JPH0448263A - Analytical method and apparatus for catecholamines - 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 analyzing catecholamines in biological fluids, and in particular to an analytical method for removing coexisting components unnecessary for analysis of residual proteins, etc. in sample biological fluids, and Regarding analysis equipment.

[Prior Art] Catecholamine is a general term for epinephrine (E), norepinephrine (NE), dopamine (DA), etc., and plays an important role as an adrenal medullary hormone and a sympathetic neurotransmitter. Catecholamines in biological fluids such as blood and urine are measured in order to diagnose pheochromocytoma and sympathoblastoma and to determine the effectiveness of treatment, and are an extremely important element in clinical medicine.

However, the concentration of catecholamines in biological fluids is low, and their measurement requires high sensitivity and high selectivity. High performance liquid chromatography (HPLC) is widely used as the most suitable method for such analysis and measurement.

Electrochemical detection methods and fluorescence methods are known as HPLC detection methods, but the former lacks specificity, making it difficult to separate catecholamines and unnecessary components [clinical testing, 32. No, l 2. 1522nd to 1527th
Page (November 191118) The latter is based on the trihydroxyindole (THI) method and the diphenylethylenediamine (
However, since the THI method has low sensitivity for dopamine (DA) and is difficult to measure blood levels, the DPE method is generally used.

Other methods include the alumina extraction method in which catechol compounds are extracted using acid-treated alumina, the cation exchange method in which ions such as amino compounds are adsorbed and eluted using a cation exchange column, and the boric acid gel method in which boric acid gel is used.

[Problems to be Solved by the Invention] The recovery rate of catecholamines by the above methods is approximately 85% for the cation exchange method, 60-80% for the boric acid gel method, and 60-70% for the alumina extraction method. The method was also inadequate.

These methods also include measurement errors due to pretreatment (protein removal operation) of the sample biological fluid.

Analyzing these catecholamines requires many steps and complicated operations, including methods for preserving sample biological fluids.
Highly skilled operators were needed [Journal of Chromatography.

344, pp. 61-70 (1985)].

All of the above-mentioned conventional techniques have low catecholamine recovery rates (85% or less) and poor reproducibility, so they are insufficient as highly accurate analytical methods. Furthermore, it is not easy to remove proteins during pretreatment (protein removal rate of 99% or more), and therefore, even in the cation exchange column method, which has the highest catecholamine recovery rate, the packing material for the column is styrene-vinylbenzene polymer gel. Therefore, there is a problem that residual proteins in the sample biological fluid are adsorbed to the packing material by hydrophobic bonds, preventing complete adsorption of catecholamines, and thus the recovery rate of catecholamines is not improved.

Furthermore, there are problems such as adsorption of cationic components and neutral substances other than catecholamines in sample biological fluids, which appear as unnecessary peaks on the chromatogram, overlapping with catecholamine peaks and interfering with analysis. be.

The purpose of the present invention is to provide an analysis method that uses a pre-column, prevents the adsorption of proteins and unnecessary coexisting components on the column, reduces peaks on these chromatograms, and improves the accuracy of catecholamine analysis, as well as the analysis device. It is about providing.

[Means for Solving the Problems] The gist of the present invention for achieving the above object is as follows.

A process to remove protein components from sample biological fluid.

A step of adding a required amount of a derivatization (labeling) reagent to the deproteinized sample biological fluid to label catecholamines in the fluid.

The sample biological fluid containing the labeled catecholamine is introduced into a precolumn filled with a methacrylate polymer gel having a hydrophobic group on the surface, the labeled catecholamine is adsorbed onto the polymer gel, and coexisting components unnecessary for analysis are removed using a pretreatment liquid. a step of washing and removing; a step of removing the labeled catecholamines adsorbed on the pre-column with an eluent and introducing the labeled catecholamines into a separation column using organic silane-treated silica gel as a packing material; adsorption onto the separation column; A method for analyzing catecholamines, the method comprising: developing labeled catecholamines with an eluent and detecting them with a probe;
and the device.

In the present invention, in order to prevent the remaining proteins in the sample biological fluid after protein removal from being adsorbed to the pre-column for pretreatment, hydrophobic groups are added to the surface of a methacrylate polymer gel whose base material is hydrophilic as a packing material for the pre-column. Use a gel with

This prevented adsorption of hydrophobic proteins. That is, the pre-column separates catecholamines and residual protein components.

The hydrophobic group on the surface of the methacrylate polymer gel is preferably an alkyl group having 1 to 6 carbon atoms or a phenyl group.

The size of the pre-column is determined based on the principle of gel filtration chromatography: VM = Vo + Vi (however, the total mobile phase volume in the vM2 column v the external volume of the mini gel particles vi the internal volume of the mini gel particles). When the length is 10 mm, the internal volume VAI is 126 μρ.

Thereby, it is possible to select the particle size, pore size, etc. of the polymer gel to suppress adsorption of unnecessary component substances including the residual protein.

The polymer gel of the present invention preferably has a particle size of 5 to 50 μm and a bore size of 50 to 500 μm.

[Function] The reason why there is little interference with the catecholamine peak on the chromatogram is that the hydrophobic groups on the surface of the polymer gel act in the same manner as in reversed phase chromatography to adsorb and concentrate catecholamines. However, the above-mentioned hydrophobic group is an adsorption gel used in reversed phase chromatography, for example,
This is thought to be because the molecular chains are not large like octadecylsilanized silica gel, so it has a relatively low adsorption capacity, so it adsorbs less protein and other unnecessary components and can be easily washed and removed with a pretreatment solution.

[Example code] The present invention will be specifically explained with reference to Examples.

[Example 1] First, the analysis process of the present invention will be described.

Step 1: Stabilization of Catecholamines in Biological Fluid A case will be described in which plasma obtained by EDTA blood collection is used as the sample biological fluid.

First, 200mM boric acid buffer (pH: 7.3).

A solution consisting of 1mM ascorbic acid and 1mM EDTA! gI, and 600 μQ of the liquid was added to 600 μQ of the sample biological fluid.
Add to μΩ and mix thoroughly.

Step 2: Deproteinization The sample solution obtained in step 1 above is centrifuged (2000G, 20 minutes) using an ultrafiltration membrane with a molecular weight cutoff of 10,000 (Centricon-10 manufactured by Amicon), and the filtrate is This is a catecholamine analysis sample (hereinafter referred to as sample).

Step 3: Labeling of catecholamine As a labeling (derivatization) reagent, 20mM 1゜2
-Diphenylethylenedimine (1,2-DPE).

A solution consisting of 12mM potassium ferricyanide, 12mM ammonium molybdate and 40% acetonitrile is prepared.

500 μΩ of the reagent and 500 μΩ of the sample obtained in step 2 above.
Ω and perform a labeling reaction at 45° C. for 3 minutes, and immediately proceed to the next step.

Step 4: Adsorption and concentration by pre-column The sample containing the labeled catecholamine obtained in step 3 is introduced into the pre-column, and the acrylate polymer gel (manufactured by Mitsubishi Kasei: Bu
tyl-CQP-30S (hydrophobic group is butyl group, bore size: 300) and concentrated, and at the same time, unnecessary coexisting components are washed and removed with a pretreatment liquid.

In this example, the precolumn size was 4 m in inner diameter.
m×length lQmm. In addition, 50mM 1ill acid, 0°1M NaCA were used for sample transfer, adsorption, and washing.
, a pretreatment solution consisting of 1mM EDTA was used. This pretreatment liquid was passed through a precolumn at 2 mΩ to concentrate labeled catecholamines in the sample and wash and remove unnecessary components.

Step 5: Analysis The labeled catecholamines adsorbed on the precolumn are separated and developed using an eluent, and chromatographic analysis is performed.

The eluent in this example was a 5/215 acetonitrile/methanol/water volume solution (50 mM boric acid buffer, 10
mM sodium dodecyl sulfonate (SDS)]
was used.

Note that one separation column has an inner diameter of 4.6 mm and a length of 80 mm.
2 mm filled with silica-DS (particle size 3 μm). Further, the liquid feeding flow rate was 1 m Q /min for both the pretreatment liquid and the eluent.

Step 6: The labeled catecholamines separated by the chromatogram separation column are subjected to excitation wavelength (Ex) of 345 nm and monitoring wavelength (Em) of 485 nm using Fluorescent Diodes (Hitachi: F-1050).
and quantitative analysis of catecholamines.

In the above method, NE, E and DA are each 1 p
It was determined by the external standard method, in which a standard sample containing M/mQ was measured and the concentration was calculated using the measured value.

As a result, the CV (41 standard deviation SD/average value did it. Incidentally, a chromatogram obtained in this example is shown in FIG.

[Example 2] FIG. 1 shows an example of a flow path system diagram of the catecholamine analyzer of the present invention.

This system consists of an autosampler 1 and a data processing device 3.
3, and an analysis section 19 having a

A sample rack 2 is attached to the sample stage 3 of the autosampler 1, and various samples 4 are mounted on the sample rack.
, a reaction reagent for fluorescent labeling 5, and an internal standard solution 6.
A standard sample 7 is housed and attached to each container. Plasma, urine, etc. used as samples have been previously subjected to protein removal treatment in step 1.2.

Further, in the vicinity of the sample stage 3, a reaction container 8, a nozzle cleaning tank 9, a drain port 10, and an injection port 11 are provided. The drive mechanism 12 of the nozzle 13 has x, y, z
The nozzle 13 has a function of being able to be freely driven in any direction, and by freely driving the nozzle 13 vertically, horizontally and vertically, it is possible to move the sample onto each container and each port.

The nozzle 13 is connected to a dispensing pump 16 and a container for cleaning liquid 17 via a three-way valve 15 with a tube 14 made of fluororesin (polytetrafluoroethylene).

The dispensing pump 16 uses a syringe pump that is driven by a pulse motor.The reaction vessel thermostat 18 is equipped with a temperature sensor to keep the reaction vessel 8 at a predetermined temperature, and the sample stage 3 is equipped with a cooling device. The temperature is controlled by a temperature sensor and control device so that samples and reagents with sample rank 2 can be kept at low temperatures.

The analysis section 19 includes a pre-column system for concentrating the sample and removing impurities, a separation column system for separating sample components, a detector, and a data processing section.

In the pre-column system, a pre-treatment liquid 20 is fed at a constant flow rate by a pump 21 and flows into a pre-column 23 via a sample injector 22 . sample injector 2
2 is provided with a measuring tube 24 for measuring a predetermined amount of the sample injected from the injection boat 11 of the autosampler 1 and introducing it into the analysis section. The precolumn 23 is provided with a constant temperature bath that maintains its temperature at a predetermined temperature.

In the separation column system, the eluent 25 is fed at a constant flow rate by the pump 26 and flows into the separation column 29 via the column switching/<lube 28. By switching the column switching valve 28, the eluent flows through the pre-column 23,
The separation column 29, which transfers the sample treated in the pre-column to the separation column 29, is provided with a constant temperature bath that maintains it at a predetermined temperature.

The detector 31 is a fluorometer that measures the fluorescence intensity of sample components eluted from the separation column 29 and has a flow cell 32 .

The data processing unit includes a data processing device 33 that performs calculation processing on the measurement results of the detector 31, calculation processing of the measurement values of various sensors,
It is equipped with a CPU 34, an output printer 35, a CRT 36, etc., which control the memory and various parts. Note that the switching cocks 37 and 38 are provided so that the pretreatment liquid 20, the eluent 25, and the liquids in the pumps 21 and 26 can be discharged as necessary.

FIG. 3 shows a flowchart of the analysis process. Based on this flow, the analysis method of this example will be described.

(1) Move the reaction vessel cleaning nozzle 13 to the position of the reaction vessel 8, and
6 and inject the cleaning liquid 17. The inside of the reaction container 8 is cleaned by repeating one or more operations several times (for example, three times) of injecting a larger amount than the capacity of the reaction container and allowing the excess cleaning liquid to overflow and be discharged from the drain boat 10.

If there is air in the pipe 14, the nozzle 13 is moved to the drain boat 10, and the cleaning liquid is sucked and discharged several times to expel the air inside the nozzle.

(2) Move the sample dispensing nozzle 13 to the position of the sample 4 containing the sample to be analyzed, and suck in a predetermined amount of the sample.

Further, at this time, in order to correct fluctuations in the recovery rate of the entire device such as the column, internal standard solution 6 (isoproterenol is used in this example) is aspirated in the same manner.

The nozzle 13 is moved to the position of the reaction vessel 8, and the inhaled sample is dispensed into the reaction vessel 8.

(3) Nozzle cleaning After moving the nozzle 13 to the drain boat 10 and discharging the cleaning liquid to wash out the sample adhering to the nozzle inner wall, move it to the nozzle cleaning tank 9 and lower it into the tank to discharge the cleaning liquid. Clean the outside of the tip of the discharge nozzle.

(4) Move the reagent dispensing and mixing nozzle 13 to the position of the reaction reagent 5 for labeling,
Inhale a predetermined amount of the reagent.

The nozzle 13 is moved to the position of the reaction container 8, and the inhaled reaction reagent is injected into the reaction container and mixed with the previously injected sample. Mixing methods include methods such as air discharge, external vibration, stirring, etc., but low viscosity liquids can be mixed by high speed discharge.

(5) Reaction The mixture of the above-mentioned sample and reaction reagent is reacted for a predetermined time in the reaction container 8 to derivatize the sample (uberization).

(6) Sample introduction The labeled sample that has undergone the reaction is inhaled through the nozzle 13, moved to the injection boat 11, and introduced into the measuring tube 24 of the sample injector 22. Next, the labeled sample is sent to the precolumn 23 by switching the sample injector 22 and injecting the pretreatment liquid 20 .

(7) Concentration and washing The labeled sample transferred to the pre-column 23 is adsorbed and concentrated by the acrylate polymer gel, and unnecessary components such as residual proteins that interfere with analysis are removed by flowing the pre-treatment liquid through the outlet port. I take a bath from 39.

(8) Switch the sample transfer column switching valve 28 to connect the pre-column 23 between the channels 40 and 41, and transfer the eluent 2S to the pre-column 23.
By flowing through the precolumn, the labeled sample concentrated in the precolumn is separated and transferred to the separation column 29. When all of the labeled sample has been transferred to the separation column 29, the column switching valve 28 is switched again (first
In the state shown in the figure), the eluent 25 flows directly to the separation column 29 without passing through the precolumn 23, and the pretreatment liquid 20 flows to the precolumn 23.

(9) The separation eluent 25 is passed through the separation column 29 to separate the labeled catecholamine of interest.

In parallel with the separation of the labeled catecholamines, a pretreatment liquid is passed through the precolumn 23 to perform a regeneration process for receiving the next labeled sample.

(10) The analytical components of the labeled catecholamine separated and eluted by the measurement separation column 29 are sequentially transferred to the flow cell 3 of the detector 31.
2 and detect the fluorescence intensity.

(11) Data processing and output The detection results are subjected to arithmetic processing in the data processing device w33 to determine the concentration of each component, and the CPU 34 outputs the above-mentioned arithmetic data and chromatogram to the CRT 36 and printer 35.

Note that the measurement of the standard sample is analyzed based on the chromatogram flowchart. In this case, the health of the reagent can be determined from the peak height, area, etc. of the chromatogram, and the shape of the peak (
The integrity of the column and eluent can be determined by the presence of cracks, leading, and tailing. If the above results are satisfactory, proceed to measurement of the unknown sample.

In addition, in the fluorescent labeling in this example, the protein-depleted sample was 500 μΩ, and the reaction reagent for fluorescent labeling was 450 μΩ.
Mix 50μQ of μΩ and internal standard solution, temperature 45℃
, for 3 minutes. Further, Table 1 shows zero analysis conditions in which the pretreatment liquid and eluent each flowed at a rate of 1 m Q /min.

Further, the chromatogram of the analysis results is shown in FIG. 4, and the reproducibility (CV value) and recovery rate when using the same sample are shown in Table 2.

As is clear from Table 2, in this example, NE
, CV value 2.5% or less for both E and DA, recovery rate 98
% or more, which is excellent.

Table X: Average value SD: Standard deviation CV = SD/X [Example 3] A flow path system diagram of another example of the catecholamine analyzer is shown in FIG.

In Example 2, as shown in FIG. 1, pumps 21 and 2 are used as transfer pumps for the pretreatment liquid and the eluent, respectively.
6 were used, but in this example, these were combined into one pump 26.
It is distinctive in that it is also used as .

Therefore, the pretreatment liquid 20 and the eluent 25 are transferred by alternately opening and closing the respective on-off valves 42 and 43. The other details are the same as those in FIG. 1 of the second embodiment.

The sample labeled by the autosampler 1 is introduced into the measuring tube 24 of the sample injector 22 from the injection port 11. By switching the sample injector 22 and the column switching valve 28, the labeled sample is transferred to the precolumn 23.

At this time, the on-off valve 42 is opened and the on-off valve 43 is closed to allow the pretreatment liquid 20 to flow in and remove unnecessary components such as residual protein.

Next, by switching the column switching valve 28, closing the on-off valve 42, and opening the on-off valve 43, the eluent 25 flows in and the labeled sample adsorbed on the pre-column 23 is transferred to the separation column 29. The labeled catecholamines desorbed and separated by the separation column are transferred to the detector 31 and the fluorescence intensity is measured.

This embodiment has the advantage that the transfer pump is not required and the amount of pretreatment liquid and eluent consumed can be reduced. Furthermore, since the washing state of the precolumn can be continuously obtained in the chromatogram, it is convenient for determining the analysis results.

In this example, the same protein-removed sample as in Example 2 was used, and the analysis conditions were the same as in Table 1. However, the packing material shown in Table 3 was used as the pre-column packing material. In addition, as a sample, 1 p
A mixture containing catecholamine M and isoproterenol as an internal standard solution was used.

FIG. 6 shows a chromatogram of a sample treated using a precolumn packed with the packing material shown in Table 3.

Particle size of each filler in Table 3: 10 μm CQP: Manufactured by Mitsubishi Kasei As is clear from Fig. 6, Butyl-CQP
The best analytical results were obtained using a pre-column containing -3O3 as a packing material.

In Collenix vs. Shite, CQP-10, and L-1180, the peak of unnecessary coexisting components overlaps with the NE peak, resulting in a decrease in analysis accuracy. In addition, CQP-30t- has a decreased peak height of catecholamines, indicating a low recovery rate.6 In addition, Phenyl-CQ P-3OS
The noise is slightly larger than that of yl-CQP-3O8.

[Example 4] Using a precolumn filled with Butyl-CQP-30S, the influence of salt added to the pretreatment liquid on the recovery rate of catecholamines was investigated.

The salts include boric acid (+NaOH), acetic acid (+N a O
H), lithium nitrate, sodium chloride, and potassium phosphate. In addition, the concentration in winter is 0.1M, p
At H7.0, 1 mM (7) EDTA was added. Other conditions are the same as in Example 3.

The measurement results are shown in Figure 7.

As is clear from the figure, the catecholamine recovery rate is closest to 100% when the pretreatment solution containing borate is used, but when other salts are used, the peaks of unnecessary coexisting components overlap and the apparent This is not preferable as it may exceed 100%.

[Example 5] Next, using a precolumn filled with Butyl-CQP-3O3, the influence of the pH of the pretreatment liquid on the recovery rate of catecholamines was investigated.

As a pretreatment solution, 0.1M boric acid and 1mM EDTA were added, and the pH was adjusted by changing the amount of NaOH added.

The measurement results are shown in FIG.

As is clear from the figure, the pH of the pretreatment liquid is preferably in the range of pH 6.5 to 7.5, as indicated by the arrow.

[Example 6] Next, the influence of the buffer added to the eluent was studied.

As an eluent, lomM SDS, 50mM in acetonitrile/methanol/water (volume ratio: 5/215)
Chromatograms were compared when ammonium borate, sodium acetate, and potassium phosphate were used as buffers at pH 7.2. In addition, as a pretreatment liquid, 0.1M lithium nitrate, 1
mM EDTA was used. The analysis was conducted using the apparatus shown in FIG. 5 under the same conditions as in Table 1.

A chromatogram of the analysis results is shown in FIG.

As is clear from the figure, there are fewer peaks of unnecessary coexisting components when borate is added to the eluent.

[Example 7] Next, the influence of the pH of the buffer in the eluent on the recovery rate of catecholamines was studied.

As a buffer, 50mM ammonium borate with N
a OH was added to adjust the pH.

Note that the measurement conditions were the same as in Example 6.

The measurement results are shown in FIG.

As is clear from the figure, the recovery rate is closest to 100% at pH 7.

[Comparative example]

Butyl-CQ P-3 as pre-column packing material
Catecholamines were analyzed in the same manner as in Example 3, except that styrene-divinylbenzene polymer gel was used as the packing material for the O8 column.

The measurement results are shown in FIG.

As can be seen from a comparison with FIG. 6-a, there are many unnecessary coexisting components, making it difficult to obtain an accurate recovery rate. By using it, unnecessary coexisting substances such as residual proteins other than catecholamines can be easily removed, and the recovery rate of catecholamines can be increased, thereby improving the accuracy of catecholamine analysis.

[Brief explanation of drawings]

Figures 1 and 5 are flow path diagrams of the catecholamine analyzer of the present invention, Figures 2, 4, 6 and 9 are chromatograms of catecholamines, and Figure 3 is a chromatogram of catecholamines of the present invention. A flow diagram showing the analysis process, Figure 7 is a graph showing the relationship between the type of salt in the pretreatment liquid and the recovery rate of catecholamine, and Figure 8 is a graph showing the relationship between the PH of the pretreatment liquid and the recovery rate of catecholamine. , Figure 10 is
FIG. 11 is a graph showing the relationship between eluent pH and catecholamine recovery rate, and is a chromatogram of catecholamine in a comparative example. 1...Auto sampler, 2...Sample rack, 3
... Sample stage, 4... Sample, 5... Reaction reagent, 6... Internal standard solution, 7... Standard sample,
8... Reaction container, 9... Nozzle cleaning tank, 1o...
Drain port. 11... Injection boat, 12... Drive mechanism, 13...
・Nozzle, 14...pipe, 15...three-way valve, 16...
・Dispensing pump, 17...Cleaning liquid, 18...Reaction container constant temperature bath, 19...Analysis section, 20...Pretreatment liquid, 21
... Pump, 22 ... Sample injector, 23
... precolumn, 24 ... measuring tube, 25 ... eluent, 26 ... pump, 28 ... column switching valve,
29...Separation column, 31...Rotator, 32...
Flow cell, 33...data processing device, 34...C
PU, 35... Output printer, 36... CRT, 3
7.38...Switching cock, 40,41...Flow path, 4
2.43...Opening/closing valve, 44...Thermostat, 45...
control line.

Claims (1)

[Scope of Claims] 1. A method for analyzing catecholamines in a biological fluid by liquid chromatography, including the steps of: removing protein components from the sample biological fluid; adding a required amount of derivatization (labeling) reagent to the deproteinized biological fluid sample; and a step of labeling the catecholamine in the liquid, introducing the sample biological fluid containing the labeled catecholamine into a precolumn packed with a methacrylate polymer gel having a hydrophobic group on the surface, and adding the labeled catecholamine to the polymer gel. A process of adsorbing and washing and removing coexisting components unnecessary for analysis with a pre-treatment solution, removing labeled catecholamines adsorbed on the pre-column with an eluent, and using organic silane-treated silica gel as a packing material. A method for analyzing catecholamines, comprising the steps of: introducing the labeled catecholamine into a separation column; developing the labeled catecholamine adsorbed on the separation column with an eluent and detecting it with a detector. 2. The method for analyzing catecholamines according to claim 1, wherein the hydrophobic group of the methacrylate polymer gel having a hydrophobic group is an alkyl group having 1 to 6 carbon atoms or a phenyl group. 3. Claim 1, wherein the catecholamine derivatization (labeling) reagent contains 1,2-diphenylethylenedimine, potassium ferricyanide, ammonium molybdate, and acetonitrile, and the detector is a fluorescence detector. Analytical method for catecholamines as described in section. 4. The method for analyzing catecholamines according to claim 1, wherein the pretreatment liquid and/or eluent contains a borate, and the pH of the pretreatment liquid is 6.5 to 7.5. 5. A reaction vessel for reacting catecholamines in the deproteinized sample biological fluid with a labeling (derivatization) reagent, and adsorbing the labeled catecholamines in the sample biological fluid and separating and removing components that do not coexist with a pretreatment liquid. A pre-column filled with a filler capable of resolving the problem, and a pre-column downstream of the pre-column, capable of adsorbing the labeled catecholamine from which unnecessary coexisting components have been removed, and sequentially developing at least epinephrine, norepinephrine, and dopamine with an eluent. It has a separation column filled with a packing material, and a detector that detects the analytical components sequentially developed from the separation column, and the reaction vessel, precolumn, separation column, and detector are connected by piping, and the The piping is equipped with a switching valve so that the pretreatment liquid or eluent can be introduced into the precolumn and the separation column as desired, and the packing material of the precolumn is a methacrylate-based packing material having an alkyl group having 1 to 6 carbon atoms or a phenyl group on the surface. 1. An analytical device for catecholamines, characterized in that the separation column is made of a polymer gel, and the packing material of the separation column is a silica-based gel treated with an organic silane.