JPS60142250A - Method and device for analyzing compound having amino group - Google Patents

Abstract

PURPOSE:To enable automatic analysis with high accuracy by bringing the compd. having the primary or secondary amino group in a sample into reaction with a labeling reagent and removing the excess labeling reagent then analyzing the prelabel deriv. with chromatography. CONSTITUTION:The analysis of the primary amine in a sample is accomplished by feeding a fluorescent reagent such as o-phthalaldehyde or the like and neutral buffer from respective vessels 1, 2 to a sample introducing device 5 and a bypass 6, mixing the same with the sample and capturing the labeling amine in a precolumn 15 as shown by the solid line of a six-way valve 8 via a reaction coil 7. A moving phase is then passed from a moving phase vessel 19 as shown by the dotted line of the valve 8 to introduce the labeling amine in the precolumn to a separating column 16 where said amine is separated with good accuracy. The fluorescent intensity thereof is measured with a detector. The sample contg. secondary amine is converted to the primary amine by an NaClO soln. and is analyzed in the above-described way. The analysis is thus made possible with the higher accuracy than with labeling after the sepn. without spreading of the band, etc. The sepn. and analysis of the secondary amine which are particularly difficult with the prior art are thus made easy.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to a method and an apparatus for analyzing compounds having a primary or secondary adano group, particularly relates to a method for analyzing amino acids having secondary amino groups, polyamines, indoleamines, imidazole amines, and aminoglycoside antibiotics, and an apparatus for the analysis.

Furthermore, the present invention provides a pre-labeling method suitable for automatic analysis of amino acids, indoleamines, imidazole amines, and aminoglycoside antibiotics in biological samples in industrial fields such as organic chemistry, biochemistry, food, pharmaceuticals, and medical devices. The present invention relates to a method for analyzing compounds having a primary or secondary amine group, particularly amino acids and derivatives thereof, and an apparatus for analyzing the same.

(b) Prior Art Fluorescence methods are often used to analyze compounds having primary amine groups, especially amino acids and their derivatives, but the Bostravel method is generally employed.

However, in the Bostravel method, for example, amino acids are separated using an ion-exchange resin and then led to a reaction coil where they are reacted with a fluorescent reagent, such as orthophthalaldehyde or fluorenosamino. In order to detect the amino acids by fluorescent derivatization, the separated amino acids are
The band is diluted by the reaction reagent, and the resulting chromatogram has poor separation.Moreover, even if the reaction can be carried out sufficiently in the reaction coil, the reaction coil must be used to prevent diffusion after separation. It cannot be made sufficiently long, the reaction tends to be insufficient, and its precision is not sufficient. Furthermore, since the fluorescent reagent is supplied by a pump just before detection, pulsation occurs and noise is likely to occur, which is a problem. There is a method of manually pre-labeling and introducing it into a separation column, but it is not easy to perform the pre-labeling reaction accurately under certain conditions and within a certain time, so automation has been desired. .

Therefore, an automated method was adopted in which a sample and a fluorescent reagent were flowed through a reaction coil to cause a reaction, and the resulting product was flowed through a separation force.

However, although this analysis method uses an autoinjector, the distance from the suction section to the separation cartridge 1 becomes long, and it is inevitable that the spread of the sample at the entrance of the separation cartridge becomes large. Furthermore, the larger the amount of sample, the more the bunt tends to spread during the suction stage, which is a problem. Furthermore, in this method (1. Since a fluorescent reagent is also introduced into the separation column, the analytical components are diluted, which is not desirable (7). In addition, the conditions for separation and the reaction conditions must be similar. There was also a problem with the condition settings.

(c) Purpose The present invention solves the problems in these conventional methods by separating a pre-labeled compound from a fluorescent reagent before introducing it into a separation column. The present invention provides a method for analyzing compounds having a primary or secondary amine group, especially amino acids, by a pre-labeling method, and an apparatus for analyzing the same.

(d) Structure The present invention involves reacting a compound having a primary or secondary amine group in a sample with a labeling reagent, and then reacting the primary or secondary amine group produced by the reaction. The pre-labeled derivative of the compound having a primary or secondary amino group is separated from the excess labeling reagent using an arbitrary solvent, and the separated pre-labeled derivative of the compound having a primary or secondary amino group is subjected to liquid chromatography. A method for analyzing a compound having a primary or secondary amine group by liquid chromatography, which is characterized by separation (-1 detection) by an analytical method, and a labeling reagent supply device and a primary or secondary amine group. A channel connecting the sample introduction device for a sample containing a compound having a secondary amine group is connected to the inlet of the reaction coil, and the outlet of the reaction coil is connected to the population of the precolumn through a channel switching valve. ,
The outlet of the precolumn is connected to a liquid chromatography separation column via a flow path switching valve to an apparatus for analyzing compounds having primary or secondary amine groups using liquid chromatography.

In the present invention, liquid chromatography means liquid chromatography, high performance liquid chromatography, and ultra high performance liquid chromatography.

In the present invention, the pre-labeling reagents include IJl,
There are visual absorbance labeling reagents for external use, fluorescent labeling reagents, and radioactive reagents labeled with IIC or (2).

Fluorescent reagents include ortho-phthalaldehyde, fluoreszamine, 1-7methylaminonaphthaleno-5-sulfonyl chloride (DNS-Cl), 7-chloro-4-1-nitropenzofuratane (NBD-CIJ), etc. However, ortho-phthalaldehyde is preferred because it is water-soluble. As a radioactive reagent, DNS-C# labeled with 1" (cy or
A solution containing acetylcysteine or 2-mercaptoethanol and buffered with sodium borate and potassium borate at pH 9 to 11 is used. 2-Mercapto 1/-ru(-) has poor R properties, so N-acetyl-
It is recommended to use cysteine.

The solvent for separating this fluorescent reagent from, for example, a pre-labeled amino acid is selected from citric acid, phosphoric acid, etc., and a neutral buffer solution such as phosphate buffer, citrate buffer, etc. is used. good. This neutral buffer solution can also contain a small amount of acetonitrile. In the case of analysis of amino acids having secondary amine groups, ascorbic acid is used.

When analyzing a compound having a secondary amine group, particularly an amino acid such as gloline or hydroxyproline, a flow path is formed to introduce a hypochlorous acid reagent. In this way, secondary amines can also be fluorescently labeled, pre-labeled derivatives of compounds having primary or secondary amine groups, especially amino acids having primary or secondary amine groups. As a pre-column for separating the pre-labeled derivative from the ortho-phthalaldehyde reagent, a resin-based reverse phase chromatography column with excellent alkali resistance, such as an octadecylsilylated column as a stationary phase column (01), is used.
S column) is used.

The flow path switching valves that can be formed at both ends of the precolumn can be installed separately, but for example, a six-way valve that can switch both flow paths at the same time has a simpler structure. It is preferable because it is lively.

(e) Example FIG. 1 is a flow path diagram showing an example of the method and apparatus for analyzing compounds having a primary amine group according to the present invention, in particular, an example of application to amino acid analysis.

FIG. 2 is a flow path diagram showing steps up to the pre-labeling step in an embodiment of the method and apparatus for analyzing compounds having primary and secondary amino groups of the present invention.

Hereinafter, the present invention will be explained in detail with reference to these drawings, but the present invention is not limited to these explanations.
Needless to say, it can take various forms.

In FIG. 1, a fluorescent reagent and an O-phthalaldehyde reagent containing N-acylcysteine are used at 17, and a weakly basic buffer solution is used as the solvent.

0-phthalaldehyde reagent container 1 and neutral buffer solution container 2
is connected to a pump 4 via a three-way valve 3 via a pipe. The flow path from the pump 4 is branched, one communicating with the sample introduction device 5 and the other communicating with the bypass flow path 6. The channels from the sample introduction device 5 and the bypass channel 6 are connected together to a reaction coil 7. The outlet of the reaction coil 7 communicates with the inlet hole 9 of the six-way valve 8, and the hole 10 also communicates with the conduit 2.
0 communicates with one port of the precolumn 15 of the ODS column (octadecylsilylated), and the other port of the precolumn 15 communicates with the hole 13 of the six-way valve 8 via a conduit 21. The mobile phase for separation is a neutral buffer containing acetonitrile;
Use the same solution as the neutral buffer in container 2. If necessary, a gradient device can be attached to change the acetonitrile concentration over time. The mobile phase container 19 for separation is connected from the pump 18 to the hole 12 of the six-way valve 8 via a conduit 23. The hole 11 of the six-way valve 8 communicates with the separation column of the ODS column via a conduit 22,
The outlet of separation column 16 communicates with detector 17 .

The procedure for amino acid analysis using the apparatus of this example will be explained below.

The three-way valve 3 and the six-way valve 8 are both connected to the flow path on the solid line side.

First, by operating the pump 4, the 0-phthalaldehyde reagent is sent from the 0-phthalaldehyde reagent container 1 to the reaction coil 7, the pipe line 20, the precolumn 15, and the pipe line 21, while the pump 18 is operated to send the 0-phthalaldehyde reagent to the reaction coil 7, the pipe line 20, the precolumn 15, and the pipe line 21. The mobile phase for separation from the mobile phase container 19 flows from the conduit 23 to the separation column 16 via the six-way valve 8 .

A sample is introduced into the sample introduction device 5. Amino acids in the sample are reacted with 0-7 taraldehyde in the reaction coil 7 to be pre-labeled and collected in the pre-column 15. After a sufficient reaction, the three-way valve 3 is switched to the flow path on the dotted line side, and the neutral buffer is passed from the neutral buffer container 2 to the reaction coil 7 via the pre-column 15 and the pipe line 21 to the six-way valve. 8, the pre-labeled amino acid derivative remaining in the reaction column 7 is sent to the pre-column 15,
While collecting, the 0-phthalaldehyde reagent is expelled from the precolumn 15.

When O-phthalaldehyde and N-acetyl cysteine are expelled from the precolumn 15, the six-way valve 8 is switched to the dotted line flow path, and the neutral buffer containing acetonitrile is passed through the pump 18, the line 23, and the six-way valve. hole 12
, through the hole 13 and the conduit 21 to the pre-column 15, and the pre-labeled amino acid visiting compound in the pre-column 15 is passed through the conduit 2, the hole 10.11 of the hexagonal valve 8 and the conduit 22, Sent to separation column 16.

The pre-labeled amino V derivative is chromatographically separated in the H1 separation column and detected with a fluorescence detector 17.

Generally, the larger the number of samples, the wider the sample band becomes during the sample introduction process. However, in the device of this embodiment, since the sample band is collected after being labeled in the pre-column 15, such a sample band becomes wider. The spread of is eliminated. Moreover, since the mobile phase for separation is sent from the pre-column 15 to the separation column 16 and detected immediately after separation, it is possible to avoid diffusion after separation, so that there is no reduction in resolution due to spread of particles. .

In FIG. 2, thorium hypochlorite reagent container 26 is connected to sample introduction device 30 through pump 29. In FIG. This sample introduction device 3o communicates with a reaction coil 32. The 0-phthalaltehyde reagent container 24 and the neutral buffer container 25 are connected to the reaction coil 33 via a three-way valve 27 and a pump 28.

The analytical procedure for an amino acid having a secondary group will be explained below.

The three-way valve 27 is connected to the flow path indicated by the solid line.

The pump 29 is operated to send the hypochlorous acid reagent from the hypochlorous acid reagent container 26 to the reaction coil 32 . The sample is introduced from the sample introduction device 30 and reacted with hypochlorous acid in the reaction coil 32 to convert the secondary amine groups into primary amine groups.

On the other hand, the 0-phthalaldehyde reagent is sent from the container 24 to the reaction coil 33 by operating the pump 28, and is reacted with the abanoic acid having a primary amine group generated in the reaction coil 32, thereby converting the amino acid into a full label. Generate derivatives.

When the amino acid pre-labeling reaction is completed, the three-way valve 27 is switched to the dotted line flow path to send the neutral buffer containing ascorbic acid from the container 25 to the reaction coil 33.
Amino acid analysis is then performed in the same manner as in Figure 1.

An example of amino acid analysis using the apparatus shown in FIG. 1 is shown below.

As a precolumn, 8WF-2600<inner diameter 4.0 dragon,
Length 30 mm) I-Shim-Pack C 1 manufactured by Sekisui Fine Chemical Co., Ltd. was used as the separation column.
Phenylalanine, tyrosine, histidine, 1-methylhistidine, 3-methyl-
Amino acids such as methylhistidine were analyzed.

The OPA reagent used contained 0.1% N-acetylcysteine and was adjusted to pH with 0.1M potassium borate solution.
A 01% OPA solution of 9.2 was used.

In addition, as a washing solution, a 10mM potassium salt solution with a pH of 7 was used, and as a mobile phase for separation, pl-
A mixed solution of 10 mM potassium phosphate solution and acetonitrile was used. This separation mobile phase was created using a gradient device, starting with a mixed solution of 5% acetonitrile, gradually increasing the concentration of acetonitrile, and finally changing to a mixed solution of 50% acetonitrile.The temperature was 40C. The flow rate for both solutions was 1. Om11
The liquid was delivered in minutes.

-t, the analysis results were good for amino acids such as phenylalanine, tyrosine, histidine, 1-methylhistidine, and 3-methylhistidine.

(f) Effects The present invention uses an ODS column for reversed phase chromatography as a precolumn to convert compounds having primary or secondary amine groups into polymers with a fluorescent reagent or the like. The derivative is once collected in the column, and the excess fluorescent reagent is separated and removed from the pre-labeled derivative, so that the pre-labeled derivative can be concentrated. The band spread that occurs when

Moreover, such primary or secondary
The function of concentrating the prelabeled derivative of a compound having an amino group at the same level allows the length of the reaction coil to be increased, so that the prelabeling reaction can be performed mechanically under the same conditions. Therefore, compared to the conventional boss label method and pre-label method, it is easier to carry out, and moreover, amino acid analysis, for example, can be performed easily and with high accuracy by operating a valve. It can be said that it is suitable for automation.

Furthermore, the present invention allows easy analysis of compounds having secondary amine groups, which has been difficult with conventional Bost column methods and the like.

Furthermore, the pre-label method of the present invention facilitates automation of not only high-performance liquid chromatography analysis but also ultra-high-performance chromatography analysis if the components to be analyzed are limited.

As described above, the present invention has many advantages and has a great influence compared to conventional compounds having a 7-mino group, especially amino acid analysis methods and analyzers thereof.

[Brief explanation of the drawing]

1M is a flow path diagram showing an embodiment of the method for analyzing a compound having 7 primary amyl groups of the present invention and its analyzer, in particular, an example of application to amino acid analysis; FIG. FIG. 2 is a flow path diagram showing steps up to the pre-labeling step in an embodiment of the method and apparatus for analyzing compounds that irritate primary and secondary amine groups of the present invention. 1 and 24 are 0-phthalaltehyde reagent containers, 2 and 2
51′i Neutral buffer container, 3 and 27 are three-way valves, 4,1
8. 28 and 29 are pumps, 5 and 3° are sample introduction devices, 6 and 31 are bypass channels, 7° are 32 and 33 are reaction coils, 8 is a six-way valve, 15 is a brake column, 16 is a separation column, 17 is 19 is a mobile phase container for separation. Procedures Amendment Store (Method) Kazuo Wakasugi, Commissioner of the Patent Office1, Indication of the case, Patent Application No. 251G51, filed in 1982,2, Name of the invention: Method for analyzing compounds having 7 amino groups and apparatus for analyzing the same d
:3. Relationship to the Ill-Stopper Case Name of Patent Applicant (199> Shimaoki Seisakusho Co., Ltd. 4, Agent)

Claims (3)

[Claims] (1) React a compound having a primary or secondary amino group in a sample with a labeling reagent, and then pre-label the compound having a primary or secondary amine group produced by the reaction. The derivative is separated from excess labeling reagent using an arbitrary solvent, and the separated pre-labeled derivative of the compound having a primary or secondary amine group is separated and detected by liquid chromatography analysis method. A method for analyzing a compound having a primary or secondary amine group by liquid chromatography, characterized in that: (2) A compound having a primary or secondary amine group,
The liquid according to claim 1, which is an amino acid having a primary or secondary amine group, a polyamine, an indoleamine, an imidazole amine, an aminoglycoside antibiotic, and a derivative thereof. A method for analyzing a compound having a primary or secondary amine group by chromatography. (3) A channel connecting the labeling reagent supply device and the sample introduction device for a sample containing a compound having a primary or secondary amine group is connected to the reaction coil inlet 11 and the reaction coil A first-class liquid chromatography method, characterized in that the outlet communicates with the inlet of a precolumn via a flow switching valve, and the outlet of the precolumn communicates with a liquid chromatography column via a flow switching valve. is an analyzer for compounds having secondary amino groups.