JP2857082B2 - New chemiluminescent labeling agent and method and apparatus for measuring catecholamines - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a chemiluminescent labeling agent for measuring catecholamines, a method for measuring catecholamines in a sample, and an apparatus for measuring catecholamines.

[0002]

2. Description of the Related Art Catecholamine is a general term for biogenic amines having a 3,4-dihydroxyphenyl skeleton. In particular, norepinephrine, epinephrine and dopamine play important roles in maintaining homeostasis in the body as corticosteroids and neurotransmitters. Running.

[0003] Therefore, the analysis of catecholamines is indispensable in biochemical and pharmacological studies of the endocrine and nervous systems and in clinical chemistry studies.

Conventionally, a method or an apparatus for analyzing catecholamines in biological samples such as plasma and urine is generally separated into fractions containing each component by high performance liquid chromatography or the like, and the separated catecholamines are further subjected to various detection methods. Quantified by

As the detection method, there are known an electrochemical detection method, a chemiluminescence method and the like in addition to a fluorescence method (natural fluorescence method and fluorescence derivatization method).

[0006] Among them, the electrochemical detection method has the highest sensitivity, but has a problem that the selectivity of catecholamines is poor, and it is difficult to detect catecholamines at a very low concentration such as a plasma sample.

[0007] Among the fluorescence methods, the method based on the detection of natural fluorescence is simple, but generally has insufficient sensitivity.

Further, as a fluorescent derivatization method, there are a method using ethylenediamine (ED method), a method using trihydroxyindole (THI method), and a method using 1,2-diphenylethylenediamine. Among them, the ED method has a problem of low sensitivity, and the THI method has advantages of high sensitivity and high selectivity, but has a problem of low detection sensitivity to dopamine. It is also known that the method using 1,2-diphenylethylenediamine has sensitivity comparable to electrochemical detection and has high selectivity.

However, the above-mentioned fluorescence method generally requires irradiation with excitation light, so that fluorescence from impurities and the like other than the substance to be detected cannot be avoided, so that fluorescence noise is increased and high sensitivity detection is performed. There is a problem of hindering.

On the other hand, the detection method of detecting luminescence is the same as the above-described fluorescence method. However, according to the chemiluminescence method in which luminescence is based on a chemical reaction, luminescence by excitation light is not detected in principle, but catecholamines Therefore, it is considered that only luminescence based on a chemical reaction with a derivative of is detected, and therefore, background noise is extremely small and high-sensitivity analysis becomes possible.

[0011]

According to the detection by the fluorescence method, the trace amount of catecholamine in a biological sample, particularly plasma, can be quantified even by using the 1,2-diphenylethylenediamine method considered to be the most sensitive. Therefore, at least about 0.5 ml of plasma is required, and a more sensitive measurement method capable of measuring with 100 μl or less of plasma is desired.

[0012] Therefore, in order to achieve this object, it is desired to develop a chemiluminescence method with extremely low background emission noise.

That is, (1) the development of a reaction system that reacts specifically and quantitatively with catecholamines to generate a chemiluminescent derivative, and (2) further reacts specifically and quantitatively with the derivative to form a chemical compound. There is a demand for the development of a reaction system that provides luminescence, the synthesis of reaction reagents, a measurement method using them, and the development of a measurement device.

[0014]

The present inventors have conducted intensive studies to achieve the above object, and as a result, 6-aminomethylphthalhydrazide as a novel chemiluminescent labeling reagent reacts with catecholamines to form a stable reagent. The present inventors have found that such a derivative is produced, and that the derivative emits strong light when oxidized, thereby completing the present invention.

That is, as shown in FIG. 1, catecholamines react almost quantitatively with 6-aminomethylphthalhydrazide in the presence of a weak oxidizing agent to produce a derivative represented by the following formula almost quantitatively. .

[0016]

Embedded image

The obtained derivative is oxidatively decomposed by a suitable oxidizing agent, and is accompanied by strong luminescence. Quantitative analysis of catecholamines becomes possible by optically detecting this luminescence.

More specifically, the present invention relates to the synthesis of 6-aminomethylphthalhydrazide as a novel chemiluminescent labeling agent for catecholamines.

Further, the present invention provides a catecholamine,
The present invention relates to a method for measuring catecholamines, comprising: a step of forming a derivative by reacting with 6-aminomethylphthalhydrazide; and a step of measuring luminescence by a reaction between the derivative and an oxidizing agent.

The present invention also provides a separation step of separating a mixture of catecholamines in a sample, and a step of forming a derivative of each of the catecholamines obtained in the separation step by reaction with 6-aminomethylphthalhydrazide. Measuring the luminescence due to the reaction between the derivative and the oxidizing agent according to the present invention.

Further, the present invention relates to a method for measuring catecholamines, wherein the mixture of catecholamines in the sample contains at least one selected from the group consisting of norepinephrine, epinephrine and dopamine.

Further, the present invention relates to a method for measuring catecholamines, wherein the derivative with 6-aminomethylphthalhydrazide is a compound represented by the following formula.

[0023]

Embedded image

Here, R ₁ represents OH or H, and R ₂
Represents CH ₃ or H.

Further, the present invention is characterized in that the oxidizing agent coexisting in the formation of a derivative by reaction with 6-aminomethylphthalhydrazide contains at least hexacyanoferrate (III), hydrogen peroxide or peroxidase. The present invention relates to a method for measuring catecholamines.

The present invention further provides a separation means for separating a mixture of catecholamines in a sample, a means for reacting each catecholamine obtained by the separation means with 6-aminomethylphthalhydrazide to obtain a derivative. The present invention relates to an apparatus for measuring catecholamines in a sample, comprising: means for measuring luminescence due to a reaction between a derivative and an oxidizing agent.

Furthermore, the present invention relates to a catecholamines measuring apparatus, wherein the catecholamines mixture in the sample contains at least one selected from the group consisting of norepinephrine, epinephrine and dopamine.

Further, the present invention relates to an apparatus for measuring catecholamines, wherein the derivative with 6-aminomethylphthalhydrazide is a compound represented by the following formula.

[0029]

Embedded image

[0030] Here, R ₁ represents OH or H, R ₂
Represents CH ₃ or H.

Further, the present invention is characterized in that the oxidizing agent coexisting in the formation of a derivative by reaction with 6-aminomethylphthalhydrazide contains hexacyanoferrate (III), hydrogen peroxide or peroxidase. It relates to a catecholamines measuring device.

Hereinafter, the present invention will be described in more detail.

(Chemiluminescent Labeling Agent, 6-Aminomethylphthalhydrazide) The method for synthesizing 6-aminomethylphthalhydrazide, which is a derivatizing reagent used in the present invention, is not particularly limited, but for example, the reaction shown in FIG. It is preferable to synthesize according to the formula. Here, the starting material, 4-methyl-N-phenylphthalimide, is a known substance. In order to further brominate the methyl group, N-bromosuccinimide (NBS) is used.
With peroxides (eg, benzoyl peroxide, BPO)
By reacting in the presence of

The method of activating the bromomethyl group of this bromomethyl-N-phenylphthalimide and converting it into an aminomethyl group is not particularly limited in the present invention. In the present invention, in particular, a quaternary salt obtained by reacting with hexamethylenetetramine is isolated, and further delepine (Delepine)
It is preferable to carry out aminomethylation by a reaction.

The obtained 4-aminomethyl-N-phenylphthalimide can be purified as white crystals having a purity of 95% or more by recrystallizing its hydrochloride from ethanol. The structure of the purified product is melting point, thin layer chromatography (TLC), liquid chromatography (HPL)
C), infrared absorption spectrum, nuclear magnetic absorption spectrum, etc.

Further, 4-aminomethyl-N-phenylphthalimide is neutralized and reacted with hydrazine.
In the present invention, there is no particular limitation on the method for converting to aminomethylphthalhydrazide. In the present invention,
Preferably, it is possible to obtain 6-aminomethylphthalhydrazide with high purity by reacting with hydrazine in ethanol.

In the present invention, the purity of 6-aminomethylphthalhydrazide may be 90% or more, preferably 95% or more. If necessary, it is most preferable to use a purity of 99.5% or more by a method such as recrystallization.

6-aminomethylphthalhydrazide is acetonitrile, ethanol, acetone, DMF, DMSO
And the like are soluble in water-miscible solvents, and the solution can be stored stably for a long period of time. For example, in the present invention, 6-aminomethylphthalhydrazide (purity of 95% or more) is dissolved in water-acetonitrile (1: 1 volume / volume) (at a concentration of about 10%).
mM), there is no substantial change in reactivity for about one month.

(Derivatization Reaction by Reaction of 6-Aminomethylphthalhydrazide with Catecholamines) In the present invention, the reaction conditions for forming a derivative by reacting 6-aminomethylphthalhydrazide with catecholamines are not particularly limited. Not done.

It is preferred to use at least about 100 equivalents of 6-aminomethylphthalhydrazide to catecholamines.

The reaction solvent is not particularly limited, but catecholamines or a mixture thereof as a sample is generally used.
It is a solution of a buffer such as Bicine, and therefore it is preferable that 6-aminomethylphthalhydrazide be dissolved in an organic solvent miscible with those buffers. For example, acetonitrile, DMSO and the like can be preferably used.

The weak oxidizing agent added to the above mixture is preferably used at a concentration of at least 1 mM.

In the present invention, usable oxidizing agents are not particularly limited, and various organic oxidizing agents or inorganic oxidizing agents can be used. For example, various organic peroxides (eg, peracetic acid, perbenzoic acid, alkyl hydroperoxide) can be suitably used as the organic oxidizing agent.
As the inorganic oxidizing agent, various inorganic peroxides (for example, hydrogen peroxide, sulfur peroxide, periodic acid and salts thereof, and metal oxidizing agents) can be suitably used. In the present invention, in particular, as the oxidizing agent, hexacyanoiron (II
I) Acid salts (for example, potassium salt, sodium salt, etc.) can be preferably used.

Further, it is desirable to complete the formation of the derivative by stirring and reacting the above mixture at about 35 ° C. to 45 ° C. The reaction time depends on the oxidizing agent used, but is preferably about 10 to 60 minutes. In order to further shorten the reaction time, the reaction temperature is preferably set to 50 ° C to 100 ° C. For example, 100 μl of norepinephrine (100 nM Bicine buffer) as catecholamines
And a mixture of 100 μl of 6-aminomethylphthalhydrazide (10 mM acetonitrile) and hexacyanoiron
(III) 10 μl of potassium acid salt (20 mM water)
When the mixture is added and mixed at 80 ° C., the formation of the derivative is completed in 5 to 40 minutes.

The obtained derivative is stable in the solution, and at a concentration of 0.1 to 10 μM, there is no substantial change even after about one week.

The structure of the derivative can be confirmed by a yellow residue obtained by removing the solvent after the reaction, by infrared absorption spectrum, nuclear magnetic resonance absorption spectrum, mass spectrometry, thin layer chromatography, liquid chromatography and the like. .

Further, in the present invention, the preparation of the above derivative may be carried out before separation of catecholamines in the sample by liquid chromatography or the like. That is, a derivative mixture of each catechol component is prepared from the catecholamines mixture in the sample by the above-described derivatization reaction, and the resulting derivative mixture of each catechol component can be separated and then subjected to a luminescence reaction described later.

Alternatively, after separation of catecholamines in a sample by liquid chromatography or the like, each of the separated fractions can be derivatized and subjected to the luminescence reaction described below. In this case, the time required for derivatization can be adjusted by selecting the reaction conditions as described above.

(Chemiluminescence) In the present invention, in order to obtain sufficient chemiluminescence by the derivative, it is preferable to use at least 1 to 100 mM of an oxidizing agent to be added. When the oxidizing agent is in excess, the light emission time is short compared to the light emission measurement time, which hinders efficient measurement.

In the present invention, usable oxidizing agents are not particularly limited, and various organic oxidizing agents or inorganic oxidizing agents can be used. For example, various organic peroxides (eg, peracetic acid, alkyl hydroperoxide) can be suitably used as the organic oxidizing agent. As the inorganic oxidizing agent, various inorganic peroxides (for example, hydrogen peroxide, sulfur peroxide, peroxidase, hemin, metal oxidizing agent) can be suitably used. In the present invention,
In particular, as the oxidizing agent, hexacyanoferrate (III) (eg, potassium salt, sodium salt, etc.) can be preferably used.

In the present invention, the chemiluminescence by the derivative has a maximum at about 500 nm. Further, in the reaction with the oxidizing agent, mixed components other than the above-mentioned derivatives (for example, buffer solution, catecholamines, 6-methylaminophthalhydrazide, and oxidation products after light emission) do not show any chemiluminescence. Therefore, in the present invention, the background noise is extremely small.

Further, 6-methylaminophthalhydrazide is a compound having chemiluminescence that is considered to be present in a biological sample, such as saccharides, keto acids, amino acids, nucleic acid bases, steroids, polyamines, carboxylic acids, alcohols and aldehydes. Not generated at all.

The reaction rate of the chemiluminescence is extremely high, for example, in the reaction of the norepinephrine derivative with 10 mM potassium hexacyanoiron (III), the luminescence is within about 10 seconds after mixing.

Further, in the present invention, the total amount of the obtained light has little variation due to the mixing method.

Therefore, in the present invention, the detector usable for detecting the chemiluminescence is not particularly limited, as long as it can detect the amount of the luminescence. For example, a chemiluminescence measuring device or the like can be preferably used, and in particular, the use of a chemiluminescence detector combined with HPLC is preferable.

(Light Emission Mechanism) The cause of the chemiluminescence according to the present invention may be considered as follows. The derivative has a specific structure of having a phthalhydrazide group and a benzoxazole group in the same molecule.
Oxidative decomposition of this derivative by the oxidizing agent generates excess energy due to the generation of nitrogen from the phthalhydrazide group, and this energy is efficiently transferred to the benzoxazole group in the same molecule and excited. Therefore, the light emission from the excited benzoxazole group is considered as one of the causes of the chemiluminescence according to the present invention. That is, as described above, the light-emitting device has a portion that generates and provides energy in the same molecule and a portion that is excited by receiving the energy and can emit the excitation energy by emitting light. Is considered to be efficiently detectable. This light emitting mechanism
This is thought to be different from the chemiluminescence associated with oxidative decomposition caused only by the phthalhydrazide group found in luminol and the like.

(Separation Means) In the present invention, the means for separating catecholamines is not particularly limited. A general high-performance liquid chromatograph can be used. The sample is generally in a state of being dissolved in a buffer, and therefore, a reversed phase system such as ODS can be preferably used as the filler, and various buffers, acetonitrile, methanol, or a mixed solvent thereof can be used as the solvent. It can be used preferably. For example, for separation of a mixture of norepinephrine, epinephrine and dopamine (Bicine buffer), three kinds of catecholamines can be separated by using an ODS column packing material, water or a phosphate buffer, acetonitrile or the like as a solvent.

[0058]

The catecholamines form derivatives by reaction with 6-aminomethylphthalhydrazide. Further, the derivative is formed also in a mixture of catecholamines to give a mixture of the respective derivatives. The derivative or the derivative mixture produces strong chemiluminescence when oxidized. This chemiluminescence has extremely low background noise in light detection, and thus enables highly sensitive quantitative analysis of catecholamines.

[0059]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist.

Example 1 (Synthesis of 6-aminomethylphthalhydrazide) 4-methyl-N-phenylphthalimide (10 g) was added to N-bromosuccinimide (NBS) (8.3 g) and benzoyl peroxide (BPO). (0.4 g) was mixed in carbon tetrachloride and refluxed for 4 hours to effect bromomethylation.

After removing the reaction solvent, the obtained bromomethylated product was subjected to column chromatography (silica gel / benzene).
(Melting point 176.2-177.9 ° C). TLC
(Silica gel, benzene, Rf 0.16), infrared absorption spectrum (IR), H, C-nuclear magnetic resonance absorption spectrum (NMR), the structure was confirmed. Further, the mass spectrum (JEOL JMS-D3 manufactured by JEOL Ltd.)
00, FAB high-speed electron impact, the same applies hereinafter), (M +
H) ⁺ (316) and (M + H + 2) ⁺ (318) peaks were observed.

Further, 5 g of bromomethylated compound was added to 250 ml of chloroform in hexamethylenetetramine (HMT).
A) (2.5 g) was reacted at room temperature for one day to obtain a precipitated quaternary salt. This crude product was purified as needle-like white crystals by recrystallization from ethanol (decomposition point 224.
3-227 ° C). The structure was confirmed by TLC (silica gel, 25% aqueous ammonia-acetone (1:49) Rf0.04), infrared absorption spectrum (IR), and H, C-nuclear magnetic resonance absorption spectrum (NMR). Further, a (M) ⁺ (376) peak was confirmed from a mass spectrum.

Further, 1 g of this salt was reacted at 50 ° C. in 250 ml of ethanol containing 5% hydrochloric acid to convert to an aminomethyl group. The reaction was almost quantitative (decomposition point 218.
2-220.6 ° C). Furthermore, TLC (silica gel, acetone Rf0.3), infrared absorption spectrum (IR),
By H, C-nuclear magnetic resonance absorption spectrum (NMR),
Purity and structure were confirmed.

Further, according to the mass spectrum, (M + H)
⁺ (253) peak confirmed.

0.5 g of the obtained aminomethylated hydrochloride
To the mixture was added 1 ml of hydrazine in 60 ml of ethanol, and the mixture was refluxed for about 30 minutes. After removing the ethanol, the crude product was recrystallized from ethanol to give 6-aminomethylphthalhydrazide with a purity of 90% or more (melting point: 256.2 to 260.4 ° C). The structure was confirmed by infrared absorption spectrum (IR), H, C-nuclear magnetic resonance absorption spectrum (NMR), mass spectrum, and elemental analysis. Further, by mass spectrum, (M +
H) ⁺ (192) peak was confirmed.

Example 2 (Quantification of norepinephrine, epinephrine or dopamine).

(1) 20 μg of catecholamine was added to Bici
Dissolve in 1 liter of ne buffer to prepare a solution of about 100 nM.

(2) 19 mg of 6-aminomethylphthalhydrazide (purity: 95% or more) was added to 10 m of acetonitrile.
and prepare a solution of about 10 mM.

(3) A solution of 66 mg of potassium hexacyanoferrate (III) in 10 ml of sodium carbonate solution (50 mM)
And prepare a 20 mM solution.

(4) Catecholamine solution 1 of (1)
00 μl and 10 μl of the 6-aminomethylphthalhydrazide solution of (2) are mixed, and 10 μl of potassium hexacyanoferrate (III) solution of (4) is further added. The resulting mixture is reacted at about 37 ° C. for 30 minutes or more. .

The completion of the derivative formation was determined by HPLC (ODS reverse phase column; solvent: acetonitrile, phosphate buffer (1:
(4 vol / vol), which confirms the almost complete disappearance of catecholamines.

(5) The reaction solution of (4) containing the obtained derivative is transferred to a luminescence detection cell, and then the cell is inserted into a luminescence measuring section (Micro Leader MLR-10, manufactured by Corona Co., Ltd.).
0).

(6) 100 μl of the potassium hexacyanoferrate (III) solution of (3) is added, and the luminescence is measured.

From these results, the detection limit (3S / N) of each catecholamine by the chemiluminescence method according to the present invention was several pM.
It is estimated to be. This value is about 10 times or more higher than the fluorescence method using 1,2-diphenylethylenediamine.

Example 3 (Analysis of catecholamine mixture) Norepinephrine,
Epinephrine and dopamine were each dissolved in Bicine buffer to a concentration of 10 mM to prepare a mixed sample for analysis.

A One embodiment of the means for measuring by a method of derivatization after separation of the obtained sample mixture is shown in FIG. That is, a high performance liquid chromatograph (HPLC) system 1 for separating a sample mixture, a reaction coil 2 for mixing each separated component with 6-aminomethylphthalhydrazide 3 to generate a derivative, It comprises a light emission detector 5 for detecting light generated by mixing the oxidizing agent 4, and a data processing computer 6.

[0077] HPLC: Shimadzu Corp., LC10 Column: TSK ODS80T _M, 150mm Eluent: acetonitrile (2% by volume), citrate buffer (pH 3) (98% volume) mixed solvent mobile phase flow rate: 0. 8 ml / min Reaction coil: inner diameter 0.5 mm stainless steel tube 10 m Mixing reaction tank temperature: 80 ° C. 6-aminomethylphthalhydrazide: mixed with acetonitrile solvent (1 mM), peristaltic pump (0.8 ml / min) Luminescence detector: Shimadzu Corporation Co., Ltd., Chemiluminescence Detector C
LD10A Oxidizing agent for emission: 1: 1 mixture of potassium hexacyanoferrate (III) and 40 mM, 100 mM hydrogen peroxide in sodium carbonate (50 mM) solvent, mixed with peristaltic pump (0.8 ml / min). Mixing of (1) After 100 μl of the sample was introduced from the sample injector 7 and separated on a separation column (retention times of norepinephrine, epinephrine and dopamine were about 5, 8, and 12 minutes, respectively), a 6-aminomethylphthalhydrazide solution was supplied to the three-way joint 10. It is introduced into a mixed reaction coil and derivatized. The light emitting oxidizing agent 4 solution is introduced into the light emitting mixing cell together with the solution containing the derivative by the three-way joint 10, and the generated light is detected.

The obtained light emission is processed by the computer data processing device 6. Under these conditions norepinephrine,
Epinephrine and dopamine each emitted as a single peak, and detection was completed in about 15 minutes.

The change in the amount of light emission due to the concentration of each component was measured. From the calibration curve prepared in advance, it was found that the detection limit at S / N = 3 was about 100 fg / injection amount.

B FIG. 4 shows one embodiment of a means for measuring the obtained sample mixture by derivatization before separation. That is, the mixture before separation is preliminarily mixed with 1,2-diphenylethylenediamine to produce a derivative, and the high-performance liquid chromatograph (HPLC) system 1 for the purpose of separating the resulting derivative mixture is used. It comprises a light emission detector 5 for detecting light generated by mixing the agent 4, and a data processing computer 6.

[0081] HPLC: Shimadzu Co., Ltd., LC10 column: TSK-gel ODS80T _M, 150m
m Eluent: mixed solution of acetonitrile, methanol, and phosphate buffer (4: 1: 5 volumes) Mobile phase flow rate: 0.8 ml / min Emission detector: Chemiluminescence detector C, manufactured by Shimadzu Corporation
LD10A Oxidizing agent for emission: 1: 1 mixture of 40 mM potassium hexacyanoferrate (III) and 100 mM hydrogen peroxide in sodium carbonate (50 mM) solvent, mixed with peristaltic pump (0.8 ml / min). Mixing of (1) A 100 μl sample and a 6-methylaminophthalhydrazide solution are mixed and reacted for derivatization. After separating the obtained derivative mixture with a separation column (retention times of the respective derivatives of norepinephrine, epinephrine and dopamine are about 4, 7, and 10 minutes, respectively), the oxidizing agent solution for luminescence 4 is connected by a three-way joint 10. The light that is introduced together with the solution containing each derivative into the mixed cell for light emission is detected.

The obtained light emission is processed by the data processor 6. Under these conditions, norepinephrine, epinephrine and dopamine each emit a single peak,
Detection was completed in about 15 minutes.

The change in the amount of light emission due to the concentration of each component was measured. From the calibration curve prepared in advance, it was found that the detection limit at S / N = 3 was about 100 fg / injection amount.

[0084]

EFFECTS OF THE INVENTION Catecholamines form derivatives by reaction with 6-aminomethylphthalhydrazide. The derivatives provide strong chemiluminescence when oxidized. Since this chemiluminescence has very little background noise in light detection, highly sensitive quantitative analysis of catecholamines becomes possible.

[Brief description of the drawings]

FIG. 1 is a diagram showing a derivative formation reaction between catecholamines and 6-aminomethylphthalhydrazide, and chemiluminescence by oxidation of the derivative.

FIG. 2. From 4-methyl-N-phenylphthalimide,
BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the synthetic route of 6-aminomethyl phthalhydrazide.

FIG. 3 is a diagram showing a measurement system for measuring a sample mixture by derivatization after separation.

FIG. 4 is a diagram showing a measurement system for separating and measuring a sample mixture after derivatization.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... HPLC system, 2 ... reaction coil, 3 ... 6-aminomethyl phthalhydrazide, oxidizing agent mixed solution, 4 ... luminescent oxidizing agent, 5 ... chemiluminescence detector, 6 ... data processing device,
7 ... sample injector, 8 ... separation column, 9 ...
Eluent, 10 ... 3-way joint.

Claims (10)

(57) [Claims] 1. A chemiluminescent labeling agent for catecholamines comprising 6-aminomethylphthalhydrazide represented by the following formula: Embedded image 2. A catecholamines comprising a step of forming a derivative of catecholamines by reacting with 6-aminomethylphthalhydrazide, and a step of measuring luminescence by a reaction between the derivative and an oxidizing agent. Measuring method. 3. A separation step of separating a mixture of catecholamines in a sample, a step of forming a derivative of each catecholamine obtained by the separation step with 6-aminomethylphthalhydrazide, and oxidizing the derivative with the derivative. Measuring a luminescence due to a reaction with the agent. 4. The method according to claim 2, wherein the mixture of catecholamines contains at least one selected from the group consisting of norepinephrine, epinephrine and dopamine. 5. The method according to claim 2, wherein the derivative is a compound represented by the following formula. Embedded image Here, R ₁ represents OH or H, and R ₂ represents CH ₃ or H. 6. The method according to claim 2, wherein the oxidizing agent is at least one selected from the group consisting of hexacyanoferrate (III), hydrogen peroxide, and peroxidase.
5. The measuring method according to any one of 4. 7. A separation means for separating a mixture of catecholamines in a sample, a means for reacting each catecholamine obtained from said separation means with 6-aminomethylphthalhydrazide to obtain a derivative, and the derivative and an oxidizing agent Means for measuring luminescence due to the reaction with catecholamines in a sample. 8. The measuring device according to claim 7, wherein the catecholamines mixture contains at least one selected from the group consisting of norepinephrine, epinephrine and dopamine. 9. The measuring device according to claim 7, wherein the derivative is a compound represented by the following formula. Embedded image Here, R ₁ represents OH or H, and R ₂ represents CH ₃ or H. 10. The method according to claim 10, wherein the oxidizing agent is hexacyanoiron (III).
8. The composition according to claim 7, comprising at least one selected from the group consisting of acid salts, hydrogen peroxide and peroxidase.
The measurement device according to any one of claims 9 to 9.