JPS5847255A - Chromatographic device for liquid - Google Patents

Abstract

PURPOSE:To enable the analysis of a substance to be detected in a short time for separation and with high sensitivity by a constitution wherein a means for making enzyme react coexistently on a separated substance is provided between the outlet of a separating column of a chromatographic device for liquid and an excitation-light applying element generating fluorescence in the separated substance. CONSTITUTION:An enzume column 10 is provided at the outlet 8 of a separating column 7 of a chromatographic device for liquid through the intermediary of a mixing pipe 9. Enzume corresponding with a substance to be detected is fixed beforehand in the column 10. A solution 11 prepared by melting in a buffer solution a compound emitting fluorescence in enzyme reaction to the substance to be detected, and an auxiliary substance, if necessary, is supplied to the mixing pipe 9 by a pump 12. A product of reaction from the enzyme column 10 is made to flow on the surface of an irradiation plate 14 from an outlet 13 and down to a drain 15. A pulsed excitation light is applied from a light source 17 for excitation onto the irradiation plate, and the fluorescence thus generated is transduced photoelectrically by an electron multiplier 24 and enters a detecting device 25. The pulsed light is emitted in synchronization with the device 25. In this way, microcomponents in blood, urine or the like can be quantified with high sensitivity and in a short time.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to improvements in liquid chromatographs used for analysis. This invention relates to a device that reacts an enzyme with a separated substance that appears at the exit of a liquid chromatograph to obtain a fluorescent substance, and detects the fluorescent substance by irradiating it with light.

It is essential for drug development to observe the effects and side effects of drugs by examining the conditions in which they are metabolized and absorbed or excreted in the body. In recent years, in particular, it has become necessary to investigate the effects and side effects of pharmaceuticals in extremely detailed ways, and technology to quantify extremely small amounts of substances contained in blood, urine, other excreta, secretions, and tissues has become necessary. . The means for this purpose are:
Liquid chromatographs are known as excellent devices.

A liquid chromatograph is a device configured so that when a sample is passed through a separation column together with a developing agent, the components in the sample are separated according to their physical and chemical properties and appear at the outlet. In order to detect trace amounts of substances contained in the substances that appear at the column outlet of this liquid chromatograph, the substance that appears at the outlet is irradiated with a light beam, and the chemical substance is excited by this irradiation and detects the fluorescence emitted. However, it is known as an excellent method for detecting particularly trace amounts of components.

Conventionally, according to this method, 10-6 g in 1 ml is generally
Although it has a sensitivity that can detect chemical substances present at about The development of superior analytical equipment is desired. In addition, conventional liquid chromatographs have the drawback that the time it takes to pass a sample through a separation column is long, resulting in poor analysis efficiency.

The present invention was made against this background, and an object of the present invention is to provide a liquid chromatograph apparatus that can improve detection ability and shorten the time required for analysis.

Furthermore, because the substance to be detected does not emit fluorescence even when irradiated with light, liquid chromatographs can detect substances that could not be detected with conventional devices of this type. The purpose is to provide equipment.

The present invention combines an enzymatic reaction with a liquid chromatography device, and is characterized by a combination of two-step separation: separation using a separation column and separation using the specificity of the enzyme.

When performing two-stage separation in this way, the separation using the separation column in the first stage only requires a coarse separation, making it possible to shorten and simplify the separation column and shorten the time required for separation. .

In addition, in order to improve the detection ability, a pulsed light source is used, and the fluorescent light excited thereby is detected by a detector that operates in synchronization with the pulses.

This configuration makes it possible to irradiate the entire target substance with a beam of light with high instantaneous energy using a small device, and also eliminates the optical noise required to detect excited fluorescence, resulting in extremely high detection performance. be able to.

That is, the present invention is provided with a means for reacting a substance appearing at the outlet of the separation column with an enzyme between the outlet of the separation column and the means for irradiating the light beam, and the means for irradiating the light beam emits light in a pulsed manner. It is characterized in that it includes an excitation light source device, and the detecting means includes a detector that operates in synchronization with the pulses emitted by the excitation light source device.

The means for allowing the enzyme to coexist and react preferably includes an enzyme column filled with a preliminarily immobilized enzyme.

This enzyme (e.g., redox-type enzyme system) contains auxiliary substances (e.g., hydrogen peroxide, NAD, etc.) that participate in the reaction.
Even if it is not possible to directly obtain a fluorescent substance from a sample by coexisting with H, etc., a fluorescent substance can be obtained by reacting the separated substance and the above-mentioned auxiliary substance with an enzyme, This can be observed.

This will be explained in detail with reference to the drawings.

FIG. 1 is a block diagram of an apparatus according to an embodiment of the present invention. The eluent 1 is sent to a pipe line 3 via a liquid pump 2 . A sample 5 to be analyzed is injected into this conduit 3 from a sample pulp 6. The liquid in the pipe line 3 is sent to the column 7 at high pressure by the action of the liquid sending pump 2. Column 7 is a separation column for chromatography containing a known packing material and eluent, and separates the substances that have passed through it according to their physical and chemical properties. appears.

A mixing tube 9 is connected to the outlet of the column 7, and the substance sent to the outlet 8 of the column 7 is guided to the next enzyme column 10, and the enzyme and auxiliary substances sent from the solution 11 by the liquid feed pump 12 are here. mixed. The substance mixed with enzymes and the like reacts in the enzyme column 10. This reaction will be described in detail later, but depending on the type of enzyme and reaction conditions, it is a good method to immobilize a portion of the enzyme in advance in the enzyme column IO. The enzyme column 10 is equipped with an electric heater, and current is passed through a terminal 32 to create optimal temperature conditions for enzyme reaction.

The substance sent out from the outlet 131C of the enzyme column 10 flows down along the surface of the irradiation plate 14 and flows into the drain 15.

The surface of this plate 14 is irradiated with a laser beam from an excitation light source device 17 through a lens 18 . An optical trap 19 is arranged in the path of this light beam, and the light beam is absorbed here. The excitation power supply device 17 is driven by a power supply 20.

Fluorescent light emitted from the substance irradiated with the laser beam on the surface of the irradiation plate 14 passes through the filter 22, lens 23 and slit, enters the electron multiplier 24, is converted into an electrical signal, and is sent to the detector 25. to go into.

The electron multiplier tube is driven by a power supply 26. The part indicated by a dashed line 28 in the figure is a dark box. Another input of the detector 25 is given a signal obtained by splitting the laser beam by a splitter 29 and converting this into an electrical signal by a photoelectric converter 30. The detection output is sent to terminal 31.

Here, the excitation light source device 17 is a nitrogen laser (wavelength 337
．． 1 nm) is optimal. Moreover, the excitation light source device 17 is driven in a pulsed manner. The detector 5 is suitably a boxcar integrator, a gate type photocounter, or the like, and performs integration or gate operation in accordance with the light emission pulse of the excitation light source device 17 to eliminate noise and perform highly sensitive detection.

As described above, the feature of the present invention is that a means for mixing and reacting enzymes is connected to the outlet of the column 7.
The substance produced by this reaction is detected by irradiating it with light, and this enzyme reaction will be explained in detail below using Examples.

In the first example carried out by the inventor, the substrate to be detected is a t-amino acid. Operations for quantifying t-amino acids are extremely necessary in pharmaceutical research and testing. Liquid chromatographs have traditionally been used for this purpose. Using the apparatus shown in FIG.
t-amino acids are successively isolated. This is mixing tube 9
sent to.

On the other hand, solution 11 is a mixture of 0.1 molar phosphate buffer for pH adjustment and 10 molar p-hydroxyphenylacetic acid, and is sent from pump 12 to mixing tube 9, and is poured into enzyme column IO together with the above amino acid. In the fermentation column 10, t-amino acid oxidase (KOl.
4.3.2) and peroxidase (KOl.11.1.
7) is fixed. Figure 2 shows the reaction process. That is, when the substrate is a t-amino acid, the first enzyme is t-amino acid oxidase (BOl.4.3.
2) is used. Due to the action of this enzyme, the substrate t-amino acid is oxidized to 2-oxoacid. To illustrate the reaction, H2 +RC! (!OOH+H2O2+NH31.

As mentioned above, this reaction utilizes oxygen (02) in the air and has the property of producing hydrogen peroxide (H2O2).

This first enzyme may be mixed in through the mixing tube 9 shown in Fig. 1, but according to the results of the inventor's experiments, it is easier and more effective to immobilize it in the enzyme column lO in advance. be.

The hydrogen peroxide (H2O2) produced in this way is
It is decomposed by the action of a second enzyme, hydrogen peroxide decomposing enzyme (peroxidase (Ec, 1, 11, 1, 7)).

The action of this second enzyme oxidizes the auxiliary substance during the decomposition of hydrogen peroxide. As this auxiliary substance, one is selected that is non-fluorescent before oxidation and becomes fluorescent when oxidized, and this is added to the solution 11 shown in FIG.
The mixture is mixed in the mixing tube 9.

The substance that is non-fluorescent before oxidation and becomes fluorescent upon oxidation may be appropriately selected depending on the enzyme to be used. Examples practiced by the inventors are selected from phenols and aromatic amines, with a particular but non-limiting example being p-hydroxyphenylacetic acid.

This second enzyme may also be sent out and mixed from solution (1), but it can also be placed in advance in the enzyme column 10 as an immobilized enzyme. As for the auxiliary substances, it is preferable to mix them in by being delivered from the solution 11 in principle.

In this way, by reacting a substrate to be detected using an enzyme and observing the resulting substance, it is possible to expand the range of objects to be observed, including substrates that do not have fluorescence. In particular, when the present invention is implemented and the enzyme is activated by connecting it to a separation column for chromatography,
The separation in a separation column is not as strict as in conventional equipment, but the target substrate can be selected depending on the enzyme to be reacted with, so the column configuration is simple and easy, making liquid chromatography equipment inexpensive. I can do it.

By this method, 5 x 10 amino acids with aromatic nuclei
It was confirmed that it was possible to quantify up to a concentration of -'gr/ml. Furthermore, it has been found that since the separation column 7 can be shortened, the time required for measurement can be significantly shortened. Therefore, one operator can measure a large number of samples using one device, which is considered to be extremely useful for measuring the effects of pharmaceuticals in the future.

Next, as a second example conducted by the inventor, an example including numerical values will be shown regarding the determination of uric acid in blood.

Take 1 mA of serum, add 10 mt of methanol to it, heat it, and separate the methanol liquid layer. This is dried under reduced pressure. The residue is dissolved in 1 mA of 80 timethanol solution. 10 μt of the sample is separated and quantified using a liquid chromatography device shown in FIG.

Column 7 is filled with RP-0,8 and its length is 3.1
mmφ×250 mm, and 0.1 M 1-potassium phosphoric acid salt is used as the eluent at a flow rate of 0.8 m4/In1n.

From the mixing tube 9 connected to the outlet of column 7, 10-4M
0.33 L of p-hydroxyphenylacetic acid solution
/'min.

The enzyme column IO is filled with uricase (u, c, 1, 7, 6, 3) and peroxidase (BOI, 11, 1, 7) immobilized on glass beads. Enzyme column lO
The size of the column is 2.0 mmφ×100 mm, and the temperature of the enzyme column is maintained at approximately 35°C.

The excitation light source device 17 is a nitrogen laser, and the filter 2
2 transmits fluorescent light of 425 nm and removes scattered light of excitation light of 337.1 nm. This reaction is shown in FIG.

This results in 5X10 g contained in 1 mA of blood.
, r uric acid could be quantified.

The third example of a reaction using an enzyme carried out by the inventor is the reaction system shown in FIG. This uses oxidoreductases as enzymes, and NAD and cotinamide adenine dinucleotide) as auxiliary substances.
or NADP (cotinamide adenine dinucleotide phosphate).

Many of the enzymes of the oxidoreductase family require the action of a common auxiliary substance, NAD or NADP. In addition, this enzyme converts the substrate into the product by converting the NA
D or NADP to MADE or NADPH respectively
change to NADH or NADPH is 540nm
It absorbs wavelengths of 460 nm and emits strong fluorescence at 460 nm.

For this reason, a nitrogen laser (337,1 nm) is used as an excitation light source.
) is extremely effective. If the excitation light is pulsed and the fluorescence is measured in synchronization with this, it is possible to measure a trace amount of NADH or NADPH.

By quantifying NADH or NADPH in this manner, the substrate, which is the object of measurement, can be quantified in a stoichiometric manner. Among oxidoreductases, many use NAD or NADP as a coenzyme, and therefore there are many types of substrates that can be measured, and the range of use is wide.

Next, as a specific example, measurement of a trace amount of lactic acid in blood will be described. The enzyme used here is lactate dehydrogenase (EC, 1.1.1.27). This enzyme requires NAD as a coenzyme and reacts as shown below.

L-lactate NAD? Pyruvic acid ten NADH This reaction is reversible, and hydrazine is added to the pyruvate produced to form a hydracene of pyruvate and prevent the reversible reaction from proceeding. NA generated by this operation
DH is proportional to the amount of lactic acid. NADH is excited with a nitrogen laser (337.1 nm), and the generated fluorescence (460 nm) is measured with an electron multiplier synchronized with the laser.

The above lactate dehydrogenase (r;c, 1, 1, 1, 27
) has a high specificity for the substrate, so in the case of short-time analysis with rapid operation or analysis that does not require sufficient precision, the separation column shown in Figure 1 can be made extremely short, and in extreme cases Measurement can be performed even if column 7 is removed.

The serum is diluted 10 times, and 10 μt of the serum is injected through the sample pulp 6. In this case, the eluent] is pl(8,
5 in 0.1 M phosphate buffer, 0.2 mM NAD.

A solution containing 50 mM hydrazine and a small amount of EDTA is used. Pump 2 has a current of approximately 0.3 mA per minute.

The enzyme column 10 is kept at 25°C. Excitation light source 17
A nitrogen laser (331.7 nm) was used.

As a result, 10 g of lactic acid is present in 1 mA of serum.
It was possible to measure r or less.

In this way, detection can be performed even when the substrate is other than an amino acid or even when the substrate is non-fluorescent. Enzymes, auxiliary substances, acceptors, etc. can be selected in various combinations depending on the substrate.

As explained above, according to the present invention, even extremely small amounts of substances can be modified into a detectable state by biochemical reaction using enzymes. By irradiating this with an excitation light source that emits pulsed light 7 and performing detection in synchronization with this, it is possible to perform measurements with extremely high sensitivity, which has not been possible in the past.

Furthermore, even if the substrate to be detected does not emit fluorescence, it can be quantified by replacing it with a substance that emits fluorescence through an enzymatic reaction as described above, broadening the range of applications of liquid chromatography.

Furthermore, the remarkable effect of combining enzyme reactions is
The point is that even if the degree of separation by conventional separation columns is coarsened, measurements can be performed with sufficiently high sensitivity. This improves measurement efficiency and allows a larger number of measurements to be taken than previously possible, making it extremely effective for investigating the properties of pharmaceuticals.

Furthermore, immobilizing enzymes on enzyme columns allows operation of this type of device without requiring special knowledge or skills for handling enzymes. A conventional liquid chromatograph was a device that was divided into several parts and had to be skillfully connected and handled, but now it can be provided as a single, easy-to-handle device. Moreover, if the enzyme column is provided as a consumable item, the present invention will greatly contribute to analytical technology.

[Brief explanation of the drawing]

FIG. 1 is a configuration diagram of an apparatus according to an embodiment of the present invention. FIG. 2 is a diagram showing an example of a reaction system using an enzyme. FIG. 3 is a diagram showing another example of a reaction system using an enzyme. FIG. 4 is a diagram showing yet another example of a reaction system using enzymes. 1... Eluent, 2... Liquid pump, 3... Piping line,
5...sample, 6...valve, 7...column, 8...
...Outlet, 9...Mixing tube, lO...Enzyme column, 1
DESCRIPTION OF SYMBOLS 1... Solution, 12... Liquid pump, 13... Outlet, 14... Irradiation plate, 15... Drain, 17...
Excitation light source device, 18... Lens, 19... Optical trap, 20... Power supply, 22... Filter, 23...
・Lens, 24... Electron multiplier, 25... Detection device, 26... Power supply, 28... Dark box, 29... Splitter, 30... Photoelectric converter, 32... Terminal . Patent applicant: Nitshin Electric Manufacturing Co., Ltd. Agent: Takashi Ideke, patent attorney

Claims (5)

[Claims] (1) A liquid equipped with a separation column through which a sample passes, a means for irradiating a substance separated by this separation column with light, and a means for converting fluorescence excited by the light into an electrical signal and detecting it. In the chromatographic apparatus, between the outlet of the separation column and the means for irradiating the light beam,
The means for causing an enzyme to coexist with and react with the substance appearing at the outlet of the separation column is provided, the irradiating means includes an excitation light source device that emits light in a pulsed manner, and the detecting means inputs the electric signal to excite the substance. A liquid chromatography device comprising a detector that operates in synchronization with pulses emitted from a light source device. (2) The liquid chromatography device according to claim (1), wherein the means for allowing the enzyme to coexist and react includes an enzyme column filled with a preliminarily immobilized enzyme. (3) Claims (1) or (2) include a means for causing an enzyme to coexist and react with the enzyme, and a means for causing one or more auxiliary substances related to the enzyme reaction to coexist with the enzyme. ) The liquid chromatography device described in item 1. (4) The enzyme reacts the separated substance and the auxiliary substance with a first enzyme, and further converts the substance produced by the reaction involving this first enzyme into a different type of auxiliary substance from the above-mentioned auxiliary substance. The liquid chromatography device according to claim 3, further comprising a second enzyme for reacting to obtain a fluorescent substance. (5) The liquid chromatography device according to any one of claims (1) to (4), wherein the means for allowing the enzyme to coexist and react includes a heating means.