JP2001027632A - Method for measuring melatonin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To reduce a maintenance frequency for an apparatus without a cross reaction to similar compounds by bringing a 5-methoxyindole compound of a specific formula into contact with a reagent of a specific formula for forming a fluorescent derivative before or after separating the 5-methoxyindole compound by a column. SOLUTION: Before or after a 5-methoxyindole compound expressed by a formula is separated by a column, the compound is brought in contact with a reagent of a formula II for forming a fluorescent derivative, whereby the compound is turned to a fluorescent derivative which is then measured by liquid chromatography (R1: a hydrogen atom or hydroxyl group, M1: a side chain, Ar: an aryl group, L: a leaving group). The 5-methoxyindole compound to be measured can exemplify melatonin having the hydrogen atom R1 and side chain M1 of a 2-methyl amide ethyl group. A phenyl group, substitution phenyl group, naphthyl group or the like is used as the aryl group Ar, and a hydrogen atom, cyano group, nitro group or the like is used as the leaving group. A general liquid chromatograph apparatus can be used as it is and the fluorescent derivative can be detected by a fluorescence detector according to the method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring 5-methoxyindole compounds contained in samples such as tissue extracts, serum, plasma, urine and the like.

[0002]

2. Description of the Related Art A compound represented by the general formula (1) (wherein R1 represents a hydrogen atom or a hydroxyl group, and M1 represents a side chain)
- Among methoxyindole compounds, R1 is hydrogen, side chain M1 2-methyl-N-ethyl-N- group (-CH ₂ -CH ₂ -N
The H-CO-CH _3), Compound referred to as melatonin,
It is an important substance in the body related to circadian rhythm.

[0003]

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[0004] Melatonin has been hitherto described by a method using an antibody (Masayoshi Yoshioka et al., Protein Nucleic Acid Enzyme VOL. 26 No.
9 (1981) p 1184), a method by liquid chromatography using an electrochemical detector (Shifumi)
Ebihara, et al. SCIENCE VO
L. 231 (1986) p491).

[0005]

The conventional method for measuring melatonin has the following problems. First, in a method using an antibody, it is difficult to avoid a cross-reaction with a similar compound, so that the value of melatonin cannot be known accurately. Next, in the method using liquid chromatography using an electrochemical detector, cross-reaction does not pose a problem unlike the method using an antibody, but the electrochemical detector requires frequent maintenance. There are issues. If this maintenance is neglected, the measurement result will be adversely affected. Therefore, after the measurement is performed for a certain period of time, the operation of the apparatus must be stopped and the detector must be maintained. As a result, the operating time per apparatus is shortened, which is not suitable for performing a rapid measurement on a large number of samples.

Accordingly, an object of the present invention is to provide a method for measuring a 5-methoxyindole compound containing melatonin without causing a cross-reaction with a similar compound and eliminating the need for frequent maintenance of equipment such as a detector. It is in.

[0007]

Means for Solving the Problems As a result of diligent studies to achieve the above object, the present inventors have determined a method for measuring melatonin or the like by a precolumn or postcolumn measurement method using a specific fluorescent derivatization reagent. Was completed.

That is, the present invention relates to the above-mentioned general formula (1) wherein
R1 represents a hydrogen atom or a hydroxyl group, and M1 represents a side chain), wherein the 5-methoxyindole compound is measured by liquid chromatography before or after separation using a column. (2) (wherein, Ar represents an aryl group and L represents a leaving group).

[0009]

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Further, the present invention provides the above-mentioned general formula (1) wherein
R1 represents a hydrogen atom or a hydroxyl group, M1 represents a side chain) and / or 5-hydroxyindole represented by the general formula (3) (wherein M2 represents a side chain). A method of measuring by liquid chromatography, in which both are separated by a column, and then both or one of them is represented by the general formula (2) (wherein, Ar represents an aryl group and L represents a leaving group). The above-described measurement method, wherein the method is performed by bringing the compound into contact with a derivatization reagent to perform fluorescence derivatization.

[0011]

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Hereinafter, the present invention will be described in detail.

Among the fluorescent derivatization reagents represented by the general formula (2) (wherein, Ar represents an aryl group and L represents a leaving group), 1,2-diphenylethylenediamine is catecholamines, vanillyl mandelic acid, homovanillin As a fluorescent derivatization reagent for acids, serotonin, 5-hydroxyindoleacetic acid, benzylamine is a serotonin,
Known as a fluorescent derivatization reagent for 5-hydroxyindoleacetic acid, represented by general formula (1) represented by melatonin (wherein R1 represents a hydrogen atom or a hydroxyl group, and M1 represents a side chain). It is not known as a reagent for fluorescently derivatizing 5-methoxyindole.

In the fluorescent derivatization reagent (arylaminomethyl derivative) represented by the general formula (2) (where Ar represents an aryl group and L represents a leaving group), the aryl group represented by Ar is phenyl Group, substituted phenyl group, naphthyl group, substituted naphthyl group (for example, methylnaphthyl group,
Methoxylnaphthyl group), pyridyl group, furyl group, biphenyl group and the like. Examples of the substituent of the substituted phenyl group include 2-, 3- or 4-position methoxyl group, ethoxyl group, hydroxyl group, methyl group, ethyl group, chloro group, fluoro group, dimethylamino group, cyano group, nitro group. , 3,4-position dimethoxyl group, dichloro group, methylenedioxyl group and the like.

The leaving group represented by L in the general formula (2) includes a hydrogen atom, a cyano group, a nitro group, a halogen group (for example, a chloro group, a fluoro group, a bromo group, etc.), a substituted acyl group, a substituted acyl group, Examples thereof include a sulfonyl group, a hydroxyl group, a thiol group, and an arylaminomethyl group. Examples of the substituent of the substituted acyl group and the substituted sulfonyl group include an alkyl group (for example, a methyl group and an ethyl group), an amino group, and an amino substituent (for example, a carboxylalkyl group, an alkoxycarbonyl group, an aminoalkylcarbonyl group,
Aminoarylsulfonyl group, sulfoaryl group, aminocarboxyaryl group or aminoalkoxycarbonylalkyl group). Here, the aryl group of the arylaminomethyl group includes a phenyl group,
Examples include a substituted phenyl group, a naphthyl group, a substituted naphthyl group (eg, a methylnaphthyl group, a methoxylnaphthyl group, etc.), a pyridyl group, a furyl group, a biphenyl group, and the like. Further, as the substituent of the substituted phenyl group, 2-, 3- or 4-position methoxyl group, ethoxyl group, hydroxyl group,
Examples thereof include a methyl group, an ethyl group, a chloro group, a fluoro group, a dimethylamino group, a cyano group, a nitro group, a 3,4-dimethoxyl group, a dichloro group, and a methylenedioxyl group.

In the 5-methoxyindole compound represented by the general formula (1) to be measured, R1 is a hydrogen atom and the side chain M1 is a 2-methylamidoethyl group (-CH
_{2 -CH 2 -NH-CO-CH} 3) melatonin can be exemplified a. Also, melatonin metabolites can be measured by the method of the present invention. For example when R1 is a hydroxyl group, the side chain M1 2-methyl-N-ethyl-N- group (-CH ₂
—CH ₂ —NH—CO—CH ₃ ), 6-hydroxymelatonin, R1 is a hydrogen atom, and the side chain M1 is a 2-aminoethyl group (—CH ₂ —CH ₂ —NH ₂ );
5- which is a carboxymethyl group (—CH ₂ —COOH)
Specific examples include methoxytryptamine and 5-methoxyindoleacetic acid.

When the 5-methoxyindole compound in the sample comes into contact with the above-mentioned fluorescent derivatization reagent, the 5-methoxyindole compound is fluorescently derivatized. The fluorescent derivatization of the 5-methoxyindole compound may be performed before or after the 5-methoxyindole compound in the sample is separated by the column for liquid chromatography. The method of bringing the sample into contact with the fluorescent derivatization reagent before separation is a so-called pre-label measurement method, and the method of contacting the sample after separation is a so-called post-label measurement method.

In the present invention, a liquid chromatograph apparatus for performing a normal pre-label measurement method or a post-label measurement method can be used as it is, and the detection of a fluorescently derivatized 5-methoxyindole compound can be performed by a conventional method. A fluorescence detector can be used.

When an arylaminomethyl derivative of the above formula (2) is used as a prelabeled fluorescent derivatization reagent and a 5-methoxyindole compound is converted to a fluorescent derivative, the fluorescent derivative is separated and measured. As the column, an ion exchange column, a reversed phase column, or the like may be used.

In the 5-methoxyindole compound in the sample, R1 in the general formula (1) is a hydrogen atom, and a 5-hydroxyindole compound of the general formula (3) having the same side chain M2 as the side chain M1 is obtained. When present in a sample, it is necessary to carry out a labeling reaction after separating the 5-hydroxyindole compound from 5-methoxyindole.
This is because the reaction mechanism between the 5-methoxyindole compound and the arylaminomethyl derivative is such that the methoxyl group at the 6-position is first oxidized to become a hydroxyl group, and a derivatization reaction occurs. That is, in the general formula (1),
When R1 of the methoxyindole compound is a hydrogen atom and a 5-hydroxyindole compound of the general formula (3) having the same side chain M2 as the side chain M1 is present, these cannot be separated when reacted with an arylaminomethyl derivative. This is because a fluorescent substance is produced.

Specifically, for example, when melatonin is measured by the prelabeling fluorescence derivatization method, it is possible to directly measure N-acetylserotonin as long as the sample does not contain N-acetylserotonin or its concentration can be ignored in the measurement. However, when the presence of N-acetylserotonin is expected, N-acetylserotonin and melatonin are first separated using, for example, a reversed-phase column or an ion-exchange column, and then fluorescent derivatization is performed. Measure by chromatograph. In the case of a post-label measurement method in which a 5-methoxyindole compound in a sample is separated by a liquid chromatography column and then the separated 5-methoxyindole compound is converted to a fluorescent derivative and measured, as described in the above example, melatonin and N- Since acetylserotonin is separated, even if both melatonin and N-acetylserotonin coexist in the sample, there is no problem in the measurement of melatonin.

The fluorescent derivatization reaction of the 5-methoxyindole compound represented by the general formula (1) with the arylaminomethyl derivative represented by the general formula (2) proceeds rapidly when an oxidizing agent is present. In both cases, it is preferable that both come into contact in the presence of an oxidizing agent.
As the oxidizing agent, for example, KIO ₄ , NaIO ₄ , NaIO
₃ , KIO ₃ , K ₃ Fe (CN) _{6 and the} like. For example, using NaIO ₄ and K ₃ Fe (CN) ₆ together,
Each final concentration is 3.3 mmol / L, 1 mmol
/ L allows the fluorescent derivatization reaction to proceed quickly. Furthermore, when a 5-methoxyindole compound is fluorescently derivatized, a fluorescent derivatization can be more quickly caused by coexisting a water-soluble organic solvent such as methanol, ethanol, acetonitrile, or 2-propanol.

The reaction temperature and time required for fluorescent derivatization are as follows:
It is in a relationship that when the reaction temperature is low, it takes a longer time than when the reaction temperature is high. According to the findings of the inventor,
If the reaction temperature is 90 ° C., the reaction time is desirably about 1 to 3 minutes. In addition to the above, in order to accelerate the fluorescence derivatization reaction, a metal oxide ion such as ammonium molybdate, tungstate, niobate or chromate may be added as a catalyst.

As described above, when a 5-methoxyindole compound and a 5-hydroxyindole compound coexist in a sample, if both are separated by a liquid chromatography column first, only the 5-methoxyindole compound can be obtained. It is possible to measure, measure only the 5-hydroxyindole compound, or measure both the 5-methoxyindole compound and the 5-hydroxyindole compound. In this case as well, it is preferable to use an oxidizing agent for accelerating the fluorescent derivatization reaction, but for the 5-hydroxyindole compound, it is sufficient to use an oxidizing agent having a final concentration of about 1 mmol / L. Similarly to the above, the addition of a water-soluble organic solvent or a metal oxide ion can promote the fluorescence derivatization reaction. Regarding the reaction temperature, for example, if the reaction temperature is 90 ° C., the reaction time is preferably 1 to 3 minutes.

The 5-hydroxyindole compound measured according to the present invention is a compound represented by the general formula (3), wherein the side chain M2 has a 2-aminoethyl group (—CH ₂ —CH ₂ —
NH ₂ ) and carboxymethyl group (—CH ₂ —COOH) are serotonin and 5-hydroxyindoleacetic acid, respectively. Serotonin and 5-hydroxyindole acetic acid in urine are known to be high in patients with carcinoid and dumping syndrome, and 5-hydroxyindole acetic acid in urine is known to be high when migraine attacks. Doing so has clinical significance.

In addition, the side chains M2 each have a 2-methylamidoethyl group (—CH ₂ —CH ₂ —NH—CO—CH
_3), aldehyde methyl group (-CH ₂ -CHO), 2-amino-3-carboxyethyl (-CH ₂ -CH (NH ₂₎
-COOH), 2-Hidokishiruechiru group (-CH ₂ -C
H ₂ -OH), compound N- acetyl serotonin, 5
-Hydroxyindolealdehyde, 5-hydroxytryptophan, 5-hydroxytryptophor and the like,
Known as 5-hydroxyindole compounds.

[0027]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the examples.

Example 1 A pre-label measurement was performed using the apparatus having the configuration shown in FIG. 1 is an eluent (pH 4.5) consisting of 10 mM sodium acetate, 150 mM ammonium nitrate and 10% acetonitrile, and 2 is an eluent (pH 4.5) consisting of 10 mM sodium acetate, 150 mM ammonium nitrate and 50% acetonitrile.

The pump 3 is a multi-pump CCPM (trade name, manufactured by Tosoh Corporation), and the mixer 4 is a dynamic mixer M
X810 (trade name, manufactured by Tosoh Corporation) and autosampler 5 are autosampler AS-8010 (trade name, manufactured by Tosoh Corporation)
Column 6 is TSKgel ODS-80Ts
(Product name, 4.6 mm ID × 15, manufactured by Tosoh Corporation)
0 mm, reversed phase column), and the fluorescence detector 7 is a fluorescence detector FS
-8010 (trade name, manufactured by Tosoh Corporation) was used.

The sample was prepared at a concentration of 0.5 μmol / ml
-Hydroxyindole acetic acid or melatonin was used.
The sample was prepared with a 0.01N hydrochloric acid solution. Two kinds of reaction reagents (A; a reaction reagent containing 40 mmol / L benzylamine, 1% sodium methylate and 50% acetonitrile, B; a reaction reagent containing 6 mmol / L potassium ferricyanide and 20 mmol / L NaIO ₄ ) were prepared, used.

100 μL of the sample, 2.5 μL of 6% perchloric acid solution, 50 μL of reaction reagent A, and reaction reagent B
Put 50 μL into a glass vial with a stopper,
The mixture was allowed to react for minutes and used for measurement. The injection volume of the autosampler was 20 μL. The eluent is sent by the pump 3 so that the eluents 1 and 2 have a ratio of 4: 6, and the flow rate is 1.0 ml / min. The fluorescence was detected at an excitation wavelength of 345 nm and a fluorescence detection wavelength of 481 nm.

The measurement results of 5-hydroxyindoleacetic acid and melatonin are shown in FIGS. 3 and 4, respectively.

Example 2 Post-label measurement was performed using the apparatus having the configuration shown in FIG. 8 is an eluent (pH 4.5) consisting of 10 mM sodium acetate, 150 mM ammonium nitrate and 10% acetonitrile, and 9 is an eluent (pH 4.5) consisting of 10 mM sodium acetate, 150 mM ammonium nitrate and 50% acetonitrile.

The pump 10 is a multi-pump CCPM (trade name, manufactured by Tosoh Corporation), the mixer 11 is a dynamic mixer MX810 (trade name, manufactured by Tosoh Corporation), and the autosampler 12 is an autosampler AS-8010 (tosoh Corporation). ), Column 13 is TSKgel OD
S-80Ts (Tosoh Corporation, trade name, 4.6 mm
I. D. X 150 mm, reverse phase column), column oven 14 is column oven CO-8010 (Tosoh Corporation)
Manufactured by Teflon tube (0.4 m)
ml. D. × 25 m), a cooling coil 16 Hateflon tube (0.4 mm ID × 2 m), a fluorescence detector 17 is a fluorescence detector FS-8010 (manufactured by Tosoh Corporation, trade name), and a pump 18 is a dual pump DP-8020 (Trade name, manufactured by Tosoh Corporation).

The standard samples were 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid,
It was prepared with a 0.01 N hydrochloric acid solution so that hydroxytryptophor, N-acetylserotonin and melatonin all became 83 nmol / ml. The plasma sample is
A half amount of a 6% perchloric acid solution was added to and mixed with plasma collected using an EDTA2K blood collection tube, and the supernatant obtained by centrifugation was used. The reaction reagent 19 contained 40 mmol / L benzylamine, 1% sodium methylate and 50% acetonitrile, the reaction reagent 2 contained 6 mmol / L potassium ferricyanide and 20 mmol / L NaIO ₄ , and the injection amount of the autosampler was 20 μL. did.

As the eluent, 100% of the eluent 8 is fed from 0 to 25 minutes, and the eluents 8 and 9 are sent from 25 to 27 minutes.
Was performed at a ratio of 6: 4, and after 27 minutes, the eluents 8 and 9 were sent by the pump 10 at a ratio of 6: 4. Although the flow rate is 1.0 ml / min, the flow rates of the reaction solutions 19 and 20 are both 0.25 ml.
/ Min. The reaction temperature (set temperature of the column oven 14) was 90 ° C. Fluorescence detection is performed at an excitation wavelength of 34
The measurement was performed at 5 nm and a fluorescence detection wavelength of 481 nm.

The measurement results of the standard sample and the plasma sample are shown in FIGS. In the plasma sample (FIG. 6), a peak of serotonin was confirmed, and the other components contained in the standard sample were lower than the sensitivity.

Example 3 The same operation as in Example 2 was carried out except that a reagent containing 40 mmol / L 1,2-diphenylethylenediamine, 1% sodium methylate and 50% acetonitrile was used as the reaction reagent 19. FIG. 7 shows the measurement results.

Example 4 The same operation as in Example 1 was carried out except that the eluents 1 and 2 were sent by the pump 3 so as to have a ratio of 7: 3. The sample was prepared with a 0.01N hydrochloric acid solution so that both 6-hydroxymelatonin and melatonin had a concentration of 250 nmol / ml. FIG. 8 shows the measurement results.

[0040]

According to the present invention, a 5-methoxyindole compound represented by melatonin is fluorescent-derivatized using a fluorescent derivatizing reagent represented by the general formula (2), and the resulting compound is subjected to fluorescence detection for ordinary liquid chromatography. It is measured with a container.

When the post-labeling measurement is carried out in the present invention, only the 5-methoxyindole compound is measured,
Measuring only the hydroxyindole compound, or 5-
A methoxyindole compound and a 5-hydroxyindole compound can be measured simultaneously by a single operation.

Compared with the measurement using a conventional antibody, the present invention does not affect the measurement accuracy by the cross-reaction with a similar compound, and it is difficult to perform the measurement using an antibody. -Methoxyindole compound and 5-
Simultaneous measurement of hydroxyindole compounds can also be realized. Also, in comparison with the conventional method using an electrochemical detector, the present invention can be implemented by a commonly used pre-label or post-label method, and requires an electrochemical detector that requires frequent maintenance. Therefore, it is easy to carry out, and it is possible to measure a large number of specimens (biological samples such as blood and urine) one after another without gaps.

As described above, the present invention makes it possible to treat a 5-methoxyindole compound and / or a 5-hydroxyindole compound in a shorter time and continuously in a larger amount, thereby reducing the measurement cost and The present invention promotes accumulation of clinical data and contributes to early detection and treatment of various diseases by measuring the above compounds.

[Brief description of the drawings]

FIG. 1 shows a schematic diagram of a pre-label measuring device used in Examples 1 and 4.

FIG. 2 is a schematic diagram of a post-label measuring device used in Examples 2 and 3.

FIG. 3 shows the measurement results of 5-hydroxyindoleacetic acid of Example 1. 5HIAA in the figure indicates 5-hydroxyindoleacetic acid.

FIG. 4 shows measurement results of melatonin in Example 1. MLT in the figure indicates melatonin.

FIG. 5 is a measurement result of a standard sample of Example 2. 5HTP, 5HT, 5HIAA, 5HTO in the figure
L, N-Ac5HT, and MLT represent 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid, 5-hydroxytryptophor, N-acetylserotonin, and melatonin, respectively.

FIG. 6 is a measurement result of a plasma sample of Example 2. 5HT in the figure indicates serotonin.

FIG. 7 is a measurement result of a standard sample of Example 3. 5HTP, 5HT, 5HIAA, 5HTO in the figure
L, N-Ac5HT, and MLT represent 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid, 5-hydroxytryptophor, N-acetylserotonin, and melatonin, respectively.

FIG. 8 is a measurement result of Example 4. 6 in the figure
HMLT and MLT indicate 6-hydroxymelatonin and melatonin, respectively.

[Explanation of symbols]

1.2.8.9 eluent, 3.10.18 pump, 4
・ 11 mixer, 5 ・ 12 Autosampler, 6 ・ 13
Column, 7.17 fluorescence detector, 10 pumps, 14
Column oven, 15 reaction coils, 16 cooling coils, 19/20 reaction reagents

Claims (3)

[Claims] 1. A compound represented by the general formula (1) wherein R1 is a hydrogen atom or a hydroxyl group, and M1 is a side chain.
A method for measuring a methoxyindole compound by liquid chromatography, wherein a 5-methoxyindole compound is converted to a compound of the general formula (2) before or after separation by a column, wherein Ar represents an aryl group and L represents a leaving group. The method according to any one of claims 1 to 3, wherein the measurement is carried out by contacting with a fluorescent derivatization reagent represented by formula (1). Embedded image Embedded image 2. A compound represented by the general formula (1) wherein R1 is a hydrogen atom or a hydroxyl group, and M1 is a side chain.
A method for measuring 5-hydroxyindole represented by a methoxyindole compound and / or 5-hydroxyindole represented by the general formula (3) (wherein M2 represents a side chain) by liquid chromatography. Alternatively, the above-mentioned measurement method is characterized in that one of them is brought into contact with a fluorescent derivatization reagent represented by the general formula (2) (wherein, Ar represents an aryl group and L represents a leaving group) to perform fluorescence derivatization. Embedded image Embedded image Embedded image 3. The method according to claim 1, wherein an oxidizing agent is present together with the fluorescent derivatizing reagent.