 10 mg / mL (solid) or 10 μL / mL (liquid). As a buffer, a pH 2.5 or 7.0 sodium phosphate buffer solution (20 mM) was used. In the derivatization with EMM, a pH 3.25 or 4.25 sodium citrate buffer solution (200 mM) and a pH 6.0 sodium phosphate buffer solution (20 mM) were also used as a buffer.

[0187] (C-2. Analysis of Derivatives) The sample solution was analyzed by LC-MS / MS under the following conditions: LC was performed using an Agilent 1200 series (Agilent), and the tandem mass spectrometer was a 3200QTRAP system (Sciex), with measurements performed in selected ion detection (SIM) mode. (LC conditions) Mobile phase A: 0.1% formic acid solution Mobile phase B: Acetonitrile Gradient conditions: 10%B (0-1.0min), 10-90%B (1.0-5.0min), 90%B (5.0-6.5min), 90-10%B (6.5-7.0min), 10%B (7.0-10.0min) Column: L-Column ODS, 3 μm, 2.1 × 50 mm (Chemicals Evaluation and Research Institute, Japan) Column temperature: 40℃ ·Flow rate: 0.4mL / min Injection volume: 10μL

[0188] (MS conditions) The experiment was carried out under the following conditions. DP(V):46 EP(V):8 CEP(V):14 The detection conditions and retention times for Cys and Cys derivatized with various derivatizing agents are shown in the table below. In the table below, X-Cys means Cys derivatized with derivatizing agent X. In addition, when detecting EBzM-Cys, two diastereomer peaks were observed, and the retention times of each are shown in the table below.

[0189] [Table 11]

[0190] (Analysis results) The analysis results are shown in the table below. The Cys reaction rate was calculated using the above formula (1). Items marked with "*" indicate that no data is available.

[0191] [Table 12]

[0192] At pH 7.0, derivatives were produced and detected for all agents. Under acidic conditions at pH 2.5, derivatives were produced when olefin compounds containing an ethylene structure with two electron-withdrawing groups (EMM, BPSE, Acry-CF3A, Acry-CF3E, EiPrM, EBzM, BEPE, CA) were used. In particular, derivatives were produced at high reaction rates when olefin compounds represented by formula (I) (EMM, BPSE, Acry-CF3A, Acry-CF3E, BEPE, CA) were used.

[0193] The results when EMM was used as the derivatizing agent and the reaction was carried out in various buffer solutions are shown in the table below.

[0194] [Table 13]

[0195] The above results show that the derivatized Cys peak area value decreases when the pH is 6.0 or higher.

[0196] [Reference Example D: Behavior of organic substances under various pH conditions] In this reference example, the stability of organic substances under acidic conditions is evaluated by observing the behavior of the organic substances under various pH conditions.

[0197] (D-1. Observation of thiol exchange reaction) (Sample preparation) L-Cys hydrochloride monohydrate was dissolved in 0.01 mol / L hydrochloric acid to a concentration of 10 mmol / L, and GSSG was dissolved in 0.01 mol / L hydrochloric acid to a concentration of 50 mmol / L. 850 μL of various sodium phosphate buffers (pH 2.5, 6.0, 7.0, or 8.0) and 100 μL of GSSG solution (50 mmol / L) were added to 50 μL of Cys solution (10 mmol / L) to prepare a mixed solution. (Finally, Cys was present at 0.5 mmol / L and GSSG was present at 5 mmol / L in the solution.) Sampling was performed over time, derivatization was performed with BPSE, and analysis was performed by LC under the following conditions, and the area values ​​of Cys and GSH were measured. Peaks were detected by an HPLC-UV detector.

[0198] (Sampling and Derivatization) To 10μL of the mixed solution, 10μL of 10% (w / v) trichloroacetic acid aqueous solution, 80μL of pH 2.5 sodium phosphate buffer, and 20μL of BPSE acetonitrile solution (5mg / mL) were added, and the mixture was left to stand at room temperature for 10 minutes. After the reaction, 100μL of pH 2.5 sodium phosphate buffer was added to prepare the sample solution.

[0199] (LC conditions) Mobile phase A: 20mmol / L sodium phosphate buffer (pH 2.5) Mobile phase B: Acetonitrile Gradient conditions: 25%B (0-8.0min), 25-90%B (8.0-9.0min), 90%B (9.0-11.0min), 90-25%B (11.0-12.0min), 25%B (12.0-15.0min) Column: Triart C18, 3 μm, 150 × 4.6 mm (YMC) Column temperature: 40℃ ·Flow rate: 1.0mL / min Injection volume: 10μL Under these conditions, Cys elutes at 6.3 minutes and GSH elutes at 6.8 minutes. (Detection conditions) Detection wavelength: 260nm

[0200] (Analysis results) The analysis results are shown in the table below and in Figures 9 to 12. Figures 9, 10, 11, and 12 are graphs showing the time course of the peak area values ​​of Cys and GSH at pH 8.0, pH 7.0, pH 6.0, and pH 2.5, respectively. Note that the data at 0 min at pH 6.0, 7.0, and 8.0 were considered to be data without buffer treatment, and were shared with the data at 0 min at pH 2.5.

[0201] [Table 14]

[0202] [Table 15]

[0203] [Table 16]

[0204] [Table 17]

[0205] The above results show that under conditions of pH 7.0 or higher, Cys reacts rapidly with GSSG, GSH produced by the exchange reaction is detected, and Cys decreases. On the other hand, under conditions of pH 6.0 or lower, the reaction between Cys and GSSG hardly proceeds, and particularly under conditions of pH 2.5, GSH, the product of the exchange reaction, is not detected even after 1 hour. These results show that thiol compounds can be accurately analyzed under acidic conditions even in samples in which disulfide compounds and thiol compounds are simultaneously present.

[0206] (D-2. Observation of thiol oxidation reaction) (Sample preparation) L-Cys hydrochloride monohydrate was dissolved in 0.01 mol / L hydrochloric acid to a concentration of 10 mmol / L, and copper sulfate pentahydrate was dissolved in 0.1 mmol / L. 50 μL of Cys solution (10 mmol / L) was mixed with 850 μL of various sodium phosphate buffers (pH 2.5, 6.0, 7.0, or 8.0) and 100 μL of copper sulfate aqueous solution (0.1 mmol / L) to prepare a mixed solution. (Finally, Cys was present at 0.5 mmol / L and copper sulfate at 0.01 mmol / L in the solution.) Sampling was performed over time, derivatization was performed with BPSE, and analysis was performed by LC under the same conditions as in (D-1. Observation of thiol exchange reaction), and the area value of Cys was measured. The same analytical and derivatization methods as in (D-1. Observation of thiol exchange reaction) were used.

[0207] (Analysis results) The analysis results are shown in the table below and in Figure 13. Figure 13 is a graph showing the change over time in the peak area value of Cys at pH 8.0, pH 7.0, pH 6.0, and pH 2.5. Note that the data at 0 min at pH 6.0, 7.0, and 8.0 are considered to be data without buffer treatment and are therefore shared with the data at 0 min at pH 2.5.

[0208] [Table 18]

[0209] These results indicate that at pH 7 or higher, Cys is rapidly decreased and rapidly oxidized. On the other hand, under conditions of pH 6.0 or lower, Cys hardly decreased, indicating that the oxidation reaction of Cys hardly progressed. These results indicate that under acidic conditions, the oxidation reaction of thiol compounds is suppressed, enabling accurate analysis of thiol compounds.

[0210] [Example E] Derivatization of sulfanyl and amino groups In this example, it was confirmed that cysteine ​​or cystine was mixed with EMM under acidic conditions, and then the pH of the solution was made basic, causing the amino group (-NH2) of cysteine ​​or cystine to react with EMM, thereby derivatizing cysteine ​​or cystine. That is, the sulfanyl group (-SH) of cysteine ​​reacted with EMM under acidic conditions to be converted into a substituent that is stable against oxidation, and then the amino group of cysteine ​​reacted with EMM under basic conditions to obtain a derivative. Under acidic conditions, amino groups do not usually react with EMM. Therefore, under acidic conditions, disulfide compounds such as cystine, which has an amino group but no sulfanyl group, do not usually react with EMM and are not derivatized. However, it was confirmed that even such disulfide compounds react with EMM under basic conditions to be derivatized. By reacting not only the sulfanyl group but also the amino group with EMM, the hydrophobicity of disulfide compounds that are not derivatized under acidic conditions can be increased, facilitating separation by liquid chromatography, and increasing the detection sensitivity of derivatives by mass spectrometry.

[0211] [ka]

[0212] (E-1. Production of Derivatives) Cysteine ​​or cystine (Sigma) was dissolved in 0.01 mol / L hydrochloric acid to a concentration of 250 μmol / L to prepare a standard. 20 μL of this standard, 20 μL of 10% trichloroacetic acid aqueous solution, and 40 μL of 0.1 mol / L hydrochloric acid were mixed and stirred well. 50 μL of this solution was mixed with 300 μL of 20 mmol / L sodium phosphate buffer (pH 2.5), 50 μL of EMM acetonitrile solution (10 μL / mL) was added, stirred, and left to stand at 40 ° C for 5 minutes. 100 μL of this solution was taken and mixed with 200 μL of 100 mmol / L sodium phosphate buffer (pH 9.0), further stirred, and left to stand at 40 ° C for 5 minutes. 15 μL of this solution was taken, 50 μL of 0.3% formic acid solution was added, and the mixture was stirred well to prepare a sample for analysis.

[0213] (E-2. Analysis of Derivatives) The sample solution was analyzed by LC-MS / MS under the following conditions: Agilent 1290 Infinity II series (Agilent) was used for LC, and QTRAP 5500 system (Sciex) was used as the tandem mass spectrometer.

[0214] (LC conditions) Mobile phase A: 0.1% formic acid, Mobile phase B: acetonitrile Gradient conditions: 30-60%B (0-6.0min), 60-90%B (6.0-7.5min), 90%B (7.5-9.0min), 90-30%B (9.0-9.1min), 30%B (9.1-10.0min) Column: L-Column ODS, 3 μm, 2.1 × 50 mm (Chemicals Evaluation and Research Institute, Japan) with Inertsil ODS-3, 3 μm, 1.5 × 10 mm Guard column for UHPLC (GL-Science) attached as a guard column Column temperature: 40℃ ·Flow rate: 0.4mL / min Injection volume: 1μL

[0215] (MS / MS conditions) The test was carried out under the conditions shown in the table below.

[0216] [Table 19]

[0217] (Analysis results) The analysis results are shown in the table below, and an example of a chromatogram is shown in Figure 14. In the table below and Figure 14, "3EMM-Cysteine" and "3EMM-Cys" refer to derivatives obtained by reacting one molecule of cysteine ​​with three molecules of EMM. "4EMM-Cystine" and "4EMM-Cys2" refer to derivatives obtained by reacting one molecule of cystine with four molecules of EMM. FIG. 14 is an example of a chromatogram showing a peak of derivatized cysteine ​​or a peak of derivatized cystine by EMM.

[0218] [Table 20]

[0219] These results indicate that by reacting cysteine ​​with EMM under acidic conditions, the sulfanyl group reacts with EMM, and then by making the solution basic, the amino group reacts with two molecules of EMM, thereby derivatizing cysteine. By making the reaction solution basic, cystine is derivatized by reacting its two amino groups with four molecules of EMM. In other words, the results show that cysteine ​​can be analyzed by LC-MS / MS as a compound that has reacted with EMM3 molecules, and cystine can be analyzed by LC-MS / MS as a compound that has reacted with EMM4 molecules.

Claims

1. (1) A sample containing an organic substance containing one or more groups selected from the group consisting of sulfanyl and sulfino, and an olefin compound, mixing under acidic conditions to obtain a treated sample containing a derivative of said organic material; and (2) A method for analyzing a sample containing an organic substance, comprising detecting a derivative of the organic substance in the treated sample, the method comprising: The olefin compound is N-ethylmaleimide or a compound containing an ethylene structure having at least two electron-withdrawing groups; said at least two electron withdrawing groups are each independently -C(=O)-OR 1 , -S(=O) 2 -R 2 or -C(=O)-NR 6 R 7 ; Where: R 1 and R 2 each independently represent a monovalent hydrocarbon group or a monovalent heterocyclic group, each of which may have a substituent; A method for analyzing a sample containing an organic substance, wherein R 6 and R 7 each independently represent a hydrogen atom, a monovalent hydrocarbon group, or a monovalent heterocyclic group, each of which may have a substituent.

2. 2. The method for analyzing a sample containing an organic substance according to claim 1, wherein the ethylene structure has two electron-withdrawing groups.

3. 3. The method for analyzing a sample containing an organic substance according to claim 1, wherein the two electron-withdrawing groups are bonded to the same carbon atom constituting the ethylene structure.

4. 4. The method for analyzing a sample containing an organic substance according to claim 1, wherein at least two of the electron-withdrawing groups are the same group.

5. The method for analyzing a sample containing an organic substance according to any one of claims 1 to 4, wherein the olefin compound is a compound represented by the following formula (I): 【Chemistry 1】 (However, EWG １ and E.W.G. ２ each independently represents the electron-withdrawing group, and may form a ring together with the carbon atom to which they are attached.

6. The method for analyzing a sample containing an organic substance according to claim 1, wherein the olefin compound is N-ethylmaleimide, diethylmethylenemalonate, or 1,1-bis(phenylsulfonyl)ethylene.

7. The method for analyzing a sample containing an organic substance according to any one of claims 1 to 6, wherein the acidic condition is a condition of pH less than 6.

0.

8. The method for analyzing a sample containing an organic substance according to any one of claims 1 to 7, wherein the organic substance is one or more selected from the group consisting of cysteine, reduced glutathione, γ-glutamylcysteine, cysteinylglycine, homocysteine, N-acetylcysteine, cysteine ​​persulfide, hypotaurine, glutathione persulfide, and peptidic compounds containing a cysteine ​​residue.

9. The analysis method comprises: (2a) further comprising isolating a derivative of the organic material from the treated sample; The detection in the step (2) (2b) detecting a derivative of the separated organic material; A method for analyzing a sample containing an organic substance according to any one of claims 1 to 8, comprising:

10. The method for analyzing a sample containing an organic substance according to any one of claims 1 to 9, wherein the sample further contains a disulfide compound.

11. 11. The method for analyzing a sample containing an organic substance according to claim 10, wherein the disulfide compound is at least one selected from the group consisting of oxidized glutathione and cystine.

12. the organic material further comprises an amino group; Step (1) is (1') A method for analyzing a sample containing an organic substance according to any one of claims 1 to 11, comprising mixing the sample with the olefin compound under acidic conditions, and then further mixing them under neutral or basic conditions to obtain a treated sample containing a derivative of the organic substance.

13. A reagent for analyzing an organic substance containing one or more groups selected from the group consisting of sulfanyl and sulfino, comprising: the organic substance is at least one selected from the group consisting of cysteine, reduced glutathione, γ-glutamylcysteine, cysteinylglycine, homocysteine, N-acetylcysteine, cysteine ​​persulfide, hypotaurine, glutathione persulfide, and a peptidic compound containing a cysteine ​​residue; The analytical reagent comprises an olefinic compound, the olefinic compound being N-ethylmaleimide or a compound comprising an ethylene structure having at least two electron-withdrawing groups; said at least two electron withdrawing groups are each independently -C(=O)-OR 1 , -S(=O) 2 -R 2 or -C(=O)-NR 6 R 7 ; Where: R 1 and R 2 each independently represent a monovalent hydrocarbon group or a monovalent heterocyclic group, each of which may have a substituent; An analytical reagent, wherein R 6 and R 7 each independently represent a hydrogen atom, a monovalent hydrocarbon group, or a monovalent heterocyclic group, each of which may have a substituent.

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