JPS5938651A - Quantitative analysis of catecholamine - Google Patents

Abstract

PURPOSE:To simultaneously measure three components in high sensitivity, by a method wherein noradrenaline, adrenaline and dopamine components in a specimen liquid from a living body are fractionated by liquid chromatography and dopamine is selectively converted to noradrenaline which is, in turn, further converted to a derivative emitting fluorescence to detect said fluorescence. CONSTITUTION:A phosphate buffer solution is supplied from an eluted liquid tank 1 by a constant flow pump 2 and a serum specimen containing catecholamine is injected from a specimen injector 3. The specimen liquid is passed through a separation column 4 to separate three components, that is, noradrenaline, adrenaline and dopamine. Dopamine beta-hydroxylase is acted on dopamine in a reaction tank 7 to convert the same to noradrenaline. A potassium ferricianide aqueous solution, an ascorbic acid aqueous solution and a sodium hydroxide aqueous solution are supplied to a reaction tank 12 by a constant flow triplet pump 8 and hydroxyindole derivatives of noradrenaline, adrenaline and noradrenaline converted from dopamine are respectively formed to be detected by a fluorescence detector 13.

Description

[Detailed description of the invention] The present invention relates to a method for quantifying catecholamines.

This invention relates to a method for quantifying three components, noradrenaline, adrenaline, and donoqmine, by separating catecholamines contained in the amount of gC in a biological sample liquid such as blood or urine using a liquid chromatograph.

Conventionally, as a method for quantifying catecholamines contained in biological sample fluids, three components, noradrenaline, adrenaline, and do/(mine) are fractionated using high-performance liquid chromatography, and the electrochemical properties of each of these components are utilized.1. (T II I The two laws of
It is shared.

However, in the VMD method, all components with electrochemical properties in a biological sample liquid are detected with the same sensitivity;
In order to detect catecholamines, it has the disadvantage of requiring quite extensive pretreatment, and the concentration of catecholamines in the song is 10-1! It is difficult to detect adrenaline and dopamine because they are in very small amounts, about p (pg). It was t. Also, T HI is extremely sensitive [VM
ITh, l: It is also possible to detect blood catecholamines such as noradrenaline and adrenaline. However, dopamine has a disadvantage in that it cannot be detected because it has poor conversion efficiency into THI substances and the measurement wavelength is similar to that of noradrenaline and adrenaline.

The present invention solves the above-mentioned drawbacks, separates each component of noradrenaline, adrenaline, and dono-shokimine in a biological test liquid by liquid chromatography, selectively converts dopamine to norato-1-naline, and further improves fluorescence. The expressed derivative +(g%y) provides a method for detecting and quantifying fluorescence;

The gist of the present invention is to combine catecholamines with giP.
Molecule f in liquid chromatography using liquid I! Power 8K) Enter Q, noradrenaline, adrenaline, 1
The three and nine components of pamine are divided into nf L, which is used to act on dopamine-β-hydroxylase to selectively convert noradrenaline into noradrenaline and then convert it from noradrenaline, adrenaline, and dopamine. The obtained noradrenaline is brought into contact with tetrahydroxyindole, and the fluorescence of each derivative obtained is detected,
This method consists in the coccygeal method for catecholamines, which is characterized by quantifying three acids, adrenaline, adrenaline, and dopamine, using VC wells.

The following method (111 Determination of Catecholamines) will be explained in more detail.

1j catecholamines are present in biological fluids such as +rn fluid and urine.
It is contained in the background in grams. Try this biological fluid
and liquid chromatography is used to quantify the catecholamines contained therein.

Biological samples subjected to liquid chromatography #;! :
, and is subjected to a pretreatment operation to remove components that inhibit the fractionation of catecholamines.

One step of liquid chromatography is not shown in the mi diagram.

], i, l: A solution tank, for example, a phosphoric acid solution is used, and a constant flow t, 1. The liquid is sent by the pump 2. 3
vi sample injector, and a sample solution containing catecholamine is supplied and mixed with solution 6#.

Reference numeral 4 denotes a separation column, which is filled with particles of, for example, porous silica into which octafsilyl is introduced, divinylbenzene-(meth)acrylic acid copolymer, or the like.

1'! '; IfE, pH value of the solution, ionic strength, type of ion, and flow rate are adjusted as appropriate keeping in mind the 40-minute σ# capacity of the separation column.

The sample solution mixed with the dissolved Nr solution passes through the minute column 4 and is sequentially separated into three components: turadrenaline, adrenaline, and dopamine.

By the way, r-pamine cannot be detected with good sensitivity by the VMD method and the THI method, which have been conventional methods for fractionating tunotecolamine. Therefore, in accordance with the present invention, dopamine β-hydroxylase is activated.

5 is an enzyme solution tank, into which a dopamine β-hydroxylase solution is supplied by a constant liquid level pump 6.

1i (I rff element J Dono E) Converts min to noradrenaline by selective t ji % 2, and the catecholamines excreted in the order of renarin, adrenaline, and F-pamine are It is converted into tll, noradrenaline, adrenaline, and dopamine to become tuladrenaline.

The enzyme solution contains a mixture of β-hydroxylated enzymes and (
・τ, e.g. λ+I H solution, auxiliary ';i' element and [7] ascorbic acid, catalase to stabilize enzyme activity,
A mixture of adenosine trifuosupate and glucose dehyde F logonose is used as the groove bottom component. As a mild blue liquid, for example, H'2 such as phosphoric acid, acetic acid, citric acid, and its sodium salt, potassium salt.

A material made of lithium salt or the like is used. In addition, since the activity is lowered by compounds having enzyme-inactive 1et-311 groups, N-ethylmaleimide, sulfuric acid, etc. are used to inactivate compounds having -8it wells. As an ingredient. Further, in order to increase the y 2 ratio from dopamine to norato 1/nalin, it is preferable to use a dicarboxylic acid such as fumaric acid as the i7 component.

The preferred pH value for the enzymatic reaction of the enzyme is 4.8 to 5.7.
It is more preferably 5.2 to 5.4, and more preferably 5.3.

Such zymogen F [in order to obtain a suitable P II value, it is necessary to adjust the pH value and ionic strength of the buffer solution. The PR value and ionic strength of the buffer solution are preferably adjusted according to the P value, ionic strength, and flow rate of the eluent after filtration through the column 4. Dopamine is converted into noradrenaline by an enzymatic reaction.

(C) Either the enzyme solution is fed from the nf elementary solution tank 5 with a constant flow pump 6 and reacted in the reaction tank 7, or the carrier on which the enzyme is immobilized at 417 K or O is filled with the enzyme solution. It is preferable to load a column with a 114J solution, adjust the pH with 114J solution, and carry out an enzyme reaction in the column.

As the reaction tank 7, for example, the reaction coil is F? The enzyme solution is supplied to a mixed component of catecholamines divided into noradrenaline, adrenaline, and dopamine flowing through the FF coil and a dissolved FFE solution to cause a selective reaction between do-1mine and the enzyme, - Pamine is converted by noradrenaline Q, and noradrenaline, adrenaline, and dopamine released from the reaction tank 7 and C51 column are converted to noradrenaline 1 and noradrenaline 1/
4iqjj of tetrahydroxyindole for Narin!
jlsab, thereby detecting the fluorescence of each derivative obtained in i(1).Tetrahydroxyindole derivative Vr
:', an aqueous solution of potassium hepterythyanide, an aqueous ascorbic acid solution, and an aqueous solution of sodium hydroxide are supplied. Each of these aqueous solutions rt reacts with noradrenaline, adrenaline, and dono (noradrenaline converted from triphosphate) to produce tetrahydroxyindole derivatives.

9 H7, Z IJ potassium cyanide aqueous solution tank, 10 ascorbic acid aqueous solution tank, 11 i-1 caustic soda aqueous solution tank, and each aqueous solution is supplied to the reaction tank 12 from these six tanks by a constant flow triple pump 8. Ru. By reacting with these three liquids, it is converted into a tetrahydroxyindole derivative of adrenaline, a tetrahydroxyindole derivative of adrenaline, and a tetrahydroxyindole derivative of noradrenaline 3r1, which is 1% p1% of noradrenaline.

Each derivative is a fluorescent substance and can be detected by a fluorescence detector 13. This is a 14t:l recorder, and the detection by the fluorescence detection unit 13 is recorded by the recorder. And based on this detection result, noradrenaline,
It is possible to know the amount of hydroxyindole derivatives of noradrenaline converted from adrenaline and dopamine, and from the information obtained from VC, it is possible to know the amount of noradrenaline and adrenaline that were originally contained in biological samples.

You can do this by smelling dopamine. .

According to Kihatsu rllll, the three components of catecholamines, adrenaline, noradrenaline, and dopamine, which are contained in only trace amounts of VC in Test 1'+1, can be measured simultaneously and with high sensitivity. Further, there are advantages in that the measurement can be carried out using a separate device and the measurement time can be shortened.

Example °1 Healthy 1! 5 me of blood from 5 human specimens was collected into a vacuum blood collection tube, left at room temperature for 30 minutes, and then centrifuged at 3000 r, p, m for 5 minutes to obtain $2 mg of blood.

1 (Idiko's serum is treated with NFL by activated alumina,
Components that inhibit the separation of catecholamines by liquid chromatography were removed to obtain a serum sample containing catecholamines.

A 0.1M phosphate buffer solution was supplied from the solution tank 1 at a flow rate of αF3me 7 minutes using a constant flow pump 2, and
A serum sample containing catecholamines was injected from syringe 3 and mixed with the phosphate buffer. As the separation column 4, a stainless steel column filled with divinylpenthene-acrylic acid copolymer particles and having an inner diameter of 5 rTlff and a length of 30 m was used.

0.2n, 17 from enzyme solution tank 5 by constant flow pump 6
The enzyme solution was supplied at a flow rate of 10 min. Composition of enzyme solution (・j
Dopamine β-hydrogenase 5 units/200me1P T
l 6.0 of 0.5 molar rex buffer, 40 mmol of N-ethylmaleimide, 1% by weight of catalase, 10
It consisted of mmol of sodium 7 malate, 10 mmol of sodium ascorbate, and 5 μmol of copper sulfate. Enzyme reaction 1+"l 7 pieces with an inner diameter of 0.5 gates 1.
．． The enzymatic reaction was carried out at 37° C. using a 10 m thick coil. Dopamine was converted to Tuluatrein K17 by an elementary reaction.

r9j 11'';4'il Noradrenaline, adrenaline, and noradrenaline converted from F-pamyri released from 7 were contacted with an aqueous solution of potassium 7-erycyanide, an aqueous ascorbic acid solution, and an aqueous solution of IJ cum, respectively. 9 tanks of potassium ferricyanide aqueous solution containing 0.05% by weight of potassium ferricyanide, 0.2 mol of potassium ferricyanide, 0.4 mol of potassium ferricyanide, 0.05% by weight of potassium ferricyanide, 0.2 mol of potassium ferricyanide, 0.4 mol of potassium ferricyanide. The composition is potassium phosphate, and the concentration Vcq is 0.2% by weight from the ascorbic acid aqueous solution tank
The caustic soda aqueous solution program 6 was adjusted to the specified concentration, and the temperature of 1 reaction tank + 2 J! It was kept at 60°C.

The reaction with each water fi ÷ liquid (τ) produced the respective hydroxyl-conductors of noradrenaline, adrenaline, and noradrenaline converted from 1°-pamily (71). Each derivative was detected by fluorescence detection M13. and was recorded by the recorder 14. P, , P2, and P3 are the peaks of the li Jp form of noradrenaline, adrenaline, and dono (noradrenaline converted from mm), respectively.

The chromatograms of the respective derivatives recorded by the C recorder 14 in this manner are shown in FIG.

Based on the results obtained in this way, standard catecholamines were added to the standard catecholamines (positive line using standard catecholamines) and the pool surface, and pretreatment was performed. The concentration in the song was obtained as shown in Table 1.

Margin below Table 1

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram showing an example of an embodiment of the method of the present invention, and FIG. 2 shows a chromatogram obtained in Example t. Description of symbols: 1 solution tank, 2, 6 constant flow pump, 3 sample injector, 4 separation column, 5 enzyme solution tank, 7.12 reaction tank,
8 Constant flow triple pump, 9 Potassium ferricyanide aqueous solution tank, 10 Ascorbic acid aqueous solution tank, 11 Caustic soda aqueous solution tank, 13 Fluorescence detector, 14. Recorder patent applicant: Kensui Kagaku Kogyo Co., Ltd. Representative: Mototoshi Tsuneuma

Claims (1)

[Claims] A sample solution containing L catecholamine is introduced into a separation column in liquid chromatography to separate three components, noradrenaline, adrenaline, and F-pamine,
Dopamine-β-hydroxylase was applied to this to selectively convert donobemin to noradrenaline, and then tetrahydroxyindole was brought into contact with noradrenaline converted from noradrenaline, adrenaline, and do/ζamine. A method for determining catecholamines, which is characterized by detecting the fluorescence of each derivative and quantifying the three components of noradrenaline, adrenaline and dopamine based on this.