JP3823473B2 - Catecholamine analyzer - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a catecholamine analyzer for performing quantitative determination of epinephrine (E), norepinephrine (NE), or dopamine (DA) using the principle of liquid chromatography.
[0002]
[Prior art]
Usually, catecholamine is separated by liquid chromatography after pretreatment of a sample (blood, plasma, urine, etc.) with a deproteinized substance, and analyzed by an electrochemical detection method or a fluorescence detection method.
[0003]
The electrochemical detection method is capable of highly sensitive analysis, but detects all of the electrochemically active components, in other words, it cannot specifically detect catecholamines. Inappropriate.
[0004]
In addition to the method using natural fluorescence, there are methods that use a fluorescent reagent specific to a catechol skeleton or an amine in order to enhance fluorescence intensity and increase selectivity. That is, as a specific fluorescent reagent, for example, THI (Trihydroxyindol), ethylenediamine, OPA (OrtoPthal Aldehyde), fluorescamine, dansyl chloride, DPE (DiPhenylEthylenediamine), etc. are used, and a method of fluorescent derivatization of catecholamine before column separation (pre-column) Method), and a method of derivatizing catecholamine after column separation (post-column method) is known.
[0005]
[Problems to be solved by the invention]
Blood and urine contain catecholamines such as E, NE, DA, etc. All of these in one operation by using liquid chromatography and pre-column (derivatization) or post-column (derivatization) methods. However, urine is characterized by a higher concentration of DA than E and NE. It is not uncommon for many samples to have a difference of about 1000 times between the lower E concentration limit and the upper DA concentration limit.
[0006]
Thus, when analyzing E, NE, and DA in a single operation using liquid chromatography, the concentration of DA is high when the gain of the detector is set in consideration of the lower concentration limit of E or NE. In a sample derived from urine, the signal derived from DA exceeds the measurement limit, and an operation such as diluting the sample and performing an analysis operation again becomes necessary. However, if the gain of the detector is set to be suitable for a high concentration DA, there is a problem that the sensitivity is insufficient for low concentrations of N and NE.
[0007]
Therefore, the present invention changes the detector settings for, for example, 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 concentration difference between E or NE and DA occurs. An object of the present invention is to provide a catecholamine analyzer that does not require a sample to be diluted for analysis.
[0008]
[Means for Solving the Problems]
The present invention made in view of the above object is a catecholamine analyzer that separates and analyzes at least epinephrine (E), norepinephrine (NE), and dopamine (DA) in a sample by a separation column, and includes at least a fluorescence detector. An apparatus and a control apparatus, the fluorescence detection apparatus has a detection sensitivity switching means for the fluorescence detector, the control apparatus stores a storage means for storing an identification signal for identifying a sample, and a time for DA to elute from the separation column A catecholamine analyzer characterized by having a function of reducing the detection sensitivity of the fluorescence detector while DA is detected by the fluorescence detector. Hereinafter, the present invention will be described in detail.
[0009]
The present invention is an apparatus for derivatizing catecholamine from its natural fluorescence, for example, with THI, ethylenediamine, OPA, fluorescamine, dansyl chloride, DPE, etc., and detecting it with a fluorescence detector. When catecholamine is fluorescently derivatized, either a so-called precolumn method or a postcolumn method may be employed.
[0010]
The device of the present invention is characterized by including a detection device including a fluorescence detector and a control device, but may include other devices as will be described later. The fluorescence detection apparatus has a detection sensitivity switching means of the fluorescence detector. Usually, the fluorescence detector is composed of a light source, an excitation light condensing system, a measurement cell, a fluorescence condensing system, and a detection element. Each of these components can be the same as a normal fluorescence detector.
[0011]
The light source may be any 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.
[0012]
The excitation light condensing system and the fluorescence condensing system may be composed of 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 condensing systems.
[0013]
The measurement cell can be exemplified by a hollow rectangular parallelepiped or hollow cylindrical body made of, for example, optical glass or quartz glass, and the hollow portion serving as a flow path of the 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 with the measurement cell, but it is preferable that the excitation light condensing system and the fluorescence condensing system are particularly irradiated or extracted so as to be orthogonal. When both are on the same axis, that is, when the light source and the detection element are arranged in the same direction, the excitation light path and the fluorescence light path may be separated using a dichroic mirror or the like.
[0014]
For example, a photodiode, an avalanche photodiode, a photoelectric tube, a photomultiplier tube, or the like may be used as the detection element, but an avalanche photodiode or a photomultiplier tube is preferable for performing highly sensitive analysis. This is because an avalanche photodiode or photomultiplier tube 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 a part of the light from the excitation light condensing system or detects the excitation light transmitted through the measurement cell Can be arranged. As these elements, one element can be used for both purposes, but may be provided separately.
[0015]
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, the intensity of excitation light from the light source of the fluorescence detector is changed, or the fluorescence intensity from the sample is changed. Or changing the sensitivity of the fluorescence detection element of the fluorescence detector. Here, the change includes increasing / decreasing the light source intensity or the like. Specifically, the light source intensity or the like sufficient for the analysis of E or NE is given, and the concentration is higher than these. When analyzing DA, it means that the light source light intensity is relatively attenuated as necessary, so that it is not necessary to dilute the sample.
[0016]
In order to change the intensity of the light source, for example, in the case of an Xe flash lamp, a capacitor having a different capacity may be switched, or a current flowing through the lamp may be changed by changing a charging voltage of the capacitor. For example, in the case of a Xe flash lamp or a mercury lamp, the current passed 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.
[0017]
In order to change the intensity of excitation light from the light source or change the intensity of fluorescence from the sample, for example, a filter, a semi-transparent plate, an aperture, a slit, a mesh, etc. What is necessary is just to mechanically put in and out the light-shielding plate that transmits only the part. For example, as long as the filter has a characteristic capable of reducing excitation light or fluorescence, there is no limitation on the spectral characteristic or the like. A liquid crystal filter that can electrically change the transmittance is particularly preferable because it does not need to be mechanically taken in and out of the light collecting system. In addition, the intensity of the excitation light or fluorescence that irradiates the measurement cell or the detection element can be changed by changing the light collection characteristics of the excitation light collection system or the fluorescence collection system. That is, if the light condensing characteristic is deteriorated, the irradiation range is expanded, and the amount of light reaching the effective volume (effective area) of the measurement cell or the detection element can be reduced. About an aperture, a slit, and a mesh, not only these can be taken in and out, but the opening diameter can be changed and the slit width can be changed.
[0018]
In order to change the sensitivity of the fluorescence detection element, for example, in the case of a photomultiplier tube, the applied voltage may be changed to change the amplification factor. Since the amplification factor per stage of the photomultiplier tube is proportional to the 0.7 to 0.8th power of the voltage, the applied voltage is 0.75 times because the 10th stage is proportional to the 7th to 8th power of the voltage. When it is reduced to 1, the amplification factor decreases to 1/10.
[0019]
The control device, which is another feature of the apparatus of the present invention, has storage means for storing an identification signal for identifying a sample, means for storing the time for DA to elute from the separation column, and timing means, While DA is detected by the fluorescence detector, it has a function of reducing the detection sensitivity of the fluorescence detector, but may be configured using a computer, a microchip, or the like.
[0020]
The storage means for storing an identification symbol for identifying a sample stores information for identifying the type of the sample, that is, whether the sample subjected to analysis is a urine sample or a plasma sample. It is. This information may be input automatically from, for example, a barcode attached with a sample, or may be input from a keyboard or the like. Note that the storage means may be configured to automatically save information relating to, for example, analysis results and whether or not an intensity change operation such as the light source described above has been performed.
[0021]
The time measuring means is a means for calculating the elapsed time from the injection of the sample into the analyzer, and as described above, the time measuring means in the computer can be used as it is.
[0022]
When the sample subjected to analysis is a urine sample, the control device calculates the elapsed time from the injection of the sample into the device by means of time measuring means, and determines the timing when the DA fraction reaches the detector. Change the intensity of the light source. The time taken for the DA fraction to reach the detector after sample injection varies depending on the separation column used, the liquid feed speed, the length of the pipe, etc., but in the same analyzer, urine samples, plasma samples, etc. Since they are the same, it can be exemplified that the controller is configured to input 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 for returning the intensity of the light source of the detector to the initial state to the fluorescence detection device after the analysis of one sample is completed.
[0023]
The apparatus of the present invention has various elements of a normal 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 transfer, a separation column, a waste liquid reservoir for discarding a sample after analysis, etc. The piping which connects can be illustrated. Each of these elements may be configured to be controlled by the above-described control device, for example. For example, it is 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 reverse phase column, an ion exchange column, or an affinity column, can be used. In addition to the separation column, a pretreatment column for concentrating or purifying the catecholamine component in the sample can be disposed upstream of the separation column. Furthermore, in addition to the above-described elements, when a precolumn method or a postcolumn method is used, a reactor can be used to cause a fluorescent derivatization reaction of catecholamine. As the reactor for the precolumn method, for example, a reactor integrated with a sample injection device such as conventionally used can be exemplified. The integrated apparatus includes a main body constituted by a sample holding means such as a container for holding a sample and a six-way valve, a reactor (reaction vessel), a container for holding a fluorescent reagent, a temperature control means for the reactor, and the like. Can be configured. This makes it possible to inject the sample after mixing the sample and the fluorescent reagent and performing a fluorescence derivatization reaction at a constant temperature for a fixed time. As the reactor for the post-label method, a hollow capillary, a capillary or a column filled with inert particles such as glass can be used. This reactor may be connected downstream of the separation column and reacted with the catecholamine separated by mixing a fluorescent reagent.
[0024]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, examples and the like will be described to describe the present invention in more detail, but the present invention is not limited to these.
[0025]
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 condensing system, 3 is a measurement cell, 4 is a fluorescence condensing system, 5 is a fluorescence detecting element, 6 is a filter for changing the intensity of excitation light from the light source, and 7 is the filter. Is a drive source for taking the light into and out of the optical path of the excitation light condensing system, 8 is a power source for driving the light source, 9 is a high voltage power source for generating a voltage to be applied to the fluorescence detection element, and 10 is a fluorescence detector (fluorescence detection device). , 11 is a control device.
[0026]
The light source is specifically a commercially available xenon flash lamp (manufactured by Hamamatsu Photonics), and the vertical length of the bright line is 3 mm and the width is 1 mm. The light source is moved in the horizontal direction using a moving mechanism and fixed at a position where the signal becomes the largest.
[0027]
Specifically, the excitation light condensing system was 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 has an elliptical shape of 24 mm × 16 mm. The interference filter selectively transmits light having a wavelength of 330 to 350 nm.
[0028]
Specifically, the fluorescence condensing system is 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 has an elliptical shape of 18 mm × 14 mm. The interference filter selectively transmits light having a wavelength of 460 to 500 nm, and can prevent the excitation light reflected by the wall of the measurement cell from reaching the fluorescence detection element.
[0029]
Specifically, the fluorescence detection element is 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. The fluorescence detection element is moved in the horizontal direction using a moving mechanism, and is fixed at a position where the fluorescence signal becomes the largest.
[0030]
Specifically, a commercially available ND filter was used as the filter. The filter has a transmission characteristic of about 10%. The drive source for putting the filter in and out of the optical path of the excitation light collecting system is specifically a rotary solenoid, and the filter can be put in and out by applying a voltage.
[0031]
FIG. 2 is a diagram showing an outline of a catecholamine analyzer by a 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 catecholamines, and 16 is an ion. A separation column filled with exchange gel, 17 is a reactor with a fluorescent reagent, and 18 is each apparatus shown in FIG. Further, 19 is a metering pump device (piston) for sucking the sample stored in the sample container up to the loop, and 20 is a loop for injecting a fixed amount of sample arranged in the 6-way valve. . In the drawing, a liquid feeding pump, a mobile phase container, a fluorescent reagent container, etc. for liquid feeding are not shown.
[0032]
Catecholamines were analyzed using the apparatus shown in FIG. 2 and a commercially available analyzer (HLC-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, while flowing at the flow rate, the mixture was mixed with diphenylethylenediamine (manufactured by Tosoh Corporation) preheated to 90 ° C., and the fluorescence derivatization reaction was performed at 90 ° C. for about 3 minutes at the same temperature. went. All the reagents used were dedicated reagents for the above-mentioned commercially available apparatus, and the use conditions were in accordance with the specified conditions.
[0033]
The catecholamine fraction that was fluorescently derivatized while passing through the liquid was subsequently passed through the measurement cell 3 of FIG. 1 to excite the fluorescence and detect the fluorescence from the catecholamine fraction. In the apparatus of the present invention, when the urine sample was subjected to measurement, the control apparatus was configured so that the filter shown in FIG. 1 was put into the excitation light optical 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.
[0034]
When plasma was analyzed, both the commercially available apparatus and the analyzer of the present invention had an E of 5-200 pg / ml, NE of 40-2000 pg / ml and DA of 100 pg / ml or less, and a fluorescence signal ratio of 1: 2: 1. In both devices, the dynamic range of the detector was 1000, so it was possible to analyze in one operation without diluting the sample.
[0035]
On the other hand, when an analysis is performed on a urine sample, E is 1 to 200 ng / ml, NE is 10 to 600 ng / ml, DA is 90 to 5000 ng / ml, and the ratio of fluorescence signals is 1: 5000 at maximum, The dynamic range of the detector was exceeded. For this reason, in a commercially available apparatus, the result shown in FIG. 3 was obtained before the sample was diluted, and DA could not be analyzed. On the other hand, in the apparatus of the present invention, prior to the analysis of DA, the detection sensitivity of the detector is reduced to 1/10 by putting the ND filter in the excitation light condensing system, so that the entire sample is not diluted. It was possible to analyze catecholamines at once. The result is shown in FIG.
[0036]
【The invention's effect】
According to the present invention, by changing the sensitivity of the fluorescence detector depending on the type of sample, it is possible to analyze all catecholamines in a single operation even for a sample showing a signal ratio exceeding the dynamic range of the detector. . For example, since a urine sample contains a high concentration of DA compared to the concentrations of E and NE, conventionally, after analysis of E and NE, it was sometimes necessary to dilute the sample and analyze DA again. 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 diagram showing the result of analyzing a urine sample without diluting with a commercially available catecholamine analyzer. In the figure, NE represents norepinephrine, E represents epinephrine, and DA represents dopamine.
FIG. 4 is a diagram showing the result of analyzing a urine sample without diluting with the catecholamine analyzer of the present invention. In the figure, NE represents norepinephrine, E represents epinephrine, and DA represents dopamine. The arrows before and after the DA peak decrease the detection sensitivity of the fluorescence detector to 1/10 (arrow a). The return timing is indicated (arrow b).
[Explanation of symbols]
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 Amplifying circuit 11 Control device 12 Sample container 13 Sample injection device 14 Pretreatment column (impurity For removal)
15 Pretreatment column (for concentration)
16 Separation column (ion exchange column)
17 Reactor 18 Fluorescence detector 19 Washing liquid injector 20 Loop

Claims (7)

Epinephrine at least in the sample (E), in norepinephrine (NE) and dopamine (DA) catecholamine analyzer that separates analyzed by separation column, comprising a detection device and a control device comprising at least a fluorescent detector, detection device The control device has a detection sensitivity switching means for the fluorescence detector, and the control device has a storage means for storing an identification signal for identifying the type of the sample, a means for storing the time when the DA is eluted from the separation column, and a time measuring means. In addition, the catecholamine analyzer has a function of reducing the detection sensitivity of the fluorescence detector while DA is detected by the fluorescence detector depending on the type of the sample . The analyzer according to claim 1, further comprising a fluorescent reagent introduction path for fluorescently labeling the sample and a sample / fluorescent reagent reactor on the upstream side of the separation column. 2. The analyzer according to claim 1, further comprising a fluorescent reagent introduction path for fluorescently labeling the sample and a sample / fluorescent reagent reactor in a flow path from the column to the detector. 2. The analyzer according to claim 1, wherein the sensitivity switching means of the fluorescence detector included in the detection device is means for changing the intensity of the light source of the fluorescence detector. 2. The analyzer according to claim 1, wherein the sensitivity switching means of the fluorescence detector included in the detection device is a means for changing the intensity of excitation light from the light source of the fluorescence detector. 2. The analysis apparatus according to claim 1, wherein the sensitivity switching means of the fluorescence detector included in the detection apparatus is a means for changing the fluorescence intensity from the sample. Sensitivity switching means of the fluorescence detector detecting device has found analyzer according to claim 1, which is a means for changing the sensitivity of the fluorescence detection device in the fluorescence detector.

Images