JPH0368859A - Determination of dopa, catecholamine and their metabolites - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for quantifying dopa, catecholamines, and their metabolites, and more specifically, for example, in a specimen such as urine, blood, cerebrospinal fluid, brain tissue, etc. The present invention relates to a method for quickly and easily quantifying dopa, catecholamines, and their metabolites contained in

[Prior art and problems to be solved by the invention] In order to eliminate catecholamine-producing tumors such as neuroblastoma and pheochromocytoma, it is possible to eliminate dopa, dopamine, norepinephrine, epinephrine, etc. contained in specimens such as urine. Concentrations of catecholamines and their metabolites, such as vanyl biruvic acid, vanillactic acid, homovanillic acid, vanillin mandelic acid, 3-methoxytyramine, norepinephrine, and metanephrine, were determined by filter paper color method, thin layer chromatography, and gas chromatography. , mass spectrometry, liquid chromatography, and other methods are known.

However, these quantitative methods are unsuitable for clinical analysis because they require complicated operations, cannot be automated, and require expensive equipment, and liquid chromatography has recently become increasingly popular in the field of clinical analysis. Although it is considered the most promising analytical method, it has the disadvantage that it takes time to separate the target substance using the column, and the quantitative results vary widely due to column deterioration and changes in characteristics, resulting in a lack of reproducibility.

The purpose of the present invention is to be able to quickly and easily quantify dopa, catecholamines, and their metabolites contained in a sample, and 6) to be able to automate the measurement and continuously process a large number of samples. , dopa, catecholamines and their metabolites.

[Means for Solving the Problems] The present inventor selectively electrochemically active substances other than dopa, catecholamines, and their metabolites contained in a sample using a constant potential electrolyzer set at a specific potential. We have discovered that it is possible to electrolyze and quantify dopa, catecholamines, and their metabolites remaining in the sample using a coulometer or amperometer set at the potential of the constant potential electrolyzer and a specific potential range, and the present invention reached.

That is, in the method for quantifying dopa, catecholamines, and their metabolites of the present invention, electrochemically active substances other than dopa, catecholamines, and their metabolites contained in a sample are measured at a working electrode potential of +0.1 to +0. Electrolysis is carried out in a constant potential electrolyzer set at 6V (silver/silver chloride sanbai electrode group), and then dopa, catecholamines, and their metabolites are electrolyzed by adjusting the potential of the working electrode to the potential of the working electrode of the constant potential electrolyzer. The feature is that electrolysis is carried out using a coulometer or amber make set at a potential 0.01 to 0.5 μ higher.

The present invention will be explained in detail below.

The quantitative method of the present invention uses electrochemically active substances other than dopa, catecholamines, and their metabolites (hereinafter simply referred to as catecholamines, etc.) contained in a sample using a constant potential electrolyzer such as a coulometer. Electrolyze in advance.

The specimen is not particularly limited, and examples include urine, blood, cerebrospinal fluid, brain tissue, and other animal tissues.

The potential of the working electrode of a constant potential electrolyzer (hereinafter sometimes simply referred to as a reactor) that electrolyzes electrochemically active substances other than catecholamines is +0.1 when a silver/silver chloride electrode is used as the electrode. ~+0.6V, preferably +
0.3 to +0.55V, more preferably +0.45 to
Set to +0.55V.

By setting such a potential or passing the sample through the actuator, it is possible to satisfactorily electrolyze electrochemically active substances other than catecholamines and the like contained in the sample.

Note that if the potential of the working electrode is less than +0.1V, electrochemically active substances other than the substance to be measured, such as catecholamines, remain without being decomposed, and if it exceeds +0.6V, The substance to be measured decomposes.

The potential of the working electrode of the present invention is specified by the potential when the reference electrode is a silver/silver chloride electrode, but if another reference electrode is used, for example, a potential corresponding to the above-mentioned potential corrected for a standard hydrogen electrode It can be carried out in

After electrochemically active substances other than catecholamines and the like contained in the sample are electrolyzed in this manner, the substances to be measured such as catecholamines remaining in the sample are electrolyzed using a coulometer or an amperometer.

Coulometer or amperometer that electrolyzes catecholamines, etc. (hereinafter sometimes simply referred to as a detector)
The potential of the working electrode is 0.01 lower than the potential of the reactor.
Set the potential to ~0.5v higher.

Note that if the potential of the working electrode of the detector is less than 0.01■, the substance to be measured cannot be detected;
If the value exceeds 100%, the coexisting substances will be electrolyzed and the measurement accuracy will decrease.

The quantitative method of the present invention electrolyzes catecholamines, etc. under the potential of such a working electrode, and for example, when the detector is a coulometer, the amount of electricity required for electrolyzing the catecholamines, etc., or when the detector is an amperometer. In some cases, catecholamines, etc. are determined based on the current during electrolysis.

Next, the measurement system used in the quantitative method of the present invention will be explained with reference to the drawings.

Examples of measurement systems used in the quantitative method of the present invention include:
As shown in FIGS. 1 to 4, the measurement is performed using a measurement system in which a coulometer a as a reactor 1 and a coulometer a and/or an amperometer b as a detector 2 are connected in series.

The coulometer used as the reactor 1 or the detector 2 is not particularly limited as long as it can be used for so-called coulometono. For example, as shown in FIG. a working electrode 11, a reference electrode 12, a counter electrode 13, an electrolytic cell 14 housing these, and a coulometer 15 incorporating a potentiostat that applies a constant potential to the working electrode 11 and the counter electrode 13 with reference to the reference electrode 12. One example is a device having the following.

The working electrode of the coulometer is not particularly limited and any known working electrode can be used, but carbon materials that are porous and have a large surface area are particularly preferred.

As the carbon material, for example, the carbon material previously proposed by the present inventors (
(Japanese Patent Application No. 1-136371), that is, the surface thereof has an average pore diameter of 0.1 to 50, preferably 1 to 30.
F pores, a specific surface area of 10 rr+''/g or more, preferably 50 rn''/g or more, and an average carbon layer spacing (d002) determined by X-ray analysis of 3.
Porous graphitic carbon compacts possessing physical properties of 35 to 3.42 mm, preferably 3.35 to 3.40 mm, can be used.

The reference electrode of the coulometer is not particularly limited and any known electrode can be used, but preferred among these are a silver/silver chloride electrode and a ferrician iron/ferrocyan iron electrode.

The counter electrode of the coulometer is not particularly limited, and for example, an electrode made of a known corrosion-resistant metal can be used, but platinum, gold, and stainless steel are particularly preferred.

The amperometer is not particularly limited as long as it can be used for so-called amperometry; for example, as shown in FIG. , based on the electrolytic cell i4 containing these and the reference electrode 12,
An example may be a device having an ammeter 16 incorporating a potentiostat that applies a constant potential to the working electrode 11 and the counter electrode 13.

There are no particular restrictions on the working electrode of an amperometer.
Although known working electrodes can be used, carbon electrodes are preferred, and more preferred are carbon electrodes.
It is a glassy carbon electrode.

There are no particular restrictions on the reference electrode of an amperometer.
Generally, a reference electrode that can be used in a coulometer can be suitably used.

There are no particular restrictions on the counter electrode of the amperometer, and a counter electrode that can normally be used in a coulometer can be suitably used.

Note that the measurement system that can be used in the quantitative method of the present invention only needs to have at least a reactor and a detector coupled in series as described above, for example, as shown in FIGS. 1 to 3. , a measurement system in which a coulometer a forming a reactor l and a coulometer a or an amperometer b forming a detector 2 are connected in series.

Alternatively, as shown in FIG. 4, for example, a measurement system may be used in which a plurality of coulometers a constituting the detector 2 are connected in parallel. In this case, by setting one of the coulometers a to the reactor potential and the other to the detector potential, and measuring the difference in current value between the two using the difference current amplifier 18, the coexistence that was not electrolyzed in the reactor 1 can be measured. A current value based on an object can be corrected.6 Alternatively, a measurement system (not shown) may be used in which a plurality of coulometers constituting a reactor are connected in parallel.

In addition to a coulometer and an amber make, the measurement system used in the quantitative method of the present invention can also include a filter or a precolumn 3 upstream of the reactor 1, as shown in FIGS. 1 to 4, for example.

Filters and precolumns are used to remove components such as proteins contained in the sample in advance to prevent them from affecting the electrochemical properties of the reactor and detector or clogging the reactor and detector. be.

The filter is not particularly limited as long as it does not have the ability to retain catecholamines, etc., and for example, sintered filters, various glass filters, etc. can be suitably used.

As the pre-column, for example, a pre-column filled with alkylated Shinoriki gel, a reversed-phase packing material such as styrene-divinylbenzene copolymer and its derivatives, a cation exchange resin, etc. can be used.

In addition, in the measurement system shown in FIGS. 1 to 4, normally,
In order to smoothly conduct the sample 4A into the reactor l and the detector 2, the carrier is pumped into the reactor l by the pump 7.
The light is flowing in two directions from the detector.

As a carrier, water (specifically, 50%
% or more) can be used, but the pH of the buffer solution is preferably 2 to 10, and the content of methanol or acetonitrile is preferably 30% or less.

The flow rate of the carrier flowing into the measurement system is usually 0.1 to 1.
10-7 minutes, preferably 0.3-2 d/min.

Further, as shown in FIGS. 1 and 4, the carrier supplied to the reactor and the detector may be disposed of in the waste liquid reservoir 9 after being conducted from the carrier reservoir 8 to the reactor 1 and the detector 2, or may be disposed of in the waste fluid reservoir 9 or As shown in the figure and FIG. 3, it may be used in cycles.

In addition, when the carrier is used in circulation, it is preferable to provide a constant potential electrolysis device as a guard cell 5 upstream of the reactor 1, as shown in FIGS. 2 and 3.

The guard cell has the function of pre-electrolyzing the electrochemically active substance contained in the circulating carrier.

As the guard cell, a coulometer, which can normally be used as a reactor or a detector, can be suitably used.

The potential of the working electrode of the constant potential electrolyzer used as a guard cell is either the same as the potential of the working electrode of a coulometer or amperometer used as a detector, or 0.
Set to a higher potential of O1 to 0.5V.

(Function) In the measurement system shown in FIGS. 1 to 4, the sample 4A introduced into the system from the front stage of the reactor 1 using the sample injector 4 etc. is set to a predetermined potential along with the carrier. The electrochemically active substances other than catecholamines and the like contained in the sample 4A are electrolyzed here.

Next, catecholamines and the like that have not been electrolyzed in the reactor 1 are led together with the carrier to a detector 2 set at a predetermined potential and are electrolyzed.

During this electrolysis, for example, when the detector is a coulometer, an electrolytic response signal such as catecholamine is detected as a coulometric value, or as a current value when an amperometer is used,
This electrolytic response signal is then input to the meter 6 or data processing device, and catecholamines and the like are quantified based on the obtained data.

[Effects of the Invention] As described above, the method for quantifying catecholamines, etc. of the present invention does not require a separation column for separating catecholamines, etc., and since the quantitative determination of catecholamines, etc. is carried out electrochemically, it can be performed quickly and easily. The concentration of catecholamines, etc. contained in a sample can be determined with excellent reproducibility.

Furthermore, measurements can be automated and a large number of specimens can be processed continuously.

[Example] The present invention will be explained in more detail with reference to Examples below.

Example 1 Using the measurement system shown in Figure 1, the sum of vanillylmandelic acid, homovanillic acid, and vanillactic acid in the urine of 3 neuroblastoma patients, 2 pheochromocytoma patients, and 10 healthy individuals was quantified, We investigated the applicability of this method to the diagnosis of catecholamine-producing tumors.

Note that a coulometer was used as the detector.

0.0 with 10% methyl alcohol as carrier.
Using a 0.05 molar phosphoric acid aqueous solution, the flow rate was 0.51 d! /min through the measurement system.

A porous graphite carbon molded body was used as the working electrode, and stainless steel was used as the counter electrode.

A silver/silver chloride electrode is used as a reference electrode, and based on this, the reactor potential is +0.5 V and the detector potential is +0.5 V.
5V, and the potential of the guard cell was set to +0.65V.

As samples, 10-fold dilutions of urine from patients with catecholamine-producing tumors and urine from healthy individuals were used.

1Ou of this sample was taken out and injected into the measurement system, and the amount of electricity required for electrolysis of catecholamines, etc. contained in the sample was determined.

In addition, the quantitative value of catecholamines etc. contained in the sample is
A separately prepared standard solution of vanillylmandelic acid was injected into the measurement system, and the amount of electrolysis was calculated in terms of vanillylmandelic acid based on the obtained electrolytic capacity.

The results are shown in Table 1.

In addition, the analysis time of the measurement system was determined from the time of sample injection to the time of output of electrolytic power, and the standard solution of vanillyl mandelic acid was injected into the measurement system 10 times, and the reproducibility of the quantitative value (R3D) was determined.
I asked for

The results are shown in Table 2.

Comparative Example 1 From a sample similar to Example 1, liquid chromatography [
Separation column: Nucleosil 5-Cre, eluent: 0.05M citric acid/acetonitrile (7:1
．． After separating vanillylmandelic acid, homovanillic acid, and vanyl lactic acid using [V/V)], conduction was conducted through the same detector as in Example 1, and the analysis time and reproducibility were determined in the same manner as in Example 1.

The results are shown in Table 2.

Table 1 Quantitative values of vanillylmandelic acid, homovanillic acid, and vanyl lactic acid (rIg/l) in urine Subjects Healthy subjects (10 people) Neuroblastoma patients (3 people) Quantitative values 1 to 10 to 35 Pheochromocytoma patients (2 Table 2 Comparison of analysis time and reproducibility Method of Example 1 30 seconds ±5% Method of Comparative Example 1 20 minutes ±10% Evaluation Vanillyl mandel in the urine of healthy people and patients with catecholamine-producing tumors. As shown in Table 1, clear differences were detected in the acids, homovanillic acid, and vanyl lactic acid, confirming the effectiveness of water washing in diagnosing catecholamine-producing tumors.

Furthermore, as shown in Table 2, compared to the conventional method, the method according to the present invention took less time for analysis and had better reproducibility, making it an excellent analytical method.

[Brief explanation of drawings]

1 to 4 are conceptual diagrams illustrating a measurement system used in the quantitative method of the present invention. FIG. 5 is a conceptual diagram illustrating the coulomake according to the present invention. FIG. 6 is a conceptual diagram illustrating an amperometer according to the present invention. 1...Reactor 2...-Detector 3...Filter or precolumn 4...
・Sample injector 4A...Sample 5...
・Guard cell 6...-Recorder 7...Pump 8...Carrier reservoir 9...-Waste liquid reservoir 11...Working electrode 12... ... Reference electrode 13 ... Counter electrode 14 ... Electrolytic cell 15 ... Coulometer 16 ... Ammeter
17...Sample channel 18...Difference current amplifier a...Coulometer b...Amperometer

Claims (1)

[Claims] (1) Electrochemically active substances other than dopa, catecholamines, and their metabolites contained in the sample are measured at a constant potential of the working electrode set to +0.1 to +0.6 V (based on the silver/silver chloride reference electrode). Electrolysis is carried out in an electrolysis device, and then dopa, catecholamines, and their metabolites are transferred to a coulometer or amperometer whose working electrode potential is set to 0.01 to 0.5 V higher than the potential of the working electrode of the potentiostatic electrolysis device. 1. A method for quantifying dopa, catecholamines and their metabolites, which comprises electrolysis.