JPH0126506B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying catecholamines,
The present invention relates to a method for separating extremely small amounts of catecholamines contained in biological sample fluids such as blood and urine using liquid chromatography, and quantifying the three components of noradrenaline, adrenaline, and dobamine. Conventional methods for quantifying catecholamines contained in biological sample fluids involve fractionating three components, noradrenaline, adrenaline, and dobamine, using high-performance liquid chromatography, and detecting them using the electrochemical properties of each component. method (VMD method) and
Two methods have been put into practical use: fractionating it into three components, noradrenaline, adrenaline, and dobamine, using high-performance liquid chromatography, then converting it into a fluorescent substance, and then detecting and quantifying the fluorescence (THI method). However, since the VMD method detects all components with electrochemical properties in a biological sample with the same sensitivity, it has the disadvantage that it requires quite complicated pretreatment to detect catecholamines. The concentration of catecholamines is
It was difficult to detect adrenaline and dobamine because the amount was extremely small, about 10 ^-12 g (pg). Furthermore, the THI method is superior in sensitivity to VMD, and can detect blood catecholamines such as noradrenaline and adrenaline. However, dobamine has the disadvantage that it has poor conversion efficiency into THI substances and cannot be detected because the measurement wavelength is different from that of noradrenaline. The present invention solves the above-mentioned drawbacks, and fractionates the components of noradrenaline, adrenaline, and dobamine in a biological sample liquid by liquid chromatography.
The purpose of the present invention is to provide a method for detecting and quantifying these using chemiluminescence. The gist of the present invention is to add dansyl chloride to a sample solution containing catecholamines, react with the catecholamines to produce catecholamine derivatives, and introduce the sample solution containing the catecholamine derivatives into a separation column in liquid chromatography. and noradrenaline derivatives,
It was separated into an adrenaline derivative and a dobamine derivative, and each separated derivative contained bis(2,4,6
- Trichlorophenyl) oxalate (hereinafter abbreviated as oxalate) and hydrogen peroxide are brought into contact to generate chemiluminescence, and this chemiluminescence is detected and quantitatively determined. Exists. Next, the method for quantifying catecholamines of the present invention will be explained in more detail. Dansyl chloride is added to a biological sample liquid such as blood or urine, and reacts with a small amount of catecholamine contained in the sample liquid to generate a dansylated catecholamine derivative. This dansylation step can be performed as a pretreatment in liquid chromatography. Dansylation of catecholamines in a sample solution can be carried out, for example, by adjusting the water content of the sample solution to 20 to 40% by weight, and adding 1% by weight under heating conditions of PH8 to 9, 40 to 50°C, and 20 minutes. It is sufficient to add an acetone solution of dansyl chloride having a concentration of at least 6 times the molar amount of the catecholamine, and thereby the dansylation reaction of the catecholamine can be carried out completely. The catecholamine derivative thus obtained, ie, the dansylated catecholamine, is introduced into a separation column in liquid chromatography and separated into a noradrenaline derivative, an adrenaline derivative, and a dobamine derivative. The liquid chromatography process in this case is shown in FIG. 1
is an eluent tank, for example, a water: methanol: triethylamine mixture is used, and a constant flow pump 2 is used.
The liquid is delivered by The pH value, ionic strength, type of ion, flow rate, etc. of the eluent are adjusted according to the separation ability of the separation column. 3 is a sample injector, into which a sample liquid containing a catecholamine derivative is supplied and mixed with an eluent. 4 is a separation column filled with particles of, for example, porous silica into which octadecyl groups have been introduced, divinylbenzene-(meth)acrylic acid copolymer, or the like. The sample liquid mixed with the eluent passes through a separation column and is separated into three components: a noradrenaline derivative (dansylated noradrenaline), an adrenaline derivative (dansylated adrenaline), and a dobamine derivative (dansylated dobamine). In the present invention, each derivative thus separated is brought into contact with oxalic acid ester and hydrogen peroxide to generate chemiluminescence. This oxalate ester is used after being dissolved in a solvent such as ethyl acetate. 5 is an oxalic acid ester solution tank, which is supplied through a flow pipe by a constant flow pump 6. 7 is a hydrogen peroxide solution tank, in which acetone, acetonitrile, alcohol having 1 to 5 carbon atoms, etc. are used as a solvent to improve the miscibility of the eluent and the oxalic acid ester solution; Supplied. In addition, since chemiluminescence shows its maximum intensity under weakly alkaline conditions, for example, a Tris-HCl buffer solution or the like is supplied from a buffer tank 9 by a constant flow pump 10.
The PH value is adjusted to slightly alkaline, preferably PH8.0. The oxalate ester solution and hydrogen peroxide solution are combined,
1,2 by reaction between oxalic acid ester and hydrogen peroxide
-Dioxetanedione is produced. This substance is a high-energy substance, which transfers energy to the separated noradrenaline derivative, adrenaline derivative, and dobamine derivative, and each highly energized derivative emits light. The reaction between oxalate ester and hydrogen peroxide and the resulting energy transfer by the high-energy substance occur very quickly. There is no problem in merging them at the same time. This chemiluminescence is then detected and quantified. 1
1 is a detector, which detects with a photomultiplier tube. It is advantageous in terms of sensitivity to process the signal from the photomultiplier tube in the form of pulses using a photon counter, but it may also be extracted as a current as in a spectrometer. The extracted signal is recorded by a recorder 12. Based on the recorded results, for example, the amount of noradrenaline derivatives, adrenaline derivatives, and dobamine derivatives can be determined by the number of pulse signals of a photon counter. , adrenaline, and dobamine can be quantified. According to the present invention, adrenaline,
Three components, noradrenaline and dobamine, can be measured simultaneously and with high sensitivity. Further, there are advantages such as being able to perform measurements with one device and shortening the measurement time. Example 1 5 ml of blood from 3 healthy subjects was collected into a vacuum blood collection tube, left at nitrogen temperature for 30 minutes, centrifuged at 3000 rpm for 5 minutes, and 2 ml of serum was added with 2 ml of 0.01M Tris-HCl buffer (PH8.0). ) and after mixing, 1% by weight
3 ml of an acetone solution of dansyl chloride was added thereto, and the mixture was heated at 50°C for 20 minutes to react, thereby dansylating the catecholamine. 100μ of the sample solution containing the catecholamine derivative thus obtained was injected into a high performance liquid chromatography system. In high performance liquid chromatography, water:methanol:triethylamine (60:30:10) was supplied as an eluent from an eluent tank 1, and was fed by a constant flow pump 2 at a flow rate of 0.2 ml/min. Separation column 4 was a 4.6 mm inner diameter, 250 mm long column filled with porous silica into which octadecyl groups were introduced.
mm was used. The dansylated derivative of catecholamine that has exited the separation column 4 is separated into a noradrenaline derivative, an adrenaline derivative, and a dobamine derivative.
A 0.1 mol Tris-HCl buffer (PH8.0) was fed to this using a constant flow pump 10 at a flow rate of 0.1 ml/min.
In addition, 5 mmol of bis(2,
A solution of 4,6-trichlorophenyl) oxalate in ethyl acetate was fed at a flow rate of 2.5 ml/min, and
A solution of millimolar hydrogen peroxide in acetone was fed by a constant flow pump 8 at a flow rate of 2.5 ml/min to cause chemiluminescence of each derivative. In this case, the screw (2,
4,6-trichlorophenyl) oxalate is
It was synthesized from 2,4,6-trichlorophenol and oxalyl chloride and recrystallized from benzene. Detection of the generated chemiluminescence was performed with a photomultiplier tube placed in close contact with the flow cell, and the obtained signal was processed with a photon counter and recorded with a recorder. As a result, chromatograms as shown in Figure 2 were obtained for all three cases, and catecholamines in the blood were quantified as the amount of chemiluminescence caused by oxalate.
Fractions were measured simultaneously. From the calibration curve using a dansylated derivative of standard catecholamine and the recovery rate obtained by adding standard catecholamine to pooled serum and performing dansylation, the blood concentration of catecholamine for each of the three samples was obtained as shown in Table 1. . 【table】

[Brief explanation of drawings]

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 an example. Explanation of symbols 1... Eluent tank, 2, 6, 8, 10
...Constant flow pump, 3. Sample injector, 4. Separation column, 5. Oxalate ester solution tank, 7. Hydrogen peroxide solution tank, 9. Buffer solution tank, 11. Detector.

Claims (1)

[Claims] 1. Adding dansyl chloride to a sample solution containing catecholamine and reacting with the catecholamine to generate a catecholamine derivative,
The sample solution containing this catecholamine derivative is introduced into a separation column in liquid chromatography and separated into a noradrenaline derivative, an adrenaline derivative, and a dobamine derivative. A method for quantifying catecholamines, which is characterized by bringing chemiluminescence into contact with (enyl) oxalate and hydrogen peroxide, and detecting and quantifying this chemiluminescence.