Derivatization reagent, derivatization kit, and method for improving sensitivity of nitrogen-containing compound in mass spectrometry

Abstract

Provided is a novel technique for improving sensitivity in mass spectrometry of nitrogen-containing compounds. The present invention can provide a derivatization reagent containing a compound represented by formula (1). RR 'CO (1) (In formula (1), R and R' may be the same or different and each represents a hydrogen atom or a substituted or unsubstituted alkyl group or aryl group having 1-11 carbon atoms.

Description

Technical Field

[0001] The present invention relates to a derivatization reagent, a derivatization kit, a method for improving the sensitivity of nitrogen-containing compounds in mass spectrometry, and the like. Background Art

[0002] Liquid chromatography-tandem mass spectrometer (LC-MS / MS) and matrix-assisted laser desorption ionization mass spectrometer (MALDI-MS) are very powerful analytical methods for quantitatively analyzing trace metabolites and contents. In these analytical methods, ionization of the analysis object compound is sometimes performed. Regarding the analysis of the ionization of the analysis object compound, according to the analysis object compound, there are analysis object compounds with low ionization efficiency, and there are also many situations where it is impossible to detect and where it is impossible to ensure the detection accuracy for quantification. Therefore, sometimes the analysis object compound is derivatized to improve the ionization efficiency.

[0003] For example, the following Non-Patent Documents 1 and 2 disclose that a fat-soluble hormone (androgen or estrogen) not containing a nitrogen atom is derivatized with a derivatizing agent containing a nitrogen atom.

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent document 1: J. Chromatogr. B 2011, 879, 1159-1165.

[0007] Non-patent document 2: Biomed. Chromatogr. 2021, 35, e5036.

[0008] Non-patent document 3: Anal. Chem. 1997, 69, 137-144.

[0009] Non-patent document 4: Rapid Commun Mass Spectrum 1999, 13.1413-1422.

[0010] Non-patent document 5: J.Am.Soc.Mass Spectrum 2017,28,1889-1900. Summary of the invention

[0011] An object of the present invention is to provide a new technique for improving the sensitivity in mass spectrometry analysis of nitrogen-containing compounds.

[0012] The present inventors have found that, as shown in the following [Examples], the sensitivity in mass spectrometry analysis of nitrogen-containing compounds can be improved by derivatizing nitrogen-containing compounds with aldehydes or ketones, thereby completing the present invention. The present invention is as follows.

[0013] The present invention can provide a derivatization reagent comprising a compound (preferably an aldehyde or a ketone) represented by the following formula (1).

[0014] RR'CO(1)

[0015] In the formula (1), R and R' are the same or different and represent a hydrogen atom or a substituted or unsubstituted alkyl or aryl group having 1 to 11 carbon atoms.

[0016] The present invention can provide a derivatization kit, which comprises the derivatization reagent and a reducing agent.

[0017] The present invention can provide a method for improving the sensitivity of nitrogen-containing compounds in mass spectrometry analysis, which comprises using the derivatization kit (preferably a derivatization reagent containing an aldehyde or ketone) to perform derivatization to N-alkylate or N-arylate the amino group of the nitrogen-containing compound to produce a secondary amine or a tertiary amine.

[0018] The derivatizing reagent can also be used to N-alkylate or N-arylate the amino group of a nitrogen-containing compound.

[0019] The nitrogen-containing compound may be one or more selected from amino acids, peptides, and catecholamines contained in biological components.

[0020] Alternatively, the derivatization reagent is used to N-alkylate or N-arylate the amino group of the nitrogen-containing compound.

[0021] (a) When the nitrogen-containing compound is an amino acid or peptide in a biological component, R and R' in the formula (1) are the same or different and are a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 8 carbon atoms, or

[0022] (b) When the nitrogen-containing compound is a catecholamine in a biological component, R and R' in the formula (1) are the same or different and are substituted or unsubstituted alkyl groups having 2 to 9 carbon atoms.

[0023] The derivatization reagent or the derivatization kit can also be used for mass spectrometry analysis.

[0024] The reducing agent may also include one or more selected from borane, a complex containing borane, and a salt of a boron hydride compound.

[0025] The present invention can provide a new technique for improving the sensitivity in mass spectrometry analysis of nitrogen-containing compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1This is a diagram schematically showing a reaction in which an aliphatic or aromatic aldehyde or ketone and a reducing agent are used to form an imine by dehydration condensation between an amine (1) and an aldehyde or ketone (2), and the resulting imine is reduced with a reducing agent (secondary amine (3) or tertiary amine (4)).

[0027] Figure 2 The diagram shows the chemical structural formulas of various amino acids (triiodothyronine (5), thyroxine (6), homocysteine ​​(7)) used in the present embodiment.

[0028] Figure 3A This is a diagram showing various peptides (angiotensin I (8), oxytocin (9), glucagon (10), and adrenocorticotropic hormone (11)) used in this embodiment.

[0029] Figure 3B This is a diagram showing various peptides (insulin (12), C-peptide (13)) used in this embodiment.

[0030] Figure 4 This is a diagram showing various catecholamines (epinephrine (14), norepinephrine (15), and dopamine (16)) used in this embodiment.

[0031] Figure 5 The derivative compound (T3-Me2) of the amino acid standard (T3) using an aldehyde reagent (formaldehyde) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph).

[0032] Figure 6 The derivative compound (T4-Me2) of the amino acid standard (T4) using an aldehyde reagent (formaldehyde) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph).

[0033] Figure 7 The derivative compound (T4-Bn2) of the amino acid standard (T4) using an aldehyde reagent (benzaldehyde) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph).

[0034] Figure 8 The derivative compound (HCys-iPr) of the amino acid standard (HCys) using the ketone reagent (acetone) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph).

[0035] Fig. 9The derivative compound (AI-Me2) of the peptide standard (AI) using an aldehyde reagent (formaldehyde) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph).

[0036] Fig.10 The derivative compound (AI-Et2) of the peptide standard (AI) using an aldehyde reagent (acetaldehyde) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph).

[0037] Fig.11 The derivatized compound (AI-iPr) of the peptide standard (AI) using a ketone reagent (acetone) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph).

[0038] Fig.12 The derivative compound (OXT-Me2) of the peptide standard (OXT) using an aldehyde reagent (formaldehyde) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph).

[0039] Fig.13 The derivative compound (OXT-Et2) of the peptide standard (OXT) using the aldehyde reagent (acetaldehyde) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph).

[0040] Fig.14 The derivatized compound (OXT-Pr2) of the peptide standard (OXT) using an aldehyde reagent (propionaldehyde) is shown. 1 Graphs showing the results of H-NMR analysis (upper panel A) and MS analysis (lower panel B).

[0041] Fig.15 The derivatized compound (OXT-iPr) of the peptide standard (OXT) using a ketone reagent (acetone) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph).

[0042] Fig.16 The derivatized compound (GN-Me4) of the peptide standard (GN) using an aldehyde reagent (formaldehyde) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph).

[0043] Fig.17The derivative compound (GN-Et2) of the peptide standard (GN) using an aldehyde reagent (acetaldehyde) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph).

[0044] Fig.18 The derivatized compound (GN-Pr2) of the peptide standard (GN) using an aldehyde reagent (propionaldehyde) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph).

[0045] Fig.19 The derivatized compound (GN-iPr) of the peptide standard (GN) using a ketone reagent (acetone) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph).

[0046] Fig. 20 The derivative compound (INS-Me6) of the peptide standard (INS) using an aldehyde reagent (formaldehyde) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph).

[0047] Fig.21 The derivative compound (INS-Et5) of the peptide standard (INS) using an aldehyde reagent (acetaldehyde) is shown. 1 Graphs showing the results of H-NMR analysis (upper graph) and MS analysis (lower graph). DETAILED DESCRIPTION

[0048] Hereinafter, the preferred embodiment for implementing the present technology is described. In addition, the embodiment described below represents an example of a representative embodiment of the present technology, and the scope of the present technology is not narrowly interpreted thereby. In this specification, the percentage is based on the volume expression unless otherwise specified. In addition, the upper limit (below) and lower limit (above) of each numerical range (~) can be arbitrarily combined as desired. In addition, in the following descriptions of "1." to "5.", etc., in each technical feature, each structure, each definition, each term, each method, etc. such as aldehyde, reducing agent, and sensitivity improvement method in mass spectrometry, sometimes the repeated description is appropriately omitted. Moreover, the descriptions of "1." to "5.", etc. can also be applied to any one of the various technologies or embodiments of "1." to "5.", and each technical feature, etc. can be appropriately adopted in each respective technology or embodiment, etc.

[0049] 1. Description of the Invention

[0050] Compounds such as peptides and proteins having nitrogen atoms (hereinafter also referred to as "nitrogen-containing compounds") generally tend to be easily ionized compared to fat-soluble hormones, and therefore, the derivatization of nitrogen-containing compounds is not necessarily a common method. On the other hand, in the sequence analysis of proteins and amino acids, examples of using phosphonium compounds having phosphorus atoms as charge labels have been reported (Non-patent document 3: Anal. Chem. 1997, 69, 137-144; Non-patent document 4: Rapid Commun Mass Spectrum 1999, 13. 1413-1422.; Non-patent document 5: J. Am. Soc. Mass Spectrum 2017, 28, 1889-1900.). However, it is not shown in these documents whether they can be applied to quantitative determination.

[0051] Liquid chromatography-tandem mass spectrometer (LC-MS / MS) and matrix-assisted laser desorption ionization mass spectrometer (MALDI-MS) are very powerful analytical methods for quantifying trace metabolites and contents. However, depending on the compound, there are many examples where the ionization efficiency is low and cannot be detected, or although it can be detected, the accuracy cannot be maintained until quantification. Although the sensitivity can be covered by increasing the sample amount as in food and supplements, there are many examples where the sample amount cannot be simply increased as in human metabolites. In order to solve this problem, not only the higher sensitivity of the mass spectrometer has been improved, but also the analysis object compound (such as derivatization) has been widely changed to improve the ionization efficiency.

[0052] Usually, biological metabolites are mainly organic compounds composed of carbon, hydrogen, oxygen, and nitrogen. Oxygen atoms and nitrogen atoms with non-shared electron pairs are easy to attach hydrogen ions (protons), and the ionization efficiency is high. In particular, nitrogen atoms have a high proton addition ability compared to oxygen atoms, and compounds containing nitrogen atoms have a tendency to be easily ionized compared to compounds that do not contain nitrogen atoms. For compounds that do not have nitrogen atoms such as fat-soluble steroid hormones, the ionization efficiency is improved by introducing nitrogen atoms by derivatization reagents. However, nitrogen-containing compounds such as amino acids, peptides, proteins, and catecholamines are easily ionized, so it is difficult or almost not studied to improve sensitivity based on derivatization reactions.

[0053] In LC-MS / MS, which is a conventional method for quantitative mass spectrometry, electrospray (ESI) or atmospheric pressure chemical (APCI) ionization is performed, so the ease of ionization of the analysis object compound does not depend on the polarity of the analysis object compound itself (the ease of proton addition) and the organic solvent composition of the mobile phase volatilized during ionization. If the compound is different, the structure is also different, and the polarity also changes, so in order to further improve the detection sensitivity, it is necessary to optimize the ionization efficiency matched with the compound. In addition, in LC-MS / MS, it is possible to perform more sensitive quantitative determination by selected reaction monitoring (SRM), so it is also an important aspect to obtain fragmentation with a lower background. Usually in mass spectrometry in positive ion mode, nitrogen atoms with non-shared electron pairs have the characteristic of being easy to be proton added, and have an ionization promoting effect. Nitrogen-containing compounds such as amino acids, peptides, and proteins have nitrogen atoms that are easy to be proton added in the molecule, so they have the property of being easy to be ionized compared with non-nitrogen-containing compounds.

[0054] For example, when a strong cationic derivatization reagent such as a phosphonium group is used to carry out derivatization, there is a tendency that the background noise from the reaction reagent becomes higher. In addition, there is a tendency that the molecular weight increases and it is difficult to ionize when derivatization is carried out, so sometimes the ionization efficiency improvement effect is offset or weakened. That is, it is considered that the common sense in the mass spectrometry of nitrogen-containing compounds is that if the sensitivity is not improved even if the time such as derivatization reaction is consumed, it is better to directly measure without derivatization. However, in order to carry out more accurate quantification of these nitrogen-containing compounds, it is also required to further improve the sensitivity. Therefore, in the high-sensitivity detection of nitrogen-containing compounds, different methodologies are needed.

[0055] The present inventors have focused on amino groups that also serve as ionization-promoting groups. Amino acids, peptides, proteins, and catecholamines that function in vivo are almost all primary amines. Amines are primary, secondary, tertiary, and quaternary according to the number of substituents, and quaternary is divided into charged ammonium. Regarding primary, secondary, and tertiary, the increase in substituents is usually accompanied by an increase in basicity (protons are more easily added) and an increase in hydrophobicity (since the solvent composition with more organic solvents is introduced into the ion source, it is easier to ionize). In addition, the increase in basicity can be judged by the acid dissociation constant (pKa) as an indicator, and the increase in hydrophobicity can be judged by the hydrophobicity (ClogP) indicator, and the hydrophobicity (ClogP) can be calculated by ChemDraw. That is, the present inventors believe that the ionization efficiency can be improved by secondary or tertiary amino groups. Moreover, a reductive amination reaction is used in the reaction of secondaryizing or tertiaryizing primary amines. The reaction involves using an aldehyde or ketone (e.g., an aliphatic or aromatic aldehyde or ketone) and a reducing agent to first form an imine by dehydration condensation of an amine (1) and an aldehyde or ketone (2), and then reducing the formed imine (e.g., Figure 1). In the present reaction, the reaction proceeds to secondary (3) and tertiary (4), but not to quaternary. There are many types of aldehydes or ketones (for example, aliphatic and aromatic aldehydes or ketones) available on the market, so it is possible to screen the best substituent according to the molecular weight and hydrophobicity of the compound to be analyzed, and it is possible to expect to improve the best ionization efficiency for each compound. In addition, in the derivatization reaction, the background increase caused by the residual reagent has so far caused problems such as a decrease in S / N. However, when using low-order aliphatic aldehydes or ketones, their boiling points are low, and the unreacted aldehydes or ketones can be distilled off by concentration or nitrogen purging, and the reducing reagent (mainly a borohydride reagent) is converted into a water-soluble borate by a hydrolysis reaction, which is not adsorbed on the reverse phase chromatography column and has the advantage of being able to be easily separated and removed.

[0056] The present invention is preferably used for highly sensitive detection and quantification of trace metabolites using mass spectrometry in the field of clinical examinations, and can be used in a wide range of fields and models regardless of the manufacturer of the mass spectrometer. In the future, in order to widely popularize the quantification of trace metabolites based on mass spectrometry in the field of clinical examinations, it is expected that the device will show higher detection sensitivity at any time and in any place, regardless of the manufacturer. The present invention can compensate for the differences between models of each manufacturer by improving the detection sensitivity, thereby supporting popularization.

[0057] Furthermore, the present inventors have discovered the following including the [Examples] described later.

[0058] [1] The present invention can provide a method for improving the sensitivity in mass spectrometry analysis based on secondary or tertiary amination of alkylated or arylated amines.

[0059] [2] In addition, the analyte compound derivatized by the derivatization reaction is more effectively ionized by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), or matrix-assisted laser desorption ionization (MALDI), but other ionization methods can also achieve the same effect. This makes it possible to more accurately and with high resolution measure and observe the quantification of the analyte compound and its distribution in tissues and cells (imaging).

[0060] [3] The derivatization reaction uses nitrogen-containing compounds such as amino acids (representative compounds: thyroid hormone, etc.), peptides (representative compounds: oxytocin, etc.), catecholamines (representative compounds: dopamine, etc.) as substrates. The substrates are substances contained in biological components (blood, serum, plasma, urine, sweat, cerebrospinal fluid, hair, nails, feces, etc.) and can be detected after appropriate pretreatment.

[0061] [4] The alkylation or the arylation is preferably performed using an aldehyde or a ketone and a reducing agent.

[0062] [5] The aldehyde or ketone is preferably an aliphatic or aromatic aldehyde having 1 to 12 carbon atoms. It is also preferred that the aldehyde or ketone further contains a substituent such as a methyl group.

[0063] [6] The reducing agent is preferably one or more selected from borane, a complex containing borane, and a salt of a boron hydride compound, but is not limited thereto as long as it can exert the same effect as these reducing agents.

[0064] [7] The present invention can provide a derivatization kit for mass spectrometry analysis comprising at least an aldehyde or a ketone and a reducing agent.

[0065] In addition, as will be seen from the [Examples] described later, by using this technology, in mass spectrometry analysis (preferably LC-MS / MS analysis), in comparison with the compounds before derivatization, it was confirmed that the sensitivity of amino acids after derivatization was increased by up to 2.5 times, peptides after derivatization was increased by up to 17 times, and catecholamines after derivatization was increased by up to 195 times. In addition, by using this technology, even nitrogen-containing compounds in living organisms such as plasma, serum, and urine were confirmed to have increased sensitivity in mass spectrometry analysis after they were derivatized.

[0066] 2. First embodiment of the present invention (derivatization reagent containing aldehyde or ketone)

[0067] The first embodiment can provide a derivatization reagent containing an aldehyde or a ketone.

[0068] In addition, as another embodiment, the present invention may provide a derivatization reagent comprising: <1> a compound represented by the formula (1): RR'CO(1) (R and R' represent a hydrogen atom or an alkyl group or an aryl group, and they may be the same or different); or <2> a compound represented by the chemical formula (2): RCHO(2) (R represents a hydrogen atom or an alkyl group or an aryl group), in which case R' of the formula (1) is a hydrogen atom; or <3> a compound represented by the formula (1) (R and R' represent an alkyl group or an aryl group, and they may be the same or different), in which case R and R' of the formula (1) are not both hydrogen atoms.

[0069] In addition, the derivatization reagent may also be any one of a composition, a derivatization composition, a derivatization agent, or a sensitivity enhancer in mass spectrometry. Thus, the derivatization of the analysis target compound can be easily performed. Furthermore, the sensitivity (e.g., detection sensitivity or measurement sensitivity, etc.) of the analysis target compound based on the derivatization can also be improved, for example, it can be expected that the sensitivity in mass spectrometry can be improved.

[0070] In the first embodiment, an aldehyde or a ketone is preferably used as a derivatization reagent. For example, one or more than one of the exemplary compounds of the aldehyde or ketone described later can be selected for use. In addition, the aldehyde or ketone preferably uses a nitrogen-containing compound as a derivatized analysis target compound, preferably for the purpose of N-alkylation or N-arylation of the nitrogen-containing compound. The derivatization reagent is preferably a reagent for mass spectrometry or a reagent for mass spectrometry. When derivatizing, it is preferred to use at least an aldehyde or a ketone and a reducing agent described later in combination with the analysis target compound.

[0071] Furthermore, by using the derivatization reagent of the first embodiment for the analysis target compound, a compound in which the analysis target compound is derivatized (preferably N-alkylated or N-arylated) can be obtained. By using the N-derivatized analysis target compound for determination or detection, the sensitivity of the determination or detection of the analysis target compound can be improved.

[0072] 2-1. Aldehydes or ketones

[0073] The aldehyde or ketone used in the present invention is not particularly limited. The aldehyde or ketone used in the present invention can be used as a compound contained in or used in a derivatization reagent or a derivatization kit, and can be used as a compound in the production of these reagents or kits.

[0074] Examples of the aldehyde or ketone include aliphatic aldehyde or ketone, alicyclic aldehyde or ketone, aromatic aldehyde or ketone, and heterocyclic aldehyde or ketone, and any one of them may be used, and one or more selected from them may be used. Among them, aliphatic aldehyde or ketone and / or aromatic aldehyde or ketone are preferred.

[0075] The number of carbon atoms of the aldehyde or ketone is not particularly limited. From the viewpoint of improving sensitivity, the preferred lower limit is preferably 1, 2 or 3 or more. In addition, the preferred upper limit is preferably 20 or less, more preferably 15 or less, further preferably 12 or less, and further preferably 10 or less. In addition, the number of carbon atoms is preferably an integer. The aldehyde or ketone used in the present invention may be a commercially available product, or may be an aldehyde or ketone produced by referring to a disclosed method for producing an aldehyde or ketone.

[0076] The aldehyde or ketone as a preferred embodiment used in the present invention includes a compound represented by the following formula (1): RR'CO(1) (in the formula (1), R and R' are the same or different and represent a hydrogen atom or a hydrocarbon group). In addition, the formula (1) can also be expressed as a compound represented by RC(=O)-R' as required. The hydrocarbon groups in R and R' in the formula (1) are the same or different and may have a substituent or may be unsubstituted. The hydrocarbon group is more preferably an alkyl group or an aryl group.

[0077] The alkyl group may also be either chain (straight chain or branched) or cyclic. More preferably, R and R' in the formula (1) are the same or different, and are preferably a hydrogen atom or a substituted or unsubstituted alkyl or aryl group having 1 or 2 to 11 carbon atoms. In addition, in the compound represented by the formula (1), when either R or R' is a hydrogen atom, the other may represent a hydrogen atom or a hydrocarbon group, and when both R and R' are not hydrogen atoms, R and R' may be the same or different, and may also represent a hydrocarbon group.

[0078] In the formula (1), the hydrocarbon groups in R and R' are not particularly limited, and examples thereof include aliphatic hydrocarbon groups, alicyclic hydrocarbon groups, aromatic hydrocarbon groups, and the like, and one or more selected from these groups can be used. The number of carbon atoms in the hydrocarbon group in R is not particularly limited, and as a preferred lower limit, it is preferably 1 or 2 or more, and as a preferred upper limit, it is preferably 20 or less, more preferably 15 or less, further preferably 12 or less, further preferably 10 or less, and further preferably 9 or 8 or less.

[0079] The aliphatic hydrocarbon group is not particularly limited, and for example, a methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, allyl or other linear or branched aliphatic hydrocarbon groups (linear or branched alkyl, alkenyl and alkynyl, etc.) etc. can be enumerated. They can be any one of linear or branched. Among them, linear or branched alkyl is preferred.

[0080] Among them, a substituted alkyl group (e.g., aralkyl group) or an unsubstituted alkyl group having a substituent (e.g., aryl group) is preferred. One or more than two of them can be selected for use. The aryl group can be the same as the aryl group described later, and a phenyl group can be cited.

[0081] The number of carbon atoms in the aliphatic hydrocarbon group is not particularly limited, but is preferably about 1, 2 to 20, and more preferably about 1, 2 to 10. One or more of these may be selected and used.

[0082] The alicyclic hydrocarbon group is not particularly limited, and examples thereof include monocyclic alicyclic hydrocarbon groups (cycloalkyl, cycloalkenyl) such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cyclooctyl, cyclodecyl, and cyclododecyl; bridged ring hydrocarbon groups, etc. Representative examples of the bridged ring in the bridged ring hydrocarbon group include adamantane ring, norbornane ring, norbornene ring, etc. Aromatic rings can also be condensed with alicyclic rings. Among them, cycloalkyl (cyclic alkyl) is preferred. The number of carbon atoms in the alicyclic hydrocarbon group is not particularly limited, and preferably has a carbon number of about 3 to 20. One or more of them can also be selected for use.

[0083] As the aromatic hydrocarbon group, for example, monocyclic aromatic hydrocarbon groups such as phenyl, tolyl, and o-xylyl; aryl groups such as polycyclic aromatic hydrocarbon groups such as naphthyl, etc., may also have substituents such as alkyl groups. Non-aromatic rings may also be condensed with aromatic rings. The number of carbon atoms in the aromatic hydrocarbon group is not particularly limited, and preferably has about 6 to 20 carbon atoms. Among them, substituted aryl groups (for example, alkylaryl groups) or unsubstituted aryl groups having substituents (preferably alkyl groups) are preferred. One or more of them can be selected for use. The alkyl group may be the same as the above-mentioned alkyl group, and methyl, ethyl, etc. may be mentioned.

[0084] The hydrocarbon group may be a substituted or unsubstituted hydrocarbon group. The "substituent" of the hydrocarbon group as the substituent may also have various substituents (e.g., substituents having 1 to 3 carbon atoms), such as halogen atoms, oxo groups, hydroxyl groups, substituted oxy groups (e.g., alkoxy groups, aryloxy groups, acyloxy groups, etc.), carboxyl groups, substituted oxycarbonyl groups, substituted or unsubstituted carbamoyl groups, cyano groups, nitro groups, substituted or unsubstituted amino groups, linear or branched alkyl groups (e.g., methyl, ethyl, etc. alkyl groups), cyclic alkyl groups, aryl groups (e.g., phenyl, naphthyl, etc.), heterocyclic groups, etc. Among them, the substituent is preferably a hydrocarbon group (unsubstituted or substituted group). As a substituent, more specifically, for example, a substituent selected from an aryl group having an unsubstituted alkyl group (the alkyl group is linear, branched or cyclic) and an aryl group having an unsubstituted aryl group or an unsubstituted alkyl group, and the carbon number of these substituents can adopt the above-mentioned carbon number. One or more of them can be selected for use.

[0085] As preferred examples of aliphatic aldehydes used in the present invention, for example, aliphatic saturated aldehydes such as formaldehyde, acetaldehyde, propionaldehyde, butylaldehyde, isobutylaldehyde, valeraldehyde (valeraldehyde), isovaleraldehyde, pivalaldehyde, hexanal, heptaldehyde, octanal, nonanal, decanal, dodecanal, tetradecanal, hexadecanal, octadecane, octadecane, etc.; aliphatic dialdehydes such as glyoxal and succinaldehyde; aliphatic unsaturated aldehydes such as acrolein, crotonaldehyde, and pentenal, etc. can be cited. One or more of them can be selected for use. As preferred examples of aliphatic ketones, acetone, methyl ethyl ketone, 3-pentanone, etc. can be cited.

[0086] Preferred examples of the alicyclic aldehyde used in the present invention include cyclopropanecarboxaldehyde, cyclopentanecarboxaldehyde, cyclohexanecarboxaldehyde, cyclooctanecarboxaldehyde, cyclododecanecarboxaldehyde, 5-norbornene-2-carboxaldehyde, etc. One or more of these can be selected and used.

[0087] Preferred examples of the aromatic aldehyde used in the present invention include benzaldehyde, o-, m-, p-tolualdehyde, salicylaldehyde, cinnamaldehyde, α-, β-naphthaldehyde, o-phthalaldehyde, terephthalaldehyde, and isophthalaldehyde, etc. One or more kinds thereof may be selected and used.

[0088] Preferred examples of the aldehyde used in the present invention include, for example, one or more selected from aliphatic aldehydes such as formaldehyde, acetaldehyde, propionaldehyde, butylaldehyde, isobutylaldehyde, valeraldehyde (valeraldehyde), isovaleraldehyde, pivalaldehyde, hexanal, and heptaldehyde; alicyclic aldehydes such as cyclopropanecarboxaldehyde; and aromatic aldehydes such as benzaldehyde. Among them, aliphatic aldehydes are preferred, and as examples thereof, one or more selected from formaldehyde, acetaldehyde, propionaldehyde, butylaldehyde, isobutylaldehyde, valeraldehyde (valeraldehyde), isovaleraldehyde, hexanal, and heptaldehyde are more preferred.

[0089] Furthermore, preferred examples of the ketone used in the present invention include one or more selected from aliphatic ketones such as acetone, methyl ethyl ketone, and 3-pentanone. Among these, acetone is more preferred.

[0090] 2-2. Analytical compounds

[0091] In the present invention, the compound to be analyzed for derivatization is not particularly limited, but is preferably a nitrogen-containing compound. The compound to be analyzed may be a matrix, or may be a target for derivatization or a target for mass spectrometry analysis.

[0092] The nitrogen-containing compound used in the present invention is preferably a nitrogen-containing compound having at least one primary amine (HHN-) in the molecular structure of the nitrogen-containing compound. In addition, in the molecular structure of the nitrogen-containing compound, it is preferred to have at least one aryl group or methylene group, and the aryl group or methylene group may be any of unsubstituted or substituted, for example, an aryl group having an alkyl group may be mentioned. The alkyl group may be any of unsubstituted or substituted, and further, a chain (straight chain, branched chain) or a cyclic group may be mentioned. The number of carbon atoms of the alkyl group is not particularly limited, for example, a carbon number of about 2 to 4 may be mentioned. As a substituent, there is no particular limitation, and alkyl groups, iodine atoms, hydroxyl groups, carbonyl groups, amino groups, etc. may be mentioned. One or more than two of them may be selected.

[0093] The nitrogen-containing compound is preferably selected from one or more of amino acids, peptides, catecholamines, proteins, etc., and can be any of synthetic or natural products, including clinical examination samples (for example, biological components, tissues and cells of animals and plants, etc.), environmental hygiene inspection samples, products (foods, medicines, quasi-drugs, cosmetics, chemicals, etc.), etc.

[0094] As a more preferred example, for example, it is preferably selected from one or more of biological components, organic synthesis products, and separated and purified products. By using this technology after appropriate pretreatment of nitrogen-containing compounds contained in biological components that are usually difficult to measure or detect, it can be well derivatized and is also suitable as a derivatized product for mass spectrometry analysis. Therefore, the present invention is preferably used for the measurement or detection of biological components, and more preferably, the nitrogen-containing compounds contained in biological components are used as the analysis object compounds.

[0095] Examples of the nitrogen-containing compounds are not particularly limited, and include amino acids (e.g., triiodothyronine, thyroxine, homocysteine, etc.), peptides (e.g., angiotensin I, oxytocin, glucagon, adrenocorticotropic hormone, insulin, C-peptide, etc.), catecholamines (e.g., epinephrine, norepinephrine, dopamine, levodopa, etc.), and the like. One or more selected from these can be used.

[0096] In the present invention, the compound to be analyzed is preferably one or two or more selected from amino acids, peptides, and catecholamines contained in biological components.

[0097] When the analyte compound is an amino acid (preferably an amino acid present in a biological component), the number of primary amines (HHN-) in its molecular structure is preferably 1 to 2, more preferably 1, and the number of aryl groups in its molecular structure is preferably 0 to 3, more preferably 0 to 2, and further preferably 2. The preferred molecular weight of the amino acid of the analyte compound is preferably 50 to 1000, more preferably 100 to 800. In addition, the molecular weight is calculated based on the sum of the atomic weights contained in the molecule. In addition, the preferred hydrophobicity (ClogP) of the amino acid of the analyte compound is preferably -4 to 16, more preferably -2 to 10.

[0098] When the target compound is a peptide (preferably a peptide present in a biological component), the number of primary amines (HHN-) in the molecular structure is not particularly limited, but is preferably 1 or more, more preferably 2 or more as a preferred lower limit, and is preferably 30 or less, more preferably 20 or less, further preferably 15 or less, and further preferably 10 or less as a preferred upper limit. When the target compound is a peptide, the number of aryl groups in the molecular structure is not particularly limited, but is preferably 1 or more, more preferably 2 or more as a preferred lower limit, and is preferably 30 or less, more preferably 20 or less, further preferably 15 or less, and further preferably 10 or less as a preferred upper limit. The preferred molecular weight of the peptide of the target compound is preferably 100 or more, more preferably 200 or more as a preferred lower limit, and is preferably 10000 or less, more preferably 9000 or less, further preferably 8000 or less, and further preferably 6000 or less as a preferred upper limit. In addition, the preferred hydrophobicity (ClogP) of the peptide of the analysis target compound is preferably -4 or more, more preferably -2 or more as its preferred lower limit value, and is preferably 10 or less, more preferably 8 or less, further preferably 6 or less, and further preferably 4 or less as its preferred upper limit value.

[0099] In the present technology, from the viewpoint of the analytical target compound whose sensitivity is easily improved, among the amino acids, triiodothyronine, thyroxine, and homocysteine ​​are preferred; among the peptides, angiotensin I, oxytocin, glucagon, insulin, and C-peptide are preferred; and among the catecholamines, epinephrine, norepinephrine, and dopamine are preferred, and one or more selected from them can be used.

[0100] As a preferred embodiment of the present invention, (a) when the nitrogen-containing compound is an amino acid or a peptide, R and R' in the formula (1) RR'CO are the same or different, preferably a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 8 carbon atoms, or (b) when the nitrogen-containing compound is a catecholamine, R and R' in the formula (1) RR'CO are the same or different, preferably a substituted or unsubstituted alkyl group having 2 to 9 carbon atoms.

[0101] In the case where the nitrogen-containing compound (a) is an amino acid or a peptide, as a more preferred embodiment, R and R' in the formula (1) RR'CO are the same or different, and are preferably a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 3 carbon atoms, and more preferably a hydrogen atom or a linear alkyl group having 1 to 2 carbon atoms, and further preferably R and R' are one or more selected from a hydrogen atom, a methyl group, an ethyl group, a propyl group, etc. The analyte compound as a sample is preferably an amino acid and / or a peptide in a biological component.

[0102] In addition, when the nitrogen-containing compound in (a) is an amino acid or a peptide, the carbon number of R and R' in the formula (1) is preferably 0 to 5, more preferably 4 or less or 3 or less, and further preferably 0, 1 and 2. When the carbon number of R and R' is 0, R and R' are preferably hydrogen atoms. In addition, the preferred aldehydes and ketones are preferably aldehydes or ketones (preferably having 1 to 3 carbon atoms) having a straight-chain C1 to C2 alkyl group or a hydrogen atom, such as a secondary or tertiary compound in which the primary or secondary amino group of the analyte compound is derivatized to N-methyl, N-ethyl, N-propyl, N-isopropyl or the like. The analyte compound as a sample is preferably an amino acid and / or a peptide in a biological component.

[0103] In the case where the nitrogen-containing compound (b) is a catecholamine, R in the formula (1) R, R'CO is preferably a substituted or unsubstituted alkyl group having 3 to 9 carbon atoms, the alkyl group preferably has 4 to 8 carbon atoms, more preferably 4 to 6 carbon atoms, the alkyl group is preferably an unsubstituted alkyl group and R' is a hydrogen atom. More preferably, R is preferably one or more selected from butyl, isobutyl, pentyl, hexyl and phenyl, among which hexyl is more preferred. The compound to be analyzed as a sample is preferably a catecholamine in a biological component.

[0104] The source of the biological component is not particularly limited, and can be any one of animals, plants or microorganisms, etc. As biological components, there is no particular limitation. Preferred biological components of animals, for example, blood, serum, plasma, urine, sweat, cerebrospinal fluid, hair, nails, feces, etc. can be cited, and one or more selected from them can be used. Animals are not particularly limited, for example, one or more selected from mammals (humans, mice, dogs, cats, rabbits, horses, pigs, cattle, etc.), birds (chickens, pigeons, etc.), reptiles, amphibians, etc. can be cited. From the perspective of diagnosis, it is preferred that humans, pets (cats, dogs, etc.), competitive animals (horses, dogs, rabbits, etc.), livestock, poultry (pigs, cattle, chickens, etc.). In addition, components such as biological components collected from animals and other organisms can be used as analytical target compounds, and the collected components can also be pre-treated before derivatization.

[0105] By the derivatization reagent used in the present invention, the primary amine contained in the analysis object compound can be converted into a secondary amine or a tertiary amine. More specifically, by the derivatization reagent used in the present invention, the primary amine contained in the analysis object compound can be N-alkylated or N-arylated. That is, by secondaryizing or tertiating the amino group of the analysis object compound, the ionization efficiency can be improved. Moreover, when compared with the parent ion peak intensity or area when the analysis object compound before derivatization is subjected to mass spectrometry analysis, the parent ion peak intensity or area when the analysis object compound after secondary amination or tertiary amination is subjected to mass spectrometry analysis becomes larger, so the sensitivity in mass spectrometry analysis can be further improved.

[0106] As the separation and purification method of the analytical object compound in the sample of the present invention, there is no particular limitation, and an appropriate separation and purification method can be adopted according to the type of sample. The separation and purification method of the biological component or the derivative, etc. is not particularly limited, and the disclosed separation and purification method of the biological component or the derivative, etc. can also be used. As an example of the separation and purification method used in the present invention, there is no particular limitation, for example, one selected from centrifugal separation, precipitation (acetonitrile precipitation, methanol precipitation, etc.), column chromatography (ion exchange, gel filtration, hydrophobicity, affinity, normal phase, reverse phase, metal chelate, etc.), ultrafiltration, antibodies, etc., or a combination of two or more thereof can be used. In addition, substances such as ion exchange and metal chelate used in the carrier of column chromatography can also be used for separation and purification methods other than column chromatography. The separation and purification method is preferably carried out before the derivatization reaction, but can also be used as a separation and purification method after the derivatization reaction.

[0107] There is no particular limitation on the ion exchanger, and any of an anion exchanger or a cation exchanger can be used. Furthermore, depending on the analyte compound collected or captured from a sample such as a biological component, any of a strong anion exchanger, a weak anion exchanger, a strong cation exchanger, and a weak cation exchanger can also be used. In addition, as examples of hydrophobic groups used in hydrophobic column chromatography containing substances having a hydrophobic group, C4 to C20 alkyl groups, phenyl groups, etc. can be cited. In addition, as examples of metal chelates, there can be cited those that can be used in Cu 2+ 、Zn 2+ 、Ni 2+ , Ca 2+ 、Co 2+ Mg 2+ Metal chelates are metal ions such as chelates in which molecules or ions called ligands are bonded to metal ions. Antibodies include antibodies specific to the compound to be analyzed. One or more selected from these can be used.

[0108] For example, a sample such as a biological component is developed on an ion exchange column, a solvent is used to wash the column and elute the compound to be analyzed, and the eluate containing the target substance is concentrated and dried. Amino acids, peptides, catecholamines, etc. can be extracted from samples such as biological components, and a disclosed extraction method can also be used in this case. Thus, the target substance used in the derivatization can be separated and purified from samples such as biological components, and the derivatization efficiency becomes better.

[0109] In addition, for example, the analyte compound contained in the sample such as the biological component can be appropriately selected from the carriers commonly used in separation and purification, and supplemented using the selected carrier. As the carrier, for example, one or more of substances selected from substances having ionic bonding, substances having affinity, substances having chemical interactions such as hydrophobic interactions (e.g., hydrophobic groups), etc. can be cited.

[0110] Furthermore, the present invention has the following advantages: the best derivatization reagent that can improve the sensitivity in mass spectrometry analysis can be screened from the aldehydes and ketones used in the present invention according to the molecular weight and hydrophobicity of the analysis target compound. In addition, since there are many commercially available aldehydes and ketones used in the present invention, it is also easy to find aldehyde and ketone derivatization reagents that can further improve the sensitivity.

[0111] In the present invention, any component may be appropriately added to the derivatization reagent. For example, one or more selected from a reducing agent, an acid, a base, etc. may be used, but no base may be used.

[0112] 3. Second embodiment of the present invention (derivative kit)

[0113] In addition, the second embodiment can provide a reagent kit for derivatization, the reagent comprising aldehydes and ketones, and the reagent comprising aldehydes and ketones can also be a derivatization reagent comprising aldehydes and ketones. In addition, as another embodiment, a reagent kit for derivatization can also be provided, the reagent comprising a compound represented by the above-mentioned formula (1) (RR'CO(1)) (the R and R' can be the same or different), or a compound represented by the chemical formula (2) (RCHO(2)) when R' of the formula (1) is a hydrogen atom.

[0114] In the kit of the second embodiment, it is preferred that a reducing agent is further included, and the reducing agent may also be a derivatization reagent or composition containing the reducing agent. The kit may be a combination or a kit, which may also be a kit product or the like. The present invention may be, for example, a single-dose composition containing an aldehyde, a ketone, and a reducing agent together, or may be a multi-dose kit such as a two-dose kit containing an aldehyde, a ketone, and a reducing agent in different containers.

[0115] As a more preferred mode in this second embodiment, a kit for derivatization containing a first reagent containing an aldehyde and a ketone and a second reagent containing a reducing agent. Furthermore, when derivatizing, it is preferred to use an acid such as acetic acid. For example, a third reagent containing an acid or an acid can also be further included in the kit for derivatization to provide. In addition, these components are made into a solution using a solvent. In addition, these components can be used for a derivatization method, a derivatization reagent or a derivatization kit, etc., and can also be used as a compound in the manufacture of these reagents or kits. In addition, in addition to aldehydes, ketones, a reducing agent and an acid, the kit can also contain a fourth derivatization reagent containing a base (alkali metals or alkaline earth metals such as ammonia and hydroxides, etc.). In this embodiment, there is an advantage that an alkaline reagent for derivatization of an object of analysis is not added in the derivatization reaction. In the reaction of this embodiment, the advantage is that even if an alkaline reagent is not used, by using a substance showing the properties of a nitrogen atom of an amino acid, a peptide, or a catecholamine as a matrix (analyzed object) as a base, a derivatization method that can improve the sensitivity of a nitrogen-containing compound can be provided.

[0116] The aldehydes, ketones and derivatization reagents containing aldehydes and ketones used in the second embodiment can appropriately adopt the above-mentioned description of "aldehydes and ketones". Preferably, the aldehydes and ketones are compounds represented by the formula (1) (RR'CO). It is further preferred that R and R' in the formula (1) are the same or different and are hydrogen atoms, substituted or unsubstituted hydrocarbon groups (preferably alkyl or aryl groups). The R and R' are not particularly limited, and are preferably hydrogen atoms and carbon atoms of 1 to 18, more preferably hydrogen atoms and carbon atoms of 1 to 11, and further preferably hydrogen atoms and carbon atoms of 1 to 9. The alkyl group is preferably a chain (straight chain, branched) or cyclic alkyl group, and the aryl group is preferably an unsubstituted aryl group. Furthermore, as a preferred embodiment, (a) when the nitrogen-containing compound is an amino acid or peptide in a biological component, R and R' in the formula (1) are hydrogen atoms or substituted or unsubstituted alkyl groups having 1 to 8 carbon atoms, or (b) when the nitrogen-containing compound is a catecholamine in a biological component, R and R' in the formula (1) are substituted or unsubstituted alkyl groups having 2 to 9 carbon atoms.

[0117] 3-1. Reducing agent

[0118] The reducing agent used in the present invention is not particularly limited and may be in liquid or solid form, and may be made into a solution by using a solvent or the like.

[0119] As the reducing agent, for example, there can be mentioned boron compounds (borane, complexes containing borane, borohydride salts, etc.); aluminum hydrides (lithium aluminum hydride, diisobutylaluminum hydride, sodium bis(2-methoxyethoxy)aluminum hydride, etc.); tributyltin hydride; hydrogen gas, etc., and one or more selected from them can be used.

[0120] Among them, the boron compounds are preferred from the viewpoint of ease of removal after derivatization.

[0121] The boron compound used in the present invention is not particularly limited as long as it has a reducing effect. Examples thereof include boranes (borane (BH3), diborane (B2H6), etc.), complexes containing borane (hereinafter also referred to as "borane complexes"), and salts of boron hydride compounds. It is preferred to use one or more selected from them.

[0122] The reducing agent used in the present invention may be a commercially available product or may be obtained by referring to a publicly available production method. Advantages of using a boron compound include the following advantages: even at a reaction temperature around room temperature, the compound to be analyzed can be easily derivatized using a derivatization reagent, and the reducing agent component after the derivatization reaction can be easily removed by reduced pressure concentration using an evaporator or the like.

[0123] As the borane complex, for example, there can be mentioned borane-2-methylpyridine complex (also known as methylpyridine borane), borane-dimethyl sulfide (DMS) complex, borane-amine complex (for example, ammonia borane (or borazane), borane trimethylamine complex, borane N,N-diisopropylethylamine complex, borane tert-butylamine complex, etc.), among which borane-2-methylpyridine complex is preferred.

[0124] The salt of the borohydride compound includes, for example, alkali metal borohydrides (lithium, sodium, potassium, etc.), alkaline earth metal borohydrides (calcium, etc.), alkali metal cyanoborohydrides (sodium, etc.), alkali metal triacetoxyborohydrides (sodium, etc.), alkali metal triethylborohydride (lithium, etc.), alkali metal tri(sec-butyl)borohydride (lithium, etc.), alkaline earth metal tri(sec-butyl)borohydride (potassium, etc.), among which alkali metal borohydrides are preferred.

[0125] 3-2.Acid

[0126] The acid used in the present invention is not particularly limited and may be in a liquid or solid state, and may be dissolved in a solvent or the like when used.

[0127] As the acid, a volatile acid is preferred from the viewpoint of being easily removed after the reaction. As the acid, for example, organic acids such as acetic acid, formic acid, propionic acid, lactic acid, and citric acid; inorganic acids such as phosphoric acid, sulfuric acid, and nitric acid; and salts thereof, and one or more selected from them can be used. Among them, organic acids are preferred. In addition, the acid used in the present invention is preferably an acid that is easily removed by an evaporator or the like (for example, acetic acid, etc.).

[0128] 3-3.Solvent

[0129] The solvent used in the present invention is not particularly limited, and preferably a volatile solvent having a boiling point of 100°C or less, for example, one or more selected from water, lower alcohols (methanol, ethanol, n-propanol, isopropanol, etc., with C1 to 3 or so), etc. can be used. The solvent can be contained in or used in the derivatization reagent or derivatization kit. The solvent can also be used as a solution in the form of a solution containing aldehydes or ketones, a solution containing a reducing agent, etc. In addition, a solvent that can be concentrated to dryness by an evaporator or the like is used, thereby removing the borane-based reducing agent together.

[0130] 3-4. Amount of each component used in the present invention

[0131] The concentration of the aldehyde or ketone used in the derivatization reaction is not particularly limited, but is preferably 5 to 100% (v / v) relative to the sample (1 to 100,000 pg).

[0132] The concentration of the reducing agent used in the derivatization reaction is not particularly limited, but is preferably 1 to 5 mg / mL relative to the sample (1 to 100,000 pg).

[0133] The concentration of the acid used in the derivatization reaction is not particularly limited, but is preferably 8 to 20% (v / v) relative to the sample (1 to 100,000 pg).

[0134] 3-5. Third embodiment of the present invention (method for using each reagent included in the kit and method for producing a derivatized nitrogen-containing compound)

[0135] The third embodiment can provide a method for derivatizing a nitrogen-containing compound or a method for producing a derivatized nitrogen-containing compound by reacting the nitrogen-containing compound with the derivatization reagent or the derivatization kit. By using aldehydes or ketones to derivatize the nitrogen-containing compound, the signal intensity or detection sensitivity of the detection device can be improved. As an advantage of the present invention, even if the nitrogen-containing compound in the sample is a small amount, the analyte compound in the sample can be detected or measured more accurately.

[0136] In addition, the present invention preferably includes separating and purifying nitrogen-containing compounds from a collected sample (preferably a biological component), and the separation and purification may also be performed as a pretreatment, thereby removing impurities contained in the sample (preferably a biological component) and improving the sensitivity of detection or measurement of the compound to be analyzed.

[0137] In addition, in the third embodiment, the derivatized nitrogen-containing compound after the reaction is preferably separated and purified, thereby removing unnecessary derivatization reagent after the reaction, thereby improving the sensitivity of detection or measurement of the analyte compound.

[0138] In addition, in the third embodiment, it is preferred that the derivatized nitrogen-containing compound be subjected to mass spectrometry.

[0139] The nitrogen-containing compound as the analysis object compound is preferably the amino acid, peptide, or catecholamine contained in the above-mentioned biological components, and is preferably selected from amino acids such as triiodothyronine, thyroxine, and homocysteine; peptides such as angiotensin I, oxytocin, glucagon, insulin, and C-peptide; and one or more catecholamines such as epinephrine, norepinephrine, and dopamine.

[0140] Preferably, the nitrogen-containing compound is reacted with the derivatization reagent or the derivatization kit to derivatize the nitrogen-containing compound.

[0141] As a more preferred mode, the sample containing the nitrogen-containing compound is preferably mixed with an aldehyde or ketone and a reducing agent to be derivatized, and more preferably, the three components of aldehyde or ketone, reducing agent and acid are added to the sample containing the nitrogen-containing compound. In addition, after the three components of aldehyde or ketone, reducing agent and acid are added to the sample containing the nitrogen-containing compound and mixed, a base may be further added. These samples and each component may also be made into a solution, for example, a derivatization kit composed of a first solution containing an aldehyde or ketone, a second solution containing a reducing agent, a third solution containing an acid, etc. may be cited; in addition, a derivatization kit having a first container containing an aldehyde or ketone, a second container containing a reducing agent, a third container containing an acid, etc. may be cited. In addition, the nitrogen-containing compound may also be derivatized in a derivatization device constructed in a manner that can implement the method of use, manufacturing method, etc. of the present invention using these components, reagents or solutions. It is preferred not to use an alkaline reagent during derivatization.

[0142] The reaction conditions of derivatization are not particularly limited. The temperature conditions of derivatization are not particularly limited, and are preferably 5 to 50°C, more preferably 10 to 40°C. As the advantages of the derivatization of the present invention, it has the following advantages: since the derivatization reaction is carried out even if it is not at a high temperature, temperature control and equipment can be omitted, so it is easy for a wide range of users to operate. In this case, it is preferably room temperature (for example, 10 to 40°C), and more preferably 20 to 30°C. The pressure conditions of derivatization are not particularly limited, and the ability to carry out the derivatization reaction at normal pressure (standard atmospheric pressure 1atm) is also an advantage of the present technology. In the present technology, the ability to carry out the derivatization of the analysis target compound at normal temperature and pressure is also an advantage. The reaction time of derivatization is not particularly limited, and as a preferred lower limit, it is preferably 0.1 hours or more, more preferably 0.5 hours or 1 hour or more, and as a preferred upper limit, it is preferably 24 hours or less, and more preferably 18 hours or less. During the reaction of derivatization, it is preferred to add an acid (for example, acetic acid, etc.). In the derivatization reaction, a solvent for dissolving or suspending the compound or the like may be used. As the solvent, an aqueous solvent (water, lower alcohol, etc.), an organic solvent, etc. may be used appropriately depending on the compound or derivatization reagent used.

[0143] The derivatized nitrogen-containing compound obtained in the third embodiment is preferably a compound in which the amino group (—NHH) of the nitrogen-containing compound is bonded to the alkyl or aryl group of the R or R′ portion contained in the aldehyde or ketone as the derivatization agent to undergo secondary or tertiary amination.

[0144] 4. Fourth embodiment of the present embodiment (mass spectrometry method and method for improving sensitivity in mass spectrometry)

[0145] As described above, the fourth embodiment can provide a mass spectrometry method, which includes a derivatization step of derivatizing the analysis target compound using a derivatization aldehyde or ketone, a derivatization reagent, or a derivatization kit. The fourth embodiment can provide a derivatized compound obtained by a derivatization reaction using each component such as an aldehyde or ketone. The fourth embodiment preferably includes a pretreatment step of pretreating the sample by a separation and purification method before the derivatization step.

[0146] The compound to be analyzed is preferably the nitrogen-containing compound described above. In addition, the derivatization can improve the measurement sensitivity or detection sensitivity of the compound to be analyzed by mass spectrometry.

[0147] Furthermore, the mass spectrometry method may include a mass spectrometry step including ionizing the mass spectrometry analysis target compound after the derivatization step.

[0148] In order to perform more efficient ionization, the ionization may be ionization based on, for example, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), matrix-assisted laser desorption ionization (MALDI), or the like, or may be ionization based on ionization methods other than these.

[0149] According to the fourth embodiment, the derivatized target compound is ionized by these ionization methods, thereby improving the measurement sensitivity or detection sensitivity of the target compound.

[0150] The mass spectrometry step can be performed, for example, by liquid chromatography-tandem mass spectrometry (LC-MS / MS) or matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), but the apparatus for performing the mass spectrometry step is not limited thereto.

[0151] The specific steps in the mass spectrometry process can be appropriately set according to the ionization method used and the device used. In this embodiment, for example, a mass spectrometry device, a pretreatment device configured to pretreat a sample by a separation and purification method, a derivatization device configured to derivatize a sample, a gas chromatography device, an HPLC device, etc. can be cited, and one or more selected from them can be used.

[0152] According to the fourth embodiment, in mass spectrometry analysis, the N-derivatized target compound and its fragment ions (characteristic fragment ions) can be detected or measured, and based on the results, it can be determined whether the sample contains the target compound, or the target compound in the sample can be detected or quantitatively measured.

[0153] In addition, in the fourth embodiment, when mass spectrometry is used to detect or measure the analyte compound contained in the biological component in the sample, the same compound as the analyte compound can be used as a standard substance and compared with the standard substance. That is, by comparing the derivative of the analyte compound in the biological component with the derivative of the standard substance, the analyte compound in the biological component can be detected or measured.

[0154] In addition, methods related to the present embodiment such as the fourth embodiment (for example, a derivatization method or a mass spectrometry method, a determination method or a detection method for judging improved sensitivity, etc.) can also be controlled by a control unit having a CPU, etc. In addition, the methods such as the fourth embodiment can use one or more of the methods selected from the public methods, such as the standard addition method, the external standard method (absolute standard method), the internal standard method, the area percentage method, the corrected area percentage method, etc., when detecting or determining. In addition, the CPU possessed by the mass spectrometry device or the control unit (computer, server, cloud, etc.) of the device can also be used, based on the data (molecular weight fragment ions, characteristic fragment ions, fragment ion patterns, peak height, area, retention time, etc.) of the derivative of the analysis object compound stored in the storage unit, and compared with the biological components in the sample, and identification, detection or quantitative determination can be performed.

[0155] 5. In addition, the present technology can appropriately adopt the following other modes or the following structures or technical features.

[0156] [1] A derivatization reagent comprising an aldehyde or a ketone. Preferably, the derivatization reagent is for mass spectrometry. Preferably, the derivatization reagent is an aliphatic or aromatic aldehyde or ketone.

[0157] [2] The derivatization reagent according to [1], wherein the aldehyde and the ketone are compounds represented by the following formula (1).

[0158] RR'CO(1): In the formula (1), R and R' are the same or different and represent a hydrogen atom or a substituted or unsubstituted hydrocarbon group.

[0159] [3] The derivatization reagent according to [2], wherein the hydrocarbon groups of R and R' in the formula (1) are the same or different and are alkyl groups or aryl groups.

[0160] [4] The derivatization reagent according to any one of [1] to [3], which is used for N-alkylating or N-arylating the amino group of a nitrogen-containing compound.

[0161] [5] The derivatization reagent according to [4], wherein the nitrogen-containing compound is one or more selected from amino acids, peptides, and catecholamines contained in a sample (preferably a biological component).

[0162] [6] The derivatization reagent according to any one of [1] to [5], wherein the derivatization reagent is used to N-alkylate or N-arylate an amino group of a nitrogen-containing compound.

[0163] (a) When the nitrogen-containing compound is an amino acid or peptide in a sample (preferably a biological component), R and R' in the formula (1) are the same or different and are a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 8 carbon atoms, or

[0164] (b) When the nitrogen-containing compound is a catecholamine in a sample (preferably a biological component), R and R' in the formula (1) are the same or different and are substituted or unsubstituted alkyl groups having 2 to 9 carbon atoms.

[0165] [7] The derivatization reagent according to any one of [1] to [6], wherein a reducing agent is used during derivatization. More preferably, an acid reagent is further used during derivatization (for example, each reagent is added simultaneously or at different times) and / or no base reagent is used during derivatization.

[0166] [8] A reducing agent, wherein the derivatization reagent according to any one of [1] to [6] is used in derivatization. It is more preferred that an acid reagent is further used in derivatization and / or that no base reagent is used in derivatization.

[0167] [9] A derivatization kit for mass spectrometry analysis, comprising the derivatization reagent (first reagent) according to any one of [1] to [7] and a reducing agent (second reagent), and preferably further comprising an acid (third reagent). The kit may be a combination or a kit. In addition, each reagent of the kit is preferably contained in an openable and closable sealed container (glass, plastic, etc.; screw-capped, etc.).

[0168]

[10] The derivatization kit according to [8] or [9], wherein the reducing agent comprises one or more selected from borane, a complex containing borane, and a salt of a boron hydride compound.

[0169]

[11] The derivatization kit according to any one of [8] to

[10] , further comprising an acid reagent.

[0170]

[12] The derivatization kit according to any one of [8] to

[11] , which does not contain an alkaline reagent or does not use an alkaline reagent. Preferably, the derivatization kit does not use an alkaline reagent during derivatization.

[0171]

[13] A method for producing a derivatized compound, a method for derivatizing an analyte compound, or a method for improving the sensitivity of mass spectrometry analysis of an analyte compound, wherein the derivatization reagent described in any one of [1] to [7] or the derivatization kit described in any one of [8] to

[12] is used for the analyte compound.

[0172]

[14] A method for producing a derivatized compound, a method for derivatizing an analyte compound, or a method for improving the sensitivity of mass spectrometry analysis of an analyte compound, comprising a derivatization step using the derivatization reagent described in any one of [1] to [7] or the derivatization kit described in any one of [8] to

[12] for the analyte compound.

[0173]

[15] The method according to

[13] or

[14] , comprising a pretreatment step of isolating the analyte compound from a sample collected from an animal or the like and the derivatization step.

[0174]

[16] The method according to any one of

[13] to

[15] , comprising the derivatization step and a post-treatment step of separating the derivatized analyte compound or removing unnecessary reagents after the reaction.

[0175]

[17] The method according to any one of

[13] to

[16] , comprising an analysis step of analyzing the derivatized target compound by device analysis.

[0176]

[18] The method according to any one of

[13] to

[17] , comprising performing a derivatization step of N-alkylating or N-arylating an amino group of a nitrogen-containing compound contained in a sample to convert the amino group into a secondary or tertiary amine using a derivatization reagent containing an aldehyde or a ketone,

[0177] The nitrogen-containing compound is one or more selected from amino acids, peptides and catecholamines contained in biological components,

[0178] The aldehyde or ketone is a compound represented by the following formula (1):

[0179] RR'CO(1)

[0180] In the formula (1), R and R' represent a hydrogen atom or a substituted or unsubstituted alkyl or aryl group having 1 to 11 carbon atoms.

[0181]

[19] An aldehyde or ketone in the manufacture of a derivatization reagent or a derivatization kit, or use thereof. More preferably, a reducing agent is further used and / or an acid reagent is used and / or no alkaline reagent is used.

[0182]

[20] An aldehyde or ketone for producing a derivatization reagent or a derivatization kit or for producing the same, or use thereof. Preferably, a reducing agent and / or an acid reagent is further used, and preferably no alkaline reagent is used.

[0183]

[21] An aldehyde or ketone for derivatization or a mixture for derivatization, the mixture comprising an aldehyde or ketone and a reducing agent, more preferably comprising an acid reagent and / or excluding a base reagent.

[0184]

[22] An aldehyde or ketone in a combination product for derivatization or a kit product for derivatization, wherein the aldehyde or ketone and the reducing agent (more preferably, the aldehyde or ketone, the reducing agent and the acid reagent) are independent of each other. It is further preferred that no alkaline reagent is used in the kit product for derivatization.

[0185] The kit product can also be a combination product or a set product.

[0186]

[23] The aldehyde or ketone according to

[22] or the combination product according to

[20] , wherein the derivatized analyte compound is a nitrogen-containing compound (preferably a nitrogen-containing compound in a biological component).

[0187]

[24] A method for derivatizing a nitrogen-containing compound or a method for mass spectrometry analysis of a nitrogen-containing compound, comprising: using an aldehyde or ketone selected as a derivatizing agent for improving the sensitivity of mass spectrometry analysis by a method for screening a derivatizing agent for improving the sensitivity of a nitrogen-containing compound in mass spectrometry analysis.

[0188]

[25] A mass spectrometry analysis method, comprising using the derivatization reagent described in any one of [1] to [7] above, or the N-alkylated or N-arylated nitrogen-containing compound obtained using the derivatization kit described in any one of [8] to

[12] above.

[0189]

[26] A method for screening a derivatization agent for improving the sensitivity of nitrogen-containing compounds in mass spectrometry analysis,

[0190] The screening method includes:

[0191] A step of mixing a reagent containing an aldehyde or a ketone and a nitrogen-containing compound to form a derivatization; and

[0192] A step of determining that the derivatization reagent is a derivatization reagent for improving the mass spectrometry sensitivity of the nitrogen-containing compound when the characteristic signal of the mass spectrometry of the nitrogen-containing compound after derivatization becomes larger than the characteristic signal of the mass spectrometry of the nitrogen-containing compound before derivatization. More preferably, in the derivatization step, a reducing agent and / or an acid reagent is used as a reagent and / or an alkaline reagent is not used.

[0193]

[27] The screening method according to

[24] , wherein the reagent containing the aldehyde or ketone is a derivatization reagent containing the aldehyde or ketone according to any one of [1] to [7].

[0194]

[28] A kit for derivatization, comprising an aldehyde or ketone identified as a derivatization reagent for improving sensitivity according to

[26] or

[27] above, or a reagent containing an aldehyde or ketone.

[0195] [Example]

[0196] Hereinafter, the present technology will be described in more detail based on examples, etc. In addition, the examples, etc. described below are examples of representative examples, etc. of the present technology, and the scope of the present technology is not to be narrowly interpreted thereby.

[0197] [Test Example 1: Production of a derivative compound of a secondary or tertiary amine using a derivatization reagent (aldehyde or ketone reagent) as a standard substance (analyte compound)]

[0198] 1. Reagents

[0199] The group of aldehydes or ketones shown in Table 1 was used for derivatization.

[0200]

Table 1

[0201] Table 1: Reagents (aldehyde: RCHO, ketone: RR'-CO)

[0202]

[0203]

[0204] 5% aldehyde or ketone aqueous solution (formaldehyde is 10% aqueous solution, hexanal and heptaldehyde, benzaldehyde is 5%-10% ethanol aqueous solution, acetone is 50% or 100% aqueous solution)

[0205] 3 mg / mL borane-2-methylpyridine complex-isopropanol solution (prepared when needed)

[0206] Acetic acid

[0207] 2. Derivatization

[0208] [Standard (analyzed compound)]

[0209] Amino acids (triiodothyronine (T3) molecular weight 651.0, hydrophobicity (ClogP) 2.63, thyroxine (T4) molecular weight 776.9, hydrophobicity (ClogP) 3.51, homocysteine ​​(HC ys) molecular weight 135.2, hydrophobicity (ClogP) -2.07)

[0210] · Peptides (angiotensin I (AI) molecular weight 1296.5, hydrophobicity (ClogP) -2.70, oxytocin (OXT) molecular weight 1007.2, hydrophobicity (ClogP) -0.65, glucagon (GN) molecular weight 3482.8, hydrophobicity (ClogP) unknown, adrenocorticotropic hormone (ACTH) molecular weight 4579.3, hydrophobicity (ClogP) unknown, insulin (Ins) molecular weight 5807.6, hydrophobicity (ClogP) unknown, C-peptide (CP) molecular weight 3020.3, hydrophobicity (ClogP) unknown)

[0211] Catecholamines (epinephrine (Ad) molecular weight 183.2, hydrophobicity (ClogP) -0.68, norepinephrine (NA) 169.2, hydrophobicity (ClogP) -0.99, dopamine (DP) molecular weight 153.2, hydrophobicity (ClogP) 0.17)

[0212] 3. Analysis conditions

[0213] [LC analysis conditions]

[0214] Equipment: Shimadzu LC-20A

[0215] Chromatographic column YMC Triart C18 (2.1×50mm)

[0216] Elution conditions: flow rate 0.4 mL / min; solvent A: 0.1% formic acid-water; solvent B: methanol

[0217] Time (min) [Solvent B %]: 0 min [0 %], 3 min [0 %], 3.5 min [100 %], 4 min [0 %], 5 min [0 %]

[0218] [MS / MS analysis conditions]

[0219] Equipment: Shimadzu LCMS8040 triple quadrupole mass spectrometer

[0220] Ionization conditions: ESI positive ion mode

[0221] The collision divergence conditions (CE) and the measurement results (SRM chromatogram) are shown in the following table.

[0222] [MS analysis]

[0223] Equipment: Shimadzu LCMS8040 triple quadrupole mass spectrometer

[0224] The ionization conditions were measured in ESI positive ion mode scanning mode, and the mass spectrum is shown. The horizontal axis of the spectrum is m / Z.

[0225] [ 1 H-NMR analysis]

[0226] Installed JNM-ECA500 NMR spectrometer from Japan Electronics

[0227] The standard substance (0.1 mg to 1.0 mg) was used and derivatized, and the spectrum was obtained by integration 1024 times (HCys integration 16 times) at room temperature (22°C). The horizontal axis of the spectrum is ppm.

[0228] [Derivatization]

[0229] Add 25 μL of aldehyde or ketone aqueous solution, 25 μL of picoline borane aqueous solution, and 5 μL of acetic acid to the standard (1 pg to 100 ng), and let stand at room temperature (20 to 30°C) for 30 minutes to perform a derivatization reaction. After derivatization, aldehyde or ketone, acetic acid, and boron are removed by condensing to dryness by an evaporator. After condensing to dryness by an evaporator, it is dissolved in a re-dissolution solvent (30% acetonitrile aqueous solution) and analyzed by LC-MS / MS.

[0230] 4. Results of derivatization of amino acid standards

[0231] A derivative compound of a secondary or tertiary amine is obtained by using an aldehyde or a ketone as a standard substance (analytical compound: an amino acid present in a biological component).

[0232] Specifically, formaldehyde is used in triiodothyronine (T3) and thyroxine (T4), respectively. In addition, the standard substance (analyzed compound) is benzaldehyde used in thyroxine (T4). Thus, derivative compounds of secondary amines or tertiary amines are obtained, respectively. Furthermore, acetone is used in homocysteine ​​(HCys). Thus, derivative compounds of secondary amines are obtained. These derivative compounds are obtained by 1 H-NMR analysis and MS analysis identified them as T3-Me2 (N,N-dimethyltriiodothyronine), T4-Me2 (N,N-dimethylthyroxine), T4-Bn2 (N,N-dibenzylthyroxine), HCys-iPr (N-isopropylhomocysteine) ( Figures 1 to 8 Thus, it was confirmed that amino acids (especially amino acids present in biological components) can be derivatized by aldehydes or ketones having 1 to 8 carbon atoms.

[0233] Furthermore, the SRM parameters of the standard (non-derivatized) and the derivative compounds based on aldehydes or ketones are shown in Tables 2 to 4 (the SRM chromatogram (10 ng) is not shown). Table 5 shows the results of the derivative compounds with high relative sensitivity improvement ratio and significantly improved detection sensitivity or measurement sensitivity. 1 The results of H-NMR analysis and MS analysis are shown in Figures 4 to 8 .

[0234] Thus, the sensitivity of the derivatized compound (especially when using aldehydes or ketones having 1 to 8 carbon atoms) obtained by using aldehydes or ketones in amino acids (especially amino acids present in biological components) is higher than that of the standard substance. Thus, by using aldehydes or ketones as derivatization reagents, the detection sensitivity is improved.

[0235]

Table 2

[0236] Table 2: SRM parameters for triiodothyronine (T3)

[0237]

[0238]

Table 3

[0239] Table 3: SRM parameters of thyroxine (T4)

[0240]

[0241]

Table 4

[0242] Table 4: SRM parameters for homocysteine ​​(HCys)*

[0243]

[0244]

[0245] *ND: Not Determined

[0246]

Table 5

[0247] Table 5: Relative sensitivity improvement of standards (amino acids)*

[0248]

[0249] *Relative sensitivity improvement when the case without derivatization is taken as 1

[0250] 5. Results of derivatization of peptide standards

[0251] A derivative compound of a secondary or tertiary amine is obtained by using an aldehyde or a ketone as a standard substance (analytical compound: a peptide present in a biological component).

[0252] Specifically, formaldehyde, acetaldehyde or acetone are used in angiotensin I (AI). Formaldehyde, acetaldehyde, propionaldehyde or acetone are used in oxytocin (OXT). Formaldehyde, acetaldehyde, propionaldehyde or acetone are used in glucagon (GN). Formaldehyde and acetaldehyde are used in insulin (INS). Thus, derivative compounds of secondary amines or tertiary amines are obtained. The derivative compounds of AI are prepared by1 H-NMR analysis and MS analysis identified them as AI-Me2 (N,N-dimethylangiotensin I), AI-Et2 (N,N-diethylangiotensin I), AI-iPr (N-isopropylangiotensin I) ( Figures 9-11 ). The OXT derivative compounds were respectively 1 H-NMR analysis and MS analysis identified them as OXT-Me2 (N,N-dimethyloxytocin), OXT-Et2 (N,N-diethyloxytocin), OXT-Pr2 (N,N-dipropyloxytocin), OXT-iPr (N-isopropyloxytocin) ( Figures 12 to 15 The derivative compounds of GN were identified as GN-Me4 (N,N,N',N'-tetramethyl glucagon), GN-Et2 (N,N'-diethyl glucagon), GN-Pr2 (N,N'-dipropyl glucagon), GN-iPr (N-isopropyl glucagon) ( Figures 16 to 19 ). The derivative compounds of INS were respectively 1 H-NMR analysis and MS analysis identified them as INS-Me6 (N,N,N',N',N',N'-hexamethyl insulin) and INS-Et5 (N,N,N',N',N'-pentaethyl insulin). Thus, it was confirmed that peptides (especially peptides present in biological components) can be produced from aldehydes having 1 to 8 carbon atoms ( Figures 20-21 ).

[0253] Furthermore, the SRM parameters of the standard (non-derivatized) and the derivative compounds based on aldehydes or ketones are shown in Tables 6 to 11 (the SRM chromatogram (10 ng) is not shown). The results of the derivative compounds with high relative sensitivity improvement ratios are shown in Table 12. These identification results are shown in Figures 9 to 21 .

[0254] Thus, the sensitivity of the derivatized compound (especially when using an aldehyde or ketone having 1 to 8 carbon atoms) obtained by using an aldehyde or ketone in a peptide (especially a peptide present in a biological component) is higher than that of the standard substance. Thus, by using an aldehyde or ketone as a derivatization reagent, the detection sensitivity is improved.

[0255]

Table 6

[0256] Table 6: SRM parameters of angiotensin I (AI)*

[0257]

[0258] *ND: Not Determined

[0259]

Table 7

[0260] Table 7: SRM parameters of oxytocin (OXT)

[0261]

[0262]

[0263]

Table 8

[0264] Table 8: SRM parameters of glucagon (GN)

[0265]

[0266]

Table 9

[0267] Table 9: SRM parameters for adrenocorticotropic hormone (ACTH)

[0268]

[0269]

Table 10

[0270] Table 10: SRM parameters of insulin (INS)

[0271]

[0272]

Table 11

[0273] Table 11: SRM parameters for C-peptide (CP)

[0274]

[0275]

Table 12

[0276] Table 12: Relative sensitivity improvement of standards (peptides)*

[0277]

[0278] *Relative sensitivity improvement when the case without derivatization is taken as 1

[0279] **10ng

[0280] 6. Results of derivatization of catecholamine standards

[0281] Derivatized compounds of secondary or tertiary amines were obtained by using aldehydes in standard samples (analyzed compounds: catecholamines present in biological components). Furthermore, the SRM parameters of the standard samples (non-derivatized) and the derivatized compounds based on aldehydes are shown in Tables 13 to 15 (SRM chromatograms (10 ng) are not shown). Specifically, Ad-Me, Ad-Et, Ad-Pr, Ad-CpM, Ad-Bu, Ad-IB, Ad-V, Ad-IV, Ad-Hx, Ad-Hp, and Ad-Bn were obtained from the adrenaline standard sample. NA-Me2, NA-Et2, NA-Pr2, NA-CpM, NA-Bu2, NA-IB2, NA-V2, NA-IV2, NA-Hx2, NA-Hp2, and NA-Bn2 were obtained from the noradrenaline standard sample. DP-Me2, DP-Et2, DP-Pr2, DP-CpM, DP-Bu2, DP-IB2, DP-V2, DP-IV, DP-Hx, DP-Hp2, and DP-Bn2 were obtained from dopamine standards.

[0282] The results of the derivatized compounds with high relative sensitivity improvement ratios are shown in Table 16. Thus, the sensitivity of the derivatized compounds (especially when aldehydes with 3 to 9 carbon atoms are used) obtained by using aldehydes in catecholamines (especially catecholamines present in biological components) is higher than that of the standard. Thus, by using aldehydes as derivatization reagents, the detection sensitivity is improved.

[0283]

Table 13

[0284] Table 13: SRM parameters of epinephrine (Ad)

[0285]

[0286]

Table 14

[0287] Table 14: SRM parameters for norepinephrine (NA)*

[0288]

[0289] *ND: Not Determined

[0290]

Table 15

[0291] Table 15: SRM parameters of dopamine (DP)

[0292]

[0293]

Table 16

[0294] Table 16: Relative Sensitivity Improvement of Standards (Catecholamines)*

[0295]

[0296] *Relative sensitivity improvement when the case without derivatization is taken as 1

[0297] [Test Example 2: Detection of oxytocin in the presence of plasma extract]

[0298] 1. Reagents

[0299] 5% aldehyde aqueous solution (propionaldehyde, butylaldehyde, valeraldehyde)

[0300] 3 mg / mL borane-2-methylpyridine complex-isopropanol solution (prepared when needed)

[0301] Acetic acid

[0302] 2. Preparation of Plasma Extracts

[0303] A weak cation exchange column (Waters, Oasis) was washed with methanol (2 mL). (R) WCX 3cc, Cartridge 60mg, 30μm), equilibrated with water (2mL). 4% phosphate buffer (300μL) and mixed plasma (300μL) were mixed and developed on the column. 5% ammonia water (2mL) and 75% acetonitrile aqueous solution (2mL) were used to wash in sequence, and 1% formic acid 75% acetonitrile aqueous solution (2mL) was used to elute. The eluate was concentrated by centrifugal evaporator to obtain a plasma extract.

[0304] 3. Derivatization

[0305] 0, 1, 10 pg of oxytocin standard was added to the plasma extract, and 25 μL of aldehyde aqueous solution, 25 μL of methylpyridine borane solution, and 5 μL of acetic acid were added, and the solution was left to stand at room temperature for 30 minutes. The derivatized aldehyde, acetic acid, and boron were removed by evaporation and concentration. After evaporation and concentration, the solvent was dissolved in a re-solvent (30% acetonitrile solution) and analyzed by LC-MS / MS.

[0306] 4. LC analysis conditions

[0307] Equipment: Shimadzu LC-20A

[0308] Chromatographic column YMC Triart C18 2.1×50mm

[0309] Elution conditions Flow rate 0.4 mL / min Solvent A: 0.1% formic acid-water B: methanol

[0310] Time (min) [Solvent B %]: 0 min [0 %], 3 min [0 %], 3.5 min [100 %], 4 min [0 %], 5 min [0 %]

[0311] 5. MS / MS analysis conditions

[0312] Equipment: Shimadzu LCMS8040 triple quadrupole mass spectrometer

[0313] Ionization conditions: ESI positive ion mode

[0314] The collision divergence conditions (CE) and the measurement results (SRM chromatogram) are shown in the following table.

[0315] 6. Measurement results

[0316] The sensitivity improvement effect based on N-alkylation is shown in Tables 17 and 18. The sensitivity improvement was confirmed to be 3.7 times in the dipropylated form (OXT-Pr2), 2.4 times in the dibutylated form (OXT-Bu2), and 1.7 times in the dicyclized form (OXT-V2). Therefore, the detection sensitivity of peptides in serum and plasma is improved by N-alkylation.

[0317]

Table 17

[0318] Table 17: SRM parameters of oxytocin (OXT)

[0319]

[0320]

Table 18

[0321] Table 18: Relative sensitivity enhancement of oxytocin in the presence of plasma extracts

[0322]

[0323] *t R :Holding time (minutes)

[0324] **Relative sensitivity improvement ratio when no derivatization was performed was taken as 1.

[0325] The numbers in the table are the peak areas on the SRM chromatograms.

[0326] [Test Example 3: Optimization of derivatization reaction]

[0327] The optimization of the catecholamine derivatization reaction using a standard substance was performed (concentration of the aldehyde reagent, presence of acetic acid).

[0328] 1. Reagents

[0329] 10% heptaldehyde-80% ethanol aqueous solution

[0330] 3 mg / mL borane-2-methylpyridine complex-isopropanol solution (prepared when needed)

[0331] Acetic acid

[0332] 2. Derivatization

[0333] Add 25 μL of aldehyde aqueous solution, 25 μL of picoline borane solution, and 5 μL of acetic acid to the catecholamine (epinephrine, norepinephrine, dopamine) standard (1 ng) and let stand at room temperature for 30 minutes. The derivatized aldehyde, acetic acid, and boron are removed by condensing to dryness with an evaporator. After condensing to dryness, dissolve in a redissolution solvent (30% acetonitrile solution) and perform LC-MS / MS analysis.

[0334] 3. Analysis conditions

[0335] LC analysis and MS / MS analysis were performed under the same analysis conditions as those of Experimental Example 1.

[0336] 4. Research Results

[0337] The sensitivity improvement effects due to N-alkylation and arylation are shown in Tables 19 and 20.

[0338] When the analysis target is catecholamine, it was confirmed that the sensitivity was improved when heptanal was used as a derivatization reagent. Therefore, the detection sensitivity of catecholamine is improved by N-alkylation.

[0339]

Table 19

[0340] Table 19: SRM parameters for catecholamines SRM parameters for adrenaline (Ad)

[0341]

[0342]

Table 20

[0343] Table 20: Relative sensitivity improvements associated with optimization of derivatization reactions

[0344]

[0345] *t R :Retention time (min) **The case without derivatization is set as 1

[0346] The numbers in the table are the peak areas on the SRM chromatograms.

[0347] [Test Example 4: Detection of oxytocin in plasma]

[0348] 1. Reagents

[0349] 20% formaldehyde aqueous solution

[0350] 3 mg / mL borane-2-methylpyridine complex-isopropanol solution (prepared when needed)

[0351] Acetic acid

[0352] 2. Extraction of Oxytocin from Plasma

[0353] A strong cation exchange column (Agilent, Bond Elut Plexa PCX 1cc Cartridge 30mg) was washed with 1% formic acid-methanol (1mL) and equilibrated with 1% formic acid aqueous solution (1mL). Mixed plasma (300μL) was added to oxytocin standard (0, 20, 200ng), and the plasma dilution diluted with water (700μL) was developed on the column. The column was washed with 1% formic acid aqueous solution (1mL) and 1% formic acid-methanol (1mL) in sequence, and eluted with 5% ammonia water (1mL). The eluate was concentrated by a centrifugal evaporator to obtain an oxytocin extract.

[0354] 3. Derivatization

[0355] 50 μL of aqueous formaldehyde solution, 50 μL of picoline borane solution, and 10 μL of acetic acid were added to the oxytocin extract and allowed to stand at room temperature for 30 minutes. The derivatized aldehyde, acetic acid, and boron were removed by condensing to dryness by an evaporator. After condensing to dryness by an evaporator, the extract was dissolved in a re-dissolving solvent (30% acetonitrile solution) and analyzed by LC-MS / MS.

[0356] 4. LC analysis conditions

[0357] Equipment: Shimadzu LC-30A

[0358] Chromatographic column YMC Triart C18 2.1×50mm

[0359] Elution conditions Flow rate 0.4 mL / min Solvent A: 0.1% formic acid-water B: methanol

[0360] Time (min) [Solvent B %]: 0 min [0 %], 3 min [0 %], 3.5 min [100 %], 4 min [0 %], 5 min [0 %]

[0361] 5. MS / MS analysis conditions

[0362] Equipment: Shimadzu LCMS8060 triple quadrupole mass spectrometer

[0363] Ionization conditions: ESI positive ion mode

[0364] The collision divergence conditions (CE) and the measurement results (SRM chromatogram) are shown in Table 21 below.

[0365] 6. Measurement results

[0366] The sensitivity improvement effect by N-alkylation (dimethylation) is shown in Table 21 and Table 22. A 2.8-fold (2.8-fold when 1 ng of OXT was added) sensitivity improvement was confirmed in the dimethylated form (OXT-Me2). Therefore, N-alkylation improves the detection sensitivity of peptides in plasma.

[0367]

Table 21

[0368] Table 21: SRM parameters of oxytocin (OXT)

[0369]

[0370]

Table 22

[0371] Table 22: Relative sensitivity improvement of oxytocin from plasma

[0372]

[0373] *t R :Holding time (minutes)

[0374] **Relative sensitivity improvement ratio when the case where no derivatization was performed was taken as 1.

[0375] The numbers in the table are the peak areas on the SRM chromatograms.

[0376] [Test Example 5: Detection of Catecholamines in Urine and Serum]

[0377] 1. Reagents

[0378] 10% valeraldehyde in 50% ethanol in water

[0379] 3 mg / mL borane-2-methylpyridine complex-isopropanol solution (prepared when needed)

[0380] Acetic acid

[0381] 2. Extraction of catecholamines from urine and serum

[0382] A strong cation exchange column (Agilent, Bond Elut Plexa PCX 1cc Cartridge 30mg) was washed with 1% formic acid-methanol (1mL) and equilibrated with 1% formic acid aqueous solution (1mL). Catecholamine (epinephrine, norepinephrine, dopamine) standards (0 or 200ng) were added to the sample (1mL of water, 1mL of urine, or a solution obtained by diluting 300μL of mixed serum with 700μL of water) and developed on the column. The column was washed with 1% formic acid aqueous solution (1mL) and 1% formic acid-methanol (1mL) in sequence, and eluted with 5% ammonia water (1mL). The eluate was concentrated by a centrifugal evaporator to obtain a catecholamine extract.

[0383] 3. Derivatization

[0384] 50 μL of valeraldehyde aqueous solution, 50 μL of picoline borane solution, and 10 μL of acetic acid were added to the oxytocin extract, and the mixture was allowed to stand at room temperature for 30 minutes. The derivatized aldehyde, acetic acid, and boron were removed by condensing to dryness with an evaporator. After condensing to dryness, the mixture was dissolved in a re-dissolving solvent (30% acetonitrile solution) and analyzed by LC-MS / MS.

[0385] 4. LC analysis conditions

[0386] Equipment: Shimadzu LC-20A

[0387] Chromatographic column OSAKA SODA CAPCELL CORE C18 2.1×75mm

[0388] Elution conditions Flow rate 0.4 mL / min Solvent A: 0.1% formic acid-water B: methanol

[0389] Time (min) [Solvent B %]: 0 min [0%], 0.5 min [0%], 2 min [100%], 3 min [100%], 3.5 min [0%], 5 min [0%]

[0390] 5. MS / MS analysis conditions

[0391] Equipment: Shimadzu LCMS8040 triple quadrupole mass spectrometer

[0392] Ionization conditions: ESI positive ion mode

[0393] The collision divergence conditions (CE) and the measurement results (SRM chromatogram) are shown in Table 23 below.

[0394] 6. Measurement results

[0395] The sensitivity improvement effect by N-alkylation (pentylation) is shown in Tables 23 to 26. Even in urine and serum, the detection sensitivity is improved by derivatization.

[0396]

Table 23

[0397] Table 23: SRM parameters of catecholamines 1. SRM parameters of epinephrine (Ad)

[0398]

[0399] 2. SRM parameters of norepinephrine (NA)

[0400]

[0401] 3. SRM parameters of dopamine (DP)

[0402]

[0403]

Table 24

[0404] Table 24: Relative sensitivity improvement of epinephrine from urine and serum

[0405]

[0406] *t R :Holding time (minutes)**ND:Not determined

[0407] ***Relative sensitivity improvement ratio when the case where no derivatization was performed was taken as 1.

[0408] The numbers in the table are the peak areas on the SRM chromatograms.

[0409]

Table 25

[0410] Table 25: Relative sensitivity improvement of norepinephrine from urine and serum

[0411]

[0412] *t R :Holding time (minutes)**ND:Not determined

[0413] ***Relative sensitivity improvement ratio when the case where no derivatization was performed was taken as 1.

[0414] The numbers in the table are the peak areas on the SRM chromatograms.

[0415]

Table 26

[0416] Table 26: Relative sensitivity improvement from dopamine in urine and serum

[0417]

[0418] *t R :Holding time (minutes)**ND:Not determined

[0419] ***Relative sensitivity improvement ratio when the case where no derivatization was performed was taken as 1.

[0420] The numbers in the table are the peak areas on the SRM chromatograms.

[0421] In addition, in this specification, "first, second, third...", "A, B, C...", "once, twice, three times...", numbers, letters, etc. are sometimes added for convenience of explanation, but the present invention does not limit the interpretation order in a narrow sense, and the order can be changed arbitrarily. In addition, in this specification, for example, "performing (a situation)" and "performing (a situation)" can also be referred to as a method, process, means, or step, etc., and these terms can also be appropriately replaced with each other. For example, "step" can also be referred to as "performing (a situation)", method, process, or means, etc., "process" can also be referred to as "performing (a situation)", method, step, or means, etc., and "means" can also be referred to as "performing (a situation)", method, process, step, mechanism, system (system), device, or part, and "part" can also be a mechanism, means, device, or system (system) or a mechanism, means, or device used for them. A combination product can also be a combination product or a combination.

Claims

1. A derivatization reagent comprising a compound represented by the following formula (1), RR'CO(1) In the formula (1), R and R' are the same or different and represent a hydrogen atom or a substituted or unsubstituted alkyl or aryl group having 1 to 11 carbon atoms. 2 . The derivatization reagent according to claim 1 , which is used for N-alkylating or N-arylating the amino group of a nitrogen-containing compound.

3. The derivatization reagent according to claim 2, wherein The nitrogen-containing compound is one or more selected from amino acids, peptides and catecholamines contained in biological components.

4. The derivatization reagent according to claim 1, wherein The derivatization reagent is used to N-alkylate or N-arylate the amino group of the nitrogen-containing compound. (a) When the nitrogen-containing compound is an amino acid or peptide in a biological component, R and R' in the formula (1) are the same or different and are a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 8 carbon atoms, or (b) When the nitrogen-containing compound is a catecholamine in a biological component, R and R' in the formula (1) are the same or different and are substituted or unsubstituted alkyl groups having 2 to 9 carbon atoms. The derivatization reagent according to claim 1 or 2, which is used for mass spectrometry analysis. 6 . A derivatization kit comprising the derivatization reagent according to claim 1 and a reducing agent.

7. The derivatization kit according to claim 6, wherein: The reducing agent comprises one or more selected from borane, a complex containing borane, and a salt of a boron hydride compound.

8. A method for improving the sensitivity of nitrogen-containing compounds in mass spectrometry, comprising using the derivatization kit according to claim 6 to perform derivatization to N-alkylate or N-arylate the amino group of the nitrogen-containing compound to produce a secondary amine or a tertiary amine.

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