DE2744812A1 - MEANS FOR DETERMINING PEROXIDES - Google Patents

Description

Ht. I. \ <;. K. WIIKSTIM) Kk. '! 0OO Μ ν N (MI H.V'llO I) H. K. ν. Ι'Κ (ΙΙΜΛΝΧ s <; m> κκι kkstkassi: s

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1A-49 667

Patent application

Applicant: Istituto Sieroterapico e Vaccinogeno Toscano ¹¹ SCLAVO "SpA Siena, Via Fiorentina, 1 Italy

Title: Means for the determination of peroxides

809815/0712

you. inc. ι ·. \ vi: i: s ι noil · · miiiii μ ι; χ rn kn iio

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1-49 867
Applicant: Instituto

Sieroterapico

Description

The invention relates to a means that the determination of peroxides that are formed by enzymatic and / or chemical reactions.

It is known that many reactions require the use of enzymes in order to achieve a higher one Achieve speed of response and, in some cases, a more immediate means of review of the reaction process: many of the enzymatic reactions themselves involve an interaction between the starting substrate and oxygen, one of the end products being hydrogen peroxide, the amount of which

,. _, (used),. ,. , is proportional to the / output connection.

On the other hand, there are numerous purely chemical reactions that produce peroxides, and also in this case the formation of peroxides is proportional to the amount of the compound used in the reaction at the beginning of the Reaction. It is also known that hydrogen peroxide and generally all peroxides can be used as Substrates for carrying out reactions using enzymes such as peroxidase and catalase. Such Enzymes catalyze the oxidation of the corresponding substrates at the expense of the peroxide, whereby this is reduced (im special case of hydrogen peroxide, water is formed) · The oxidized substances that are formed are proportional to the amount of peroxide present and thus also to the amount of initial substrate that is involved in the original reaction participates with the formation of peroxide.

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A reaction system has now been found which allows the determination of peroxides and thus the measurement of the starting substrate.

Such a reagent system according to the invention exists from a substance that acts as a reduced substrate when reacting with the peroxide in the presence of peroxidase acts and a reagent, which is described in more detail below and at the expense of the chemical substance which has been oxidized in the first reaction with the peroxide. The resulting oxidized substance has such a structure, generally a quinone structure, which has an intense color, so that you Presence and, most importantly, their amount can easily be determined.

According to an alternative embodiment according to the invention, it is even possible that no reduced substance is reacted in the first stage with the peroxide, i.e. it can be used together with the reagent used, such as indicated above, to be added at the end of a colorimetric Measurement.

In a special case, in the case of the invention Process the reduced substance, in the previous enzymatic reaction, brought about by the peroxide Is catalyzed, ferric cyanide: it is oxidized to ferricyanide, which in a second stage with a reagent is brought together, the following detection reagent which reacts as indicated above to form a colored substance.

As can be seen, the method of determining peroxide comprises two steps, the first being an enzymatic one Level is. Bas two-stage work can be avoided,

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as stated obea, by initially not adding any ferrocyanide is used and the determination of the peroxide is carried out as a simple reaction with the detection reagent, i. e. that the ferrocyanide is an integral part of the detection reagent can be.

The substances that make up the detection reagent must contain special reactive groups so that substances can be applied that appear to be very different 3ind.

In order to obtain the required properties, it must Detection reagent consist of

a) a component which contains at least one aromatic ring and at least one phenolic ring in the molecule -OH group,

b) a component that contains at least one reactive group bound to a structure, which is derived from i-phenyl-5-pyrazolinone.

The detection means according to the invention can be specially designed are represented by a phenol or an analogue thereof and 4-aminoantipyrine or an analogue thereof.

Such a reagent can be in a form of a solution which can be used directly as well as in the form of Phenol and 4-aminoantipyrine are produced in individual concentrated, separate solutions that are

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application can be mixed and diluted. The reagent can can also be produced in such a way that the substances in in dry form and dissolved in water to obtain the solution to be used.

The combination of phenol and 4-aminoantipyrine is just one of the possible combinations that can be used for the Agents or methods according to the invention are suitable: Many substances can be used instead of 4-aminoantipyrine or the phenol can be used, taking into account the essential theoretical conditions that are necessary for a chemical substance are required in the system according to the invention.

Compounds that can be used in place of phenol are phenols, naphthols, and hydroxyquinolines Kydroxychinaldine and other compounds, as will be explained in more detail later.

Also for substances that instead of 4-aminoantipyrine can be used, examples are given later.

Many phenols can be used for the end-redox system to maintain the chromogenic structure.

Such a structure that can be obtained with the phenols can be symbolized by the following formula be given:

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ο - =

NO

1A-49 867 27U812

whose substituents will be explained in more detail later «

If you start from the structure of the phenol and with

R ,,

and R _n the various substituents

₍ » ^1V 2» ^ll ₃ » ^XV A

designated, a list can be drawn up which is exemplary of the possible according to the invention applicable substances for the method according to the invention.

(R ₁ to Rr = H> phenol) TABEL link according to the following CH, R ₂ R ₃ R ₄ ^R 5 Tabel. phenol _ _ - o-cresol
m-cresol I. CH ₅ CH ₅ 5-hydroxy-1,3-dimethyl-
benzene CH,
^C 6 ^H 5 CH ₅ CH ₅ 3-hydroxy-1,4-
dimethylbenzene _ CH ₅ Thymol
Carvacrol
o-hydroxybiphenyl - CH ₅ -
- ° 3 ^H 7 C ₅ H ₇

809815/0712

β -
2 R ₁ R ₂ Cl OCH, OCH ₅ COOH - 1-49 867
27U812 - _ Cl Br OCH ₅ SO ₅ SO ₅ H - - COOH link ° 6 ^Η 11 " - SO ₅ H - - "D Tj π
R ₅ R ₄ R ₅ Cl Br - Br _ _ _ -. COOCH ₅ - o-cyclohexylphenol COCH, - Cl - COOCH, _ _ - Br - _ - - - o-hydroxyacetophenone COC ₆ H ₄ OH- - COOH - 2,4'-dihydroxy
benzophenol ί "ΊΤΤ Br _ - _ 2-chloro-5-hydroxytoluene Br Cl o-chlorophenol OCH, p-chlorophenol 2,4-dichlorophenol p-bromophenol 2,6-dibromophenol 2,4; 6-tribromophenol Guaiacol Monomethyl ether of
Resorcinol Monomethyl ether of
Hydroquinone Guaiacol sulfonate o-phenolsulfonic acid p-phenolsulfonic acid Salicylic acid m-hydroxybenzoic acid p-hydroxybenzoic acid Methyl p-hydroxybenzoate Methyl salicylate

Phenyl salicylate (Salol) COOC ₆ H ₅ -

Salicylsalicylic acid

Salicylamide

Salicylaldehyde

Vanillic acid

3,5-dibromo-4-hydrobenzoic acid

Catechol
Resorcinol
5-methylresorcinol
Epinephrine

COOC ₆ H ₄
COOH - CONH ₂ - CHO - OCH ₅ - Br - OH OH - OH

OH

CHO

COOH

CH NHCH

Br

Phloroglucine

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CH ₂ CHOH OH

Connection

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27U812

Pyrogallol m-aminophenol

OH OH NH,

Many different naphthols can also be used in the final redex system to

to preserve gene substance.

/ Structures The 'obtained with naphthols can be represented

as, for example, derivatives of 1-naphthol:

R R

NO

On the basis of this naphthalene structure, in which the

Substituents R. ,, R ₀ , R ^, R., R,

and R ₀ ver

^L - | » "2 ' ^XV 3 ♦ ^iV A * 5» 6' 7 8 represent different groups, an exemplary list can be drawn up of reactive substances that are also suitable for the reaction systems according to the invention.

suitable are:

(R ₁ to R ₈ table.

= H-

Naphthalene) according to the following

809815/0712

link

R.

1-naphthol I acid

1-naphtholic acid Chromotropic acid H-acid

1-1 sulfonic acid

2-naphthol R acid

G-SaIz (dipotassium salt) Schaffer's acid

3-hydroxy-2-naphthoic acid

N-substituted 3-hydroxy-2-naphthamide

OH OH

OH OH OH

OH

SO H OH - OH SO H
ό OH - OH - OH COOH OH CONHR

SO H 3

SO H

SO H

so 3 ^H. so 3 ^K so 3 ^H.

OH

PO ->. I. VD 812 867

It is also possible to use compounds of the hydroxyquinoline and hydroxyquinoline groups for the redox system to obtain the chromogenic substance.

A atruktur obtained with this compound in ^came f olgendermaßen symbolizes:

NO

RR
(this example is derived from 8-hydroxyquinoline).

Starting from the quinoline structure and _{denoting the various substituents with R 1} , Rp, R * R., Rc, Rg> R7, the following list of exemplary compounds can be drawn up which can be used for the system according to the invention.

(R ₁ to R ₇ = H —-> quinoline) according to the following table.

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Connection K, ^R 2 - 3-hydroxyquinaldine CU Oil - 5-hydroxyquinoline - - 6-hydroxyquinoline - - 7-hydroxychiaolin - 8-hydroxyquinoline - 7-methyl-8-hydroxy- ~
quinoline -

8-hydroxyquinolines-3-sulfonic acid

on

Oil

on

cn on

on

In addition to the groups of chemical compounds indicated above, other compounds can also be used instead of phenol become, such as

Barbituric acid and its derivatives (not in the 5-position substituted);

- pyrazolones

rl

- Dikctohydrind.cn

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- 2,4-diamino-6-hydroxypyrimidine:

4-Aminoantipyrine can also be replaced by other compounds, although it has proven to be particularly effective and for that purposes according to the invention has proven particularly suitable.

The most important compounds in the invention System can be used instead of 4-aminoantipyrine,

belong:

Aminophenazone (4-dimethylamino-2,3-dimethyl-l-phenyl ~ 3-

pyrazolin-5-one);

Sulfamylpyrine: (sodium salt of 2,3-dimethyl-l-phenyl-

5-pyrazolone-4-aminomethanesulfonic acid),

Dibupyrene: (Na salt of (1-phenyl-2,3-dimethyl-5-pyrazolone-

4-yl) isobutylamino) methanesulfonate,

Propylphenazon: ^ -Isopropyl ^^ - dimethyl-l-phenyl-S-

pyrazolin-3-one),

Thozalinone: (2-dimethylamino-5-phenyl-2-oxazolin-4-one), Methampyrone: (Na-1-phenyl-2,3-dimethyl-5 ~ pyrazolon-4-

methylaminomethanesulfonate).

As indicated above, the peroxide to be determined can originate from an enzymatic and / or chemical reaction or it can exist in some other way and interest its multitude.

For example, hydrogen peroxide can be produced in the following way:

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Al * 27U812

example 1 Formation of hydrogen peroxide through enzymatic reactions.

1) uric acid + O ₂ - allantoin + CO ₂ + H ₂ O ₂ (I)

2) cholesterol + O ₂ 4-cholestan-3-one + H ₂ O ₂ (II)

3) D-galactose + O ₂ D-galacto-hexadialdose + H ₂ O ₂ (III)

In all three of the above cases, the hydrogen peroxide produced in the reaction can be according to the following general implementation, which is the same for all considered systems, can be measured.

H ₉ O ₉ + reduced chromogen ■ HoO + oxidized ^{ά ά} ^ eroxiaase; ί chromogenic (IV)

The POD (peroxidase) is not only effective against hydrogen peroxide, but also against methyl peroxide and Ethyl peroxide.

With regard to the chromogen, two basic distinctions can be made be made, namely:

a) It is formed by a substance (ferrocyanide) which is oxidized (ferricyanide) and which then reacts in this form with other reactive components without the action of enzymes.
Ferricyanide + 4-hydroxybenzoate + 4-aminophenazone)

Ferrocyanide + colored quinone (V)

b) It is only formed by 4-hydroxybenzoate and 4-aminophenazone, which occurs directly in the reaction catalyzed by POD reacted.

+ 4-hydroxybenzoate + 4-aminophenazone

H ₂ O + colored quinone (VI)

In both cases the end product that can be measured photometrically (i.e. the quinone) is the same. In case a), the reaction proceeds in two stages and there are two different reagents required. In case b) the reaction takes place in a single step and only one reagent is required, which simplifies the system « 809815/0712

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The use of 4-II-hydroxybenzoate or another solid reagent with the properties indicated and as indicated in Tables I, II and III in addition, the production of the reagent in powder form.

Example 2

Formation of hydrogen peroxide through chemical reactions or measurement of the hydrogen peroxide present «

1) The hydrogen peroxide is already present in the solution and its amount should be determined. In this Case it is sufficient to apply the system according to reactions IV, V or VI directly to the solution in which

the hydrogen peroxide is to be determined.

2) The peroxide is formed by chemical reactions of the classic type, e.g. as used industrially to form these Substance is applied.

BaO ₂ + 2 H ⁺ + SO ₄ ""*> BaSO ₄ + H ₂ O ₂

3) The peroxide is formed by the action of ionizing radiation on water that contains dissolved oxygen (cfr. Nouveau Traite de Ghimie Minerale, by P. Pascal, Masson & Cie, publishers, XIII, 542) «

Numerous reactions occur, as follows can be summarized:

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+ ° 2 "2 O l · H "2 O H l · H OH l II O
2 2 OH 2 + 2 ° 2 OH 2 +] 2 HO HO 2 HO HO ₂

1A-49 867

H + OH

- £ - 11 O _n -h £ 11 ^ 2 2 * 2 H + JH _n O

H ₂ O -H HC H ₂ O + Π

° 2

1 H 0 "H- I II 0 _Λ + £ O + -i- O _n

² 22 ² 22 ² 2 ²

4) The hydrogen peroxide is found in rainwater and snow (see: Nouveau Traite de Ghimie Minerale, op. P. 543).

5) The hydrogen peroxide can be formed under certain conditions, e.g. by the decomposition of persulfates, Perborates, percarbonates ^ corresponding to the following reactions:

> H ₂ SO ₄ +

KaDO -I- Π, O y KaDO '+ HO

3 2 2 2Z

^K 2 ^C 2 ° 6 · '· ^{2 1!} 2 ° > ²

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Claims (3)

Claims 1 .J means for the determination of peroxides, containing a reagent alone or together with a chromogenic agent, characterized in that the reagent a) a component which contains at least one aromatic ring and at least one phenolic ÜH group, and b) a component having at least one reactive group attached to a structure that is derived is from i-phenyl-5-pyrazolinone, includes. 2. Agent according to claim 1, characterized in that the chromogenic agent ferrocyanide is. 3. application of the agent according to claim 1 or 2, characterized in that the mixture in which the Peroxide is to be detected, directly or after prior reaction with a chromogenic agent with the reagent in contact brings. 6231 809815/0712