JPH029307B2 - - Google Patents

Description

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

Catecholamines are a group of biogenic amines having a catechol (3,4-dihydroxyphenol) skeleton, and among these, 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, electrochemical detectors can detect particles immediately after column elution, and have a detection sensitivity of 1 ng or less, which is averaged with high sensitivity. 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 the longest time (approximately 2 hours), and there was a risk that the sample would decompose 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 provides a flow potentiostatic electrolysis cell that can further improve the high sensitivity, high precision, and high reproducibility of electrochemical analysis in catecholamine analysis, and also provides sample adsorption and
The purpose is to connect separation column channels.

That is, the present invention focuses on the fact that the electrode reaction of catecholamines occurs reversibly. In an electrochemical detector consisting of a cell, the extreme surface of a first working electrode is exposed on the upstream side of a flow channel formed in a thin layer, and the extreme surface of a second working electrode is exposed on the downstream side. After exposing the surface and setting the potentials of the first and second working electrodes (versus silver-silver chloride electrode potential) to the potentials necessary for oxidation and reduction of catecholamine, respectively, oxidizing the catecholamine with the first working electrode. Only catecholamines in various electroactive substances are selectively detected by recording the current flowing during re-reduction at the second working electrode. Since this allows the presence of other components that react irreversibly, operations from sample preparation to catecholamine adsorption/separation column can be simplified and automated as described below.

(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 to 8.5, Teflon (registered trademark name)
The catecholamines are adsorbed onto the alumina by passing them through a micro-alumina column (a Teflon tube with an inner diameter of 0.5 mm and a length of approximately 2 cm) at a flow rate of several tens of μ/min, and then the ions Wash the column by sending only exchange water.

(2) Switch the flow path of the micro-alumina column adsorbing 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 μm).
Britton column with a pH of approximately 1.8 as the mobile phase.
- Robinson's buffer solution is pumped at a flow rate of about 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 for detection and quantification. The catecholamine automatic analyzer of the present invention, which is constructed based on such a principle, requires only about 100μ or less of a biological sample;
Furthermore, catecholamines can be automatically analyzed rapidly 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 reagent 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 the sample into the catecholamine adsorption column, desorb the catecholamine adsorbed in the column 22, transfer it to the separation column 23, and perform other valve operations necessary for liquid chromatography. 22, 23 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 8 mm.
A glassy carbon disk is embedded in the lower Daiflon (registered trademark) resin plate 32, and its surface is well polished to make it glass-like. 28 is a reference electrode, and a silver-silver chloride electrode is fixed to the upper Daiflon resin plate 31 with a screw. Reference numeral 29 denotes a counter electrode, which is a stainless steel tube fixed to the Diflon resin plate 31 with screws, and also serves as a cell outlet. 30 is a spacer for these resin plates 31 and 32, which is made by cutting out a small piece in the center of a Teflon film 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 directly fixed to the Daiflon resin plate 31 with screws, thereby making the empty volume due to communication extremely small to 0.3 microns or less.

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 switching valve 8,
15 and 16 are set in the positions shown in FIG. 7, 13 and 14. 0.25% EDTA (2Na) is used as a stabilizer for catecholamines as the sample pH adjustment solution 34 necessary for selectively adsorbing catecholamines on the alumina column.
and a Tris buffer with a pH of about 8.8 containing 0.05% _NaHSO3 . 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, sample injector 5
Set the valve in the position shown in Figure 3A,
A urine sample of 50 to 100μ is collected into the microsyringe 4 and injected into the sample loop. Next, the three-way flow switching valves 8, 15, 16, the valve of the sample injector 5, and the hexagonal two-way flow switching valve 21
is switched to the position shown in Figure 3B, and the microfeeders 6, 11, and 12 are operated in the forward direction to condition the alumina column, adsorb and concentrate catecholamines in the sample onto the microalumina column, and wash. Conditioning of the microseparation column is carried out in parallel with the operation. Conditioning of the alumina column is carried out 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. The concentration of adsorption of catecholamines on the microalumina column is approximately 30μ when the sample is ion-exchanged with water.
/min to remove solids in the sample through a solution filter 19 filled with quartz wool, and mix it with the pH adjustment solution at the same flow rate in the mixing joint 20 to adjust the pH to about 8.5. This is then carried out by passing it through a micro-alumina column. Note that the flow path from the mixing joint 20 to the micro alumina column 22 has an inner diameter of 0.5
Use a Teflon tube with a length of about 12 cm. 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. Meanwhile, the valve of the sample injector 5 is switched as shown in FIG. 3A, and the next sample is simultaneously injected into the sample loop in parallel. and,
After the catecholamines have been completely desorbed from the alumina column, the concentration operation of catecholamines in the next sample can be carried out during the separation operation using the micro-separation column by switching each valve again to the position shown in Figure 3B. 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 transferred to the working electrode 36.
and 37 potentials of 0.8V and 0.2V vs. silver, respectively.
Two-electrode thin-layer electrolytic cell 2 set to a silver chloride electrode
Each catecholamine is selectively detected and quantified by measuring the reduction current flowing through the working electrode 37. Catecholamine is oxidized at the working electrode 36 to become a quinone type, and the working electrode 3
In step 7, it is reduced again and returns to its original catechol form.

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

In addition, when approximately 8 μ/min is used as the flow path of the mobile phase, approximately 60% of the catecholamines oxidized at the working electrode 36 in the present 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 A
The figure corresponds to the response of an electrochemical detector using a conventional thin-layer electrolytic cell with one working electrode.

NA, AD, DA and LD in the figure are noradrenaline, adrenaline, dopamine and L, respectively.
- Indicates dopa. 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 accurately quantify them 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.
It is clear that by using two working electrodes, only catecholamines can be selectively detected and quantified 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. Figure B is a plan view of the spacer layer; Figures 3A, B, and C are flow path diagrams showing flow path switching states of the system shown in Figure 1; Figures 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 flow channel having an opening formed in a thin layer spacer in a flow type dual working electrode type thin layer electrolytic cell for measuring catecholamines separated and eluted into components from a separation column by constant potential electrolysis. The extreme surface of the first working electrode is exposed on the upstream side of the electrode, and the extreme surface of the second working electrode is exposed on the downstream side of the electrode. A catecholamine analyzer characterized in that a potential sufficient to reduce the oxidized catecholamine is applied to each of the second working electrodes. 2. The apparatus according to claim 1, characterized in that a catecholamine separation column is loaded at the inlet to the flow channel.