JPH03252555A - Biological sample analysis method and analyzer - Google Patents

Abstract

PURPOSE:To allow measurement with high reproducibility and high sensitivity by using a pretreating column into which a hydrophilic gel introduced with phenyl boric acid is packed and utilizing this column for component adsorption and derivation reaction. CONSTITUTION:The hydrophilic gel 201 introduced with the phenyl boric acid 202 is packed as a fixing phase packing material into the pretreating column. Catecholamine 204 as the biochemical component included in a biosample is adsorbed and immobilized to the packing material when the biosample is introduced into this column. DEP (1, 2-diphenyl ethylenediamine) 205 is supplied as a reagent for derivation into the pretreating column in this state. The flow of the liquid is stopped and the catecholamine 204 is brought into reaction with the DPE 205 when the inside of the column is filled with this reagent. The adsorption is then released from the packing material and the liquid contg. the DPE is transferred to a separating column. The eluate is subjected to a chromatography sepn. according to the supply thereof and is detected by a detector.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a biological sample analysis method and an analysis device,
In particular, the present invention relates to an analytical method and an analytical apparatus for derivatizing a test component and then separating the components.

[Conventional technology]

Liquid chromatography separates components in solution and then
It is characterized by the ability to selectively analyze specific components, and there are many important analysis items in the field of clinical testing. However, on the other hand, many analytical methods are complex and take a long time to analyze, which hinders the increase in the number of samples analyzed in routine work operations such as clinical tests, where a large number of samples must be processed within a limited time. This is the cause of the slow spread of this type of automatic machines.

Analysis items for which application to routine work has been delayed include analysis of catecholamines and analysis of glycated hemoglobin. When these analytical items are separated and analyzed by liquid chromatography, the sample is generally reacted with a fluorescent labeling agent for derivatization, and then measured using a fluorometer. Two known labeling methods are the pre-labeling method, in which components are separated using a separation column and then reacted with a labeling agent, and the post-labeling method, in which components are separated using a separation column and then reacted with a labeling agent. High-sensitivity detection is difficult because of the large diffusion of components after column elution, and the pre-label method is advantageous when measuring trace components in biological samples.

Labeling catecholamines by pre-labeling method (
As prior art for derivatization) and chromatographic analysis, JP-A No. 61-88148 and JP-A No. 60-143
The methods described in No. 766 and JP-A-1-126544 are known.

Among these, in JP-A-61-88148, alumina is added to the sample to adsorb catecholamines on the alumina.
Catecholamines are derivatized by reacting with dansyl chloride during adsorption. Next, the derivatized sample is removed from the alumina, and the liquid is concentrated by evaporation. The sample thus prepared is injected into the channel of a liquid chromatograph, its components separated, and fluorescence detected.

In JP-A-60-143766, after a biological sample is injected into a flow path, it is reacted with a potassium ferricyanide solution in a first reaction section, then reacted with an ascorbic acid solution in a second reaction section, and then reacted with an ascorbic acid solution in a third reaction section. In the reaction section, catecholamine is converted to trihydroxyindole by reacting with sodium hydroxide solution. A configuration is shown in which the resulting solution containing trihydroxyindole is concentrated by being guided to a reverse phase column for concentration using an acidic buffer, and then the components are separated using a separation column for fluorescence analysis.

Furthermore, in JP-A-1-126544, a biological sample is introduced into an adsorption column filled with an adsorbent made of an ion exchange resin to adsorb catecholamines in an acidic state, and then a derivatization reagent is supplied to the adsorption column to remove catecholamines. It is derivatized in the adsorbed state. Next, the adsorption column is washed with a weakly acidic adjustment solution to discharge decomposed components, and then a neutral desorption solution is introduced into the adsorption column to release the catecholamine adsorption state and lead to the separation column, where the components are separated. It has been shown that fluorescence can be measured using

On the other hand, JP-A-55-51354 discloses that catecholamines are separated and analyzed by only concentrating them without pre-labeling them. In this prior art, the sample is injected into a concentration column to adsorb and concentrate catecholamines, and then
The adsorbed catecholamines are desorbed from the concentration column using an acidic buffer, the components are separated using a separation column, and the autofluorescence of the catecholamines is detected. This JP-A-5
No. 5-51354 uses a concentrating column only for concentrating catecholamines, and points out that glycol gel containing boric acid is preferred as the adsorption packing material.

[Problem to be solved by the invention]

Among the above-mentioned prior art, the method disclosed in Japanese Patent Application Laid-open No. 61-88148 takes time to prepare the sample to be injected into the channel of the liquid chromatograph, and is difficult to automate, making it difficult to perform clinical tests. It is not suitable for routine work such as

Furthermore, in the method disclosed in JP-A-60-143766, the labeling reaction is carried out while the sample is flowing through three types of reaction coils, resulting in a complex structure and poor reproducibility of analysis results. The method disclosed in JP-A-1-126544 has a difficulty in eliminating the influence of interfering components such as proteins in biological samples, and it is difficult to carry out the derivatization reaction with good reproducibility.

Furthermore, since JP-A-55-51354 measures the autofluorescence of catecholamines, it is difficult to perform high-sensitivity measurements.

In addition, pre-labeling has been frequently used in recent years as a method for increasing sensitivity, but side reactions may occur in addition to the desired reaction.
There are problems such as interference with the reaction due to coexisting components.

An object of the present invention is to reduce the decomposition of biochemical components to be tested and to obtain measurement results with high reproducibility even when the biochemical components to be tested are derivatized before being separated by chromatography. Another object of the present invention is to provide a biological sample analysis method and an analysis device that can perform highly sensitive measurement even if the content of biochemical components to be tested is minute.

[Means to solve the problem]

In the biological sample analysis method based on the present invention, a pretreatment column filled with a hydrophilic gel into which phenylboric acid has been introduced is used, and this pretreatment column is utilized for component adsorption and derivatization reaction. In the operation of this analysis method, a biological sample is supplied to a pretreatment column, and a biochemical component to be detected is adsorbed to the pretreatment column. filling the inside with a derivatization reagent solution, and reacting the derivatization reagent with the biochemical component to be detected in the pretreatment column while stopping the flow of the liquid in the pretreatment column, and causing the derivatization reagent to react with the derivatization reagent in the pretreatment column. deadsorbing the biochemical components to be tested from the packing material; transferring a liquid containing the derivatized biochemical components from the pretreatment column to the separation column; and transporting the derivatized biochemical components by the separation column. separating chemical components;
It is characterized by

A biological sample analysis device according to the present invention comprises a liquid chromatograph configured to analyze a biological sample,
It is configured to be able to execute the above analysis method.

[Effect]

The present invention is based on the invention that by adsorbing biochemical components, especially catecholamines, on a specific pretreatment column and then acting on a specific derivatization reagent, the biochemical components can be derivatized without much promotion of decomposition. It is based on what researchers have found.

The pretreatment column used in the present invention is filled with a hydrophilic gel containing phenylboric acid as a stationary phase packing material. The adsorption and derivatization reactions of biochemical components in this pretreatment column will be explained with reference to the schematic diagram in FIG. FIG. 3(a) shows the adsorption phenomenon, and FIG. 3(b) shows the derivatization reaction.

In FIG. 3(a), phenylboric acid 202 is introduced into a hydrophilic gel matrix 201. The liquid in the pretreatment column filled with such a packing material is made weakly alkaline (pH 8).
9) and introduce a biological sample containing biochemical components such as catecholamines, the catecholamines 204 are adsorbed by the filler. That is, the hydroxyl group of phenylboric acid 202 and the cis-diol (Cis -Di) of the catechol nucleus
al ) forms a chelate, thereby immobilizing catecholamines on the filler.

However, most of the proteins, amino acids and peptides in biological samples (e.g. serum, urine) do not have cis-diols and therefore cannot be adsorbed onto the packing material, so the pretreatment column is By flowing the pretreatment liquid for a predetermined period of time following the introduction of the biological sample, unadsorbed components can be discharged from the pretreatment column. That is, biochemical components having cis-diol are selectively trapped in the pretreatment column, and components that inhibit the derivatization reaction of the test component are removed from the pretreatment column.

Generally, biochemical components such as catecholamines decompose over time, but since the functional groups that are easily decomposed are combined with the filler during adsorption, the functional groups can be protected from decomposition.

After discharging reaction-interfering components from the pretreatment column, a derivatization reagent solution is supplied to the pretreatment column adsorbing biochemical components. In Figure 3(b), the fluorescent reagent 1,2-diphenylethylenediamine (DPE) is used as the derivatization reagent.
05 is an example.

Immediately after the pretreatment column is filled with the derivatization reagent solution, the flow of the solution in the pretreatment column is stopped by stopping the liquid pump or closing the flow path with a valve. This starts the derivatization reaction, but the liquid inside the pretreatment column at this time is neutral (pH 6.5 to 7.5).

The adsorbed catecholamine undergoes a condensation ring-closing reaction with DPE to become a derivatized product 206.

As a result of this reaction, catecholamines are released from the filler and released. Therefore, during the derivatization reaction, the liquid in the pretreatment column is kept from flowing out. The reason why the derivatized product 206 becomes free is thought to be that the stability constant of the bond between DPE and catecholamine is higher than the stability constant of adsorption of catecholamine to the packing material, but it is not clear that the pretreatment column By maintaining the temperature at 30 to 60°C, the derivatization reaction can be completed after a predetermined time (within 10 minutes).

Next, the pretreatment column and the separation column are communicated, and an eluent or pretreatment solution is fed to introduce the derivatized biochemical component into the separation column. The separation column is preferably filled with a reversed-phase packing material. The components chromatographically separated by the separation column as the eluent is supplied are detected by a detector (e.g. fluorescence detector).
[Example] Fluorescent labeling agents as derivatization reagents for catecholamines include, for example, 1,2-diphenylethylenediamine (
DPE) can be used, and the fluorescent labeling agent solution prepared includes 60 mM DPE.

Contains 2mM potassium ferricyanide, 40% acetonitrile, etc. The liquid nature of this derivatization reagent solution is neutral (pH 6.5 to 7.5). In addition, the eluent supplied to the separation column to separate catecholamines is
For example, acetonitrile, methanol and aqueous solution at 5:2:
A solution containing 4 parts is used. In this case, the aqueous solution has
Contains 0.1M sodium hydrogen phosphate and 10mM sodium dodecyl sulfate. The liquid nature of this eluent is acidic (for example, pH 3.0).

The pretreatment column employed in the analysis device to which the present invention is applied is filled with a filler made of hydrophilic gel into which phenylboric acid is introduced. The stationary phase packing material is preferably a polymer gel, but is not limited thereto, and may also be silica gel. Further, the separation column is filled with a reverse phase type packing material (for example, silica-0DS, etc.). The biochemical components to be tested, such as catecholamines, are adsorbed onto the pretreatment column.
In addition, a weakly basic (pH 8 to 9) buffer is used as the pretreatment liquid that is passed through the pretreatment column to discharge albumin, globulin, amino acids, and the like.

An example of an automatic catecholamine analyzer to which the present invention is applied will be described below with reference to the drawings.

FIG. 1 is a schematic diagram showing the overall configuration of an automatic catecholamine analyzer. This device has an autosampler 26 equipped with various containers and a dispensing mechanism, and a concentration/separation section 44 that performs sample concentration and separation operations within a flow path system. Hereinafter, the concentration/separation section 44 will be referred to as an analysis section for convenience.

A sample rack 27 is loaded onto the sample stage 28 of the autosampler 26 . The sample rack 27 is
It holds multiple sample containers containing plasma samples. The sample rack 27 also includes derivatization reagent containers 30 for fluorescent labeling. Internal standard solution container 31. A standard sample container 32 is arranged. In this autosampler 26, a mixing container 33, a nozzle cleaning tank 34. A drain port 35° and an injection port 36 are provided.

Dispensing nozzle 38 serves to transfer sample or reagent to injection port 36 . The drive mechanism 37 has x and y.

It has a driving function in the Z direction, and can freely drive the dispensing nozzle 38 vertically, horizontally, vertically, and up and down to move it to the position of the container or port on the sampler. The slider 5 holding the dispensing nozzle 38 provides a lateral movement. The upper end of the dispensing nozzle 38 is connected to a three-way valve 40 by a thin tube 39 such as a plastic tube.

The dispensing pump 41 and the six dispensing pumps 41 connected to the cleaning liquid tank 42 are syringe pumps driven by a pulse motor. Furthermore, a cooling device is incorporated in the sample stage 28 to keep the samples and reagents on the sample rack 27 at a low temperature during analysis.

The analysis section 44 includes a pretreatment column channel system for concentrating the sample and removing unnecessary substances such as proteins, a separation column channel system for separating sample components, and a measurement calculation section. In the pretreatment column flow path system, the pretreatment liquid in the pretreatment liquid tank 45 is fed at a constant flow rate by a pump 46 and flows into the pretreatment column 48 via a sample introduction valve 47 . Sample introduction valve 4
7 is provided with a measuring tube 49 for measuring a predetermined amount of the sample liquid injected from the injection port 36 of the autosampler and introducing it into the analysis section.

In the separation column flow path system, the eluent in the eluent tank 50 is pumped through the pump 5.
1 at a constant flow rate, and flows to a single separation column 53 via a flow path switching valve 52. Flow path switching valve 52
By switching, the eluent flows through the pretreatment column 48, and the liquid containing the biochemical components derivatized in the pretreatment column 48 is transferred to the separation column 53. The measurement calculation section includes a fluorometer 54 for measuring the fluorescence intensity of sample components eluted from the separation column 53, an A/D conversion section 55 for processing and displaying the measurement results, and a control section 56.
Printer57. The fluorometer 54 is composed of a CRT 58 or the like, and has a flow cell 59. The switching valves 60.61 are provided so that the liquid in the pumps 46.51 can be purged when necessary. The pretreatment column 48 is heated by the constant temperature device 43.
It is maintained at a predetermined temperature within the range of 0 to 60°C and is configured to perform a stable derivatization reaction.

FIG. 4 shows a control system diagram in the analyzer of FIG. 1. Analysis control is performed mainly by the control unit 56 based on input from the operation panel 77. First, when the power switch is turned on, the temperature control device 78 operates and the fluorometer 54
The light source lights up.

Next, when the analysis preparation switch is turned on, the pump 46 and the pump 51 start feeding liquid. After attaching the sample rack 27 to the autosampler 26, turn on the analysis start switch.
, autosampler 26, sample introduction valve 47. The flow path switching valve 52 starts operating, and an analysis operation is performed according to a predetermined analysis procedure. The measurement results are data processed by a signal from the fluorometer 54 in the control unit via the A/D converter 55, outputted to the printer 57, and displayed on the CRT 58.

Analytical operations using the analyzer shown in FIG. 1 include (1) collecting a biological sample into a dispensing nozzle on an autosampler;

Step of injecting the collected sample into the measuring tube, (2) Transferring the sample in the measuring tube to the pretreatment column with a buffer solution, adsorbing the biochemical components to be tested on the pretreatment column, and adding the buffer solution to the pretreatment column. (3) The derivatized biochemical components are separated in the separation column, and each The steps of measuring the components are carried out in this order.

These three steps are performed in parallel, and are performed sequentially on the sample introduced into the flow path at different timings in each step.

The analysis operation by the analyzer shown in FIG. 1 will be explained with reference to the flowchart shown in FIG.

Soil 2j 5-hole 1 When the analysis operation is started, a sample dispensing operation is performed using the dispensing nozzle 38 in step 101. The dispensing nozzle 38 is moved to the position of the sample container 29 to be analyzed next, and after sucking and holding a predetermined amount of sample into the dispensing nozzle, the nozzle 38 is moved to the position of the injection port 36. With the sample introduction valve 47 in the state shown in FIG. 1, the sample liquid is pushed out from the nozzle 38 and injected into the sample measuring tube 49. When it is desired to mix the internal standard solution with the sample, a mixing container is provided on the sample stage 28, and a predetermined amount of the sample and internal standard solution are once transferred into the mixing container using the dispensing nozzle 38 and mixed. Injection port 36 from the mixing vessel
The dispensing nozzle 38 is operated to inject the mixed liquid into the dispensing nozzle 38. Isoprotenol can be used as a standard substance.

Susu Goshiji In step 102, nozzle 38 is cleaned.

In this case, move the nozzle 38 to the drain port 35,
After discharging the cleaning liquid (usually pure water) to wash away the dirt on the inner wall of the nozzle caused by the sample, the nozzle is moved to the cleaning tank 34 and lowered into the tank, and the cleaning liquid is discharged to the nozzle 38.
Clean the outside of the tip.

The waste liquid is discharged from the drain 80.

In Step 103, the biological sample in the measuring tube 49 is supplied to the pretreatment column 48. When the sample introduction valve 47 is switched after the metering tube 49 is filled with sample liquid, the metering tube 49 connected to the port of the switching valve 47 is connected to the flow path 62 and the flow path 6.
3, and a weakly alkaline pretreatment liquid (pH 8,
7) A predetermined amount of sample liquid flows into the pretreatment column 4.
Transferred to 8. The size of the pretreatment column is 40m in inner diameter.
.

The length is 30cm.

In the protein production step 104, catecholamines in the biological sample are adsorbed and captured in the pretreatment column 48, and protein removal is performed. The pretreatment liquid from the pretreatment liquid tank 45 is transferred to the measuring tube 4.
Even after all the sample liquid in the pretreatment column 9 has been sent to the pretreatment column 48, the sample liquid is continued to be sent to the pretreatment column 48. The pretreatment liquid continues to flow for about 10 minutes. During this time, catecholamines are adsorbed and captured and accumulated and concentrated in the pretreatment column. At the same time, protein components and derivatization reaction inhibiting components that interfere with the measurement are discharged from the system through the drain 64 by the flow of the pretreatment liquid.

The pretreatment liquid is fed by the liquid feeding pump 46 at a flow rate of 1 mQ/win. The pretreatment solution consisted of a pH 8.7 buffer containing 0.2M diammonium hydrogen phosphate and 10mM EDTA. The phenylboric acid bonded to the filler matrix in the pretreatment column 48 is negatively charged in weakly alkaline liquids, and the cis-
Adsorption occurs when the diol forms a chelate. On the other hand, albumin, globulin, etc. are not adsorbed by the filler, so
The adsorbed catecholamines discharged from the pretreatment column 48 are purified. Such adsorption protects the functional groups of catecholamines.

In the K east partial pressure step 105, the derivatization reagent solution on the sample rack 27 is collected into the measuring tube 49. sample rack 27
In the reagent container 30 installed above, 60mM of 1,2
- A derivatization reagent solution containing diphenylethylenediamine, 2mM potassium ferricyanide, and 40% acetonitrile (CHsCN) is contained, and this reagent solution is adjusted to pH 7.0.

The dispensing nozzle 38 is moved above the reagent container 30 based on a control signal from the control unit 56, and is lowered into the reagent container 30, and the dispensing nozzle 38 is moved by the suction operation of the dispensing pump 41.
A predetermined amount of derivatization reagent solution is sucked and held in the chamber. Subsequently, the dispensing nozzle 38 is moved to the position of the injection port 36, and the extrusion operation of the dispensing pump 41 pushes out the reagent liquid held from the injection port 36 into the flow path system, so that it flows into the measuring tube 49 with a capacity of 200 μΩ. The state of the sample introduction valve 47 at this time when it is filled with the derivatization reagent solution is the state shown in FIG.

In step 106, a derivatization reaction of the biochemical component to be tested is performed in the pretreatment column 48. The pretreatment column 48 is
It is constantly maintained at 50° C. by a temperature-controlled constant temperature block 43. When the sample introduction valve is rotated by 60 degrees from the state shown in FIG.
The derivatization reagent liquid in the metering tube 49 is pushed out by the pretreatment liquid from 2 and introduced into the pretreatment column 48 via the flow path 63 and the switching valve 52. At the beginning of the introduction of the derivatization reagent solution, catecholamines remain adsorbed on the packing material in the pretreatment column 48. When the derivatization reagent liquid transfer operation is continued for 15 seconds at a flow rate of 1 mQ/in, the pretreatment column 48 is filled with the derivatization reagent liquid.

At the timing when the pretreatment column 48 is filled with the derivatization reagent solution (15 seconds after switching the valve 47), the control unit 5
The liquid feeding operation of the liquid feeding pump 46 is stopped by the control signal from 6, and this stopped state continues for 3 minutes. During this time, the catecholamines adsorbed on the filler are mixed with the DPE.
Derivatization is carried out by the reaction shown in Figure (b). 50℃
Under the conditions of 3 minutes, the adsorbed catecholamines are almost completely derivatized, and the state of adsorption with the filler is released. Since there are no proteins, SH compounds, etc. that inhibit the derivatization reaction in the pretreatment column 48, the reaction efficiency is extremely high.

In the sample to column step 107, a liquid containing derivatized biochemical components to be tested is transferred from the pretreatment column 48 to the separation column 53. When the liquid feeding pump 46 is brought back into the liquid feeding state after being stopped for 3 minutes, the pretreatment liquid pushes out the liquid containing the derivatized catecholamine in the pretreatment column 48 . Before this catecholamine-containing liquid reaches the flow path switching valve 52, the angle of the flow path switching valve 52 is rotated by 60 degrees to change the flow path connection state. As a result, the catecholamine-containing liquid is transferred to the separation column 53 by the eluent from the eluent tank 50.

In the bottom light separation step 108, the 0 separation column 53 in which components are separated by the separation column 53 is filled with silica-0DS (particle size: 3 μm), which is a reverse phase type packing material. This separation column 53 has an inner diameter of 4.6 mm and a length of 60 m. The eluent is a phosphate buffer (pH 3.0) containing acetonitrile and methanol.

When the liquid containing the derivatized components flowing out from the pretreatment column 48 reaches the separation column 53, the flow path switching valve 52 is switched by a control signal from the control unit 56 to the solid line state shown in FIG. . In this state, the eluent from the eluent tank 50 continues to flow through the separation column 53. By feeding this eluent, catecholamines are separated into components, such as norepinephrine (NE), epinephrine (E), and dopamine (
DA) forms a component band and is eluted from the separation column 53. After component separation, the separation column 53 is regenerated.

With the start of component separation in step 108, the flow path switching valve 5
2, the pretreatment liquid is passed through the pretreatment column 48. This is step 109. By passing this pretreatment liquid, the pretreatment column 48 is restored to a state in which it can receive the next biological sample. The time for step 109 is shorter than the time for step 108.

In step 110, the eluate from the separation column 53 flows into the flow cell 59 and is observed by the fluorometer 54. The fluorescence intensity of each derivatized component is measured,
The concentration is calculated from the area of each component peak. The excitation wavelength of the fluorometer is 345 nm and the fluorescence wavelength is 485 nm.

At the second left step 111, the data obtained at step 110 is sent to the printer 57. Output to CRT58, etc.

In the printer 57, the chromatogram figure and the numerical value of the concentration value of each component to be tested are displayed for each biological sample on print paper. A similar display can also be performed on the screen of the CRT 58. In step 112, it is determined whether all the samples on the autosampler 26 have been processed;
If there are still samples remaining, the process returns to step 101 to sample the next two samples. If all samples have been processed, the analysis operation of the apparatus ends.

In the flowchart of Fig. 2, for the sake of simplification of explanation,
Although it only shows the flow of analysis operations for one biological sample,
In the analyzer shown in FIG. 1, three biological samples are processed simultaneously. These samples are processed in a staggered manner so that the operational steps do not overlap. That is, if a particular sample is
During the processing at 8, the sample introduced one preceding this particular sample is separated by the separation column 53.

Meanwhile, the sample introduced one sample later than the specific sample is processed in the autosampler 26 and in the measuring tube 4.
9 is introduced.

For example, when performing the component separation step using the separation column 53 and the regeneration step of the separation column 53 for the first sample in 15 minutes, the second sample and the third sample are also processed in parallel. . In this case, the pretreatment column 48 performs regeneration with the pretreatment liquid, reception and adsorption of the second sample, reception and reaction of the derivatization reagent liquid, transfer of the derivatized component to the separation column, etc. . With respect to sampler 26 and metering tube 49 within the same 15 minute period,
Introducing the reagent solution for the second sample into the measuring tube 49, supplying the reagent solution to the pretreatment column 48, washing the inside of the measuring tube, collecting the third sample using the dispensing nozzle 38, and Introduction into the metering tube 49 is performed.

FIG. 5 is a chromatogram showing an example of analysis of a biological sample obtained in the example shown in FIG. In Figure 5,
NE is norepinephrine, E is epinephrine, DA is the catecholamine component of dopamine, and IP is isoprotenol added as an internal standard substance. FIG. 5 shows the results obtained when each of these components was fed together at 50 picograms (p g) to pretreatment column 48. Dopamine, which was previously considered difficult to measure with high sensitivity, has been detected with sufficient sensitivity to quantify it. The numerical values in FIG. 5 indicate retention times.

Next, experimental examples measured using the analysis method according to the present invention and the analysis method according to the prior art will be explained.

Experimental example Pooled plasma from healthy individuals was used as a biological sample.

The analysis procedure of the prior art was based on the analysis method using the pre-labeling method shown in JP-A-1-126544, but the same derivatization reagent solution as in the example of the present invention was used.

The above-mentioned pooled plasma was measured 10 times each by method C according to the embodiment of the present invention (method of this embodiment) and by a method according to the prior art (conventional method), and the reproducibility of the measurements was examined. Table 1 shows the results.
As shown in Table 1.

With regard to norepinephrine (NE) and dopamine (DA), the method of this example provided significantly superior standard deviation ratios (CV).

Table 1 Table 2 Next, norepinephrine (NE) was added to the pooled plasma described above.
, epinephrine (E), and dopamine (DA) at 50 pg/m each,
The recovery rates were determined by the method of this example and the conventional method. The results are shown in Table 2. As shown in Table 2, based on the method of this example, a higher recovery rate was obtained compared to the conventional method.

〔Effect of the invention〕

According to the present invention, there is little decomposition of the biochemical components to be tested during analysis operations, and the biochemical components to be tested can be derivatized with high efficiency, so that highly sensitive and accurate measurements can be performed.

[Brief explanation of drawings]

FIG. 1 is a schematic configuration diagram of an analyzer according to an embodiment of the present invention, FIG. 2 is a flowchart showing an operation procedure for explaining the analysis operation by the analyzer shown in FIG. 1, and FIG. 3 is a pre-processing diagram. A schematic diagram to explain the adsorption and derivatization reaction of biochemical components in the column, Figure 4 is a control system diagram of the analyzer shown in Figure 1, and Figure 5 is an analysis example obtained by the analyzer shown in Figure 1. This is a chromatogram showing. 26... Auto sampler, 29... Sample container, 30
... Derivatization reagent liquid container, 36 ... Injection boat, 3
8...Dispensing nozzle, 47...Sample introduction valve, 48...
・Pretreatment column, 49...Measuring tube, 52...Flow path switching valve, 53...Separation column, 54...Fluorometer,
56... Control part, 201... Hydrophilic gel base material, 20
2...Phenylboric acid. Fig. 2-3 ((b) I!ll conductorization 201 Fig. 0 TomiC 01 Fig. 7 5 4

Claims (1)

[Scope of Claims] 1. Supplying a biological sample to a pretreatment column filled with a hydrophilic gel containing phenylboric acid as a filler to adsorb a biochemical component to be tested; Filling the pretreatment column with a derivatization reagent solution while the biochemical components to be analyzed are adsorbed; reacting the component with a derivatization reagent, causing the biochemical component to be tested to be released from adsorption from the packing material as a result of the derivatization reaction, and transferring the liquid containing the derivatized biochemical component from the pretreatment column to the separation column. to transport;
and separating the derivatized biochemical components using the separation column. 2. In the analysis method according to claim 1, the liquid property when the biochemical component to be tested is adsorbed onto the pretreatment column is weakly alkaline, and the liquid property during the derivatization reaction is neutral. A biological sample analysis method characterized by: 3. The method for analyzing a biological sample according to claim 1, wherein the derivatization reagent solution is a solution containing 1,2-diphenylethylenediamine. 4. A first step of collecting a sample from inside the sample container with a dispensing nozzle and supplying the sample collected into the dispensing nozzle into the measuring tube connected to the flow path switching valve, and introducing phenylboric acid. The sample in the measuring tube is transferred with a buffer to a pretreatment column filled with hydrophilic gel as a filler to adsorb the biochemical components to be tested in the sample, and then the pretreatment column is derivatized. A second step of filling with a reagent solution and stopping the flow of the solution in the pretreatment column to perform a derivatization reaction, and separating the derivatized biochemical components received from the pretreatment column into the separation column. and a third step of detecting the specific sample, while the specific sample is undergoing the second step, the third step is performed on the sample preceding the specific sample, and the third step is performed on the sample preceding the specific sample, and A method for analyzing a biological sample, characterized in that the first step is performed on a sample subsequent to the sample. 5. A liquid chromatograph equipped with a pretreatment column that can adsorb biochemical components in a biological sample, a separation column that can separate the biochemical components, and a detector that detects fluorescence based on the components flowing out from the separation column. In the graph analyzer, a column packed with a hydrophilic gel into which phenylboric acid has been introduced is provided as the pretreatment column, a column packed with a reverse phase type packing material is provided as the separation column, and a derivatization reagent solution is provided in the pretreatment column. A liquid chromatography analyzer, characterized in that it is provided with means for supplying derivatization reagent solution and stopping the flow of the liquid in the pretreatment column when the pretreatment column is filled with the derivatization reagent liquid. 6. The liquid chromatography analyzer according to claim 5, further comprising means for keeping the pretreatment column at a temperature of 30 to 60°C. 7. The analyzer according to claim 5, further comprising means for introducing the biological sample into the measuring tube, and means for transporting the biological sample in the measuring tube to the pretreatment column by a flow of buffer solution. A liquid chromatography analyzer featuring: 8. Supplying the biological sample to a pretreatment column filled with a hydrophilic gel containing phenylboric acid as a filler, allowing biochemical components containing diols to be adsorbed to the filler, and pouring a buffer solution into the pretreatment column. filling the pretreatment column with a liquid containing a derivatization reagent capable of bonding with a functional group involved in the adsorption of the biochemical components; 1. A method for analyzing a biological sample, comprising derivatizing chemical components, and introducing the derivatized biochemical components in the pretreatment column to a separation column to separate the components. 9. The method for analyzing a biological sample according to claim 8, wherein the reaction for derivatizing the biochemical component is carried out while the flow of the liquid in the pretreatment column is stopped. 10. The method for analyzing a biological sample according to claim 8 or 9, wherein the biochemical component having a diol is a catecholamine, and the derivatization reagent is 1,2-diphenylethylenediamine. Analysis method. 11. The analysis method of claim 8 or 9, wherein the reaction for derivatizing the biochemical component is a ring-closing reaction between the biochemical component and the derivative reagent. Method.