JPH0769314B2 - Method and apparatus for analyzing biological sample - Google Patents

Description

TECHNICAL FIELD The present invention relates to a biological sample analysis method and analyzer,
In particular, the present invention relates to an analysis method and an analysis device for derivatizing a test component and then separating the components.

[Conventional technology]

Liquid chromatographs are used to separate 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 complicated and time-consuming to analyze, which will increase the number of analyzed samples in routine work, which requires processing many samples such as clinical tests within a limited time. This is a cause of the delay in the spread of this type of automatic machine.

Analytical items for which catecholamines and glycated hemoglobin have been lately applied to such routine works are listed. When these analytical items are separated and analyzed by liquid chromatography, it is common to react the sample with a fluorescent labeling agent for derivatization and measure with a fluorometer. As a labeling method, there are known a pre-labeling method of reacting with a labeling agent before separation of components by a separation column and a post-labeling method of separating components by a separation column and then reacting with a labeling agent. In that case, since the diffusion of the components after the elution with the separation column becomes large, it is difficult to perform highly sensitive detection, and the prelabeling method is advantageous when measuring trace components in a biological sample.

As prior art for labeling (derivatizing) catecholamines by a prelabeling method and performing chromatographic analysis, it is described in JP-A-61-88148, JP-A-60-143766 and JP-A-1-126544. There are known methods.

Among them, in JP-A-61-88148, alumina is added to a sample to adsorb catecholamine on alumina, and dansyl chloride is reacted during the adsorption to derivatize catecholamine. The derivatized sample is then desorbed from the alumina and the liquid is concentrated by evaporation. The sample thus prepared is injected into the flow channel of the liquid chromatograph to separate the components and detect fluorescence.

In Japanese Patent Application Laid-Open No. 60-143766, after injecting a biological sample into a channel, it is reacted with a potassium ferriciyanide solution in the first reaction section, then with an ascorbic acid solution in the second reaction section, and then with the third reaction section. Catecholamine is converted to trihydroxyindole by reacting with sodium hydroxide solution in the reaction part. A configuration is shown in which a liquid containing trihydroxyindole thus produced is guided to a concentration reverse-phase column for concentration with an acidic buffer and concentrated, and then the components are separated in a separation column to perform fluorescence analysis.

Further, in JP-A-1-126544, a biological sample is introduced into an adsorption column filled with an adsorbent made of an ion exchange resin, catecholamines are adsorbed in an acidic state, and then a derivatization reagent is supplied to the adsorption column to catecholamines. Derivatized in the adsorbed state. Next, the adsorption column is washed with a weakly acidic adjusting solution to discharge the decomposed components, and then a neutral desorption solution is introduced into the adsorption column to release the adsorbed state of catecholamine and guide it to the separation column for component separation. Fluorescence measurements have been shown.

On the other hand, Japanese Unexamined Patent Publication (Kokai) No. 55-51354 discloses that catecholamines are not pre-labeled but only concentrated to be separated and analyzed. In this prior art, after injecting a sample into a concentration column to adsorb and concentrate catecholamine, the adsorbed catecholamine is desorbed from the concentration column using an acidic buffer solution, and the components are separated in a separation column to cause autofluorescence of catecholamine. Is being detected. This Japanese Patent Laid-Open No. 55-5135
No. 4 uses a concentrating column only for concentrating catecholamines and points out that glycol gels containing boric acid as adsorbent packing are preferred.

[Problems to be Solved by the Invention]

Among the above-mentioned prior arts, the method according to Japanese Patent Laid-Open No. 61-88148 requires a long time for the operation for preparing the sample to be injected into the flow channel of the liquid chromatograph, and is difficult to automate. It is not suitable for such routine works.

Further, in the method according to JP-A-60-143766, the labeling reaction is carried out while the sample is passed through the three types of reaction coils, so that the structure is complicated and the reproducibility of the analysis result is not good. The method according to JP-A-1-126544 has a difficulty in eliminating the influence of interfering components such as proteins in a biological sample, and it is difficult to carry out the derivatization reaction with good reproducibility. Further, since JP-A-55-51354 measures autofluorescence of catecholamine, high sensitivity measurement is difficult.

In addition, pre-labels have been widely used in recent years as a method of increasing sensitivity, but side reactions may occur in addition to the intended reaction,
There is a problem with the interference of the reaction due to coexisting components.

An object of the present invention is to reduce the decomposition of a biochemical component to be tested and obtain a measurement result with high reproducibility even when the biochemical component to be tested is derivatized before chromatographic separation. An object of the present invention is to provide a method and an analyzer for analyzing a biological sample which can be measured with high sensitivity even when the content of a biochemical component to be tested is minute.

[Means for Solving the Problems]

In the method for analyzing a biological sample according to the present invention, a pretreatment column packed with a hydrophilic biogel into which phenylboric acid is introduced is used, and this pretreatment column is used for component adsorption and derivatization reaction. In the operation of this analysis method, a biological sample is supplied to a pretreatment column to adsorb a biochemical component to be detected, and the pretreatment column is adsorbed with the biochemical component to be detected in the pretreatment column. Filling the inside with the derivatization reagent solution, reacting the test biochemical component in the pretreatment column with the derivatization reagent in a state where the flow of the liquid in the pretreatment column is stopped, and with the derivatization reaction, Desorbing the test biochemical component from the filler,
A liquid containing a derivatized biochemical component is transferred from the pretreatment column to a separation column, and the derivatized biochemical component is separated by the separation column.

A biological sample analyzer according to the present invention comprises a liquid chromatograph configured to analyze a biological sample,
It is arranged to be able to carry out the analysis method described above.

[Action]

According to the present invention, when a biochemical component, particularly catecholamine, is adsorbed on a specific pretreatment column and a specific derivatizing reagent is allowed to act on the column, the biochemical component can be derivatized without being promoted so much. It is based on what they found.

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

In FIG. 3 (a), phenyl boric acid 202 is introduced into the hydrophilic gel matrix 201. Liquidity in the pretreatment column packed with such a packing material is weakly alkaline (pH 8-9)
When a biological sample containing a biochemical component such as catecholamine is introduced, the catecholamine 204 is adsorbed by the filler. That is, the catecholamine is immobilized on the filler by forming a chelate between the hydroxyl group of phenyl boric acid 202 and the cis-diol (Cis-Diol) of the catechol nucleus.

However, since most of proteins, amino acids and peptides in biological samples (eg serum, urine) do not have cis-diol, adsorption to the packing material does not occur. Therefore, a component that is not adsorbed can be discharged from the pretreatment column by flowing the weakly alkaline pretreatment liquid into the pretreatment column for a predetermined time following the introduction of the biological sample. That is, the biochemical component having cis-diol is selectively trapped in the pretreatment column, and the component that inhibits the derivatization reaction of the test component is removed from the pretreatment column. In general, biochemical components such as catecholamines undergo decomposition over time, but the functional groups that are easily decomposed are bonded to the filler during adsorption, and as a result, the functional groups can be protected from decomposition. .

After the reaction-interfering component is discharged from the pretreatment column, the derivatization reagent solution is supplied to the pretreatment column which adsorbs the biochemical component. In FIG. 3 (b), 1,2-diphenylethylenediamine (DPE) 205, which is a fluorescent reagent, is illustrated as a derivatizing reagent. When the pretreatment column is filled with the derivatization reagent solution, the flow of the solution in the pretreatment column is stopped by immediately stopping the liquid feed pump or closing the flow path with a valve. As a result, the derivatization reaction is started, and the liquid property in the pretreatment column at this time is neutral (pH 6.5 to 7.5).

The adsorbed catecholamine undergoes a condensation ring closure reaction with DPE to become a derivatized product 206. With this reaction,
Catecholamine is released from the filler by releasing the constraint. Therefore, keep the liquid in the pretreatment column from flowing out during the derivatization reaction. It is not clear that the derivatized product 206 becomes free because the stability constant of the bond between DPE and catecholamine is higher than the stability constant of the adsorption of catecholamine to the filler.
If the pretreatment column is maintained at a temperature of 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 with each other, and the eluent or the pretreatment liquid is sent to introduce the derivatized biochemical component into the separation column. As the separation column, one packed with a reverse phase type packing material is preferably used. The components chromatographically separated by the separation column with the supply of the eluent are detected by a detector (for example, a fluorescence detector).

〔Example〕

Fluorescent labeling agents as derivatizing reagents for catecholamines include, for example, 1,2-diphenylethylenediamine (DP
E) can be used, and the prepared fluorescent labeling agent solution contains 60 mM DPE, 2 mM potassium ferricyanide, 40% acetonitrile and the like. The liquid property of this derivatization reagent solution is neutral (pH 6.5 to 7.5). The eluent supplied to the separation column for separating the catecholamines is, for example, 5: 2: acetonitrile, methanol, and an aqueous solution.
A solution containing 4 is used. In this case, the aqueous solution
It contains 0.1 M sodium hydrogen phosphate and 10 mM sodium dodecyl sulfate. The liquid property of this eluent is acidic (for example, pH 3.0).

The pretreatment column employed in the analyzer to which the present invention is applied is filled with a packing material made of a hydrophilic gel into which phenylboric acid is introduced. The stationary phase filler is preferably a polymer gel, but is not limited to this and may be silica gel. Further, the separation column is packed with a reverse phase type packing material (for example, silica-ODS). The pretreatment liquid to be adsorbed to the biochemical component such as catecholamine to the pretreatment column and to be discharged to the pretreatment column in order to discharge albumin, globulin, amino acid, etc. is a weakly basic (pH 8-9) buffer. A liquid is used.

An example of a catecholamine automatic 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 apparatus has an autosampler 26 equipped with various containers and a dispensing mechanism, and a concentrating / separating unit 44 for concentrating and separating the sample in the flow path system. Below,
The concentration / separation unit 44 is referred to as an analysis unit for convenience.

The sample rack 27 is loaded on the sample stage 28 of the autosampler 26. The sample rack 27 holds a plurality of sample containers containing plasma samples. In addition, the sample rack 27 contains a derivatization reagent container for fluorescent labeling.
30, an internal standard solution container 31, and a standard sample container 32 are arranged. In this auto sample 26, the sample stage 28
A mixing container 33, a nozzle cleaning tank 34, a drain port 35, and an injection port 36 are provided at a fixed position close to.

The dispensing nozzle 38 functions to transfer the sample or reagent to the injection port 36. The drive mechanism 37 has a drive function in X, Y, and Z directions, and can drive the dispensing nozzle 38 vertically and horizontally to move it to the position of the container or port on the sampler. The slider 5 holding the dispensing nozzle 38 provides lateral movement. The upper end of the dispensing nozzle 38 is connected to a dispensing pump 41 and a cleaning liquid tank 42 via a three-way valve 40 by a thin tube 39 such as a plastic tube. The dispensing pump 41 uses a syringe pump driven by a pulse motor. A cooling device is incorporated in the sample stage 28 so that the samples and reagents on the sample rack 27 are kept at a low temperature during the analysis.

The analysis unit 44 includes a pretreatment column flow channel system for concentrating the sample and removing unwanted substances such as proteins, a separation column flow channel system for separating the sample components, and a measurement calculation unit. In the pretreatment column channel system, the pretreatment liquid in the pretreatment liquid tank 45 is pumped.
The solution is sent at a constant flow rate by 46 and flows into the pretreatment column 48 via the sample introduction valve 47. The sample introduction valve 47 is provided with a measuring pipe 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 sent by the pump 51 at a constant flow rate and flows into the single separation column 53 via the flow path switching valve 52. By switching the flow path switching valve 52, the eluent flows through the pretreatment column 48, and the liquid containing the biochemical component derivatized in the pretreatment column 48 is transferred to the separation column 53. The measurement calculation unit is a fluorometer that measures the fluorescence intensity of the sample components eluted from the separation column 53.
54 for calculating and displaying measurement results
The A / D conversion unit 55, the control unit 56, the printer 57, the CRT 58, and the like are included, and the fluorometer 54 has a flow cell 59. Switching valve 60,6
1 is provided so that the liquid in the pumps 46 and 51 can be purged when necessary. The pretreatment column 48 is 30 to 60 depending on the thermostat 43.
The temperature is maintained at a predetermined temperature within 0 ° C., and a stable derivatization reaction is performed.

FIG. 4 shows a control system diagram in the analyzer of FIG. Analysis control is performed by inputting from the operation panel 77.
It is performed around 56. First, when the power switch is turned on, the temperature control device 78 operates and the light source of the fluorescence photometer 54 is turned on. Next, when the switch for analysis preparation is turned on, the pump
46, Pump 51 starts liquid delivery. After mounting the sample rack 27 on the autosampler 26 and turning on the analysis start switch, the autosampler 26, the sample introduction valve 47, and the flow path switching valve 52
Starts to perform an analysis operation according to a predetermined analysis procedure. The measurement result is obtained by converting the signal from the fluorometer 54 into an A / D converter 5
The data is processed in the control unit via 5 and sent to the printer 57 and displayed on the CRT 58.

The analysis operation by the analyzer of FIG. 1 is (1) a step of collecting a biological sample in a dispensing nozzle on an autosampler and injecting the collected sample into a measuring tube, (2) a measuring tube in a pretreatment column The sample is transferred with a buffer solution, the biochemical components to be tested are adsorbed to the pretreatment column, and the buffer solution is flown through the pretreatment column to discharge proteins and the like,
After that, a step of supplying a derivatization reagent solution to the pretreatment column to derivatize the biochemical component, (3) a step of separating the derivatized biochemical component with a separation column, and measuring each component, in that order. Done.

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

The analysis operation by the analyzer of FIG. 1 will be described with reference to the flow chart of FIG.

When the sample weighing and analyzing operation is started, the sample dispensing operation by the dispensing nozzle 38 is performed 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 in 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 extruded from the nozzle 38 and injected into the sample measuring pipe 49. If you want to mix the internal standard solution with the sample, set the mixing container to the sample stage.
It is provided on the 28, a predetermined amount of sample and the internal standard solution is once moved into the mixing container by the dispensing nozzle 38 and mixed, and the mixed solution is fed from the mixing container to the injection port 36 by the dispensing nozzle 38. Operate to inject. Isoprotenol can be used as the standard substance.

Nozzle Cleaning In step 102, the nozzle 38 is cleaned. In this case, the nozzle 38 is moved to the drain port 35, and a cleaning liquid (usually pure water) is discharged to wash away dirt on the inner wall of the nozzle due to the sample, and then the nozzle is moved to the cleaning tank 34 and lowered into the tank. Then, the cleaning liquid is discharged to clean the outside of the tip of the nozzle 38. The waste liquid is discharged from the drain 80.

Introduction of Sample to Pretreatment Column At 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 measuring pipe 49 is filled with the sample liquid, the measuring pipe 49 connected to the port of the switching valve 47 is connected between the flow passage 62 and the flow passage 63,
A predetermined amount of the sample liquid is transferred to the pretreatment column 48 by the flow of the weak alkaline raw pretreatment liquid (pH 8.7). The pretreatment column has an inner diameter of 40 mm and a length of 30 mm.

Concentration / Deproteinization / Purification In step 104, catecholamine in the biological sample is adsorbed and captured in the pretreatment column 48 to deproteinize. The pretreatment liquid from the pretreatment liquid tank 45 is continuously sent to the pretreatment column 48 even after all the sample liquid in the measuring pipe 49 has been sent to the pretreatment column 48. The time for which the pretreatment liquid is kept flowing is about 10 minutes. During this period, catecholamines are adsorbed and captured, accumulated in the pretreatment column, and concentrated. At the same time, the protein component and the derivatization reaction inhibiting component that interfere with the measurement are discharged from the drain 64 to the outside of the system by the flow of the pretreatment liquid.

The pretreatment liquid is sent by the liquid sending pump 46 at a flow rate of 1 ml / min. The pretreatment solution consists of a buffer solution of pH 8.7 containing 0.2 M diammonium hydrogen phosphate and 10 mM EDTA. Phenyl boric acid bound to the filler matrix in the pretreatment column 48 is negatively charged in the weakly alkaline liquid state, and the hydroxyl group of boric acid and the cis-diol of the catechol nucleus form a chelate to cause adsorption. On the other hand, since albumin, globulin, etc. are not adsorbed by the packing material, they are discharged from the pretreatment column 48 and the adsorbed catecholamines are purified. The functional group of catecholamine is protected by such adsorption.

At the reagent dispensing step 105, the derivatization reagent solution on the sample rack 27 is collected in the measuring pipe 49. The reagent container 30 installed on the sample rack 27 contains a derivatization reagent solution containing 60 mM 1,2-diphenylethylenediamine, 2 mM potassium ferriciyanide, and 40% acetonitrile (CH ₃ CN). , This reagent solution is adjusted to pH 7.0.

The dispensing nozzle 38 is moved onto the reagent container 30 based on a control signal from the control unit 56, is lowered into the reagent container 30, and is sucked by the dispensing pump 41 so that a predetermined amount of the derivative is introduced into the dispensing nozzle 38. Inhalation and holding of the chemical reagent solution. Subsequently, the dispensing nozzle 38 is moved to the position of the injection port 36, and the reagent liquid held in the flow path system is pushed out from the injection port 36 by the pushing operation of the dispensing pump 41, and the inside of the measuring tube 49 having a volume of 200 μ is discharged. Fill with derivatization reagent solution. The state of the sample introduction valve 47 at this time is the state shown in FIG.

Derivatization Reaction In step 106, the derivatization reaction of the biochemical component to be tested is performed in the pretreatment column 48. The pretreatment column 48 is always kept at 50 ° C. by the temperature controlled constant temperature block 43. When the sample introduction valve is rotated by an angle of 60 degrees from the state shown in FIG. 1, the connection state of the flow channel is switched, and the pretreatment liquid from the flow channel 62 pushes out the derivatization reagent liquid in the measuring pipe 49,
It is introduced into the pretreatment column 48 via the flow path 63 and the switching valve 52. When starting the introduction of derivatization reagent solution, pretreatment column 48
Catecholamines are still adsorbed on the filler inside. When the transfer operation of the derivatization reagent solution is continued for 15 seconds at a flow rate of 1 ml / min, the pretreatment column 48 is filled with the derivatization reagent solution.

At the timing when the pretreatment column 48 is filled with the derivatization reagent solution (15 seconds after the switching of the valve 47), the liquid feed operation of the liquid feed pump 46 is stopped by the control signal from the control unit 56, and this stop is performed. The state is continued for 3 minutes. During this period, the catecholamine adsorbed on the filler reacts with DPE as shown in FIG. 3 (b), and is derivatized. The catecholamine adsorbed under the condition of 50 ° C. for 3 minutes is almost completely derivatized and the adsorbed state with the filler is released. Since there is no protein or SH compound that inhibits the derivatization reaction in the pretreatment column 48, the reaction efficiency is extremely high.

Sample Transfer to Separation Column At step 107, the liquid containing the derivatized test biochemical component is transferred from the pretreatment column 48 to the separation column 53. When the liquid-sending pump 46 is changed from the stopped state for 3 minutes to the liquid-sending state again, the pretreatment liquid pushes out the liquid containing the derivatized catecholamine in the pretreatment column 48. Before the liquid containing the catecholamine reaches the flow path switching valve 52, the flow path switching valve 52
Rotate the angle of 60 degrees to change the flow path connection state. As a result, the liquid containing catecholamine is transferred to the separation column 53 by the eluent from the eluent tank 50.

In the component separation step 108, the component separation by the separation column 53 is performed. The separation column 53 is filled with silica-ODS (particle size 3 μm) which is a reverse phase type packing material. The separation column 53 has an inner diameter of 4.6 mm and a length of 60 mm. The eluent is a phosphate buffer (pH 3.0) containing acetonitrile and methanol.

When the liquid containing the derivatized component flowing out from the pretreatment column 48 reaches the separation column 53, the flow path switching valve 52 is switched by the 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 is kept flowing into the separation column 53. The catecholamine is separated into components by feeding the eluent, and norepinephrine (N
E), epinephrine (E), and dopamine (DA) form component bands and are eluted from the separation column 53. After the component separation, the separation column 53 is regenerated.

Pretreatment column regeneration The pretreatment liquid is passed through the pretreatment column 48 by switching the flow path switching valve 52 with the start of the separation of the components of the step 108. This is step 109. By passing the pretreatment liquid, the pretreatment column 48 is restored to a state in which the next biological sample can be received. The time of step 109 is shorter than the time of step 108.

In the measuring 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, and 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.

Data display / output In step 111, the data obtained in step 110 is printed by the printer 5.
7, output to CRT58, etc. In the printer 57, a graphic of a chromatogram and numerical values of concentration values of each component to be tested are displayed on a print sheet for each biological sample. Similar display can be made on the screen of CRT58. In step 112, it is judged whether or not all the samples on the autosampler 26 have been processed, and if they still remain, the process returns to step 101 to sample two samples later. If all have been processed, the analysis operation of the device ends.

In the flow chart of FIG. 2, for simplification of description,
Only the flow of analytical operation for one biological sample is shown,
In the analyzer shown in FIG. 1, three biological samples are processed at the same time. These samples are processed with deviation so that the operating steps do not overlap. That is, a particular sample is pretreated in column 48.
The sample introduced one before this particular sample is separated by the separation column 53 while being treated with, and the sample introduced one behind the particular sample in the meantime is Processing in the autosampler 26 and introduction processing to the measuring pipe 49 are performed.

For example, when the component separation step by the separation column 53 and the regeneration step of the separation column 53 are performed for the first sample in 15 minutes, the second sample and the third sample are also processed in parallel. . In this case, regarding the pretreatment column 48,
Regeneration by 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. are performed. Within the same 15-minute period,
As for the sampler 26 and the measuring tube 49, the reagent solution for the second sample is introduced into the measuring tube 49, the reagent solution is supplied to the pretreatment column 48, the inside of the measuring tube is washed, and the third sample dispensing nozzle.
The sampling by 38, the introduction of the third sample into the measuring pipe 49, etc. are performed.

FIG. 5 is a chromatogram showing an analysis example of the biological sample obtained by the example of FIG. In FIG.
NE is norepinephrine, E is epinephrine, and DA is the catecholamine component of dopamine. IP is isoprotenol added as an internal standard. FIG. 5 shows the results obtained by supplying each of these components to the 50 picogram (pg) pretreatment column 48 together. Dopamine, which has been difficult to measure with high sensitivity in the past, is also detected with sufficient sensitivity that can be quantified. The numerical value in FIG. 5 shows the retention time.

Next, examples measured by the analysis method according to the present invention and the analysis method according to the related art will be described.

Experimental Example A pooled plasma of a healthy person was used as a biological sample. The analysis procedure of the prior art was based on the analysis method using the perlabeling method disclosed in JP-A-1-126544, but the same derivatization reagent solution as in the examples of the present invention was used.

By using the above-mentioned pooled plasma according to the method of the present invention (method of this example) and the method according to the prior art (conventional method)
The measurement was repeated 10 times and the reproducibility of the measurement was examined. The results are shown in Table 1. As shown in Table 1, the standard deviation rate (CV) obtained by the method of this example was remarkably excellent for norepnephrine (NE) and dopamine (DA).

Next, the above-mentioned pool plasma was added to norepinephrine (NE),
A solution containing epinephrine (E) and dopamine (DA) at 50 pg / ml was added, and the recoveries 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 as compared with the conventional method.

〔The invention's effect〕

According to the present invention, the biochemical components to be tested are less decomposed during the analysis operation, and the biochemical components to be tested can be derivatized with high efficiency. Therefore, highly sensitive measurement and accurate measurement can be performed.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of an analyzer according to an embodiment of the present invention, FIG. 2 is a flow chart showing an operation procedure for explaining an analysis operation by the analyzer of FIG. 1, and FIG. FIG. 4 is a schematic diagram for explaining adsorption and derivatization reaction of biochemical components in the treatment column, FIG. 4 is a control system diagram in the analyzer of FIG. 1, and FIG. 5 is obtained by the analyzer of FIG. 3 is a chromatogram showing an analysis example. 26 …… Autosampler, 29 …… Sample container, 30 …… Derivatization reagent solution container, 36 …… Injection port, 38 …… Dispensing nozzle,
47 …… Sample introduction valve, 48 …… Pretreatment column, 49 …… Measuring tube, 52 …… Flow path switching valve, 53 …… Separation column, 54 …… Fluorometer, 56 …… Control section, 201 …… Hydrophilic gel matrix, 202 ...
… Phenyl boric acid, 204 …… Catecholamine, 205 …… DP
E.

Claims (11)

[Claims] 1. A biological sample is supplied to a pretreatment column filled with a hydrophilic gel containing phenylboric acid as a packing material to adsorb a biochemical component to be inspected, and the biochemistry to be inspected in the pretreatment column. Filling the pretreatment column with a derivatization reagent solution in a state where the components are adsorbed, and the test biochemical components and derivatives in the pretreatment column in a state where the flow of the solution in the pretreatment column is stopped. Reacting a derivatization reagent, desorbing the biochemical component to be detected from the packing material along with the derivatization reaction, and transferring a liquid containing the derivatized biochemical component from the pretreatment column to the separation column And a method for analyzing a biological sample, wherein the derivatized biochemical component is separated by the separation column. 2. The analytical method according to claim 1, wherein the liquid property when the biochemical component to be tested is adsorbed on the pretreatment column is weak alkaline, and the liquid property during the derivatization reaction is medium. A method for analyzing a biological sample, which is characterized by being sexual. 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 a sample container by a dispensing nozzle and supplying the sample collected in the dispensing nozzle into a measuring pipe connected to a flow path switching valve; The sample in the measuring tube was transferred by a buffer solution to a pretreatment column filled with a hydrophilic gel introduced with boric acid as a packing material to adsorb the biochemical components to be detected in the sample, and then the pretreatment column. And a derivatization biochemical component received from the pretreatment column into the separation column, the second step of filling the liquid with the derivatization reagent liquid and stopping the flow of the liquid in the pretreatment column to carry out the derivatization reaction. And performing a third step on a sample preceding the specific sample while the specific sample is performing the second step. Subsequent trials of the above specific sample Method for analyzing a biological sample, characterized in that executing the first step to. 5. A pretreatment column capable of adsorbing a biochemical component in a biological sample, a separation column capable of separating the biochemical component into components, and a detector for detecting fluorescence based on the component flowing out from the separation column. In the liquid chromatograph analyzer, a column filled with a hydrophilic gel containing phenylboric acid is provided as the pretreatment column, a column filled with a reverse phase type packing material is provided as the separation column, and the pretreatment column is a derivative. A liquid chromatograph analysis apparatus, comprising means for supplying a derivatization reagent solution and for stopping the flow of the solution in the pretreatment column when the pretreatment column is filled with the derivatization reagent solution. 6. The liquid chromatographic 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 a buffer solution. A liquid chromatograph analyzer characterized by being provided. 8. A biological sample is supplied to a pretreatment column packed with a hydrophilic gel containing phenylboric acid as a packing material so that a biochemical component having a diol is adsorbed to the packing material, and the pretreatment column is buffered. A liquid is flown to discharge the component having no diol from the pretreatment column, and the pretreatment column is filled with a liquid containing a derivatizing reagent capable of binding to a functional group involved in adsorption of the biochemical component. A method for analyzing a biological sample, comprising derivatizing the biochemical component, and introducing the biochemical component derivatized in the pretreatment column to a separation column to separate the component. 9. The analysis method according to claim 8, wherein the reaction for derivatizing the biochemical component is carried out with the flow of the liquid in the pretreatment column stopped. Method. 10. The analytical method according to claim 8 or 9, wherein the biochemical component having the diol is catecholamine, and the derivatization reagent is 1,2-diphenylethylenediamine. Method for analyzing biological sample. 11. The method according to claim 8 or 9, wherein the reaction for derivatizing the biochemical component is a ring closure reaction between the biochemical component and the derivative reagent. Sample analysis method.