JPS5848064B2 - Analysis method for urine constituent substances - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for analyzing urine constituents by ion exchange chromatography, and particularly to improvements in eluents used in the method.

It is said that there are approximately 2,000 components of urine, and many of these components are closely related to the pathological conditions of the human body, and are known to increase or decrease specifically in response to specific diseases.

Therefore, analysis of urine constituent substances is expected to greatly contribute to the diagnosis and treatment of patient conditions, the development of artificial organs, and the like.

As conventional analytical methods, the absorbance measurement method for quantifying individual constituent substances in urine and anion exchange chromatography using a buffer solution of acetic acid and ammonium acetate as an eluent are generally known.

However, when using the former absorbance measurement method, it may be interfered with by other coexisting substances, and it may take a long time to pre-process the sample. The disadvantage is that it is not always possible to clarify the correlation between

On the other hand, the latter method, anion exchange chromatography, requires almost no pretreatment of the sample and can be analyzed in its original state. Although it has the advantage of being able to analyze the correlation between the composition of urine constituents and diseases as well as the qualitative and quantitative analysis of urine, the following drawbacks are still unavoidable at present.

That is, one of these methods is a concentration gradient method in which the concentration of the eluent is linearly increased using a buffer solution of acetic acid and ammonium acetate as the eluent and an ultraviolet absorption spectrophotometer as the detector. A method of separating and developing urine constituents by elution method is known, but C C t D
t S cott Outside 3: Am, J, Ct: n, Pa
th, 53, 701 (1868)], when this method is used, the analysis time is very long as it takes 40 hours to analyze one sample, and the acetic acid-based buffer used as the eluent is ultraviolet. Since it exhibits absorption, it inevitably suffers from the disadvantages of a decrease in detection accuracy as the baseline rises and a limitation on the detection wavelength.

Improves the above disadvantages, for example, reduces urine constituents by about 100% in 2 hours.
A method for separating and detecting components has also been developed [Setagai 4: Differentiation, 73, 27 (1978)], but even with this method, it still takes time to use the analysis results to understand pathological conditions. In addition to being too hot, the eluent used is an acetic acid-based buffer, which still poses a problem in terms of ultraviolet light absorption.

Regarding the above-mentioned problems regarding ultraviolet absorption, the advantages of ultraviolet absorption spectrometers include that they are easier to handle than other detectors, such as fluorescence detectors, and do not require reaction reagents. However, the detection wavelength is usually 2 5 4 when the eluent is an ultraviolet absorbing acetic acid buffer.
It has been impossible to detect creatinine, which has an absorption maximum at 234 nm, and other wavelengths, such as creatinine, which has an absorption maximum at 234 nm.

Therefore, there were limitations in pursuing in more detail the relationship between the composition of urine constituents and pathological conditions.

An object of the present invention is to provide a method for analyzing urine constituent substances using ion exchange chromatography, which eliminates the drawbacks of the conventional techniques, improves detection accuracy, expands the detection wavelength range, and significantly shortens analysis time. There is something to do.

In order to achieve the above object, the present invention provides a method for analyzing urine constituent substances applying so-called gradient elution ion exchange chromatography in which the concentration of the eluent changes with the passage of analysis time, and in which chloride is used as the eluent. Using an aqueous solution containing acetonitrile and acetonitrile, the concentrations of chloride and acetonitrile in the aqueous solution were adjusted continuously or stepwise to 0.00, respectively.
1M to 3, OM and O, 1 volume to 30 volume%t
It is characterized by changing.

According to the present invention, the urine constituents to be analyzed broadly include urine constituents, biologically related substances, and various organic acids such as nucleic acids.

As for the analysis method of the residue, conventional gradient elution ion chromatography techniques and apparatuses may be applied to the residue, except that the eluent is configured according to the present invention.

However, modifications such as adding a separation column packed with a cation exchange resin upstream of a separation column packed with a commonly used anion exchange resin may be carried out as necessary.

To prepare the eluent, prepare an aqueous solution containing chloride and acetonitrile at a low concentration (in some cases, the acetonitrile is O) and a high concentration (final solution), respectively, according to the conventional method. A method may be employed in which the concentration of the eluent is continuously increased over the course of the analysis time by processing the eluent in an apparatus, or a method in which eluent having a stepwise concentration is prepared and used sequentially is also possible.

Prior to adjusting or feeding the eluent, it is desirable to feed water that does not contain chloride or acetonitrile for a certain period of time in order to maintain the equilibrium of the separation column.

The chloride used as a component of the eluent is widely selected from water-soluble inorganic chlorides, with ammonium chloride, sodium chloride, and potassium chloride being particularly preferred.

The chloride concentration in the eluent decreases to 0 over the course of analysis time.
001M to 3. It is desirable to change it at 0 Mt.

0. At a concentration lower than OOIM, the effect of the present invention is not sufficient,
Further, a concentration higher than 3.0M is not favorable because post-processing including column regeneration takes time.

1. The concentration of acetonitrile increases from 0.1 volume to 30 volumes over the course of analysis time! It is desirable to change the

The effect of the present invention was sufficient at a concentration lower than 0.1 volume.
Even if the capacity exceeds 1 or 30 φ, there is no particular improvement in the effect.

In addition to the above-mentioned essential components, other substances such as methyl alcohol and ethyl alcohol may be added as adjustments or auxiliaries as necessary.

The amount of the alcohol added may be changed in the range of 0.5 volumes to 30 volumes over the course of the analysis time.

Hereinafter, the present invention will be explained in more detail with reference to embodiments shown in the drawings.

The apparatus shown in FIG. 1 shows conventional liquid chromatography that can be applied to the present invention, including a storage tank IA for an aqueous solution (initial solution) containing chloride and acetonitrile at low concentrations, and an aqueous solution containing high concentrations of chloride and acetonitrile. A gradient device 8 is used to mix and adjust the eluent in the (final solution) storage tank IB, storage tanks 1A and 1B according to the passage of analysis time, and the liquid led from the gradient device is sent to the separation column 4 through the sample injection port 3. a detector 5, generally represented by an ultraviolet absorbance meter, for sequentially detecting the liquid discharged from the separation column 4;
It mainly consists of a recorder 6 that operates based on the signal from the detector 5.

In the figure, 7 indicates the discharged liquid after detection, and the casing 9 indicates a water inlet.

When the device configured as described above is pressed, a sample of urine or the like supplied from the sample inlet 3 + 4 is introduced into a separation column that has been equilibrated in advance with water sent through the inlet 9 and the pump 2, and its constituent components are It is absorbed by the anion exchange resin packed in the column.

Thereafter, the gradient device 8 is started, whereby the urine constituents absorbed by the anion exchange resin are separated into their respective components and reach the detector 5 in sequence, where they are separated according to the amount of each component. A signal** signal was sent out, and a total of 6 records were recorded.
recorded in

Next, in place of the apparatus shown in FIG. 7 and the combination of each storage tank and gradient device shown in FIG.
The configuration is the same except that H and a switching valve 10 for each tank are provided, and even in the case of an apparatus with such a configuration, the eluent in each tank can be sequentially changed from low concentration to high concentration by the switching valve 10. Therefore, it is possible to analyze in the same way.

Hereinafter, the effects of the present invention will be explained in more detail with reference to Examples.

Comparative Example Based on the apparatus shown in FIG. 1, urine was analyzed according to the method of Seta et al. and under the conditions shown in Table 1.

As a result of the analysis, the chromatogram shown in FIG. 2 was obtained.

In the figure, A shows the peaks of uric acid, B shows the peaks of hippuric acid, and C shows the peaks of indoleacetic acid.

It has been known that the method has disadvantages such as significantly lower accuracy because the baseline of absorbance increases with the passage of time, and that the analysis time is too long, requiring 2 hours.

Example 1 In place of the initial solution consisting of 0.006M ammonium acetate monoacetate, O. 6. Add 1 liter of ammonium chloride solution of OOIM. The experiment was carried out under the same conditions as in the comparative example, except that instead of the final solution consisting of OM ammonium acetate monoacetate, a mixed aqueous solution consisting of 2, OM ammonium chloride and acetonitrile in a 30 solvable amount was used, and the chromatogram shown in Fig. 3 was obtained. I got it.

In the figure, A shows the peaks of uric acid, B shows the peaks of hippuric acid, and C shows the peaks of indoleacetic acid.

As is clear from FIG. 3, according to this example, there was no increase in the baseline over time and the analysis time was significantly shortened to 1 hour.

The reason why the baseline does not increase is considered to be that the present invention does not use an ultraviolet-absorbing acetic acid eluent.

Incidentally, when we determined the relationship between ultraviolet transmittance and wavelength when an acetic acid buffer was used as the eluent and when the eluent of the present invention was used, the results shown in Figures 4 and 5 were obtained, respectively. It was done.

Na button, 20 in the figure indicates the concentration of ammonium acetate monoacetate is 0.
．． 0 0. In the case of IM (initial solution), 21 is the same as 0.
1 M, 22 indicates the case of 6.0 M (final solution),
1, 30 is when the ammonium chloride concentration is 0.001M (initial solution), 31 is when the ammonium chloride concentration is 0.75
M, acetonitrile concentration: 5 Capacity Hiroshi 32 has an ammonium chloride concentration of 3, O M, acetonitrile concentration is 1
The case of 5 volumes φ (final liquid) is shown.

As is clear from each figure, in any case, as the eluent concentration increases, the wavelength at which absorption increases shifts to the longer wavelength side, but in the present invention (see Figure 5), even the final solution has a wavelength of 2 20
On the other hand, the conventional method has the characteristic that absorption is large even at 250 nm, whereas it does not show large absorption up to about 250 nm.

This means that, according to the present invention, even if the concentration of the eluent increases (in other words, even with the passage of time), the baseline rises at commonly used wavelengths, such as 254 nm or 280 nm. This suggests that not only does it not cause a problem, but it is also possible to use lower wavelengths.

Furthermore, considering the fact that the analysis time is shortened by the present invention, the chloride ion generated from the chloride used in the present invention has a selectivity coefficient of about 1 for strongly basic anion exchange resins compared to the conventional acetate ion. This is thought to be due to the synergistic effect of increasing the solubility of the solute in the mobile phase due to the action of acetonitrile coexisting with chloride and the 10 times larger amount.

Example 2 0.03M instead of 0.001M ammonium chloride aqueous solution
3. Dilute the sodium chloride aqueous solution. 2. Replaced with a mixed aqueous solution of OM ammonium chloride-15 volumes of acetonitrile.
The procedure was carried out in the same manner as in Example 1 except that a mixed aqueous solution of 0 M netrium chloride and 30 volumes of acetonitrile was used, and the chromatogram shown in FIG. 6 was obtained.

In the figure, A shows the peaks of uric acid, B shows the peaks of hippuric acid, and C shows the peaks of indoleacetic acid.

As is clear from FIG. 6, according to this example, Example 1
A similar effect was achieved.

Example 3 After filling each storage tank 1C to 1H of the apparatus shown in FIG. 7 with each eluent in the set shown in Table 4, the switching valve 10 was operated to feed each liquid for the liquid delivery time shown in the table. Analysis of urine from patients with renal disease was carried out in the same manner as in Examples 1 and 2, except that the liquids were passed sequentially, and the chromatogram shown in FIG. 8 was obtained.

As is clear from FIG. 8, it has been found that the same effects as in Examples 1 and 2 can be achieved in this example as well.

As explained above, according to the present invention, it is possible to detect urine constituent substances that absorb ultraviolet rays in the wavelength range of 250 nm or shorter, which was impossible with conventional analysis methods, and that it is possible to detect Even if the flow rate is the same as that of the conventional method, the analysis time can be significantly shortened from the conventional 2 hours to about 1 hour.
Since the baseline does not rise as the analysis time passes, the quantitative accuracy of each component is improved, and the sensitivity of the detector can be greatly increased (several tens of times), making it possible to detect trace components. Various effects are achieved.

[Brief explanation of drawings]

Fig. 1 is a system diagram of an example of conventional liquid chromatography applicable to the present invention, Fig. 2 is a chromatogram obtained by the conventional method, Fig. 3 is a chromatogram obtained by the embodiment of the present invention, and Fig. 4 is a system diagram of an example of conventional liquid chromatography applicable to the present invention. FIG. 5 is an ultraviolet absorption characteristic diagram using a conventional eluent, FIG. 5 is an ultraviolet absorption characteristic diagram using an eluent of the present invention, and FIG. 6 is a chromatogram obtained by another example of the present invention.
FIG. 7 is a system diagram of an example of liquid chromatography according to the present invention, and FIG. 8 is a chromatogram obtained by another example of the present invention. 1A...Initial liquid storage tank, 1B...Final liquid storage tank, 1C,
1D, IE, 1F, IG, IH... Eluent storage tank, 2.
... Pump, 3... Sample injection port, 4... Separation column, 5... Detector, 6... Recorder, 7... Effluent,
8... Gradient device, 9... Water inlet, 10.
...Switching valve, 20...Ammonium acetate monoacetate 0.0
01M, 21... Ammonium acetate monoacetate 0. 1
M, 22...Ammonium acetate monoacetate 6.0M, 30
...Ammonium chloride 0.0 0 1M, 3 1
...Ammonium chloride 0.75M-acetonitrile 5
%, 3 2...Ammonium chloride 3, OM-acetonitrile 15 cups.

Claims (1)

[Claims] 1. In a method for analyzing urine constituent substances applying gradient elution ion exchange chromatography in which the concentration of the eluent changes with the passage of analysis time, an aqueous solution containing chloride and acetonitrile is used as the eluent, and the aqueous solution The chloride concentration in the solution was varied continuously or stepwise from 0.001 mol to 3.0 mol, and the acetonitrile concentration was continuously varied from 0.1 volume percent to 30 volume percent. A method for analyzing urinary composition or substances characterized by stepwise change.