JP2001165858A - Method and apparatus for measuring 5-alkoxyindoles - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measuring method which can measure 5-alkoxyindoles contained in a biological specimen or the like with high sensitivity and high accuracy without application of a complicate pretreatment. SOLUTION: This measuring method comprises: (1) an oxidization process for generating a quinoneimine derivative by electrolytically oxidizing 5- alkoxyindoles contained in a biological specimen, (2) a reduction process generating 5-hydroxyindoles by electrolytically reducing a quinoneimine derivative, (3) a derivative generation process to react 5-hydroxyindoles with aromatic aminomethans to thereby generate a fluorescent derivative, and (4) a fluorescence detection process to irradiate the fluorescent derivative with stimulating light and to detect fluorescent light by electron transition within a particle of the fluorescent derivative.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method and an apparatus for measuring 5-alkoxyindoles.

[0002]

2. Description of the Related Art 5-Alkoxyindoles are a general term for compounds in which an alkoxyl group is bonded to the 5-position of an indole skeleton. Among them, melatonin is one of the hormones in the brain and mainly He is involved in adjusting sleep, daily rhythm, etc. Melatonin is known to have a route synthesized from tryptophan as a starting material in the living body via serotonin which is a physiologically active substance.In its biosynthesis and metabolic pathway, 5-methoxyindole which is a metabolite of melatonin is known. Produced. Melatonin is widely used for adjusting the daily rhythm of aircrew and night shift workers due to its utility, but its mechanism of action has not yet been fully elucidated.

[0003] In particular, the melatonin concentration in human blood is extremely low, and during the daytime active period when the amount of secretion is relatively small, several pg are required.
/ ML only. As an analytical method for such an extremely small amount of melatonin, for example, literatures DCSanders, AK
Chaturvedi and JRHordinsky, Journal of Analytica
l Toxicology, 32,159-167 (1999) includes a radioimmunoassay (hereinafter referred to as "RIA") method and a gas chromatography-mass spectrometry (hereinafter referred to as "GC-MS").
Method and high performance liquid chromatography (hereinafter referred to as "HP
LC ”) method has been reported.

[0004] However, RIA is simple in operation and high in sensitivity, but is limited by the equipment and waste associated with the use of radioisotopes, and there are many analogous compounds that cause cross-reaction in biological samples. RIA may overestimate the amount of melatonin (ie, increase the positive error). On the other hand, the GC-MS method has high specificity and relatively excellent selectivity and sensitivity, but derivatization to a volatile substance is complicated, which requires a long time for analysis and increases costs. There is a tendency.

[0005] On the other hand, the HPLC method is an analysis method that is widely used for the analysis of biological substances. As the detection method, a fluorescence detection method using natural fluorescence and an electrochemical detection method are known. It is simple and economical. However, 5-alkoxyindoles such as melatonin have a large unipolar potential (electrode potential) required for the electrode reaction, and the electrochemical detection method needs to increase the decomposition voltage, but this increases noise and increases sensitivity. It tends to decrease significantly.

In addition, in a biological sample, there is a problem that background noise increases due to interference peaks caused by reducing substances such as catechols and other indoles. In addition, it is difficult to directly convert 5-alkoxyindoles such as melatonin into a fluorescent substance, and the fluorescence detection method using natural fluorescence has a problem that aromatic amino acids and interference peaks derived from other indoles are generated. It appears and the background noise tends to increase. And H
There is also a problem that the PLC method requires a relatively large amount of sample.

[0007] As described above, conventionally, 5-toxin such as melatonin has been used.
Sensitivity, accuracy,
No method has been fully satisfactory in terms of selectivity, speed and economy. Therefore, in order to measure 5-alkoxyindoles contained in a biological sample or the like with high sensitivity and high accuracy, complicated pretreatments such as highly-separating and purifying the 5-alkoxyindoles are required. I had to rely on Therefore, a simpler, more sensitive and accurate method for measuring 5-alkoxyindoles has been desired.

Therefore, the present invention has been made in view of such circumstances, and without performing complicated preprocessing,
5-alkoxyindoles contained in biological samples and the like,
It is an object of the present invention to provide a method and an apparatus for measuring 5-alkoxyindoles, which can be measured with higher sensitivity and higher precision than before.

[0009]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have conducted intensive studies. As a result, 5-oxyindoles are oxidized under predetermined conditions, and then reduced to give 5-hydroxyindoles. We found that it can be converted to indole. In addition, it has been found that by causing a predetermined labeling reagent to act on the obtained 5-hydroxyindoles, fluorescence can be emitted by excitation light, and an extremely stable fluorescent derivative is quantitatively generated. Was completed.

That is, the method of the present invention for measuring 5-hydroxyindoles is a method for measuring 5-alkoxyindoles in a subject, which comprises subjecting the 5-alkoxyindoles to electrolytic oxidation to form 5-alkoxyindoles. An oxidation step of producing a quinone imine derivative corresponding to
A reduction step of electrolytically reducing a quinone imine derivative to form a 5-hydroxyindole corresponding to a 5-alkoxyindole, and a reaction with a 5-hydroxyindole, an aromatic aminomethane, and an oxidizing agent to form a fluorescent derivative. The method is characterized by including a derivative generation step of generating a derivative, and a fluorescence detection step of irradiating the fluorescent derivative with excitation light and detecting fluorescence from the fluorescent derivative due to the irradiation of the excitation light.

Here, the 5-alkoxyindole is preferably represented by the following formula (1):

[0012]

Embedded image [Wherein, R ¹ represents an alkyl group, R ² represents an acetamido group, a carboxyl group, or the following formula (2) or (3)
Represents a group represented by

[0013]

Embedded image (Wherein, R ²¹ represents a methylene group, an alkylene group or a polymethylene group, R ²¹ may be different or the same, R ²² represents a hydrogen atom, a carboxyl group, an aldehyde group or an acyl group, and R ²³ represents A carboxyl group, an aldehyde group or a hydroxyl group.)].

More preferably, as 5-alkoxyindoles, 5-methoxytryptophan, 5-methoxytryptamine, 5-methoxyindole-3-acetic acid,
Examples include at least one selected from the group consisting of 5-methoxyindole-3-acetamide, 5-methoxytryptophor and melatonin.

The quinone imine derivative and 5-hydroxyindole are represented by the following formulas (4) and (5), respectively:

[0016]

Embedded image [In the formula, R ² represents an acetamido group, a carboxyl group, an aldehyde group or a group represented by the above formula (2) or (3). ] Are represented.

More preferably, 5-hydroxyindoles corresponding to the above-mentioned 5-alkoxyindoles include 5-hydroxytryptophan, serotonin, and 5-hydroxyindole.
It produces at least one selected from the group consisting of hydroxyindole-3-acetic acid, 5-hydroxyindole-3-acetamide, 5-hydroxytryptophor or N-acetylserotonin.

Further, aromatic aminomethanes include:
Each of the following formula (6);

[0019]

Embedded image [Wherein, X represents an aromatic hydrocarbon, an aromatic heterocyclic hydrocarbon, or an aromatic condensed polycyclic hydrocarbon. ] Can be preferably used.

As the fluorescent derivative, specifically,
Formula (7) below;

[0021]

Embedded image [Wherein, X represents an aromatic hydrocarbon, an aromatic heterocyclic hydrocarbon, or an aromatic condensed polycyclic hydrocarbon. ] Are represented.

According to such a method for measuring 5-alkoxyindoles, first, 5-alkoxyindoles contained in a subject (solution) are electrolytically oxidized to produce a quinone imine derivative. At this time, the 5-alkoxyindole is oxidized directly or indirectly at the anode, and the quinone imine derivative, which is a product, exists in the solution without being deposited or fixed on the anode. Next, the quinone imine derivative in this solution is electrolytically reduced to produce 5-hydroxyindoles. At this time, the quinone imine derivative is
The product, 5-hydroxyindole, which is reduced directly or indirectly at the cathode, exists in solution without deposition or sticking to the cathode.

Next, 5-hydroxyindoles in this solution are reacted with aromatic aminomethanes in the presence of an oxidizing agent to quantitatively generate a fluorescent derivative. When the obtained fluorescent derivative is irradiated with excitation light having a predetermined wavelength such as ultraviolet light, it emits fluorescence having a wavelength specific to the fluorescent derivative due to electron transition in the molecule. And
By detecting this fluorescence, 5-alkoxyindoles in the subject can be selectively measured with low background noise.

When the specimen contains a plurality of types of 5-alkoxyindoles, separation of each of the 5-alkoxyindoles and / or the respective 5-hydroxyindoles corresponding to the respective 5-alkoxyindoles is performed. Preferably, the method further comprises a step. In this way, even if the specimen is a sample containing a plurality of types of 5-alkoxyindoles, discrimination of each 5-alkoxyindole can be performed by detecting the fluorescence from the fluorescent derivative derived from each 5-alkoxyindole. Measurement becomes possible. In this separation step, each 5-alkoxyindole is preferably separated from other indoles, proteins, lipids, and the like.

Preferably, in the oxidation step, the oxidation potential during electrolytic oxidation is set to +1.0 to +2.0 V, more preferably +1.5 to +2.0 V, and in the reduction step,
It is preferable that the reduction potential at the time of performing the electrolytic reduction is -2.0 to 0.0V, more preferably -2.0 to -1.0V. In this case, the electrolytic oxidation and the electrolytic reduction can proceed steadily, and the current efficiency can be improved.

In the present invention, “oxidation potential” means
The temperature of the solution containing the subject is 25 ° C, and the pH is 5.5 to 7.
The measured value of the potential difference between the anode during electrolytic oxidation (in an acetate buffer or the like) and a silver / silver chloride electrode as a reference electrode is shown. The “reduction potential” refers to a cathode during electrolytic reduction at a temperature of 25 ° C. and a pH of 5.5 to 7 (such as an acetate buffer) of a solution containing an analyte, and a cathode serving as a reference electrode. The measured value of the potential difference from the silver / silver chloride electrode is shown. At this time, the salt concentration in the buffer was 50 m
mol / L or more.

The apparatus for measuring 5-alkoxyindoles of the present invention is an apparatus for suitably implementing the method for measuring 5-alkoxyindoles of the present invention. That is, the apparatus for measuring 5-alkoxyindoles of the present invention is a method for measuring 5-alkoxyindoles in a subject, wherein the subject containing the 5-alkoxyindoles is contained, Is electrolytically oxidized to form a quinoneimine derivative corresponding to a 5-alkoxyindole, and electrolytic reduction is performed when the quinoneimine derivative is electrolytically reduced to form a 5-hydroxyindole corresponding to the 5-alkoxyindole Part, a 5-hydroxyindole, an aromatic aminomethane, and a derivative generating part in which a fluorescent derivative is generated by a reaction with the oxidizing agent; and irradiating the fluorescent derivative with excitation light, and irradiating the fluorescent derivative with the excitation light. And a fluorescence detector for detecting fluorescence emitted from the fluorescent derivative.

Here, the 5-alkoxyindoles include those represented by the above formula (1), and more specifically, 5-methoxytryptophan, 5-methoxytryptamine, 5-methoxyindole-3- Acetic acid, 5-
It is at least one selected from the group consisting of methoxyindole-3-acetamide, 5-methoxytryptophor and melatonin. Examples of the quinone imine derivative include those represented by the above formula (4).

Further, examples of 5-hydroxyindoles include those represented by the above formula (5), and more specifically, 5-hydroxytryptophan, serotonin,
It is at least one selected from the group consisting of 5-hydroxyindole-3-acetic acid, 5-hydroxyindole-3-acetamide, 5-hydroxytryptophor and N-acetylserotonin. Further, as the aromatic aminomethanes, those represented by the above formula (6) can be preferably used, and examples of the fluorescent derivatives include those represented by the above formula (7).

When the subject contains a plurality of types of 5-alkoxyindoles, a separating unit for separating each 5-alkoxyindole and / or each 5-hydroxyindole corresponding to the 5-alkoxyindole is provided. It is more preferable to provide further. Further, the electrolytic oxidizing unit may
The oxidation potential of the alkoxyindoles is from +1.0 to +
2.0 V, and the electrolytic reduction unit can reduce the reduction potential of the quinone imine derivative from -2.0 to 0.0
It is preferable that V can be V.

[0031]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described.
This will be described in detail with reference to the drawings. Note that the same components are denoted by the same reference numerals, and redundant description will be omitted.

FIG. 1 shows an electrolytic oxidation reaction of a 5-alkoxyindole, an electrolytic reduction reaction of a quinone imine derivative, a reaction of forming a fluorescent derivative of a 5-hydroxyindole, and a fluorescent emission by irradiating excitation light to the fluorescent derivative in the present invention. FIG. 3 is a view showing a reaction route of the present invention. As shown in FIG. 1, in the method for measuring 5-alkoxyindoles according to the present invention,
Electrolytic oxidation of the 5-alkoxyindole contained in the subject to produce a quinoneimine derivative (oxidation step), electrolytic reduction of the quinoneimine derivative to produce a 5-hydroxyindole (reduction step), And aromatic aminomethanes in the presence of an oxidizing agent to produce a fluorescent derivative capable of emitting fluorescence by irradiation with excitation light (derivative generating step),
The fluorescent derivative is irradiated with excitation light to detect fluorescence emission due to intramolecular electron transition of the fluorescent derivative (fluorescence detection step).

Hereinafter, the analyte, 5-alkoxyindoles, quinone imine derivatives, 5-hydroxyindoles, aromatic aminomethanes, and oxidizing agents will be described.
Next, embodiments of the method and apparatus for measuring 5-alkoxyindoles according to the present invention will be described in detail.

<Analyte> The analyte that can be measured by the method for measuring 5-alkoxyindoles of the present invention is not particularly limited as long as it contains 5-alkoxyindoles. It may be semi-solid or solid. Specifically, a sample from a living body includes a urine sample, a blood sample, a living tissue, and the like, and a pharmaceutical includes an bulk powder and a preparation.

It is preferable that a test solution obtained by adding, dissolving or dispersing the test sample in an organic or inorganic solvent is used as a sample for measurement. In this way, there is an advantage that pretreatment such as removal of residues and interfering components, extraction, and adjustment of liquid properties such as pH is facilitated. In the present invention, 5-
Since the electrolytic oxidation of the alkoxyindoles and the electrolytic reduction of the electrolytic oxidation products are performed, the preparation of the sample in a liquid mixture in advance eliminates the necessity of newly adjusting the electrolytic solution at the time of electrolysis, thereby improving workability.

Further, the subject may contain 5-hydroxyindoles in advance. 5-Hydroxyindoles such as N-acetylserotonin and serotonin are precursors of 5-alkoxyindoles such as melatonin in a living body. In a biological sample, 5-alkoxyindoles and 5-hydroxyindoles are usually used. And are mixed. 5-hydroxyindoles are oxidized and reduced by electrolysis (electrolytic oxidation, electrolytic reduction),
As a result of the reaction, the original 5-hydroxyindoles are formed. Therefore, it is possible to simultaneously measure 5-alkoxyindoles and 5-hydroxyindoles contained in the subject in advance.

When the subject is a biological sample, it usually contains proteins and lipids in many cases, and it is necessary to protect the electrode for electrolysis and to sufficiently prevent the reaction of generating a fluorescent derivative. It is preferable to remove coexisting proteins and lipids in advance. Examples of the pretreatment for this include addition of an acid or an organic solvent, filtration with an ultrafiltration membrane, etc., and solid phase extraction. In addition, some protein components emit fluorescence by themselves when irradiated with excitation light, and removal of protein is a preferable treatment because it may become background light when detecting fluorescence. .

Further, other low molecular components may be mixed in the subject. Since the reaction of fluorescently derivatizing 5-hydroxyindoles with the above-mentioned aromatic aminomethanes has extremely high selectivity, other low-molecular components considered to be present in biological samples other than 5-hydroxyindoles, for example, saccharides The possibility that a fluorescent derivative is produced from keto acids, amino acids, nucleic acid bases, peptides, oligonucleotides, steroids, polyamines, carboxylic acids, alcohols and aldehydes is extremely low. Therefore, even if these components coexist in the subject,
There is little risk that the measurement of alkoxyindoles and 5-hydroxyindoles will be disturbed.

<5-Alkoxyindoles> Examples of the 5-alkoxyindoles that can be measured according to the present invention include those represented by the following formula (1), but are not particularly limited thereto.

[0040]

Embedded image [Wherein, R ¹ represents an alkyl group, R ² represents an acetamido group, a carboxyl group, or the following formula (2) or (3)
Represents a group represented by

[0041]

Embedded image (Wherein, R ²¹ represents a methylene group, an alkylene group or a polymethylene group, R ²¹ may be different or the same, R ²² represents a hydrogen atom, a carboxyl group, an aldehyde group or an acyl group, and R ²³ represents It represents a carboxyl group, an aldehyde group or a hydroxyl group.)]

Here, the number of carbon atoms of the alkyl group of R ¹ in the formula (1) is preferably 1 to 5, more preferably 1 to 3, and from the viewpoint of containing a bio-related substance, R ¹
Is a methyl group, that is, a compound in which a methyloxy group is bonded to an indole skeleton is important. Further, when R ² in the formula (1) is represented by the formula (2) or (3), R 2
The carbon number of ²¹ is preferably 1 to 5, more preferably 1 or 2, and when R ²² is an acyl group, those having preferably 1 to 5, more preferably 1 or 2 are mentioned.

More specifically, 5-alkoxyindoles represented by the formula (1) include 5-methoxytryptophan, 5-methoxytryptamine, 5-methoxyindole-3-acetic acid, 5-methoxyindole-3-acetamide , 5-methoxytryptophor or melatonin, which may be used alone or in combination of two or more.

These are particularly important as biologically-related substances, and 5- (5-)
The conversion to hydroxyindoles is performed well, and the selectivity in fluorescent derivatization with aromatic aminomethanes is excellent. Further, among the 5-alkoxyindoles represented by the formula (1), although they do not exist in vivo, they have high selectivity in fluorescent derivatization, and
-Methoxyindole-3-carboxylic acid or 5-methoxyindole-aldehyde is also suitable as a measurement target in the present invention.

<Quinonimine Derivative> In the present invention, a quinoneimine derivative is produced by electrolytically oxidizing a 5-alkoxyindole by electrolyzing a solution containing the above-mentioned test sample containing the 5-alkoxyindole at an anodic oxidation potential. Let me know. The quinone imine derivative is a quinone imine derivative in which the R ¹ group is removed from the 5-alkoxyindole molecule represented by the formula (1), that is, a quinone imine derivative represented by the following formula (4).

[0046]

Embedded image [Wherein, R ² is the same as in the above formula (1). ]

Here, R ² in the formula (4) corresponding to the above-mentioned suitable 5-alkoxyindoles is -C
_{H 2 CH 2 NHCOOH, -CH 2} CH 2 NH 2, -CH 2 C
_{OOH, -NHCOCH 3, -CH 2 CH} 2 OH, -CH 2
A group represented by CH ₂ NHCOCH ₃ ;

<5-Hydroxyindole> In the present invention, the solution containing the quinoneimine derivative is electrolyzed at the cathodic reduction potential, whereby the quinoneimine derivative is electrolytically reduced to produce 5-hydroxyindole.
Examples of the 5-hydroxyindoles include those represented by the following formula (5) in which a hydrogen atom is bonded to an oxygen atom and a nitrogen atom in the molecular skeleton of the quinone imine derivative.

[0049]

Embedded image [Wherein, R ² is the same as in the above formula (1). ]

Here, 5-hydroxyindoles corresponding to the preferred 5-alkoxyindoles described above include 5-hydroxytryptophan, serotonin, and 5-hydroxyindole.
-Hydroxyindole-3-acetic acid, 5-hydroxyindole-3-acetamide, 5-hydroxytryptophor or N-acetylserotonin, which may be used alone or in combination of two or more. .

<Aromatic Aminomethanes> In the present invention, aromatic aminomethanes and an oxidizing agent are added to a measurement sample containing the above-mentioned 5-hydroxyindoles to oxidize the 5-hydroxyindoles. Fluorescent derivatization. As the aromatic aminomethanes used in the present invention, those represented by the following formula (6) can be preferably used.

[0052]

Embedded image [Wherein, X represents an aromatic hydrocarbon, an aromatic heterocyclic hydrocarbon, or an aromatic condensed polycyclic hydrocarbon. ]

Among these aromatic aminomethanes, those in which X in the above formula (6) is a substituted or unsubstituted aromatic hydrocarbon, that is, benzylamines represented by the following formula (8) are used. And more preferably, references J. Ishida, R. Iizuk
a, M. Yamaguchi, Clin. Chem., 39, 2355-2356 (1933) reports a highly sensitive method for fluorescent derivatization of 5-hydroxyindoles with such benzylamines.

[0054]

Embedded image [Wherein R ³ represents a hydrogen atom, an alkyl group, an alkoxyl group, a halogen group, a hydroxyl group, a sulfamoyl group,
An alkylamino group; n represents an integer of 1 to 5;
When R is 2 or more, R ³ may be the same or different. ]

Further, among these benzylamines, for example, benzylamine, 4-methoxybenzylamine, 3,4-dimethoxybenzylamine, 4-
Methylbenzylamine, 4-hydroxybenzylamine, 4-chlorobenzylamine, 4-dimethylaminobenzylamine, 4-sulfamoylbenzylamine (R
³ is NH ₂ SO ₂ —, and n is 1).

As the aromatic aminomethanes other than benzylamines, for example, in the formula (6), X represents an aromatic heterocyclic ring from the viewpoint of the reactivity of the fluorescent derivative formation reaction and industrial applicability. 2-aminomethylpyridine, 3-aminomethylpyridine, 4-aminomethylpyridine, 2-aminomethylbenzimidazole, pyridoxamine and the like which are formula hydrocarbons are useful, and in the formula (6), X is an aromatic condensed polycyclic ring. As a compound of formula hydrocarbon, 1-aminomethylpyrene and the like can be preferably used.

These aromatic aminomethanes are generally easy to obtain with high reagent purity, and are rich in the reactivity of the formation of the fluorescent derivative in the presence of an oxidizing agent. It is a suitable labeling reagent.
It is preferable that the aromatic aminomethanes are dissolved in a solvent that is miscible with the solution containing the analyte and then added to the solution containing the analyte. In particular, when a solution in which the analyte is added, dissolved, or dispersed in a buffer solution or the like is used, it is more preferable to use a predetermined organic solvent miscible with the buffer solution or the like as the solvent for the aromatic aminomethanes. Specific examples of such a predetermined organic solvent include, for example, acetonitrile, methanol, ethanol, 2-propanol, acetone, dimethylformamide, dimethylsulfoxide and the like.

<Oxidizing Agent> The oxidizing agent usable in the present invention is not particularly limited, and various organic oxidizing agents or inorganic oxidizing agents can be used. For example, various organic peroxides such as peracetic acid, perbenzoic acid, and 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 are suitable. Among these oxidizing agents, it is preferable to use hexacyanoferrate (III) (for example, potassium salt, sodium salt, etc.), hydrogen peroxide, organic peroxide, inorganic peroxide and the like.

<Fluorescent Derivative> The fluorescent derivative obtained by reacting the above-mentioned 5-hydroxyindole with an aromatic aminomethane in the presence of an oxidizing agent is specifically represented by the following formula (7). Such a material is capable of absorbing light having a predetermined wavelength (for example, ultraviolet light) and emitting fluorescence by electronic transition in a molecule.

[0060]

Embedded image [Wherein, R ² is the same as in the above formula (1);
X is the same as in the above formula (6). ]

These fluorescent derivatives are very stable in solution, and at a concentration of 0.01 to 10 μM, no substantial change is observed even for about one week. Further, the molecular structure of the fluorescent derivative can be confirmed by infrared absorption spectrum, nuclear magnetic resonance absorption spectrum, mass spectrometry, thin-layer chromatography, liquid chromatography, etc., for the residue obtained by removing the solvent after the derivatization reaction. .

FIG. 2 is a block diagram showing a schematic configuration of a first embodiment of a 5-alkoxyindole measuring apparatus according to the present invention. As shown in FIG.
0 (a measuring device for 5-alkoxyindoles) is arranged in an oxidation cell 6 (electrolytic oxidation section) to which an analyte solution and an eluent such as a buffer are mixed and supplied, and in a stage subsequent to the oxidation cell 6. An electrolysis device 8 is connected to the reduction cell 7 (electrolysis reduction section), and the reduction cell 7 is provided with a reaction tank 11 (derivative generation section) and a cooling tank 12 in order, and a fluorescence detection section 20 is arranged at the latter stage thereof. It was done. The eluent is supplied from the eluent supply unit 1 to the guard cell 2 by the pump P1, and an analyte solution is injected from an analyte injection unit 3 provided downstream of the guard cell 2 and added thereto. Then, it is successively eluted into the oxidation cell 6 by flowing down the HPLC column 5 (separation section) sequentially.

The oxidation cell 6 has an electrode for electrolysis,
A predetermined potential is applied from the electrolysis device 8 to the anode, and the 5-alkoxyindole contained in the mixed solution is electrolytically oxidized. The electrolysis device 8 may be any device that can independently adjust the electrode potentials of the oxidation cell 6 and the reduction cell 7. For example, the electrolysis device 8 has a plurality of potentiometers or a coulometric electrochemical detection with high electrolysis efficiency. A vessel or the like can be used. In order to prevent the oxidation cell 6 from being contaminated with a reducing substance (substance to be oxidized) mixed as an impurity in the mobile phase, a voltage substantially equal to the anode potential in the oxidation cell 6 is also applied from the electrolysis device 8 to the guard cell 2. ing.

The material of the anode provided in the oxidation cell 6 is not particularly limited, but an electrode having a large oxygen overvoltage is preferable in order to suppress generation of oxygen from the anode. In addition, it is desirable to use an insoluble electrode in order to suppress a decrease in current efficiency or electrolytic efficiency due to dissolution of the anode. Examples of such an anode material include graphite (such as porous graphite), platinum, platinum-coated titanium or zirconium, and lead peroxide. In particular, since the oxidation potential of 5-alkoxyindoles is high, it is preferable to use an insoluble electrode to increase current efficiency.

The quinone imine derivative produced by the electrolytic oxidation is present in the mixed solution without being fixed or deposited on the anode, and this mixed solution is sent to the reduction cell 7 and electrolyzed in the reduction cell 7. The reduction cell 7 has an electrode for electrolysis, a predetermined potential is applied to the cathode from the electrolysis device 8, and the quinone imine derivative contained in the mixed solution is electrolytically reduced. The 5-hydroxyindole produced by the electrolytic reduction exists in the mixed solution without being fixed or deposited on the cathode, and the mixed solution is sent out from the reduction cell 7.

A three-way joint 9 is provided between the reduction cell 7 and the reaction tank 11.
, A mixed reagent of an aromatic aminomethane, its solvent and an oxidizing agent is added to the above mixed solution from a reagent supply unit 10 by a pump P 2, and this mixed solution is supplied to a reaction coil 11 a disposed in a reaction vessel 11. Supplied to The reaction tank 11 contains a heat medium such as water, and the heat medium is heated by a heating unit (not shown). Reaction coil 11
The mixture containing 5-hydroxyindoles passing through a is heated indirectly by a heated heat medium, and the 5-hydroxyindoles are oxidized to fluorescent derivatives.

The cooling tank 12 provided at the subsequent stage of the reaction tank 11
Is for cooling the mixed liquid sent through the reaction coil 11a of the reaction tank 11, and contains therein a refrigerant such as water, and the cooling coil 12a is arranged in the refrigerant. The cooling tank 12 has a cooling unit (not shown) for cooling the refrigerant, and the liquid mixture containing the fluorescent derivative passing through the cooling coil 12a is indirectly cooled by the cooled refrigerant. In addition, depending on the cooling temperature, air cooling may be used without particularly requiring a cooling unit.

The fluorescence detector 20 provided at the subsequent stage of the cooling tank 12 emits from an excitation light source 21 for emitting excitation light L1 such as ultraviolet light, and a fluorescent derivative in the mixed solution by irradiation with the excitation light L1. A light detector 22 for detecting the fluorescence L2 is provided. The control analysis unit 13 connected to the excitation light source 21 and the photodetector 22 of the fluorescence detection unit 20
1 controls the emission of the excitation light L1 and the operation of the photodetector 22, and analyzes and records the fluorescence signal detected by the photodetector 22, and outputs the result.

A first embodiment of the method for measuring 5-alkoxyindoles according to the present invention using the measuring apparatus 100 configured as described above will be described. First, a mixed solution of an organic solvent and a buffer is accommodated in the eluent supply unit 1 as an eluent, and the eluent is supplied under pressure to the guard cell 2 and thereafter by operating the pump P1. A sample solution containing a plurality of 5-alkoxyindoles and a plurality of 5-hydroxyindoles is injected from the sample injection section 3 and mixed with an eluent, and the mixed solution is placed in a guard column 4 and an HPLC column 5. Let flow down at the flow rate. At this time, it is preferable to adjust the pH of the mixture of the analyte solution and the eluent to be 5.5 to 7.

The guard column 4 and the HPLC column 5 are provided as an adsorbent, for example, silica gel used in ordinary HPLC or silica gel with a functional group bonded to a base material, for example, octadecyl silica gel (O
DS) or the like. As the adsorbent, those having a polarity and a pore size suitable for 5-alkoxyindoles and 5-hydroxyindoles can be appropriately selected and used. As the form of the column, a distribution / adsorption column or the like can be used, and for example, an ODS reverse phase column is preferably mentioned. As a solvent for the mobile phase (the above-described eluent), various buffers, acetonitrile, methanol, a mixed solvent thereof, and the like can be preferably used.

In this way, the mixed solution was applied to the HPLC column 5
To separate each 5-alkoxyindole and each 5-hydroxyindole contained in the mixture (separation step). Here, the guard column 4 and the HP
The LC column 5 is housed in a thermostat, and the temperature of the HPLC column 5 is preferably room temperature to 80 ° C, more preferably 40 to 80 ° C.
It is preferable to keep the temperature constant within the range of 60 ° C.

Each separated 5-alkoxyindole and each 5-hydroxyindole were separated by HPLC column 5
And sequentially sent to the oxidation cell 6 for each separated fraction. Next, the oxidation potential of the electrode of the oxidation cell 6 is preferably +1.0 to +2.0 V, and more preferably ++.
A voltage is applied so as to be 1.5 to +2.0 V to electrolytically oxidize 5-alkoxyindoles. At this time, it is preferable that the temperature of the mixed solution in the oxidation cell 6 be a temperature at which the electrolysis efficiency or the current efficiency is maintained high depending on the electrolysis conditions. Thus, the above-mentioned quinone imine derivatives are sequentially generated from each 5-alkoxyindole (oxidation step).

Next, the mixed solution containing the quinone imine derivative is continuously sent to the reduction cell 7. A voltage is applied to the electrode of the reduction cell 7 so that the reduction potential is preferably −2.0 to 0.0 V, more preferably −2.0 to −1.0 V, and the quinone imine derivative is electrolytically reduced. At this time, it is preferable that the temperature of the mixed solution in the reduction cell 7 be a temperature at which the electrolysis efficiency or the current efficiency is maintained high according to the electrolysis conditions. In this way, the above-mentioned 5-hydroxyindoles are sequentially generated from the quinone imine derivative (reduction step).

Next, the mixed solution containing 5-hydroxyindoles obtained by the electrolytic reduction is supplied from the reagent supply unit 10 via the three-way joint 9 by the pump P2 to the aromatic aminomethanes, their solvents and their oxidation. Mix the reagent with the agent. Here, the amount of aromatic aminomethane used is
At least about 10 to 5-hydroxyindoles
It is preferable that the amount is equal to or more than 0 times equivalent. The oxidizing agent is desirably used at a concentration of at least 1 mmol / L.

Further, the volume mixing ratio of the mixed solution and the mixed reagent delivered from the reduction cell 7 varies depending on the concentrations of 5-alkoxyindoles and 5-hydroxyindoles in the sample, the type of eluent, and the like. At this time, it is preferable that the pH of the mixed solution after mixing the mixed reagents is 8 to 10. Then, the mixed solution is supplied to the reaction tank 11.
Is sent to the reaction coil 11a disposed therein. The reaction tank 11 contains water as a heat medium, and is heated and maintained at a constant temperature of preferably room temperature to 100 ° C, more preferably 60 to 80 ° C by a heating unit. By doing so, 5-hydroxyindoles are oxidized to fluorescent derivatives for each fraction separated by the HPLC column 5 (derivative generation step).

Next, the mixed solution containing the fluorescent derivative is sent to the cooling coil 12 a of the cooling tank 12. 0 ° C. in the cooling tank 12
Cooled water is stored in the vicinity, and the mixed liquid is cooled, and then the mixed liquid is introduced into the fluorescence detector 20. Then, the mixed light is irradiated with the excitation light L1 from the excitation light source 21.
The excitation light L1 has, for example, a peak wavelength of 345 nm.
And the excitation light source 21 can emit such excitation light L1.
It is not particularly limited, and may be a lamp light source or a laser light source. The irradiation of the excitation light L1 may be either pulse irradiation or continuous (CW) irradiation.

When ultraviolet light having a wavelength of 345 nm is irradiated as the excitation light L1, a wavelength of 481 nm
(In the case of a fluorescent derivative derived from melatonin or its metabolite)
Is emitted. The photodetector 22 is not particularly limited as long as it can detect such wavelength light in the visible region. For example, a normal fluorescence detector or a fluorescence measurement device, in particular, fluorescence combined with HPLC A detector can be used. Further, in order to increase the detection resolution of the specific wavelength light, a band-pass filter, a spectroscope, and the like may be disposed in front of the detection unit of the photodetector 22. Thus,
The mixture is irradiated with the excitation light L1 from the excitation light source 21, and the fluorescence emitted from the mixture is detected by the photodetector 22 (fluorescence detection step).

The fluorescence detection signal detected by the photodetector 22 is converted into an electric signal and output to the control analyzer 13. The control analysis unit 13 obtains the detection signal intensity and the like at each measurement time, and outputs detector response data in the form of a chromatogram usually obtained by chromatography such as HPLC, and calculates the area under the peak. Will be Then, based on these results, 5-
Measurement such as qualitative or quantitative determination of alkoxyindoles or 5-hydroxyindoles is performed.

The measuring apparatus 10 having the configuration as described above
According to the method for measuring 5-alkoxyindoles of the present invention, 5-hydroxyindoles that can be fluorescently derivatized by a simple method of electrolytically oxidizing 5-alkoxyindoles and then electrolytically reducing them. Is generated. By detecting the fluorescence from the fluorescent derivative generated from the 5-hydroxyindole, 5-
Indirect measurements of alkoxyindoles can be made. Therefore, simple and highly sensitive measurement of 5-alkoxyindoles, which was difficult to perform with high sensitivity by the conventional HPLC method, becomes possible.

Further, the fluorescence derivatization reaction of 5-hydroxyindoles is excellent in selectivity, and even if other low-molecular substances are mixed, there is very little possibility that they are fluorescently derivatized. Therefore, it becomes possible to selectively measure 5-alkoxyindoles. Furthermore, since the emission of fluorescence having a wavelength specific to the fluorescent derivative is detected by irradiating the fluorescent derivative with excitation light, the selectivity of 5-alkoxyindoles is further enhanced and the background noise is sufficiently reduced. In addition, extremely sensitive measurement becomes possible.

Further, since highly selective and highly sensitive measurement of such 5-alkoxyindoles can be easily performed, it is not necessary to use a radioisotope as in the conventional RIA. Therefore, there are no restrictions such as disposal of equipment and radioactive waste. Furthermore, as described above, interference with other substances can be sufficiently suppressed, so that the risk of overestimating the amount of 5-alkoxyindoles by RIA can be sufficiently reduced. Therefore, highly accurate measurement is possible. In addition, the responsiveness of the luminescence reaction by irradiating the fluorescent derivative with excitation light is fast, and high-sensitivity measurement by light detection is performed, so that the analysis (measurement) time is significantly reduced as compared with the conventional GC-MS method. It does not require complicated operations such as derivatization to volatile substances. As a result, the cost required for measurement and the required amount of the subject can be reduced.

In addition, 5-alkoxyindoles are
-Electrooxidation and electrolytic reduction are used in combination to convert to hydroxyindoles, so no oxidizing agent or reducing agent is required. Therefore, as compared with the case where these reagents are used, the processing can be simplified and the cost can be reduced. Moreover, as compared to using an oxidizing agent or a reducing agent, stable oxidation and reduction reactions can be easily maintained, and the purity of the reaction product can be increased. Therefore, measurement excellent in quantitative correlation between the finally generated fluorescent derivative and the 5-alkoxyindole can be performed.

In the oxidation step, the oxidation potential during electrolytic oxidation is set to +1.0 to +2.0 V, and in the reduction step, the reduction potential during electrolytic reduction is set to −2.0 to 0.0 V.
In this case, there are the following advantages. That is, when the oxidation potential is +1.0 V or more, the oxidation reaction at the anode can proceed steadily and the current efficiency can be improved. On the other hand, when the oxidation potential is set to +2.0 V or less, the increase in overvoltage can be suppressed, and the decomposition of 5-alkoxyindoles can be prevented. When the reduction potential is 0.0 V or less, the reduction reaction at the cathode can proceed steadily and the current efficiency can be improved. On the other hand, when the oxidation potential is -2.0 V or more, the increase in overvoltage can be suppressed, and the decomposition of the quinone imine derivative can be prevented.

Further, the oxidation cell 6 and / or the reduction cell 7
If an electrode having a large oxygen overvoltage or an insoluble electrode is used as the electrode, particularly the anode constituting the oxidation cell 6, the anode potential can be made extremely noble, so that the above-mentioned suitable oxidation potential can be easily achieved, and the current efficiency can be maintained well. it can. Further, before the electrolytic oxidation step, 5-alkoxyindoles and 5-hydroxyindoles are separated using the HPLC column 5, so that a plurality of types of 5-alkoxyindoles and / or 5-hydroxyindoles are contained in the sample. When hydroxyindoles are included, they can be separated and measured separately.

Furthermore, even if 5-hydroxyindoles are contained in the sample, it is possible to discriminate the 5-alkoxyindoles from the 5-hydroxyindoles formed by electrolytic reduction and electrolytic oxidation. Therefore, each 5-
The measurement accuracy of the alkoxyindoles and each of the 5-hydroxyindoles is improved, and simultaneous measurement using the same test sample becomes possible, so that quick measurement can be performed. Therefore, it is possible to improve the processing performance related to the measurement of 5-alkoxyindoles.

FIG. 3 is a block diagram showing a schematic configuration of a second embodiment of the measuring apparatus for 5-alkoxyindoles according to the present invention. As shown in FIG.
0 (a measuring device for 5-alkoxyindoles) is HP
LC system, ie guard cell 2, guard column 4 and HP
Except for not having the LC column 5, it has the same configuration as the measuring device 100 shown in FIG.

In such a measuring device 200, the total amount of 5-alkoxyindoles contained in the mixture of the test solution and the eluent is measured. In addition, 5-
Even when hydroxyindoles are contained, 5-
The total amount of alkoxyindoles and 5-hydroxyindoles can be measured. At this time, the separation time by the HPLC method is reduced, so that the measurement time can be further reduced. Therefore, for example, a demand for quickly and accurately measuring the total amount of melatonin-related substances contained in a trace amount in a biological sample as a subject can be sufficiently satisfied.

FIG. 4 is a block diagram showing a schematic configuration of a third embodiment of the measuring apparatus for 5-alkoxyindoles according to the present invention. As shown in FIG.
0 (a measuring device for 5-alkoxyindoles) is shown in FIG.
The guard column 4 and the HPLC column 5 between the reduction cell 7 and the three-way joint 9 in the measuring device 200 shown in FIG.
Are arranged in this order.

Using the measuring device 300 configured as described above, when a plurality of types of 5-alkoxyindoles are contained in a subject, it is possible to separate and measure each of the 5-alkoxyindoles separately. It is. Further, when 5-hydroxy indoles to the subject is further included can be determined based on R ² shown in among expression of 5-alkoxy indoles and 5-hydroxy indoles (1) can be discriminated different.

In the measuring devices 100, 200, and 300, a deaerator may be provided between the pump P1 and the eluent supply unit 1. In this case, the baseline of the HPLC response peak due to the gas contained in the eluent can be sufficiently stabilized. Further, the measuring device 300 shown in FIG.
In this case, the guard column 4 and the HPLC column 5 may be arranged between the three-way joint 9 and the reaction tank 11 or between the cooling tank 12 and the fluorescence detector 20.

[0091]

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> [Measurement of a test solution containing melatonin and 5-methoxyindole-3-acetic acid which is a main metabolite of melatonin] FIG. 2
The melatonin and 5-methoxyindole-
Analysis of 3-acetic acid was performed. (1) Sample solution: Equivalent mixed solution of melatonin and 5-methoxyindole-3-acetic acid (200 nmol each)
/ ML) 20 μL (2) HPLC column 5: manufactured by Shiseido Co., Ltd .; SHISEIDO C
APCELL PAK C18 UG80 (inner diameter 4.6mmφ x effective height 1
(3) Eluent: a mixture of acetonitrile (15 vol%), methanol (10 vol%), and 250 mmol / L sodium acetate buffer (pH 6.0, 75 vol%) (4) Mobile phase flow rate in HPLC column 5: 1.0 mL /
(5) Electrolyzer 8: manufactured by Esa; Coulochem II, potential of guard cell 2 +1.5 V, anode of porous cell 6 (porous graphite electrode) oxidation potential +1.5 V, cathode of reduced cell 7 (porous graphite electrode) Reduction potential -1.5 V (6) Temperature of mixture of analyte solution and eluent: room temperature, p
H: 6.0 (7) Mixed reagent: acetonitrile (50 vol%) containing 20 mmol / L benzylamine and 3 mmol / L potassium hexacyanoferrate (III), and 10 mm
ol / L borate buffer (pH 11, 50 vol%) mixed solution, supply flow rate 0.5 mL / min. (8) Reaction tank 11: hot water bath temperature 70 ° C., reaction coil 11 a
(Inner diameter 0.5mm, length 10m, material is stainless steel, Teflon or polyetheretherketone (PE
EK)) (9) Cooling bath 12: water bath temperature 0 ° C, cooling coil 12a
(Inner diameter 0.5mm, length 10m, material is stainless steel, Teflon or polyetheretherketone (PE
EK)) (10) Fluorescence detector 20: manufactured by Hitachi, Ltd .; fluorescence detector L-7480, peak wavelength of excitation light L1 (excitation wavelength) 34
5 nm, wavelength 481 nm of fluorescence L2

Under such conditions, the detector response signal obtained by the photodetector was processed by the control analyzer 13 to obtain a chromatogram shown in FIG. 5 (in the figure, the vertical axis represents an arbitrary unit (a.
u. ). The same applies hereinafter). As shown in FIG. 5, melatonin and 5-methoxyindole-3-acetic acid were well separated and each was detected as a single peak. The measurement was completed in a very short time of about 12 minutes. Further, the concentration of each component in the test solution was changed to perform quantification based on the amount of fluorescence emission, and the detection limit was determined from a calibration curve prepared in advance. As a result, the detection limit at S / N = 3 (3σ detection) The limit was 1 pmol or less per 20 μL injection volume of the test solution. From this, it was confirmed that the measurement of 5-alkoxyindoles with extremely high sensitivity was possible according to the present invention.

Further, various saccharides,
When keto acids, amino acids, nucleic acid bases, peptides, oligonucleotides, steroids, polyamines, carboxylic acids, alcohols, aldehydes, etc. were injected, no corresponding fluorescence emission peak was observed (below the detection limit). T). From this, according to the present invention, 5-
It has been confirmed that measurement with high selectivity of alkoxyindoles is possible.

<Example 2> [Measurement of a sample solution containing melatonin, serotonin which is a precursor of melatonin, and N-acetylserotonin]
As an analyte solution, an equivalent mixed solution of melatonin, serotonin and N-acetylserotonin (each 25 nmo)
1 / mL) in the same manner as in Example 1 except that 20 μL of melatonin, serotonin and N
-Analysis of acetylserotonin was performed. FIG. 6 shows a chromatogram obtained by processing the detector response signal obtained by the photodetector under such conditions by the control analyzer 13.
As shown in FIG. 6, melatonin, serotonin and N-acetylserotonin were well separated and detected as single peaks. The measurement was completed in a very short time of about 12 minutes.

When the detection limit (3σ detection limit) at S / N = 3 was determined in the same manner as in Example 1, it was 1 pmol or less per 20 μL injection amount of the test solution.
From this, it was confirmed that according to the present invention, 5-alkoxyindoles and 5-hydroxyindoles can be measured simultaneously and with extremely high sensitivity. further,
As in Example 1, when various sugars, keto acids, amino acids, nucleobases, peptides, oligonucleotides, steroids, polyamines, carboxylic acids, alcohols, aldehydes, and the like were injected into the HPLC column 5, fluorescence corresponding to these was injected. Was not observed (below the detection limit).

Example 3 The same sample solution as used in Example 1 was injected into the measuring device 200 shown in FIG.
Analysis of melatonin and 5-methoxyindole-3-acetic acid in the test solution was performed under the same conditions as in Example 1 except that the component separation by the LC column 5 was not performed.
The total amount of melatonin and 5-methoxyindole-3-acetic acid determined from the amount of fluorescence emission was determined individually from the chromatogram shown in FIG.
It was equivalent to the total amount of methoxyindole-3-acetic acid.

Example 4 The same sample solution as used in Example 2 was injected into the measuring device 200 shown in FIG.
Under the same conditions as in Example 1 except that the component separation by the LC column 5 was not performed, melatonin in the test solution,
Serotonin and N-acetyl serotonin were analyzed. The total amount of melatonin, serotonin and N-acetylserotonin determined from the amount of fluorescence emission was obtained by using FIG.
Melatonin individually quantified from the chromatogram shown in
It was equivalent to the total amount of serotonin and N-acetyl serotonin.

<Comparative Example 1> Example 1 was repeated except that no voltage was applied to the oxidation cell 6 and the reduction cell 7 of the measuring apparatus 100 shown in FIG. 2, that is, electrolytic oxidation and electrolytic reduction were not performed. The analysis of melatonin and 5-methoxyindole-3-acetic acid in the test solution was attempted in the same manner as described above. However, no fluorescence emission peak as shown in FIG. 5 appeared on the chromatogram, and it was impossible to measure melatonin and 5-methoxyindole-3-acetic acid.

Comparative Example 2 Melatonin and 5-methoxyindole in a test solution were prepared in the same manner as in Example 1 except that the mixed reagent was not added and the derivatization step for generating a fluorescent derivative was not performed. An attempt was made to analyze 3-acetic acid. However, no fluorescence emission peak as shown in FIG. 5 appeared on the chromatogram, and it was impossible to measure melatonin and 5-methoxyindole-3-acetic acid.

[0101]

As described above, according to the present invention, 5-
According to the method for measuring alkoxyindoles, 5-alkoxyindoles contained in a biological sample or the like can be measured with higher sensitivity and higher precision than before, without performing complicated pretreatment. Further, according to the present invention, 5-alkoxyindoles that can be measured with high sensitivity and high accuracy compared to conventional methods can be used for 5-alkoxyindoles contained in biological samples without the need for complicated pretreatment. A measuring device can be obtained.

[Brief description of the drawings]

FIG. 1 shows an electrolytic oxidation reaction of a 5-alkoxyindole, an electrolytic reduction reaction of a quinone imine derivative, and 5
It is a figure which shows the fluorescent derivative production | generation reaction of-hydroxyindole, and the reaction path | route of fluorescence emission by excitation light irradiation to a fluorescent derivative.

FIG. 2 is a block diagram showing a schematic configuration of a first embodiment of a 5-alkoxyindole measuring device according to the present invention.

FIG. 3 is a block diagram showing a schematic configuration of a second embodiment of a 5-alkoxyindole measuring device according to the present invention.

FIG. 4 is a block diagram showing a schematic configuration of a third embodiment of the measuring device for 5-alkoxyindoles according to the present invention.

FIG. 5 is a chromatogram of 5-alkoxyindoles obtained in Example 1.

FIG. 6 is a chromatogram of 5-alkoxyindoles and 5-hydroxyindoles obtained in Example 2.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Eluent supply part, 2 ... Guard cell, 3 ... Object injection part, 4 ... Guard column, 5 ... HPLC column (separation part), 6 ... Oxidation cell (electrolytic oxidation part), 7 ... Reduction cell (electrolytic reduction part) ), 8 ... electrolyzer, 9 ... three-way joint, 10
... Reagent supply unit, 11 ... Reaction tank (derivative generation unit), 11a
... Reaction coil, 12 ... Cooling tank, 12a ... Cooling coil, 1
3: Control analysis unit, 20: Fluorescence detection unit, 21: Excitation light source,
22 photodetector, 100, 200, 300 measuring device (measuring device for 5-alkoxyindoles), L1 excitation light, L2 fluorescence, P1, P2 pump.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 27/52 F term (Reference) 2G042 AA01 BD03 BD12 BD16 BD19 CA10 DA03 DA10 EA20 FA04 FB02 2G045 AA40 BA20 BB60 CA25 CB01 CB03 DA54 FA27 FA29 FB12 GC15 2G054 AA06 BB04 CA21 CB01 CD01 EA03 EB01 GA04 GB02 JA06

Claims (10)

[Claims] 1. A method for measuring 5-alkoxyindoles in a subject, the method comprising electrolytically oxidizing the 5-alkoxyindoles to obtain the 5-alkoxyindoles.
An oxidation step of producing a quinone imine derivative corresponding to the alkoxyindole; a reduction step of electrolytically reducing the quinone imine derivative to produce a 5-hydroxyindole corresponding to the 5-alkoxyindole; A derivative generating step of generating a fluorescent derivative by reacting an aromatic aminomethane with an oxidizing agent; irradiating the fluorescent derivative with excitation light; and detecting fluorescence from the fluorescent derivative due to the irradiation of the excitation light. A fluorescence detection step;
A method for measuring 5-alkoxyindoles, comprising: 2. The 5-alkoxyindole is represented by the following formula (1): [Wherein, R ¹ represents an alkyl group, R ² represents an acetamido group, a carboxyl group, or the following formula (2) or (3)
Represents a group represented by Embedded image (Wherein, R ²¹ represents a methylene group, an alkylene group or a polymethylene group, R ²¹ may be different or the same, R ²² represents a hydrogen atom, a carboxyl group, an aldehyde group or an acyl group, and R ²³ represents A carboxyl group, an aldehyde group or a hydroxyl group.)], Wherein the quinone imine derivative and the 5-hydroxyindole are represented by the following formulas (4) and (5), respectively: [In the formula, R ² represents an acetamido group, a carboxyl group, an aldehyde group or a group represented by the above formula (2) or (3). Wherein each of the aromatic aminomethanes is represented by the following formula (6): [Wherein, X represents an aromatic hydrocarbon, an aromatic heterocyclic hydrocarbon, or an aromatic condensed polycyclic hydrocarbon. Wherein the fluorescent derivative is represented by the following formula (7): [Wherein, X represents an aromatic hydrocarbon, an aromatic heterocyclic hydrocarbon, or an aromatic condensed polycyclic hydrocarbon. 5. The method according to claim 1, wherein
Method for measuring alkoxyindoles. 3. When the subject contains a plurality of types of 5-alkoxyindoles, the respective 5-alkoxyindoles and / or the respective 5-hydroxyindoles corresponding to the 5-alkoxyindoles are separated. The method for measuring 5-alkoxyindoles according to claim 1 or 2, further comprising a separation step. 4. In the oxidation step, the oxidation potential when performing electrolytic oxidation is +1.0 to +2.0 V, and in the reduction step, the reduction potential when performing electrolytic reduction is −2.0 to 0.0 V.
The method for measuring 5-alkoxyindoles according to any one of claims 1 to 3, characterized in that: 5. The method according to claim 1, wherein the subject is selected from the group consisting of 5-methoxytryptophan, 5-methoxytryptamine, 5-methoxyindole-3-acetic acid, 5-methoxyindole-3-acetamide, 5-methoxytryptophor or melatonin. The one containing at least one selected 5-alkoxyindole is used, and as the 5-hydroxyindole, 5-hydroxytryptophan, serotonin, 5-hydroxyindole-3-acetic acid, 5-hydroxyindole-3-acetamide, 5 At least one selected from the group consisting of -hydroxytryptophor and N-acetylserotonin.
5. The method for measuring 5-alkoxyindoles according to any one of 4. 6. An apparatus for measuring 5-alkoxyindoles in a subject, wherein the quinoneimine corresponding to the 5-alkoxyindole is accommodated by accommodating the subject and electrolytically oxidizing the 5-alkoxyindole. An electrolytic oxidation unit in which a derivative is produced; an electrolytic reduction unit in which the quinone imine derivative is electrolytically reduced to produce 5-hydroxyindoles corresponding to the 5-alkoxyindoles; the 5-hydroxyindoles; A derivative generating unit that generates a fluorescent derivative by a reaction between an aromatic aminomethane and an oxidizing agent; irradiating the fluorescent derivative with excitation light; and detecting fluorescence emitted from the fluorescent derivative by the irradiation of the excitation light. A fluorescence detector, comprising: a device for measuring 5-alkoxyindoles. 7. The 5-alkoxyindole is represented by the formula (1), the quinone imine derivative is represented by the formula (4), and the 5-hydroxyindole is A compound represented by the formula (5), wherein the aromatic aminomethane is a compound represented by the formula (6), and the fluorescent derivative is a compound represented by the formula (7):
The apparatus for measuring 5-alkoxyindoles according to claim 6, wherein: 8. When the subject contains a plurality of types of 5-alkoxyindoles, the respective 5-alkoxyindoles and / or the respective 5-hydroxyindoles corresponding to the 5-alkoxyindoles are separated. The device according to claim 6, further comprising a separation unit.
A device for measuring alkoxyindoles. 9. The electrolytic oxidizing section is capable of setting the oxidation potential of the 5-alkoxyindole to +1.0 to +2.0 V. The electrolytic reducing section is configured to reduce the quinone imine derivative. Can be set to -2.0 to 0.0V.
An apparatus for measuring 5-alkoxyindoles according to any one of claims 6 to 8, characterized in that: 10. The 5-alkoxyindole,
At least one selected from the group consisting of 5-methoxytryptophan, 5-methoxytryptamine, 5-methoxyindole-3-acetic acid, 5-methoxyindole-3-acetamide, 5-methoxytryptophor and melatonin; At least the 5-hydroxyindole is selected from the group consisting of 5-hydroxytryptophan, serotonin, 5-hydroxyindole-3-acetic acid, 5-hydroxyindole-3-acetamide, 5-hydroxytryptophor and N-acetylserotonin. The apparatus for measuring 5-alkoxyindoles according to any one of claims 6 to 9, wherein the apparatus is one.