JPH06160366A - Measuring method and device for catecholamine in biological fluid - Google Patents

Abstract

PURPOSE:To properly measure main components of catecholamine, changing-over the sensitivity of a measuring process from high to low after norepinephrine and epinephrine are eluted in a separation process. CONSTITUTION:Shift phase 20 is, through a separation column 12, openings 32a and 32b of a turn-over cock 18, a reactive coil 14, openings 32e and 32f, and a fluorescence detector 16, made conductive and stable in system, and then a to-be-examined sample is injected from an autosampler 22. At this time, a DE reagent tank 26 supplies a coil 14 with reactive solution, so the sample is separated in a column 12, norepinephrine, epinephrine and dopamine eluted in this sequence. The norepinephrine and epinephrine are respectively made to be ethylene diamine derivative, for fluorescence detection in high sensitive measurement of the first mode. In about 10 minutes after sample injection, dopamine eluate from the column 12 is, through the openings 32a and 32f, directly introduced into the detector 16, for fluorescence detection in the second mode of low-sensitive measurement. Thus main contents of catechol amine are quantified under proper conditions.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for measuring catecholamines in biological fluids, and more particularly to a method and an apparatus for separating and measuring various catecholamines by fluorescence measurement.

[0002]

BACKGROUND ART Catecholamine has an important function as a neurotransmitter for maintaining homeostasis in a living body. Therefore, it is not possible to separate and quantify catecholamine in a biological fluid such as urine, hydrolyzed urine or blood. It is an extremely useful tool for pathological analysis of Conventional methods for measuring catecholamines in biological fluids include natural fluorescence method, electrochemical detection method,
Derivatization methods and the like are known. Although the electrochemical detection method has high sensitivity, it has disadvantages in that the detection selectivity is poor and the sensitivity changes greatly with electrode deterioration. On the other hand, the natural fluorescence method is a simple method, but its sensitivity is low. Derivatization methods include the trihydroxyindole (THI) method and the ethylenediamine (ED) method. The THI method is H
This is the most commonly used method for measuring catecholamines using PLC. Moreover, it has the characteristics that it is highly sensitive to epinephrine and norepinephrine and that the detection sensitivity of dopamine is low. Here, the concentration of epinephrine and norepinephrine is low and the concentration of dopamine is high in urine. That is, the standard values of catecholamine 3 components in urine (values that are often used in clinical tests, average values that healthy people show), necessary quantification ranges and detection limits are as follows.

[0003]

[Table 1] ──────────────────────────────────── Catecholamine Reference value (ng / ml) Quantitative range (ng / ml) Detection limit (ng / ml) ──────────────────────────────────── Norepinephrine 61 -68 10-500 5.0 Epinephrine 16.1-17.3 1-600.5 Dopamine 722-797 100-10,000 50 ────────────────── Therefore, the sensitivity characteristic of the THI method is extremely convenient when urine is used as the sample.

[0004]

However, the THI
The method requires three kinds of reaction solutions, and further requires 4-5N sodium hydroxide solution in the reaction solutions.
It's difficult to handle. In addition, there is a problem in that maintenance and adjustment of the device also take time and labor because a pump for liquid supply is required for each reaction liquid. On the other hand, unlike the THI method, the ED method requires only one type of reaction solution and has good selectivity, but it was difficult to use when the above-mentioned three types of catecholamines were simultaneously measured. That is, when urine is used as the sample, dopamine is contained 100 times or more that of epinephrine. Therefore, if the injection amount, reaction conditions, etc. are set so that the conditions are suitable for the quantification of epinephrine, the quantification range of dopamine deviates from the linear range of the calibration curve, and accurate quantification cannot be performed. In addition, if the measurement conditions are adjusted to dopamine, epinephrine cannot be measured due to insufficient sensitivity. Therefore, the ED method alone is not suitable for separating and quantifying catecholamines in biological fluids. The present invention has been made in view of the above-mentioned problems of the prior art, and an object thereof is a catecholamine measuring method and measuring apparatus in a biological fluid capable of appropriately measuring epinephrine, norepinephrine and dopamine which are catecholamines in a biological fluid. To provide.

[0005]

In order to achieve the above object, a method for measuring catecholamine in biological fluid according to the present invention includes a separation step, a high sensitivity measurement step, and a low sensitivity measurement step. Then, the separation step separates the catecholamine mixture resulting from the biological fluid. In the high sensitivity measurement step, the norepinephrine fraction and the epinephrine fraction obtained in the separation step are measured. In the low sensitivity measurement process,
The dopamine fraction obtained in the separation step is measured. Then, after the norepinephrine fraction and the epinephrine fraction are eluted from the separation step, the high sensitivity measurement step is switched to the low sensitivity measurement step. Further, in the high-sensitivity measurement step, it is preferable that after derivatizing norepinephrine and epinephrine with ethylenediamine by the ethylenediamine derivatization method, measurement is performed by the fluorescence method, and in the low-sensitivity measurement step, dopamine is measured by the natural fluorescence method. . Further, the apparatus for measuring catecholamine in biological fluid according to the present invention is characterized by including a separation means, a derivatization means, a fluorescence measurement means, and a switching means. Here, the separation means separates the catecholamine mixture derived from the biological fluid. The derivatization means uses norepinephrine and epinephrine in the eluate obtained by the separation means as ethylenediamine derivatives. The fluorescence measuring means measures the fluorescence of catecholamine or its derivative. The switching means introduces the eluate from the separation means into the fluorescence measurement means via the derivatization means or directly. Then, the norepinephrine fraction and the epinephrine fraction are introduced as an ethylenediamine derivative into the fluorescence measuring means, and the dopamine fraction is directly introduced into the fluorescence means, and fluorescence is measured respectively.

[0006]

The method and apparatus for measuring catecholamines in biological fluids according to the present invention introduces the norepinephrine and epinephrine fractions into the high-sensitivity measuring step, while the dopamine fraction into the low-sensitivity measuring step, as described above. As a result, even when the amount of dopamine in a biological fluid such as urine is significantly higher than that of epinephrine, epinephrine, norepinephrine and dopamine can be appropriately quantified from one sample solution. Specifically, in the high-sensitivity measurement step, since epinephrine and norepinephrine are each converted into an ethylenediamine derivative by the ethylenediamine derivatization method and then measured by the fluorescence method, the measurement can be performed with high selectivity and high sensitivity. In the low-sensitivity measurement step, dopamine is directly measured by the fluorescence method, but the sensitivity is low.However, since the amount of dopamine itself is large, the same fluorescence measurement conditions as in the high-sensitivity measurement step without deviating from the linear range of the calibration curve. Can be used for quantitative measurement.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will be described below with reference to the drawings. FIG. 1 shows a schematic configuration of a device for measuring catecholamine in biological fluid according to an embodiment of the present invention.
A catecholamine measuring apparatus 10 shown in the same drawing is provided with a separation column 12 as a separating means, a reaction coil 14 as a derivatizing means, and a fluorescence detector 16 as a fluorescence measuring means.
And a 6-way switching cock 18 as switching means.
Then, from the mobile phase tank 20, a mobile phase (5 mM SDS;
0.1M NaH ₂ PO ₄ ; 20% MeCN; pH 3.0
H ₃ PO ₄ ) is supplied to the autosampler 22, and the test sample from the autosampler 22 is supplied to the separation column 12 at a flow rate of 1.0 ml / mn. The separation column 12 is made of CrestPac C18S manufactured by JASCO Corporation, and the separation column 12 is kept at 40 ° C. by a column thermostat 24. In the reaction coil 14, the test sample liquid separated from the separation column 12 via the cock 18 and the reaction liquid (0.1M ethylenediamine;
2M Tris) is mixed and then supplied at 70 ° C.
It is arranged in the reaction tank 28. The reaction coil was formed with an inner diameter of 0.5 mm × 10 m and the reaction liquid flow rate was 1.
It is 0 ml / min. The fluorescence detector 16 has two modes, the first mode being the excitation wavelength. 325 nm, fluorescence wavelength. 510nm,
gain × 100, the second mode is the excitation wavelength. 285nm,
Fluorescence wavelength. 325 nm, gain × 1.

The output of the fluorescence detector 16 is subjected to desired data processing by the computer 30, and is output to a display, a printer or the like (not shown). The 6-way switching cock 18 has openings 32a, 32b, ... 32f, and the opening 32a is the outlet of the separation column 12,
The opening 32b is the inlet of the reaction coil 14, the opening 32c is the closing plug 34, the opening 32d is the waste liquid pipe 36, and the opening 32e.
Is connected to the outlet of the reaction coil 14 and the opening 32f is connected to the inlet of the fluorescence detector 16, and the openings 32 can be connected to each other by the instruction of the computer 30 or manually.

The catecholamine measuring apparatus 10 according to the present embodiment is constructed as described above, and its operation will be described below. First, with the reagent not supplied from the ED reagent tank 26, the switching cock 18 is operated as shown in FIG. 2 to separate the mobile phase from the mobile phase tank 20 into the separation column 1.
2. Conducting through the openings 32a and 32b of the switching cock 18, the reaction coil 14, the openings 32e and 32f of the switching cock 18, and the fluorescence detector 16 to stabilize the system. Next, 50 μl of a test sample (catecholamine standard sample: 0.2 to 25 ng of norepinephrine, 0.04 to 5.0 ng of epinephrine, 3.9 to 500 ng of dopamine) is injected from the autosampler 22. At this time, the reaction liquid is supplied from the ED reagent tank 26 to the reaction coil 14. As a result, the test sample is sequentially separated by the column 12 and eluted in the order of norepinephrine, epinephrine, and dopamine. Then, the norepinephrine fraction and the epinephrine fraction are each made into an ethylenediamine derivative and introduced into the fluorescence detector 16 for fluorescence detection. In this case, the fluorescence detection condition is the first mode adapted to the fluorescence measurement of the ethylenediamine derivative according to the instruction from the computer 30. Next, 10 minutes after the injection of the sample, since the norepinephrine and epinephrine fractions have already been eluted, the computer 30 gives an instruction to the switching cock 18 to elute from the separation column 12 as shown in FIG. The liquid is directly introduced into the fluorescence detector 16 through the openings 32a and 32f of the switching cock 18 to detect fluorescence. At this time, the fluorescence detector 16 is set to the second mode suitable for the natural fluorescence measurement of dopamine according to the instruction of the computer 30.

As described above, according to the catecholamine measuring apparatus according to the present embodiment, it is possible to quantify norepinephrine, epinephrine and dopamine which are the main components of catecholamine in the biological fluid under appropriate conditions. The chromatograms measured under the above conditions are shown in FIGS. 4 and 5, and FIG. 4 shows norepinephrine 1.
56 ng, epinephrine 0.31 ng, dopamine 31.3
An example of measurement of ng, FIG. 5 is an example of measurement of 6.25 ng of norepinephrine, 1.25 ng of epinephrine, and 125 ng of dopamine (each 4-fold amount in the case of FIG. 4). In contrast to FIG. 4, in FIG. 5, the peak area and height corresponding to each component are 3.9 to 4.0.
It was doubled, and a linear relationship was recognized in the calibration curve in all cases. On the other hand, in FIGS. 6 and 7, except that all catecholamines including dopamine are ethylenediamine derivatives,
The chromatogram measured under the above conditions is shown, FIG. 6 shows a measurement example of norepinephrine 1.56 ng, epinephrine 0.31 ng, dopamine 31.3 ng, and FIG. 7 shows norepinephrine 6.25 ng, epinephrine 1.25 ng,
It is an example of measurement of 125 ng of dopamine (4 times the amount in the case of FIG. 6). In contrast to FIG. 6, in FIG. 7, the area and peak height for norepinephrine are 3.6 times and that for epinephrine is 3.4 times, but the area is 2.0 for dopamine.
The peak height was 1.6 times, and the peak height was 1.6 times, and no linear relationship was found in the calibration curve.

Incidentally, FIG. 8 shows a calibration curve of each catecholamine in the case of using this embodiment, and FIG. 9 shows a calibration curve obtained by converting all of them into an ethylenediamine derivative. Figure 8
Shows linearity for all catecholamines, but it is understood in FIG. 9 that the linearity of dopamine is particularly problematic. As described above, according to the catecholamine measuring apparatus of the present embodiment, it is possible to perform appropriate quantitative measurement of each catecholamine using one measuring apparatus and one detector. In this example, an example in which elution was carried out in the order of norepinephrine-epinephrine was explained, but it may be reversed depending on column conditions and the like. Further, the switching instruction to the switching means can be performed, for example, by the count number of the elution peak.

[0012]

As described above, according to the catecholamine measuring method and measuring apparatus of the present invention, ethylenediamine derivatives are formed for norepinephrine and epinephrine, and dopamine is directly measured for fluorescence. It becomes possible to carry out the fractional quantification of the medium catecholamine under the condition suitable for each component.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of a catecholamine measuring apparatus according to an embodiment of the present invention.

[Fig. 2]

FIG. 3 is an explanatory diagram of a fractionated quantitative state of norepinephrine, epinephrine, and dopamine by the catecholamine measuring device shown in FIG. 1.

[Fig. 4]

5 is a chromatograph of norepinephrine, epinephrine, and dopamine by the catecholamine measuring device shown in FIG.

FIG. 6,

FIG. 7 is a chromatograph when the ethylenediamine derivatization method is applied to all of norepinephrine, epinephrine, and dopamine.

FIG. 8 is a calibration curve of each catecholamine when the present invention is used.

FIG. 9 is a calibration curve when each catecholamine is converted to ethylenediamine.

[Explanation of symbols]

 10 Biological fluid catecholamine measuring device 12 Separation column (separation means) 14 Reaction coil (derivatization means) 16 Fluorescence detector (fluorescence detection means) 18 6-way switching cock (switching means)

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[Procedure amendment]

[Submission date] November 2, 1992

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Correction target item name] All drawings

[Correction method] Change

[Correction content]

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[Figure 5]

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[Figure 1]

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[Figure 8]

[Figure 9]

Claims (3)

[Claims] 1. A separation step of separating a catecholamine mixture derived from a biological fluid, a high-sensitivity measurement step of measuring a norepinephrine fraction and an epinephrine fraction obtained by the separation step, and a dopamine obtained by the separation step. A low-sensitivity measurement step for measuring a fraction, and characterized by switching from the high-sensitivity measurement step to the low-sensitivity measurement step after the norepinephrine fraction and the epinephrine fraction are eluted from the separation step Method for measuring catecholamines. 2. The method according to claim 1, wherein in the high sensitivity measurement step, norepinephrine and epinephrine are derivatized with ethylenediamine derivatization method by ethylenediamine derivatization method, and then measured by a fluorescence method, and in the low sensitivity measurement step, natural fluorescence is measured. A method for measuring catecholamine in a biological fluid, which comprises measuring dopamine by a method. 3. Separation means for separating a catecholamine mixture originating from biological fluid, derivatization means for converting norepinephrine and epinephrine in the eluate obtained by the separation means into ethylenediamine derivatives, and fluorescence of catecholamine or its derivatives A fluorescence measuring means for measuring, and a switching means for introducing the eluate from the separating means through the derivatizing means or directly into the fluorescence measuring means, and the norepinephrine fraction and the epinephrine fraction as an ethylenediamine derivative. An apparatus for measuring catecholamines in a biological fluid, which is introduced into the fluorescence measuring means, and the dopamine fraction is directly introduced into the fluorescence means to measure fluorescence.