JPH03255955A - Catechol amine metabolyte analyzing apparatus - Google Patents

Abstract

PURPOSE:To eliminate the labor for sorting samples and to make improvement in efficiency by using an auto-sampler with a pretreating function to replace a microplate reader. CONSTITUTION:The sample to be measured is sucked and discharged from a sample bottle 14 into an empty reaction bottle 15 by a syringe 5. After a syringe 5 is internally washed, reaction liquids (two kinds 12, 13) are successively added to the sample. A specified volume of the reaction liquid is taken after stirring and reacting and is measured with a visible detector 16. The sample is introduced to a liquid chromatogram of a sepn. column 28 and electrochemical detector 29 which measure VMA, VLA, HVA by using the microsyringe 5 of the auto-sampler 1. The reaction is effected successively according to the reaction program incorporated into the sampler 1 in order to measure only the creatinine from the same sample and the resulted reaction product is sent via a sampling valve 21 by the syringe 5 to a visible photometer 16, by which the absorbance thereof is measured. The VMA, HVA, VLA, and creatinine are exactly measured from the one sample by the operation thereof.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention targets all newborn infants at health institutes or public health centers in each prefecture, and analyzes urine components using a liquid chromatograph or other models. This device is capable of early detection of childhood cancer and involves a device that analyzes all urine components using liquid chromatography.

[Conventional technology]

Conventional equipment measures VMA, HVA, and VLA using a high-performance liquid chromatograph, and measures creatinine using a high-performance liquid chromatograph.

The value is calculated by measuring with a microplate reader and finding the correlation between them.

[Problem to be solved by the invention]

In the above-mentioned conventional technology, a sample is divided into two parts, and one part is distributed in a liquid chromatograph and the other part is distributed in a microplate. This method may cause handling errors when handling a large number of samples, increases the size of the entire device, and is a burden on the customer in terms of price.

An object of the present invention is to replace a microplate reader with an autosampler with a pretreatment function.

[Means to solve the problem]

In order to achieve the above objective, an autosampler with a pretreatment function was used in place of a microplate reader.

This autosampler with pretreatment is capable of dispensing several types of reaction reagents, diluting them, adding samples, stirring (by inhaling air with a syringe and expelling air into the reaction sample bottle many times, stirring by bubbling, or After the reaction, switch the injection valve of the autosampler and measure with a visible photometer connected to the drain valve.

On the one hand, from undiluted urine sample bottles without any pretreatment.

Because it is analyzed directly using a liquid chromatograph, time and
Less sample loss.

[Function] An autosampler with a pretreatment function for a liquid chromatograph can perform the same process as a microplate reader.

For example, accurately aspirate and dispense the sample to be measured into an empty reaction bean using a syringe, wash the inside of the syringe, add reaction solutions (2 types) sequentially, stir, and after 2 reactions, take a certain amount of the reaction solution and visually detect it. Measure at 505 nm of the instrument (this step is all done with a microsyringe).

At this time, the sample to be measured is subjected to VMA and VLA using the microsyringe of the autosampler.

The reaction is carried out sequentially according to the reaction program set in the separation column and electrochemical detector (or fluorescence detector) for measuring HVA, and in the autosampler to measure only creatinine from the same sample. Same method)'4 The reactant is sent into a visible photometer through a valve using a syringe of an autosampler, and its absorbance is measured. Through these operations, VMA, HVA, VLA, and creatinine can be accurately measured from a single sample.

〔Example〕

An embodiment of the present invention will be described below with reference to FIGS. 1 to 5.

FIG. 1 shows the flow path configuration of the apparatus used to carry out the present invention.

An eluent 24 and a pump 23 for delivering it. Auto sampler 1
．． Column 28. It consists of a detector (detector 299 spectrometer 16) and data processing bags W117, 18, and 30.

First, to analyze VMA, HLA, and HVA,
95 with 5% acetonitrile as eluent 24
50 mmol tartaric acid, 0.01 percent EDTA-disodium per minute with pump 23.
Flow at a flow rate of 8mA.

For the column 28, a Hitachi backed column #3056 (silica gel ○D8 5 microns) with an inner diameter of 4 mm and a length of 150 mm was installed in a column open 27 maintained at 40 degrees Celsius. Place the standard sample in sample bottle 1
A certain amount is taken with the needle 11, introduced into the sampling valve 21 from the injection port 20, and after separating the three commands in the column 28, the detector 29 (EC
Detected by D).

On the other hand, the system for measuring creatinine is as follows, and the detection principle is as follows.

Creatinine + sodium hydroxide + picric acid↓ creatinine picrate (Red - absorption at visible wavelength 505 nm) The above method is performed using an autosampler with a pretreatment function.

Take 20 microliters of sample from the sample bottle 14 and put it into the reaction bottle 15.

Put 160 mmol of sodium hydroxide and a nonionic surfactant in the reaction reagent bottle 12, take 600 microliters from there, put it in the reaction bottle 15, and insert the syringe 5 with the needle 11 in the reaction bottle. 150 per second
Stir and mix by adding and removing 500 microliters from the reaction bottle at microliter speed.

5 first to prevent the inside of the syringe 5 from becoming contaminated with samples and reagents.
A better method is to introduce approximately 0 microliters of air into the needle, and then aspirate and discharge 450 microliters of the reaction solution.

5.6 mmol of picric acid is placed in the reaction reagent bottle 13, from which 140 microliters is sucked in with a needle and the entire amount is discharged into the reaction bottle 15.

As above, place the needle in the reaction bottle and stir thoroughly to mix.
React at 40 degrees C for minutes.

The analysis method is specifically described in Figure 2.

First, in order to obtain a chromatogram of creatinine, add 30% diluted solution (for dispensing) 33 into the needle with syringe 5.
Aspirate 0 microliters.

Next, 50 microliters of the reaction solution is aspirated from the reaction bottle 15, and further 100 microliters of the diluent (for dispensing) are aspirated to sandwich the reaction solution in the needle.

As shown in FIG. 3, the injection valve 21 is connected to an injection boat and a spectrometer for measuring creriatinin.

Put the needle into the injection boat and syringe 5
is discharged at a rate of 3 milliliters per minute.

A chromatogram is obtained as shown in FIG. 4(C), and its area value is stored in the data processing device 17.

The results of creatinine, VMA, VLA, and HVA obtained are shown in FIG.

(A) is a chromatogram of standard samples of VMA, VLA, and HVA, and the unknown sample is quantified by converting each area value.

(B) is an example of analysis of pediatric urine in which VMA and HVA were detected.

(C) is the analysis result of creatinine.

Each component obtained in FIG. 4 is further corrected for the creatinine value by the data processing device (upper-level computer) 18, and the final result is calculated.

FIG. 5 shows the flowchart.

〔Effect of the invention〕

(b) By incorporating a preprocessing function into the autosampler, there is no need to separate samples and efficiency can be improved. Simplification and labor-saving are important, especially in the field of use in testing centers and public health centers, where there is a need to measure new diagnostic tests.

(b) In the present invention, since a dedicated microplate reader for measuring creatinine is not required, the cost can be reduced to 1/2 to 1/3 by using a photometer.

(c) Space is reduced by the size of the microplate reader.

[Brief explanation of drawings]

Fig. 1 is a flow diagram of an embodiment of the present invention, Fig. 2 is a flow chart of creatinine measurement, Fig. 3 is a diagram showing a modification of the present invention, and Fig. 4 (A) is a flow diagram of VMA, HLA, HVA, standard FIG. 5 is a diagram showing a chromatogram of a sample, (B) is a diagram showing a chromatogram of pediatric urine, (C) is a diagram showing a chromatogram of creatinine, and FIG. 5 is a diagram showing a data processing flowchart. l... Auto sampler, 2... Syringe valve control device, 3... Syringe flow path switching motor, 4... Syringe flow path switching valve, 5... Syringe, 6... Syringe motor, 7... Syringe control device, 8... Teflon tube, 9... Needle height control motor,
DESCRIPTION OF SYMBOLS 10... Needle height control device, 11... Needle, 1-2... Reaction reagent bottle, 13... Reaction reagent bottle, 1
4... Sample bottle, 15... Reaction bottle, 16... Spectrometer, 17... Data processing device, 18... Data processing device, 19... Waste liquid bottle, 20... Sample, 21... Sampling valve, 22... Loop, 23... Pump, 24... Eluent, 25... Sampling valve control device, 26... Sampling valve flow path switching motor, 27... Column open, 28...Column, 2
9...Detector, 30...Data processing device, 31...
・Pump cam, 32... Pump drive motor No. 4 No. 2E21 #3 Diagram difficult to understand

Claims (1)

[Claims] 1. In a system consisting of a liquid pump, an autosampler with automatic dilution, reaction, and dispensing functions, a separation column and a column open, a plurality of detectors, and a data processing section, the autosampler has two components. By providing a flow path switching valve, a flow path system is configured that directly flows from the separation column and the detector or sampler to the other detector. During the analysis, other samples are mixed with the reaction solution in the autosampler, and then the reactants are introduced into the detector flow system. By this, creatinine is analyzed, and these two types of analysis are performed through one autosampler, and at the same time,
A catecholamine metabolite analyzer, characterized in that a data processing section allows the amount of catecholamine metabolites to be corrected using creatinine data.