JPH1183828A - Catecholamine analyzer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To permit all catecholamine analysis with a single operation for a specimen showing its signal ratio exceeding a dynamic range of a detector by changing sensitivity of a fluorescent detector according to specimen type. SOLUTION: This apparatus separates epinephrine(E), norepinephrine(NE), and dopamine(DA) by a separation column, and performs analysis. A fluorescent detecting apparatus has detection sensitivity switching means of a fluorescent detector 18, a control apparatus has storing means for storing an identification signal for a specimen, DA has means for storing a time of flowing out from the separation column and clock means, and has a function for lowering sensitivity of the fluorescent detector 12 while DA is detected by the fluorescent detector 18.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a catecholamine analyzer for quantifying epinephrine (E), norepinephrine (NE) or dopamine (DA) using the principle of liquid chromatography.

[0002]

2. Description of the Related Art Normally, catecholamines are separated by liquid chromatography after pretreatment of a sample (blood, plasma, urine, etc.) with proteins, etc., and analyzed by an electrochemical detection method or a fluorescence detection method. Is done.

[0003] The electrochemical detection method is capable of high-sensitivity analysis but detects all electrochemically active components, in other words, cannot specifically detect catecholamines. Not suitable for analysis.

As a fluorescence detection method, there is a method using a fluorescent reagent specific to a catechol skeleton or an amine in order to enhance fluorescence intensity and increase selectivity, in addition to a method using natural fluorescence. That is, as a specific fluorescent reagent, for example, THI (T
rihydroxyindol), ethylenediamine,
OPA (OrtoPthalAldehyde), fluorescamine, dansyl chloride, DPE (DiPh
There is known a method of preparatively derivatizing catecholamine before separation using a column (pre-column method), or a method of post-separation of catecholamine after separation using a column (post-column method) using an ethylethylamine or the like.

[0005]

The blood and urine contain E, N
It contains catecholamines such as E and DA, and by using liquid chromatography and pre-column (derivatization) method or post-column (derivatization) method, all of them can be analyzed in one operation. ,
Urine is characterized by a higher DA concentration than E and NE. It is not uncommon for many samples to have a difference of about 1000 times between the lower limit of the concentration of E and the upper limit of the concentration of DA.

As described above, when E, NE and DA are analyzed in one operation using liquid chromatography, the gain of the detector is set by setting the gain of the detector in consideration of the lower limit of the concentration of E or NE. For urine samples with high concentration, D
The signal derived from A exceeds the measurement limit, and an operation such as diluting the sample and performing the analysis operation again is required. However, if the gain of the detector is set to be suitable for high-density DA, there is a problem that the sensitivity is insufficient for low-density N and NE.

Therefore, the present invention provides a method for setting a detector for a blood-derived sample (hereinafter referred to as a blood sample) and a urine-derived sample (hereinafter referred to as a urine sample) when a difference in concentration between E and NE and DA occurs. It is an object of the present invention to provide a catecholamine analyzer that does not need to be changed or to perform an analysis operation by diluting a sample.

[0008]

Means for Solving the Problems The present invention made in view of the above object provides at least epinephrine (E) in a sample,
A catecholamine analyzer for separating and analyzing norepinephrine (NE) and dopamine (DA) by a separation column, comprising a detection device including at least a fluorescence detector and a control device, wherein the fluorescence detection device is a detection sensitivity switching means of the fluorescence detector The control device has storage means for storing an identification signal for identifying the sample, means for storing the time when DA elutes from the separation column, and time counting means, and DA is detected by the fluorescence detector. A catecholamine analyzer having a function of reducing the detection sensitivity of a fluorescence detector during the measurement. Hereinafter, the present invention will be described in detail.

The present invention provides a method for converting catecholamines from their natural fluorescence, for example, to THI, ethylenediamine, OP
A, a device for fluorescence derivatization with fluorescamine, dansyl chloride, DPE, etc., and detection with a fluorescence detector. When catecholamine is fluorescently derivatized, any of the so-called pre-column method and post-column method may be employed.

[0010] The apparatus of the present invention is characterized by including a detection apparatus including a fluorescence detector and a control apparatus, but may also include an apparatus described later. The fluorescence detector is
It has means for switching the detection sensitivity of the fluorescence detector. Usually, the fluorescence detector is composed of a light source, an excitation light focusing system, a measuring cell, a fluorescence focusing system, and a detection element. These components can be the same as those used in a normal fluorescence detector.

The light source may be a light source capable of obtaining a wavelength and intensity necessary for fluorescence excitation of a catecholamine component or a fluorescent derivative thereof, such as a Xe lamp, a Xe flash lamp, a mercury lamp, an incandescent lamp, and a laser.

The excitation light condensing system and the fluorescence condensing system may be constituted by a condensing element such as a lens or a mirror and a spectroscopic element such as a filter or a diffracted photon. If necessary, an aperture for limiting the aperture ratio can be arranged in these light collection systems.

The measuring cell is, for example, a hollow rectangular parallelepiped or hollow cylindrical body made of optical glass, quartz glass, or the like, in which a hollow portion serves as a flow path of a sample to be analyzed. The excitation light condensing system and the fluorescence condensing system are arranged on the same axis or on an axis intersecting the measurement cell, but it is particularly preferable that the irradiation or extraction is performed so as to be orthogonal. If both have the same axis,
That is, when the light source and the detection element are arranged in the same direction, the excitation light optical path and the fluorescent light path may be separated using a dichroic mirror or the like.

As the detection element, for example, a photodiode, an avalanche photodiode, a photoelectric tube, a photomultiplier, or the like may be used. For high-sensitivity analysis, an avalanche photodiode or a photomultiplier is preferable. This is because an avalanche photodiode or a photomultiplier can obtain an amplification factor depending on the applied voltage. In order to correct the light intensity fluctuation of the light source, in addition to the detection element that measures the fluorescence intensity, an excitation light detection element that extracts part of the light from the excitation light condensing system and detects the excitation light transmitted through the measurement cell Can be arranged. As for these elements, one element can be used for both purposes, but they may be provided separately.

As the detection sensitivity switching means of the fluorescence detector included in the fluorescence detection device, for example, the intensity of the light source of the fluorescence detector is changed, or the intensity of the excitation light from the light source of the fluorescence detector is changed, or For changing the fluorescence intensity or for changing the sensitivity of the fluorescence detection element of the fluorescence detector. Here, the change includes increasing or decreasing the light source intensity or the like. Specifically, a sufficient light source intensity or the like is provided for the analysis of E or NE, and the concentration is higher than these. In the DA analysis, the light source light intensity is relatively attenuated as necessary, which means that the sample does not need to be diluted.

In order to change the intensity of the light source, for example, in the case of a Xe flash lamp, it is only necessary to switch capacitors having different capacities or to change the current flowing through the lamp by changing the charging voltage of the capacitors. For example, in the case of a Xe flash lamp or a mercury lamp, the current flowing through the lamp may be changed. For example, in the case of an incandescent lamp, the voltage applied to the lamp or the current flowing through the lamp may be changed.

In order to change the intensity of the excitation light from the light source or the intensity of the fluorescence from the sample, for example, a filter, a semi-permeable plate, an aperture, a slit, a mesh, etc. A light-shielding plate that transmits only part of light may be mechanically moved in and out. For example, the filter is not limited in spectral characteristics and the like as long as the filter has a characteristic capable of reducing excitation light or fluorescence. A liquid crystal filter whose transmittance can be changed electrically does not need to be mechanically moved into and out of the light collecting system, and is particularly preferable. In addition, the intensity of the excitation light or the fluorescence that irradiates the measurement cell or the detection element can be changed by changing the light collection characteristics of the excitation light focusing system or the fluorescence focusing system. That is, if the light-collecting characteristics are deteriorated, the irradiation range is widened, and the amount of light reaching the effective volume (effective area) of the measurement cell or the detection element can be reduced. For the aperture, slit, and mesh, not only can they be taken in and out, but also the aperture diameter can be changed and the slit width can be changed.

In order to change the sensitivity of the fluorescence detecting element, for example, in the case of a photomultiplier tube, the applied voltage may be changed to change the amplification factor. The amplification factor per stage of the photomultiplier tube is
Since the voltage is proportional to the power of 0.7 to 0.8, the voltage is proportional to the power of 7 to 8 in 10 stages. Therefore, when the applied voltage is reduced to 0.75 times, the amplification factor is reduced to 1/10. .

The control device, which is another feature of the device of the present invention, has a storage means for storing an identification signal for identifying a sample, a means for storing a time when the DA is eluted from the separation column, and a timing means. Although the function of reducing the detection sensitivity of the fluorescence detector while DA is detected by the fluorescence detector is provided, it may be configured using a computer, a microchip, or the like.

The storage means for storing an identification code for identifying the sample stores information for identifying the type of the sample, that is, whether the sample subjected to the analysis is a urine sample or a plasma sample. It is something to memorize. This information may be automatically input from, for example, a barcode attached to the sample, or may be input from a keyboard or the like. The storage unit may be configured to automatically save, for example, the analysis result and information on whether or not the intensity change operation of the light source or the like has been performed as described above.

The time measuring means is means for calculating the elapsed time from the injection of the sample into the analyzer, and the time measuring means in the computer can be used as it is as described above.

When the sample subjected to the analysis is a urine sample, the control device calculates the elapsed time from the injection of the sample into the device by a timer, and the DA fraction reaches the detector. The intensity of the light source and the like are changed at a certain timing. The time required for the DA fraction to reach the detector after sample injection varies depending on the separation column used, the liquid feeding speed, the length of the piping, etc., but in the same analyzer, urine samples, plasma samples, etc. Since they are the same, it can be exemplified that the control device is configured to be inputted from the outside, and the input value is compared with the time measurement result by the time measuring means. The control device is configured to issue a command to the fluorescence detection device to return the intensity of the light source of the detector to the initial state after the analysis of one sample is completed.

The apparatus of the present invention has various elements of a conventional liquid chromatography apparatus in addition to the characteristic apparatus described above. For example, a mobile phase container for holding a mobile phase, a sample injection device for injecting a sample, a liquid pump for liquid sending, a separation column, a waste liquid reservoir for discarding a sample after analysis, and the like, Can be exemplified. Each of these elements may be configured to be controlled by the control device described above, for example. For example, it is also possible to place the sample injection device under the control of the control device and to inject the sample in synchronization with the start of the timing means of the control device. As the separation column, a separation column conventionally used for catecholamine analysis, such as a reversed phase column, an ion exchange column, or an affinity column, can be used. Further, in addition to the separation column, a pretreatment column for concentrating or purifying the catecholamine component in the sample can be arranged upstream thereof. Furthermore, when a pre-column method or a post-column method is used in addition to the above-described components, a reactor can be used to cause a fluorescent derivatization reaction of catecholamine. As the reactor for the precolumn method, for example, a reactor integrally formed with a sample injection device as conventionally used can be exemplified. The integrated apparatus comprises a main body composed of a container for holding a sample and sample injection means such as a six-way valve, a reactor (reaction container), a container for holding a fluorescent reagent, and a means for adjusting the temperature of the reactor. Can be configured. This makes it possible to mix the sample and the fluorescent reagent, perform a fluorescence derivatization reaction at a constant temperature for a certain period of time, and then inject the sample. As a reactor for the post-label method, a hollow thin tube,
A capillary or column filled with inert particles such as glass can be used. This reactor may be connected downstream of the separation column, and may be mixed with a fluorescent reagent to react with the separated catecholamine.

[0024]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the present invention is not limited thereto.

FIG. 1 shows an outline of the apparatus of the present invention. In the figure, 1 is a light source, 2 is an excitation light focusing system, 3 is a measurement cell, 4 is a fluorescence focusing system, 5 is a fluorescence detection element, 6 is a filter for changing the intensity of excitation light from the light source, and 7 is the filter. Drive source for moving the light into and out of the optical path of the excitation light focusing system, 8
Is a power supply for driving the light source, 9 is a high-voltage power supply for generating a voltage to be applied to the fluorescence detection element, 10 is a fluorescence detector (fluorescence detection device), and 11 is a control device.

The light source is specifically a commercially available xenon flash lamp (manufactured by Hamamatsu Photonics), and the length of the bright line in the vertical direction is 3 mm and the width is 1 mm. The light source is moved in the horizontal direction by using a moving mechanism, and is fixed at a position where the signal is maximized.

The excitation light condensing system was specifically composed of two plano-convex lenses, an interference filter, and an aperture. The plano-convex lens has a diameter of 25 mm and the aperture is 24 mm x 16 m
m is an ellipse. Further, the interference filters are 330 to 35
It selectively transmits light having a wavelength of 0 nm.

The fluorescence condensing system was specifically composed of two plano-convex lenses, an interference filter, and an aperture. The plano-convex lens has a diameter of 20 mm and the aperture is 18 mm x 14 mm
Oval. Further, the interference filter is 460-500.
Since the light having a wavelength of nm is selectively transmitted, it is possible to prevent the excitation light reflected by the walls of the measurement cell from reaching the fluorescence detecting element.

The fluorescence detecting element is specifically a commercially available photomultiplier (manufactured by Hamamatsu Photonics), and a slit having a width of 1 mm is arranged on the front surface thereof so that scattered light from the measurement cell wall does not enter. did. The fluorescence detection element is moved in the horizontal direction by using a moving mechanism, and is fixed at a position where the fluorescence signal is maximized.

Specifically, a commercially available ND filter was used. The filter has a transmission characteristic of about 10%. The drive source for moving the filter into and out of the optical path of the excitation light focusing system is specifically a rotary solenoid, and the filter can be moved in and out by applying a voltage.

FIG. 2 is a diagram schematically showing a catecholamine analyzer by the post-column method. In the figure, 12 is a sample container, 13 is a sample injection device using a 6-way valve, 14 is a pretreatment column for removing impurities in the sample, 15 is a pretreatment column for concentrating catecholamine, 16
Is a separation column filled with an ion exchange gel, 17 is a reactor with a fluorescent reagent, and 18 is each device shown in FIG. Reference numeral 19 denotes a metering pump device (piston) for aspirating the sample stored in the sample container up to the loop, and reference numeral 20 denotes a loop arranged in the six-way valve for injecting a fixed amount of the sample. . In this figure, a liquid sending pump, a mobile phase container, a fluorescent reagent container and the like for liquid sending are not shown.

The apparatus shown in FIG. 2 and a commercially available analyzer (H
Catecholamine was analyzed using LC-725CA (manufactured by Tosoh Corporation). The catecholamine fraction in the sample was eluted from the separation column 16 at a flow rate of 1 ml / min. Thereafter, in the reactor 17, diphenylethylenediamine (manufactured by Tosoh Corporation) preheated to 90 ° C. is mixed while passing the solution at the above flow rate, and at 90 ° C. for about 3 hours at the same temperature.
The fluorescence derivatization reaction was performed for minutes. The reagents used were all dedicated reagents for the above-mentioned commercially available equipment, and the conditions for use were in accordance with the specified conditions.

The catecholamine fraction which has been fluorescently derivatized while passing the solution is subsequently passed through the measuring cell 3 of FIG.
Fluorescence was excited and fluorescence from the catecholamine fraction was detected. In the apparatus of the present invention, when the urine sample was used for measurement, the control device was configured so that the filter shown in FIG. 1 was inserted into the excitation light path at the timing when the DA fraction reached the detector. Specifically, the rotary solenoid was driven 19 minutes after the urine sample was injected.

When plasma was analyzed, E was 5 to 200 pg / m in both the commercially available apparatus and the analyzer of the present invention.
1, NE is 40-2000 pg / ml and DA is 10
Below 0 pg / ml, the ratio of the fluorescent signals was 1: 2: 1. Both devices have a detector dynamic range of 1000
Therefore, it was possible to analyze in a single operation without diluting the sample.

On the other hand, when the urine sample was analyzed, E was 1 to 200 ng / ml and NE was 10 to 600 ng / ml.
ng / ml and DA is 90-5000 ng / ml,
The ratio of the fluorescence signals was up to 1: 5000, exceeding the dynamic range of the detector. For this reason, in a commercially available device, the result as shown in FIG. 3 is obtained before the sample is diluted,
It was not possible to analyze for DA. On the other hand, in the apparatus of the present invention, before the DA analysis, the detection sensitivity of the detector is reduced to 1 by inserting an ND filter into the excitation light focusing system.
By reducing to / 10, it was possible to analyze all catecholamines at once without diluting the sample. The result is shown in FIG.

[0036]

According to the present invention, all catecholamines can be analyzed by a single operation even for a sample having a signal ratio exceeding the dynamic range of the detector by changing the sensitivity of the fluorescence detector according to the type of the sample. It is possible. For example, since a urine sample contains a higher concentration of DA than the concentrations of E and NE, it has conventionally been necessary to dilute the sample and analyze DA again after the analysis of E and NE. According to the present invention, not only the operation and time required for such sample dilution but also the time required for reanalysis can be saved.

[Brief description of the drawings]

FIG. 1 is a diagram showing an outline of a catecholamine analyzer of the present invention.

FIG. 2 is a diagram showing an outline of a catecholamine analyzer of the present invention.

FIG. 3 is a view showing the results of analyzing a urine sample without dilution using a commercially available catecholamine analyzer. In the figure, NE indicates norepinephrine, E indicates epinephrine, and DA indicates dopamine.

FIG. 4 is a view showing the results of analyzing a urine sample without dilution using a catecholamine analyzer of the present invention.
In the figure, NE indicates norepinephrine, E indicates epinephrine, and DA indicates dopamine. Arrows before and after the peak of DA decrease the detection sensitivity of the fluorescence detector to 1/10 (arrow a), and decrease the detection sensitivity after DA detection. The timing of the return is shown (arrow b).

[Description of sign]

 DESCRIPTION OF SYMBOLS 1 Light source 2 Excitation light condensing system 3 Measurement cell 4 Fluorescence condensing system 5 Fluorescence detection element 6 ND filter 7 Rotary solenoid 8 Power supply 9 High voltage power supply 10 Amplifier circuit 11 Controller 12 Sample container 13 Sample injection device 14 Pretreatment column (impurity 15) Pretreatment column (for concentration) 16 Separation column (ion exchange column) 17 Reactor 18 Fluorescence detector 19 Washing liquid injector 20 Loop

Claims (7)

[Claims] (1) at least epinephrine (E) in a sample,
A catecholamine analyzer for separating and analyzing norepinephrine (NE) and dopamine (DA) by a separation column, comprising a detection device including at least a fluorescence detector and a control device, wherein the fluorescence detection device is a detection sensitivity switching means of the fluorescence detector The control device has storage means for storing an identification signal for identifying the sample, means for storing the time when DA elutes from the separation column, and time counting means, and DA is detected by the fluorescence detector. A catecholamine analyzer having a function of reducing the detection sensitivity of a fluorescence detector during the measurement. 2. The analyzer according to claim 1, further comprising a fluorescent reagent introduction passage for fluorescently labeling the sample and a reactor for the sample and the fluorescent reagent, on the upstream side of the separation column. 3. The method according to claim 1, further comprising a fluorescent reagent introduction path for fluorescently labeling the sample and a reactor for the sample and the fluorescent reagent in a flow path from the column to the detector.
Analyzer. 4. The analyzer according to claim 1, wherein the sensitivity switching means of the fluorescence detector included in the fluorescence detector is means for changing the intensity of the light source of the fluorescence detector. 5. The analyzer according to claim 1, wherein the sensitivity switching means of the fluorescence detector included in the fluorescence detector is means for changing the intensity of the excitation light from the light source of the fluorescence detector. 6. The analyzer according to claim 1, wherein the sensitivity switching means of the fluorescence detector included in the fluorescence detector is means for changing the intensity of the fluorescence from the sample. 7. The analyzer according to claim 1, wherein the sensitivity switching means of the fluorescence detector included in the fluorescence detector is means for changing the sensitivity of the fluorescence detection element in the fluorescence detector.