JPS6049262A - Automatic analysis device for adenine - Google Patents

Abstract

PURPOSE:To eliminate the need for laborious pretreatment and to enable analysis with high sensitivity by incorporating bromoacetoaldehyde which is a fluorescence reagent into an eluate from a high-speed chromatograph column emitted therefrom after sepn. of adenine or the like in said column then passing the same through a reaction vessel and detecting the formed fluorescent material. CONSTITUTION:A BrCH2CHO soln. 6 is added from a pump 5 to the eluate emitted from a column 4 held at an optimum temp. by a thermostatic chamber 4' after separating adenine or the like in said column by feeding, for example, an eluate 2 from a pump 1 to a sample feeding part 3 and passing the same through said column with a device for separating and analyzing the nucleic acid component in a living body by using a high-speed chromatograph. Said liquid is fed to a reaction tube 7 in a thermostatic chamber (100 deg.C) 7' and the resultant product of reaction is cooled in a cooling pipe 8 and is fed to a fluorescence detector 9, by which the fluorescence intensity is detected. The result thereof is recorded and displayed on a recorder 10. The automatic analysis of 1 Pmol adenini with high accuracy is thus made possible. The BrCH2CHO may be added prelimarily into the eluate 2 and be mixed with the sample in the sample feeding part.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating and analyzing adenine derivatives found in animal and plant tissues or body fluids using high-performance liquid chromatography, and in particular, the present invention relates to a method for separating and analyzing adenine derivatives found in animal and plant tissues or biological body fluids, and in particular, uses bromoacetaldehyde as a fluorescent reagent to separate and analyze adenine derivatives. This relates to a method and device for automatically analyzing adenines online with high sensitivity through a fluorescent reaction with adenine, and is used to quantify δ1 adenines, which are found in a wide variety of natural products such as animals and plants, and in body fluids of living organisms. , quantification of synthetic adenine, further applications include analysis of freshness of fish, organs, etc., and structural analysis of nucleic acids (DNA
It can be widely used for sequence analysis).

Prior Art Since many types of adenine derivatives exist in trace amounts in vivo or in natural products, specific and highly sensitive analysis is required.

Conventionally, a number of methods have been used to analyze adenine compounds using ultraviolet absorption, but since there are many substances that absorb ultraviolet rays, they are not specific and selective, and the sensitivity is limited because it is measured using a light-absorbing method. Not.

Recently, a method (J,
Chromatogr, 123.220, 1976
) have been proposed, but the chloroacetaldehyde used in these reactions has various drawbacks, such as poor reactivity, high temperatures and long reaction times, and insufficient reaction rates. .

Overcoming these above-mentioned drawbacks, a fluorescent substance is produced by reacting bromoacetaldehyde with adenine as a fluorescence analysis method for adenine which has high fluorescence reactivity, therefore high sensitivity and excellent selectivity. An analytical method for detecting the fluorescence has been filed as Japanese Patent Application No. 53-22556.

However, although this method is excellent in sensitivity, it has various problems in operation. In other words, a pre-labeling method in which a bromoacetaldehyde reagent is reacted with adenine and a buffer solution is used before separating and detecting adenines using high-performance liquid chromatography, which precludes online automation due to the troublesome pre-labeling process. In addition, labeled components change over time, resulting in detection errors, and since the BJFF moacetaldehyde reagent is an irritating reagent, care must be taken in pre-labeling.

Purpose The present invention has been made based on the current situation, and takes advantage of the advantage that when bromoacetaldehyde is reacted with adenine, highly sensitive fluorescent coloring can be obtained and highly sensitive detection can be performed.
An object of the present invention is to provide a method and apparatus for automatic adenine analysis, which eliminates the need for complicated pretreatment before separation and analysis using a liquid D-madograph, and enables automatic analysis by conducting reactions online.

In order to achieve the object of the present invention, the method of the present invention employs two different mixing methods to analyze adenine compounds with the fluorescent reagent bromoacetaldehyde.

One of the configurations of the present invention is that after adenine is separated in a column of a high-performance liquid chromatograph, a fluorescent reagent bromoacetaldehyde is mixed into the column solution (by a separate reaction reagent pump) and sent to the column solution. The entire column eluate is passed through the reaction tank and the generated fluorescent substance is detected by a fluorescence detector for analysis.

Another configuration is to include the fluorescent reagent bromoacetaldehyde in the eluent before separation in a high-performance liquid chromatography column, pass the column eluate through a reaction tank, and transfer the generated fluorescent substance to a fluorescent detector. It is used for detection and analysis.

Either of these two configurations can be used to analyze adenine compounds in the same way.

Examples of the present invention are shown below.

Example An embodiment of the present invention is shown in Zorrock diagrams in FIGS. 1 and 2.

The same numbers in 1 indicate the same thing. Fig. 1 shows an example of a method in which a fluorescent reagent, bromoacetaldehyde, is mixed into a column eluate separated by a column using a gold reagent pump. 1 is a pump for feeding the eluent, 5 is a pump for feeding the reaction reagent, 2 is the solvent, 4 is Calano, 4' is a constant temperature bath, 3 is a sample injection part, 6 is a fluorescent reagent bromoacetaldehyde solution, 7
1 is a reaction tank, 7' is a constant temperature bath, 8 is a cooling device, 9 is a fluorescent detector, 10 is a recorder, and 11 is a drain liquid. 4' is a constant temperature bath for keeping the column temperature at optimum temperature conditions, and 7' is for keeping the reaction tank at optimum temperature conditions.

FIG. 2 shows an example in which bromoacetaldehyde is sent together with the eluent. Fluorescent reagent bromoacetaldehyde,
Before passing through the column, the sample is placed in solvent 2 and injected through sample injection port 3 for analysis. Column 4, constant temperature bath 4'
, reaction 4 carbon dioxide 7, constant temperature bath 7', cooling pipe 8, detector 9,
Record 1110 is similar to FIG. Adenines are fluorescent reagent bromoacetaldehyde (BrCIL2CILO
) reacts with the fluorescent material to induce I N'-etheno. Generate a body and irradiate 240~320cm of light: 380~
Analysis by detecting 500 screams of fireflies 8 i'L
Ru.

The measurement results obtained by separating and analyzing the present invention using the configuration shown in FIG. 2 are shown in FIG. 3 and subsequent figures.

The measurement conditions are 9 below.

Solvent: 0.025 M citric acid-0.05 M sodium hydrogen phosphate-0.3 M salt buffer solution (PH
5,0) Near-human nitrile (4:IV/V)0.
1M glomoacetaldehyde flow rate: 0.1 m for 2 minutes Column: Hitachi Dal 30/2 N (4,6 m IDX 3
5m+L) Temperature 45C Reaction coil: 0.1m IDX 30mL Temperature 1
00C detection: JASCO FP-110 Excitation 253.7]m Emission 400 nm The F3 diagram shows the effect of the acetonitrile concentration in the eluent on the capacity factor.

The horizontal axis is the concentration of acetonitrile, and the vertical axis is the capacity and city.
The factor is the logarithm of ``togK'', where ``ha''
−(tr to )/to is defined. Here t.

is the retention time of adenosine, and tr is the retention time of other components. Generally, to elutes without being retained in the column during the retention time of components such as sample solvent.

The opening in the figure is AMP (adenine monophosphate)
P (adenine triphosphate) is shown. Adeni/ is considered to be eluted without being retained. From FIG. 3, it can be seen that if acetonitrile is selected for separation, the retention times will be different and separation will be achieved.

Figure 4 shows the sol column reaction of adenine and bromoacetaldehyde (reaction before separation on the column) and post-column reaction (reaction before separation on the column).
A chromatogram of the reaction after separation on a column is shown.

A: Bromoacetaldehyde, a component silalabeled by the adenine I P +r+ot, no I/column reaction, was added to the anti-LiS coil 100C'. with the eluent. Separation analysis example B when passing through r
Analysis example C through a reaction coil Adenine I without column reaction
An example of separating and analyzing bromoacetaldehyde by sending bromoacetaldehyde together with the eluent through a reaction coil at 100C, and an example of analysis when the reaction coil was kept at a constant temperature of 5r and 20C. Shows temperature effects. The curves in the figure are the reaction yields when 3P mot adenine is separated using an eluent containing bromoacetaldehyde and passed through reaction coils at various temperatures, and the components of 1], × 80, · are It is similar to FIG.

The higher the temperature, the higher the peak value.

Figure 6 shows the calibration curve for adenines. The horizontal axis is the injection amount and the vertical axis is the peak value. The components of mouth, x, ○, and . are the same as in FIG. 3.

Figure 7 shows a chromatogram of ATP.
This is an example of analyzing ATP of P mat.・Figure 8 shows the chromatogram of the brain extract of rat (m
Ale Wlster 2 10 S' type fresh brain was homogenized in 3mt of 0.4MHCω4, a portion of the centrifuged supernatant was diluted 50 times with eluent, and the diluted solution 1 μtf was injected and separated and analyzed. This is a chromatogram.

It is clear from the measurement results in Examples that when bromoacetaldehyde is reacted with adenine, highly sensitive fluorescent coloring can be obtained and detection with madder sensitivity can be performed. In addition, online automatic analysis is now possible since the analysis is performed by omitting the troublesome procedure of fluorolabeling, in which the bromoacetaldehyde reagent, adenine, and buffer are all reacted.

The apparatus for separating and analyzing adenine by highly sensitive automatic analysis according to the present invention can be used in a variety of fields, such as the determination of aranine compounds in the body fluids of animals and plants, and the structural analysis of nucleic acids, making it a great contribution to the Japanese industry.

[Brief explanation of drawings]

FIGS. 1 and 2 show a block diagram of the automatic adenine analyzer of the present invention. Figure 1 shows bromoacetaldehyde sample = 1g, 1?
Fig. 2 is an example in which bromoacetaldehyde is sent together with solution 61#, Fig. 3 is a measurement example showing the effect of the acetonitrile concentration in the eluent on the capacity factor, Fig. 4 The figure shows the chromatogram of the pre-column reaction and post-column reaction of adenine and bromoacetaldehyde.
fl [, Figure 7 is a chromatogram of ATP, and Figure 8 is a chromatogram of brain oil exudate. 1... Pump, 2... Eluent, 3... Sample injection port, 4... Column, 5... Reagent 7j? pump, 6...reaction liquid, 7...reaction coil, 8...cooling/guizo, 9.
...Fluorescence detector, 10...Record, 11...Drainage. Patent Applicant JASCO Corporation Agent Yukio Maruyama No. 3 Figure 10 30 50V/VO/. ACetO=tti'le Figure 4 J) lpl Ge・1 6 Figure IJ Yuhekoshi 7 Figure 8'/11 0-

Claims (2)

[Claims] (1) In a device that separates and analyzes adenines using high-performance liquid chromatography, a new reaction reagent is fed into the column eluate separated by the column, I? A fluorescent reagent, bromoacetaldehyde, is mixed in the liquid and sent, the mixed column eluate is passed through a reaction tank, and the generated fluorescent product is detected and analyzed using a fluorescent detector. Characteristic automatic adenine analyzer. (2) In a device that separates and analyzes nucleic acid components using high-performance liquid chromatography, a fluorescent reagent bromoacetaldehyde is contained in the eluent, the column eluate is passed through a reaction tank, and the resulting fluorescent product is fluoresced. An automatic adenine analyzer that is completely characterized by detection and analysis using a detector.