Method for producing cysteine ​​derivatives, a derivatizing agent for cysteine ​​under acidic conditions, and a reagent for the analysis of cysteine.

Abstract

To provide a novel method for producing a derivative of an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino.SOLUTION: A method for producing a derivative of an organic substance, the method including 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 to obtain the derivative of the organic substance, wherein the olefin compound includes an ethylene structure having at least two electron-withdrawing groups, excluding halogen atoms.SELECTED DRAWING: None

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 Art

[0002] In vivo, thiols play an important role. For example, they play roles such as maintaining the redox state in vivo, controlling the function of proteins, and detoxifying foreign substances in the body 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 using an electrochemical detector by utilizing the redox reaction on the electrode surface (Non-Patent Documents 1 and 2). Also, attempts have been made to convert thiols into derivatives and analyze them (Non-Patent Documents 3 to 8). As a reaction of thiols, a reaction with a carbon-carbon double bond is known (Non-Patent Documents 9 and 10).

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

[0004] However, in the analytical methods described in Non-Patent Documents 1 and 2, thiols are analyzed without derivatization, making them susceptible to oxidation by dissolved oxygen in the sample or eluent used for analysis, resulting in difficulty in obtaining stable analytical results. Furthermore, electrochemical detectors do not have sufficient selectivity for thiols, and when measuring samples containing amino acids, sugars, or other complex interfering components, the measurement may be interfered with by these components. In addition, electrochemical detectors require maintenance such as electrode cleaning, and their detection sensitivity easily fluctuates and becomes unstable with use, making them difficult to handle.

[0005] In the techniques described in Non-Patent Documents 3-6, thiol derivatization is performed under neutral to basic conditions. However, the inventors have found that under neutral to basic conditions, the oxidation reaction of thiols and the exchange reaction between thiols and disulfide compounds proceed easily, making thiol analysis difficult. Therefore, in the techniques described in Non-Patent Documents 3 to 6, which perform thiol derivatization under neutral to basic conditions, accurate analysis of thiols may be difficult.

[0006] On the other hand, the techniques described in Non-Patent Documents 7 and 8 involve derivatizing thiols under acidic conditions (pH 3.5), but this results in poor reactivity and requires special conditions such as long reaction times or microwave application. [Means for solving the problem]

[0007] As a result of diligent research, the inventors have discovered 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 sulfanil, seranil, and sulfino under acidic conditions is useful for the analysis of organic substances containing one or more groups selected from the group consisting of sulfanil, seranil, and sulfino, and have completed the present invention. The above prior art neither teaches nor suggests (a) the reaction of a specific olefin compound with an organic substance containing one or more groups selected from the group consisting of sulfanil, seranil, and sulfino under acidic conditions, nor (b) that a derivative of an organic substance obtained by such a reaction is useful for the analysis of organic substances containing one or more groups selected from the group consisting of sulfanil, seranil, and sulfino.

[0008] In other words, the present invention provides the following:

[0009] [1] The method involves reacting an organic substance containing one or more groups selected from the group consisting of sulfanil, seranil, and sulfino with an olefin compound under acidic conditions to obtain a derivative of the organic substance. Herein, the olefin compound comprises an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms), and the method for producing a derivative of an organic substance. [2] A 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 two electron-withdrawing groups are bonded to the same carbon atom constituting the ethylene structure. [4] A 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).

Chemical formula

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

[11] The analysis in step (2) is (2a) Separating the derivative of the organic substance from the treated sample, (2b) To detect the derivative of the separated organic substance, A method for analyzing a sample containing the organic substance described in

[10] , including the following.

[12] A 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 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 and 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 derivatizer for organic substances under acidic conditions, comprising an olefin compound having an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms), and containing one or more groups selected from the group consisting of sulfanyl, seranyl, and sulfino.

[16]

[15] A reagent for the analysis of organic substances comprising one or more groups selected from the group consisting of sulfanil, seranil, and sulfino, comprising the derivatizing agent described in

[15] . [Effects of the Invention]

[0010] The present invention provides a novel method for producing derivatives of organic substances containing one or more groups selected from the group consisting of sulfanil (-SH), seranil (-SeH), and sulfino (-S(=O)-OH); and a novel method for analyzing samples containing such organic substances. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows an example of a chromatogram that shows a cysteine ​​peak. [Figure 2] Figure 2 shows an example of a chromatogram illustrating the NEM-Cys peak. [Figure 3] Figure 3 is an example of a chromatogram showing the EMM-Cys peak. [Figure 4] Figure 4 is an example of a chromatogram showing the peak of derivatized hypotaurine by EMM. [Figure 5] Figure 5 is an example of a chromatogram showing the peaks of cysteine ​​and persulfide cysteine ​​derivatized by EMM. [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. [Figure 7] Figure 7 shows an example of a chromatogram of a thiol derivative mediated by BPSE. [Figure 8] Figure 8 shows an example of a chromatogram of a derivatized chiralthiol. [Figure 9] Figure 9 shows graphs illustrating the changes over time in the peak area values ​​of Cys and GSH at pH 8.0. [Figure 10] Figure 10 is a graph showing the time course of the peak area values ​​of Cys and GSH at pH 7.0. [Figure 11] Figure 11 is a graph showing the time course of the peak area values ​​of Cys and GSH at pH 6.0. [Figure 12] Figure 12 is a graph showing the time course of the peak area values ​​of Cys and GSH at pH 2.5. [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. [Figure 14] Figure 14 is an example of a chromatogram showing the peak of derivatized cysteine ​​or derivatized cystine as determined by EMM. [Modes for carrying out the invention]

[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. Furthermore, the "agent" may be a single substance or a composition composed of two or more substances.

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

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

[0015] In this invention, sulfanil is -(S) n -SH is a base represented by (n represents a non-negative integer). According to the method for producing derivatives according to this embodiment, -(S) such as -S-SH and -SS-SH n Organic substances having a group represented by -SH (where n is an integer of 1 or more) (hereinafter also referred to as persulfides) can also be derivatized with olefin compounds. Such persulfides (e.g., persulfide cysteine) play an important role in oxidation-reduction in living organisms. According to the method for producing derivatives of this embodiment, persulfides can be derivatized while maintaining their oxidation-reduction state, making it possible to understand the oxidation-reduction state in living organisms. From the viewpoint of the number of types and abundance of sulfanil-containing organic substances (especially natural organic substances in biological samples such as blood, saliva, urine, and feces), -(S) n The n of the group represented by -SH 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 this invention, seranyl is also a group represented by -SeH. The selenium atom is an atom belonging to Group 16 of the periodic table, the same as the sulfur atom, and exhibits similar reactivity to the sulfur atom. Therefore, the reactivity of the selenium atom in an organic substance to an olefin compound is similar to the reactivity of the sulfur atom in an organic substance to an olefin compound. Thus, the olefin compounds described later, which have an ethylene structure that can react well with the sulfur atom in an organic substance, can also react well with the selenium atom in an organic substance.

[0017] One or more groups selected from the group consisting of sulfanyl, seranyl, and sulfino (-S(=O)-OH) may be present in multiples within a single molecule of an organic substance. Furthermore, the organic substance may have one or more (e.g., two, three, or four) functional groups in addition to one or more groups selected from the group consisting of sulfanyl, seranyl, and sulfino. Examples of functional groups, though not particularly limited, include 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 group A. Here, the amino groups that the organic substance may contain in addition to group A include unsubstituted amino groups (-NH2), monosubstituted amino groups, and disubstituted amino groups. The amino groups that the organic substance may contain in addition to group A are preferably one or more selected from the group consisting of unsubstituted amino groups and monosubstituted amino groups, with unsubstituted amino groups being more preferred. The organic substance containing group A may contain only one amino group per molecule, or it may contain multiple amino groups. The number of amino groups contained in one molecule of the organic substance may be one, two, or three, preferably one or two, and more preferably one.

[0019] The organic material may originate from the sample described later. The organic substance may be a high-molecular-weight compound or a low-molecular-weight compound. Preferably, the organic substance is a low-molecular-weight compound. Low molecular weight compounds are compounds with a molecular weight of 1500 or less. The molecular weight of low molecular weight compounds 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 low molecular weight compounds may also be 30 or more, 40 or more, or 50 or more. The low molecular weight compound may be an amino acid (e.g., cysteine, selenocysteine), a peptide compound, or a salt thereof. Here, a peptide compound is a compound having a structure obtained by the condensation of two or more amino acid molecules. The organic substance may be a peptide compound, which is a high-molecular-weight compound. Examples of peptide compounds that are high-molecular-weight compounds include proteins such as albumin.

[0020] The organic substance may be a natural compound or a synthetic compound.

[0021] Specific examples of organic substances include cysteine, reduced glutathione, γ-glutamylcysteine, cysteinylglycine, homocysteine, N-acetylcysteine, persulfurized cysteine ​​(e.g., S-mercaptocysteine, S-disulfanylcysteine, S-trisulfanylcysteine), hypotaurine, persulfurized glutathione, peptide compounds containing cysteine ​​residues, 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-methylbutan-2-thiol. Organic substances may be a single type or a combination of two or more types. The organic substance is preferably one or more selected from the group consisting of cysteine, reduced glutathione, γ-glutamylcysteine, cysteinylglycine, homocysteine, N-acetylcysteine, persulfurized cysteine ​​(e.g., S-mercaptocysteine, S-disulfanylcysteine, S-trisulfanylcysteine), hypotaurine, persulfurized glutathione, and peptide compounds containing cysteine ​​residues. Here, the peptide compound containing the cysteine ​​residue can be a low-molecular-weight compound (e.g., an oligopeptide) or a high-molecular-weight compound (e.g., a protein).

[0022] [1.2. Olefin Compounds] Olefin compounds are compounds that contain an ethylene structure (ethene structure) having at least two electron-withdrawing groups, and can react with sulfanil, seranil, or sulfino in organic substances via this ethylene structure. However, in this specification, the electron-withdrawing groups do not include halogen atoms.

[0023] The ethylene structure typically has two or more electron-withdrawing groups, usually four or fewer, preferably two or more and three or fewer, and more preferably two. Having the number of electron-withdrawing groups within this range improves the reactivity between the olefin compound and organic substances.

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

[0025] In olefin compounds, it is preferable that two electron-withdrawing groups are bonded to the same carbon atom constituting the ethylene structure. This can improve the reactivity of the carbon atom other than the carbon atom 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 an ethylene structure, and when electron-withdrawing groups are bonded to adjacent carbon atoms in an ethylene structure, these two electron-withdrawing groups may form a ring together with the carbon atoms to which they are bonded.

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

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

[0029] It is even more preferable that the olefin compound has an ethylene structure with two identical electron-withdrawing groups, and that these two identical electron-withdrawing groups are 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 prevents the organic substance from being converted into two diastereomer derivatives through the reaction between the olefin compound and the organic substance.

[0030] The ethylene structure may have groups other than electron-withdrawing groups. Examples of groups other than electron-withdrawing groups that the ethylene structure may have include monovalent hydrocarbon groups, preferably one or more selected from the group consisting of monovalent linear hydrocarbons and monovalent aromatic hydrocarbon groups, and more preferably one or more selected from the group consisting of alkyl and aryl groups. Preferably, the ethylene structure does not have any groups other than electron-withdrawing groups.

[0031] Examples of electron-withdrawing groups 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 These are some examples. Here, R 1 , R 2 , R 3 , and R 4 Each of these independently represents a monovalent hydrocarbon group or a monovalent heterocyclic group, which may or may not have substituents. R 5 , R 6 , and R 7 Each of these independently represents a hydrogen atom, a monovalent hydrocarbon group, or a monovalent heterocyclic group, which may or may not have substituents.

[0032] Examples of monovalent hydrocarbon groups include monovalent linear hydrocarbon groups, monovalent alicyclic hydrocarbon groups, and monovalent aromatic hydrocarbon groups.

[0033] A monovalent linear hydrocarbon group refers to a hydrocarbon group composed solely of a linear structure, and whose main chain does not contain a cyclic structure. However, the linear structure may be linear or branched. Examples of monovalent linear hydrocarbon groups include alkyl, alkenyl, and alkynyl groups. Alkyl, alkenyl, and alkynyl groups may be linear or branched.

[0034] As alkyl groups, alkyl groups having 1 to 12 carbon atoms are preferred, alkyl groups having 1 to 6 carbon atoms are more preferred, and alkyl groups having 1 to 4 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of alkyl groups 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] As the alkenyl, alkenyls having 2 to 12 carbon atoms are preferred, alkenyls having 2 to 6 carbon atoms are more preferred, and alkenyls having 2 to 4 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of alkenyls having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0036] As for the alkynyl, alkynyls having 2 to 12 carbon atoms are preferred, alkynyls having 2 to 6 carbon atoms are more preferred, and alkynyls having 2 to 4 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of alkynyls having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0037] Alkyl groups are preferred as monovalent chain hydrocarbon groups.

[0038] A monovalent alicyclic hydrocarbon group refers to a hydrocarbon group that contains only alicyclic hydrocarbons as its ring structure and does not contain an aromatic ring. The alicyclic hydrocarbon may be monocyclic or polycyclic. However, it does not need to be composed solely of alicyclic hydrocarbons; it may contain a chain-like structure as part of it. Examples of monovalent alicyclic hydrocarbon groups include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be monocyclic or polycyclic.

[0039] As for cycloalkyls, cycloalkyls having 3 to 12 carbon atoms are preferred, cycloalkyls having 3 to 6 carbon atoms are more preferred, and cycloalkyls having 5 to 6 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of cycloalkyls having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0040] As the cycloalkenyl, cycloalkenyls having 3 to 12 carbon atoms are preferred, cycloalkenyls having 3 to 6 carbon atoms are more preferred, and cycloalkenyls having 5 to 6 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of cycloalkenyls having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0041] As the cycloalkynyl, cycloalkynyls having 3 to 12 carbon atoms are preferred, cycloalkynyls having 3 to 6 carbon atoms are more preferred, and cycloalkynyls having 5 to 6 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of cycloalkynyls having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0042] As the monovalent alicyclic hydrocarbon group, cycloalkyl groups are preferred.

[0043] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not need to consist solely of an aromatic ring; it may also contain a chain structure, an alicyclic hydrocarbon structure, or other structures as part of it. Therefore, a monovalent aromatic hydrocarbon group can be an aralkyl group. The aromatic ring may be monocyclic or polycyclic. Preferred monovalent aromatic hydrocarbon groups are aryl groups having 6 to 12 carbon atoms, more preferably aryl groups having 6 to 10 carbon atoms, and even more preferably aryl groups having 6 carbon atoms. The carbon atom count does not include the carbon atoms of substituents. Examples of aryl groups having 6 to 12 carbon atoms include phenyl and naphthyl.

[0044] Phenyl is preferred as the monovalent aromatic hydrocarbon group.

[0045] Among these, alkyl, cycloalkyl, and aryl groups are preferred as monovalent hydrocarbon groups, with alkyl or aryl groups being more preferred.

[0046] A monovalent heterocyclic group is a group obtained by removing one hydrogen atom from a cyclic compound containing a heterocycle. Preferably, the heterocyclic group contains one or more atoms selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably, one or more atoms selected from the group consisting of oxygen, sulfur, and nitrogen atoms. A monovalent heterocyclic group is either a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group.

[0047] A monovalent aromatic heterocyclic group refers to a heterocyclic group containing an aromatic ring. Preferred monovalent aromatic heterocyclic groups are those having 1 to 15 carbon atoms, more preferably those having 1 to 9 carbon atoms, and even more preferably those having 1 to 6 carbon atoms. The carbon atoms of substituents are not included in the above carbon number calculation. Examples of monovalent aromatic heterocyclic groups include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridadinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, prinyl, anthraquinolyl, carbazonyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, acridinyl, coumarinyl, xanthenyl, and phthalazinyl.

[0048] A monovalent non-aromatic heterocyclic group is a heterocyclic group that does not contain an aromatic ring. Preferred monovalent non-aromatic heterocyclic groups are those having 2 to 15 carbon atoms, more preferably those having 2 to 9 carbon atoms, and even more preferably those having 2 to 6 carbon atoms. The carbon atoms of substituents are not included in the above carbon number calculation. Examples of monovalent non-aromatic heterocyclic groups include oxylanil, azilidinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranil, tetrahydrofuranil, dioxolanil, tetrahydrothiophenyl, pyrrolinil, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranil, tetrahydropyranil, tetrahydrothiopyranil, morpholinil, thiomorpholinil, piperazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

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

[0050] Examples of substituents 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 (Here, R s1 (This represents a hydrogen atom or a monovalent hydrocarbon group.) -(C=O)-R s2 (Here, R s2 (This represents a hydrogen atom or a monovalent hydrocarbon group.) -(C=O)-OR s3 (Here, R s3 (This represents a hydrogen atom or a monovalent hydrocarbon group.) -O-(C=O)-R s4 (Here, R s4 (This represents a hydrogen atom or a monovalent hydrocarbon group.) -N(R s5 )2(Here, there are multiple R s5 Each of these independently represents a hydrogen atom or a monovalent hydrocarbon group. -(C=O)-N(R s6 )2(Here, there are multiple R s6 Each of these independently represents a hydrogen atom or a monovalent hydrocarbon group. -N(R s7 )-(C=O)-R s8 (Here, R s7 R represents a hydrogen atom or a monovalent hydrocarbon group. s8 (This represents a monovalent hydrocarbon group.) -SO2-R s9 (Here, R s9 (This represents a hydroxyl or monovalent hydrocarbon group.) -S(=O)-R s10 (Here, R s10 (This represents a hydroxyl or monovalent hydrocarbon group.) Nitro, Cyano, and halogen atom These are some examples. Examples of halogen atoms include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms, with fluorine atoms or chlorine atoms being preferred.

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

[0052] Electron-withdrawing groups are, Preferably, -C(=O)-OR 1 -S(=O)2-R 2 , -P(=O)(-OR 3 )2, cyano, alkyl, carboxy, nitro, -S(=O)-R substituted with halogen atoms 4 -C(=O)-R 5 , and -C(=O)-NR 6 R 7 One or more species selected from the group consisting of, More preferably, -C(=O)-OR 1 -S(=O)2-R 2 , -P(=O)(-OR 3)2. One or more selected from the group consisting of cyano, alkyl substituted with halogen atoms, and carboxyl, More preferably, -C(=O)-OR 1 -S(=O)2-R 2 , -P(=O)(-OR 3 )2 and one or more selected from the group consisting of cyano. Here, R 1 , R 2 , R 3 , R 4 , R 5 , R 6、 and R 7 This is the same as the definition above.

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

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

[0055] [ka]

[0056] In equation (I), EWG 1 and EWG 2 Each of these groups independently represents an electron-withdrawing group, and together with the carbon atom to which they are bonded, they may form a ring.

[0057] Preferred examples of electron-withdrawing groups are the same as those described above. EWG 1 and EWG 2 Preferably, these are the same group. This prevents the organic derivatives produced by the reaction of the olefin compound with the organic substance from becoming two diastereomers.

[0058] Olefin compounds can be produced by previously known methods. Alternatively, commercially available olefin compounds can be used.

[0059] [1.3. Reaction Conditions] In the manufacturing method of this embodiment, an organic substance and an olefin compound are reacted under acidic conditions. This can suppress the oxidation reaction of group A present in the organic substance. Furthermore, if oxides such as disulfide compounds are present 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] "Acid conditions" means that the reaction between organic substances and olefin compounds is carried out in an acidic solution. The organic substances and olefin compounds do not need to be completely dissolved in the acidic solution; the reaction may be carried out with the organic substances and olefin compounds dispersed in the acidic solution. The acidic solution typically has a pH of less than 7, preferably 6.5 or lower, more preferably 6.0 or lower, even more preferably less than 6.0, and most preferably 5.5 or lower, 5.25 or lower, 5.0 or lower, 4.75 or lower, 4.5 or lower, or 4.25 or lower, preferably 0.0 or higher, more preferably 2.0 or higher. By setting the pH of the acidic solution within the above range, side reactions such as oxidation of organic substances and exchange reactions with disulfide compounds can be effectively suppressed while inhibiting the decomposition of derivatives. Here, the pH of the acidic solution and the pH of the neutral or basic solution described later can be measured using a pH meter employing the glass electrode method at a temperature of 25°C.

[0061] The acidic solution is preferably a solution containing water. The acidic solution may also contain an organic solvent in addition to water. The organic solvent is preferably a solvent that can be mixed with water in any proportion. Examples of such organic solvents include alcohol solvents such as methanol and ethanol; nitrile solvents such as acetonitrile; and aprotic polar solvents such as dimethyl sulfoxide, dimethylformamide, dioxane, and tetrahydrofuran. When the acidic solution is a solution containing water, the weight of the organic solvent in the solution relative 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] Acidic solutions typically contain acids. Examples of acids include inorganic acids and organic acids. Examples of inorganic acids include phosphoric acid, hydrochloric acid, sulfuric acid, and perchloric acid. Examples of 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 with the pH 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, preferably 5°C or higher, more preferably 10°C or higher, and 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, even more preferably 15 minutes or less, and usually 0 minutes or more.

[0065] Typically, reactions under acidic conditions are carried out by mixing organic substances and olefin compounds in an acidic solution. There are no particular limitations on the method of mixing organic substances and olefin compounds in an acidic solution. For example, 1) a method of preparing an acidic solution containing an organic substance and adding an olefin compound to it and mixing them, and 2) a method of preparing a solution containing an organic substance, adding a buffer solution or the like to adjust the pH to a desired range, and then adding an olefin compound and mixing it. From the viewpoint of suppressing the oxidation reaction of organic substances, it is preferable to prepare an acidic solution containing organic substances and then add and mix an olefin compound to the resulting solution. 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 organic substances in the acidic solution is not particularly limited, but may be, for example, 0.1 μmol / L to 100 mmol / L. The amount of olefin compound relative to the organic substance is not particularly limited, but for example, it may be 1 to 100 in molar ratio.

[0067] If the organic substance contains an amino group in addition to group A, the organic substance may be reacted with the olefin 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. Subsequently, under neutral or basic conditions, the amino group contained in the organic substance (which may be an unsubstituted amino group, a monosubstituted amino group, or a disubstituted amino group) reacts with the olefin compound to be converted into a substituted amino group (or a substituted ammonia group if the amino group contained in the organic substance is a disubstituted amino group) having a substituent derived from the olefin compound. By this manufacturing method, both group A contained in the organic substance and the amino group that the organic substance may contain react with the olefin compound, and a derivative is obtained in which both group A and the amino group are converted.

[0068] Derivatives containing more substituted amino groups offer advantages in manufacturing and analysis compared to derivatives containing the unsubstituted amino group. Generally, more substituted amino groups exhibit improved hydrophobicity compared to the unsubstituted amino group. Therefore, separation conditions for organic derivatives can often be easily established in liquid chromatography. 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, resulting in weak retention in reversed-phase liquid chromatography and extremely strong retention in hydrophilic interaction chromatography (HILIC), which can make it difficult to easily establish separation conditions. Furthermore, derivatives containing more substituted amino groups often have higher detection sensitivity in mass spectrometers compared to derivatives containing the unsubstituted amino group.

[0069] Neutral or basic conditions mean 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 need to be completely dissolved in the solution; the reaction may be carried out with the acidic derivative and the olefin compound dispersed in the solution.

[0070] The neutral or basic solution typically has a pH of 7 or higher, preferably 7.5 or higher, more preferably 8 or higher, even more preferably 8.5 or higher, preferably 11 or lower, more preferably 10 or lower, and even more preferably 9.5 or lower. By setting the pH of the neutral or basic solution within the above range, the reaction between amino groups that may be present in organic substances and olefin compounds can be promoted, and the decomposition of derivatives can be suppressed.

[0071] Neutral or basic solutions are preferably solutions containing water. Neutral or basic solutions may also contain organic solvents in addition to water. Preferred organic solvents are those that can be mixed with water in any proportion. Examples of such organic solvents include those exemplified in the description of acidic solutions. If the neutral or basic solution is a solution containing water, the range of the weight of the organic solvent in the solution relative to the water may be the same as the preferred range given in the description of the acidic solution. More preferably, the neutral or basic solution is an aqueous solution.

[0072] Basic solutions typically contain a base. Examples of bases include inorganic bases and organic bases. Examples of inorganic bases 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 organic bases include amines (e.g., trimethylamine, triethylamine). A buffer solution with a pH adjusted to a desired range may be used as a neutral or basic solution.

[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, preferably 5°C or higher, more preferably 10°C or higher, and 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, even more preferably 15 minutes or less, and usually 0 minutes or more.

[0075] Typically, reactions under neutral or basic conditions are carried out by mixing the derivative and the olefin compound in a neutral or basic solution under acidic conditions.

[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. Alternatively, the derivative under acidic conditions may be mixed with the olefin compound in a neutral or basic solution without isolating it from the acidic solution, by adjusting the pH of the acidic solution to a desired neutral or basic pH.

[0077] As described above, when the derivative is mixed with the olefin compound in solution without isolating it under acidic conditions, additional olefin compound may be added to the solution. Alternatively, an excess amount of olefin compound may be added during the reaction under acidic conditions, and the surplus olefin compound may be used in the reaction under neutral or basic conditions.

[0078] The formation of derivatives of organic substances can be confirmed by methods such as liquid chromatography and mass spectrometry.

[0079] The method for producing the derivative of this embodiment may include optional steps in addition to the reaction step of reacting an organic substance with an olefin compound. Examples of optional steps include, for example, 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 method, group A is converted to a group that is more stable against oxidation. Therefore, derivatives of organic substances can be analyzed more accurately compared to analyzing the organic substance itself.

[0081] [2. Analytical methods for samples containing organic substances] A method for analyzing a sample containing an organic substance according to one embodiment of the present invention includes (1) a sample containing an organic substance having one or more groups selected from the group consisting of sulfanil, seranil, and sulfino, An olefin compound containing an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms), To obtain a treated sample containing a derivative of the organic substance by mixing under acidic conditions, and (2) The method includes analyzing the derivative of the organic substance in the treated sample.

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

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

[0084] The concentrations of organic substances and olefin compounds in the sample are not particularly limited, as long as they react to produce derivatives of organic substances and the products can be analyzed. The individual concentrations of organic substances and olefin compounds 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 one that has undergone pretreatment, such as deproteinization. In the analytical method of this embodiment, since the sample is processed under acidic conditions, it is not necessary to adjust the sample's pH to neutral or basic after performing an acidic treatment such as deproteinization as a pretreatment. The sample can be mixed with the olefin compound while maintaining its acidic pH. Under acidic conditions, side reactions such as the oxidation reaction of group A and the exchange reaction between group A and disulfide are less likely to proceed. Therefore, the progress of side reactions can be suppressed between the time of pretreatment and analysis. As a result, organic substances containing group A can be analyzed more accurately.

[0086] The sample may contain disulfide compounds. Disulfide compounds are compounds containing a group represented by -SS-. Disulfide compounds can be produced by the oxidation of thiols. For example, in biological samples, information on the relative abundance of thiols (e.g., cysteine, reduced glutathione) and disulfide compounds (e.g., cystine, oxidized glutathione) is useful for understanding the redox state of the biological sample. In the analytical method of this embodiment, easily oxidizable group A, such as sulfanil, contained in the organic substance is reacted with an olefin compound to convert the organic substance into a derivative before analysis. Furthermore, in the analytical method of this embodiment, the sample is treated under acidic conditions. As mentioned above, under acidic conditions, the exchange reaction between thiols and disulfide compounds is less likely to occur. Therefore, the amount of organic substance contained in the sample can be accurately determined.

[0087] Examples of disulfide compounds that the sample may contain include compounds in which the sulfanil present in the organic substance is converted to a disulfide through intermolecular reaction. More specifically, examples 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 groups that the disulfide compound may contain include unsubstituted amino groups (-NH2), as well as monosubstituted amino groups and disubstituted amino groups. The amino groups that the disulfide compound may contain are preferably one or more selected from the group consisting of unsubstituted amino groups and monosubstituted amino groups, with unsubstituted amino groups being more preferred. The disulfide compound may contain only one amino group in one molecule, or it may contain multiple amino groups. The number of amino groups that may be contained in one molecule of the disulfide compound may be one, two, three, or four, preferably one, two, or three, and more preferably one or two. In one embodiment, the sample includes one or more selected from the group consisting of oxidized glutathione and cystine.

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

[0089] A sample containing an organic substance and an olefin compound are mixed under acidic conditions to obtain a treated sample containing a derivative of the organic substance. Preferably, the mixture of the sample containing organic matter and the olefin compound is carried out in an acidic solution. The type of acidic solution and its pH can be the same as in the preferred example described in [1.3. Reaction Conditions].

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

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

[0092] If the organic substance further contains an amino group in group A, it is preferable that step (1) 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 the derivative of the organic substance. As described above, group A in the organic substance in the sample reacts with the olefin compound under acidic conditions to be converted into a substituent that is stable against side reactions such as oxidation. Then, under neutral or basic conditions, the amino group (which may be an unsubstituted amino group, a monosubstituted amino group, or a disubstituted amino group) in the organic substance reacts with the olefin compound to be converted into a substituted amino group (or a substituted ammonia group if the amino group in the organic substance is a disubstituted amino group), yielding a derivative of the organic substance. As described above, the more substituted amino group has improved hydrophobicity, so derivatives in which a less substituted amino group has been converted into a more substituted amino group can usually be easily separated by setting the separation conditions in liquid chromatography. In addition, derivatives in which a less substituted amino group has been converted into a more substituted amino group usually have increased detection sensitivity in mass spectrometers.

[0093] If a sample contains a compound with a low degree of substitution (for example, a disulfide compound containing an unsubstituted amino group), the compound is highly hydrophilic, resulting in weak retention in reversed-phase liquid chromatography and extremely strong retention in hydrophilic interaction chromatography (HILIC), which can make it difficult to easily set separation conditions. Even when a sample contains compounds with a low degree of substitution, compounds containing low-degree-of-substitution amino groups, such as disulfide compounds containing unsubstituted amino groups, will undergo a reaction with the olefin compound under neutral or basic conditions, converting the amino group into a more substituted amino group (or a substituted ammonia group if the amino group is a disubstituted amino group). Similar to derivatives of organic substances, compounds such as disulfide compounds in which the amino group has been converted into a more substituted amino group can usually be easily separated using liquid chromatography, as separation conditions can be easily set. Furthermore, the detection sensitivity in mass spectrometers is usually increased.

[0094] For example, we will describe an analytical method when 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 dimerized form of cysteine. It is thought that in the acidic treated sample obtained by mixing the olefin compound and the sample under acidic conditions, a cysteine ​​derivative (first derivative) and cystine are present, which are obtained when the sulfanyl group of cysteine ​​reacts with the olefin compound.

[0095] Cysteine ​​derivatives have one unsubstituted amino group, while cystine has two unsubstituted amino groups. This difference results in a significant difference in hydrophobicity between cysteine ​​and cystine derivatives. Therefore, setting up conditions for simultaneous analysis of cysteine ​​and cystine using liquid chromatography can sometimes be challenging.

[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, it is considered that the treated sample contains a cysteine ​​derivative (second derivative) obtained by the reaction of sulfanil groups and amino groups with the olefin compound, and a cystine derivative obtained by the reaction of amino groups 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 smaller, making it relatively easy to set conditions for simultaneous analysis of cysteine ​​derivatives and cystine during liquid chromatography.

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

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

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

[0101] The processing temperature and processing 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 can be mixed in an acidic solution, and then further mixed in a neutral or basic solution to obtain a treated sample containing a derivative of the organic substance.

[0103] [2.2. Process (2)] In step (2), derivatives of organic substances in the treated sample are analyzed. Step (2) may include steps (2a) and (2b) in that order. [2.2.1. Process (2a)] In step (2a), derivatives of organic substances are separated from the sample treated in step (1). The separation can be carried out by any method that can separate the derivative from other substances (e.g., disulfide compounds) contained in the sample being treated. Examples of separation methods, though not limited to them, include 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, as well as the type and properties of the derivatives of organic substances. Liquid chromatography is preferred as the separation method, and high-performance liquid chromatography (HPLC) is more preferred. These separation methods can be carried out 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 in reversed-phase liquid chromatography include, but are not limited to, silica gel modified with hydrophobic compounds such as octadecylsilane. Examples of mobile phases include, but are not limited to, organic solvents, aqueous solutions, and mixtures thereof. Preferred examples of organic solvents include acetonitrile, methanol, ethanol, and isopropanol. 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.

[0106] Examples of stationary phases in normal-phase liquid chromatography include, but are not limited to, silica gel and alumina. 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 mixtures thereof. Preferred examples of organic solvents include acetonitrile, methanol, ethanol, and isopropanol. 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 targets that have poor retention in the stationary phase of reversed-phase chromatography. For example, disulfide compounds such as cystine and oxidized glutathione, which are not derivatized by 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 method, by using a column loaded with chiral packing material (chiral column), chiral compounds can be separated when an organic substance is a mixture of chiral compounds. For example, derivatives of D-cysteine ​​(which is rare in nature) and derivatives of L-cysteine ​​can be separated using a chiral column.

[0110] [2.2.2. Process (2b)] In step (2b), derivatives of the separated organic substance are detected. Examples of detection methods, though not limited to them, include mass spectrometry using a mass spectrometer, detection of ultraviolet absorption (UV), photodiode array detection (PDA), fluorescence detection (FL), detection of visible light absorption, inductively coupled plasma atomic emission spectrometry (ICP), detection of corona charged particles, differential refractive index detection (RI), and evaporative light scattering detection (ELSD). The detection method may be appropriately selected depending on the type and properties of impurities contained in the sample, as well as the type and properties of derivatives of organic substances. Preferred detection methods include mass spectrometry, ultraviolet absorption detection, fluorescence detection, visible light absorption detection, and inductively coupled plasma atomic emission spectroscopy, with mass spectrometry and ultraviolet absorption detection being more preferred. These detection methods may be used individually or in combination. These separation methods can be carried out by previously known methods.

[0111] The mass spectrometry method is not particularly limited, and may be a mass spectrometry method that combines various ionization methods, various ion separation methods, and various detectors. 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 (e.g., Orbitrap® mass spectrometers).

[0112] [2.3. Optional Steps] The analysis method of this embodiment may include any steps other than steps (1) and (2) described above. An example of an optional step is the derivatization of amino acids using an amino acid derivatization reagent such as ninhydrin or o-phthalaldehyde, when a sample containing amino acids is used. It is preferable to perform the amino acid derivatization step after step (1) and before step (2). Including such optional steps provides the following advantages, for example: For example, cysteine ​​and cystine are usually difficult to analyze simultaneously 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 performing step (1), only cysteine ​​reacts with the olefin compound and is converted into a derivative. As a result, the retention time of cysteine ​​after derivatization with an amino acid derivatizing agent is different from that of cystine. Therefore, with the analytical method of this embodiment, cysteine ​​and cystine can be analyzed simultaneously using an amino acid analyzer.

[0113] [3. Derivatizers for organic substances under acidic conditions] An agent according to one embodiment of the present invention is a derivatizing agent for an organic substance containing one or more groups selected from the group consisting of sulfanil, seranil, and sulfino, under acidic conditions, 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 described above. Similarly, examples and preferred examples of organic substances are the same as those described above. The conditions for producing derivatives using a derivatizing agent are acidic. Preferably, the derivatization of organic substances is carried out in an acidic solution containing the derivatizing agent. The type of acidic solution and the pH of the acidic solution can be the same as those described in the preferred examples in [1.3. Reaction Conditions].

[0115] The concentration of the olefin compound in the derivatizing agent depends on the dilution ratio of the derivatizing agent, but is, 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. Reagents for the Analysis of Organic Substances] A reagent according to one embodiment of the present invention is an analytical reagent for organic substances containing one or more groups selected from the group consisting of sulfanil, seranil, and sulfino, and includes the derivatizing agent.

[0117] Examples and preferred examples of olefin compounds are the same as those described above. Similarly, examples and preferred examples of organic substances are the same as those described above.

[0118] The concentration of the olefin compound in the analytical reagent depends on the dilution ratio of the analytical reagent, but is, 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 steps (1) and (2) described above. [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. Unless otherwise specified, the following operations were carried out under atmospheric pressure and at room temperature.

[0121] [Explanation of abbreviations] In the following, abbreviations have the following meanings. (Olefin compounds (derivatives)) 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" represents phenyl and "Et" represents ethyl.

[0124] (Organic substances (chalcogen-containing compounds)) • Thiols Cys: cysteine Cys2: Cystine GSH: Reduced Glutathione GSSG: Glutathione (oxidized form) 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, analytical methods, etc.) LC-MS / MS: Liquid Chromatography-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 residual Cys after the reaction. Furthermore, the EMM-derivative Cys peak was confirmed by LC-MS / MS.

[0129] [ka]

[0130] (A1-1. Manufacturing of derivatives) L-cysteine ​​hydrochloride (Sigma) was dissolved in 0.1% formic acid solution (Fujifilm Wako Pure Chemical Industries, prepared by dilution with pure water) to a concentration of 10 mmol / L. The derivatizing agent EMM (Asta Tech) was dissolved in acetonitrile (Fujifilm Wako Pure Chemical Industries) to a concentration of 10 μL / mL, and the derivatizing agent NEM (Wako Pure Chemical Industries) was dissolved in acetonitrile to a concentration of 10 mg / mL. 10 μL of the derivatizing 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 the mixture was thoroughly stirred to obtain the sample solution.

[0131] (A1-2. Analysis of derivatives) The sample solution was analyzed by LC-MS / MS under the following conditions. An Agilent 1200 series LC spectrometer (manufactured by Agilent) and a 3200QTRAP system tandem mass spectrometer (manufactured by Sciex) were used.

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

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

[0134] [Table 1]

[0135] (Analysis results) A peak for non-derivativeized Cys was observed at 1.36 min, NEM-Cys peaks at 2.00 and 2.14 mins (two peaks were observed due to diastereomer formation), and an EMM-Cys peak at 8.23 ​​min. Each peak was integrated. The measurement was performed twice, and the result was the average of the two measurements. The derivatization rate was calculated using the following formula (1).

[0136]

number

[0137] The analysis results are shown in the table below. Examples of chromatograms are also shown in Figures 1-3. Figure 1 shows an example of a chromatogram that shows a cysteine ​​peak. Figure 2 shows an example of a chromatogram illustrating the NEM-Cys peak. Figure 3 is an example of a chromatogram showing the EMM-Cys peak.

[0138] [Table 2]

[0139] The results above indicate that Cys can be derivatized with high reaction rates by NEM or EMM under acidic conditions and mild temperature conditions (room temperature). In particular, EMM showed a higher reaction rate compared to NEM. Furthermore, when Cys, a chiral compound, was derivatized with NEM, a diastereomer was formed, and the NEM-Cys peak split into two. On the other hand, when Cys was derivatized with EMM, no diastereomer was formed, and no peak splitting occurred.

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

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

[0142] (A2-2. Analysis of derivatives) Sample solutions were analyzed using LC-MS / MS. A Nexera LC system (Shimadzu Corporation) was used, and a Triple Quad 6500 tandem mass spectrometer (Sciex) was employed.

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

[0144] (MS / MS conditions) The procedure 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] Based on these results, it can be seen that various thiols can be derivatized by EMM, the derivatized thiols can be simultaneously separated by reverse-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 acid (-SO2H) are nucleophilic and can be derivatized by an EMM agent and analyzed.

[0150] [ka]

[0151] (A3-1. Manufacturing of derivatives) Hypotaurine (Sigma) was dissolved in 0.01 mol / L hydrochloric acid to a concentration of 1 mmol / L. 10 μL of the 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. The mixture was stirred and left at room temperature for 10 minutes. 920 μL of 0.1% formic acid solution was added and thoroughly stirred to obtain the sample solution.

[0152] (A3-2. Analysis of derivatives) The sample solution was analyzed by LC-MS / MS under the following conditions. An Agilent 1200 series LC spectrometer (manufactured by Agilent) and a 3200QTRAP system tandem mass spectrometer (manufactured by Sciex) were used.

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

[0154] (MS / MS conditions) The procedure 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. An example of a chromatogram is shown in Figure 4. Figure 4 is an example of a chromatogram showing the peak of derivatized hypotaurine by EMM.

[0157] [Table 6]

[0158] Based on these results, it can be seen 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, we confirmed that persulfide derivatization and analysis are possible. Persulfide cysteine ​​(Cys-SH and Cys-SSH) was used as the persulfide. Cysteine ​​persulfide plays an important role in oxidation-reduction reactions in living organisms. Therefore, if it is possible to derivatize persulfides while maintaining their oxidation-reduction state in the present invention, it would be useful for understanding the oxidation-reduction state in living organisms.

[0160] (A4-1. Manufacturing of derivatives) To 70 μL of pH 6.7 ammonium formate solution (manufactured by Fujifilm Wako Pure Chemical Industries), 10 μL of 100 mmol / L cysteine ​​solution, 10 μL of 100 mmol / L sodium sulfide aqueous solution (manufactured by Fujifilm Wako Pure Chemical Industries, diluted with pure water), and 10 μL of 0.1% hydrogen peroxide aqueous solution (manufactured by Kanto Chemical Industries, diluted with pure water) were mixed and reacted at 30°C for 30 minutes to generate persulfurized cysteine ​​in the system. To 10 μL of the above solution, 60 μL of 20 mmol / L sodium phosphate buffer (pH 2.5) and 10 μL of EMM acetonitrile solution (10 μL / mL) were added, stirred, and left at room temperature for 10 minutes. 420 μL of 0.1% formic acid solution was added and stirred well to obtain the sample solution.

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

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

[0163] (MS / MS conditions) Performed under the conditions shown in the following table.

[0164]

Table 7

[0165] (Analysis results) The analysis results are shown in the following table. Also, an example of the chromatogram is shown in Figure 5. Figure 5 is an example of a chromatogram showing the peaks of cysteine and persulfidated cysteine derivatized by EMM.

[0166]

Table 8

[0167] Based on these results, it can be seen that persulfides such as persulfide 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, we 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, then amino acids were separated from the samples using an ion exchange column. Subsequently, the amino acids were derivatized with ninhydrin using a post-column derivatization method, and Cys, Cys2, and amino acids were simultaneously analyzed. In amino acid analyzers, Cys and Cys2 typically elute from the column simultaneously, making it difficult to analyze Cys and Cys2 at the same time.

[0169] (A5-1. Manufacturing of derivatives) A mixed standard solution was prepared by adding 25 μL of 10 mmol / L cysteine ​​hydrochloride aqueous solution and 125 μL of pure water to 100 μL of amino acid mixed standard solution, type H (manufactured by Fujifilm Wako Pure Chemical Industries) (a solution containing 2.5 mmol / 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). To 100 μL of this solution, 200 μL of 20 mmol / L sodium phosphate buffer (pH 2.5) and 50 μL of EMM acetonitrile solution (5 μL / mL) were added, the mixture was 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 the sample solution.

[0170] (A5-2. Analysis of derivatives) The sample solution was analyzed using the following analytical instrument. • Analytical instrument: Hitachi L-8900 high-speed amino acid analyzer • Analytical method: The "Biological Fluid Analysis Method" from the method package with an analysis time of "148 minutes" was used.

[0171] (Analysis results) Cysteine ​​derivatives eluted after a retention time of 40.3 minutes, and Cys2 eluted after a retention time of 48.8 minutes. Other amino acids eluted at the retention time of 148 minutes, which is the standard retention time for "biofibreeding fluid analysis". An example of a 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 these results, it can be seen that by processing a sample containing Cys, Cys2, and various amino acids under acidic conditions and analyzing it with a standard amino acid analyzer, Cys, Cys2, and various amino acids can be analyzed simultaneously.

[0172] [Example B: Example using BPSE] [Example B1] Derivatization and analysis using BPSE In this example, we confirmed that thiols can be derivatized and analyzed using BPSE. The following shows an example of a reaction between BPSE and thiols.

[0173] [ka]

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

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

[0176] [Table 9]

[0177] From the above results, it can be seen that under acidic conditions, thiols can be derivatized by BPSE, separated by column chromatography, and detected using a UV detector. Amino acids having sulfanyl are expected to be converted into highly hydrophobic amino acids by derivatization and to be well separated and detected from other amino acids with low hydrophobicity by column chromatography using an ODS (C18) column.

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

[0179] (B2-1. Manufacturing of derivatives) A mixed standard solution of D,L-cysteine ​​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), and 10 μL of BPSE acetonitrile solution (10 mg / mL) was added. The mixture was stirred and left at room temperature for 10 minutes. 420 μL of 20 mmol / L sodium phosphate buffer (pH 2.5) was added and thoroughly stirred. This solution was further diluted 10-fold in a mobile phase to obtain the sample solution.

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

[0181] (Analysis results) The analysis 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 chiralthiol.

[0182] [Table 10]

[0183] The above results show that chiral thiols can be derivatized with BPSE under acidic conditions, separated using a chiral column, and detected. Therefore, the method described in the examples allows for the efficient detection of D-Cys, which is naturally present in small amounts, 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 (derivatives) containing EMM and BPSE with Cys was evaluated. The olefin compounds used in this example and reference example, and their abbreviations, 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. To 10 μL of this Cys solution, 60 μL of buffer at various pH levels and 10 μL of derivatizing agent solution (or acetonitrile) were added, and the mixture was allowed to stand at room temperature for 10 minutes. After the reaction, 920 μL of 0.1% formic acid aqueous solution was added to prepare the sample solution. The acetonitrile solution of the derivatizing agent was prepared at a concentration of 10 mg / mL (solid) or 10 μL / mL (liquid). A pH 2.5 or 7.0 sodium phosphate buffer solution (20 mM) was used as the buffer. For derivatization by 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 further used as buffers.

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

[0188] (MS conditions) The following conditions were met. 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 refers to Cys derivatized with derivatizing agent X. Furthermore, in the detection of EBzM-Cys, two peaks were observed as diastereomers, and their respective retention times are listed in the table below.

[0189] [Table 11]

[0190] (Analysis results) The analysis results are shown in the table below. The Cys response rate was calculated using formula (1) above. Items marked with "*" indicate that data is unavailable.

[0191] [Table 12]

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

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

[0194] [Table 13]

[0195] Based on these results, it can be seen 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 their behavior under various pH conditions.

[0197] (D-1. Observation of the 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. To 50 μL of Cys solution (10 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 prepare a mixed solution. (Finally, the solution contained 0.5 mmol / L of Cys and 5 mmol / L of GSSG.) Samples were taken over time, derivatization was performed by BPSE, and analysis was performed by LC under the following conditions to measure the area values ​​of Cys and GSH. Peaks were detected by an HPLC-UV detector.

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

[0199] (LC condition) Mobile phase A: 20 mmol / L sodium phosphate buffer (pH 2.5) Mobile phase B: Acetonitrile • Gradient conditions: 25%B (0-8.0 min), 25-90%B (8.0-9.0 min), 90%B (9.0-11.0 min), 90-25%B (11.0-12.0 min), 25%B (12.0-15.0 min) • Column: Triart C18, 3μm, 150×4.6mm (manufactured by YMC) Column temperature: 40°C ·Flow rate: 1.0mL / min Injection volume: 10 μL 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 and Figures 9-12 below. Figures 9, 10, 11, and 12 are graphs showing the time-dependent changes in 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 0-minute data for pH 6.0, 7.0, and 8.0 is assumed to be from samples that have not undergone buffer treatment and is therefore shared with the 0-minute data for pH 2.5.

[0201] [Table 14]

[0202] [Table 15]

[0203] [Table 16]

[0204] [Table 17]

[0205] Based on these results, it can be seen that under conditions of pH 7.0 or higher, Cys and GSSG react rapidly, and the GSH produced by the exchange reaction is detected, indicating a decrease in Cys. On the other hand, under conditions of pH 6.0 or lower, the reaction between Cys and GSSG hardly proceeds, and in particular, under conditions of pH 2.5, the product of the exchange reaction, GSH, is not detected even after 1 hour. These results demonstrate that even in samples containing both disulfide and thiol compounds, thiol compounds can be accurately analyzed under acidic conditions.

[0206] (D-2. Observation of the thiol oxidation reaction) (Sample preparation) L-Cys hydrochloride monohydrate and copper sulfate pentahydrate were dissolved in 0.01 mol / L hydrochloric acid to a concentration of 10 mmol / L and 0.1 mmol / L, respectively. To 50 μL of Cys solution (10 mmol / L), 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) were added to prepare a mixed solution. (Finally, the solution contained 0.5 mmol / L of Cys and 0.01 mmol / L of copper sulfate.) Samples were taken over time, derivatization was performed by BPSE, and the area values ​​of Cys were measured by LC under the same conditions as in (D-1. Observation of Thiol Exchange Reaction). 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 time course of the peak area values ​​of Cys at pH 8.0, pH 7.0, pH 6.0, and pH 2.5. Note that the 0-minute data for pH 6.0, 7.0, and 8.0 are assumed to be untreated and are therefore shared with the 0-minute data for pH 2.5.

[0208] [Table 18]

[0209] Based on these results, it can be seen that under conditions of pH 7 or higher, Cys decreases rapidly, indicating that Cys is rapidly oxidized. On the other hand, under conditions of pH 6.0 or lower, Cys hardly decreases, indicating that the oxidation reaction of Cys hardly proceeds. These results indicate that under acidic conditions, the oxidation reaction of thiol compounds is suppressed, allowing for accurate analysis of thiol compounds.

[0210] [Example E] Derivatization of sulfanyl and amino groups In this example, it was confirmed that when cysteine ​​or cystine was mixed with EMM under acidic conditions, and then the solution was made basic, the amino group (-NH2) of cysteine ​​or cystine reacted with EMM, resulting in the derivatization of cysteine ​​or cystine. Specifically, 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 typically do not react with EMM. Therefore, under acidic conditions, disulfide compounds such as cystine, which have an amino group but no sulfanyl group, typically do not react with EMM and are not derivatized. However, it has been confirmed that even such disulfide compounds can be derivatized under basic conditions when the amino group reacts with EMM. 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. Furthermore, the detection sensitivity of derivatives in mass spectrometers can be improved.

[0211] [ka]

[0212] (E-1. Manufacturing 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 thoroughly stirred. 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, and the mixture was stirred and allowed 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), and the mixture was further stirred and allowed 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 thoroughly stirred to prepare the analytical sample.

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

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

[0215] (MS / MS conditions) The procedure 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. 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 the reaction of one molecule of cysteine ​​with three molecules of EMM. "4EMM-Cystine" and "4EMM-Cys2" refer to derivatives obtained by the reaction of one molecule of cystine with four molecules of EMM. Figure 14 is an example of a chromatogram showing the peak of derivatized cysteine ​​or derivatized cystine as determined by EMM.

[0218] [Table 20]

[0219] Based on these results, it can be seen that when cysteine ​​is reacted 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, resulting in the derivatization of cysteine. By making the reaction solution basic, cystine is derivatized when its two amino groups react with four molecules of EMM. In other words, the above results indicate that cysteine ​​can be analyzed by LC-MS / MS as a compound reacted with EMM3 molecules, and cystine can be analyzed by LC-MS / MS as a compound reacted with EMM4 molecules.

Claims

1. A method for producing a cysteine ​​derivative, comprising reacting cysteine ​​with 1,1-bis(phenylsulfonyl)ethylene under acidic conditions to obtain a cysteine ​​derivative represented by the following formula (II). 【Chemistry 1】 (In formula (II), Ph represents a phenyl group.)

2. The method for producing the derivative according to claim 1, wherein the acidic conditions are conditions where the pH is less than 6.

0.

3. A derivatizer for compounds represented by the following formula (II), including 1,1-bis(phenylsulfonyl)ethylene, under acidic conditions of cysteine. 【Chemistry 2】 (In formula (II), Ph represents a phenyl group.)

4. A reagent for the analysis of cysteine, comprising the derivatizing agent described in claim 3.

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