DD249154A3 - TEST EQUIPMENT FOR DETERMINING SUBSTANCES IN BODY FLUIDS - Google Patents

Abstract

The invention relates to the improvement of the storage stability of test agents for the determination of substances in body fluids. The object of the invention is to improve the storage stability of test agents in order to avoid economic losses without at the same time substantially impairing the indicator reaction. The invention has for its object to produce test agents with reduced autoxidation. According to the invention, the object is achieved by adding one or more sulfites or a mixture of hydroxylamine and one or more sulfites to the test agents, since it has been found, surprisingly, that these additives can suppress autoxidation without significantly impairing the indicator reaction. Taking advantage of the inventive effect test agents can be produced with improved shelf life. These test agents can be used for example in medical diagnostics and in therapy control for the determination of substances in body fluids.

Description

Field of application of the invention

The invention relates to the improvement of the storage stability of test agents in which autoxidizable indicator systems are used for the determination of substances in body fluids. They should preferably be used in medical diagnostics and in therapy control.

Characteristic of the known technical solutions

In the previously known test agents, which use an oxidase / peroxidase reaction to determine substances in body fluids (eg test agents for the detection of glucose, uric acid, amino acids, triglycerides, cholesterol, pyruvate, etc.), are often used to detect the intermediary resulting hydrogen peroxide substituted benzidines (DE-OS 2460903) or substituted p-phenylenediamines used in conjunction with a color coupler.

A disadvantage of such indicator systems is their high sensitivity to light, atmospheric oxygen and moisture. In particular, the substituted p-phenylenediamines are increasingly attacked, especially at higher pH values, which are used for example for a fast color coupling or for certain enzymatic reactions. Therefore, even during the storage of the test agent, the indicator reaction partially proceeds without the action of the sample, whereby this test center! can only be used for a limited time.

It is known from photochemistry that solutions of substituted phenylenediamines can be stabilized by the addition of, for example, hydroxylamine and sodium sulfite against attack by atmospheric oxygen (DRP 743262, Meyer, K., Brune, W .: Z.wiss Photogfaphie46 [1951], 169). However, a z.T. considerable disturbance of the color reaction during the development process can be accepted.

It is also known that hydrogen oxide oxidizes both hydroxylamine and alkali metal sulfites very rapidly (Gmelin's Handbook of Inorganic Chemistry, Verlag Chemie, Berlin 1936, 23, 722, Gmelin's Handbook of Inorganic Chemistry, Verlag Chemie, Weinheim 1960, 9, 544) these substances to stabilize a system with the help of hydrogen peroxide is to be detected, appear impossible.

Object of the invention

The object of the invention is to improve the shelf life of test media using autoxidizable indicator systems to avoid economic losses, without significantly affecting the indicator reaction.

Explanation of the essence of the invention

The invention has for its object to produce test agents with reduced autoxidation and thus improved storage stability.

The test agents may be based either on templates impregnated with the reagents or on films containing the reagents. Before being used, they are converted into handy and thus material-saving formats (e.g.

Test strips) cut.

According to the invention the object is achieved in that the test agents on the basis of the oxidase / peroxidase reaction and with autoxidizable indicator systems one or more sulfites or a mixture of hydroxylamine and one or more sulfites are added, because it was surprisingly found that by these additives the autoxidation can be suppressed without significant impairment of the indicator reaction (see Table 1, test agents Nos. 2,3,4, 5,7,8,9,10 and compilation in Example 3), whereby the shelf life of the test means without sensitivity loss significantly improved (see Table 1). This undisturbed effect of the indicator reaction by the stabilizing additives of the present invention is particularly important for two reasons. On the one hand, in this case the degradation of the stabilizing additives occurring during storage can not lead to an increase in the sensitivity, ie. H. lead to false positive values. On the other hand, in an undisturbed indicator reaction, the water absorption of film-based test agents can be minimized, which is known to have an advantageous effect on the hematocrit dependence of the test results when using whole blood as the examination material.

Table 1: Influence of the amounts of stabilizing additives on the autoxidation and the color yield of various test agents (except the amount of oxidase used and the antioxidant concentration same formulations and properties at No. 1 to 5 or No. 6 to 10)

Glucose detection test reagent, pH 7.2 ... 7.4

No. Na 2 O 3 / hydroxylamine (each

in mol% based on substituted p-phenylenediamine)

1 0 2 10 3 50 4 100 5 200

relative increase in blank value within

Half a year in cooler

and dry storage (in%,

related to unstabilized

Movie)

! ΊθΟ

Test agent for detecting uric acid, pH 8.0 ... 8,2

No. Na ₂ SO ₃ (in mol% based on

substituted p-phenylenediamine)

6 0 7 10 8th 50 9 100 10 200

Relative blank increase within one year in cool and dry storage%, relative to unstabilized

Movie) __

TÖO

75:

47

40

34

Color yield (in%, based on unstabilized film, use of whole blood, 30 mmol / l glucose)

100 97 98 89, 69

Color yield (in%, based on unstabilized film, use of whole blood, 500μu.mol / L uric acid)

100 98 98 92 75

The suitability of these additives to prevent autoxidation was not foreseeable, since in addition to the Autöxydationsschutzwirkung and the high reactivity of these compounds to hydrogen peroxide, which is intermediately formed in the test agents with oxidase / peroxidase reaction and quantitatively detected, is well known (Gmelins Handbuch the inorganic chemistry, publishing house chemistry, Berlin 1936,23,572; Gmelin's manual of the inorganic chemistry, publishing house chemistry, Weinheim 1960,3,514f.). Conveniently, the additives are used in a stoichiometric ratio to the developer, without the effect achieved is limited to this concentration. By using the additives of the invention, test agents having improved storage stability can be prepared.

embodiments

In the following, the invention will be explained in more detail with reference to embodiments.

Example 1:

Test films for the detection of glucose

There are produced 5 test films with the following composition:

gelatin

HS'-sulfo ^ '- phenoxyphenyll-S-carboxyheptadecyl-amidopyrrazolone-io)] 4- (N, N-ö-sulfobutyl-butylammonium) -anitium sulfate Glucose oxidase: peroxidase

6g

368 mg 360 mg 15000U 1500U

and in addition to the usual excipients such as wetting agents, plasticizers and hardeners per gassing solution the following amounts of stabilizing additives:

Hydroxylamine [mg] Na ₂ SO ₃ -7H ₂ O [mg] sulfate O 0 7.5 23 37 116 74 232 148 464

FiIm no.

The mixtures are adjusted to pH 7.3 to 7.4 and applied to transparent cellulose acetate films, which are provided with an adhesive layer, and dried. Subsequently, the test films are conveniently cut into strips. The test films have the properties summarized in Tab. 1 with regard to their storage stability and color yield.

Example 2:

Test films for detecting uric acid

There are produced 5 test films with the following composition:

Gelatin i-tS'-sulfo ^ '- phenoxyphenyl J-S-carboxyheptadecyl-amidopyrrazolone-io)] 4- (N, N-8-sulfobutyl-butylammonium) anilinium sulfate uricase peroxidase

Na-borate buffer (SÖRENSEN-CLARK)

25 g 230 mg 150 mg

75 U 5000U

75 ml

As well as the usual auxiliaries, such as wetting agents, plasticizers and hardeners per gassing solution, the following amounts of sodium sulfite:

FiIm no. Na ₂ SO ₃ -7H ₂ O [mg] 6 0 7 10 8th 48 9 96 10 192

The mixtures are adjusted to pH 8.0... 8.2 and applied to transparent cellulose acetate films provided with an adhesive layer and dried. Subsequently, the test films are conveniently cut into strips. The test films have the properties summarized in Tab. 1 with regard to their storage stability and color yield. The above examples are not limiting of the solution according to the invention. They should demonstrate their generality for various enzymatic reactions and reactions at different pH values.

Example 3:

Test agent for the detection of glucose: -

There are produced 5 test films with the following composition:

Gelatin.

1- [3'-sulfo-4'-phenoxyphenyl] -3- [carboxyheptadecylamidopyrrazolone (5)] 4- (N, N-8-sulfobutyl-butylammonium) -silinium-sulfate glucose oxidase peroxydase

6g

368 mg 360 mg 15000U 1500U

and in addition to the usual excipients such as wetting agents, plasticizers and hardeners per gassing solution the following amounts of stabilizing additives:

FiIm no.

11 12 13 14 15 16 17 18 19 20 21 22 23

Hydroxylamine [mg]

7.5 37 74 148

(NH ₄ I ₂ SO ₃ K ₂ SO ₃ -2H ₂ O [Mg] [Mg] 0 0 10.5 0 53 0 107 0 214 0 0 18 0 89 0 179 0 357 10.5 0 53 0 107 0 214 0

The mixtures are adjusted to pH 7.3 to 7.4 and applied to transparent cellulose acetate films, which are provided with an adhesive layer, and dried. Subsequently, the test agents are conveniently cut into strips.

The test agents have the properties listed in the following list with regard to their storage stability and color yield.

Compilation of the properties of the test agents

FiIm no. relative increase in blank within one year Color yield (in%, based on the unstabili in cool and dry storage (in%, based film, use of whole blood, on the unstabilized film) 30 mmol / l glucose) 11 100 100 12 76 99 13 47 93 14 41 90 15 37 71 16 80 97 17 46 95 18 42 89 19 33 66 20 71 98 21 39 93 22 36 88 23 25 62

Claims (3)

1. A test agent for the determination of substances in body fluids based on the oxidase / peroxidase system and using aromatic diamines or benzidines as color developer and a color coupler or dye fixing agent at pH values of> 5, characterized in that in the test agents one or more alkali or ammonium sulfites or a mixture of hydroxylamine and one or more alkali or ammonium sulfites are included. 2. Test agent according to item 1, characterized in that the one or more alkali or ammonium sulfites in a total concentration of 2 to 10OOMol-% sulfite, preferably 50 to 200 mol% sulfite, based on the color developer, are included. 3. Testmittef according to item 1, characterized in that hydroxylamine in a concentration of 0 to 1000 mol%, preferably 50 to 200 mol%, based on the color developer, is included.