JPS5827062A - Liquid chromatograph for measuring catecholamine - Google Patents

Abstract

PURPOSE:To reduce a level of noise due to pulsatory flow and to permit detection of adrenalin of small amount by a construction wherein fluorescence sensitive reaction solutions are supplied into a flow tube for the column effluent of the chromatograph using a reciprocative plural-pump so that those solutions are applied to the flow tube at plural positions. CONSTITUTION:An effluent from a separation column 2 of the liquid chromatograph for measuring catecholamine is sent to a flow tube in a fluorescence sensitive portion 3. Potassium ferricyanide aqueous solutionI, ascorbic acid aqueous solution II and NaOH aqueous solution III are respectively supplied through line tubes 14, 15 and 16 to plural positions of the flow tube 4 using a reciprocative plural-pump 5, thus avoiding occurrnce of pulsatory flow. In this way, a level of noise produced in the fluorescence detecting portion due to the pulsatory flow is reduced so much to permit detection of catecholamine, particularly adrenalin of very small amount (4 pico gram).

Description

DETAILED DESCRIPTION OF THE INVENTION To meet the needs of this liquid chromatograph for measuring otepuramine.

Body leverage #Amine is adrenaline, noradrenadan,
It is a general term for dopamine, and cathepoolamine OMZ + is used to diagnose lateral renal medulla and sympathetic ganglion production, shingonblastoma, pheochromocytoma, etc., and to diagnose recent essential hypertension and psychosis. I've also been benefited from it. To measure catecholamine O, amine Oat is measured in urine and blood.
In the measurement, the detection sensitivity is set to the highest level because the amount is very small.
IiK adrenaline exists only in extremely small amounts and is often undetectable.

Conventionally, cathepool amine has been measured by converting cathepool amine into a fluorescent substance using a trihythosekidiindole method using a high-speed chamber, and then detecting this using an electrophotometer. Call liquid chromatoguts 7
In flCsI, there is a liquid feeding part that supplies an eluent, a separation part in which a packing material for couple amine separation is placed in a column Km, and a reaction liquid Kli, which is generated and transferred into the flow pipe of the ArIL outlet pipe. It consists of a photo-sensitizer that carries out fluorescence sensitization within the flow tube, and a fluorescence detection section that detects catecholamines that have been sensitized to fluorescence.

In this case, the reaction solution for photosensitization is an aqueous solution of zerichanide and an aqueous ascorbic acid solution.

Three liquids, an aqueous sodium hydroxide solution, are used to treat cholera, and a peristaltic pump as shown in Figure 117 is used! IIIj
Liquid is being pumped from 1K to 1K.

In the pedaster pump, the fluorescence sensitizing reaction liquid supplied to the elastic tube 1' is fed by a rotary roll 2', using pulsation while Ixp. When separating from 1', a part of the liquid is returned due to anti-elasticity, and the liquid 3 becomes 1! The reaction solution does not flow smoothly into the flow tube, and the burr is detected by the fluorescence detection unit.
There were cases where the noise level was higher than the peak of catecholamines, especially adrenaline, and p was impossible to measure when measuring CFi specimens.

The present inventor #I conducted various studies on ways to reduce the noise repetition in the conventional liquid coumadog for measuring catecholamines, and as a result, the fluorescent sensitization reaction solution t was sent to the flow pipe of the column effluent. It has been found that a method that does not cause pulsation when feeding is effective. As a result of further studies, Kj discovered that the noise level could be significantly reduced by feeding the fluorescence sensitizing reaction solution into the flow tube by operating a reciprocating multiple pump, thereby completing the present invention. ! It was.

The gist of the present invention is to provide a liquid feeding section for supplying an eluent, a separation section in which a force-cooled amine separation charge IAIIJ is housed in a cup, and a fluorescent sensitizing effect on a flow tube for flowing liquid. A liquid chromatograph for measuring kanafuramine consists of a fluorescence sensitization section that feeds the reaction solution and sensitizes the O catecholamine in the effluent to fluorescence, and a fluorescence detection section that is sensitized to fluorescence and detects nine catecholamines. te, rental ff1l11 work-
The catecholamine measurement IIa liquid flow pipe 7 is characterized in that O is supplied to the flow pipe of the reaction liquid at a few points in the flow pipe 011 by operating a reciprocating multiple pump O.

*About the liquid chromatograph for measuring kanafuramine of the present invention! will be explained in detail.

In the liquid feeding section that feeds the IFi liquid, the eluent is fed to the column by a constant flow pump. As an eluent,
For example, an aqueous phosphoric acid-calcium solution having a concentration of 1/1 G/l and a hydrogen value of 4 is used.

Reference numeral 2 denotes a separation section, in which a filler for catecholamine separation is filled in a container. Examples of the filler include styrene, nitylstyrene, divinylpeneyne, beer toluene, vinyl natsutareji, N-vinylcarbaso #, etc.
Ibiz-based monomers having an enyl group, acrylic acid, methacdaric acid, polyethylene glycol di(meth)acrylate, polypropylene glycol di(meth)acrylate, and Fa xyethyl methacrylate, tetramethylol blown (mono, su, tri)acrylate, (meth)acrylamide, alkoxymethacrylate I&I (
O copolymers with meth)acrylamide, N,N-dimethylaminoethyl (meth)acrylate, etc. are suitable for use.

When serum, plasma, and other components containing θ catecholamines are fed into the chamber, the catecholamines are separated in the separation section.

S is a fluorescence sensitization section, which feeds the fluorescence sensitization reaction solution into the flow tube 4 of the p% effluent, and is kept at the reaction temperature, and the fluorescence is generated in the flow tube 4, which is kept at the reaction temperature. Sensitization takes place. For this purpose, O is supplied to the four circulation pipes of the reaction liquid from a plurality of locations by the operation of a reciprocating multiple pump s. As the reaction liquid, for example, an aqueous potassium chloride solution is used as the first liquid, an ascorbic acid aqueous solution is used as the @I liquid, and a water-soluble sodium hydroxide solution is used as the carbon dioxide solution. For example, adrenaline (in the case of adrenaline), fluorescence is sensitized by the following reaction.

Fluorescence sensitization occurs in the same way in adrenadan and do/4min.

In the example shown in the second tg1, the If/
1 is fed from the front end of the distribution pipe 4o, and the third liquid and the third liquid are combined in the middle at the rear of this and are fed into the distribution pipe 4o.
), the No. 1 liquid and the No. 1 liquid are respectively sent to the distribution pipe 4.
Alternatively, the first liquid, the second liquid, and the second liquid may be separately fed from the 3° I part of the flow pipe 4.

A reciprocating pump is used to supply K to the reaction liquid tgL passage tube 4. Bonda5Fi! As shown in 5INK, the crankshaft 6 is driven by the motor 1, and the crankshaft 6 has cams I and 9.
．． 10 and is Kfk to move the plunger 11.12.131 alternately up and down fi11. l
When pump 2 is activated, 111j[, I! The dosing liquid is sent to the flow q14 through the upward movement of the plunger 11, 11F (from the liquid supply pipe 14.1@), and this mixed liquid is sucked from the liquid tank by the downward movement of the granger 120. 11,110 Downward operation (first liquid, first heating is liquid)
The 11M liquid is fed to the flow pipe 4 by the movement of the planar rear 1fO.

Top v! , In 0 cases, a Fis pump is used for convenience, but 1
A continuous pump may also be used, and in the case of fuel, for example, the first liquid is pumped from the liquid supply pipe to the distribution pipe 4 by the upward movement of the plunger O.
The liquids IIK, 1t, and 11111 are sucked into the upward movement of the plunger O, and the liquids No. 2 and 11111 are fed into the upward movement of the plunger O from the liquid supply pipe to the distribution pipe 4. The first liquid is sucked into the pot by the downward movement of the plunger and is trapped.

In this way, when the pump 5 is operating, the supply of the reaction liquid to the flow pipe 4 is not disrupted, and the flow is opened smoothly (
By doing this, the nozzle rate in the fluorescence detection unit 11 is significantly lowered compared to when using a PEX pump, and it becomes possible to measure extremely small amounts of O catecholamines.

For example, a fluorometer is used as the fluorescence detection device. 1. Obtain the detection results of catecholamines using fluorescence detection 1117 from Kujima Dogutsu AK).

In this book - Ming Catefu - #A7j1MI liquid chromatography -, the O equipped with the 0111E tube is the conventional 04
There is no backflow of the reaction liquid O fed into the KfIL tube, unlike when feeding the reaction liquid using a 1-star pump with a pulsation rate of 1 s, and the flow is made possible by the operation of the reaction liquid reciprocating pump. Since it is fed into the tube without interruption and the fluorescence sensitization reaction takes place circularly, the nozzle number in the fluorescence detection IIK is significantly lowered, making it possible to measure extremely small amounts of O catecholamines.

As a result, catecholamines in the blood, which could not be detected in conventional specimens, can be detected with high precision, which is of great significance in terms of clinical diagnosis.

Implementation-1 A catecholamine aqueous solution containing 10 bipgt^ of each of Ato V-narin and Nor-Ato Vf-n was used as a sample solution, and the liquid was transferred to IIKs? As an eluent, 1/10 mol 1]n111[t4! ! The eluent can be delivered to the separation IIO cut using a constant flow pump. Full*m in the separated city
Divinylbenzene-acid and 4sy acid copolymer @
The polymerized particles were packed into a stainless steel column with a length of 5 mm.

In fluorescence sensitization II, arrange the liquid supply tube as shown in Figure 62, and! I! As a liquid, an aqueous solution of 1 nitride, g
11? Mu aqueous solution #! Using a reciprocating pump, each of the Ill liquid, first wave, and sight was delivered to the armpit without interruption. Mayuka Suga controlled the reaction temperature by maintaining the reaction-O temperature at 4S'CK.

The fluorescence detection section has an excitation wavelength of 420 μm and a fluorescence wavelength of 1
A 20 amO fluorometer was used. The detection results for fluorescence detection 11 were obtained in a timely manner. P in FIG. 4 indicates the absorption peak of adrenaline.

The S1M ratio in Chromadogutsum is 100%, and the detection limits Fi for noradrenaline and adrenaline are both 3 and 9.

Jl theory protein manipulation and 1st mina treatment on 100 blood samples of Yasuto O's blood using conventional methods! ξ, these samples were measured, and the results showed that norepinephrine and epinephrine could not be quantified in one sample.

Implementation #I! Same as Example 1 of 11I except that a reciprocating triple pump K was used instead of the reciprocating triple pump K in Example IK, and the first liquid was combined with Kfl1 before the @8@tl pump sucked,
An aqueous catecholamine solution containing 10 picograms of adrenaline and noradrenaline each was subjected to liquid chromatography.The chromatograph in this case is shown in FIG.

r, noradrenaline, P, Fi adrenaline and a starch pump were used, and a catecholamine aqueous solution containing adrenaline and noradrenaline at 50 pkg each was prepared in the same manner as in Example 1 except for 9. In the case of Jin, the glue cost is shown in Figure 6.

P, shows the light absorption peak of adrenaline.

The detection limits for noradrenaline and epinephrine in the obtained somatogatum were 30 picodads (S/N ratio), respectively.

When O2 blood samples from overseas patients were subjected to protein removal and alumina treatment in the same manner as in Example 1, and these samples were measured, 5 samples were found to have no adrenaline detected. Ta.

[Brief explanation of drawings]

1811! ll is the liquid liquid for measuring cathepolyte amines 0III diagram, *zl! An explanatory diagram showing 140 examples of the 3F1 separation unit and fluorescence sensitization, Fig. 3 is an explanatory diagram showing a reciprocating three-stroke pump Of4, Fig. 114 is a chromadogtum in Example 111, and Fig. 5 is an example of a chromadogram in snow. , Figure 6 shows the comparison example.
! ! FIG. 7 is an explanatory diagram showing an example of a conventional Verister pump. Explanation of symbols: 1. Dosei algae, goose separation section, 3. fluorescence sensitization section, 4.g. tube.
2 reciprocating multiple pump, 11. 13.1 mm plunger 14, Xi, 1@liquid pipe, 11 fluorescence detection algae, 1 recording section

Claims (1)

[Claims] 1. A liquid feeding part that supplies an eluent, a separation part in which a packing material for Kabulamine separation is placed in the cuff, and a cuff A.
a fluorescence sensitization unit that feeds a fluorescence sensitization reaction solution into the flow pipe of the effluent to sensitize the licatefuramine in the effluent to fluorescence;
In the liquid chromatograph 7 for measuring cathetoulamine, which is composed of a fluorescent detection section that is sensitized to fluorescence and detects nine catecholamines, the reaction liquid is fed into the flow tube by the operation of a reciprocating pump. (Multiple locations on the Yoni Seittsu tube) 1
Liquid chroma for measuring catechol amines [graph