JP2001242174A - Method for analyzing histamine or histidine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an analysis method whereby histamine or histidine can be detected specifically with high sensitivity. SOLUTION: A derivative obtained, by making an aromatic fused ring- containing compound including an acid hydroxyimidyl ester functional group or the like such as 4-(1-pyrene) succin imidyl butyrate, 4-(1-pyrene) butanoly chloride or the like react with histamine or histidine is subjected to excimer fluorometic analysis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for excimer fluorescence analysis of histamine or histidine.

[0002]

2. Description of the Related Art Physiologically active amines such as histamine and histidine have been quantified in trace amounts in the fields of physiology and medicine. Among these bioactive amines,
In particular, histamine is important for evaluating allergic reactions. However, since the amount of this physiologically active amine is very small, its detection and quantification are extremely difficult.

Therefore, a radioimmunoassay method has been developed as a method for performing highly sensitive analysis. However, this method has problems in that a special reagent or device is required, the cost is high, and the operation is complicated.

Conventionally, high performance liquid chromatography (HP
A fluorescence detection HPLC method using orthophthalaldehyde, which is incorporated into the LC) method, has been developed.

In the fluorescence detection HPLC method using this orthophthalaldehyde, histamine is reacted with orthophthalaldehyde in a pre- or post-column to convert the histamine into a fluorescent substance, which is detected.

However, in this fluorescence detection HPLC method, the condensate of histamine and orthophthalaldehyde is unstable and easily broken. Also, the detection sensitivity of histamine is low. Moreover, as compared with the above radioimmunoassay method, the above-mentioned fluorescence detection HPLC method basically solves the problem of operability, although it is partially solved.

[0007]

While the present inventors have made various studies to solve the above problems, the reaction product of a compound containing an aromatic condensed ring with histamine or histidine exhibits strong excimer fluorescence. The present inventors have found that they can be used for highly sensitive and specific detection of these physiologically active substances, and that they can solve the above problems when combined with the liquid chromatography, and have completed the present invention. Therefore, an object of the present invention is to provide an analysis method capable of detecting histamine or histidine with high sensitivity and specificity.

[0008]

Means for Solving the Problems To achieve the above object, the present invention provides [1] a derivative obtained by bonding two molecules of an aromatic condensed ring-containing compound to one molecule of histamine or histidine; The present invention proposes a method for analyzing histamine or histidine, which is characterized by performing fluorescence analysis.

The present invention also relates to [2] histamine or a derivative obtained by binding two molecules of an aromatic condensed ring-containing compound to one molecule of histidine, by liquid chromatography equipped with a fluorescence detector and eluted with an excimer. The present invention proposes a method for analyzing histamine or histidine, which is characterized by performing fluorescence detection.

Further, [3] the aromatic condensed ring-containing compound according to [1] or [2] comprises an aromatic condensed ring group and an amine-reactive functional group; A compound in which the amine-reactive functional group is linked with an alkylene group having 1 to 10 carbon atoms, [4]
[5] [3] wherein the aromatic fused ring group according to [3] is a pyrenyl group, a benzopyrenyl group, a naphthyl group, an indenyl group, a fluorenyl group, a phenanthrenyl group, an anthryl group, and a coronenyl group. The described amine-reactive functional group is a carboxylic acid hydroxyimidyl ester functional group, a carboxylic acid halide functional group, an aldehyde functional group, a phenylisothiocyanate functional group, a halobenzofurazan functional group, a sulfobenzenesulfonic acid halide functional group. , An azide functional group, or a triazinyl group, and [6] [1] or [2]
Is a compound containing 4- (1-pyrene) butyric acid succinimidyl ester or chloride 4- (1-
Pyrene) butanoyl.

Hereinafter, the present invention will be described in detail.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION As the compound containing an aromatic condensed ring of the fluorescent reagent used in the present invention, a compound containing pyrene (1) in the molecule can be exemplified.

[0013]

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The present inventors have discovered a technique for fluorescently derivatizing a physiologically active amine such as histamine or histidine using the compound containing an aromatic condensed ring as a fluorescent reagent.

As shown in the reaction scheme schematically shown by the following pyrene group, a fluorescent derivative (3) formed by a physiologically active amine and two fluorescent reagents per one molecule of the biologically active amine is: Emits fluorescence by excitation with ultraviolet light. On the other hand, the fluorescent reagent itself (2) which does not form a derivative with the physiologically active amine also emits fluorescence when excited by ultraviolet light.

However, the fluorescence of the fluorescent derivative (3) formed with the above-mentioned physiologically active amine and the fluorescent reagent and the fluorescence of the fluorescent reagent itself (2) without forming the above-mentioned derivative with the biologically active amine are different from each other. Since the wavelengths of the fluorescent light are greatly different, the fluorescent light can be detected with high sensitivity and selectively.

[0017]

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Next, the most characteristic operation of fluorescent derivatization in the analysis method of the present invention will be described.

To the solution containing histamine or histidine, a compound containing an aromatic condensed ring-containing compound or a compound containing an aromatic condensed ring-containing compound as a fluorescent reagent is added to convert the histamine or histidine into a fluorescent derivative.

After the above-mentioned fluorescence derivatization, the histamine or histidine which has been fluorescence-derivatized is eluted and detected by liquid chromatography equipped with a fluorescence detector.

The aromatic condensed ring-containing compound of the present invention comprises an aromatic condensed ring group and an amine-reactive functional group, wherein the aromatic condensed ring group and the amine-reactive functional group are It is preferable to use a compound linked by an alkylene group having 1 to 10 carbon atoms.

Examples of the aromatic condensed ring group include a pyrenyl group, a benzopyrenyl group, a naphthyl group, an indenyl group, a fluorenyl group, a phenanthrenyl group, an anthryl group, and a coronenyl group.

The above-mentioned amine-reactive functional groups include a carboxylic acid hydroxyimidyl ester functional group (4), a carboxylic acid halide functional group (5), an aldehyde functional group (6), and a phenyl isothiocyanate functional group (7). , Halobenzofurazan functional group (8), sulfobenzenesulfonic acid halide functional group (9), benzoic acid halide functional group (10), azide functional group (11), and triazinyl group (12). can do.

[0024]

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[0025]

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Here, X represents a halogen atom.

[0027]

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[0028]

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[0029]

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Here, X represents a halogen atom.

[0031]

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Here, X represents a halogen atom.

[0033]

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Here, X represents a halogen atom.

[0035]

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[0036]

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As described above, any compound having an aromatic condensed ring capable of performing excimer fluorescence analysis on a derivative obtained by reacting with histamine or histidine can be used as the fluorescent reagent of the present invention. Among them, succinimidyl 4- (1-pyrene) butyrate and 4- (1-pyrene) butanoyl chloride are particularly preferred from the viewpoint of availability.

Next, a case where histamine (14) is converted to a fluorescent derivative using succinimidyl 4- (1-pyrene) butyrate (13) as a fluorescent reagent will be described by the following reaction formula.

[0039]

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As shown in the above reaction formula, the acid hydroxyimidyl ester functional group of 4- (1-pyrene) butyric acid succinimidyl ester (13) is converted to the amino group and aminoethyl group of the imidazole ring of histamine (14). To produce the pyrene derivative (15). In the derivative (15), it is considered that the paired pyrenyl group bonded to the amino group of the histamine (14) interacts and emits excimer fluorescence when excited by ultraviolet light.

Therefore, it is considered that a physiologically active amine such as histamine (14) can be detected with high sensitivity and selectively by a fluorescence detector.

4- (1-pyrene) butanoyl chloride (1
When 6) is used as a fluorescent reagent, the amino group of the imidazole ring of histamine (14) can be obtained as in the case of succinimidyl 4- (1-pyrene) butyrate (13).
And the amino group of the aminoethyl group to produce a pyrene derivative.

[0043]

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When histidine (17) is used as a detection target instead of histamine (14), similarly to the case of histamine (14), the amino group and the side chain of the imidazole ring of histidine (17) are used. An amino group and 4-
(1-pyrene) succinimidyl butyrate (13)
Acid hydroxyimidyl ester functional group of
It reacts with the acid chloride functional group of (1-pyrene) butanoyl (16) to produce a pyrene derivative.

[0045]

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Histamine (14) or histidine (1
As a solvent used for excimer fluorescence derivatization of 7), water or an organic solvent such as methanol, ethanol, propanol, tetrahydrofuran, acetonitrile, acetone, dioxane, sulfolane, sulfoxide, or a mixture of these solvents as appropriate Are preferred.

The amount of the aromatic condensed ring-containing compound used in the excimer fluorescent derivatization is determined by the above-mentioned histamine (1).
4) or 1 μmol of histidine (17), and 0.
A ratio of 1 to 3 mmol is preferred.

Separately, when a solution of an aromatic condensed ring-containing compound is prepared and used, the solvent may be water or a lower alcohol such as methanol, ethanol or propanol, or a water-soluble solvent such as acetonitrile, acetone, dioxane, sulfolane or sulfoxide. Solvents or those obtained by appropriately mixing these solvents are preferable.

In the reaction of histamine or histidine with a compound containing an aromatic condensed ring, the reaction temperature is from room temperature to 1
20 ° C is preferable, and particularly preferably 60 to 100 ° C. The reaction time is preferably 30 minutes to 3 hours. The reaction is preferably performed in a sealed stopper.

In a solution containing a fluorescent derivative obtained by the above-mentioned reaction of histamine or histidine with a compound containing an aromatic condensed ring, the fluorescent derivative is stably present in the pH range of 4 to 9, so that a buffer solution is used. Does not reach.

For this reason, in the analysis by high performance liquid chromatography (HPLC), it is not necessary to change the pH of the eluent to flow the eluent, so that the operation is extremely easy.

Histamine or histidine, which has been derivatized with fluorescence as described above, emits excimer fluorescence when excited with ultraviolet light, so that it can be detected with high sensitivity and selectively by a fluorescence detector.

[0053]

EXAMPLES The present invention will be described more specifically with reference to the following examples.

Example 1 (Preparation of 1 × 10 ⁻⁶ M histamine solution) 3.2 mg of histamine hydrochloride (FW 184.07) was dissolved in the following solvent A, and the solvent A was added. The volume of the solution was 5.00 mL. 1 mL of this histamine solution and 2.48 mL of solvent A were mixed to obtain a 1 × 10 ⁻⁴ M histamine solution. This 1 × 10 ⁻⁴ M histamine solution was diluted 100-fold with solvent A to obtain a 1 × 10 ⁻⁶ M histamine solution.

The solvent A is tetrahydrofuran,
Dimethyl sulfoxide and water are 1: 2 by volume:
These were mixed at a ratio of 1.

(Preparation of 1 mM 4- (1-pyrene) butyric acid succinimidyl ester (PSE)) PSE (F.
W. 385.42) 5.8 mg was dissolved in acetonitrile, and the acetonitrile was further added to adjust the volume of the solution to 5.00 mL (3 mM). This PSE solution was diluted 3-fold with acetonitrile to obtain a 1 mM PSE solution.

(Fluorescent derivatization of histamine) 1 × 10 ^-6
M histamine solution 100 μL, 1 mM MPSE solution 10
0 μL was added and the mixture was heated at 100 ° C. for 90 minutes to obtain a histamine fluorescent derivative solution. After cooling this fluorescent derivative solution in ice water, 10 μL of the solution was injected into a high-performance liquid chromatography (HPLC) column described below.

(High performance liquid chromatography (HPL)
C) Conditions) The HPLC column used was wako as a packing material.
A column (150 × 2.0 mm, id) packed with sil II 5C18-100 (particle diameter 5 μm) was used.

The eluent of the mobile phase was 0.5% by volume of triethylamine in water-acetonitrile (20/80 = v /
v) In solution, the flow rate was 200 μl / min.
The excitation wavelength (Ex) of the fluorescence detector is 345 nm,
The fluorescence wavelength (Em) was 485 nm.

FIG. 1 shows the obtained chromatogram.

Under the above HPLC elution conditions, the PSE derivative of histamine was detected within 15 minutes with a single elution solvent. The detection limit (S / N = 3) was extremely high at 1 fmol / injection amount. The repetition accuracy was good within 5%.

Example 2 Example 1 was repeated except that 4- (1-pyrene) butanoyl chloride (PBC) was used instead of 4- (1-pyrene) butyric acid succinimidyl ester (PSE) as a fluorescent reagent. The histamine was subjected to an HPLC elution test according to the method described above.

Example 3 Histidine HPLC was carried out in the same manner as in Example 2 except that histidine was used instead of histamine as a detection target.
Was subjected to an elution test.

[0064]

According to the analytical method of the present invention, histamine or histidine is derivatized by using a compound containing an aromatic condensed ring as a fluorescent reagent, so that these compounds can be detected with high sensitivity and selectively.

Further, the analysis method of the present invention uses the pH of the eluent
It is not necessary to change the flow rate and flow the eluent, so that the operation is extremely easy.

[Brief description of the drawings]

FIG. 1 is a chromatogram showing the elution state of histamine in Example 1.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Takashi Yoshitake 3-822 Ishii-cho, Hita-shi, Oita Prefectural Institute for Evaluation of Chemical Substances, Hita Works (72) Inventor Fumio Ichinose 3-822, Ishii-cho, Hita-shi, Oita Address F-term in Hita Works of the Chemical Substance Evaluation and Research Institute (Reference)

Claims (6)

[Claims] 1. A method for analyzing histamine or histidine, wherein excimer fluorescence analysis is performed on a derivative obtained by binding two molecules of a compound containing an aromatic fused ring to one molecule of histamine or histidine. 2. A derivative obtained by binding two molecules of an aromatic condensed ring compound to one molecule of histamine or histidine is eluted by liquid chromatography equipped with a fluorescence detector, and excimer fluorescence is detected. For analyzing histamine or histidine. 3. The aromatic condensed ring-containing compound comprises an aromatic condensed ring group and an amine-reactive functional group, wherein the aromatic condensed ring group and the amine-reactive functional group are each composed of one or more carbon atoms. 1 to
The method for analyzing histamine or histidine according to claim 1 or 2, which is a compound linked by 10 alkylene groups. 4. The method for analyzing histamine or histidine according to claim 3, wherein the aromatic fused ring group is a pyrenyl group, a benzopyrenyl group, a naphthyl group, an indenyl group, a fluorenyl group, a phenanthrenyl group, an anthryl group, and a coronenyl group. . 5. The amine-reactive functional group is a carboxylic acid hydroxyimidyl ester function, a carboxylic acid halide function, an aldehyde function, a phenylisothiocyanate function, a halobenzofurazan function, a sulfobenzenesulfonic acid halogen. Functional group, azide functional group,
4. The method for analyzing histamine or histidine according to claim 3, which is a triazinyl group. 6. The method according to claim 6, wherein the aromatic condensed ring-containing compound is 4- (1-
Pyrene) succinimidyl butyrate or 4-chloro chloride
The method for analyzing histamine or histidine according to claim 1 or 2, which is (1-pyrene) butanoyl.