JP4835036B2 - Highly sensitive detection method for organic compounds - Google Patents

Description

  The present invention relates to a novel highly sensitive detection method for organic compounds and an apparatus used therefor. More specifically, high-sensitivity detection of an organic compound, including labeling the organic compound to be detected with a metal-containing labeling reagent capable of binding to the organic compound to be detected, and analyzing the labeled organic compound by inorganic analysis The present invention relates to a method and an apparatus used therefor.

  As a method for analyzing organic compounds such as amino acids and organic acids, a derivatizing reagent such as o-phthalaldehyde (OPA), dansyl chloride, phenyl isothiocyanate (PITC) is reacted with the target compound, A method of indirectly detecting or quantifying a target compound by measuring fluorescence is common. However, since the detection sensitivities of these techniques vary greatly depending on the type of the target compound, the concentration of the target compound in the sample cannot be compared or quantified without a purified standard sample. However, metabolites represented by amino acids and organic acids have many unstable substances and very many kinds, so it is difficult to obtain standard samples. For these reasons, it has been extremely difficult to compare the concentrations of organic compounds.

  Further, as a method for analyzing an organic compound other than the derivatizing reagent as described above, there is a luminescent probe analysis using a metal chelate reagent. Luminescent probe analysis can analyze compounds such as amino acids and organic acids in a sample by detecting chemiluminescence of a chelate compound supported mainly on a metal. However, this technique has a detection limit on the order of pmol, and requires several hundreds of thousands of cells to detect intracellular metabolites such as amino acids and organic acids. For this reason, since it is necessary to collect a large amount of samples, the burden on the living body is large, and what can be analyzed is limited to those that can be obtained in large quantities such as blood and tissues. As a result, samples with a small amount of sample could not be analyzed, and could not be used for clinical examinations or biological analysis over time.

  Non-patent documents 1 to 4 and the like are cited as documents relating to the detection of organic compounds using a derivatizing reagent, and these documents all use light such as ultraviolet absorption and fluorescence as detection means.

  Patent Document 1 describes a reagent for chromatographic detection by ruthenium complex chemiluminescence. According to this reagent, carboxylic acid, primary amine and alcohol can be detected, but the detection sensitivity is at the pmol level, and it cannot be said that sufficient detection sensitivity is obtained.

  Patent Document 2 describes a reagent for detecting a ruthenium complex chemiluminescence of a carboxylic acid containing a benzoquinolizidine derivative. According to this reagent, carboxylic acid in a sample can be detected. However, like Patent Document 1, the detection sensitivity is at the pmol level, and it cannot be said that sufficient detection sensitivity is obtained.

Non-Patent Document 5 uses a platinum complex as a probe for a DNA / RNA assay. In this document, the emission line produced when a compound is reacted with an oligonucleotide is detected. However, the method described in this document has low detection sensitivity, and is not a method for the purpose of separation detection, so detection for each compound is impossible.
JP 9-318545 A JP-A-11-230910 Ross, M. (Roth, M.), “Analytical Chemistry”, 1971, Vol. 43, p. 880 Meffin, Pee. Je. (Meffin, PJ) et al., “Journal of Pharmaceutical Sciences”, 1977, 66, p. 583 Cary, M. A. (Cary, M.A.) and Persinger, A.C. F. (Persinger, A. F.), “Journal of Chromatographic Science”, 1972, Vol. 10, p. 537 Newberg, Sea. (Newberg, C.) et al., “Analytica Chimica Acta, 1952, Vol. 7, p.238. “Nucleic Acids Research”, 2002, Vol. 30, No. 21, p. e114

  As described above, it cannot be said that the conventional detection method of organic compounds has sufficient detection sensitivity, and the creation of a more sensitive detection method of organic compounds has been desired. In view of such circumstances, an object of the present invention is to provide a method for analyzing a trace amount of an organic compound with high sensitivity and speed as compared with a conventional method.

The gist of the present invention is as follows.
[1] (1) A step of producing a conjugate of an organic compound and a metal element-containing compound;
(2) A method for analyzing an organic compound, comprising: subjecting the conjugate to an inorganic analysis and analyzing a metal element contained in the conjugate.
[2] The metal element-containing compound has the formula:
[M _v (R) _m (L1) _n (L2) _o (L3) _p (L4) _q (L5) _r (L6) _s ] _{t Su}
[Where,
M is a metal element detectable by inorganic analysis,
R is a multidentate ligand of M;
L1, L2, L3, L4, L5 and L6 are ligands of M, which may be the same or different,
S is a group having a reactive site capable of binding to the organic compound, and is covalently bonded to one or more of R, L1, L2, L3, L4, L5 and L6;
m, t, u and v are each independently an integer of 1 or more,
n, o, p, q, r and s are each independently 0 or an integer of 1 or more.]
The method according to [1] above, represented by:
[3] The method according to [2] above, wherein M is a transition element.
[4] The method according to [2] above, wherein M is selected from the group consisting of ruthenium, indium, terbium, thulium, europium, rhodium and gold.
[5] The method according to [1] above, wherein the formation of the conjugate of the organic compound and the metal element-containing compound is caused by bonding the metal element-containing compound to the organic compound.
[6] In the above [1], the formation of a conjugate of an organic compound and a metal element-containing compound is caused by binding a metal element after binding a multidentate ligand ligand or a ligand to the organic compound. The method described.
[7] The method according to any one of [1] to [6], wherein the inorganic analysis is ICP-MS, ICP-AES, or AAS.
[8] The method according to any one of [1] to [7], further including a chromatography purification step.
[9] An organic compound analyzer comprising an inorganic analysis means for analyzing a metal element in a combined body of an organic compound and a metal element-containing compound.
[10] The apparatus according to [9], further comprising a chromatography means for purifying a conjugate of the organic compound and the metal element-containing compound.

  The present invention makes it possible to detect trace amounts of organic compounds that were difficult to analyze in the past, and is effective in analysis in the fields of life sciences, medical treatments, and environmental sciences such as in vivo metabolite analysis. Means can be provided. Moreover, since this invention analyzes an organic compound indirectly using a metal element containing compound as a label | marker, an analysis sensitivity is not influenced by the kind of organic compound. Furthermore, since the present invention can be quantified without a standard product to be analyzed, an unknown sample can be analyzed.

The organic compound analysis method of the present invention includes the following two steps.
(1) A step of generating a conjugate of a target organic compound and a metal element-containing compound (2) A step of subjecting the conjugate to an inorganic analysis and analyzing a metal element contained in the conjugate

  In the present invention, any organic compound can be analyzed because a ligand portion that serves as a binding portion between the metal element-containing compound and the organic compound may be appropriately selected. The bond between the organic compound and the metal-containing compound may be any bond, and examples thereof include a covalent bond, an ionic bond, and a coordinate bond.

  In the present invention, a conjugate of an organic compound and a metal element-containing compound can be produced by combining a metal element-containing compound with an organic compound to produce a conjugate, or a multidentate ligand ligand (R ) Or ligands (L1, L2, L3, L4, L5, and L6), and then the metal element (M) is bonded to form a final form in which the organic compound and the metal element-containing compound are bonded. It also includes the case of generating a body.

  Organic compounds that can be analyzed particularly preferably according to the present invention include macromolecules such as amino acids, amines, nucleobases, nucleosides, nucleotides, organic acids, aldehydes, ketones, thiols, hydroxy compounds, peptides, polypeptides, nucleic acids, and proteins. Compounds.

  Examples of amino acids include glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, cystine, methionine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, histidine, proline, oxy Examples include proline.

  Examples of the amine include ethaneamine, benzylamine, ethylenediamine, diethylamine, trimethylamine, dichlorodiethylamine, N-ethyl-N-methylbutan-1-amine, hydroxylamine, naphthylhydrazine, diazoaminobenzene, and the like.

  Examples of the nucleobase include adenine, guanine, cytosine, uracil, thymine and the like.

  Examples of the nucleoside include adenosine, guanosine, cytidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, thymidine, inosine, nicotinamide, ribotide, riboflavin and the like.

  Examples of nucleotides include adenylic acid, guanylic acid, cytidylic acid, uridylic acid, deoxyadenylic acid, deoxyguanylic acid, deoxycytidylic acid, thymidyl acid, inosinic acid, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, flavin mononucleotide Examples thereof include nucleotides and flavin adenine dinucleotide.

  Examples of organic acids include formic acid, acetic acid, ethyl acetate, monochloroacetic acid, stearic acid, acrylic acid, oleic acid, linolenic acid, pyruvic acid, cyclohexanecarboxylic acid, benzoic acid, maleic anhydride, dimethyl terephthalate, lactic acid, phthalate An acid etc. are mentioned.

  Examples of the aldehyde include formaldehyde, acetaldehyde, benzaldehyde, aldehyde ether, vanillin, paraformaldehyde, acrylic aldehyde, citral, aldol, and the like.

  Examples of the ketone include acetone, methyl ethyl ketone, camphor, cyclohexane, phenylacetone, anthraquinone, phorone, and acetol.

  Examples of the thiol include methyl mercaptan, butyl mercaptan, pentyl mercaptan, and thiophenol.

  Examples of the hydroxy compound include methanol, propanol, pentanol, octanol, terpene alcohol, ethylene glycol, pentaerythritol, D-glucitol, glycerin, ethochlorbinol, phenol, xylenol, naphthol, hydroquinone, and the like.

  Examples of the peptide include glycine-valine, phenylalanine-glycine, alanine-phenylalanine, tyrosine-leucine, glycine-valine-phenylalanine and the like.

  Examples of the polypeptide include Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly (SEQ ID NO: 1).

  Examples of the nucleic acid include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

  Examples of the protein include albumin, globulin, keratin, collagen, fibrinogen and the like.

  The metal element-containing compound in the present invention has an inorganic atom mainly composed of metal atoms other than carbon, nitrogen, oxygen and hydrogen, which can be specifically detected by inorganic analysis, in a reagent structure, and is quantified as an organic compound. A substance with the ability to bind physically.

As the metal element-containing compound in the present invention, the formula:
[M _v (R) _m (L1) _n (L2) _o (L3) _p (L4) _q (L5) _r (L6) _s ] _{t Su}
The compound represented by these is preferable.

The definition of each symbol in the above formula is as follows.
M is a metal element that can be detected by inorganic analysis. Examples of such metals include ruthenium, gold, europium, terbium, thulium, copper, ytterbium, indium, selenium, tin, rhodium, aluminum, arsenic, osmium, gadolinium, etc., among others, ruthenium, indium, Terbium, thulium, europium, rhodium and gold are particularly suitable for the present invention.
Moreover, as M, the transition metal of the 3rd group-the 11th group after the 4th period of a periodic table is also a metal which can become bivalent or trivalent, and since it has the property which is easy to make a chelate compound, it is suitable. is there.

  R is one or more M multidentate ligands. When the metal element-containing compound has two or more multidentate ligands, the plurality of multidentate ligands may be the same as or different from each other. Multidentate ligands include aromatic and aliphatic ligands. Suitable aromatic multidentate ligands are aromatic multidentate ligands including nitrogen, oxygen, phosphorus, sulfur and the like. Preferred aromatic multidentate ligands are, for example, bipyridyl, bipyrazyl, terpyridyl, porphyrin, calixarene, phenanthroyl and the like. Preferred aliphatic multidentate ligands are, for example, ethylenediamine, benzylethylenediamine, diethylenetriamine, triethylenetetramine, ethylene glycol, and their acetic acids, phosphoric acids and mercaptans.

  The multidentate ligand can be unsubstituted or substituted with any of a number of substituents known to those skilled in the art. Examples of suitable substituents are alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxy, imino, hydroxycarbonyl, aminocarbonyl, amidine, guanidinium, Ureido, sulfur-containing groups, phosphorus-containing groups and carboxylic acid esters of N-hydroxysuccinimide.

  The metal element-containing compound may have one or more monodentate ligands. A number of monodentate ligands are known to those skilled in the art. Suitable monodentate ligands include, for example, carbon monoxide, cyanide, isocyanide, halide, and aliphatic, aromatic and heterocyclic phosphines, amines, stibine, arsine.

  R may be appropriately selected depending on the type of M. For example, when gold is used as M, if a group containing a sulfur atom such as —SH or —S—S— is used as R, R can be bonded to M using the monolayer self-forming ability of the sulfur atom. . When ruthenium is used as M, if bipyridyl is used as R, R can be bonded to M by a coordinate bond between ruthenium and the nitrogen atom of the pyridine ring. When indium is used as M, if acetic acid of benzylethylenediamine is used as R, R can be bonded to M by a coordinate bond between a nitrogen atom or a carboxyl group contained in acetic acid of benzylethylenediamine and indium.

  L1, L2, L3, L4, L5 and L6 are ligands for M, and each may be the same or different. Specific examples of L1 to L6 include ammonia, chlorine, bipyridine, anthracene, ethylenediamine, nitrile, oxime, acetone, fluorine, and the like. In order to improve the stability of the reagent or add a function such as water solubility, the metal element-containing compound preferably contains L1 to L6.

  S is a group having a reactive site capable of binding to the target organic compound, and is covalently bonded to one or more of R, L1, L2, L3, L4, L5 and L6. Since S needs to be appropriately changed depending on the type of the target organic compound, it is difficult to specify it in general. For example, when the target organic compound is an amino acid, S has an activity having reactivity with an amino group. A carbamate group may be used.

m, t, u and v are each independently an integer of 1 or more, preferably 6 or less, more preferably 2 or less, and more preferably 1.
n, o, p, q, r and s are each independently 0 or an integer of 1 or more, preferably an integer of 6 or less.
A compound in which m is 1 and the sum of n, o, p, q, r, and s is an integer of 0 to 5 is particularly preferable.
v is an integer of 1 or more, and an integer of 1 to 3 is preferable.

  Specific examples of particularly preferred metal element-containing compounds include bis (2,2'-bipyridine) -4'-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis (hexafluoro-phosphate). The correspondence between this compound and the above general formula is as follows.

  A specific example of another particularly preferable metal element-containing compound is a substance in which indium is bound to 1- (4-isothiocyanobenzyl) ethylenediamine-N, N, N ′, N′-tetraacetic acid. The correspondence between this compound and the above general formula is as follows.

The inorganic analysis used in the present invention is not particularly limited as long as it is an analysis method capable of separating / detecting a metal element in a metal element-containing compound. Examples of inorganic analysis include inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma emission spectroscopy (ICP-AES), microwave induced plasma mass spectrometry (MIP), X-ray fluorescence analysis (XRF), and glow discharge. Examples include mass spectrometry (GDMS), ion selective electrode, atomic absorption analysis (AAS), and nuclear magnetic resonance analysis (NMR), and a plurality of analysis methods may be used in appropriate combination. Of these, inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma emission spectroscopy (ICP-AES) or atomic absorption spectrometry (AAS) is preferred.
In the present invention, a detector other than an inorganic analyzer such as a UV detector can be used together as necessary.

It is more preferable to include a chromatographic purification step from the viewpoint of improvement in separation / detection ability. In the present invention, such chromatography may be performed before the formation of a conjugate of an organic compound and a metal element-containing compound, or after the formation of a conjugate. If the target organic compound contains a large amount of contaminants and it is considered difficult to form a conjugate with the metal element-containing compound, the conjugate with the metal element-containing compound is used for rough purification of the organic compound. It is preferably performed before production. However, it is usually convenient to produce a conjugate, then purify the conjugate by chromatography, and then subject it to an inorganic analysis.
The chromatography that can be used is not particularly limited as long as it is a chromatography means commonly used in the field of chemical analysis. For example, gas chromatography, liquid chromatography, partition chromatography, adsorption chromatography, ion exchange chromatography Gel chromatography, paper chromatography, thin layer chromatography, column chromatography, capillary electrophoresis, and the like, and a plurality of chromatography may be used in combination.
Among these, high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) is particularly preferable from the viewpoint of simplicity and resolution.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited at all by this.

Examples 1 and 2
The equipment and reagents used in Examples 1 and 2 are as follows.

<Used equipment>
ICP-MS Yokogawa Analytical Systems' HP4500plus Environmental Specification Chamber Bergner's Mini CE Chamber
Ari Mist Nebulizer manufactured by Nebulizer Bergner
Inorganic analysis system manufactured by HPLC DIONEX
AS50 Autosampler
LC30 Chromatography Oven
AD25 Absorbance Detector
GP50 Gradient Pump
column
Made by SHISEIDO
CAPCELLPACK Ph column (Example 1)
NOMURA CHEMICAL made
DEVELOSIL C30 column (Example 2)
Block heater Reacti-Therm Heating made by PIERCE
Module

<Reagent used>
Standard solution Atomic absorption standard solution manufactured by Wako Pure Chemical Industries, Ltd.
(Each 1,000 ppm is diluted as appropriate)
Ultrapure water Collected from Millipore's pure water production system DMF (dimethylformamide) Ultrapure grade, acetonitrile (for reaction), anhydrous high purity reagent, acetonitrile (for mobile phase) Amino acid for chromatography, manufactured by Wako Pure Chemical Industries All Sigma-Aldrich Asp: Aspartic acid Gly: Glycine Tyr: Tyrosine Val: Valine Met: Methionine Ile: Isoleucine Leu: Leucine Phe: Phenylalanine Sodium dihydrogen phosphate Special grade disodium hydrogen phosphate Kanto Chemical Co., Ltd. Special reagent grade sodium carbonate Special grade reagent made by Kanto Chemical Co., Inc. Sodium hydrogen carbonate Special grade reagent made by Kanto Chemical Co., Ltd. Ru (bpy) ₂ (mcbpy-O-Su-ester) (PF ₆ ) ₂
Bis (2,2′-bipyridine) -4′-methyl-4-carboxybipyridine
Ruthenium N-succinimidyl ester-bis (hexafluoro-
Phosphate), manufactured by Fulka

<Experimental procedure>
Preparation of phosphate buffer solution 50 mmol of sodium dihydrogen phosphate and 50 mmol of disodium hydrogen phosphate were weighed and made up to a volume of 1 L with pure water. This was filtered to remove insoluble matters, and a phosphate buffer solution of 100 mM pH 6.8 was obtained.
Preparation of carbonate buffer solution 5 mmol of sodium carbonate and 5 mmol of sodium bicarbonate were weighed, dissolved in pure water, adjusted to pH 9.0 with hydrochloric acid, and then adjusted to 100 mL. This was filtered to remove insoluble matter, and a carbonate buffer solution of 100 mM pH 9.0 was obtained.
Reaction of amino acid with metal-containing reagent About 100 μmol of amino acid was weighed and dissolved in 1 mL of pure water to prepare each amino acid solution. 1 mg of a metal reagent (Ru (bpy) ₂ (mcbpy-O-Su-ester) (PF ₆ ) ₂ ) was dissolved in 250 μL of DMF to obtain a reagent solution. Next, 190 μL of carbonate buffer solution was placed in the reaction vial, 10 μL of amino acid solution was added, and 50 μL of reagent solution was further added and shaken vigorously. The reaction vial was heated at 55 ° C. overnight using a block heater. After cooling, the reaction solution was appropriately diluted to give an analytical sample (Example 1). In addition, the reaction solutions were mixed in equal amounts and appropriately diluted to obtain an analysis sample (Example 2).
As the analysis mobile phase of the amino acid reactant , phosphate buffer solution is used as solution A, acetonitrile is used as solution B, Ph column (Example 1) or C30 column (Example 2) is used as the stationary phase, and the analytical sample is HPLC / ICP- The sample was introduced into the MS system, and the mass number of Ru, m / z = 102, was detected.

<result of analysis>
[Example 1]
An amino acid reactant was analyzed using a 1.0 × 250 mm Ph column as the stationary phase of HPLC. The results are shown in FIG. A peak corresponding to 100 amol of Gly was detected.
The method of the present invention can obtain a detection limit of 10 amol, and achieves 1 million times higher sensitivity than 10 pmol which is the detection limit of the conventional metal probe analysis.
[Example 2]
Amino acid reactants were analyzed using a 1.0 × 150 mm C30 column for the HPLC stationary phase. The results are shown in FIG.
The method of the present invention was able to separate / detect peaks corresponding to Asp, Gly, Tyr, Val, Met, Ile, Leu, and Phe amino acids. The detected amounts of each amino acid are shown in Table 1. The detection limit for each amino acid was 10 amol.

Example 3
The equipment and reagents used in Example 3 are as follows.

<Used equipment>
ICP-MS Yokogawa Analytical Systems' HP4500plus Environmental Specification Chamber Bergner's Mini Chamber
Nebulizer Coaxial Nebulizer manufactured by Yokogawa Analytical Systems
Inorganic analysis system manufactured by HPLC DIONEX
AS50 Autosampler
LC30 Chromatography Oven
AD25 Absorbance Detector
GP50 Gradient Pump
column
Made by GL Science
Inertsil ODS C18 column block heater Reacti-Therm Heating made by PIERCE
Module

<Reagent used>
Ultrapure water Collected from Millipore pure water production equipment Acetonitrile (for reaction) Anhydrous high purity reagent Acetonitrile (for mobile phase) Wako Pure Chemicals chromatography Leucine Wako Pure Chemicals Phenylalanine Wako Pure Chemicals Angiotensin II Sigma Aldrich Tuftsin (Thr-Lys-Pro-Arg)
Peptide Laboratory Ammonium Acetate Wako Pure Chemical Reagent Special Grade Acetic Acid Kanto Chemical Reagent Special Grade Boric Acid Wako Pure Chemical Reagent Special Grade Sodium Hydroxide Wako Pure Chemical Reagent Special Grade Ethylenediaminetetraacetate Sodium (EDTA)
Wako Pure Chemicals reagent special grade indium nitric acid solution Wako Pure Chemicals atomic absorption analysis (1000ppm)
1- (4-Isothiocyanobenzyl) ethylenediamine-N, N, N ′, N′-tetraacetic acid
(Isothiocyanobenzyl EDTA)
Made by DOJINDO

<Experimental procedure>
Preparation of mobile phase 100 mmol of ammonium acetate was weighed, dissolved in pure water, adjusted to pH 5.8 with acetic acid, added with 30 mL of acetonitrile, made up to 1 L, and 3% acetonitrile with 100 mM pH 5.8. / 100 mM acetate buffer solution. Next, pure water was added to 600 mL of acetonitrile, and the volume was adjusted to 1 L to obtain a 60% acetonitrile solution. 5 mmol of EDTA was weighed and dissolved in 100 mL of pure water to obtain a 50 mM EDTA solution.
Preparation of borate buffer solution 20 mmol of boric acid was weighed, dissolved in pure water and adjusted to pH 9.0 with sodium hydroxide, and then adjusted to 100 mL to obtain a 200 mM pH 9.0 borate buffer solution.
Reaction of amino acid, peptide and isothiocyanobenzyl EDTA Approximately 0.1 mmol of each amino acid and peptide was weighed, dissolved in 1 mL of 0.1N hydrochloric acid, and further diluted 100 times with pure water to obtain a sample solution of 1 μmol / mL. . 1 μmol of isothiocyanobenzyl EDTA was weighed and dissolved in 500 μL of 20% acetonitrile solution to obtain a 2 μmol / mL reagent solution.
10 μL of the sample solution was dispensed into a sample tube, and 60 μL of borate buffer was added and stirred well. Further, 20 μL of acetonitrile was added, 10 μL of the reagent solution was added, and the mixture was stirred and heated at 40 ° C. for 4 hours. Thereafter, 890 μL of pH 5.8 acetate buffer was added and stirred, 10 μL of 1000 ppm indium solution was added and stirred again, and then cooled at 4 ° C. for 30 minutes to prepare an analytical sample.
As the analysis mobile phase of amino acid / peptide reaction , 3% acetonitrile / 100 mM acetate buffer solution was used for solution A, 60% acetonitrile was used for solution B, 50 mM EDTA solution was used for solution C, and a 1.0 × 250 mm ODS column was used for the stationary phase. The analytical sample was introduced into the HPLC / ICP-MS system, and m / z = 115, which is the mass number of indium, was detected.

<result of analysis>
Using the method of the present invention, peaks corresponding to amino acids and peptides were separated / detected. Thereby, it was shown that the coupling | bonding mode of the detection element in a reagent, and the kind of detection element are also possible other than a bipyridine-ruthenium complex. In addition, this time, indium was used as a detection element, but since EDTA compounds are well known to form chelate compounds with many metals, any other element that forms a chelate compound can be selected as a detection element. It is considered possible.

In the method of the present invention, the detection sensitivity hardly changes depending on the type of compound to be analyzed, so there is no need to worry about the ionization rate as in LC-MS, and actually leucine (FIG. 3), phenylalanine (FIG. 4) All of tuftsin (FIG. 5) and angiotensin II (FIG. 6) were able to obtain almost the same detection sensitivity.
In this experiment, the detection limit of each compound was about 1 pmol (about 10 times that of the metal probe analysis method). However, if the excess metal added during the reaction was removed, the detection limit was higher as in Examples 1 and 2. Sensitivity can be expected.

According to the present invention, it is possible to analyze an organic compound with a smaller amount of sample than conventional ones at fmol, amol or zmol level. Thereby, the influence on the living body and the environment by the analysis can be greatly reduced. In particular, when the organic compound is a metabolite, the burden on patients and animals can be reduced, and various things can be easily analyzed. For example, a blood test, which has been required to collect a large amount of blood until now, requires a small amount of sample. Therefore, it is not necessary to sample by injection, and only one drop of blood can be collected. In addition, if an organ such as the liver is not incised and the whole tissue is taken out, but a single cell is taken out with a capillary or the like, it is possible to analyze the organic compound in the cell to analyze the health condition. In addition, sampling can be easily performed many times from various parts, so detailed analysis can be performed, and slight changes and abnormalities in the analysis target that could not be found so far can be detected early. It becomes possible to do.
In addition, since the necessary reagents and solvents are reduced as compared with the prior art, it is possible to reduce the risk of contamination by chemical substances, and it is possible to reduce the cost.

2 is a chromatogram showing the results of Example 1. 2 is a chromatogram showing the results of Example 2. It is a chromatogram which shows the result of Example 3 at the time of using leucine as an analysis object compound. It is a chromatogram which shows the result of Example 3 at the time of using phenylalanine as an analysis object compound. It is a chromatogram which shows the result of Example 3 at the time of using a tuftsin as an analysis object compound. It is a chromatogram which shows the result of Example 3 at the time of using angiotensin II as an analysis object compound.

  SEQ ID NO: 1: This peptide is a substrate for tyrosine protein kinase and is useful for analyzing the activity of tyrosine protein kinase.

Claims (9)

(1) a step of generating a conjugate of an organic compound and a metal element-containing compound;
(2) subjecting the conjugate to chromatographic purification;
(3) The step of subjecting the conjugate to an inorganic analysis to analyze a metal element contained in the conjugate is performed successively in the order of (1), (2), and (3). An analysis method,
The organic compound is at least one polymer compound selected from the group consisting of amino acids, amines, nucleobases, nucleosides, nucleotides, organic acids, aldehydes, ketones, thiols, hydroxy compounds, peptides, polypeptides, nucleic acids, and proteins. Yes,
The metal element-containing compound has the formula:
[M _v (R) _m (L1) _n (L2) _o (L3) _p (L4) _q (L5) _r (L6) _s ] _{t Su}
[Where,
M is a transition element,
R is a multidentate ligand of M;
L1, L2, L3, L4, L5 and L6 are ligands of M, and each may be the same or different;
S is a group having a reactive site capable of binding to the organic compound, and is covalently bonded to one or more of R, L1, L2, L3, L4, L5 and L6;
m, t, u and v are each independently an integer of 1 or more,
n, o, p, q, r and s are each independently 0 or an integer of 1 or more.]
The analysis method represented by   The method according to claim 1, wherein the bond between the organic compound and the metal element-containing compound is a covalent bond. The method according to claim 1 or 2, wherein S is an N-succinimidooxycarbonyl group or an isothiocyanato group.   The method according to any one of claims 1 to 3, wherein M is selected from the group consisting of ruthenium, indium, terbium, thulium, europium, rhodium and gold.   The method of any one of Claims 1-4 that the production | generation of the coupling | bonding body of an organic compound and a metal element containing compound is based on making a metal element containing compound couple | bond with an organic compound.   The production | generation of the coupling | bonding body of an organic compound and a metal element containing compound is based on combining a metal element, after combining a multidentate ligand ligand or a ligand to an organic compound. 2. The method according to item 1.   The method according to any one of claims 1 to 6, wherein the inorganic analysis is ICP-MS, ICP-AES, or AAS.   The method according to claim 1, further comprising a chromatographic purification step.   The metal element-containing compound is bis (2,2′-bipyridine) -4′-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis (hexafluoro-phosphate) or 1- (4-isothiocyanobenzyl) The method according to any one of claims 1 to 8, which is a substance in which indium is bound to ethylenediamine-N, N, N ', N'-tetraacetic acid.

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