JPH11160241A - Method for determining quantitatively compound with using periodic acid oxidation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To permit colorimetry for a compound without using arsenious acid or its salt by making a measurement compound and periodic acid react with each other, decomposing excess periodic acid with thiosulfuric acid or its salt, and adding a color former. SOLUTION: A measurement compound and periodic acid are made to react with each other in a periodic acid oxidation, thereby producing aldehyde. An excessive amount of thiosulfuric acid or its salt is added to the residual periodic acid and mixed to quench the residual periodic acid. A color former is then added to bring about a reaction and a coloration. A colorimetric analysis is made and the compound is measured by a working curve. At this time, the measurement compound is a compound of 1, 2 amino alcohol, 1,2-diol, 1,2- hydroxylaldehyde, 1,2-hydroxyketone, 1,2-diketone, 1,2-hydroxy acid or 1,2-keto acid, e.g. saccharide such as sialic acid, etc., an amino acid such as serine, etc. Metaperiodic acid or the like is utilizable as the periodic acid, and sodium thiosulfate or the like can be the thiosulfuric acid or its salt. The color former is preferably, e.g. thiobarbituric acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for quantitatively determining a compound comprising a step of generating an aldehyde by periodate oxidation and then decomposing excess periodate with thiosulfuric acid or a salt thereof.

[0002]

2. Description of the Related Art 1,2-diol, 1,2-amino alcohol, 1,2-hydroxy aldehyde, 1,2-hydroxy ketone, 1,2-diketone, 1,2-hydroxy acid or 1,2-keto. A method using periodate oxidation is known as one of the methods for quantifying a compound having an acid or the like. Compounds determined by periodate oxidation are compounds such as certain sugars and amino acids that produce aldehydes by periodate oxidation, such as sialic acid, 2-deoxy sugar,
Examples include sugars such as 3-deoxy sugars and amino acids such as serine and threonine.

The method of quantifying a compound by periodate oxidation is briefly described as follows. First, periodic acid or a salt thereof is added to a solution of the compound to be quantified to generate an aldehyde from the compound to be quantified, and after the reaction, excess periodate in the reaction system is decomposed and then formed. A reagent (e.g., thiobarbituric acid) that condenses with an aldehyde to form a color-forming compound (e.g., thiobarbituric acid) is added to the reaction solution to generate a color-forming compound, and the resulting color-forming compound is quantified by colorimetry.

Heretofore, in this quantitative method, arsenous acid or a salt thereof has been used to decompose an excessive amount of periodic acid generated by periodate oxidation. For example,
J. Biol. Chem., Vol.234, p.1971, (1959).
have produced an aldehyde called β-formylpyruvic acid from N-acetylneuraminic acid, which is a kind of sialic acid, by periodate oxidation, and color development with thiobarbituric acid for colorimetric determination. By N
-Determination of acetylneuraminic acid. In this determination method, Warren et al. Decompose excess periodate in periodate oxidation with sodium arsenite.

However, it goes without saying that arsenic is highly toxic, and the conventional quantitative method using arsenous acid or its salt is
The burden of waste liquid treatment after measurement occurs, which is dangerous for the experimenter. Therefore, the conventional quantification method is not always a good method for measuring a large amount of sample.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for quantifying a compound using periodate oxidation without using arsenous acid or a salt thereof.

[0007]

Means for Solving the Problems The present inventors, in a step of decomposing excess periodate generated in a reaction system after a reaction for generating an aldehyde by periodate oxidation, comprise:
The present inventors have intensively searched for a safe compound that can be substituted for arsenous acid or a salt thereof which has been conventionally added to the reaction solution. As a result, they have found that thiosulfuric acid or a salt thereof exhibits sufficient measurement accuracy and is suitable as a substitute compound, and has completed the present invention.

That is, the present invention relates to 1,2-aminoalcohol, 1,2-diol, 1,2-hydroxyaldehyde, 1,2-hydroxyketone, 1,2-diketone,
A compound of 1,2-hydroxy acid or 1,2-keto acid,
This is a method for quantification using periodate oxidation, and is a method comprising the following steps. (1) 1,2-amino alcohol, 1,2-diol,
Reacting a compound of 1,2-hydroxyaldehyde, 1,2-hydroxyketone, 1,2-diketone, 1,2-hydroxy acid, or 1,2-keto acid with periodic acid;
(2) Next, excess periodate is decomposed with thiosulfuric acid or a salt thereof, and (3) a color former is further added and reacted for colorimetric determination.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The compounds measured by the quantification method of the present invention include 1,2-aminoalcohol, 1,2-diol, 1,2-hydroxyaldehyde, 1,2-hydroxyketone, and 1,2-hydroxyketone. A compound of a diketone, a 1,2-hydroxy acid or a 1,2-keto acid, which is a compound that generates an aldehyde by periodate oxidation. Examples of such compounds include sugars such as sialic acid, 2-deoxy sugar and 3-deoxy sugar, and amino acids such as serine and threonine. Specifically, as the 1,2-amino alcohol, for example, amino acids such as serine and threonine and N-
Sialic acid such as acetylneuraminic acid; Examples of the 1,2-diol include sugars such as 2-deoxy sugar and 3-deoxy sugar.

In the present invention, as the periodic acid, metaperiodic acid (HIO ₄ ) or orthoperiodic acid (H ₅ IO ₆ ) and salts of these compounds can be used.

The thiosulfuric acid or a salt thereof used in the method of the present invention is, for example, sodium thiosulfate.

The color-forming agent of the present invention is not particularly limited as long as it is a compound capable of forming a color-forming compound by condensing with an aldehyde generated from a compound to be quantified by periodate oxidation. It is one selected from an acid, phenylhydrazine, or acetylacetone and ammonium acetate.

The quantification method of the present invention is as follows. First step: The compound to be measured is reacted with periodic acid. 0.1 μM to 10 m of the compound to be quantified
M in an aqueous solution containing M
An aqueous solution containing 0 to 10,000 times the amount of periodic acid is added and mixed, and the reaction pH is adjusted to pH 1 to 12 and the reaction temperature is adjusted to 10 to 10.
The reaction is carried out at a temperature of ８０80 ° C. and the reaction is carried out for 1 to 60 minutes to oxidize periodate to produce an aldehyde.

Second step: Next, excessive periodic acid is added by adding thiosulfuric acid or a salt thereof in an excess amount as compared with the above periodate and mixed, and the periodate remaining unconsumed in the reaction in step 1 is added. And disappear. At this time, the molar ratio of periodic acid to thiosulfuric acid or a salt thereof is preferably 1: 2 to 1:20, more preferably 1: 8 to 1:12. At this time, the reaction temperature is 10 to 80 ° C., the reaction pH is pH 1 to 12, and the periodate can be eliminated for 1 to 5 minutes. After this reaction,
Stop the reaction.

Third step: Further, a color former is added to the solution, which has been stopped as described above, to cause a reaction. After color development, colorimetry is performed and the compound contained in the sample is quantified by using a calibration curve.

[0016]

EXAMPLES Examples of the present invention will be shown below to specifically explain the present invention, but these examples do not limit the present invention. Example 1 To 0.2 ml of a sample solution containing N-acetylneuraminic acid, 25 μl of 0.2 M metaperiodic acid (9 M phosphoric acid solution) was added, mixed, and allowed to stand at 37 ° C. for 5 minutes. Next, 125 μl of 0.4 M sodium thiosulfate (prepared with respect to a 0.5 M sodium sulfate aqueous solution) was added to the solution and mixed, and after confirming that the yellow color had disappeared, 0.1 mL of a coloring reagent was added.
750 μl of 6% thiobarbituric acid (prepared against a 0.5 M aqueous sodium sulfate solution) was added. Then, the color was maintained for 7 minutes in a boiling water bath. After cooling in running water for 2 minutes, 1 ml of cyclohexanone was added, mixed well, and the dye was transferred to the cyclohexanone layer and colorimetrically determined (wavelength 5
49 nm). The results are shown in FIG. 1 (FIG. 1). A calibration curve could be prepared even when sodium thiosulfate was used to decompose excess periodate, and it was found that measurement was possible in almost the same manner as when arsenous acid or a salt thereof was used.

REFERENCE EXAMPLE 1 The same measurement as in Example 1 was performed using the same concentration of sodium arsenite instead of the sodium thiosulfate of Example 1.
The results are shown in FIG. 2 (FIG. 2).

[0018]

According to the method of the present invention, it is possible to quantify the compound without using arsenous acid containing arsenic, which is highly toxic, or its salt, ensuring the safety of the experimenter, and simplifying the waste liquid treatment after the measurement. There are advantages. Therefore, the method of the present invention has an advantage that it can be applied to the quantification of a large amount of sample.

[Brief description of the drawings]

FIG. 1 is a standard curve of N-acetylneuraminic acid when sodium thiosulfate is used.

FIG. 2 is a standard curve of N-acetylneuraminic acid when sodium arsenite is used.

Claims (5)

[Claims] 1. A 1,2-amino alcohol, 1,2-diol, 1,2-hydroxy aldehyde, 1,2-hydroxy ketone, 1,2-diketone, 1,2-hydroxy acid or 1,2-keto. A method for quantifying an acid compound using periodate oxidation, comprising the following steps. (1) 1,2-amino alcohol, 1,2-diol,
Reacting a compound of 1,2-hydroxyaldehyde, 1,2-hydroxyketone, 1,2-diketone, 1,2-hydroxy acid, or 1,2-keto acid with periodic acid;
(2) Next, excess periodate is decomposed with thiosulfuric acid or a salt thereof, and (3) a color former is further added and reacted for colorimetric determination. 2. The method according to claim 1, wherein the compound is a compound of 1,2-aminoalcohol. 3. The method according to claim 1, wherein the compound is a 1,2-diol compound. 4. The color former is thiobarbituric acid, phenylhydrazine, or one selected from acetylacetone and ammonium acetate.
The method according to any one of claims 1 to 3. 5. The amount of thiosulfuric acid or a salt thereof added in the step (2) is 1: 2 to 2 in molar ratio between the periodic acid added in the step (1) and thiosulfuric acid or a salt thereof.
The method according to any one of claims 1 to 4, wherein the ratio is 1:20.