JPH0575066B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application This invention relates to a method and apparatus for analyzing histamine. More specifically, the present invention relates to a histamine analysis method and apparatus that can qualitatively and quantitatively quantify histamine in various samples such as blood plasma and blood cells with high sensitivity.

(b) Prior Art Conventionally, methods using thiol group-containing compounds such as orthophthalaldehyde and mercaptoethanol have been used to analyze compounds having amino groups such as amino acids. In this method, for example, each amino acid component separated by a column, especially each component separated by high performance liquid chromatography, is reacted with orthophthalaldehyde and mercaptoethanol to produce a fluorescent compound, and the fluorescence intensity of this compound is Qualitative and absorbance based on
This is a method for quantitative determination. Since histamine, which is the object of the present invention, is also a compound having an amino group, a method of reacting it with orthophthalaldehyde and a thiol group-containing compound and analyzing it is adopted in the same manner as above.

However, in this method, the background of fluorescence and light absorption is high due to the influence of impurities in the reagent solvent that reacts with ortho-phthalaldehyde and thiol group-containing compounds, which are the reaction reagents, and highly sensitive analysis cannot be performed. In particular, it is not possible to perform microanalysis of histamine on the pg order.

In this regard, before performing separation using high-performance liquid chromatography, etc., a fluorescent compound is produced by reacting with only the above two reaction reagents or orthophthalaldehyde reagent, and then separation is performed to generate the fluorescent compound. Methods for detecting fluorescent compounds based on fluorescence or absorbance have also been proposed. however,
In this method, multiple peaks were generated due to side reactions caused not only by the amino group of histamine but also by the imidazole group, making quantitative determination extremely difficult.

This invention was made in view of the above situation, and it is an object of the present invention to provide an analysis method that can analyze histamine with high sensitivity.

As a result of extensive research, the present inventors have found that when histamine is used as a target, under specific conditions, the fluorescent property can be reduced by simply reacting it with orthophthalaldehyde without using the above two reaction reagents. A solution is obtained, and by dissolving orthophthalaldehyde in an acidic buffer and feeding it separately from a buffer with a pH of 7 to 12, background fluorescence and light absorption are reduced compared to the conventional method using two reaction reagents. We found that there was a significant decrease in

(C) Structure of the Invention Thus, according to the present invention, a fluorescent reaction solution is obtained by mixing and reacting a histamine-containing solution with orthophthalaldehyde in the presence of a buffer solution with a pH of 7 to 12 at a mild temperature. , a method for analyzing histamine is provided, which comprises analyzing histamine by measuring the fluorescence intensity or absorbance of this reaction solution.

The most distinctive feature of this invention is that a fluorescent compound is obtained by mixing histamine with orthophthalaldehyde in solution without using any compound containing a thiol group (SH group) such as mercaptoethanol. It is in. In the case of ordinary amino acids, such fluorescent compounds cannot be easily produced without using a thiol group-containing compound. Therefore, the reaction between histamine and orthophthalaldehyde is considered to be specific. This reaction is thought to be mainly based on the reaction between the amino group and imidazole ring of histamine and orthophthalaldehyde, and a fluorescent compound different from conventional fluorescent compounds is generated, but the details are not clear. . However, by allowing histamine and orthophthalaldehyde to coexist at least under the above conditions, a fluorescent compound can be obtained, and by using this, it becomes possible to analyze histamine.

The reaction between histamine and orthophthalaldehyde in the present invention is carried out in the presence of a buffer solution with a pH of 7 to 12, preferably in the presence of a buffer solution with a pH of 7 to 8. In addition, as a buffer solution, it is usually suitable to apply a borate buffer or a phosphate buffer; for example, in the case of the former, the concentration of borate should be 50 to 200mM in the mixed reaction system. is preferable.

On the other hand, ortho-phthalaldehyde as a reaction reagent is suitable for adjusting to 0.001 to 0.1 wt% in the mixed reaction system, and preferably 0.01 to 0.02 wt%. less than 0.001wt% or
If the concentration exceeds 0.1 wt%, the fluorescence intensity will decrease, which is not preferable. Orthophthalaldehyde is usually subjected to a mixed reaction in a state dissolved in a phosphoric acid, acetic acid, or citric acid buffer having a pH of 2 to 5 (which may contain an organic solvent).

Further, the mixing reaction is carried out at a mild temperature, suitably at a temperature of at least 40°C or higher, preferably at a temperature of 40°C to 70°C.

The reaction is rapid, and a fluorescent reaction solution is usually obtained in 5 to 10 seconds. Since the fluorescence intensity and absorbance of this reaction solution both correspond to the histamine concentration, histamine can be quantified by measuring these intensities. In this case, it is suitable to set the photometric wavelength to around 440 nm for fluorescence intensity and around 360 nm for absorbance intensity. However, in order to perform microanalysis on the PG order, it is necessary to measure the fluorescence intensity.

In practice, it is preferable that the histamine-containing liquid is usually separated into its components by liquid chromatography, particularly high-performance liquid chromatography, and then subjected to a mixing reaction. As the separation column and mobile phase for liquid chromatography used in this case, it is suitable to use a separation column and mobile phase for general reversed phase or cation exchange chromatography. Columns and mobile phases include, for example, Simpack CLC-ODS and acidic phosphate buffer containing octane sulfonic acid; examples of columns and mobile phases for cation exchange chromatography include Simpack WCX-1 and neutral phosphate buffer. Examples include liquids. These separations are particularly preferred from the standpoint of highly sensitive analysis of histamine.

When using the above liquid chromatography,
A coiled mixing reaction tube is set in the eluent flow path, and orthophthalaldehyde solution and PH
By providing a liquid feeding section capable of feeding 7 to 12 buffer solutions, analysis can be performed efficiently in one step. Therefore, the present invention provides a mobile phase flow path connected to a separation column for liquid chromatography via an inlet for a histamine-containing sample, and a detection device extending from the separation column and equipped with fluorescence or absorbance measurement means. an eluent flow path connected to the
A coiled mixing reaction tube is set in the eluent flow path, and an orthophthalaldehyde solution and a PH
The present invention also provides a histamine analyzer characterized in that it is equipped with 7 to 12 buffer solution feeding sections. In this case, the coiled mixing reaction tube has an inner diameter of 0.3~
It is preferable to use a 0.5 mm one with an internal capacity of 200 to 500 μm. Further, in order to carry out the mixing reaction efficiently, it is suitable to provide a heating means capable of heating the mixture to 40° C. to 70° C. in the eluent flow path and in the vicinity of the mixing reaction tube.

(e) Example 1 shown in FIG. 1 shows a histamine analyzer for carrying out the method of the present invention, which basically consists of a pump 2
2, the mobile phase channel 2 transfers the mobile phase 23 to the separation column 3 for liquid chromatography via the sample introduction part 21, and the eluent channel 2 transfers the eluent from the separation column 3 to the fluorometer 5. 4, a coiled mixing reaction tube 41 is set in a part of the eluent flow path 4, and in front of the coiled mixing reaction tube 41, reagents of an orthophthalaldehyde solution 43 and a buffer solution 44 with a pH of 7 to 12 are placed. An introduction pipe 42 is attached. In addition,
The separation column 3 is heated to an appropriate separation temperature by a constant temperature bath 31, and a part of the eluent flow path including the mixing reaction tube 41 and the introduction tube 42 are heated to 40℃ by a constant temperature bath 47.
It is heated to between ℃ and 70℃. Further, 46 indicates a coiled mixing tube for pre-mixing the orthophthalaldehyde solution 3 and the buffer solution 44.

In this apparatus 1, first, a mobile phase 23 is flowed at a constant flow rate by a pump 22 through a separation column 3 and an eluent flow path 4 to a drain 52, and an ortho-phthalaldehyde solution 43 and a buffer solution 44 are supplied to an eluate by a pump 45 at a constant flow rate. It is injected into the channel 4. The output of the fluorometer 5 at this time is recorded by the recorder 51 and becomes the baseline output. By injecting a histamine-containing sample into the channel 2 from the sample introduction part 21 in this state, histamine is separated from other components and is sufficiently mixed and reacted with orthophthalaldehyde in the mixing reaction tube 41 of the eluent channel 4. Histamine is converted into a fluorescent derivative, and the fluorescence intensity of this fluorescent eluent is measured by a fluorometer 5 and output as a peak to a recorder.
Note that the reagent introduction tube 42 may be provided separately for the orthophthalaldehyde solution 43 and the buffer solution 44 and connected to the channel 4. In any case, by injecting the reagents into the channel 4 separately or by mixing and injecting them immediately before injection into the channel 4, the reaction reagents can be prepared just before use, further reducing the background. It is also possible to prevent fluctuations.

The results of analyzing a histamine-containing sample using the apparatus 1 described above will be explained below. The device is a high performance liquid chromatograph device LC-5A (Shimadzu Corporation).
The mixing reaction tube 41 was a stainless steel coiled tube with an inner diameter of 0.5 mm and a length of 2 m. In addition, the pump 45
BRR-2A (manufactured by Shimadzu Corporation) was used, and the fluorometer was RF-540 (manufactured by Shimadzu Corporation), and the excitation wavelength was
Fluorescence detection was performed at 360 nm and a photometric wavelength of 440 nm.
The detailed conditions are as follows.

Separation column 3: Simpack CLC-ODS (inner diameter 6.0
mm x 150 mm length) Column temperature: 55°C Mobile phase 23: 10 mM sodium acetate (PH
4.4) and ethanol, flow rate 1.5 ml/min Orthophthalaldehyde solution 43: 0.3% ethanol solution of orthophthalaldehyde and
6:100 mixture of 10mM sodium acetate (PH4.4), injection volume 0.25ml/min Buffer 44: 500mM borate buffer at pH 9.2, injection volume 0.25ml/min Sample: 100nM histamine dihydrochloride Inject 10 μ (1 pmol) of the solution (0.1N hydrochloric acid). Sodium acetate and boric acid were purchased from Wako Pure Chemical Industries for amino acid analysis, acetic acid was for reagent grade, and ethanol was for liquid chromatography. there was. Orthophthalaldehyde used was for biochemistry manufactured by Wako Pure Chemical Industries. As the histamine standard substance, dihydrochloride manufactured by Tokyo Kasei Kogyo was used.

This result is used as a recorder by the data processing device C-
Figure 2 shows a chromatogram obtained using R2AX (manufactured by Shimadzu Corporation). In the figure, A is the peak of histamine, and it can be seen that due to the reduction of the background, a clear peak was obtained despite the injection of a small amount. Note that B is a system peak and is considered to be mainly caused by the solvent. From these results, it was found that highly sensitive analysis of histamine can be performed in trace amounts on the pg order.

(f) Effects of the invention According to this invention, it is possible to perform highly sensitive analysis of histamine, and in particular, it has been possible to overcome the quantification limit, which was conventionally on the order of ng at most when fluorescence was used.
It can be improved to PG order. Furthermore, it is based on the reaction caused by the amino group and imidazole group of histamine, and because it is not easily interfered with by contaminant components (such as amino acids) that simply have an amino group, the retention time in chromatography is similar to that of other amino groups. It is not easily affected by the presence of contained components, and it is possible to selectively analyze histamine. Furthermore, since mercaptoethanol, etc., which emit a bad odor, is not used, it is advantageous in terms of operation, and analysis costs can also be reduced.

[Brief explanation of drawings]

FIG. 1 is a configuration explanatory diagram illustrating a histamine analyzer implementing the method of the present invention, and FIG. 2 is a chromatogram diagram illustrating the analysis results of histamine obtained by the method of the present invention. 1... Histamine analyzer, 2... Mobile phase channel, 3... Separation column for liquid chromatography,
4...Eluent channel, 5...Fluorometer, 21...
Sample introduction part, 23...Mobile phase, 41...Mixing reaction tube, 42...Reagent introduction tube, 43...Orthophthalaldehyde solution, 44...Buffer solution of pH 7 to 12, 4
7... Constant temperature bath.

Claims (1)

[Claims] 1. A fluorescent reaction solution is obtained by mixing and reacting a histamine-containing solution with orthophthalaldehyde in the presence of a pH 7-12 buffer solution at a mild temperature, and the fluorescence of this reaction solution is A method for analyzing histamine, which comprises analyzing histamine by measuring light intensity or absorbance. 2. The analysis method according to claim 1, wherein the buffer solution is a buffer solution with a pH of 7 to 8. 3. The analytical method according to claim 1, wherein the mixing reaction is carried out at a temperature of 40°C to 70°C. 4. The analytical method according to claim 1, wherein the concentration of orthophthalaldehyde during the mixing reaction is 0.001 to 0.1 wt%. 5. The analytical method according to claim 1, wherein the solvent for ortho-phthalaldehyde is a buffer solution with a pH of 5 or lower which may contain an organic solvent. 6. The analysis method according to claim 1, wherein the histamine-containing liquid is separated by a separation column of high performance liquid chromatography and then subjected to a mixing reaction. 7 A mobile phase flow path connected to a separation column for liquid chromatography via an introduction section for a histamine-containing sample, and a detection section extending from the separation column and equipped with a means for measuring fluorescence or absorbance. The method is characterized in that it has an eluent flow path, a coiled mixing reaction tube is set in the eluent flow path, and a liquid feeding section for an orthophthalaldehyde solution and a buffer solution with a pH of 7 to 12 is attached upstream thereof. Histamine analyzer. 8. Claim 7, wherein the separation column and mobile phase for liquid chromatography are reversed phase or separation column and mobile phase for cation exchange chromatography.
Analyzer as described in section. 9. The analysis device according to claim 7, wherein the eluent flow path is provided with a heating means capable of heating the eluent to 40 to 70°C.