JPS6142826B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying urea and its derivatives.

There are very few practical methods for colorimetric determination common to urea and its derivatives, and colorimetry is performed by developing color with diacetylmonoxime, diacylmonoxime and antipyrine, or diacetylmonoxime and thiomicarbazide in an acidic solution. I can only give you a method. However, all of these methods require heating to 100°C in a concentrated phosphoric acid solution or prolonged heating to 37°C in a fairly concentrated sulfuric acid solution, and diacetyl monooxime has a peculiar odor. and may cause discomfort to some people. Therefore, these methods cannot be said to be good methods that can withstand frequent use.

In view of these points, the present inventors conducted various studies and found that urea and its derivatives are N-(1-naphthyl)-
It has been found that N'-diethylethylenediamine and 0-phthalaldehyde cause a sharp color development.

The present invention was completed based on these new findings, and is characterized by measuring the absorbance of the reaction solution by reacting N-(1-naphthyl)-N'-diethylethylenediamine and 0-phthalaldehyde with a sample. This is a method for quantifying urea and its derivatives.

This reaction is carried out in 1N hydrochloric acid at 37°C by heating for 15 minutes, so the operation is extremely easy, there is little danger in handling reagents, and there is no odor. The reaction completes in a short time, and the reaction solution changes from red to orange-brown, so it is possible to not only perform colorimetric determination by measuring the absorbance (465 nm) after the reaction, but also use the initial velocity method (520 nm).
It can also be quantified using Furthermore, the reagent solution is stable, making this method valuable as a practical method for quantifying urea and its derivatives.

0-phthalaldehyde and N for the analysis of urea
- A method using (1-naphthyl)ethylenedian has been proposed (Clinical Chemistry Vol.
21, No. 8, p. 1136, 1975), this method also uses a large amount of concentrated sulfuric acid and has the disadvantages of high blank values, but the present invention solves these disadvantages at once. and has the above advantages.

Example 1 Preparation of Reagent 1 0.854 g of N-(1-naphthyl)-N'-diethylethylenediamine oxalate is dissolved in 500 ml of water by heating, and after cooling, 1 g of polyoxyethylene lauryl ether (Brij35) is dissolved.

Preparation of Reagent 2 Dissolve 1.34 g of 0-phthalaldehyde in 500 ml of 2N hydrochloric acid by heating.

Analysis method: Add 2 portions each of reagents 1 and 2 to 50μ of urea solution.
Add ml at a time, warm at 37℃ for 15 minutes, and rinse with running water.
Cool for a minute and measure the absorbance at a wavelength of 465 nm using the blank test solution as a control. By this method, urea 0.05 ~
Since there is a linear relationship between the amount of urea and the absorbance in the range of 1.0 μM, the amount of urea is determined from a calibration curve prepared in advance.

If the same procedure is performed for 50μ of phenyl urea solution, the same results as for urea will be obtained. Example 2 The same procedure as in Example 1 is carried out using 50μ of methyl urea solution, and the absorbance is measured at a wavelength of 520 nm. Methylurea has a linear relationship with absorbance in the range of 0.01 to 0.3 μM.

If the same procedure is performed with ethyl urea solution or citrulline solution, the same results as in the case of methyl urea are obtained.

Example 3 Preparation and quantitative determination of reagent 3 Dissolve 1.34 g of 0-phthalaldehyde in 50% ethanol to make 25 ml.

Add 2 ml of Reagent 1 and 2 ml of 2N hydrochloric acid to 50 μl of urea solution, and heat at 37°C for 10 minutes. Add 100μ of reagent 3 to this, and at 37℃ at a wavelength of 520nm,
Measure the value of increasing absorbance for minutes. With this method, there is a linear relationship between the amount of urea and the change in absorbance in the range of 0.05 to 1.5 μM of urea, and urea can be quantified from a calibration curve prepared in advance.

Example 4 A citrulline solution of 50μ is operated in the same manner as in Example 3, and the change in absorbance at a wavelength of 520 nm is measured. Citrulline has a linear relationship with the change in absorbance in the range of 0.02 to 0.4 μM.

If a methylurea or ethylurea solution is used and the same procedure is performed, the same results as for citrulline will be obtained.

Claims (1)

[Claims] 1. A method for quantifying urea and its derivatives, which comprises reacting a sample with N-(1-naphthyl)-N'-diethylethylenediamine and 0-phthalaldehyde and measuring the absorbance of the reaction solution.