JP6134164B2 - Mass spectrometry using matrix additives - Google Patents

Description

  The present invention relates to mass spectrometry that can be applied in the medical and drug discovery fields, and in particular to MALDI-MS (Matrix Assisted Laser Desorption / Ionization Mass Spectrometry) applications. More particularly, the present invention relates to mass spectrometry using a specific compound as a matrix additive, and a matrix additive for mass spectrometry.

  In MALDI (Matrix Assisted Laser Desorption / Ionization) mass spectrometry, conditions for realizing efficient ionization of molecules to be measured have been searched. For example, in JP-A-2005-326391 (Patent Document 1), a hydrophobic peptide is previously modified with a 2-nitrobenzenesulfenyl group, and α-cyano-3-hydroxycinnamic acid (3-CHCA), 3-hydroxy By performing mass spectrometry using -4-nitrobenzoic acid (3H4NBA) or a mixture thereof as a matrix, α-cyano-4-hydroxycinnamic acid (4-CHCA), which is a general matrix, or 2,5 -A method for efficiently ionizing hydrophobic peptides as compared to dihydrobenzoic acid (DHB) is described.

JP 2005-326391 A

  In the above MALDI mass spectrometry method, when the molecule to be measured is modified, a certain degree of ionization promoting effect is obtained, but when the modification is not performed, the ionization efficiency is not sufficient. Thus, molecular species that are difficult to ionize MALDI, such as hydrophobic peptides, have a problem of low ionization efficiency in MALDI mass spectrometry.

  An object of the present invention is to use a matrix additive that can easily and efficiently improve ionization efficiency in mass spectrometry without modifying a molecule to be measured (specifically, labeling or labeling). The object is to provide mass spectrometry and a matrix additive for mass spectrometry. In particular, the object of the present invention is to add a matrix that can easily and efficiently improve ionization efficiency in MALDI mass spectrometry without modifying the molecule to be measured (specifically, labeling or labeling). It is to provide a mass additive using an agent and a matrix additive for MALDI mass spectrometry.

  As a result of intensive studies, the present inventors have found that a specific dialkoxybenzoic acid derivative does not function as a matrix in mass spectrometry, but uses a molecular species that is difficult to ionize, such as a hydrophobic compound, by using it as a matrix additive. Has been found that ionization can be efficiently performed, and the present invention has been completed.

（式中、Ｍｅはメチル基を表し、Ｒ¹¹及びＲ¹²は、同一又は異なっていてもよく、炭素数２〜８のアルキル基を表す。）； 又は、
下記一般式（II）：

（式中、Ｒは水素原子又はメチル基を表し、Ｒ²¹及びＲ²²は、同一又は異なっていてもよく、炭素数２〜８のアルキル基を表す。）
で示されるジアルコキシ安息香酸誘導体をマトリックスの添加剤として用いる、疎水性化合物の質量分析法。 The present invention includes the following inventions.
(1) The following general formula (I):

(Wherein Me represents a methyl group, R ¹¹ and R ¹² may be the same or different and represent an alkyl group having 2 to 8 carbon atoms);
The following general formula (II):

(In the formula, R represents a hydrogen atom or a methyl group, and R ²¹ and R ²² may be the same or different and represent an alkyl group having 2 to 8 carbon atoms.)
A method for mass spectrometry of a hydrophobic compound using a dialkoxybenzoic acid derivative represented by the formula:

Analysis target, Ru hydrophobic compound der.

( 2 ) The mass spectrometry method according to (1 ), wherein the hydrophobic compound as an analysis target is a hydrophobic peptide. In the present specification, peptides include proteins.

( 3 ) As the matrix, α-cyano-3-hydroxycinnamic acid (3-CHCA), α-cyano-4-hydroxycinnamic acid (4-CHCA), 3-hydroxy-4-nitrobenzoic acid (3H4NBA) ), 2,5-dihydroxybenzoic acid, sinapinic acid, and a compound selected from the group consisting of 1,5-diaminonaphthalene, the mass spectrometry according to (1) or (2) above.

(4) Oite the general formula (I), R ¹¹ and R ^12, which may be identical or different, represent an alkyl group having 4-8 carbon atoms, or, in the general formula (II), R ²¹ and R ²² are, which may be identical or different, represent an alkyl group having 4-8 carbon atoms, mass spectrometry according to any one of (1) to (3).

（式中、Ｍｅはメチル基を表し、Ｒ¹¹及びＲ¹²は、同一又は異なっていてもよく、炭素数２〜８のアルキル基を表す。）； 又は、
下記一般式（II）：

（式中、Ｒは水素原子又はメチル基を表し、Ｒ²¹及びＲ²²は、同一又は異なっていてもよく、炭素数２〜８のアルキル基を表す。）
で示されるジアルコキシ安息香酸誘導体である、質量分析用マトリックスの添加剤。 -The following general formula (I):

(Wherein Me represents a methyl group, R ¹¹ and R ¹² may be the same or different and represent an alkyl group having 2 to 8 carbon atoms);
The following general formula (II):

(In the formula, R represents a hydrogen atom or a methyl group, and R ²¹ and R ²² may be the same or different and represent an alkyl group having 2 to 8 carbon atoms.)
The additive of the matrix for mass spectrometry which is the dialkoxy benzoic acid derivative shown by these.

-Additive of the above-mentioned matrix for mass spectrometry used for mass spectrometry of hydrophobic compounds.

-The additive of the said matrix for mass spectrometry used for the mass spectrometry of hydrophobic peptide.

Α-cyano-3-hydroxycinnamic acid (3-CHCA), α-cyano-4-hydroxycinnamic acid (4-CHCA), 3-hydroxy-4-nitrobenzoic acid (3H4NBA), 2,5- The additive for a matrix for mass spectrometry described above, which is added to a matrix for mass spectrometry selected from the group consisting of dihydroxybenzoic acid, sinapinic acid, and 1,5-diaminonaphthalene.

In the general formula (I), R ¹¹ and R ¹² may be the same or different and represent an alkyl group having 4 to 8 carbon atoms, or in the general formula (II), R ²¹ and R ²² may be the same or different and represents an alkyl group having 4 to 8 carbon atoms.

  In the present invention, the dialkoxybenzoic acid derivative represented by the general formula (I) or the general formula (II) is used as a matrix additive for mass spectrometry. When the specific dialkoxybenzoic acid derivative is used as a matrix additive, the ionization efficiency of a hydrophobic compound, particularly a molecular species that is difficult to ionize, such as a hydrophobic peptide, can be improved. Therefore, according to the present invention, there are provided a mass spectrometric method using a matrix additive capable of improving the ionization efficiency of a molecule to be measured, and a mass spectrometric matrix. The present invention is particularly directed to MALDI mass spectrometry, and is suitable when the analysis target is a hydrophobic compound and is particularly suitable when the analysis target is a hydrophobic peptide.

  According to the present invention, an improvement in detection sensitivity can be achieved by mass spectrometry measurement of a molecule to be measured (particularly a hydrophobic peptide).

  In the present invention, the dialkoxybenzoic acid derivative represented by the general formula (I) or the general formula (II) is used as a matrix additive for mass spectrometry.

In formula (I), Me represents a methyl group, R ¹¹ and R ¹² may be the same or different and each represents an alkyl group having 2 to 8 carbon atoms. That is, the compound represented by the formula (I) is 3,5-dialkoxybenzoic acid methyl ester. Examples of the alkyl group having 2 to 8 carbon atoms represented by R ¹¹ and R ¹² in formula (I) include an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, and an octyl group. These alkyl groups may be linear or branched. Examples of the branched one include isopropyl group, isobutyl group, sec-butyl group, tert-butyl group, isopentyl group, 2-ethylhexyl group and the like. Among these, a C4-8 alkyl group is preferable, a C4-6 alkyl group is more preferable, and a C4 alkyl group is particularly preferable. When the analysis target is a hydrophobic compound, it is considered that the alkyl group represented by R ¹¹ and R ¹² needs to have a certain degree of hydrophobicity for ionization of the hydrophobic compound. The total carbon number of the alkyl group represented by R ¹¹ and R ¹² is preferably 8 or more.

In the formula (II), R represents a hydrogen atom or a methyl group, and R ²¹ and R ²² may be the same or different and each represents an alkyl group having 2 to 8 carbon atoms. That is, the compound represented by the formula (II) is 2,5-dialkoxybenzoic acid or a methyl ester thereof. Examples of the alkyl group having a carbon number of C2~8 representing R ²¹ and R ²² are in Formula (II), an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, heptyl group, octyl group. These alkyl groups may be linear or branched. Examples of the branched one include isopropyl group, isobutyl group, sec-butyl group, tert-butyl group, isopentyl group, 2-ethylhexyl group and the like. Among these, a C4-8 alkyl group is preferable, a C4-6 alkyl group is more preferable, and a C4 alkyl group is particularly preferable. When the analysis target is a hydrophobic compound, it is considered that the alkyl group represented by R ²¹ and R ²² needs to have a certain degree of hydrophobicity for ionization of the hydrophobic compound. The total number of carbon atoms of the alkyl group represented by R ²¹ and R ²² is preferably 8 or more. Even when R is a hydrogen atom, the free degree of the carboxyl group is presumed to be small due to the hydrogen bond with the oxygen atom of the 2-position alkoxyl group.

  The dialkoxybenzoic acid derivative represented by the above formula (I) or formula (II) can be synthesized by methods known to those skilled in the art.

  The dialkoxybenzoic acid derivative represented by the above formula (I) or formula (II) is useful as a matrix additive in matrix laser desorption ionization (MALDI) mass spectrometry. By using the dialkoxybenzoic acid derivative compound in a mixture with a matrix, the ionization ability of the measurement target by the matrix can be enhanced and the detection limit can be improved.

  In the present invention, the matrix to which the additive is to be added is not particularly limited. Although it may be appropriately selected by a person skilled in the art from known matrices, for example, α-cyano-3-hydroxycinnamic acid (3-CHCA), α-cyano-4-hydroxycinnamic acid (4-CHCA), 3- A matrix selected from the group consisting of hydroxy-4-nitrobenzoic acid (3H4NBA), 2,5-dihydroxybenzoic acid, sinapinic acid, and 1,5-diaminonaphthalene can be used.

  The mass spectrometric object using the matrix additive of the present invention is not particularly limited. For example, it may be a molecule having a molecular weight of 500 to 30,000, preferably 1,000 to 10,000. Preferably, the matrix additive of the present invention can be used for mass spectrometry of hydrophobic substances because it can promote ionization of hydrophobic substances. In this case, in addition to the hydrophobic substance to be analyzed, other substances (for example, hydrophilic substances) may be mixed in the sample. As shown in the examples, the matrix additive of the present invention, when added to α-cyano-4-hydroxycinnamic acid (4-CHCA) as a matrix, compared to the case of 4-CHCA alone, The ionization of the hydrophobic substance can be selectively promoted without promoting the ionization of the hydrophilic substance. For this reason, even if a hydrophobic substance and a hydrophilic substance are mixed in the sample, the hydrophobic substance can be easily analyzed. From this point, the matrix additive of the present invention can be suitably applied to mass spectrometry of hydrophobic substances.

  The degree of hydrophobicity is not particularly limited as long as it can be determined to be hydrophobic based on various known hydrophobicity indices and hydrophobicity calculation methods. For example, the degree of hydrophobicity of the hydrophobic substance may be such that a person skilled in the art can determine that the substance is hydrophobic by the BB index (Bull and Breese Index). More specifically, the BB index can be, for example, 1000 or less, preferably -1000 or less.

  Alternatively, the degree of hydrophobicity of the hydrophobic substance may be such that a person skilled in the art can determine that the substance is hydrophobic by the HPLC index. The HPLC index was an acetonitrile aqueous solution containing 0.13% heptafluoro-n-butyric acid (HFBA) reported by CABrownw, HP JBennett, S. Solomon in Analytical Biochemistry, 124, 201-208, 1982 as eluent. Hydrophobic index based on reverse phase HPLC retention time, also referred to as HPLC / HFBA retention. More specifically, the HPLC index can be, for example, 40 or more, such as 40 to 10,000, preferably 100 to 1,000.

  Furthermore, the degree of hydrophobicity of the hydrophobic substance in the present invention is not limited as long as those skilled in the art can determine that it is hydrophobic by SSRCal Hydrophobity. SSRCal Hydrophobicity is reported by Analytical Biochemistry, 78, 7785-7795, 2006 by Oleg V. Krokhin. SSRCalc Hydrophobicity is a hydrophobicity index based on a peptide sequence-specific algorithm sequence-specific retention calculator (SSRCalc) for the retention time of RP-HPLC (reverse phase high performance liquid chromatography) of peptides. While the HPLC index or BB index estimates the retention time based only on the amino acid composition information, SSRCal Hydrophobicity estimates the retention time including the primary structure and secondary structure of the peptide. In the present invention, when the analysis target is a hydrophobic peptide, it is suitable for the degree of hydrophobicity to use SSRCal Hydrophobity as an index. More specifically, SSRCal Hydrophobicity may be 30 or more, preferably 40 to 70, for example.

  In the present invention, the effect of enhancing ionization ability of hydrophobic peptides (in the present invention, peptides include proteins) is particularly high. Whether or not it is a hydrophobic peptide can be determined by using BB index, HPLC index or SSRCal Hydrophobity, preferably SSRCal Hydrophobity or HPLC index as an index. Specifically, the amino acid constituting the peptide is hydrophobic. It may contain more amino acids of higher degrees. For example, examples of the hydrophobic amino acid include isoleucine, leucine, valine, alanine, phenylalanine, proline, methionine, tryptophan, and glycine. It may also contain cysteine, tyrosine and the like. The hydrophobic peptide may have a higher-order structure with a higher degree of hydrophobicity, not just the primary structure of such a peptide. For example, a peptide having a structure that is likely to interact with a hydrophobic stationary phase surface on which a reverse phase HPLC column is used.

  The crystal for mass spectrometry includes a step of forming, on the target plate for mass spectrometry, a droplet of a mixed solution containing at least an analysis target, a matrix, and a matrix additive in a solvent, and a droplet of the formed mixed solution. And the step of removing the solvent from the mixture to obtain the non-volatile content (that is, at least the analyte, the matrix, and the matrix additive) in the mixed solution as a residue. The obtained residue is a crystal for mass spectrometry. In this specification, the crystal for mass spectrometry and the residue are synonymous.

  As the target for mass spectrometry, a conductive metal plate usually used for MALDI mass spectrometry can be used. Specifically, a stainless or gold plate can be used.

  A specific method for preparing the liquid droplets on the target plate is not particularly limited. For example, first, a sample solution containing an analysis target, a matrix solution, and an additive solution are prepared separately, or a sample solution containing an analysis target and a matrix solution mixed with an additive are prepared separately. Next, these solutions are mixed to obtain a mixed solution, and the obtained mixed solution is dropped onto the target plate. Alternatively, the solutions are dropped on the same position on the target plate to mix on the target plate (on-target mix method). In the case of the on-target mix method, the dropping order of the solution is arbitrary.

  The ratio of the additive to the matrix can be, for example, a molar ratio of 0.01 to 50 times, preferably 0.05 to 0.5 times that of the matrix.

  More specifically, the additive can be prepared in a solution of, for example, 0.5 to 50 mg / mL, preferably 5 to 10 mg / mL, such as 5 mg / mL. The matrix can be prepared, for example, in a solution of 1 mg / mL to a saturated concentration, preferably 1 to 10 mg / mL, for example 10 mg / mL. These additive solution and matrix solution can be mixed, for example, in a volume ratio of 10: 1 to 1:10, preferably 1: 1 to 1:10, for example 1:10.

  The solvent of the mixed solution is selected from the group consisting of acetonitrile (ACN), trifluoroacetic acid (TFA), methanol (MeOH), ethanol (EtOH), tetrahydrofuran (THF), dimethyl sulfoxide (DMSO) and water. sell. More specifically, examples of the solvent of the mixed solution include ACN-TFA aqueous solution, ACN aqueous solution, MeOH-TFA, MeOH aqueous solution, EtOH-TFA aqueous solution, EtOH aqueous solution, THF-TFA aqueous solution, THF aqueous solution, DMSO-TFA aqueous solution. DMSO aqueous solution or the like is used, and more preferably, an ACN-TFA aqueous solution or an ACN aqueous solution can be used. The concentration of ACN in the ACN-TFA aqueous solution is, for example, 10 to 90% by volume, preferably 25 to 75% by volume, and the concentration of TFA is, for example, 0.05 to 1% by volume, preferably 0.05 to 0.1% by volume. It can be.

  The volume of the droplet of the mixed liquid is not particularly limited, and can be determined as appropriate by those skilled in the art. When a well is provided on the target plate, a droplet of the mixed solution can be formed in the well. In this case, the droplet is formed with a volume that can be accommodated in the well. Specifically, a droplet of about 0.1 μL to 2 μL, for example, about 0.5 μL can be formed.

  Next, the solvent is removed from the droplets of the mixed solution on the target plate. Solvent removal includes spontaneous evaporation of the solvent. The amount of the matrix contained per residue (that is, crystals for mass spectrometry) generated by evaporation can be, for example, 1 to 1,000 nmol, preferably 10 to 100 nmol. The amount of the analysis target is allowed in the range of 50 amol to 100 pmol, or 100 amol to 50 pmol, for example, with respect to 30 nmol of the matrix.

  The residue has a substantially circular shape on the contact surface with the target plate. That is, the outer edge of the residue has a substantially circular shape. The average diameter of the approximate circle may vary depending on the sample amount, the dripping amount, the matrix amount, the solvent composition, and the like, but is, for example, 1 to 3 mm, preferably 1 to 2 mm. The average diameter is the average length of line segments obtained by cutting a straight line passing through the center of gravity of the substantially circular shape at the outer edge of the residue in one residue.

  The mass spectrometer used in the present invention is not particularly limited as long as it is combined with a MALDI (Matrix Assisted Laser Desorption / Ionization) ion source. For example, MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization-Time of Flight) Mass Spectrometer, MALDI-IT (Matrix-Assisted Laser Desorption / Ionization-Ion Trap) Mass Spectrometer, MALDI-IT-TOF (Matrix-Assisted Laser Desorption) Examples include a deionization-ion trap-time-of-flight mass spectrometer and a MALDI-FTICR (matrix-assisted laser desorption / ionization-Fourier transform ion cyclotron resonance) mass spectrometer.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[Example 1]
In this example, α-cyano-4-hydroxycinnamic acid (4-CHCA, Laser Bio) was used as a matrix, and methyl 3,5-di-n-butoxy represented by the following chemical formula was used as an additive. Benzoic acid (Methyl 3,5-di (n-butoxy) benzoate, expressed as C4-M3,5-DBB), or 2,5-di-n-butoxybenzoic acid (3,5-di (n-butoxy) benzoate, expressed as C4-2,5-DBB).

(1-1) 10 mg / mL solution of 4-CHCA (50% ACN / 0.1% TFA water) (% is based on volume. The same applies hereinafter) and 5 mg / mL of C4-M3,5-DBB. The mL solution (50% ACN / 0.1% TFA water) was mixed at 10: 1 (v / v) to prepare additive mixed matrix solution 4-CHCA + C4-M3,5-DBB.
(1-2) Similarly, a 10 mg / mL solution of 4-CHCA (50% ACN / 0.1% TFA water) and a 5 mg / mL solution of C4-2,5-DBB (50% ACN / 0.1% TFA) water) was mixed at 10: 1 (v / v) to prepare additive mixed matrix solution 4-CHCA + C4-2,5-DBB.

(2) A 20 amol to 2 pmol / μL solution of hydrophobic peptide Humanin (50% ACN / 0.1% TFA water) was prepared as a sample solution. Similarly, 20 amol-2 pmol / microliter solution (50% ACN / 0.1% TFA water) of hydrophilic peptide (beta) -amyloid 1-11 was created.

(3) On the MALDI target plate (MALDI plate: sample plate 2.8 mm ring x 384 well (Shimadzu / Kratos, UK), (2) the hydrophobic peptide sample solution or the hydrophilic peptide sample solution; The additive mixed matrix solution of 1-1) or the additive mixed matrix solution of (1-2) was added dropwise in an amount of 0.5 μL and mixed (on-target mix method).
(4) Measured with AXIMA Performance (registered trademark) (Shimadzu Corporation) linear TOF and positive ion mode. And the detection limit was evaluated.

(For comparison)
(1) A 10 mg / mL solution (50% ACN / 0.1% TFA water) of 4-CHCA was prepared as a matrix solution.

(2) A 20 amol to 2 pmol / μL solution of hydrophobic peptide Humanin (50% ACN / 0.1% TFA water) was prepared as a sample solution. Similarly, 20 amol-2 pmol / microliter solution (50% ACN / 0.1% TFA water) of hydrophilic peptide (beta) -amyloid 1-11 was created.

(3) On the MALDI target plate (MALDI plate: sample plate 2.8 mm ring x 384 well (Shimadzu / Kratos, UK), (2) the hydrophobic peptide sample solution or the hydrophilic peptide sample solution; 0.5 μL each of the matrix solution of 1) was dropped and mixed (on-target mix method).
(4) Measured with AXIMA Performance (registered trademark) (Shimadzu Corporation) linear TOF and positive ion mode. And the detection limit was evaluated.

  As shown in Table 1, when the additive mixed matrix 4-CHCA + C4-M3,5-DBB or 4-CHCA + C4-2,5-DBB is used as compared with the case where 4-CHCA is used alone, the hydrophobic peptide Humanin The detection limit of 1/100 was confirmed, that is, the sensitivity was improved by 100 times. On the other hand, regarding the detection limit of the hydrophilic peptide β-amyloid 1-11, even when 4-CHCA is used alone, an additive mixed matrix 4-CHCA + C4-M3,5-DBB or 4-CHCA + C4-2,5- No change was seen using DBB.

  Thus, the matrix additive does not promote MALDI ionization of the hydrophilic peptide β-amyloid 1-11, but promotes MALDI ionization of the hydrophobic peptide Humanin, so that the hydrophobic peptide Humanin can be detected with high sensitivity. It has been shown.

[Comparative Example 1]
In this comparative example, α-cyano-4-hydroxycinnamic acid (4-CHCA, Laser Bio) is used as a matrix, and 3,5-di-n-butoxybenzoate represented by the following chemical formula is used as an additive. Acid (3,5-di (n-butoxy) benzoate, expressed as C4-3,5-DBB) was used.

(1) 10 mg / mL solution of 4-CHCA (50% ACN / 0.1% TFA water) and 10 mg / mL solution of C4-3,5-DBB (50% ACN / 0.1% TFA water) : 1 (v / v) to prepare additive mixed matrix solution 4-CHCA + C4 −3 , 5-DBB.

(2) A 20 amol to 2 pmol / μL solution of hydrophobic peptide Humanin (50% ACN / 0.1% TFA water) was prepared as a sample solution. Similarly, 20 amol-2 pmol / microliter solution (50% ACN / 0.1% TFA water) of hydrophilic peptide (beta) -amyloid 1-11 was created.

(3) On the MALDI target plate (MALDI plate: sample plate 2.8 mm ring x 384 well (Shimadzu / Kratos, UK), (2) the hydrophobic peptide sample solution or the hydrophilic peptide sample solution; 0.5 μL each of the additive-mixed matrix solution of 1) was dropped and mixed (on-target mix method).
(4) Measured with AXIMA Performance (registered trademark) (Shimadzu Corporation) linear TOF and positive ion mode. And the detection limit was evaluated.

  As shown in Table 2, no improvement in sensitivity was observed even when the additive mixed matrix 4-CHCA + C4-3,5-DBB was used compared to the case where 4-CHCA was used alone. The results of 4-CHCA alone in Table 2 are shown for comparison with Example 1 above.

Claims (4)

The following general formula (I):

(Wherein Me represents a methyl group, R ¹¹ and R ¹² may be the same or different and represent an alkyl group having 2 to 8 carbon atoms);
The following general formula (II):

(In the formula, R represents a hydrogen atom or a methyl group, and R ²¹ and R ²² may be the same or different and represent an alkyl group having 2 to 8 carbon atoms.)
A method for mass spectrometry of a hydrophobic compound using a dialkoxybenzoic acid derivative represented by the formula: The mass spectrometry method according to claim 1 , wherein the hydrophobic compound is a hydrophobic peptide. As the matrix, α-cyano-3-hydroxycinnamic acid (3-CHCA), α-cyano-4-hydroxycinnamic acid (4-CHCA), 3-hydroxy-4-nitrobenzoic acid (3H4NBA), 2 , 5-dihydroxybenzoic acid, a compound selected from the group consisting of sinapinic acid, and 1,5-diaminonaphthalene, mass spectrometry according to claim 1 or 2. Oite the general formula (I), R ¹¹ and R ^12, which may be identical or different, represent an alkyl group having 4-8 carbon atoms, or, in the general formula (II), R ²¹ and R The mass spectrometry method according to any one of claims 1 to 3 , wherein ²² may be the same or different and each represents an alkyl group having 4 to 8 carbon atoms.