JPS5845557A - Reagent for quantitative determination of hydrogen peroxide - Google Patents

Abstract

PURPOSE:To obtain a reagent easy to prepare a uniform aqueous solution and having no influence on the activity of enzyme and also, having good reproducibility, by using specific triaryl imidazole derivative etc. having a sulfonic acid radical or carboxyl group. CONSTITUTION:A compound expressed by the general formula in the figure (wherein R<1> is -H, -OCH3-CH3, R<2> is -CH3,-C2H5, R<3> is -(CH2)n-SO3H or -(CH2)n- COOH and n=1-3) or its salt, is incorporated as a color forming agent. For example, a reagent is constituted by desired reagent components participating in a series of reaction which produce hydrogen peroxide finally by acting to a substance and peroxidase or the substance acting like peroxidase and also, said compound. Said compound is used effectively for the determination of trace components because it is easily soluble in water and the maximum absorption wavelength of coloring matter produced by reacting with hydrogen peroxide is 610mm. and said compound is not affected by the substance having an absorption band in a short wavelength e.g. bilirubin, hemoglobin etc. at measurement and moreover, it has a high molecular absorption coefficient.

Description

[Detailed description of the invention] The present invention relates to a reagent K for quantifying hydrogen peroxide.

Detection and quantification of hydrogen peroxide has traditionally been carried out in various fields using various methods, but in the field of clinical testing, its overlapping nature has increased significantly over the past few years.

That is, a widely used method is to apply fermentation to a component in a biological fluid sample, measure the generated hydrogen peroxide, and quantify the component. This makes it easy to measure trace components such as glucose, cholesterol, uric acid, phospholipids, neutral fats, and free fatty acids in biological fluids, and its effectiveness has been fully demonstrated by using it for clinical diagnosis. has been done.

The reagent used in such hydrogen peroxide assay and quantitative methods involves reacting hydrogen peroxide in the presence of an oxidase or a substance that acts like peroxidase, leading to color development or fluorescence. A number of developments have been made regarding these hydrogen donors. Broadly divided: (1) A method that uses redox dyes, in which the reduced dye is oxidized to develop color for measurement. For example, reduced type 2.
Examples include 6-cyclophenol indophenol and leucomalachite green.

(2) A method of measuring color development due to oxidative condensation. For example, 4-methoxynaphthol is used to measure the color development due to its own oxidative condensation, and 4-methoxynaphthol is used as a developer.
-aminoantipyrine or 3-methyl-2-benzothiazolone hydrazone, or use an aniline compound or a phenol compound as a coupler to carry out oxidative condensation between the two, and there is a method in which color development is constant.

However, none of these methods is perfect, and there are issues with sensitivity, stability, and
They have some drawbacks in terms of measurement wavelength, water solubility, and toxicity.

As a result of various studies to eliminate these drawbacks, the present inventors have developed a color former that can be measured with high sensitivity and long wavelengths in the presence of peroxidase, has good color stability, and is water-soluble. Synthesized. From this K, we have developed a reagent that enables the preparation of a uniform aqueous solution without using a dispersant or the like and has good reproducibility without affecting enzyme activity, thereby completing the present invention.

According to the present invention, a reagent for quantifying hydrogen peroxide in a liquid, which is characterized by containing a compound represented by the following general formula or a salt thereof as a coloring agent: (where R1 is -H, -0CR, - CH.

R'' bar CHs, -Ct Is R field is provided with -PCB, circle 5osH or +CH2+nCool (representing n = 1 to 3).

As shown in the general formula, the color former compound in the present invention has a sulfonic acid group or a carboxyl group. It is a triarylimidazole derivative having a xyl group, and a triarylimidazole dye precursor similar to this is disclosed in Japanese Patent Application Publication No. 53-26-1981.
188. However, as is clear from the publication, the triarylimidazole dye precursor does not contain any compound having a sulfonic acid group or a xyl group. Moreover, the compounds described in this publication are insoluble or poorly soluble substances in water, and the names of specific water bathing substances are not indicated.

Generally, in most cases, a sample in which hydrogen peroxide is present or generated is reacted in a water bath state, and the quantitative reagent is preferably an aqueous solution for handling purposes. Furthermore, it can be said that a method of mixing and reacting a liquid and a liquid is faster than a method of reacting a liquid and a solid, and has poor reproducibility, which is why the inventors of the present invention placed importance on water solubility.

The compound of the present invention has extremely good water solubility, and the maximum absorption wavelength of the dye produced by reacting with hydrogen peroxide is 610 nm.
m, so when measuring, a substance with short wavelength sum absorption,
For example, it is not affected by bilirubin, hemoglobin, etc.

In addition, its molecular extinction coefficient is 40,000 to 60,000 eR''/-oI M
/l, so it is effective for quantifying trace components.

The compounds of the present invention can be synthesized by the following generally known methods.

for example, (1) 120~125cm 4 hours KF/dioxane CtCFI, cn, so, α αCM, CH, 80, F (2) (COα).

It was obtained through the synthesis process of

Table IK shows test results for the water solubility of the compounds of the present invention in comparison with some similar triarylimidazole dye front materials.

As is clear from Table 1, 2, 3, 4, 5゜6
, 7 was water-soluble, and the color tone of the reaction product was also stable. When detecting or determining ilK of hydrogen peroxide, the pn of the buffer when using the compound of the present invention is suitably within the range of about 6 to 8.5, such as phosphate buffer, borate buffer, Citrate buffer, succinate buffer, Tris buffer, glycine buffer, triethanolamine buffer, maquipine buffer, Gud buffer, etc. can be used.

When measuring glucose, it is preferable to allow glucose oxidase and peroxidase to interact in a phosphate buffer solution of pn 6 to 7.

When measuring cholesterol, it is preferable to allow cholesterol oxidase and peroxidase to interact in a phosphate buffer solution having a pH of 6 to 7.

When measuring phospholipids, it is preferable to allow phos-7 orinogase D to interact with choline oxidase and noya-oxidase in a p118 Tris buffer.

Preferably, the measurement of neutral fat is carried out in a pH 7 buffer solution with the action of lipoprotein lipid, glycerokinase, L-α-glycerophosphate oxidase, and peroxidase.

When measuring free fatty acids, it is preferable to carry out the measurement in a good buffer solution having a pH of 6 to 8, in which acyl-CoA synthetase, acyl-CoA oxidase, and/or oxidase are allowed to act.

When measuring uric acid, it is preferable to allow uricase and /f-oxidase to act in a borate buffer solution of pH 6 to 7.

In addition, Shimamoto said it is possible to determine 8I11 by using cholinesterase activity, pensoylcholine as a substrate, and choline oxidase and peroxidase.

Next KiJ! The invention will be explained by examples.

(1) Reagents: 70 mmol/L borate buffer (pH 6), 0.5 U/d of oxidase, and the compound of the present invention (A4 (see Table 1) > 50 μmol/1 L
fc.

(2) Sample solution: Commercially available, accumulated water peroxide was used at 2.0°1.
It was diluted with water to give 6 e l-4e O08g O-4m mol/l.

(3) Operation: 20μ each of reagent 3.0- and sample solution
was added, heated at 37°C for 5 minutes, and absorbance at a wavelength of 610 nm was measured.

(4) Results: As is clear from FIG. 1, the calibration curve for hydrogen peroxide showed good linearity. Furthermore, the stability of color development was also sufficient.

Example 2 Quantification of uric acid: (1) Principle: Uricase is reacted with uric acid to quantitatively produce hydrogen peroxide, which is measured using peroxidase and the compound of the present invention, and uric acid is determined from that value.

(2) Reagents: 70 mmol/l borate buffer (-6) K uricase 0.2 U/d, /#-oxidase 0.5 U/sd, present compound A 450 μmol/
The concentration was adjusted to 1.

(3) Operation: Reagent 3. Add 20μt of OdK serum,
It was heated at 37° C. for 15 minutes, and the absorbance at a wavelength of 610 nm was measured.

Uric acid was quantified using a separately prepared calibration curve.

(4) Preparation of calibration curve: uric acid with water [ff! 1.5゜L2 * 0-9 @ 0-6 * Oa3 m mo
A uric acid sample solution was prepared to be 1/L K.

This was used in the same manner as in the case of serum, and the respective absorbances were measured to obtain the calibration curve shown in Figure 2. It showed good linearity. It has also been confirmed that the reagent has sufficient storage stability for use.

[Brief explanation of drawings]

FIG. 1 shows a calibration curve for hydrogen peroxide measurement using the reagent of the present invention, and FIG. 2 P shows a calibration curve for urine fII measurement using the reagent of the present invention. 'cJll Tashoso 4 Shini address l (room nozB urination 1 (,,,,J/l)

Claims (2)

[Claims] (1) A reagent for quantifying hydrogen peroxide in a liquid, which contains a compound represented by the following general formula or a salt thereof as a coloring agent. (Here, R1 is -H, -QC island, -C and R2 is -σ horse,
To C, I'l. Rj Ha+CHt+, 80s Hf? , = it
+CH1+IC0OH represents H== l ~ 3) (2) All reagent components that act on a substance and participate in a series of reactions that ultimately produce hydrogen peroxide, substances that have peroxidase or arm oxidase-like action, and the claims set forth in claim 1. A reagent for quantifying the substance consisting of a compound.