SE430104B - TEST COMPOSITION FOR DETERMINING A PEROXIDATIVE ACTIVE SUBSTANCE IN A SAMPLE AND ITS USE - Google Patents

Description

7so1154-0 2.

Clinical Chemistry Principles and Techniques (Hagerstown, Maryland; Harper and Row, 1974, pp. 1124-1125). This process involves using glacial acetic acid (buffer), diphenylamine (indicator) and hydrogen peroxide. Although such wet procedures have been found to be analytically useful, they are also encumbered with obvious nuances, the least notable of which is poor reagent stability and insufficient sensitivity. Such reagent solutions suffer from the inherent weakness, that the stability (and thus the sensitivity) decreases so fast that new reagent solutions have to be prepared after a few days of storage, which necessarily results in both too much time being required by the analytical staff and in poor economy. you have to throw away expensive reagents.

A second method for the determination of peroxidatively active substances, which is the method currently preferred by clinical examination and analysis personnel, is the use of so-called reagent strips of the "dip and read" type. A typical such device is a reagent strip manufactured by the Ames Company Division of Miles Laboratories, Inc. and marketed under the name Hemastix. This reagent strip essentially comprises a porous paper matrix attached to a strip or a plastic handle. The paper is impregnated with a buffered mixture of an organic hydroperoxide and o-tolidine. When the strip is immersed in a liquid containing hemoglobin, myoglobin, erythrocytes or other pseudoperoxidases, a blue color develops in the paper, the intensity of which is proportional to the concentration of the peroxidatively active substance in the sample. By comparing the developed color in the reagent strip with a standard color scale, the person performing the test can make a semi-quantitative determination of the unknown amount in the sample. The advantages of reagent strips over wet chemical procedures are mainly of two kinds: strips are easier to use because they do not require the preparation of reagents or any associated equipment, and the reagents exhibit greater stability, resulting in greater accuracy, sensitivity and economy. 7 However, despite the increased stability and sensitivity, the inherent advantages of strips over wet chemical processes still require further improvement. Although these properties of existing known strips for the determination of pseudoperoxidases have been considerably improved over wet chemical processes, it would nevertheless be a major advance if such strips could be made even more stable during storage and 7801164-0. 5 even more sensitive to peroxidatively active substances. It was to achieve such improvements that the research work that resulted in the present invention was initiated.

At least three attempts to achieve the above objectives have been reported in the prior art. An abstract in Chemical Abstracts, Volume 85, p. 186 (1976) describes a double dip process for the preparation of reagent strips containing o-tolidine and phenylisopropyl hydroperoxide. In this process, a solution of the indicator (o-tolidine - 2HCl) and polyvinylpyrrolidone in ethanol is prepared. To this solution was added a small amount of surfactant and a sufficient amount of citrate buffer to achieve pH 5.7. Filter paper strips impregnated with ethylcellulose were dipped in this solution and dried. The filter paper so impregnated was then dipped in a second solution containing 1,4-diazabicyclo [2.2.2] octane, phenylisopropyl hydroperoxide and polyvinylpyrrolidone, dissolved in a mixture of ethanol and toluene. The purpose of this experiment was to stabilize the combination of the peroxide and the indicator by using the bicyclooctane derivative and polyvinylpyrrolidone.

A second such process is described in U.S. Patent No. 5,855,471. This patent describes the use of phosphoric or phosphonic acid amides, in which the substituent amido groups are primarily N-morpholine radicals.

In addition to these experiments, there is also an experiment described in U.S. Patent No. 5,252,762, in which the organic hydroperoxide is physically entrapped within a colloidal material, such as gelatin. When such a test strip is used, the aqueous sample dissolves the gelatin capsules, thereby exposing the hydroperoxide to react with the indicator in the presence of a peroxidatively active substance.

Each of these previous experiments aimed to stabilize the reagents so that the potentially incompatible reactive constituents (hydroperoxide and indicator) would not be combined prematurely, thereby making the test strip less sensitive. It can be said, however, that these known methods did not aim at the combined purposes of simultaneously increasing stability and sensitivity, but rather tried to preserve prevailing sensitivity by preventing decomposition of the reagents during storage.

Another known publication of interest is U.S. Patent No. 5,256,850. This patent describes the stabilization of organic hydroperoxides which are used as catalysts and oxidizing agents. The patent describes the use of primary, secondary or tertiary amine salts with organic peroxides. This publication in no way relates to test strips containing reagents. Realizing that none of the procedures described above would achieve the degree of stability and sensitivity desired in a test strip for the determination of peroxidatively active substances, it was decided to approach the problem in a completely different way. This approach to the problem was discovered during research which led to the present invention and resulted in a composition and apparatus which fully fulfilled the desired objectives of increased stability and sensitivity. However, even more surprisingly, an additional advantage was obtained in this work, namely an improved process for the manufacture of the device referred to here, whereby the preparation becomes considerably simpler than the methods indicated in the above-mentioned known publications, and more particularly a method with a single dip.

Briefly, the present invention consists in a test composition and a device for determining the presence of a pseudoperoxidase in a sample. The test composition comprises an organic hydroperoxide, an indicator capable of forcing a determinable response in the presence of a hydroperoxide and a peroxidatively active substance, and either or both of the compound (s): diluent with pre-main OTR R1 or NR1R2 or mixtures thereof, where B1 and R2 are the same or different and represent alkyl or alkoxy containing 1 to about 6 carbon atoms, or aryl and R1 (2) a borate ester of the formula / cc fl zïnl- o \ N_ (cuz) n_ 0- B (CH2) p * -0 7801164--0 in which m, n and p, which may be the same or different, are integers from l to about 4.

The device of the present invention comprises the above composition incorporated in a carrier matrix. The process comprises preparing the test composition and incorporating it into the matrix.

The organic hydroperoxide to be used in the test composition can be selected from many well known organic hydroperoxides.

However, it must be able to react with a peroxidatively active substance in the presence of a peroxide sensitive indicator to produce a detectable response, such as a color change or a change in the amount of light absorbed or reflected by the test composition. Among the hydroperoxides which have been found to be suitable are tert-butyl hydroperoxide, cumene hydroperoxide, diisopropylbenzene hydroperoxide, 2,5-dimethylhexane-2,5-dihydroperoxide, paramentan hydroperoxide or mixtures thereof. Of these, cumene hydroperoxide has proven to be the most suitable.

There are many indicators which are capable of producing a determinable response in the presence of a hydroperoxide and a pseudoperoxidase, and which are therefore suitable for use according to the invention. These include compounds of so-called "benzidine type", and typical examples of such are benzidine, o-tolidine, 5,3 ', 5,5'-tetramethylbenzidine, 2,7-diaminofluorene or mixtures thereof in various proportions.

The borate esters, which are currently considered to contribute to increased stability and sensitivity, have the above formula. Among compounds which fall under this general formula, it has been found that trimethanolamine borate, triethanolamine borate and tri (n-propanol) amine borate are particularly suitable. These compounds correspond to the above general formula when m, n and p are the same integer and are 1, 2 and 3, respectively.

The amount of borate tests to be used in the present composition and apparatus can vary widely. This is reflected in the following examples. Thus, the molar ratio of triethanolamine borate equivalents to cumene hydroperoxide equivalents is 4.71.

Conversely, Example 3 gives a molar ratio of 2.83 between borate and hydroperoxide (a normality ratio of 1.42 if one considers that the peroxide is difunctional).

However, equivalent ratios between about 1.4 to about 5, as set forth in the examples, are not limiting with respect to the amount of boron to be used in the present invention. ... .- '' h which is sufficient to achieve the desired degree of stability and _. The sensitivity of the test composition can be used, and this degree is still easily determined at the laboratory table in accordance with the present description of the invention.

In a preferred embodiment of the invention, the composition comprises cumene hydroperoxide, o-tolidine, a mixture of dimethyl sulfone and dimethyl sulfoxide and triethanolamine borate. The test composition is typically prepared by dissolving or suspending each ingredient to be used in water or any other suitable suspending or solvent agent. Such solvents or suspending agents include chloroform, methanol, ethanol, methylene chloride, cyclohexane, etc. The test device can be prepared by a single dipping method. Accordingly, a portion of the carrier matrix material is immersed in the solution or suspension and then dried. The test devices thus obtained show very little loss of reactivity even after storage under difficult conditions, such as about 60 ° C to about 7000 for 1-5 days and longer. For the sake of comparison, similar test devices were prepared but without the presence of borate tests and / or diluents. When these strips were stored under essentially identical, difficult conditions, a significant loss in reactivity and sensitivity was observed.

The carrier matrix used in the manufacture of the test device can take a variety of forms. "Thus, U.S. Patent No. 5,846,247 discloses the use of felt, porous ceramic strips and woven or carpeted glass fibers. U.S. Pat. No. 3,552,938 further discloses the use of wood sticks, cloth, sponge materials and clayey substances. The use of synthetic resin and fiberglass blankets as a carrier matrix is proposed in British Pat. No. 1,369,139. Another British Pat. No. 1,349,623 proposes the use of Polyamide fibers are disclosed in French Patent Specification 2,170,397. Despite these proposals, the material which is preferably used as a carrier matrix in known devices and which is particularly suitable for the present invention is a liquid-absorbent paper, such as filter paper, From the above it will be appreciated that there is a large clearance in the choice of suitable material for use as a carrier matrix, and that the matrix can take various physical forms. All of these types are considered to be within the scope of the present invention.

The mechanism by which the present solution to the problem of providing improved stability and sensitivity is obtained is not known for sure. However, the unique chemical properties of the borate ester described and for which protection is claimed in accordance with the present invention provide a sufficient basis for speculation. It is known that peroxides are generally unstable compounds, or at least less stable than most compounds found in nature.

Some are explosive. Others, such as organic hydroperoxides (namely, cumene hydroperoxide) are relatively stable, but are readily dissociated in the presence of acids, such as those commonly used in occult blood sensitive devices. When this decomposition occurs in the presence of an oxidizable indicator (such as those described herein), a redox reaction occurs. It is assumed that this premature reaction is the cause of the reduced sensitivity of peroxidatively sensitive reaction strips. On the other hand, borate esters of the type described here are unique to the geometry of the nitrogen and boron atoms in the bicyclic structure, in that these atoms each form a bridgehead. The nitrogen atom at one bridgehead is electron-rich and contains an undivided pair of electrons, which protrude in the direction from the axis of the molecule.

The second bridgehead, the boron atom, which is located at the other axial end of the molecule, lacks electrons and tends to coordinate with electron-rich anions.

Due to its electron richness, it is thus quite probable that the nitrogen end of the molecule can bind a proton, while the electron-poor boron atom at the other bridgehead can be coordinatively coupled with an anionic peroxide residue. Thus, it is believed that the unique electron distribution of the bicyclic molecules discussed herein stabilizes the organic peroxide of the present test composition by chemically positioning it between the peroxidic proton and the oxygen atom to form a coordinately coupled ion pair.

Thus, in the present test composition, the surprisingly increased stability is assumed to arise by preventing the organic hydroperoxide from ionically reacting with the indicator until the solvating ability of the sample destroys the peroxide-borate complex and releases the peroxide for oxidation of the indicator in the presence of a peroxidative agent. and subject.

.But even though the marked increase in stability and sensitivity is the result of the use of borate esters, it has surprisingly been found that even greater stability and sensitivity can arise when the borate ester and the above solvents are both included in the test composition. The diluent compounds believed to contribute to the increased stability and sensitivity of the present invention have the formulas set forth above. Of the compounds encompassed by these general formulas, it has been found that N, N-dimethylformamide, dimethylsulfoxide, dimethylsulfone or mixtures thereof are particularly suitable. Other diluents which have been found to be effective are benzyl sulfoxide, 4-chlorophenyl sulfone, 4-fluoro-3-nitrophenyl sulfone and others. These compounds correspond to the above formulas, if R 1 and R 2 are each methyl.

The scope of the designations R1 and R2 according to the invention is quite wide. Thus, B1 and R2 refer to substituted and unsubstituted alkyl containing 1 to about 6 carbon atoms. Examples of alkyl groups are methyl, letyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl and other isomers, including the isomers of hexane. These alkyl groups are also illustrative of the alkoxy groups which fall under the above definition of R1 and R2. When R 1 and R 2 are aryl, the scope is likewise at, and the term aryl, as used herein, includes substituted and unsubstituted aryl groups, such as phenyl, benzyl, tolyl, anilino, naphthyl, etc. Typical groups, with which B1 and H2 may be substituted are amino, nitro, amido, nitrile, hydroxyl, alkoxy, halogen, phenyl, sulfonic acid, carboxylic acid, etc. The amount of diluent used in the present composition, apparatus and method may vary widely. and can be easily determined at the laboratory table. When the diluent is a liquid, such as dimethyl sulfoxide (see Table II below), the amount may vary from about 10% to about 100%, based on the volume of diluent added to the composition, compared to the volume of solvent or suspending agent (in the case of Example 12). water). A preferred amount is from about 25% to about 100%.

Similarly, since the diluent is a mixture of more than one compound of the type disclosed herein, the ratio of the amounts of the various compounds used in the diluent may vary widely, and these can also be readily determined in the laboratory. The following examples are intended to further illustrate the invention without, therefore, limiting the same. 7801164-0 9 A. Preparation of test composition and apparatus.

Example 1. The test composition.

A test composition was prepared by dissolving the following ingredients in 150 ml of deionized water. The ingredients were added to the water in the order in which they are specified. trisodium citrate 3.2 g citric acid 2.2 g tetrasodium salt of ethylenediaminetetraacetic acid 0.1 g dimethyl sulfone 1.0 g sodium lauryl sulfate 1.0 g 6-methoxyquinoline 0.5 g dimethyl sulfoxide 25.0 ml acetone 25.0 ml cumene hydroperoxide 2.0 g triethanolamine borate (Aldrich Chemical Co., Inc.) 10.0 g 6-tolidine 0.4 Example 2. Test device.

Strips of filter paper Whatmann EMM were dipped in the test composition according to Example 1. The dipped strips were dried at 7000 for about 15 minutes to form carrier matrices impregnated with test composition. The dried carrier matrices are then attached to strips or handles of plastic (polystyrene) by means of double-sided adhesive tape, purchased from EM Company. The test devices thus obtained can be used for the determination of peroxidatively active substances by immersion in a sample, such as urine which is suspected to contain such substances, and observation of any color development in the carrier matrix.

B. Testing the stability of different test compositions.

Example 2. Vermæiav boratestern. The purpose of this experiment was to demonstrate the effect of triethanolamine borate on test compositions for determining occult blood, compared to other known additives. The following solution was prepared. chloroform 100 ml of o-tolidine 0.5 g of 2,5-dimethylhexane-2,5-dihydroperoxide 2.0 g of poly (N-vinylpyrrolidone) 10.0 g To samples of this solution (20 ml each) were added 1 g of the additives in subsequent table. These test composition solutions were allowed to stand at room temperature for about 5 hours and the observed results are summarized in Table I below. t73o11e4-o 10 Sample No. Additive l _ Result l hexamethylenetetramine Very dark, almost black solution. f g Poor sensitivity. 2 l¿4-diazabicyclo- Dark green / brownish discoloration. Mi- - ¿2.2.g / octane nimai sensitivity to Occult blood 1 f urine. 3 triethanolamine borate Weak browning. High sensitivity to occult blood in urine. 4 * control (no g Became black within about 2 hours. Unused additive g bar for analysis of occult blood.

Example gel 4. Effect of diluent. To a beaker containing 500 ml were added the following ingredients: chloroform - 100 ml of cumene hydroperoxide 4.0 g of totolidine 0.4 g of dimethyl sulphoxide to 25.0 ml The test composition thus obtained, which was slightly yellow when mixed with the above ingredients, was transferred to an Erlenmeyer flask, which was corked and allowed to stand on the laboratory table at room temperature (18.3 ° C) overnight. After 24 hours, the test composition had become only slightly darker than when it was prepared.

Exem el. Comparison with Example 4.

A test composition for control purposes, ie. for comparison with the present invention, was prepared according to the same procedure as in Example 4 except that the dimethyl sulfoxide was replaced with 25 ml of additional chloroform. This test composition without dimethyl sulfoxide was introduced into an Erlenmeyer flask, which was sealed and allowed to stand on the laboratory bench as in Example 4 (ie at 18.3 ° C for 24 hours). In contrast to the composition of Example 4, the control composition darkened to an almost black color, indicating that the test composition obtained improved stability by the diluent.

C. Stabilization of the test devices by the borate ester. _ExemQel 6. Control test.

A solution of the following test composition was prepared to prepare a reagent strip sensitive to pseudoperoxidases.

This composition does not contain the borate ester of the present invention. in 7801164-0 11 H 2 O 150 ml trisodium citrate 3.2 g citric acid 2.2 g tetrasodium salt of ethylenediaminetetraacetic acid 0.1 g dimethyl sulfone 10.0 g sodium lauryl sulfate 1.0 g 6-methoxyquinoline 0.5 g dimethyl sulfoxide 25.0 ml acetone 25, 0 ml cumene hydroperoxide 2.0 g o-tolidine 0.4 g A piece of filter paper Whatmann EMM was dipped in the above test composition solution and dried at 7000. The dried paper was cut into squares of about 5 mm side, and these were attached to plastic handles using double-sided adhesive tape (from 3M Company), forming test devices.

Example Z. The present invention.

A solution was prepared as in Example 6 above except that 10 g of triethanolamine borate was added before the addition of the cumene hydroperoxide. Test devices were prepared from this solution in the same manner as in Example 6.

Example 8. iA-diazabicyclo fi zz.g7oktan.

A solution was prepared as in Example 6 above except that 10 g of 1,4-diazabicyclo [2.2.1] octane was added prior to the addition of the cumene hydroperoxide. This solution was used to make test devices in the same manner as in Example 6.

Example 2. Hexamethylenetetramine.

A solution was prepared as in Example 6 above except that 10 g of hexamethylenetetramine was added before the addition of cumene hydroperoxide. Test devices were prepared from this solution in the same manner as in Example 6 above.

Example 10. Comparative tests on stability and sensitivity.

The test devices of Examples 6-9 were subjected to severe conditions to determine their relative stability and sensitivity. Devices from Examples 6-8 were stored for three days at about 6,000 and those of Example 9 for one day at about 7,000. These test devices subjected to severe conditions were then tested for their sensitivity by dipping in urine containing one millionth of blood.

Only the device containing triethanolamine borate (Example 7) showed a color change at this concentration of occult blood (namely 0.015 mg / dl). The remaining devices were insensitive to this concentration of occult blood.t _ D. Stabilization of the test devices by the diluent.

Exemgel ll. Six test devices were prepared from six test composition solutions containing varying amounts of dimethyl sulfoxide according to Table II below. The six test composition solutions were all prepared with the following composition: water 75 ml trisodium citrate 3.2 g citric acid a 4.5 g criethanolamine borate a 10.0 g tetrasodium salt of ethylene diamine tetraacetic acid 0.1 g dimethyl sulfone 5.0 g of sodium lauryl sulfate 1.0 g of 6-mecoxycinoline 0.5 g of cumene hydroperoxide 2.0 g of 0-toliain 0.4 g To each of these six test composition solutions were added the following amounts of dimethyl sulfoxide and methanol: Table II, Test composition Dimethyl sulfoxide Methanol no. ml) (ml) ii 0 75 2 5 70 3 10 '65 4 25 50 5 40 35 6 60 15 Test devices were then prepared from each of these test composition solutions by dipping a piece of filter paper Whatmann EMM in each solution. The dipped strips of filter paper were taken out of the respective solution, dried and subjected to intensive conditions in an oven at 7000 for 18 hours. After this treatment, each strip was tested in a test solution comprising a dilution of 1,000,000 fresh whole blood in urine.

The intensity of the color which developed in each strip constituted a comparator. The results are summarized in Table III below.

Table III Test device Color change x * nrx l 0 (no staining) 2 l (traces) 3 5 4 5 5 6 6 7 (almost no loss in reactivity) K The number of the test devices corresponds to the number of the test compositions in Table II. The color assessment was based on a scale fi from 0 to 8, where 8 denotes the color developed by a newly manufactured, unconditionally treated test device prepared from test composition No. 6 (Table II). A rating of 0 implies no staining, a rating of 7 indicates almost no loss in reactivity and a t rating of l indicates only traces of staining. It is quite obvious from the data in Table III that the sensitivity increases with the amount of diluent added (here dimethyl sulfoxide) in the test composition.

E. increased stability of the test composition (with borate tests) with the addition of various diluents.

Example 12. Eight test compositions were prepared in the same manner as in Example 11. Seven of these contained different diluents and one (control) did not contain any diluent. The purpose of this experiment was to determine the ability of various diluents to increase the stability of test compositions that were sensitive to peroxidatively active substances, while keeping the amount of borate tests in the composition constant.

A solution was prepared containing the following ingredients: water 50 ml trisodium citrate 3.2 g citric acid 2.2 g sodium lauryl sulfate 1.0 g 6-methoxyquinoline 1.0 g methanol 50.0 ml triethanolamine borate 5.0 g cumene hydroperoxide 2,0 g-o-tolidine 0.4 g A small sample of this solution was added to each of eight test tubes. Thereafter, small amounts of the diluents listed in Table IV were added to each of the test tubes, which were then sealed and allowed to stand at room temperature on the laboratory table for one week. At the end of this week, the test tubes were examined for relative dark staining, with the test composition being less stable the darker the paint. The results are given in Table IV in the descending order of stability (ie test composition 1 is the most stable and test composition 8 is the least stable).

Table IV Test composition Diluent no. Dimethyl sulfoxide N, N-dimethylformamide dimethyl sulfone Benzyl sulfoxy 4-chlorophenyl sulfone 4-fluoro-3-nitrophenyl sulfone 72 in 2-imidazolidone 8 (control) ^ no O '\ U1 \ -I =' \ NI IV samples 1 and 2 show excellent stability, solution 3 a lesser but nevertheless good stability, solutions 4-7 were moderately stable and solution 8, the control sample, was considerably less stable than solutions 1-7. F. Preferred Embodiments. iExample l fi.

The following ingredients are mixed with 150 ml of water: trisodium citrate 3.2 g of citric acid. 7 to 2.2 g of tetrasodium salt of ethylenediaminetetraacetic acid 0.1 g of ethyl sulfone 2 '10.0 g of sodium lauryl sulphate 1.0 g of 6-methoxyquinoline 0.5 g of dimethyl sulphoxide 25.0 ml of acetone. 25.0 ml triethanolamine borate 10.0 g cumene hydroperoxide 2 2.0 g o-tolidine 0.4 g g Strips of Whatmann 3MM filter paper were dipped in the above solution and dried at 70 ° C. After the filter paper has dried, it is cut into squares with a 4 mm side. These squares were then mounted on polystyrene handles measuring 8 x 15 mm using double sided adhesive tape (3M company). Test devices prepared in the above manner were subjected to intense conditions at 60 ° C for three days and were found to be sensitive to occult blood in urine at concentrations of at least as low as 0.015 mg%.

Example 14.

This example illustrates a second preferred embodiment of the test device according to the invention.

A test composition was prepared by mixing the following ingredients in the following order in a beaker with stirring: water 75 ml trisodium citrate 5.2 g citric acid 4.15 g triethanolamine borate 10.0 g ethylenediaminetetraacetic acid 0.1 g sodium lauryl sulfate 1.5 g dimethylsulfone 10.0 6-methoxyquinoline 0.6 g N, N-dimethylformamide 75 ml cumene hydroperoxide 3.0 g o-tolidine 0.8 g Strips of filter paper Whatmann EMM were dipped in the above composition and dried for about 11 minutes at about 90 ~ 92 ° C. The dried strips were then mounted on plastic handles using double-sided adhesive tape.

Some of the strips were subjected to intensive conditions by storage at 70 ° C for one day and others by storage at 40 ° C for twelve weeks. Both batches of strips developed a blue / green color when brought into contact with a 1: 1 x 10 dilution of fresh whole blood in urine.

In summary, the above examples show how to prepare and use the composition and test device of the invention (Examples 1 and 2), comparative results regarding stability and sensitivity (Examples 3-12) and describe preferred embodiments (Examples 13 and 14). The effect of the borate ester and the diluent, separately and in combination with each other, on the test composition is shown in Examples 3-5, unless the Effects When the test device is shown in Example 6-1. The use of different diluents is exemplified in Example 12.

Claims (8)

7so1àe4-o 16 P a t e n t k r a v A test composition for determining a peroxidatively active substance in a sample, comprising an organic hydroperoxide and an indicator capable of producing a determinable response in the presence of the hydroperoxide and a peroxidatively active substance, characterized in that the composition further comprises either or both of (a) a diluent of the formula go RT 1> sæo, R1 __. s ___. R2, 122 å, / o in R1 _._ c NR1R2 or mixtures thereof, wherein R1 and R2 are the same or different and represent hydrogen , alkyl or alkoxy, containing 1-6 carbon atoms, or aryl, and (b) a borate ester of the formula in (cnzh 2 o - N - (CH 2) n - -O 2 B (CH 2) - -O in which m, n and p, which are the same or different, are integers from T to 4. Composition according to Claim 1, characterized in that it comprises diluents consisting of dimethyl sulphoxide, dimethyl sulphone, N, N-dimethylformamide, benzyl sulphoxide, 4-chlorophenyl sulphon, 4-fluoro-3-nitrophenylsulphone or mixtures of d-sulfonates. A composition according to claim 1 or 2, characterized in that it comprises borate ester, which consists of trimethanolamine borate, triethanolamine borate, tri (n-propanol) amine borate or mixtures thereof. Composition according to any one of Claims 1 to 3, characterized in that the organic hydroperoxide consists of tert-butyl hydroperoxide, cumene hydroperoxide, diisopropylbenzene hydroperoxide, 2,5-dimethylhexane-2,5-dihydroperoxide, paramentan hydroperoxide or mixtures thereof. § 5. Composition according to any one of claims 1-4, characterized in that the indicator is benzidine, o-tolidine, 3,3 ', 5,5'-tetramethylbenzidine, 2,7-diaminofluorene or mixtures thereof . Composition according to any one of claims 1-5, characterized in that it is incorporated in a carrier matrix. Composition according to Claim 6, characterized in that the carrier matrix consists of liquid-absorbent paper. Use of a test compound of a peroxidatively active substance in a sample, in that the sample is brought into contact with the composition and a definable response is observed, said test composition comprising on the one hand an organic hydroperoxide and an indicator, which is capable of producing said response in the presence of the hydroperoxide and the peroxidatively active substance, and in addition either or both of (a) comprises a diluent of the formula o R. R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, R 2, 1-6 carbon atoms, or aryl, and (b) a borate ester of the formula (CH 2) 2 O / 2 m 2 N - - (CH 2) I 1-- O - B 2 / (c fi 2) pv which m, n and p, which are the same or different, are integers from 1-4.