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

Abstract

To provide a novel technique for improving the sensitivity of a nitrogen-containing compound in mass spectrometry.SOLUTION: The invention can provide a derivatization reagent comprising a compound represented by the formula (1): RR'CO, where R and R' are identical or different and each represent a hydrogen atom, a substituted or unsubstituted, alkyl or aryl group having 1 to 11 carbon atoms.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] Liquid chromatography tandem mass spectrometry (LC-MS / MS) and matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) are extremely powerful analytical techniques for quantifying trace amounts of metabolites and other substances. In these analytical techniques, the target compound may be ionized. In analyses in which the target compound is ionized, some target compounds have low ionization efficiency, and there are many cases in which the target compound cannot be detected or the detection accuracy for quantification cannot be ensured. Therefore, the target compound may be derivatized to increase the ionization efficiency.

[0003] For example, the following Non-Patent Documents 1 and 2 disclose the derivatization of fat-soluble hormones (male and female hormones) that do not contain nitrogen atoms with a derivatization reagent that contains a nitrogen atom. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] J. Chromatogr. B 2011, 879, 1159-1165. [Non-Patent Document 2] Biomed. Chromatogr. 2021, 35, e5036. [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. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a new technique for improving the sensitivity in mass spectrometry of nitrogen-containing compounds. [Means for solving the problem]

[0006] The present inventors have found that the sensitivity of a nitrogen-containing compound in mass spectrometry can be improved by derivatizing the nitrogen-containing compound with an aldehyde or a ketone, as shown in the Examples below, and have thus completed the present invention.

[0007] The present invention can provide a derivatization reagent comprising a compound (preferably an aldehyde or ketone) represented by the following formula (1). RR'CO (1) [In the formula (1), R and R' are the same or different and each represents a hydrogen atom, or a substituted or unsubstituted alkyl group or aryl group having 1 to 11 carbon atoms.]

[0008] The present invention can provide a derivatization reagent kit comprising the derivatization reagent and a reducing agent.

[0009] The present invention can provide a method for improving the sensitivity of a nitrogen-containing compound in mass spectrometry, which comprises derivatizing an amino group of a nitrogen-containing compound by N-alkylating or N-arylating it using the derivatization reagent kit (preferably a derivatization reagent containing an aldehyde or ketone) to convert it into a secondary amine or tertiary amine.

[0010] The derivatizing reagents may be used to N-alkylate or N-arylate an amino group of a nitrogen-containing compound. The nitrogen-containing compound may be one or more selected from amino acids, peptides, and catecholamines contained in biological components. The derivatization reagent is used for N-alkylating or N-arylating an amino group of a nitrogen-containing compound, (a) when the nitrogen-containing compound is an amino acid or a peptide in a biological component, R and R' in the formula (1) are the same or different and each is 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) may be the same or different and each may be a substituted or unsubstituted alkyl group having 2 to 9 carbon atoms. The derivatization reagent or the derivatization reagent kit may be used for mass spectrometry. The reducing agent may contain one or more selected from borane, a complex containing borane, and a salt of a boron hydride compound. Effect of the Invention

[0011] The present invention provides a new technique for improving the sensitivity in mass spectrometry of nitrogen-containing compounds. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows an outline of a reaction in this embodiment in which an aliphatic or aromatic aldehyde or ketone and a reducing reagent are used to form an imine by dehydration condensation of an amine (1) and an aldehyde or ketone (2), and the formed imine is reduced with a reducing reagent (secondary amine (3) or tertiary amine (4)). [Diagram 2] FIG. 1 shows the chemical structural formulas of various amino acids (triiodothyronine (5), thyroxine (6), and homocysteine ​​(7)) used in this embodiment. [Figure 3A] FIG. 1 shows various peptides (angiotensin I (8), oxytocin (9), glucagon (10), adrenocorticotropic hormone (11)) used in this embodiment. [Figure 3B] FIG. 1 shows various peptides (insulin (12), C-peptide (13)) used in this embodiment. [Figure 4] FIG. 1 shows various catecholamines (adrenaline (14), noradrenaline (15), and dopamine (16)) used in this embodiment. [Diagram 5] FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a derivative compound (T3-Me2) of an amino acid preparation (T3) using an aldehyde reagent (formaldehyde). [Figure 6] FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a derivative compound (T4-Me2) of an amino acid preparation (T4) using an aldehyde reagent (formaldehyde). [Figure 7] FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a derivative compound (T4-Bn2) of an amino acid sample (T4) using an aldehyde reagent (benzaldehyde). [Figure 8] FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a derivative compound (HCys-iPr) of an amino acid sample (HCys) using a ketone reagent (acetone). [Figure 9] FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a derivatized compound (AI-Me2) of a peptide preparation (AI) using an aldehyde reagent (formaldehyde). [Figure 10] FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a derivatized compound (AI-Et2) of a peptide preparation (AI) using an aldehyde reagent (acetaldehyde). [Figure 11] FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a compound (AI-iPr) derived from a peptide preparation (AI) using a ketone reagent (acetone). [Figure 12]FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a derivative compound (OXT-Me2) of a peptide sample (OXT) using an aldehyde reagent (formaldehyde). [Figure 13] FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a derivative compound (OXT-Et2) of a peptide sample (OXT) using an aldehyde reagent (acetaldehyde). [Figure 14] FIG. 1 shows the results of 1H-NMR analysis (top panel A) and MS analysis (bottom panel B) of a derivative compound (OXT-Pr2) of a peptide sample (OXT) using an aldehyde reagent (propionaldehyde). [Figure 15] FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a derivative compound (OXT-iPr) of a peptide sample (OXT) using a ketone reagent (acetone). [Figure 16] FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a derivative compound (GN-Me4) of a peptide sample (GN) using an aldehyde reagent (formaldehyde). [Figure 17] FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a derivative compound (GN-Et2) of a peptide preparation (GN) using an aldehyde reagent (acetaldehyde). [Figure 18] FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a derivative compound (GN-Pr2) of a peptide sample (GN) using an aldehyde reagent (propionaldehyde). [Figure 19] FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a compound (GN-iPr) derived from a peptide preparation (GN) using a ketone reagent (acetone). [Figure 20] FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a compound (INS-Me6) derived from a peptide sample (INS) using an aldehyde reagent (formaldehyde). [Figure 21]FIG. 1 shows the results of 1H-NMR analysis (upper panel) and MS analysis (lower panel) of a compound (INS-Et5) derived from a peptide sample (INS) using an aldehyde reagent (acetaldehyde). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] A preferred embodiment for carrying out the present technology will be described below. The embodiment described below shows an example of a representative embodiment of the present technology, and the scope of the present technology will not be narrowly interpreted by this. In this specification, percentages are expressed by volume unless otherwise specified. In addition, the upper limit (or less) and the lower limit (or more) of each numerical range (~) can be arbitrarily combined as desired. In addition, in the explanations of "1." to "5." etc. described below, overlapping explanations of each technical feature, each configuration, each definition, each term, each method, etc., such as aldehyde, reducing agent, and method for improving sensitivity in mass spectrometry, may be omitted as appropriate. The explanations of "1." to "5." etc. can be applied to each of the technologies or each embodiment of "1." to "5.", and each technical feature, etc. can be appropriately adopted in each of the technologies or each embodiment, etc.

[0014] 1. Description of the Invention

[0015] Compounds such as peptides and proteins having nitrogen atoms (hereinafter also referred to as "nitrogen-containing compounds") generally tend to be more easily ionized than fat-soluble hormones, and for this reason, derivatization of nitrogen-containing compounds is not necessarily a common method. On the other hand, in the analysis of protein and amino acid sequences, examples have been reported in which phosphonium compounds having phosphorus atoms have been used as charge tags (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, these documents do not indicate whether they can be applied to quantitative measurement.

[0016] Liquid chromatography tandem mass spectrometry (LC-MS / MS) and matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) are extremely powerful analytical methods for quantifying trace amounts of metabolites and substances. However, there are many cases where the ionization efficiency is low for some compounds, making them impossible to detect, or where they can be detected but the accuracy of quantification cannot be maintained. Although it is possible to cover the sensitivity aspect by increasing the amount of sample, such as in the case of foods and supplements, there are many cases where it is not possible to simply increase the amount of sample, such as human metabolites. To solve this problem, not only have there been improvements in the equipment, such as increasing the sensitivity of mass spectrometers, but it has also been widely practiced to modify the target compounds (e.g., derivatize) to increase the ionization efficiency.

[0017] In general, biological metabolites are organic compounds mainly composed of carbon, hydrogen, oxygen, and nitrogen, and oxygen and nitrogen atoms with unshared electron pairs are easily added with hydrogen ions (protons), resulting in high ionization efficiency. Nitrogen atoms in particular have a higher proton addition ability than oxygen atoms, and compounds containing nitrogen atoms tend to be more easily ionized than compounds without nitrogen atoms. For compounds that do not have nitrogen atoms, such as fat-soluble steroid hormones, the ionization efficiency has been improved by introducing nitrogen atoms using derivatization reagents. However, nitrogen-containing compounds such as amino acids, peptides, proteins, and catecholamines are easily ionized, so improving sensitivity through derivatization reactions has been difficult or has hardly been considered.

[0018] In LC-MS / MS, the standard method of quantitative mass spectrometry, electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) is used, so the ease of ionization of the analyte depends on the polarity of the analyte itself (ease of protonation) and the composition of the organic solvent in the mobile phase that evaporates during ionization. Since different compounds have different structures and polarities, it is necessary to optimize the ionization efficiency to suit the compound in order to further increase the detection sensitivity. In addition, since LC-MS / MS enables more sensitive quantitative measurement by selected reaction monitoring (SRM), it is also important to obtain fragmentation with lower background. In general, in mass spectrometry in positive ion mode, nitrogen atoms with unshared electron pairs have the characteristic of being easily protonated, which has the effect of promoting ionization. Nitrogen-containing compounds such as amino acids, peptides, and proteins have nitrogen atoms in their molecules that are easily protonated, so they are easier to ionize than non-nitrogen-containing compounds.

[0019] For example, when derivatization is performed using a strong cationic derivatization reagent such as a phosphonium group, the background noise from the reaction reagent tends to be high, and derivatization tends to increase the molecular weight and make ionization difficult, which may offset or reduce the effect of improving ionization efficiency. In other words, it has been considered common knowledge in mass spectrometry of nitrogen-containing compounds that if the sensitivity is not increased even after the effort of derivatization, it is better to measure directly without derivatization. However, further improvement in sensitivity is required for more accurate quantification of these nitrogen-containing compounds. Therefore, a different methodology is needed for high-sensitivity detection of nitrogen-containing compounds.

[0020] The present inventors focused on the amino group, which is also an ionization-promoting group. Most of the amino acids, peptides, proteins, and catecholamines that act in vivo are primary amines. Amines are classified as primary, secondary, tertiary, and quaternary depending on the number of substituents, and quaternary amines are classified as ammonium, which has an electric charge. As for primary, secondary, and tertiary amines, the increase in the number of substituents generally leads to an increase in basicity (protons are more easily added) and an increase in hydrophobicity (the ionization is more likely because the ionization is more easily carried out because it is introduced into the ion source in a solvent composition with a higher organic solvent content). The increase in basicity can be determined using the acid dissociation constant (pKa) as an index, and the increase in hydrophobicity can be determined using hydrophobicity (ClogP), which can be calculated using ChemDraw. That is, the present inventors thought that the ionization efficiency could be increased by converting the amino group into a secondary or tertiary group. Then, a reductive amination reaction was used to convert a primary amine into a secondary or tertiary amine. This reaction is performed by first forming an imine by dehydration condensation between an amine (1) and an aldehyde or ketone (2) using an aldehyde or ketone (e.g., an aliphatic or aromatic aldehyde or ketone) and a reducing reagent, and then reducing the formed imine with a reducing reagent (e.g., Figure 1). In this reaction, the reaction proceeds to secondary (3) and tertiary (4), but not to quaternary. Since many types of aldehydes or ketones (e.g., aliphatic and aromatic aldehydes or ketones) are commercially available, it is possible to screen the optimal substituent according to the molecular weight and hydrophobicity of the analyte, and it is expected that the ionization efficiency can be optimized for each compound. In addition, in the derivatization reaction, there have been problems such as a decrease in S / N due to an increase in background due to residual reagents. However, when lower aliphatic aldehydes or ketones are used, they have a low boiling point, and unreacted aldehydes or ketones can be distilled off by concentration or nitrogen gas purging. In addition, reducing reagents (mainly borohydride-based reagents) are converted into water-soluble borates by hydrolysis, which do not adsorb to reversed-phase columns and can be easily separated and removed.

[0021] The present invention is preferably for detecting and quantifying trace metabolites with high sensitivity using mass spectrometry in the field of clinical testing, and can be used in a wide range of fields and models, regardless of the manufacturer of the mass spectrometer. In order for the quantification of trace metabolites by mass spectrometry to become widespread in the field of clinical testing in the future, it is desirable to be able to achieve high detection sensitivity at any time, anywhere, and with any manufacturer's device, and the present invention can improve detection sensitivity to bridge the differences between models from each manufacturer, thereby supporting its widespread use.

[0022] The present inventors have further discovered the following, as will be described later in the Examples. [1] The present invention can provide a method for improving sensitivity in mass spectrometry by converting amines into secondary or tertiary amines by alkylation or arylation. [2] Furthermore, the analyte derivatized by the above derivatization reaction can be ionized more efficiently by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorption ionization (MALDI), but similar effects may be obtained with other ionization methods as well. This allows for more accurate and high-resolution measurement and observation of the quantification of the analyte and its distribution (imaging) in tissues and cells. [3] The derivatization reaction uses nitrogen-containing compounds such as amino acids (representative compounds: thyroid hormone, etc.), peptides (representative compounds: oxytocin, etc.), and catecholamines (representative compounds: dopamine, etc.) as substrates. The substrates can also be those contained in biological components (blood, serum, plasma, urine, sweat, cerebrospinal fluid, hair, nails, feces, etc.), and can be detected after appropriate pretreatment. [4] The alkylation or arylation is preferably carried out using an aldehyde or ketone and a reducing agent. [5] The aldehyde or ketone is preferably an aliphatic or aromatic aldehyde having 1 to 12 carbon atoms. It is preferable that the aldehyde or ketone has a substituent such as methyl. [6] The reducing agent is preferably one or more selected from the group consisting of borane, complexes containing borane, and salts of boron hydride compounds, but is not limited to this as long as the reducing agent has the same effect as these. [7] The present invention can provide a derivatization reagent kit for mass spectrometry, which comprises at least an aldehyde or ketone and a reducing agent.

[0023] As will be apparent from the Examples below, by using this technology, in mass spectrometry (preferably LC-MS / MS analysis), the sensitivity was improved by up to 2.5 times after derivatization for amino acids, up to 17 times after derivatization for peptides, and up to 195 times after derivatization for catecholamines, compared to compounds before derivatization. Furthermore, by using this technology, even nitrogen-containing compounds in living organisms such as plasma, serum, and urine were derivatized, and then the sensitivity of the nitrogen-containing compounds was improved in mass spectrometry.

[0024] 2. First embodiment of the present invention (derivatization reagent containing an aldehyde or ketone) This first embodiment can provide a derivatization reagent that includes an aldehyde or a ketone. In another embodiment, the present invention provides <1> A compound represented by the formula (1): RR'CO (1) (R and R' represent a hydrogen atom, an alkyl group, or an aryl group, which may be the same or different), or <2> When R' in the formula (1) is a hydrogen atom, a compound represented by the chemical formula (2): RCHO (2) (R represents a hydrogen atom, an alkyl group, or an aryl group), or <3> A derivatization reagent containing a compound represented by formula (1) (R and R' represent an alkyl group or an aryl group, which may be the same or different), in which both R and R' in formula (1) are not hydrogen atoms, may be provided. The derivatization reagent may be any of a composition, a derivatization composition, a derivatization agent, and a sensitivity enhancer in mass spectrometry. This allows the derivatization of the target compound to be easily performed. Furthermore, the sensitivity of the target compound to be analyzed by derivatization (e.g., detection sensitivity or measurement sensitivity, etc.) can be improved, and for example, the sensitivity in mass spectrometry can be expected to be improved.

[0025] In the first embodiment, it is preferable to use an aldehyde or ketone as the derivatization reagent, and for example, one or more of the exemplified aldehyde or ketone compounds described later can be selected and used. In addition, it is preferable that the aldehyde or ketone is used for N-alkylating or N-arylating a nitrogen-containing compound as the target compound for derivatization. It is preferable that the derivatization reagent is a reagent for mass spectrometry or a reagent for mass spectrometry. During derivatization, it is preferable to use at least an aldehyde or ketone in combination with a reducing agent described later for the target compound.

[0026] By using the derivatization reagent of the first embodiment for the analyte compound, the analyte compound can be derivatized (preferably N-alkylated or N-arylated). By using this N-derivatized analyte compound for measurement or detection, the sensitivity of the measurement or detection of the analyte compound can be improved.

[0027] 2-1. Aldehyde or ketone 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 reagent kit, and can be used as a compound in the production of these reagents or reagent kits.

[0028] Examples of the aldehyde or ketone include, for example, an aliphatic aldehyde or ketone, an alicyclic aldehyde or ketone, an aromatic aldehyde or ketone, and a heterocyclic aldehyde or ketone, and any of these may be used, and one or more selected from these may be used. Among these, an aliphatic aldehyde or ketone and / or an aromatic aldehyde or ketone are preferred. The number of carbon atoms of the aldehyde or ketone is not particularly limited, but from the viewpoint of improving sensitivity, a suitable lower limit is preferably 1, 2 or 3 or more, and a suitable upper limit is preferably 20 or less, more preferably 15 or less, further preferably 12 or less, and more preferably 10 or less. 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 one produced by referring to a known method for producing an aldehyde or ketone.

[0029] Aldehydes or ketones suitable for use in the present invention include compounds 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), and the formula (1) can also be expressed as a compound represented by RC(=O)-R', if necessary. The hydrocarbon groups in R and R' in the formula (1) may be the same or different and may have a substituent or may be unsubstituted, and the hydrocarbon group is more preferably an alkyl group or an aryl group. The alkyl group may be either chain-like (straight-chain or branched-chain) or cyclic. More preferably, R and R' in the formula (1) are the same or different and represent a hydrogen atom, or a substituted or unsubstituted alkyl group or aryl group having 1 or 2 to 11 carbon atoms. In addition, in the compound represented by formula (1), when either R or R' is a hydrogen atom, the other may represent a hydrogen atom or a hydrocarbon group, and when neither R nor R' is a hydrogen atom, R and R' may be the same or different and represent a hydrocarbon group.

[0030] In the formula (1), the hydrocarbon group in R and R' is not particularly limited, but examples thereof include aliphatic hydrocarbon groups, alicyclic hydrocarbon groups, aromatic hydrocarbon groups, etc., and one or more selected from these can be used. The number of carbon atoms in the hydrocarbon group in R is not particularly limited, but the preferred lower limit is preferably 1 or 2 or more, and the preferred upper limit is preferably 20 or less, more preferably 15 or less, even more preferably 12 or less, more preferably 10 or less, and more preferably 9 or 8 or less.

[0031] The aliphatic hydrocarbon group is not particularly limited, and examples thereof include linear or branched aliphatic hydrocarbon groups (linear or branched alkyl groups, alkenyl groups, and alkynyl groups, etc.) such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, and allyl. These may be linear or branched. Among these, linear or branched alkyl groups are preferred. Among these, substituted alkyl groups (e.g., arylalkyl groups) having a substituent (e.g., aryl group) or unsubstituted alkyl groups are preferred. One or more of these can be selected and used. The aryl group may be the same as the aryl group described below, and examples thereof include a phenyl group. The number of carbon atoms in the aliphatic hydrocarbon group is not particularly limited, but is preferably about 1 or about 2 to 20, and more preferably about 1 or about 2 to 10. One or more of these can be selected and used.

[0032] The alicyclic hydrocarbon group is not particularly limited, but examples thereof include monocyclic alicyclic hydrocarbon groups (cycloalkyl groups, cycloalkenyl groups) such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cyclooctyl, cyclodecyl, and cyclododecyl groups; bridged ring hydrocarbon groups, and the like. Representative examples of the bridged ring in the bridged ring hydrocarbon group include an adamantane ring, a norbornane ring, and a norbornene ring. An aromatic ring may be condensed to the alicyclic ring. Among these, a cycloalkyl group (cyclic alkyl group) is preferred. The number of carbon atoms in the alicyclic hydrocarbon group is not particularly limited, but is preferably about 3 to 20 carbon atoms. One or more of these can be selected and used.

[0033] Examples of the aromatic hydrocarbon group include monocyclic aromatic hydrocarbon groups such as phenyl, tolyl, and o-xylyl groups; and aryl groups such as polycyclic aromatic hydrocarbon groups such as naphthyl groups, which may have a substituent such as an alkyl group. A non-aromatic ring may be condensed to the aromatic ring. The number of carbon atoms in the aromatic hydrocarbon group is not particularly limited, but is preferably about 6 to 20. Among these, substituted aryl groups (e.g., alkylaryl groups) having a substituent (preferably an alkyl group) or unsubstituted aryl groups are preferred. One or more of these can be selected and used. The alkyl group may be the same as the alkyl group described above, and examples thereof include a methyl group and an ethyl group.

[0034] The hydrocarbon group may be a substituted or unsubstituted hydrocarbon group. The "substituent" of the hydrocarbon group of the substituent may have various substituents (e.g., a substituent having 1 to 3 carbon atoms, etc.), such as a halogen atom, an oxo group, a hydroxyl group, a substituted oxy group (e.g., an alkoxy group, an aryloxy group, an acyloxy group, etc.), a carboxyl group, a substituted oxycarbonyl group, a substituted or unsubstituted carbamoyl group, a cyano group, a nitro group, a substituted or unsubstituted amino group, a linear or branched alkyl group (e.g., an alkyl group such as a methyl group or an ethyl group), a cyclic alkyl group, an aryl group (e.g., a phenyl group, a naphthyl group, etc.), a heterocyclic group, etc. Among these, the substituent is preferably a hydrocarbon group (unsubstituted or substituted). More specifically, the substituent is preferably, for example, an unsubstituted alkyl group (the alkyl group is linear, branched or cyclic), and an unsubstituted aryl group or an aryl group having an unsubstituted alkyl group, and the number of carbon atoms of these groups may be appropriately selected from the above-mentioned number of carbon atoms. One or more of these may be selected and used.

[0035] Suitable examples of the aliphatic aldehydes used in the present invention include aliphatic saturated aldehydes such as formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde, valeraldehyde (pentanal), isovaleraldehyde, pivalaldehyde, hexanal, heptanal, octanal, nonanal, decanal, dodecanal, tetradecanal, hexadecanal, and octadecanal; aliphatic dialdehydes such as glyoxal and succindialdehyde; and aliphatic unsaturated aldehydes such as acrolein, crotonaldehyde, and pentenal. One or more of these can be selected and used. Suitable examples of the aliphatic ketones include acetone, methyl ethyl ketone, and 3-pentanone.

[0036] Suitable examples of the alicyclic aldehyde used in the present invention include cyclopropanecarboxaldehyde, cyclopentanecarbaldehyde, cyclohexanecarbaldehyde, cyclooctanecarbaldehyde, cyclododecanecarbaldehyde, 5-norbornene-2-carboxaldehyde, etc. One or more of these can be selected and used.

[0037] Suitable examples of the aromatic aldehyde used in the present invention include benzaldehyde, o-, m-, and p-tolualdehyde, salicylaldehyde, cinnamaldehyde, α- and β-naphthaldehyde, phthalaldehyde, terephthalaldehyde, and isophthalaldehyde, etc. One or more of these may be selected and used.

[0038] Suitable examples of the aldehyde used in the present invention include one or more selected from aliphatic aldehydes such as formaldehyde, acetaldehyde, propionaldehyde, butylaldehyde, isobutyraldehyde, valeraldehyde (pentanal), isovaleraldehyde, pivalinaldehyde, hexanal, and heptanal; alicyclic aldehydes such as cyclopropanecarboxaldehyde; and aromatic aldehydes such as benzaldehyde. Among these, aliphatic aldehydes are preferred, and examples thereof include one or more selected from formaldehyde, acetaldehyde, propionaldehyde, butylaldehyde, isobutyraldehyde, valeraldehyde (pentanal), isovaleraldehyde, hexanal, and heptanal. Preferred examples of the ketones used in the present invention include one or more aliphatic ketones such as acetone, methyl ethyl ketone, and 3-pentanone, and among these, acetone is more preferred.

[0039] 2-2. Analytical compounds In the present invention, the target compound to be derivatized is not particularly limited, but is preferably a nitrogen-containing compound. The target compound to be analyte may be a substrate, or may be an object to be derivatized or an object to be subjected to mass spectrometry.

[0040] The nitrogen-containing compound used in the present invention preferably has at least one primary amine (HHN-) in the molecular structure of the nitrogen-containing compound. In addition, it is preferable that the nitrogen-containing compound has at least one aryl group or methylene group in the molecular structure, and the aryl group or methylene group may be either unsubstituted or substituted, for example, an aryl group having an alkyl group. The alkyl group may be either unsubstituted or substituted, and may be chain-like (straight-chain or branched-chain) or cyclic, and the number of carbon atoms of the alkyl group is not particularly limited, but may be, for example, about 2 to 4 carbon atoms. The substituent is not particularly limited, and may be an alkyl group, an iodine atom, a hydroxyl group, a carbonyl group, an amino group, etc. One or more of these may be selected.

[0041] The nitrogen-containing compound is preferably one or more selected from amino acids, peptides, catecholamines, proteins, etc., and may be either synthetic or natural. Examples of the nitrogen-containing compound include samples for clinical testing (e.g., biological components, tissues and cells of animals and plants, etc.), samples for environmental hygiene testing, products (food and beverages, pharmaceuticals, quasi-drugs, cosmetics, chemicals, etc.), etc. More preferred examples include one or more selected from biological components, organic synthetic products, and separated and purified products. Nitrogen-containing compounds contained in biological components that are generally difficult to measure or detect can be derivatized well by using the present technology after appropriate pretreatment, and are also suitable as derivatives for mass spectrometry. For this reason, the present invention is preferably used for measuring or detecting biological components, and more preferably, nitrogen-containing compounds contained in biological components are used as the analysis target compounds.

[0042] Examples of the nitrogen-containing compound include, but are not limited to, amino acids (e.g., triiodothyronine, thyroxine, homocysteine, etc.), peptides (e.g., angiotensin I, oxytocin, glucagon, adrenocorticotropic hormone, insulin, C-peptide, etc.), and catecholamines (e.g., adrenaline, noradrenaline, dopamine, levodopa, etc.), and one or more selected from these can be used.

[0043] In the present invention, the compound to be analyzed is preferably one or more selected from amino acids, peptides, and catecholamines contained in biological components.

[0044] When the target compound is an amino acid (preferably an amino acid present in a biological component), the number of primary amines (HHN-) in the molecular structure is preferably 1 to 2, more preferably 1, and the number of aryl groups in the molecular structure is preferably 0 to 3, more preferably 0 to 2, and even more preferably 2. The preferred molecular weight of the target compound amino acid is preferably 50 to 1000, more preferably 100 to 800. The molecular weight is calculated from the sum of the atomic weights contained in the molecule. The preferred hydrophobicity (ClogP) of the target compound amino acid is preferably -4 to 16, more preferably -2 to 10.

[0045] When the analyte 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, and the preferred lower limit is preferably 1 or more, more preferably 2 or more, and the preferred upper limit is preferably 30 or less, more preferably 20 or less, even more preferably 15 or less, and even more preferably 10 or less. When the analyte compound is a peptide, the number of aryl groups in the molecular structure is not particularly limited, and the preferred lower limit is preferably 1 or more, more preferably 2 or more, and the preferred upper limit is preferably 30 or less, more preferably 20 or less, even more preferably 15 or less, and even more preferably 10 or less. The preferred molecular weight of the analyte compound peptide is preferably 100 or more, more preferably 200 or more, and the preferred upper limit is preferably 10,000 or less, more preferably 9,000 or less, even more preferably 8,000 or less, and even more preferably 6,000 or less. In addition, the preferred hydrophobicity (ClogP) of the peptide of the analyte compound is preferably -4 or more, more preferably -2 or more, as the preferred lower limit, and preferably 10 or less, more preferably 8 or less, even more preferably 6 or less, and even more preferably 4 or less, as the preferred upper limit.

[0046] From the viewpoint of the analyte compounds for which the sensitivity is likely to be improved in this technology, 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, adrenaline, noradrenaline, and dopamine are preferred, and one or more selected from these can be used.

[0047] 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 preferably 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, R and R' in the formula (1) RR'CO are preferably the same or different and are a substituted or unsubstituted alkyl group having 2 to 9 carbon atoms.

[0048] In a more preferred embodiment, when the nitrogen-containing compound (a) is an amino acid or a peptide, R and R' in the formula (1) RR'CO are preferably the same or different and are a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 3 carbon atoms, more preferably a hydrogen atom or a linear alkyl group having 1 to 2 carbon atoms, and even more 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 to be used as a sample is preferably an amino acid and / or a peptide in a biological component.

[0049] In addition, when the nitrogen-containing compound (a) is an amino acid or a peptide, the carbon numbers of R and R' in the formula (1) are preferably 0 to 5, more preferably 4 or less or 3 or less, and even more preferably 0, 1, and 2. When the carbon numbers of R and R' are 0, R and R' are preferably hydrogen atoms, and more preferably, the aldehyde or ketone is an aldehyde or ketone (preferably having 1 to 3 carbon atoms) having a linear C1 to C2 alkyl group or a hydrogen atom, such that the primary or secondary amino group of the analyte compound is a secondary or tertiary compound derivatized to N-methyl, N-ethyl, N-propyl, N-isopropyl, or the like. The analyte compound to be used as a sample is preferably an amino acid and / or peptide in a biological component.

[0050] When 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 number of carbon atoms of the alkyl group is preferably 4 to 8, more preferably 4 to 6, and the suitable alkyl group is an unsubstituted alkyl group, and R' is a hydrogen atom. More preferably, R is one or more selected from a butyl group, an isobutyl group, a pentyl group, a hexyl group, and a phenyl group, and among these, a hexyl group is more preferable. The analyte compound to be used as a sample is preferably a catecholamine in a biological component.

[0051] The origin of the biological component is not particularly limited, but may be any of animals, plants, microorganisms, etc. The biological component is not particularly limited. Animal biological components are preferred, and examples thereof include blood, serum, plasma, urine, sweat, cerebrospinal fluid, hair, nails, feces, etc., and one or more selected from these can be used. The animal is not particularly limited, and examples thereof include one or more selected from mammals (humans, mice, dogs, cats, rabbits, horses, pigs, cows, etc.), birds (chickens, pigeons, etc.), reptiles, amphibians, etc., and from the viewpoint of medical examinations, etc., humans, pet animals (cats, dogs, etc.), sports animals (horses, dogs, rabbits, etc.), and livestock and poultry (pigs, cows, chickens, etc.) are preferred. In addition, components such as biological components collected from living organisms such as animals can be used as the target compound for analysis, and the collected components may be pretreated before derivatization.

[0052] The derivatization reagent used in the present invention can convert a primary amine contained in an analyte compound into a secondary amine or a tertiary amine. More specifically, the derivatization reagent used in the present invention can N-alkylate or N-arylate a primary amine contained in an analyte compound. That is, the ionization efficiency can be increased by secondary or tertiaryizing an amino group of an analyte compound. The parent ion peak intensity or area obtained by mass spectrometry of a secondary or tertiary amine-converted analyte compound is larger than the parent ion peak intensity or area obtained by mass spectrometry of an analyte compound before derivatization, and therefore the sensitivity in mass spectrometry can be further improved.

[0053] The method for separating and purifying the analyte compounds in the sample in the present invention is not particularly limited, and a suitable separation and purification method can be adopted depending on the type of sample. The method for separating and purifying the biological components or their derivatives is not particularly limited, and a known separation and purification method for components such as biological components or their derivatives may be used. Examples of the separation and purification method used in the present invention are not particularly limited, and may be one or a combination of two or more selected from centrifugation, precipitation (acetonitrile precipitation, methanol precipitation, etc.), column chromatography (ion exchange, gel filtration, hydrophobicity, affinity, normal phase, reverse phase, metal chelate, etc.), ultrafiltration, antibody, etc. Note that substances such as ion exchange and metal chelate used as carriers for column chromatography may be used in separation and purification methods other than column chromatography. The separation and purification method is preferably performed before the derivatization reaction, but may be used as a separation and purification method after the derivatization reaction.

[0054] The ion exchanger is not particularly limited, and either an anion exchanger or a cation exchanger can be used. Furthermore, any of a strong anion exchanger, a weak anion exchanger, a strong cation exchanger, and a weak cation exchanger can be used depending on the analyte compound to be collected or captured from a sample such as a biological component. Examples of hydrophobic groups used in hydrophobic column chromatography containing a substance having a hydrophobic group include C4-C20 alkyl groups and phenyl groups. Examples of metal chelates include Cu 2+ , Zn 2+ , Ni 2+ , Ca 2+ , Co 2+ , Mg 2+ Examples of the metal chelates include those capable of binding molecules or ions called ligands to metal ions such as those mentioned above. Examples of the antibodies include antibodies specific to the target compound. One or more of the above can be used.

[0055] For example, a sample such as a biological component is loaded onto an ion exchange column, the column is washed with a solvent, and the compound to be analyzed is eluted, and the eluate containing the target substance is concentrated to dryness. Amino acids, peptides, catecholamines, etc. may be extracted from the sample such as a biological component, and a known extraction method may be used for this purpose. This allows the target substance to be used for derivatization to be separated and purified from the sample such as a biological component, and improves the efficiency of derivatization.

[0056] Alternatively, for example, a target compound to be analyzed contained in a sample such as a biological component may be captured using a carrier appropriately selected from carriers generally used for separation and purification. The carrier may be one or more selected from substances having ionic bonding action, substances having affinity, substances having chemical interactions such as hydrophobic interactions (e.g., hydrophobic groups), and the like.

[0057] Furthermore, the present invention has the advantage that it is possible to screen the aldehydes and ketones used in the present invention for optimal derivatization reagents capable of improving the sensitivity in mass spectrometry, depending on the molecular weight and hydrophobicity of the analyte compound. Moreover, since many of the aldehydes and ketones used in the present invention are commercially available, it is also advantageous to easily find aldehyde and ketone derivatization reagents capable of further improving the sensitivity.

[0058] In the present invention, an optional component may be appropriately blended with the derivatization reagent. For example, one or more selected from a reducing agent, an acid, a base, etc. may be used, but a base may not be used.

[0059] 3. Second embodiment of the present invention (derivatization reagent kit) In addition, the second embodiment may provide a derivatization reagent kit including a reagent containing an aldehyde or ketone, and the reagent including an aldehyde or ketone may be a derivatization reagent including an aldehyde or ketone. In another embodiment, a derivatization reagent kit may be provided that includes a reagent including a compound represented by the above formula (1) (RR'CO (1)) (wherein R and R' may be the same or different) or, when R' in formula (1) is a hydrogen atom, a compound represented by chemical formula (2) (RCHO (2)). The reagent kit of the second embodiment preferably further includes a reducing agent, and the reducing agent may be a derivatization reagent or composition containing the reducing agent. The kit may be a combination or set, and may be a product such as a kit product. The present invention may be, for example, a one-component composition containing an aldehyde, a ketone, and a reducing agent together, or may be a multi-component reagent kit such as a two-component reagent kit in which the aldehyde, the ketone, and the reducing agent are contained in separate containers.

[0060] A more preferred embodiment of the second embodiment is a derivatization reagent kit including a first reagent including an aldehyde and a ketone, and a second reagent including a reducing agent. In addition, it is preferable to use an acid such as acetic acid during derivatization. For example, a third reagent including an acid or an acid may be further included in the derivatization reagent kit. These components may be made into a solution using a solvent. These components may be used in a derivatization method, a derivatization reagent, a derivatization reagent kit, or the like, or may be used as a compound in the manufacture of these reagents or reagent kits. The reagent kit may further include a fourth derivatization reagent including a base (such as an alkali metal or an alkaline earth metal, such as ammonia or hydroxide) in addition to the aldehyde, ketone, reducing agent, and acid, but this embodiment has the advantage that a base reagent for derivatization of the analyte does not need to be added to the derivatization reaction. The reaction of the present embodiment has an advantage in that it provides a derivatization method capable of improving the sensitivity of nitrogen-containing compounds by using, as a base, a substance that exhibits the properties of the nitrogen atom of the substrate (analyte), that is, an amino acid, a peptide, or a catecholamine, without using a base reagent.

[0061] The aldehyde, ketone and derivatization reagent containing the same used in the second embodiment can be appropriately described in the above description of "aldehyde, ketone". A preferred aldehyde and ketone are compounds represented by the formula (1) (RR'CO), and more preferably, R and R' in the formula (1) are the same or different and are a hydrogen atom or a substituted or unsubstituted hydrocarbon group (preferably an alkyl group or an aryl group). The R and R' are not particularly limited, and are preferably a hydrogen atom and a group having 1 to 18 carbon atoms, more preferably a hydrogen atom and a group having 1 to 11 carbon atoms, and even more preferably a hydrogen atom and a group having 1 to 9 carbon atoms. The alkyl group is preferably a chain (straight-chain or branched-chain) 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 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 a substituted or unsubstituted alkyl group having 2 to 9 carbon atoms.

[0062] 3-1.Reducing agent The reducing agent used in the present invention is not particularly limited. The reducing agent may be in either liquid or solid form, and may be made into a solution with a solvent or the like when used. Examples of the reducing agent include boron compounds (borane, complexes containing borane, boron hydride compound salts, etc.); aluminum hydrides (lithium aluminum hydride, diisobutylaluminum hydride, sodium bis(2-methoxyethoxy)aluminum hydride, etc.); tributyltin hydride; and hydrogen gas. One or more selected from these may be used. Among these, the boron compounds are preferred from the viewpoint of ease of removal after derivatization.

[0063] The boron compound used in the present invention is not particularly limited as long as it has a reducing action, and examples thereof include borane (borane (BH3), diborane (B2H6), etc.), complexes containing borane (hereinafter also referred to as "borane complexes"), and salts of boron hydride compounds, and it is preferable to use one or more selected from these. The reducing agent used in the present invention may be a commercially available product or may be obtained by referring to a known manufacturing method. The advantage of using a boron compound is that the derivatization of the target compound by the derivatization reagent can be easily carried out even at a reaction temperature of about room temperature, and the reducing agent component after the derivatization reaction can be easily removed by vacuum concentration using an evaporator or the like.

[0064] Examples of the borane complex include borane-2-methylpyridine complex (also known as picolylborane), borane-dimethylsulfide (DMS) complex, and borane-amine complex (e.g., ammonia borane (or borazane), borane trimethylamine complex, borane N,N-diisopropylethylamine complex, and borane tert-butylamine complex), and among these, borane-2-methylpyridine complex is preferred.

[0065] Examples of the salts of borohydride compounds include 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 triethylborohydrides (lithium, etc.), alkali metal tri(sec-butyl)borohydrides (lithium, etc.), and alkaline earth metal tri(sec-butyl)borohydrides (potassium, etc.), and among these, alkali metal borohydrides are preferred.

[0066] 3-2. Acid The acid used in the present invention is not particularly limited. The acid may be in either liquid or solid form, and may be made into a solution with a solvent or the like when used. The acid is preferably a volatile acid from the viewpoint of easy removal after the reaction. Examples of the acid include 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 these can be used. Among these, organic acids are preferred, and the acid used in the present invention is preferably an acid that is easily removed by an evaporator or the like (e.g., acetic acid, etc.).

[0067] 3-3.Solvent The solvent used in the present invention is not particularly limited, but is preferably a volatile solvent having a boiling point of 100° C. or less, and for example, one or more solvents selected from water and lower alcohols (C1 to C3 such as methanol, ethanol, normal propanol, isopropanol, etc.) can be used. The solvent can be contained in or used in the derivatization reagent or derivatization reagent kit. The solvent may be used as a solution, for example, a solution containing an aldehyde or ketone, or a solution containing a reducing agent. In addition, by using a solvent that can be concentrated to dryness using an evaporator, etc., the borane-based reducing agent can be removed together.

[0068] 3-4. Amount of each component used in the present invention 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).

[0069] 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).

[0070] 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).

[0071] 3-5. Third embodiment of the present invention (method of using each reagent included in the reagent kit, and method of producing a derivatized nitrogen-containing compound)

[0072] The third embodiment can provide a method for derivatizing a nitrogen-containing compound or a method for producing a derivatized nitrogen-containing compound, which comprises reacting the nitrogen-containing compound with the derivatization reagent or the derivatization reagent kit. By derivatizing the nitrogen-containing compound with an aldehyde or a ketone, the signal strength or detection sensitivity to a detection instrument can be improved. An advantage of the present invention is that even if the amount of the nitrogen-containing compound in a sample is small, the analyte compound in the sample can be detected or measured more accurately. Furthermore, 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 be carried out as a pretreatment, which can remove impurities contained in the sample (preferably a biological component), thereby improving the sensitivity of detection or measurement of the compound to be analyzed. In addition, in the third embodiment, it is preferable to separate and purify the derivatized nitrogen-containing compound after the reaction, which makes it possible to remove the derivatization reagent that is no longer needed after the reaction, thereby improving the sensitivity of detection or measurement of the target compound. In the third embodiment, it is preferable to subject the derivatized nitrogen-containing compound to mass spectrometry.

[0073] The nitrogen-containing compound to be analyzed is preferably the amino acids, peptides, and catecholamines contained in the biological components described above, and is preferably one or more selected from amino acids such as triiodothyronine, thyroxine, and homocysteine; peptides such as angiotensin I, oxytocin, glucagon, insulin, and C-peptide; and catecholamines such as adrenaline, noradrenaline, and dopamine.

[0074] It is preferable that the nitrogen-containing compound is derivatized by reacting the nitrogen-containing compound with the derivatization reagent or the derivatization reagent kit. In a more preferred embodiment, 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 the aldehyde or ketone, the reducing agent and the acid are added to the sample containing the nitrogen-containing compound and mixed. Alternatively, the three components of the aldehyde or ketone, the reducing agent and the acid may be added to the sample containing the nitrogen-containing compound and mixed, and then a base may be added. These samples and each component may be in the form of a solution, and examples of such solutions include a derivatization reagent kit consisting of a first solution containing an aldehyde or ketone, a second solution containing a reducing agent, and a third solution containing an acid; and a derivatization reagent kit having a first container containing an aldehyde or ketone, a second container containing a reducing agent, and a third container containing an acid. Alternatively, the nitrogen-containing compound may be derivatized using each component, each reagent, or each solution in a derivatization device configured to be able to carry out the method of the present invention, such as the method of use or the method of production of the present embodiment. It is preferable not to use a base reagent during derivatization.

[0075] The reaction conditions of the derivatization are not particularly limited. The temperature conditions of the derivatization are not particularly limited, and are preferably 5 to 50°C, more preferably 10 to 40°C. The advantage of the derivatization of the present invention is that the derivatization reaction proceeds without high temperatures, so that temperature control or equipment is not required, making it easy to handle for a wide range of users. In this case, room temperature (for example, 10 to 40°C) is preferable, and 20 to 30°C is more preferable. The pressure conditions of the derivatization are not particularly limited, and the derivatization reaction can proceed at normal pressure (standard atmospheric pressure 1 atm), which is also an advantage of the present technology. It is also an advantage of the present technology that the target compound can be derivatized at room temperature and normal pressure. The reaction time of the derivatization is not particularly limited, and the preferred lower limit is preferably 0.1 hours or more, more preferably 0.5 hours or 1 hour or more, and the preferred upper limit is preferably 24 hours or less, more preferably 18 hours or less. It is preferable to add an acid (for example, acetic acid, etc.) during the derivatization reaction. In addition, a solvent for dissolving or suspending a compound or the like may be used during the derivatization reaction. As the solvent, an aqueous solvent (water, a lower alcohol, etc.), an organic solvent, etc. can be appropriately used depending on the compound or derivatization reagent used.

[0076] The derivatized nitride-containing compound obtained in the third embodiment is preferably a compound in which an amino group (-NHH) of the nitrogen-containing compound is bonded to an alkyl group or aryl group in the R or R' portion contained in the aldehyde or ketone that is the derivatization reagent, and the compound is converted to a secondary amine or a tertiary amine.

[0077] 4. Fourth embodiment of the present invention (method of mass spectrometry and method of improving sensitivity in mass spectrometry)

[0078] As described above, the fourth embodiment can provide a mass spectrometry method including a derivatization step of derivatizing an analyte compound using a derivatization aldehyde or ketone, a derivatization reagent, or a derivatization reagent kit, etc. 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 a sample by a separation and purification method, etc., prior to the derivatization step. The target compound is preferably the nitrogen-containing compound described above. The derivatization can improve the measurement sensitivity or detection sensitivity of the target compound in mass spectrometry.

[0079] The mass spectrometry method may further include, after the derivatization step, a mass spectrometry step including ionizing the compound to be analyzed by mass spectrometry. The ionization may be performed by, for example, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), matrix-assisted laser desorption ionization (MALDI), or the like for more efficient ionization, or may be performed by another ionization method. By ionizing the analyte compound derivatized according to the fourth embodiment by these ionization methods, the measurement sensitivity or detection sensitivity of the analyte compound can be improved.

[0080] The mass analysis step may be performed, for example, by a liquid chromatography tandem mass spectrometer (LC-MS / MS) or a matrix-assisted laser desorption / ionization mass spectrometer (MALDI-MS), but the device performing the mass analysis step is not limited thereto. The specific procedure in the mass spectrometry step can be appropriately set depending on, for example, the ionization method employed 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 chromatograph, an HPLC device, etc. can be used, and one or more selected from these can be used.

[0081] According to the fourth embodiment, an N-derivatized analyte compound and its fragment ions (characteristic fragment ions) can be detected or measured in mass spectrometry, and based on the results, it can be determined whether or not the analyte compound is contained in a sample, and measurements such as detection or quantification of the analyte compound in the sample can be performed.

[0082] In addition, in the fourth embodiment, when mass spectrometry is used to detect or measure an analyte compound contained in a biological component in a sample, a compound identical to the analyte compound may be used as a standard substance for comparison with the standard substance. In other words, the analyte compound in the biological component can be detected or measured by comparing a derivatized product of the analyte compound in the biological component with a derivatized product of the standard substance.

[0083] In addition, the methods related to the present embodiment such as the fourth embodiment (for example, a derivatization method or a mass spectrometry method, a measurement method or a detection method for determining an improvement in sensitivity, etc.) may be controlled by a control unit having a CPU or the like. In addition, the methods of the fourth embodiment and the like may use one or more known methods selected from, for example, a standard addition method, an external standard method (absolute standard method), an internal standard method, an area percentage method, a modified area percentage method, etc., during detection or measurement. In addition, a CPU provided in a mass spectrometer or a control unit (computer, server, cloud, etc.) of the device may be used to compare the data of the derivatized product of the analyte compound stored in the memory unit (molecular weight fragment ions, characteristic fragment ions, fragment ion patterns, peak heights and areas, retention times, etc.) with the biological components in the sample, and perform identification, detection, or measurement such as quantification.

[0084] 5. In addition, the present technology may also adopt the following other aspects, configurations, or technical features as appropriate. [1] A derivatization reagent containing 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. [2] The derivatization reagent according to [1] above, wherein the aldehyde or the ketone is a compound represented by the following formula (1): RR'CO (1) [In the formula (1), R and R' may be the same or different and each represent a hydrogen atom or a substituted or unsubstituted hydrocarbon group.] [3] The derivatizing reagent according to [2] above, wherein the hydrocarbon groups of R and R' in formula (1) are the same or different and each is an alkyl group or an aryl group. [4] The derivatization reagent according to any one of [1] to [3] above, which is used for N-alkylating or N-arylating an amino group of a nitrogen-containing compound. [5] The derivatization reagent according to [4] above, wherein the nitrogen-containing compound is one or more selected from amino acids, peptides, and catecholamines contained in a sample (preferably a biological component). [6] The derivatization reagent is used for N-alkylating or N-arylating an amino group of a nitrogen-containing compound, (a) When the nitrogen-containing compound is an amino acid or a peptide in a sample (preferably a biological component), R and R' in the formula (1) are the same or different and each is a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 8 carbon atoms, or (b) The derivatization reagent according to any one of the above [1] to [5], wherein R and R' in the above formula (1) may be the same or different and each represents a substituted or unsubstituted alkyl group having 2 to 9 carbon atoms, when the nitrogen-containing compound is a catecholamine in a sample (preferably a biological component). [7] The derivatization reagent according to any one of the above [1] to [6], wherein a reducing agent is used during the derivatization. More preferably, an acid reagent is further used during the derivatization (for example, the respective reagents are added simultaneously or at different times), and / or a base reagent is not used during the derivatization.

[0085] [8] A reducing agent, in which the derivatization reagent according to any one of [1] to [6] is used during derivatization. More preferably, an acid reagent is further used during the derivatization, and / or no base reagent is used during the derivatization. [9] A derivatization reagent kit for mass spectrometry, comprising the derivatization reagent (first reagent) according to any one of [1] to [7] above and a reducing agent (second reagent), and it is preferable that the reagent kit further comprises an acid (third reagent). The kit may be a combination or a set. Moreover, each reagent in the reagent kit is preferably contained in an openable and closable airtight container (made of glass, plastic, etc.; screw cap type, etc.).

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

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

[10] above, further comprising an acid reagent.

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

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

[0086]

[13] A method for producing a derivatized compound, a method for derivatizing a target compound, or a method for improving the sensitivity of mass spectrometry of a target compound, which uses, for a target compound, the derivatization reagent according to any one of [1] to [7] above, or the derivatization reagent kit according to any one of [8] to

[12] above.

[14] A method for producing a derivatized compound, a method for derivatizing a target compound, or a method for improving the sensitivity of mass spectrometry of a target compound, the method comprising a derivatization step of using, on a target compound, the derivatization reagent according to any one of [1] to [7] above, or the derivatization reagent kit according to any one of [8] to

[12] above.

[15] The method according to

[13] or

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

[16] The method according to any one of

[13] to

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

[17] The method according to any one of

[13] to

[16] above, further comprising an analysis step of analyzing the derivatized compound by instrumental analysis.

[18] A method for derivatizing an amino group of a nitrogen-containing compound contained in a sample into a secondary amine or a tertiary amine by N-alkylating or N-arylating the amino group with a derivatization reagent containing an aldehyde or a ketone, the nitrogen-containing compound is one or more selected from amino acids, peptides, and catecholamines contained in biological components, The aldehyde or ketone is represented by the following formula (1); RR'CO (1) (wherein, in the formula (1), R and R' represent a hydrogen atom, or a substituted or unsubstituted alkyl group or aryl group having 1 to 11 carbon atoms); The method according to any one of the above

[13] to

[17] .

[0087]

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

[20] An aldehyde or ketone, or use thereof, for producing or used in producing a derivatization reagent or a derivatization reagent kit. Furthermore, it is preferable to use a reducing agent and / or an acid reagent, and it is preferable not to use a base reagent.

[0088]

[21] An aldehyde or ketone for derivatization, or a mixture for derivatization, comprising an aldehyde or ketone and a reducing agent, more preferably comprising an acidic reagent and / or not comprising a basic reagent.

[22] An aldehyde or ketone for use in a combination product for derivatization, or an aldehyde or ketone and a reducing agent (more preferably an aldehyde or ketone, a reducing agent, and an acid reagent) for use in a kit product for derivatization. Even more preferably, the kit product for derivatization does not use a base reagent. The kit product may be a combination product or a set product.

[23] The aldehyde or ketone according to

[22] or the combination product according to

[20] , wherein the compound to be analyzed for derivatization is a nitrogen-containing compound (preferably a nitrogen-containing compound in a biological component).

[0089]

[24] A method for derivatizing a nitrogen-containing compound, or a method for mass spectrometry of a nitrogen-containing compound, using an aldehyde or a ketone selected as a derivatization reagent for improving the sensitivity of mass spectrometry by a method for screening derivatization reagents for improving the sensitivity of nitrogen-containing compounds in mass spectrometry.

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

[12] above.

[0090]

[26] A method for screening a derivatization reagent for improving the sensitivity of nitrogen-containing compounds in mass spectrometry, comprising: The screening method includes: A step of mixing a reagent containing an aldehyde or ketone with a nitrogen-containing compound to derivatize the compound; and 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 the derivatization is larger than the characteristic signal of the mass spectrometry of the nitrogen-containing compound before the derivatization. More preferably, a reducing agent and / or an acid reagent is used as the reagent in the derivatization step, and / or a base reagent is not used.

[0091]

[27] The screening method according to

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

[28] A reagent kit for derivatization, comprising an aldehyde or ketone determined to be a derivatization reagent for improving sensitivity in

[26] or

[27] above, or a reagent containing the same. EXAMPLES

[0092] The present technology will be described in more detail below based on examples, etc. Note that the examples, etc. described below are examples of representative examples, etc. of the present technology, and are not intended to narrow the scope of the present technology.

[0093] [Test Example 1: Preparation of secondary amine or tertiary amine derivative compounds using a derivatization reagent (aldehyde or ketone reagent) on a sample (analyte compound)] 1. Reagents The aldehyde or ketone groups shown in Table 1 were used for derivatization.

[0094] [Table 1]

[0095] 2. Derivatization [Standard (analyte compound)] Amino acids (triiodothyronine (T3) molecular weight 651.0, hydrophobicity (ClogP) 2.63, thyroxine (T4) molecular weight 776.9, hydrophobicity (ClogP) 3.51, homocysteine ​​(HCys) molecular weight 135.2, hydrophobicity (ClogP) -2.07) 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) Catecholamines (Adrenaline (Ad) molecular weight 183.2, hydrophobicity (ClogP) -0.68, Noradrenaline (NA) 169.2, hydrophobicity (ClogP) -0.99, Dopamine (DP) molecular weight 153.2, hydrophobicity (ClogP) 0.17)

[0096] 3.Analysis conditions

[0097] [LC analysis conditions] Equipment: Shimadzu LC-20A Analytical column: YMC Triart C18 (2.1x50mm) Elution conditions: Flow rate 0.4 mL / min. Solvent A: 0.1% formic acid-water, Solvent B: methanol Time (min) [Solvent B%]: 0 min [0%], 3 min [0%], 3.5 min [100%], 4 min [0%], 5 min [0%]

[0098] [MS / MS analysis conditions] Equipment: Shimadzu LCMS8040 triple quadrupole mass spectrometer Ionization conditions: ESI positive ion mode The collision dissociation conditions (CE) and the measurement results (SRM chromatogram) are shown in the table below.

[0099] [MS analysis] Equipment: Shimadzu LCMS8040 triple quadrupole mass spectrometer Ionization conditions: ESI positive ion mode Measurements were performed in scan mode, and mass spectra were displayed. The horizontal axis of the spectrum indicates m / Z.

[0100] [ 1 H-NMR analysis] Equipment JEOL JNM-ECA500 nuclear magnetic resonance spectrometer Using standard samples (0.1 mg to 1.0 mg), after derivatization, spectra were obtained by 1024 accumulations (HCys was accumulated 16 times) at room temperature (22°C). The horizontal axis of the spectrum is ppm.

[0101] [Derivatization] To the sample (1 pg-100 ng), 25 μL of an aldehyde or ketone aqueous solution, 25 μL of a picolylborane aqueous solution, and 5 μL of acetic acid were added, and the mixture was left to stand at room temperature (20-30°C) for 30 minutes to carry out the derivatization reaction. After the derivatization, the aldehyde or ketone, acetic acid, and boron were removed by concentrating and drying using an evaporator. After concentrating and drying using an evaporator, the mixture was dissolved in a redissolution solvent (30% acetonitrile aqueous solution) and analyzed by LC-MS / MS.

[0102] 4. Results of derivatization of amino acid preparations Aldehydes or ketones were used to prepare secondary or tertiary amine derivatives of samples (analyte compounds: amino acids present in biological components). Specifically, formaldehyde was used for triiodothyronine (T3) and thyroxine (T4), respectively. Also, benzaldehyde was used for the standard (analyte compound): thyroxine (T4). In this way, a secondary amine or tertiary amine derivative compound was obtained, respectively. Furthermore, acetone was used for homocysteine ​​(HCys). In this way, a secondary amine derivative compound was obtained. These derivative compounds were, 1By H-NMR analysis and MS analysis, they were identified as T3-Me2 (N,N-dimethyltriiodothyronine), T4-Me2 (N,N-dimethylthyroxine), T4-Bn2 (N,N-dibenzylthyroxine), and HCys-iPr (N-isopropylhomocysteine), respectively (Figures 1 to 8, etc.). This confirmed that amino acids (especially those present in biological components) can be derivatized with aldehydes or ketones with carbon numbers of 1 to 8.

[0103] Furthermore, the SRM parameters of the standard (without derivatization) and the compounds derivatized with aldehydes or ketones are shown in Tables 2 to 4 (SRM chromatograms (10 ng) are not shown). Table 5 shows the results of the derivatized compounds with high relative sensitivity improvement ratios and extremely improved detection or measurement sensitivity. 1 The results of the H-NMR analysis and MS analysis are shown in Figures 4 to 8. Thus, the sensitivity of the derivatized compounds (especially when aldehydes or ketones having 1 to 8 carbon atoms are used) obtained by using aldehydes or ketones for amino acids (especially amino acids present in biological components) was higher than that of the authentic samples. This shows that the detection sensitivity was improved by using aldehydes or ketones as derivatization reagents.

[0104] [Table 2]

[0105] [Table 3]

[0106] [Table 4]

[0107] [Table 5]

[0108] 5. Results of derivatization of peptide preparations Aldehydes or ketones were used to obtain secondary or tertiary amine derivatives of samples (analyte compounds: peptides present in biological components). Specifically, formaldehyde, acetaldehyde, or acetone was used for angiotensin I (AI). Formaldehyde, acetaldehyde, propionaldehyde, or acetone was used for oxytocin (OXT). Formaldehyde, acetaldehyde, propionaldehyde, or acetone was used for glucagon (GN). Formaldehyde or acetaldehyde was used for insulin (INS). As a result, secondary amine or tertiary amine derivative compounds were obtained. The derivative compounds of AI were: 1 H-NMR and MS analyses identified them as AI-Me2 (N,N-dimethylangiotensin I), AI-Et2 (N,N-diethylangiotensin I), and AI-iPr (N-isopropylangiotensin I), respectively (Figures 9 to 11). The derivative compounds of OXT are 1 By H-NMR analysis and MS analysis, the compounds were identified as OXT-Me2 (N,N-dimethyloxytocin), OXT-Et2 (N,N-diethyloxytocin), OXT-Pr2 (N,N-dipropyloxytocin), and OXT-iPr (N-isopropyloxytocin) (Figures 12 to 15). The derivative compounds of GN were identified as GN-Me4 (N,N,N',N'-tetramethylglucagon), GN-Et2 (N,N'-diethylglucagon), GN-Pr2 (N,N'-dipropylglucagon), and GN-iPr (N-isopropylglucagon) (Figures 16 to 19). The derivative compounds of INS were identified as 1 H-NMR and MS analyses identified the insulin as INS-Me6 (N,N,N',N',N'',N''-hexamethylinsulin) and INS-Et5 (N,N,N',N',N''-pentaethylinsulin). This confirmed that peptides (especially those present in biological components) can be produced using aldehydes with 1 to 8 carbon atoms (Figures 20 to 21).

[0109] Furthermore, the SRM parameters of the standard (without derivatization) and the compounds derivatized with aldehydes or ketones are shown in Tables 6 to 11 (SRM chromatograms (10 ng) are not shown). Table 12 shows the results of the derivatized compounds with high relative sensitivity improvement ratios, and the identification results of these are shown in Figures 9 to 21. Thus, the sensitivity of the derivatized compounds (especially when aldehydes or ketones having 1 to 8 carbon atoms were used) obtained by using aldehydes or ketones on peptides (especially peptides present in biological components) was higher than that of the authentic samples. This shows that the detection sensitivity was improved by using aldehydes or ketones as derivatization reagents.

[0110] [Table 6]

[0111] [Table 7]

[0112] [Table 8]

[0113] [Table 9]

[0114] [Table 10]

[0115] [Table 11]

[0116] [Table 12]

[0117] 6. Results of derivatization of catecholamine samples A secondary or tertiary amine derivatized compound was obtained from the standard (analyte compound: catecholamine present in a biological component) using an aldehyde, and the SRM parameters of the standard (without derivatization) and the derivatized compound with an aldehyde are shown in Tables 13 to 15 (SRM chromatogram (10 ng) is 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. 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. Dopamine preparations yielded DP-Me2, DP-Et2, DP-Pr2, DP-CpM, DP-Bu2, DP-IB2, DP-V2, DP-IV, DP-Hx, DP-Hp2, and DP-Bn2.

[0118] Table 16 shows the results of the derivatized compounds with a high relative sensitivity improvement ratio. Thus, the sensitivity of the derivatized compounds (especially when an aldehyde having 3 to 9 carbon atoms was used) obtained by using an aldehyde for catecholamines (especially catecholamines present in biological components) was higher than that of the standard sample. This shows that the detection sensitivity was improved by using an aldehyde as a derivatization reagent.

[0119] [Table 13]

[0120] [Table 14]

[0121] [Table 15]

[0122] [Table 16]

[0123] [Test Example 2: Detection of oxytocin in the presence of plasma extract]

[0124] 1. Reagents 5% aldehyde aqueous solution (propionaldehyde, butyraldehyde, valeraldehyde) 3mg / mL Borane-2-picoline complex-isopropanol solution (prepared fresh) Acetic acid

[0125] 2. Preparation of Plasma Extracts Weak cation exchange column (Waters, Oasis (R) WCX 3cc, Cartridge 60mg, 30μm) was washed with methanol (2mL) and equilibrated with water (2mL). 4% phosphate buffer (300μL) and pooled plasma (300μL) were mixed and applied to the column. The column was washed with 5% ammonia water (2mL) and 75% acetonitrile solution (2mL) in sequence, and eluted with 1% formic acid 75% acetonitrile solution (2mL). The eluate was concentrated using a centrifugal evaporator to obtain a plasma extract.

[0126] 3. Derivatization 0, 1, and 10 pg of oxytocin standard was added to the plasma extract, 25 μL of aldehyde aqueous solution, 25 μL of picolylborane solution, and 5 μL of acetic acid were added, and the mixture was left to stand at room temperature for 30 minutes. The aldehyde, acetic acid, boron, etc. after derivatization were removed by concentrating and drying using an evaporator. After concentrating and drying using an evaporator, the mixture was dissolved in a redissolving solvent (30% acetonitrile solution) and analyzed by LC-MS / MS.

[0127] 4.LC analysis conditions Equipment: Shimadzu LC-20A Analytical column YMC Triart C18 2.1x50 mm Elution conditions: Flow rate 0.4 mL / min Solvent A: 0.1% formic acid-water B: methanol Time (min) [Solvent B%]: 0 min [0%], 3 min [0%], 3.5 min [100%], 4 min [0%], 5 min [0%]

[0128] 5.MS / MS analysis conditions Equipment: Shimadzu LCMS8040 triple quadrupole mass spectrometer Ionization conditions: ESI positive ion mode The collision dissociation conditions (CE) and the measurement results (SRM chromatogram) are shown in the table below.

[0129] 6.Measurement results The effect of improving sensitivity by N-alkylation is shown in Tables 17 and 18. The dipropylated form (OXT-Pr2) showed a 3.7-fold increase in sensitivity, the dibutylated form (OXT-Bu2) showed a 2.4-fold increase, and the divalerated form (OXT-V2) showed a 1.7-fold increase in sensitivity. Thus, N-alkylation improved the detection sensitivity of peptides in serum and plasma.

[0130] [Table 17]

[0131] [Table 18]

[0132] [Test Example 3: Optimization of derivatization reaction] The catecholamine derivatization reaction using the standard sample was optimized (aldehyde reagent concentration, with acetic acid). 1. Reagents 10% heptanal-80% ethanol solution 3mg / mL Borane-2-picoline complex-isopropanol solution (prepared fresh) Acetic acid

[0133] 2. Derivatization Catecholamine (adrenaline, noradrenaline, dopamine) samples (1 ng) were mixed with 25 μL of aldehyde aqueous solution, 25 μL of picolylborane solution, and 5 μL of acetic acid, and left to stand at room temperature for 30 minutes. The aldehyde, acetic acid, and boron after derivatization were removed by concentrating and drying using an evaporator. After concentrating and drying, the mixture was dissolved in a redissolution solvent (30% acetonitrile solution) and analyzed by LC-MS / MS.

[0134] 3.Analysis conditions The LC analysis and MS / MS analysis were performed under the same analytical conditions as those of the LC analysis and MS / MS analysis in Test Example 1.

[0135] 4. Results of the study The effect of improving sensitivity by N-alkylation and arylation is shown in Tables 19 and 20. When the analyte was a catecholamine, the sensitivity was improved when heptanal was used as the derivatization reagent, and thus the detection sensitivity of catecholamines was improved by N-alkylation.

[0136] [Table 19]

[0137] [Table 20]

[0138] [Test Example 4: Detection of oxytocin in plasma]

[0139] 1. Reagents 20% formaldehyde solution 3 mg / mL Borane-2-picoline complex-isopropanol solution (prepared fresh) Acetic acid

[0140] 2. Extraction of oxytocin from plasma 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). Pooled plasma (300μL) was added to oxytocin preparations (0, 20, 200ng), and the diluted plasma solution was further diluted with water (700μL) and applied to the column. The column was washed sequentially with 1% formic acid aqueous solution (1mL) and 1% formic acid-methanol (1mL), and eluted with 5% ammonia water (1mL). The eluate was concentrated using a centrifugal evaporator to obtain an oxytocin extract.

[0141] 3. Derivatization 50 μL of formaldehyde solution, 50 μL of picolylborane solution, and 10 μL of acetic acid were added to the oxytocin extract and left to stand at room temperature for 30 minutes. Aldehydes, acetic acid, boron, etc. after derivatization were removed by concentrating and drying using an evaporator. After concentrating and drying using an evaporator, the mixture was dissolved in a redissolution solvent (30% acetonitrile solution) and analyzed by LC-MS / MS.

[0142] 4.LC analysis conditions Equipment: Shimadzu LC-30A Analytical column YMC Triart C18 2.1x50 mm Elution conditions: Flow rate 0.4 mL / min Solvent A: 0.1% formic acid-water B: methanol Time (min) [Solvent B%]: 0 min [0%], 3 min [0%], 3.5 min [100%], 4 min [0%], 5 min [0%]

[0143] 5.MS / MS analysis conditions Equipment: Shimadzu LCMS8060 triple quadrupole mass spectrometer Ionization conditions: ESI positive ion mode The collision dissociation conditions (CE) and the measurement results (SRM chromatogram) are shown in Table 21 below.

[0144] 6.Measurement results The effect of improving sensitivity by N-alkylation (dimethylation) is shown in Tables 21 and 22. The dimethylated form (OXT-Me2) showed a 2.8-fold improvement in sensitivity (when 1 ng of OXT was added). Therefore, N-alkylation improved the detection sensitivity of peptides in plasma.

[0145] [Table 21]

[0146] [Table 22]

[0147] [Test Example 5: Detection of catecholamines in urine and serum]

[0148] 1. Reagents 10% valeraldehyde 50% ethanol solution 3mg / mL Borane-2-picoline complex-isopropanol solution (prepared fresh) Acetic acid

[0149] 2. Extraction of catecholamines from urine and serum 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 (adrenaline, noradrenaline, dopamine) preparations (0 or 200ng) were added to the sample (1mL water, 1mL urine, or a solution of 300μL pooled serum diluted with 700μL water) and developed on the column. The column was washed sequentially with 1% formic acid aqueous solution (1mL) and 1% formic acid-methanol (1mL), and eluted with 5% ammonia water (1mL). The eluate was concentrated using a centrifugal evaporator to obtain the catecholamine extract.

[0150] 3. Derivatization 50 μL of valeraldehyde aqueous solution, 50 μL of picolylborane solution, and 10 μL of acetic acid were added to the oxytocin extract, and the mixture was left to stand at room temperature for 30 minutes. The aldehyde, acetic acid, and boron after derivatization were removed by concentrating and drying using an evaporator. After concentrating and drying, the mixture was dissolved in a redissolution solvent (30% acetonitrile solution) and analyzed by LC-MS / MS.

[0151] 4.LC analysis conditions Equipment: Shimadzu LC-20A Analytical column OSAKA SODA CAPCELL CORE C18 2.1x75 mm Elution conditions: Flow rate 0.4 mL / min Solvent A: 0.1% formic acid-water B: methanol Time (min) [Solvent B%]: 0 min [0%], 0.5 min [0%], 2 min [100%], 3 min [100%], 3.5 min [0%]. 5 min [0%]

[0152] 5.MS / MS analysis conditions Equipment: Shimadzu LCMS8040 triple quadrupole mass spectrometer Ionization conditions: ESI positive ion mode The collision dissociation conditions (CE) and the measurement results (SRM chromatogram) are shown in Table 23 below.

[0153] 6.Measurement results The effect of improving sensitivity by N-alkylation (barrelization) is shown in Tables 23 to 26. The detection sensitivity was improved by derivatization in both urine and serum.

[0154] [Table 23]

[0155] [Table 24]

[0156] [Table 25]

[0157] [Table 26]

[0158] In this specification, for convenience, the description may be made by adding numbers or letters such as "first, second, third...", "A, B, C...", "primary, secondary, tertiary...", etc., but this does not limit the interpretation of the present invention in a narrow sense, such as in terms of order, and the order may be changed arbitrarily. In addition, in this specification, for example, "to (do)" such as "to (derivatize)" may be a method, process, means, or step, and these terms may be appropriately replaced with each other, for example, "step" may be "to (do)", method, process, or means, etc., "process" may be "to (do)", method, step, or means, etc., "means" may be "to (do)", method, process, or step, mechanism, system, device, or part, and "part" may be a mechanism, means, device, or system, or a mechanism, means, or device for providing them. A combination product may be a combination product or a combination.

Claims

1. A derivatization reagent comprising a compound represented by the following formula (1): R R'CO (1) [In the 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 to 11 carbon atoms.]

2. The derivatizing reagent according to claim 1, which is used for N-alkylating or N-arylating an 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 the group consisting of amino acids, peptides, and catecholamines contained in biological components.

4. The derivatization reagent is used for N-alkylating or N-arylating an amino group of a 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 each is 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) may be the same or different and each represents a substituted or unsubstituted alkyl group having 2 to 9 carbon atoms; The derivatization reagent according to claim 1 .

5. The derivatization reagent according to claim 1 or 2, which is used in mass spectrometry.

6. A derivatization reagent kit comprising the derivatization reagent according to any one of claims 1 to 4 and a reducing agent.

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

8. A method for improving the sensitivity of a nitrogen-containing compound in mass spectrometry, comprising: carrying out derivatization by N-alkylating or N-arylating an amino group of the nitrogen-containing compound to a secondary amine or a tertiary amine using the derivatization reagent kit according to claim 6.

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