JPH05113438A - Method and apparatus for simultaneously analyzing catecholamines and metabolite of catecholamine - Google Patents

Abstract

PURPOSE:To obtain the method and the apparatus effective for the diagnosis for neuroblatoma, sympathetic neuroblatoma, pheochromocytoma and the like by efficiently separating and analyzing dopamine and L-dopa contained in liquid under test such as urine together with vanillylmandelic acid, homovanillic acid and creatinine at the same time. CONSTITUTION:An anion exchange resin column 7 and a negative-phase column 8 are connected in series. Eluate switching valves 1A-1C, a flow-path switching valve 9, a creatinine detector 10, and a detector 11 for the components other than creatinine are provided in a device in a high-speed liquid chromatography apparatus. Liquid under test is injected into the chroamtography apparatus. The liquid under test is sequentially introduced into the anion exchange resin column 7 and the negative-phase column 8. At first, creatinine in the liquid under test is separated and analyzed. Then, L-dopa, vanillylmandelic acid, dopamine and homovanillic acid are sequentially separated and analyzed.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to catecholamine metabolites used for diagnosis of neuroblastoma, such as vanillyl mandelic acid (hereinafter abbreviated as VMA), homovanillic acid (hereinafter abbreviated as HVA) and creatinine. (Less than,
Abbreviated as CRN. ), Etc., is mainly used for diagnosis of sympathetic neuroblastoma and pheochromocytoma, and determination of therapeutic hardening.
L-dopa, which is a catecholamine precursor (hereinafter referred to as DO
It is abbreviated as PA. ) And catecholamines such as dopamine (hereinafter abbreviated as DA) and the like, a simultaneous analysis method for catecholamines and catecholamine metabolites, which can be separated and analyzed from a test liquid containing catecholamines such as And an analyzer therefor.

[0002]

2. Description of the Related Art Neuroblastoma (hereinafter abbreviated as NB) is the most common malignant solid tumor in childhood and has a very poor prognosis. However, since the cure rate is higher in the case of early detection, early detection and diagnosis are desired. Mass screening of NB has been carried out for infants about 6 months old at a nationwide level since January 1985, and the high performance liquid chromatography method was introduced from 1988, and this method is currently used. It is the subject.

In this disease, a large amount of catecholamine metabolites VMA and HVA are excreted in urine, resulting in NB.
In mass screening, the measurement of these two components contained in urine is mainly performed, but when quantifying the components in urine, the CRN concentration in urine is also measured to measure CRN.
It is general to make a correction for the change in urine volume based on the ratio. In this way, by adopting the method of measuring VMA and HVA in urine by high performance liquid chromatography and correcting them with the CRN ratio, the method based only on the qualitative analysis of VMA performed at the beginning of the mass screening of NB What can be found only in about 1 in 20,000 people, can now be found in 1 in 5000 to 6000 people, and 90 to 95% of patients have been found.

However, it is not possible to completely detect a patient with NB even by the above-mentioned method. Therefore, the measurement items for NB mass screening have begun to be reexamined, and until now, mainly sympathetic neuroblastoma and pheochromocytoma have been reported. It is also considered to measure DOPA and DA, which are used for diagnosing tumors and determining therapeutic effects.

[0005]

In addition to the target component, the test liquid such as urine contains an acidic component such as uric acid and organic acid and a basic component such as polyamine, and the quantitative determination is inhibited by these contaminants. Therefore, pretreatment for removing contaminants is necessary. The Applicant has performed a pretreatment for removing contaminants,
A method capable of simultaneously measuring MA, HVA and CRN by high performance chromatography was developed and proposed previously (Japanese Patent Laid-Open No. 63-212861). However, even though VMA and HVA are metabolites of DOPA and DA, even if the above method is adopted, VMA and HV
It was difficult to analyze DOPA and DA together with A and CRN efficiently.

The present invention has been made in view of the above points,
An object of the present invention is to provide a simultaneous analysis method for catecholamines and catecholamine metabolites capable of simultaneously and efficiently analyzing VMA, HVA, CRN, DOPA and DA in a test solution, and an analyzer therefor.

[0007]

That is, the simultaneous analysis method for catecholamines and catecholamine metabolites of the present invention comprises:
Vanillyl mandelic acid, homovanillic acid, dopamine, L
-Introducing a test solution containing dopa and creatinine into an anion exchange resin column for high performance liquid chromatography, and then introducing an eluate from the column into a reverse phase column to separate and analyze creatinine in the test solution. , Then L-DOPA,
It is characterized in that vanillyl mandelic acid, dopamine and homovanillic acid are sequentially separated and analyzed. The analyzer for catecholamines and catecholamine metabolites of the present invention is an analyzer for simultaneously analyzing catecholamines and catecholamine metabolites by high performance liquid chromatography, which comprises creatinine, L
An anion exchange resin column and a reverse phase column connected in series for sequentially separating dopa, vanillyl mandelic acid, dopamine, and homovanillic acid, and an eluent switching valve for separating and eluting each component, and a separation A channel for creatinine which is eluted from the reversed phase column and a channel for switching between L-dopa, vanillyl mandelic acid, dopamine and homovanillic acid channels, a detector for detecting creatinine, and L-dopa And a detector for detecting vanillyl mandelic acid, dopamine, and homovanillic acid.

[0008]

In the method of the present invention, CRN, DOPA, DA and VM in the test liquid in the anion exchange resin column
A and HVA are partially fractionated and other strongly acidic components are trapped. Since CRN, DOPA, and DA have little interaction with the packing material of the anion exchange resin column, VM
A and HVA are eluted from the anion exchange resin column before being introduced into the reverse phase column.

Since the packing material of the reversed phase column interacts with the hydrophobic substance, the more hydrophilic substance is eluted from the reversed phase column earlier. C by the above anion exchange resin column
By combining the partial fractionation action of RN, DOPA, DA and VMA, HVA with the property that a hydrophilic substance elutes from a reversed-phase column as quickly as possible, CRN, DOP
A, VMA, DA, and HVA can be sequentially separated.

[0010]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the analyzer of the present invention will be described below with reference to the drawings. In the device of the present invention shown in FIG.
1C is the first, second, and third eluent tanks, 2A, 2B, respectively
Is an eluent switching valve for switching the eluent tanks 1A to 1C, 3 is a degasser for degassing the gas in the eluent,
Reference numeral 4 denotes a liquid feed pump for feeding the eluent, and the sample injected from the sample injection device 5 is passed through the prefilter 6 which protects the column by the eluent fed by the liquid feed pump 5, and the anion exchange resin column 7 and the reverse phase column 8 are sequentially introduced. 9 is CRN separated by the anion exchange resin column 7 and the reverse phase column 8
Flow path switching valve for switching the flow path of VMA, HVA, DOPA, and DA, and 10 is a CRN detector, 11 is a VMA, HVA, DOPA, and DA detector,
12 is a waste liquid tank.

The eluent used in the present invention may be a commonly used buffer such as a citrate buffer, a phosphate buffer or the like, or a buffer obtained by adding an organic solvent to these buffers. As the eluent of the first eluent tank 1A,
A citrate buffer solution or a phosphate buffer solution containing no organic solvent is preferable, and the eluent solution of the second elution tank 1B and the eluent solution of the third elution tank 1C are sequentially adjusted so that the content of the organic solvent increases. It is preferable to use an acid buffer or a phosphate buffer. In carrying out a particularly efficient analysis, an eluent containing no organic solvent is used until CRN is eluted from the reverse phase column 8, and after the CRN is eluted from the reverse phase column 8, VMA,
To elute HMA, DOPA, DA, an eluent containing about 5 to 20% of an organic solvent is used, and VMA, HMA, D
It is preferable to use an eluent containing about 30 to 60% of an organic solvent in order to wash the channel and the column after the detection of OPA and DA is completed.

When performing analysis in the apparatus of the present invention, first, the eluent flow path is set to the initial state as shown in FIG. still,
In FIG. 2, the eluent flow path is shown by a solid line (the same applies to FIG. 3 and subsequent figures). In the initial state, the eluent in the first eluent tank 1A reaches the detector 10 from the sample injection device 5 through the prefilter 6, the anion exchange resin column 7, the reverse phase column 8 and the flow path switching valve 9. Also, the liquid is fed through the flow path switching valve 9 to the detector 11 without passing through the sample introduction device 5.

The sample injected from the sample injection device 5 is introduced into the anion exchange resin column 7 through the pre-filter 6 by the eluent, the strongly acidic component is trapped in the column 7, and CRN, DOPA, DA, and V
MA and HVA are partially fractionated and then introduced into the reverse phase column 8. From the reverse phase column 8, CRN is first eluted. As a packing material for the anion exchange resin column,
Weakly basic anion exchange resins are preferred. Examples of the weakly basic anion exchange resin filler include a styrene-divinylbenzene copolymer, hydrophilic silica gel, or a hydrophilic polymer gel into which a diethylaminoethyl (DEAE) group is introduced. As the packing material for the reversed phase column 8, a styrene-divinylbenzene copolymer, hydrophilic silica gel or hydrophilic polymer gel having an octadecyl group or a phenyl group introduced therein is preferable.

After the CRN has passed through the flow path switching valve 9, the flow path switching valve 9 is switched so that the CRN is introduced into the detector 10 as shown in FIG.
To detect the CRN. An ultraviolet absorption detector is preferable as the CRN detector 10, and the measurement wavelength is 22
0 to 260 nm is preferred. The flow path switching valve 9 is shown in FIG.
After switching as shown in FIG. 2, the eluent switching valves 2A and 2B are switched so as to send the eluent in the second eluent tank 1B, and the eluent in the second eluent tank 1B as shown in FIG. From the sample injection device 5 to the flow path switching valve 9 and the detector 10, and from the flow path switching valve 9 without passing through the sample injection device 5 to the pre-filter 6, anion exchange resin column 7, and reverse phase column. The solution is sent through the route reaching the detector 11 via 8.

DOPA, VMA, DA and HVA are the first
The eluent in the elution tank 1A and the second elution tank 1 used thereafter
The eluent in B is sequentially separated in the reverse phase column 8,
It is introduced into the detector 11 and detected. DOPA, VM
A fluorescence detector is preferable as the detector 11 for detecting A, DA, and HVA, and the excitation wavelength is 280 nm and the fluorescence wavelength is 3
It is preferable to measure at 20 nm.

After the detection of DOPA, VMA, DA, and HVA is completed, as shown in FIG. 4, the eluent switching valves 2A and 2B are switched so as to feed the eluent in the third eluent tank 1C, and the third elution is carried out. The channel, the anion exchange resin column 7, and the reverse phase column 8 are washed with the eluent in the liquid tank 1C.
After cleaning, as shown in FIG. 5, the eluent switching valve 2
After switching between A and 2B and sending the eluent in the first eluent tank 1A to fill the channels and the columns 7 and 8 with the eluent in the first eluent tank 1A, as shown in FIG. The flow path switching valve 9 is switched to the initial state.

The switching of the above operation steps is performed by computer control based on the outflow time of each component obtained in advance.

The present invention will be described in more detail with reference to specific examples. Example 1 In the apparatus shown in FIG. 1, an anion exchange resin column 7 was used.
Weakly basic TSK gel VMA-Anion (4.6
mmID × 120 mm: manufactured by Tosoh Corp., TSK gel VMA-RP (4.6 mmID × 150 mm) as the reverse phase column 8:
Tosoh Corp. was used. An ultraviolet absorption detector was used as the CRN detector 10, and the measurement was performed at a wavelength of 254 nm. Also, the detector 11 for DOPA, VMA, DA, HVA
A fluorescence detector was used for the measurement as an excitation wavelength of 280 nm and a fluorescence wavelength of 320 nm. Eluent in the eluent tank 1A (hereinafter,
It is abbreviated as eluent 1A. ) As a 50 mM sodium citrate buffer (pH = 4.8), and as an eluent in the eluent tank 1B (hereinafter abbreviated as eluent 1B), a 50 mM sodium citrate buffer (pH = 10%) containing 10% acetonitrile.
4.6), 50m containing 40% acetonitrile as an eluent in the eluent tank 1C (hereinafter abbreviated as eluent 1C)
M sodium citrate buffer (pH = 5.5) was used. As a sample, CRN of 500 ng and DO
PA, VMA, HVA 5ng each, DA 2.5n
The test liquid used was a single urine sample of a healthy male. The flow rate of each eluent was set to 1 ml / min, the flow path switching valve 9 was switched after the CRN passed through the flow path switching valve 9, and the eluent switching valves 2A and 2B were switched to change from the eluent 1A. The eluent 1B is switched to send the liquid, and DOPA, VMA, DA,
HVA was sequentially separated and detected. Eluent 1 after detection
After washing by flowing C, the eluent 1A was caused to flow to fill the channel and the column with the eluent 1A. Time for each step, type of eluent flowing through each column for each step,
Table 1 shows the numbers of the figures corresponding to the states of the respective valves and the states at that time. The chromatogram of the standard sample is shown in Fig. 6.
The chromatogram of the test liquid is shown in FIG.

[0019]

[Table 1]

In Table 1, the state where the valve 2A is OFF means that the eluent 1A flows through the pump to the line, and the state where it is ON means that the eluent 1B or 1C flows to the pump. The state in which the valve 2B is OFF means that the eluent 1B flows through the pump to the line (however, the valve 2B
A is in the ON state) and the ON state means that the eluent 1C flows through the pump to the line (however, the valve 2A
Is ON). Further, when the valve 9 is OFF, the line in which the eluent flows from the sample injection device 5 through the columns 7 and 8 to the detector 10 and the eluent is the detector 1
1 is a state in which a line flowing to 1 is formed, and an ON state is a line in which the eluent flows from the sample injection device 5 to the detector 10 and a line in which the eluent flows to the detector 11 through the columns 7 and 8. Is a state in which is formed.

[0021]

As described above, according to the analysis method and apparatus of the present invention, DA, which is a catecholamine, DOPA, which is a precursor of catecholamine, and VHA, MVA, and CRN, which are catecholamine metabolites, are simultaneously separated and analyzed to obtain a CRN ratio. Since it can be determined by correction, meaningful measurement results can be provided not only by NB mass screening, but also by diagnosing tumors derived from the sympathetic nerve system and adrenal medulla and determining the therapeutic effect. Moreover, it is possible to perform analysis in a short time, and there is an effect that labor saving can be achieved by computer control.

[Brief description of drawings]

FIG. 1 is a schematic view showing an embodiment of the analyzer of the present invention.

FIG. 2 is a schematic view showing a state of one operation step of the analyzer of FIG.

3 is a schematic diagram showing states of different operating steps of the analyzer of FIG. 1. FIG.

FIG. 4 is a schematic view showing a state of still another operation step of the analyzer of FIG.

5 is a schematic view showing a state of yet another operation step of the analyzer of FIG. 1. FIG.

FIG. 6 is a chromatogram of the standard sample obtained in the example.

FIG. 7 is a chromatogram of the test liquid obtained in the example.

[Explanation of symbols]

 2A Eluent switching valve 2B Eluent switching valve 7 Anion exchange resin column 8 Reverse phase column 9 Flow path switching valve 10 Detector 11 Detector

Claims (2)

[Claims] 1. Vanillyl mandelic acid, homovanillic acid,
A test solution containing dopamine, L-dopa and creatinine was introduced into an anion exchange resin column for high performance liquid chromatography, and then the eluate from the column was introduced into a reverse phase column to separate creatinine in the test solution. A method for simultaneous analysis of catecholamines and catecholamine metabolites, which comprises sequentially separating and analyzing L-dopa, vanillyl mandelic acid, dopamine, and homovanillic acid. 2. An analyzer for simultaneously analyzing catecholamines and catecholamine metabolites by high performance liquid chromatography, the device sequentially separating creatinine, L-dopa, vanillyl mandelic acid, dopamine and homovanillic acid in a test solution. An anion exchange resin column and a reverse phase column connected in series to achieve, and an eluent switching valve for separating and eluting each component, and a flow path of creatinine that is separated and eluted from the reverse phase column. , L-dopa, vanillyl mandelic acid, dopamine, homovanillic acid and a flow path switching valve for switching the flow path, and a detector for detecting creatinine,
An analyzer for catecholamines and catecholamine metabolites, which comprises at least a detector for detecting L-dopa, vanillyl mandelic acid, dopamine, and homovanillic acid.