JPH0113059B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an automatic analyzer for directly and selectively measuring catecholamines in biological samples with high precision and high sensitivity.

Catecholamine is a general term for biogenic amines having a catechol (8,4-dihydroxyphenol) skeleton, among which dopamine, noradrenaline, and adrenaline are known as adrenal medullary hormones and chemical transmitters. These measurements are extremely important for diagnosis in clinical medicine. Liquid chromatography is suitable for the separation and analysis of chemically unstable compounds with similar structures, such as catecholamines.

Conventionally, in the analysis of catecholamines using liquid chromatography, ultraviolet absorption photometers, fluorophotometers, and electrochemical detectors have been used as detectors. However, ultraviolet absorption photometers have low sensitivity;
Not suitable for trace analysis. Fluorophotometers are widely used, but they require a reaction reagent to turn catecholamine into a fluorophore, require several minutes for the reaction, and cause peak broadening within the reaction coil after column elution. becomes a problem. Regarding detection sensitivity, the fluorescence detection method has high sensitivity for noradrenaline and adrenaline, but low sensitivity for L-dopa and dopamine. On the other hand, with an electrochemical detector, detection can be performed immediately after column elution, and the detection sensitivity is high, with detection sensitivity of 1 ng or less being averaged. However, conventional catecholamine analysis methods using electrochemical detectors require complex sample pretreatments such as ion exchange separation, alumina adsorption, centrifugation, and solvent extraction before liquid chromatography. The operation required a long time (about 2 hours), and there was a risk that the sample would separate during that time. Furthermore, in order to obtain highly accurate analysis results, skilled technicians are essential for this pretreatment, and furthermore, a considerable amount of valuable biological samples are required for analysis.

The present invention not only further improves the high sensitivity and high reproducibility of electrochemical detection in catecholamine analysis, but also eliminates sample pretreatment for liquid chromatography, which is necessary for liquid chromatography. The aim is to provide an automated analyzer.

The above-mentioned objects of the present invention are achieved on the premise of using a novel potentiostatic electrolytic measurement cell that focuses on the reversible electrode reaction of catecholamines.

That is, the present inventor created an electrochemical detector consisting of a thin layer electrolytic cell (hereinafter referred to as a two-electrode thin layer electrolytic cell) having two working electrodes based on flow potentiostatic electrolytic current measurement, and The potentials of the two working electrodes (vs. silver-silver chloride electrode potential) are set to values necessary for oxidation and reduction of catecholamines, respectively, and after oxidation is performed at the first working electrode, the potential is reduced again at the second working electrode. We discovered that it is possible to selectively detect catecholamines in various electroactive substances by recording the current flowing through them. As a result, even if other components that react irreversibly are included, they will not appear in the reduction curve, so sample pretreatment can be omitted and the catecholamine analyzer can be configured with the following switching flow path system. became possible.

(1) The sample is pumped through ion-exchanged water and merged with the flow of Tris buffer with a pH of approximately 9 to automatically lower the pH to approximately
After adjusting the temperature to 8.5, a micro alumina column (alumina with a particle size of 5 μm in inner diameter
The catecholamines are adsorbed onto the alumina by passing it through a column packed in a 0.5 mm, approximately 2 cm long Teflon tube at a flow rate of several tens of μ/min, and then only ion-exchanged water is sent to wash the column.

(2) Switch the flow path of the micro-alumina column that adsorbed the catecholamines with a valve, and connect it to a micro-separation tube (ODS with a particle size of 5 μm and an inner diameter of 0.5 mm,
Connected to a column packed in a Teflon tube with a length of about 15 cm, the pH obtained by mixing phosphoric acid, boric acid, acetic acid, and sodium hydroxide as a mobile phase
A so-called Britton-Robinson buffer solution of 1.8 is pumped at a flow rate of approximately 8 μ/min to desorb and simultaneously separate catecholamines from the micro-alumina column, and the catecholamines are introduced into the two-electrode thin-layer electrolytic cell of the electrochemical detector described above for detection. Quantify.

The catecholamine automatic analyzer of the present invention, which is constructed based on such a principle, analyzes biological samples approximately.
It requires less than 100 microns and can perform automatic catecholamine analysis quickly with high sensitivity and accuracy without sample pretreatment.

Hereinafter, one embodiment of the apparatus of the present invention will be described with reference to the drawings.

In FIG. 1 illustrating the apparatus of the present invention and its associated flow channel configuration, a sample feeding mechanism 1, a first buffer supply mechanism 2, and a second buffer supply mechanism 3 serve as channels before introducing and discharging a sample into a column. is placed. The sample feeding mechanism 1 includes a sample injection row consisting of a microsyringe 4 and a sample injector 5, and a row consisting of a microfeeder 6, a microsyringe 7, a three-way flow switching valve 8, and a reagent container 9 containing ion-exchanged water. The remaining port of the switching valve 8 is connected to the sample injector 5. A sample measuring tube 5a and a waste liquid container 10 are connected to the sample injector 5. Next, the first buffer supply mechanism 2 and the second buffer supply mechanism 3 each include a row of microfeeders 11 and 12, microsyringes 13 and 14, three-way flow switching valves 15 and 16, and reagent containers 17 and 18. It is made up of In this case, a PH adjustment buffer with a pH of approximately 8.8 is placed in the reagent container 17 in the first buffer column.
The reagent containers 18 in the buffer column each contain a buffer solution with a pH of about 1.8 as a mobile phase for column separation of catecholamines.

The sample sending channel of the sample injector 5 is connected to the mixing joint 2 via the solution filter 19.
0, and the other inlet of the mixing joint 20 is directly connected to the remaining one port (outlet) of the three-way flow valve 15 for the first buffer solution. A merging outlet of the mixing joint 20 is connected to one port of a hexagonal two-channel switching valve 21. This switching valve 21 is used to introduce a sample into a catecholamine adsorption column 22, desorb catecholamines adsorbed on the column 22, transfer them to a separation column 23, and perform other valve operations necessary for liquid chromatography, as shown in the figure. Column 22,2
3. The remaining one port (outlet) of the three-way passage valve 16 for the second buffer solution and the waste liquid container 27 are connected.

The elution channel of the separation column 23 is connected to the inlet of the two-electrode thin-layer electrolytic cell 24 described above. Each electrode of the cell 24 is connected to a dual potentiostat 25, and a detection signal from the potentiostat 25 is supplied to a Nippen recorder 26. 27 connected to the cell 24 is a waste liquid container.

Figure 2 shows the structure of the two-electrode thin-layer electrolytic cell and how it is connected to the micro-separation column.
shows the front side of the cell spacer. 3
6 and 37 are working electrodes, each with a diameter of 3 mm.
The glassy carbon disk is embedded in the lower resin plate 32 made of trifluorochloroethylene polymer (hereinafter referred to as "Dyflon") commonly known by the trade name "Dyflon", and its surface is well polished to form a glassy state. Use the one that has been prepared. 28 is a reference electrode, and a silver-silver chloride electrode is fixed to the upper Daiflon resin plate 31 with a screw. 29 is the opposite,
Attach the stainless steel pipe to the Daiflon resin plate 3 using screws.
It is fixed at 1 and also serves as a cell exit. 30 is a spacer for these resin plates 31 and 32, which is made by cutting out the center part of a Teflon membrane with a thickness of 50 μm or less to form a flow channel F, and inserting it between the Diflon resin plates 31 and 32 with four screws. It's complicated. In a preferred embodiment, the micro-separation column 25 is screwed onto the Daiflon resin plate 31.
Contact the empty volume by directly fixing the
It should be extremely small, less than 0.3μ.

FIG. 3 shows the flow path configuration of the apparatus of the present invention, and will be explained using catecholamine analysis in a urine sample as an example. First, the three-way flow valve 8, 15
and 16 are set in the positions shown in FIG. 1
3 and 14. The sample PH adjustment solution 34 necessary for selectively adsorbing catecholamines onto the alumina column includes 0.25% EDTA (2Na) and 0.25% EDTA (2Na) as catecholamine stabilizers.
Use a Tris buffer with a pH of approximately 8.8 containing _0.05NaHSO3 . A Britton-Robinson buffer with a pH of about 1.8 containing 0.5 mM sodium 1-heptanesulfonate as an ion pairing agent is used as the mobile phase 35 for rapid desorption of adsorbed catecholamines from an alumina column and mutual separation by a microseparation column. . On the other hand, the valve of the sample injector 5 is set to the position shown in FIG. 3A, and a urine sample of 50 to 100 microns is collected into the microsyringe 4 and injected into the sample loop. Next, the three-way flow switching valves 8, 15, 16, the sample injector 5 valve, and the six-way two-flow switching valve 21 are switched to the positions shown in FIG. 3B, and the microfeeders 6, 11, and 12 are moved in the forward direction. Activate it,
Conditioning of the micro separation column is performed in parallel with conditioning of the alumina column, adsorption concentration of catecholamines in the sample onto the micro alumina column, and washing operations. Conditioning of the alumina column is performed by combining the pH adjustment solution and ion-exchanged water at a flow rate of about 30 μ/min and passing them through the micro alumina column. Catecholamines are adsorbed and concentrated on the micro-alumina column using ion-exchanged water at approximately 30μ/min.
The solids in the sample are removed through a solution filter 19 filled with quartz wool.
The above PH adjustment liquid and mixing joint 2 at the same flow rate
This is done by mixing in 0°C to adjust the pH to about 8.5 and then passing through a micro-alumina column.
Note that a Teflon tube with an inner diameter of 0.5 mm and a length of about 12 cm is used for the flow path from the mixing joint 20 to the microalumina column 22. After carrying out this operation for about 15 minutes, the feeding of the pH adjusting solution was stopped, and only ion-exchanged water was fed for another 15 minutes to wash the alumina column. Thereafter, the hexagonal two-channel switching valve 21 is switched to the position shown in FIG. Perform separation.
On the other hand, set the valve of sample injector 5 to
Switch as shown in Figure A to simultaneously inject the next sample into the sample loop in parallel. After the catecholamines have been completely desorbed from the alumina column, each valve can be switched again to the position shown in FIG. Concentration operations can be carried out. Note that the alumina column can be reused semi-permanently any number of times. The catecholamines separated by the micro-separation column are separated by the working electrodes 36 and 3.
7 into the two-electrode thin-layer electrolytic cell 24 set at 0.8 V and 0.2 V versus the silver-silver chloride electrode, respectively, and measuring the reduction current flowing through the working electrode 37. selectively detect and quantify. Catecholamine is the working electrode 36
It is oxidized to the quinone type, and is reduced again at the working electrode 37 to return to the original catechol type.

Taking adrenaline as an example, the electrode process is as follows.

Note that when approximately 8 μ/min is used as the flow rate of the mobile phase, approximately 60% of the catecholamines oxidized at the working electrode 36 in this electrolytic cell 24 is transferred to the working electrode 3.
At 7, it is reduced again and returns to the original state.

Figures 4 and 5 are chromatograms obtained by injecting 100μ of a urine sample into the device of the present invention.
Each figure, A and B, records the current flowing through the working electrodes 36 and 37, respectively. In addition, each figure A
corresponds to the response of an electrochemical detector using a conventional thin-layer electrolytic cell with one working electrode. In the diagram
NA, AD, DA and LD represent noradrenaline, adrenaline, dopamine and L-dopa, respectively. In the samples shown in FIGS. 4 and 5, noradrenaline and L-dopa are incompletely separated from electroactive substances other than catecholamines, making it difficult to quantify them accurately using only the working electrode 36.
However, since the electrode reactions of many electroactive substances other than catecholamines are irreversible, they are not reduced at the working electrode 37 as shown in each figure B, and if two working electrodes are used, only catecholamines can be selectively detected. It is clear that quantification can be performed with high accuracy.

[Brief explanation of drawings]

FIG. 1 is a flow and block diagram of an analysis system including an embodiment of the device of the present invention, and FIG. 3. A, B and C are flow path diagrams showing the flow path switching states of the system shown in FIG. 1, and FIGS. 4 and 5. are two examples of chromatograms measured by the apparatus of the present invention. 1... Sample feeding mechanism, 2... First buffer supply mechanism, 3... Second buffer supply mechanism, 4, 7, 13,
14...Micro syringe, 5...Sample injector, 20...Mixing joint, 22
... Micro alumina column, 23 ... Micro separation column, 24 ... Two-electrode thin layer electrolytic cell, 28 ...
Reference electrode, 29... Counter electrode and cell exit, 36, 37
...Working electrode.

Claims (1)

[Scope of Claims] 1. A sample pressure-feeding mechanism consisting of an ion-exchanged water supply unit connected to a measuring loop and for delivering the sample in the loop; a mechanism for supplying a buffer solution for pH adjustment; the pressure-feeding mechanism; a mechanism for combining the liquids flowing out from the buffer solution supply mechanism; an alumina-filled column for selectively adsorbing catecholamines in a dissolved sample by introducing the mixed flow formed in the combining mechanism; a separation column for separating catecholamines eluted from the packed column into components; a supply mechanism for a second buffer solution used as a mobile phase in the elution and separation of catecholamines from both columns; one column and the second
A catecholamine adsorption channel connected to a buffer supply mechanism and for guiding at least the mixed flow to the alumina-filled column, and a catecholamine for guiding the second buffer to the separation column via the alumina-filled column. Desorption/separation channel,
What is claimed is: 1. A catecholamine analyzer comprising: a flow path switching valve mechanism for selectively forming the separation column; and an electrolytic cell for measuring the components eluted and separated from the separation column by potentiostatic electrolysis. 2. The electrolytic cell consists of a thin layer electrolytic cell with two working electrodes, the first working electrode has a potential sufficient to oxidize the catecholamine, and the second working electrode has a potential R sufficient to reduce the oxidized catecholamine. 2. The apparatus according to claim 1, wherein: