JPS5880556A - Chromogen for colorimetry of peroxide - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a reagent material that allows the reduction of peroxides, in particular peroxides produced by enzymatic and/or chemical reactions.

Many reactions are performed in order to achieve high reaction rates or
In rare cases, enzyme contributions are known to be utilized to enable endpoint control.

(Many of these enzymatic reactions themselves involve interactions between the carrier and oxygen, and one of the products is dissipated hydrogen, the amount of which is proportional to the starting compound. On the other hand, it is also purely chemical and the reaction process produces peroxide as the final product, the amount of which is also proportional to the amount of product introduced in the reaction at the initial conditions. There are several reactions.

Additionally, hydrogen peroxide, and generally all peroxides, are used as carriers to effect reactions that utilize the use of specific enzymes such as periomusidase (POD) and catalase.

Such enzymes catalyze the oxidation of suitable substances by consuming peroxide, which is then reduced (in the specific case of hydrogen peroxide, reduction results in water on the other hand, the oxidized material produced is proportional to the amount of peroxide present and proportional to the amount of starting support involved in the preliminary reaction to form peroxide.

A number of reagents have been proposed for the colorimetric determination of hydrogen peroxide according to the general format described below.

(Colorless) Acceptor in oxidized state (Colored) Various substances are used as acceptors in reduced state, such as:

Q-dianisidine, 0-tolidine, 0-phenylenediamine, guaiac oil, adrenaline, nolphthalein, muBTlii (co, a'-7 dinozi (3-ethyl-benzoguazoline-6-sulfonic acid) ferrocyanide, There are other substances.

/9J74 to 19113, g1aenst
a@at (and subsequently icmeraon) is
It has been shown that daamino-antivilline gives rise to a colored reaction product in the presence of a large number of aromatic or complex compounds, for example by the action of oxidizing substances such as ferrocyanide. .

Subsequently, Trindθr retested these facts and tentatively provided a colorimetric method for determining glucose in biological fluids according to the following format: The acid (POI) can be condensed with aminoantipyrine in the presence of an oxidizing agent. He pointed out that among aromatic compounds, there are aromatic amines.

Based on these teachings, many researchers have created a series of chromogens that can be substituted on the hydrogen atom, including aromatic amino (N,
M-dimethylaniline, N,N-diethylaniline)%
Furthermore, Ill, N-diethyltoluidine, N-ethyl-N-ethylacetamide-! - It was proposed to use amines substituted with aromatic rings, such as toluidine.

We have discovered that many aromatic amino acids can be condensed with 41-aminoantipyrine or similar compounds in the presence of oxidizing substances, resulting in colored compounds, and this is the technical essence of the invention. be.

Such compounds offer many advantages as they replace the aromatic amines mentioned above.

Namely: 1. The colored condensation product obtained has a higher molar extinction coefficient than the colored product obtained with the corresponding aromatic amine.

Although the aromatic amino acids mentioned above are congeners and can therefore be included in solid mixtures, they are liquid h
<Different from the aromatic amine of u.

3. This aromatic amino acid is not toxic, unlike similar aromatic amines.

The applicant is entitled to U.
K, Patent, /158j to 41. No. 67, the patent relates to certain chromogenic compounds, also suitable for the determination of peroxides, such as deramino-antibilin (and some similar compounds) and polcure. It consists of a component containing at least an aromatic ring having at least one phenol-type 10H group in the (molecule).

In the determination of enzymatically produced hydrogen peroxide in biological fluids, the above compounds make it possible to obtain satisfactory results.

However, in many cases, high sensitivity of a single chromogen is essential for quantifying hydrogen peroxide enzymatically produced by metabolites present in trace concentrations in substances and biological fluids. It is.

Tei-amino-antipyrine and phenol or p.

A chromogenic system for some of the compositions exemplified herein is about three times more sensitive than a chromogenic system using dipenzoate in hydrochloride, which chromogen system (while the reagents used are sufficiently accurate and reliable), this also makes it possible to use large quantities of the sample simultaneously present in the sample to be tested in the reaction mixture. This may have the added benefit of reducing the exposure to electrically interfering substances.

Therefore, a first object of the present invention is to provide fT-rich chromogenic compositions suitable for the determination of peroxides, and more particularly, to provide fT-rich chromogenic compositions suitable for the determination of peroxides, and more particularly, The object of the present invention is to provide a chromogen composition suitable for quantifying hydrogen peroxide produced.
It consists of a compound containing at least a reactive group bonded to a chemical structure derived from 5-pyrazolinone, and an aromatic amino acid having the following structure: (wherein E and P7 are the same or different from each other) , may be hydrogen or a hydrocarbon group, and may be hydrogen or a monovalent cation.) Among these compounds listed below, ortho- or meta-aminoandhizoic acid and N-furkylmethamino benzoin derivatives,
All of the above meta-substituted rust conductors turned out to be of particular interest. Among these compounds '4kJcD, which will be described later, the most excellent effect was shown by the acid in which the silica was completely substituted.

Among the main compounds used in systems replacing daramino-antivirin, the following can be listed: Aminophenazine (Targetylaminoco Hayashi).

3,-dinothyl-7-7enyl-3-bi519y-! −
on; sulfur-based biline (coJ3-dimethyl-1-2uni-5-pyrazolone-hiro-7 minomethanesulfonic acid sodium salt); dibupyrene ((/-phenyl-2,3-dimethyl-5-
virazolone-dayl sodium salt) inbutylaminomethane sulfonate); probylphenazine (4I-
Isoprobiluko.

3-dimethyl-7-7enyl-3-pyrazolin-3-one);
t'yzolone-tei-methylaminomethanesulfonate sodium salt).

In the following, the general pattern of the oxidation reaction of the chromogen system described here is shown, where hydrogen peroxide is used in the presence of peroxidase as the oxidation agent for the chromogen. However, other peroxides and suitable thickening agents are also suitable for this purpose.

C,)16 The principle of the reaction is as follows: Mix a solution containing the peroxide of the appropriate fb&stable and II&gt; containing one of the chromogens mentioned herein.

When the color is completely developed, the color measured at an appropriate wavelength is proportional to &ML of the peroxide. A reagent can be prepared containing the following ingredients.

Powder reagents containing the following O%&) ~ d): S) Phosphorous Wi salt, TR, aminopropanediol acetate and other derivatives or other similar substances, 1+) Antiseptic and fungal properties C) chromogenic systems containing antipyrine-type substances and aromatic amino acids; d) peroxidases.

The components of the uric acid quantitative application lid are as follows: The substance to be tested is mixed manually or automatically together with reagents including oxygen (uricase and peroxidase) and chromogens, all in a buffer. After the urine 1 has been oxidized by enzymes and the color of the reaction removed, optical readings are taken either manually or automatically at the appropriate power wavelength.

The resulting color is proportional to the amount of uric acid present in the sample.

According to this reaction pattern, h for this reagent
The parent is: In a single mixture, all the compounds necessary for the reaction of the W prime and the chromogen, or the enzymes 9 licase and PO.
D% experimental agent (phosphorus salt, TRl 5. acetate, γmino 70 pandiol and derivatives and other substances),
Aromatic amino acids and aminoantipyrine (or similar compounds), antimicrobial agents, surfactants and suitable compounds to prevent potential interference may be included.

Prepared reagents in phosphate buffered sOV
h.

IN; Vermilion 1w type! The correlation between the concentration of mucosa and uric acid at 10 nm was type 1 up to the urine concentration/dl in the sample.

Comparison Fosaati, P., Prenoipe
h L-(Quad, 5clav.

A comparative test with the method according to Diagn, Correct, 1.1111) gave the following result: (Y -m Fosaati's method) y-/,0θ/
r-θ,? ff! 4r, Mean Tei, 6 Iv/di 8
D 000g, 1llj/dlO, V, S dew 1
．． The bower.

1 Accuracy during a series of operations, mean time, y sII/d1B, D, o, tco■
/a IC, V, S-ko, tda・

Claims (1)

[Claims] /, /-phenyl-! - a chromogenic composition suitable for the colorimetric determination of peroxides, comprising a metal compound containing at least a reactive group attached to a chemical structure derived from pyrazolinone, and an aromatic amino acid of the formula: (Here, R and R7 may be the same or different from each other and may be hydrogen or a hydrocarbon group, and ! may be hydrogen or a monovalent cation.) The chromogen composition according to claim 1, wherein the chemical structure derived from 5-pyrazolinone is danoantevirin.