JPS61254852A - Method and instrument for analyzing histamine - Google Patents

Abstract

PURPOSE:To analyze histamine with high sensitivity with the simple operation alone by adding an orthophthalaldehyde soln. to the eluate of a liquid chromatography. CONSTITUTION:A mobile phase 21 is first passed through a flow passage 3, a column 4 and a flow passage 5 into a drain 54 by a pump 22 and the orthophthalaldehyde soln. 71 is injected by a pump 72 into the passage 5. The output from a fluorophotometer 52 is recorded by a data processor 53. Histamine is separated from the other components in the column 4 when a histamine-contg. sample is injected in this state from an introducing part 31 into the passage 3. The histamine is then mixed with the orthophthalaldehyde so as to react therewith in a mixing reaction tube 6, by which the histamine is converted to a fluorescence generative deriv. The fluorescent intensity of the eluate is measured by the fluorophotometer 52 and is detected as a peak by the processor 53.

Description

[Detailed description of the invention] (b) Industrial application fields The present invention relates to a histamine analysis method and apparatus.

More specifically, the present invention relates to a histamine analysis method and apparatus that can qualitatively and quantitatively quantify histamine in various biological 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 and absorbance of this compound are This method performs qualitative and quantitative measurements based on luminous intensity. 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.

(c) Problems to be Solved by the Invention However, in such a method, the fluorescence and absorption background is reduced due to the influence of impurities in the reagent solvent that reacts with the ortho-phthalaldehyde and thiol group-containing compound used as the reaction reagents. This makes it impossible to perform highly sensitive analysis, and in particular, it is not possible to perform microscopic analysis of histamine on the order of 9 g.

In this regard, before performing separation such as high performance liquid chromatography, a fluorescent compound is generated by reacting with the above two reaction reagents or orthophthalaldehyde reagent alone, and then separation is performed to detect the fluorescent compound. Methods for detecting 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 found that when histamine is used as a target, under specific conditions, the fluorescent property can be reduced by simply reacting the mixture with orthophthalaldehyde without using the above two reaction reagents. A stable solution can be obtained, and by dissolving orthophthalaldehyde in an acidic buffer, feeding it separately with a pH 7-12 buffer, and mixing it with a histamine-containing solution, two types of background fluorescence and light absorption can be obtained. It has been found that the cost is significantly lower than that of the conventional method using reactive reagents.

This invention was made by further developing the above knowledge.

(d) Means and action for solving the problems Thus, according to the present invention, a histamine-containing sample,
Subject to liquid chromatography using a -7 to 9 buffer as a mobile phase and a cation exchange chromatography column or a reversed phase chromatography column as a separation column, and mix an orthophthalaldehyde solution with the eluent from the separation column. A fluorescent reaction solution is obtained by reacting with histamine that may be present under the above-mentioned conditions and at a mild temperature, and histamine is analyzed by measuring the fluorescence intensity or absorbance of this reaction solution. A method for analyzing histamine is provided.

One of the most characteristic points of this invention is that histamine is mixed with orthophthalaldehyde in a specific solution of - without using any compound containing a thiol group (SHI) such as mercaptoethanol. The point is to obtain a compound of

The other most characteristic feature of this invention is that the above reaction is applied to the separation and analysis of histamine by liquid chromatography and analysis by post-column derivatization, and a buffer solution of pH 7 to 9 is used as the mobile phase of the liquid chromatography. As a result, the ortho-phthalaldehyde solution was directly added to the eluate from the separation column without adding a preparative solution for the mixing reaction, and the mixture reaction was carried out.

Since the analysis method of the present invention has the above characteristics, it is possible to perform highly sensitive analysis of histamine by simply adding an orthophthalaldehyde solution to the eluent of liquid chromatography.

In addition, in the case of a normal amino acid, the above-mentioned fluorescent compound is not easily produced without using a thiol group-containing compound. Therefore, the reaction between histamine and orthophthalaldehyde is considered to be specific. It is considered that this reaction is mainly based on the reaction of the amino group and imidazole ring of histamine with orthophthalaldehyde, and a fluorescent compound different from conventional fluorescent compounds is generated.

The buffer used as the mobile phase in this invention has a pH of 7.
A buffer with a pH of ~9 is used in terms of intensity of fluorescence or absorption.
7 to 8 are preferred. As such a buffer, it is usually preferable to use a phosphate buffer or a borate buffer.

Further, it is preferable that the concentration of these is 50 mM to 500 elyl from the viewpoint of buffering capacity and separation efficiency. Note that an organic solvent miscible with water may be added to the buffer solution in terms of separation efficiency.

As the separation column of the present invention, a general cation exchange chromatography column or a reverse phase chromatography column is used, and an example of the former is S1m-pack.
WCX-1 (weakly acidic cation exchange silica gel packed column manufactured by Shimadzu Corporation), and an example of the latter is S
him-paclt CL C-ODS (manufactured by Shimadzu Corporation).

On the other hand, the ortho-phthalaldehyde solution mixed with the eluent and reacted with is 15 or higher in terms of reagent stability, and it is usually preferable to use a -2 to 5 buffer solution. Examples include phosphate buffer solution, citrate buffer solution, acetate buffer solution and the like. The salt concentration of these buffer solutions is preferably 101M or more. 1
If it exceeds 0.01 M, it is not preferable because it may adversely affect the - of the mixed reaction system (eluent). Further, the amount of orthophthalaldehyde used as a reaction reagent is 0.3 to 2.0% in the mixed reaction system.
It is suitable to adjust it so that it becomes 0■M, and from 0.5 to
Preferably it is 1.5 mM. 0.31 in the reaction system
If the concentration is less than M or more than 2.0-M, the fluorescence intensity will decrease, which is not preferable. Note that the orthophthalaldehyde solution may contain an organic solvent miscible with water, such as acetonitrile, in terms of the solubility of orthophthalaldehyde.

The mixed reaction between histamine and orthophthalaldehyde in the eluent is carried out at a mild temperature, preferably at a temperature of at least 40°C or higher, preferably at a temperature of 40°C to 70°C, preferably at a temperature of 50°C or higher. is most preferred.

The reaction is rapid, and a fluorescent reaction solution is usually obtained in 5 to 20 seconds. Since the fluorescence intensity and absorbance of this reaction solution both correspond to the histamine concentration, histamine can be determined by measuring these intensities. In this case, it is suitable to set the photometric wavelength to be around 440 nm with an excitation wavelength of 360 nm in the case of fluorescence intensity, and around 360 rv in the case of absorbance. However, in order to perform trace analysis on the order of pg or rg, it is necessary to measure the fluorescence intensity.

In carrying out the method of the present invention, a coiled mixing reaction tube is set in the eluent flow path of liquid chromatography, and a liquid feeding section is provided upstream of the coiled mixing reaction tube to introduce and mix the above-mentioned orthophthalaldehyde solution. It is preferable to use from the viewpoint of analysis efficiency. Therefore, this invention includes a mobile phase supply section that supplies the m* solution of p141 to 9, and a mobile phase supply section that is connected to a cation exchange chromatography column or a reversed phase chromatography column via an introduction section for a histamine-containing sample. It has a phase flow path and an eluent flow path extending from the column and connected to a detection unit equipped with a fluorescence or absorption photometry measurement means, and a coiled mixing reaction tube is set in the elution flow path. The present invention also provides a histamine analyzer characterized in that an orthophthalaldehyde solution feeding section is attached upstream thereof.

The flow rate of the mobile phase when using such a device is 0.5~
A flow rate of about 1 g/min is usually suitable, and the flow rate of the orthophthalaldehyde solution mixed with the eluent is 0.2 to 0.5 g/min.
x1/min is usually suitable. In addition, the coiled mixing reaction tube at this time has an inner diameter of 0.3 to 0.511.
It is preferable to use one with a rating of 5- or so. 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 DEG C. to 70 DEG C. in the eluent flow path and in the vicinity of the mixing reaction tube.

Note that the liquid chromatography in this invention is preferably carried out by so-called high-performance liquid chromatography, and the above-mentioned apparatus can also be easily constructed by using a high-performance liquid chromatography apparatus.

(e) Example (1) shown in FIG. 1 shows a histamine analyzer for carrying out the method of this invention, and basically the mobile phase is PH7.
~9 buffer storage tank (21: 40ai M sodium phosphate buffer with pH 6,9) and liquid pump (22: IC-6
A, manufactured by Shimadzu Corporation), a mobile phase supply unit ('2J), and a column for cation exchange chromatograph 7-1'(4; 3hiw-pack WCX-1, inner diameter 4.0mm x 50nwn length, particle size A mobile phase flow path (3) that sends the above buffer solution to the column (packed with weakly acidic cation-exchange silica gel) and a fluorophotometer (52: RF-540, Shimadzu Corporation) to transfer the eluent from the column (4). It consists of an eluent flow path (5) that sends liquid to the mobile phase flow path (3).
) is located in the middle of the sample introduction section (31:5IL-6A).

A coiled mixing reaction tube (6) is attached to a part of the eluent flow path (5), and in front of it is a coiled mixing reaction tube (6) from which the orthoaldehyde solution is fed (7). The reagent inlet tube is connected. Liquid feeding section (71 is an orthoaldehyde solution storage tank (71:1% orthophthalaldehyde acetonitrile solution and 4iM sodium phosphate! lW
Mixed liquid with J liquid, Fk42.8) and liquid pump (72:
LC-6A).

The coiled mixing reaction tube (6) has an inner diameter of 0.3 mm and a length of 21
It consists of a stainless steel tube and is kept at about 50°C together with the column (4) in a constant temperature bath (8). And the amount of mobile phase sent is 11! /min, and the feeding rate of the orthophthalaldehyde solution is set to 0.5 zl/min. In the figure, (51) and (73) each consist of a stainless steel tube with the same shape as the mixing reaction tube, (51) is a coil for cooling the reaction liquid, and (52) is a coil for preheating the orthophthalaldehyde solution. Functions as a coil. Further, (53) is a data processing device, and (54) is a drain.

In this device (13), first, a mobile phase (sodium phosphate buffer with pH 6.9) is pumped at a constant flow rate through channel (3), column (4) and channel (5) to drain (54). and the ortho-phthalaldehyde solution is injected into the channel (5) at a constant flow rate by the pump (72).The output of the fluorometer (52) at this time is recorded by the data processing device (53) and used as a baseline. In this state, by injecting a histamine-containing sample into the channel (3) from the sample introduction part (31), histamine is separated from other components in the column (4), and then the sample is injected into the channel (5). Histamine is sufficiently mixed and reacted with orthophthalaldehyde in the mixing reaction tube (6) to convert it into a fluorescent derivative.
The fluorescence intensity of this fluorescent eluent is measured using a fluorometer (52).
, and is detected as a peak by the data processing device t (53).

In such an apparatus (1), a histamine solution of i ploI is used as a standard sample, EX-360nj El-4
The results of analysis using fluorescence intensity at 40 nl were as shown in Figure 2. It can thus be seen that the histamine peak A clearly appears after the system peak B. The detection limit for histamine (S/N ratio = 2) is 5 f.
It was also found that mol.

Next, an example in which histamine in plasma was analyzed using the method and apparatus of the present invention in the same manner as described above will be described. The pretreatment of the sample is as follows.

The blood sample was immediately centrifuged at 3000 rpm for 10 minutes and plasma 11! 1M'' AMA elementary acid saturated salt solution 5
00A was added, shaken, and centrifuged at 12,000 rpm+ for 2 minutes to remove protein. 2.5 n-butanol in the supernatant
xi and 1M sodium hydroxide saturated saline 11! 10. was added to the lower layer, shaken thoroughly, and left to stand. , J was used.

The results are shown in FIG. As described above, a clear peak A of histamine was observed after the peak D of contaminants, and it was found that the peak of histamine was not affected at all by the contaminants such as amino acids and amines in the sample. It was also found that repeated analysis every 5 minutes is possible.

(f) Effects of the invention According to this invention, it is possible to perform highly sensitive analysis of histamine, and in particular, when fluorescence is used, the limit of orientation, which was conventionally limited to the order of ng, can be improved to the order of pg or 1 g. be able to. Furthermore, it is based on the reaction caused by the amine group and imidazole group of histamine,
It is useful as a specific detection method for histamine because it is not easily interfered with by contaminant components (amino acids, etc.) that simply have amino groups, and it is also advantageous in terms of operation because it does not use mercaptoethanol or the like, which has a bad odor.

Moreover, it has the advantage that the above analysis can be carried out simply by adding an orthophthalaldehyde solution to the eluent of liquid chromatography.

[Brief explanation of drawings]

FIG. 1 is a configuration explanatory diagram illustrating a histamine analyzer that implements the method of the present invention, and FIGS. 2 and 3 are chromatogram diagrams illustrating the histamine analysis results obtained by the method of the present invention. . (1)...Histamine analyzer, (a...
... Mobile phase supply section, (3) ... Mobile phase flow path, (4) ... Cation exchange chromatography column, (5) ... Eluent flow path , (6)・
...Mixing reaction tube, (7) ...Orthophthalaldehyde solution feeding section, (8) ...Thermostatic chamber, &I+ ...Buffer solution storage tank, (52)...
...Fluorometer, (71) ...Orthophthalaldehyde solution storage tank.

Claims (1)

[Scope of Claims] 1. A histamine-containing sample is subjected to liquid chromatography using a buffer solution with a pH of 7 to 9 as a mobile phase and a column for cation exchange chromatography or a column for reversed phase chromatography as a separation column; An orthophthalaldehyde solution is mixed with the eluent from the column and reacted with any histamine that may be present under the above pH conditions and mild temperature to obtain a fluorescent reaction solution, and the fluorescence or absorbance of this reaction solution is measured. A method for analyzing histamine, which comprises analyzing histamine by measuring it. 2. The analytical method according to claim 1, wherein the reaction is carried out at a temperature of 40°C to 70°C. 3. The analytical method according to claim 1, wherein the orthophthalaldehyde solution has a pH of 5 or less. 4. The analytical method according to claim 1, wherein the solvent for orthophthalaldehyde is a buffer solution which may contain an organic solvent. 5. A mobile phase supply section that supplies a buffer solution with a pH of 7 to 9, and a mobile phase channel connected from the supply section to a cation exchange chromatography column or a reversed phase chromatography column via an introduction section for a histamine-containing sample. and an eluent flow path extending from the column and connected to a detection unit equipped with a fluorescence or absorbance measuring means, a coiled mixing reaction tube set in the elution flow path, and upstream thereof. A histamine analyzer characterized in that it is equipped with a liquid feeding section for orthophthalaldehyde solution. 6. The analysis device according to claim 5, wherein the eluent flow path is provided with a heating means capable of heating the eluent to 40 to 70°C.