JPH04204054A - Analysis device of catechol amine metabolic object and creatinine - Google Patents

Abstract

PURPOSE:To enable catechol amine metabolic object and creatinine to be analyzed with one sample-pouring operation by connecting two sample-pouring loops, introducing same samples over both loops, and then separating both loops for flowing to each corresponding analysis part. CONSTITUTION:Connected 6-way valve 3 and 16-way valve 4 are switched synchronously when pouring a sample, an eluting solution 12 flows in the order of a pump 1, the valve 3, a column 5, and a detector 8, and a carrier liquid 13 flows in the order of a pump 2, the valve 4, a reaction coil 6, and a detector 9. On the other hand, samples fill a sample loop within the valve 3 and then that within the valve 4. At the same time, a reagent is fed to a reagent loop within the valve 4 by an accessory pump. pouring operation is completed, and then both valves are switched to analysis state, thus enabling catechol amine metabolic object and creatinine to be analyzed with one sample pouring and preventing erroneous replacement of the reagent.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an analyzer capable of simultaneously analyzing and measuring catecholamine metabolites and creatinine in a biological sample, such as urine.

[Prior Art] Conventional techniques for analyzing catecholamine metabolites in urine include, for example, "mass screening for neuroblastoma using high-performance liquid chromatography."

Preventive Medicine Journal, No. 209, pp. 26-32 (1986)'' is known. In this conventional technique, catecholamine metabolites are measured by liquid chromatography, and creatinine is measured by a microplate reader. There is.

[Problems to be Solved by the Invention] In the above-mentioned conventional technology, sampling is performed at least twice, the sample detected in the first sampling is injected into a liquid chromatograph, and the sample detected in the second sampling is injected into a liquid chromatograph. is placed in a separate location and distributed to a microplate reader. According to this conventional technique, the sampling operation is complicated and there is a high risk of sample mix-up.

An object of the present invention is to provide an analyzer that can properly analyze and measure both catecholamine metabolites and creatinine with a single sample injection operation.

[Means for Solving the Problems] The present invention provides a catecholamine metabolite analysis section comprising a liquid chromatograph having a first sample injection loop and a separation column, and a creatinine analysis section having a second sample injection loop and a reaction coil. The flow reaction analysis section communicates with a first sample injection loop and a second sample injection loop at the time of sample injection, and then independently communicates the first sample injection loop with the separation column and connects the second sample loop with the second sample injection loop. It is characterized by being equipped with a valve system that communicates independently with the reaction coil.

[Function] In the present invention, catecholamine metabolites are detected using a liquid chromatograph, and creatinine is detected using a flow reaction analysis section (flow injection analysis section). During sample injection, the first sample injection loop and the second sample injection loop are connected in series, and the same sample is introduced across both loops. Next, the valve system is switched to separate both loops and allow the sample introduced into each loop to flow to the corresponding analysis section. Thereby, samples for multiple types of analysis sections can be collected with a single sample injection operation.

[Example] Urinary creatinine is measured by a flow injection method using the Jaffe reaction.

That is, creatinine reacts with picric acid in an alkaline solution to produce an orange-red substance.

A carrier liquid is continuously fed into the tube, and a sample and a reaction reagent (picric acid) are injected into the flowing liquid. The sample and reagent flow through the tube while undergoing a chemical reaction, and the target component is detected and quantified by a detector.

Urinary catecholamine metabolites are analyzed by liquid chromatography as in the conventional method.

Sample injection is performed using an autosampler for liquid chromatography. Since the two sample loops are connected in series by a valve system, both sample loops can be filled with sample in one injection operation. In a preferred embodiment, when the sample-reaction liquid valve is in the injection state, the reaction liquid is pumped using a straining pump to fill the reagent loop with the reaction liquid. After the sample and reaction solution injection operations are completed, switch the sampling valve system to return to the analysis state. This results in
Samples can be injected into the diagonal channels in a single injection operation, making simultaneous analysis possible.

An embodiment of the present invention will be described below with reference to FIGS. 1 to 3. FIG. 1 is a schematic diagram of the flow path of this analyzer. 1.2
is a liquid sending pump, 3 is a six-way bell, 4 is a sixteen-way valve for sample and reaction solution injection, 5 is a separation column, 6 is a reaction coil, 7 is a column oven, 8 is an electrochemical detector or fluorescence detector, 9 is an ultraviolet-visible detector, 10 is a glomato data processing device, 12 is an eluent for analyzing catecholamine metabolites, 13 is a carrier liquid, and 14 is a host computer.

Analysis of catecholamine metabolites is performed by the following method. The eluent 12 is pumped by a pump 1, and a sample is introduced from a six-way valve 3 by an autosampler, separated by a column 5, detected by a detector 8, and identified and quantified by a data processing device 10.

For the measurement of creatinine, the carrier liquid 13 is pumped by the pump 2, and the sample and reaction reagent are introduced into the channel through the 16-way valve 4. A sample and a reaction reagent are reacted in a reaction coil 6, detected by a detector 9, and identified and quantified by a data processing device 10.

Samples for quantifying catecholamine metabolites and creatinine are simultaneously introduced from the autosampler 1 and analyzed simultaneously.

The measurement results of catecholamine metabolites and creatinine are
The result is sent to the host computer 14, and after correcting the metabolite concentration using the creatinine value, the result is output. A flowchart of the analysis is shown in FIG.

FIG. 2 shows details of the connection between the six-way valve and the sixteen-way valve in FIG. 1. FIG. 2(A) shows the state at the time of analysis, and FIG. 2(B) shows the state at the time of sample injection. 51 is an injection boat attached to the six-way valve 3;
53 is a straining pump for feeding a reaction reagent, and 54 is a reaction reagent. 6o is a sample loop for catecholamine metabolite analysis, 61A.

61B is a reagent loop for measuring creatinine, and 62 is a sample loop for measuring creatinine. ■■■■■■ indicates the position of the six-way valve 3. ■■ of the six-way valve 3 and the inlet side of the sample loop 62 of the sixteen-way valve 4 are connected in advance.

During analysis, the liquid flows as follows. The eluent 12 is pumped
The liquid is sent from ■→■→sample loop 6 inside the six-way valve 3.
It flows in the order of 0→■→■, passes through the column 5, and is guided to the detector 8. On the other hand, the carrier liquid 13 sent by the pump 2 flows through the 16-way valve 4 from the reagent loop 61A to the sample loop 62.
It flows in the order of reagent loop 61B, passes through reaction coil 6, and flows to detector 9.

When injecting a sample, the six-way valve 3 and the sixteen-way valve 4 are set as shown in Figure 2 (
They can be switched synchronously as shown in B). At this time, the eluent 12 is pump 1 → six-way valve 3 - column 5 → detector 8
The carrier liquid 13 flows in the order of pump 2 - 16-way valve 4 (passing through) -> reaction coil 6 -> detector 9. A fixed amount of sample is aspirated from the sample bottle by the autosampler and discharged by the injection boat 51. - The sample flows through the six-way valve 3 in the order ■-■→sample loop-■→■, and then fills the sample loop 62 of the sixteen-way valve 4 connected in series. Loop the sample 60.6
At the same time as filling the reagent 54 into the reagent loop 61A, the straining pump 53 is operated and the reagent 54 is filled into the reagent loop 61A.

Fill within 61B. After the injection operation is completed, both valves 3 and 4 are switched back to the state shown in FIG. 2(A). The eluent pushes out the sample in the sample loop and introduces it into column 5 to separate catecholamine metabolites. On the other hand, the carrier liquid 13 sends the reagent and sample in the 16-way valve 4 to the reaction coil 6, and creatinine is determined colorimetrically.

FIG. 3 shows an example of analysis of standard samples of vanylmandelic acid (VMA) and small mobanilic acid (HVA), which are catecholamine metabolites. As an eluent, 5% acetonitrile,
95% 50mM tartrate buffer, 0.01% EDTA,
A solution containing 2Na was used.

Separation column 5 is Hitachi gel 83056 (Shinska-OD
S) 4111 mL D, Xl 50M was used, and the column temperature was 50°C. The flow rate was 0.8 mQ/min, a fluorescence detector was used as the detector 8, and the excitation wavelength was 28 On'm.
, the fluorescence wavelength was 315 nm. FIG. 4 is an example of analysis of a standard sample of creatinine (CRE).

Distilled water was used as the carrier liquid, and the flow rate was 1. ○ mQ/mi
n. A tetrafluoroethylene tube having an inner diameter of 0.5 M and a length of 5 m was used as the reaction coil 6, and the reaction coil 6 was placed in the same column oven 7. For reagents, 28.4m
A mixture of M picric acid solution and 0.2N sodium hydroxide solution in the ratio of 3:2 was used. Two reagent loops with a capacity of 100 uQ are used for the reagent loops 61A and 61B.
Sample loops 60 and 62 had a capacity of 40 uQ.

Figure 5 shows the analysis results and output examples of urine samples. In Table 1, the concentration of catecholamine metabolites is divided by the concentration of creatinine and is expressed as the amount of catecholamine metabolites per unit creatinine.

Example of Output of Results FIG. 6 is a connection diagram when a two-way six-way valve is used as the sample-reagent injection valve for creatinine measurement as a valve system.

Moreover, FIG. 7 is a connection diagram when a two-way six-way valve is used as the sample-reagent injection valve in the valve system. In this case, there will be one sample loop 62 and one reagent loop 6 for measuring creatinine.

According to the above-described embodiment, catecholamine metabolites and creatinine can be analyzed simultaneously from one sample bottle, thereby making it possible to automate the analysis and save labor. It also has the effect of eliminating human errors such as mixing up samples. Furthermore, since catecholamine metabolites and creatinine are analyzed in separate channels, each analysis condition can be changed individually, and even if one of the analysis conditions is changed for some reason, simultaneous analysis is possible.

〔Effect of the invention〕

According to the present invention, samples for both the catecholamine metabolite analysis section and the creatinine analysis section can be collected with a single injection operation.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a flow path system in an embodiment of the present invention, FIGS. Figure 3 shows an example of analysis of a standard sample of catecholamine metabolites, Figure 4 shows an example of analysis of a standard sample of creatinine, Figure 5 shows an example of analysis of a urine sample, and Figure 6 shows the valve system. Figure 7 shows the second modification of the valve system. Figure 8
The figure is a diagram showing the flow of analysis operation of the apparatus of FIG. 1. 3...Six-way valve, 4...16-way valve, 5...
Separation column, 6... Reaction coil, 8, 9... Inspection device, 10... Data processing device 12... Eluent, 13.
...Carrier liquid, 54...Reagent, 60.62...Sample loop, 61°C? 1. IC,S '1.50
Correction T (l 0FFS 0C) (, I C,s
s,oo ATT '7 0FF5 s荊5fig.

Claims (1)

[Claims] 1. A catecholamine metabolite analysis section consisting of a liquid chromatograph having a first sample injection loop and a separation column; a flow reaction analysis section for creatinine having a second sample injection loop and a reaction coil; The first sample injection loop is connected to the second sample injection loop, and then the first sample injection loop is independently connected to the separation column, and the second sample loop is independently connected to the reaction coil. An analytical device characterized by being equipped with a valve system.