CS219888B2 - Testing means for fixing the pressence of peroxiding active substances - Google Patents

Description

The present invention relates to a test composition for determining the presence of poroxidizing agents in; of the test sample. In particular, the invention relates to test devices for qualitative and semi-quantitative analysis of a sample with peroxidizing agents such as hemoglobin, secret blood and peroxidases.

Numerous analytical methods are currently available to detect the presence of peroxidation active agents, for example in urine samples, faecal suspensions and gastrointestinal contents. Hemoglobin and its derivatives are such typical 'peroxidation active' substances because their behavior is close to that of the enzyme peroxidase. Such substances are also referred to as pseudoperoxidases. Peroxidizing agents are similar to enzymes in that they catalyze the non -ox reaction between peroxides and benzidine, o-tolidine, 3,3 ‘, 5,5‘-tetramethylbenzidine,

With 2,7-diaminofluorene or similar indicators, thereby creating a detectable reaction, such as a discoloration. Thus, most methods of determining the presence of occult blood in the samples examined are related to this pseudoperoxidase activity.

Over time, various methods have been developed which relate to the catalysis of peroxidation oxidation, which is similar to the enzymatic catalysis of staining generators. These methods include, in particular, wet chemical processes and strips that carry a "dip and evaluation" reagent. A typical example of the prior art is given by Richard M. Henry et al. in Clinioal Chemistiry Principles and Techniques (Hagerstown, Maryland: Harper and Row, 1974), pp. 1124-1125. This method involves the use of concentrated acetic acid (buffer), diphenylamine (indicator), and hydrogen peroxide. Although these engine methods have shown analytical applicability, they are nevertheless burdened with obvious shortcomings, of which not least are poor reagent stability and poor sensitivity. Such reagent solutions possess such a rapid decrease in stability (i.e. sensitivity) that fresh reagents must be prepared after a few days of storage, a necessity which results in unnecessary waste of time for the analytical staff and low economy because of the wastage of expensive reagents.

The second method for the determination of peroxidation active substances, which is one of the methods currently preferred by clinical chemists and analysts, involves the use of reagent papers such as "immersion and evaluation". Such a typical aid is reagent paper manufactured by Ames Oompany's Divislon of Miles Laboratories, Inc. and is sold under the name HEMASTIX ^(R) . The reagent paper comprises a substantially porous paper material mounted on a strip or plastic handle. The paper is impregnated with a buffered mixture of organic peroxide and σ-tolidine. After immersion in a liquid containing hemoglobin, myoglobin, erythnocytes or other pseudoperoxidases, a blue color develops on the paper, the intensity of which is proportional to the concentration of peroxidation active substance in the sample. By comparing the coloration produced on the reagent strip with the standard colored paper, the investigator can determine (on a semi-quantitative basis) the amount of unknown substance present in the sample.

Thus, the benefits of reagent papers over wet chemical methods are predominantly of two kinds: papers are easier to use because neither reagent preparation nor equipment operation is required, and reagent stability is increased, resulting in greater accuracy, sensitivity and economy.

Despite the advantages of paper over wet chemical methods, the stability and sensitivity properties still require further improvement. Although these properties in the prior art papers for the determination of pseudoperoxidases significantly exceed those of wet chemical methods. However, there would be great progress in the art if such papers could be made even more stable in storage and more sensitive to peroxidation active substances. The research activity resulting in the present invention was directed to achieving this improvement.

At least three attempts to achieve the above objectives are known in the art. Reference in Chemical Abstracts, Vol. 85, hundred. 186 (1976) discloses a double dip method for preparing reagent papers containing o-tolidine and phenylisopropyl peroxide. This method prepares a solution of the indicator (dihydrochloride and tolidine) and polyivinylpyrrolidone in ethanol, to which a small amount of a surfactant and a sufficient amount of citrate buffer is added to reach a pH of 3.7. The impregnated filter paper was then immersed in a second solution containing 1,4-diazabicyclo [2.2.2] octane, phenynyl isoprapyl peroxide and polyvinylpyrrolidone dissolved in an ethanol-toluene mixture. an indicator using a bicyclic octane derivative and polyvinylpyrrolidone.

Another such method is disclosed in U.S. Pat. No. 3,853,471. This patent describes the use of phosphoric or phosphonic acid amides in which the amido substituents are primarily N-morpholine radicals.

In addition to these experiments, there is a reference to U.S. Pat. No. 3,252,762, wherein the organic peroxide is physically encapsulated in a colloidal material such as gelatin. When using such a test strip, the aqueous solution of the test sample dissolves the gelatin capsules, releasing the peroxide for further reaction with the indicator in the presence of the peroxidation agent.

Each of these earlier attempts has attempted to stabilize the reagents so that potentially incompatible reactants (peroxide and indicator) do not combine prematurely, thereby rendering test papers less sensitive, so it can be said that prior art methods are not directed towards combinations that would they increased both stability and sensitivity at the same time, but rather tried to protect the existing sensitivity by preventing the decomposition of the reagent during storage.

Another important reference to the prior art is U.S. Pat. No. 3,326,850. The patent relates to the stabilization of organic peroxides used as catalysts and oxidizing agents. The authors in this reference describe the use of salts of primary, secondary or tertiary amines with organic peroxides. This reference does not relate in any way to the reagent papers.

After evaluating that none of the above methods would achieve the stability and sensitivity required for the test paper for the detection of peroxidation active substances, the present inventor decided to take a completely different direction. This different approach was found during the research that led to the invention and resulted in a blend and equipment that perfectly met the requirements for increased stability and sensitivity.

However, and even more surprisingly, the result of this work was yet another advantage - an improved method of manufacturing the apparatus than previously known, and production is dramatically simpler than the methods mentioned in the prior art references - a single dip method.

Testing agent for the presence of peroxidising agents such as hemoglobin, secret blood and peroxidase in a test sample containing an organic hydroperoxide, for example t-butyl hydroperoxide, cumene hydroperoxide, diisopropylbenzene hydroperoxide, 2,5-dimethylhexane-2,5-dihydroperoxide, paramonthanhydroperoxide or mixtures thereof and an indicator capable of providing a detectable response in the presence of said hydroperoxide and a peroxidizing agent such as benzidine, o-tolidine, 3,3 ', 5,5'-tetramethylbenzidine,

2,7-diaminolfluorene or mixtures thereof, according to the invention, also comprises a compound of the following formulas:

O

Rj -— C o

NR, _and R ⁰ \ ~~ O

XCH & -O

or mixtures thereof; in the above general formulas

R1 and R2, which are the same or different, are hydrogen, aryl, alkyl or alkoxy, in each case having 1 to 6 carbon atoms in each of said radicals, and m, n and p, which are the same or different, are integers 1 to 4.

The test composition according to the invention preferably contains dimethyl sulfoxide, dimethylsulfion, id, Ν-dimethylformamide, benzylsulfoxide, 4-chlorophenylsulfone, 4-fluoro-3-nitrophenylsulfone and mixtures thereof, especially for example dimethylsulfone. and N, N-dimethylfortramide.

The test composition according to the invention may also contain trimethanolamine, triethanolamine, tri (n-propainol) amine or mixtures thereof.

The test composition according to the invention may comprise as said compound triethanolamine borate, dimethylsulfone, Ν, Ν-dimethylformamide or mixtures thereof, and trimethanolamine, triethanolamine, triethepropanol amine or mixtures thereof, or a mixture of dimethylsulfone, N, N-dimethylformamide and triethanolamine borate. .

The test composition of the invention may preferably contain such compositions incorporated into a carrier matrix.

The organic peroxide contemplated for use in the test mixture can be selected from many well known organic peroxides. It shall, however, be capable of reacting with the peroxidation active substance in the presence of a peroxide-sensitive indicator to produce a detectable reaction, for example, color changes or changes in the amount of absorbed or reflected light of the test mixture. Useful peroxides found include tert-butyl peroxide, cumene peroxide, diisopropylbenzene peroxide, 2,5-dimethylhexane-2,5-diperoxide, paramethane peroxide, or mixtures thereof. Rumenperoxide was found to be the most advantageous.

There are numerous indicators that are capable of generating a detectable reaction in the presence of peroxide and pseudoperoxidase and are therefore suitable for use in the present invention. They include compounds of the so-called 'berizidine type', of which benzidine, o-tolidine, 3,3 ‘, 5,5‘-tetramethylhenzidine, 2,7-diaminofluorene or mixtures thereof in various proportions are typical.

Borate esters currently believed to contribute to increased stability and sensitivity have the above structure. Among the compounds included in this general structure, 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 wherein m, n and p are the same or different and are integers 1, 2 and 3.

The amount of borate ester needed in this mixture and apparatus can be varied widely. This is reflected in the examples below. For example, in Example 1, the molar ratio of triethanolamine borate equivalents to cumene peroxide equivalents is 4.71. On the other hand. Example 3 illustrates a 2.83 molar ratio of borate to peroxide (1.42 ratio of normality when considering the peroxide difunctionality).

However, as shown in the examples, the ratio of equivalents of the order of 1.4 to about 5 does not in any way limit the amount of borate useful in the present invention. Any amount sufficient to achieve the desired degree of stability and sensitivity of the test mixture can be used and can be readily determined experimentally.

In a preferred embodiment of the invention, the mixture comprises cumene peroxide, o-tolidine and a mixture of dimethylsulfone and dimethylsulfoxide and triethanolamine borate.

The test mixture is typically prepared by dissolving or suspending portions of each component used in water or other suitable suspension medium or solvent. Such a solvent includes chloroform, methanol, ethanol, methyl chloride, cyclohexane, etc.

The test device can be manufactured by a single dip process. According to this method, a portion of the carrier material is immersed in a solution or suspension and then dried. The devices thus prepared exhibit little loss of reactivity even after storage under stressed conditions, for example at a temperature of about 60 ° C to about 70 ° C for three days or longer. For comparison, test devices are similarly prepared but in the absence of borate ester and / or diluent. When these strips are stored under substantially identical stress conditions, a dramatic loss of reactivity and sensitivity can be observed.

The carrier material used in the manufacture of the test device can take many forms. Ame. U.S. Patent No. 3,846,247 describes the use of felt, porous ceramic strips and woven or felted glass fibers. In addition, U.S. Pat. No. 3,552,938 discloses the use of wood sticks, tissue, foam, and clay. The use of synthetic resin yarn and fiberglass felt as a carrier material is suggested in British Patent No. 1,369,139. Another British Patent No. 1,349,623 suggests the use of a translucent thin-fiber mesh as a backing for paper backing material. Polyamide fibers are disclosed in French Patent 2,170,397. Despite these suggestions, however, the material which is mainly used in the art as a carrier material and which is particularly suitable for the present invention is absorbent paper, such as filter paper. It can be seen that there is a large amount in the selection of a suitable material for use as a carrier material, and the material may take various physical forms. All of these types are intended to be within the scope of this invention.

The mechanism by which this procedure produces increased stability and sensitivity is not safely known. However, the exceptional chemical properties of the borate ester described and protected ester described and protected in the present invention provide sufficient grounds for consideration, it is known that proxides are generally unstable or at least less stable than most naturally occurring substances. Some are explosive. Others, such as organic peroxides (such as cumene peroxide), are relatively stable, but are readily dissociated in the presence of acids as commonly used in an occult blood sensitive assay device. When such decomposition occurs in the presence of an oxidizable indicator (as described herein), a redox reaction occurs. This premature reaction is believed to cause decreased sensitivity of peroxidatively sensitive reagent papers.

On the other hand, the borate esters described herein are unique in terms of the geometry of the nitrogen and boron atoms in the bicyclic structure, each containing a separate bridging atom. One bridge nitrogen atom is rich in electrons and contains an unshared electron pair projecting out of the molecular axis. The other bridging atom, the boron atom, situated at the other end of the axis, is electron-poor and tends to coordinate bonding and anion-rich electrons.

As a result of the electron richness, the nitrogen end of the molecule, as is conceivable, could bind a proton, while the electron-poor bridge boron atom could coordinate an anonone peroxide residue.

It is believed that the unique electron distribution of these discussed bicyclic molecules stabilizes the organic peroxide in the presence of the test mixture by chemically inserting it between the peroxide proton and the oxygen atom, forming a coordinated bonded ion pair.

Surprisingly, the increased stability in this test mixture is believed to result from the organic peroxide being eliminated from ionic interaction with the indicator until the solvating activity of the test mixture disrupts the peroxide-borate complex and releases peroxide to oxidize the indicator in the presence of the peroxidatively active substances.

Despite the considerable increase in stability and sensitivity resulting from the use of borate esters, it has surprisingly been found that even greater stability and sensitivity can be achieved if the borate esters and the above diluent are included in the test mixture.

Diluents currently believed to contribute to the increased stability and sensitivity of the present invention have the above structures. Among the compounds included in these formulas, N, N-dimethylformamide, dimethylsulfoxide, dimethylsulfonate or mixtures thereof have been found to be particularly suitable. Other diluents which may be used are benzylsulfoxide, 4-chlorophenylsulfone, 4-fluoro-3-nitrophenylsulfone and others. These compounds correspond to the above structural formulas wherein R 1 and R 2 each represent a methyl group.

The scope of R 1 and R 2 contained in this invention is broad. R 1 and R 2 are substituted or unsubstituted C 1 -C 6 alkyl. Illustrative examples of alkyl groups are methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, non-opentyl, tert-pentyl, and other isomeric groups, including derived from hexane. These alkyl groups are also illustrative of the pro-alkoxy groups contemplated to satisfy the preceding definition for R 1 and R 2.

When R 1 and R 2 denote an aryl group, the range is similarly broad and the term aryl group as used herein includes substituted and unsubstituted airyl groups such as phenyl, benzyl, tolyl, aniline, naphthyl, and the like.

Typical groups with which R 1 and R 2 may be substituted are amino, nitro, amide, nitrile, hydroxyl, alkoxy, halogen, and the like. a phenyl group, a sulfonic acid, a carboxylic acid, etc.

The amount of diluent used in the composition, composition and method of the invention can be varied within wide limits and can be readily determined experimentally. If the diluent is a liquid such as dimethylsulfoxide (see Table II, below), the amount may range from about 10% to about 100%, based on the volume of diluent added to the mixture, as compared to the volume of solvent or suspending agent. (in the case of Example 12, water). The preferred range is about 25% to about 100%.

Similarly, when the diluent is a mixture of more than one diluent according to the invention, the ratio of the amounts of the various substances used can be varied within wide limits and these can also be readily determined experimentally.

In order to more clearly explain how the invention is to be practiced and used, and to illustrate preferred embodiments thereof, the following examples are included.

Examples

A. Production of test mixture and composition

Example 1

Test mixture

A test mixture was prepared by dissolving the following components in 150 mL (milliliters) of deionized water. The ingredients were added to the water in descending order as indicated:

trisodium salt of citric acid 3.2 g citric acid 2,2 g tetrasodium salt ethylenediaminotetraacetic 0.1 g dimethylsulfone 10,0 g sodium lauryl sulphate 1.0 g 6-methoxyquinoline 0.5 g dimethylsullfoxid 25.0 ml acetone 25.0 ml cumene peroxide 2,0 g triethanolamine borate (Aldrich Chemical Co., Inc.) 10,0 g o-tolidine 0.4 g

Example 2

Testing equipment

The 3MM Whatmann filter paper strips were immersed in the test mixture of Example 1. The submerged strips were dried at 70 ° C for about 15 minutes to form the carrier material impregnated with the test mixture. The dried carrier material was then bonded to plastic strips or handles (polystyrene) using a double-sided adhesive tape manufactured by 3M Company. The test equipment obtained can be used to detect peroxidation active substances by immersion in a test sample, for example urine, suspected of containing such substances, and it is observed whether any color develops in the carrier material.

B. Evaluation of stability of various test mixtures

Example 1 a d 3

Influence of borate ester

The purpose of this experiment was to show the effect of triethanolamine borate on mixtures for the evaluation of occult blood compared to other known additives. The following chloroform solution was prepared with 100 ml o-tolidine 0.5 g

2,5-dimethylhexane-2,5-dipoxide 2.0 g poly- (N-vinylpyrrolidone) 10.0 g

To an aliquot of this solution (20 ml each) was added 1 g of the additive from the following table. These test mixture solutions were allowed to stand at room temperature for about 5 hours and the observed results are summarized in Table I below.

TABLE I

Sample No. Additive

Example 4

Effect of thinner

The following ingredients were added to a 500 ml beaker:

hexamethyenteramine 1,4-diazabicyclo [2.2.2] octane borate triethanolamine control (no additives) chloroform

100 ml

Results

Very dark, almost black solution.

Low sensitivity.

Dark green / brown color. Minimally sensitive to occult blood in urine. Faintly brownish. High sensitivity to occult blood in urine.

It darkens within two hours. Not suitable for occult blood analysis.

cumene peroxide 4.0 g o-tolidine 0.4 g dimethylisulfoxide 25.0 ml

The obtained test mixture, which was faintly yellow after mixing the above ingredients, was transferred to an Erlenmeyer flask, which was stoppered and left on a la219888 boron table at normal temperature (about 18.3 ° C) overnight. After one day, the test mixture darkened only slightly more than when prepared.

Example 5

Comparison with Example 4

A test mixture for control purposes, i.e. for comparison with the invention, was prepared according to the procedure of Example 4, except that 25 ml of chloroform was substituted for the test mixture. This test mixture, without dimethylsulfoxide, was sealed in an Erlenmeyer flask and left on the laboratory bench as in Example 4 (i.e. at 18.3 ° C for 1 day). In contrast to the mixture of Experiment 4, the control mixture darkened to almost black, indicating the improved stability that the diluent imparts to the test mixtures.

and

C. Stabilization of test equipment with a magnesium ester

Example 6

Control

For the 'preparation of the pseudoperoxidase-sensitive reagent strip', a solution of the following test mixture was prepared. This sample does not contain the borate ester of the invention as a component.

water 150 ml trisodium salt of citric acid 3.2 g citric acid 2,2 g Ethylene- diaminotetraactové 0.1 g dimethylsulfone 10,0 g sodium lauryl sulphate 1.0 g ? -methoxyquinoline 0.5 g dimethylsulfoxide 25.0 ml acetone 25.0 ml cumene peroxide 2,0 g o-tolidine 0.4 g

A 3MM Whatmann filter paper clip was dipped in a solution of the above test mixture and dried at 70 ^° C. The dried paper was cut into squares of about 5 mm and attached to the plastic handles using double-sided adhesive tape (available from 3M Company) to produce a test device.

Example 7

Invention

The solution was prepared as in Example Θ above except that 10 g of triethanolamine borate was added before addition of cumene peroxide. The test apparatus was prepared from this solution as in Example Θ.

Example 8 1,4-Diiazabicyclo [2.2.2] octane

The solution was prepared as in Example 6 above, except that 10 g of 1,4-palzalbicyclo [2.2.2] octane was added before addition of cumene peroxide. This solution was used to prepare the test device in the same manner as in Example 6.

Example 9

Hexamethylenetetramine

The solution was prepared as in Example 6 above, except that 10 g of hexamethylenetetramine was added before addition of cumene peroxide. In the same manner as in the above example, a test apparatus was prepared from this solution.

Example 10

Comparison of stability and sensitivity

The test devices of Examples 6-9 were stored under stress conditions to determine their relative stability and sensitivity. The apparatus of Examples 6-8 was stored for three days at about 60 ° C and the apparatus of Example 9 for one day at about 70 ° C. These test devices exposed to severe conditions were then evaluated for sensitivity by immersion in urine containing 1 millionth of blood. The color change at this occult blood concentration (i.e. 0.015 mg / l dl) was induced only by the triethanolamine borate-containing device (Example 8). Other facilities were insensitive to this occult blood level.

D. Stabilization of test equipment with diluent

Example 1 a d 11

Six test compositions were prepared from six test solutions of mixtures containing varying amounts of dimethyl sulfoxide according to Table II below.

All six test solutions © prepared as follows: mixtures water 75 ml trisodium salt of citric acid 3.2 g citric acid 4.5 g triethanolamine borate 10,0 g tetrasodium and ethylacetate 0.1 g dimethylsulfone 5.0 g sodium lauryl sulphate 1.0 g 6-methoxyquinoline 0.5 g cumene peroxide 2,0 g o-tolidine 0.4 g

The following amounts of dimethylsulfoxide and methanol were added to each solution of the six test mixtures:

TABLE II

Test mixture No ΟΙιηβίΐΊγΐΒ-ηΙΐοχίΰ (ml) Methanol (ml)

0 75

5 70

10 65

25 50

40 35

60 15

By immersing a piece of 3MM Whatmann filter paper in each solution, test devices were prepared from each of the above test mixture solutions. Submerged strips of filter paper were removed from the respective solutions and dried and loaded in an oven for 18 hours at 70 ° C.

The loading of each strip was evaluated in a test solution containing 1: 1,000,000 fresh whole blood in urine at a dilution. The intensity of the developed color on each strip allows comparison. The results are summarized in Table III.

TABLE III

Testing facility No * * Coloring rating **

0 (no coloring)

1 (feet)

3

5

6

7 (virtually no loss of reactivity) * The test equipment numbers correspond to the test mixture numbers in Table II.

** The color evaluation was based on (Scale 0 to 8, with 8 being a color induced by freshly prepared unstressed test equipment prepared from Test Mix No. 6 (Table II). A rating of 0 indicates that no coloring was produced and scores of 1 indicate only trace amounts Coloring.

It is apparent from the data in Table III that its sensitivity increases with increasing amount of diluent (here dimethylisulfoxide) used in the composition of the test composition.

E. Increase in stability of the test mixture (with borate ester) due to various diluents.

Example 12

As in Example 11, eight test mixtures were prepared, of which seven contained different diluents and one (control) was without diluent. The purpose of this experiment was to determine the efficacy of various diluents in increasing the stability of peroxidation-sensitive test compositions where a constant amount of borate ester is maintained.

A solution was prepared containing the following components:

water 50 ml tritium citrate salt 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 kumenperoxld 2,0 g o-tolidine 0.4 g

A small portion of this solution was added to each of the eight test mixtures. Then, a small amount of the diluent listed in Table IV was added to each tube, the tubes were sealed and allowed to stand at room temperature on the laboratory bench for 1 week. Towards the end of the week, the tubes were assessed for color darkening, the darker the color, the less stable the test mixture. The results are shown in Table IV according to decreasing stability (i.e. Test Mix 1 is the most stable and Test Mix 8 is the least stable).

TABLE IV

Test Mix No. Diluent

7 (control) dimethylsulfoxide

N, N-dimethylformamide

-dimethylsulfone benzylisulifoxide

4-chloro-phenylsulfone

4-fluoro-3-nitrophenylsulfone

2-imidazo-lidone none

Samples 1 and 2 in Table IV show excellent stability; solution 3, however, has good stability; solutions 4 to 7 were on average stable; and solution 8, controls 218888, was substantially less stable than solutions 1 to 7.

F. Preferred Embodiments

Example 13

The following ingredients were mixed in 150 ml of water:

trisodium citrate 3.2 g citric acid 2.2 g ethylenedlaminotetraacetic acid quaternary salt 0.1 g dimethylsulfone 10.0 g sodium lauryl sulphate 1.0 g

6-methoxyquinoline 0.5 g dimethylsulfoxide 25.0 ml acetone 2'5.0 ml triethanolamine borate 10.0 g cumene peroxide 2.0 g o-tolidine 0.4 g

Strips of Whatmann 3MM filter paper were dipped into the above solution and dried at 70 ^Q C. After drying, the filter paper was cut into squares having a side of .4 mm. These squares were then mounted on polystyrene handles measuring about 8 x 15 mm using double-sided adhesive tape (3M Company).

The test compositions made according to the above experiment were loaded for 60 days at 60 ° C and found to be sensitive to occult blood in urine at concentrations less than 0.015 milligram, percent.

P fikl a d 14

This example illustrates a second preferred embodiment of said test apparatus.

The test mixture is prepared by mixing the following components in the following sequence, in a beaker as well as from:

water 75 ml Triple citric acid salt 3.2 g citric acid 4,15 g triethanolamine borate 10,0 g ethylenediaminotetraacetic acid 0.1 g sodium lauryl sulphate 1.15 g dimethylsulfone 10,0 g 6-methoxyquinoline 0.6 g N, N-dimethylformamide 75 ml cumene peroxide 3,0 g o-tolidine 0.8 g

5 ^. j

The 3MM Whatmann filter paper strips were immersed in the above mixture and dried for about 11 minutes at about 90 to 92 ° C. The dried strips were then attached to the plastic handle using double-sided adhesive tape.

Several strips were stressed by · storage at 70 ° C for 1 day; further storage at 40 ° C for 12 weeks. Both stripe sets developed a blue / green color when they were contacted with fresh whole blood in urine at a 1: 1 x 10 ⁶ dilution.

In conclusion: the foregoing examples show how the compositions and test apparatus of the invention (Examples 1 and 2) are prepared and used, give the results of stability and sensitivity comparisons (Examples 3 to 12), and simultaneously describe preferred embodiments (Examples 13 and 14). The effects of the barate ester and diluent, unified, even in combination, on the test mixture are described in Examples 3-5, while the effects on the test apparatus are shown in Examples 6-11. The use of various diluents is included in Example 12.

Claims (8)

1. Test device for determining the presence of peroxidising agents such as hemoglobin, secret blood and peroxidase in a test sample containing an organic hydroperoxide, for example t-butyl hydroperoxide, cumene hydroperoxide, diisopropylbenizene hydroperoxide, 2,5-dimethylhexane-2,5-dihydroperoxide, paramenthanhydroperoxide or mixtures thereof, and an indicator capable of providing a detectable response in the presence of said hydroperoxide and a peroxidizing agent such as benzidine, o-tolidine, 3,3 ', 5,5'-tetramethylbenzidine, 2,7-diamino-fluorene or mixtures thereof, characterized in that it also contains a compound of the general formula R. S -—> o, O « -so · N, ICH) _W IC Jp0. o o or mixtures thereof in which the general formulas R 1 and R 2, which are the same or different, represent hydrogen, aryl, alkyl or alkoxy, in each case having 1 to 6 carbon atoms in each of said radicals, and m and n and p, which are the same or different, are integers from 1 to 4. 2. The test composition of claim 1, wherein said compound comprises dlmethylsulfoxide, dimethylsulfone,,, Ν-dimethylformamide, benzylsulfoxide, 4-chlorophenylsulfone, 4-chloro ^| or-3-nitrophenylsuliphon or mixtures thereof. 3. A test composition according to claim 1, wherein said compound comprises a mixture of dimeithylsulfone and N, N-dimethylformamide. 4. The test composition of claim 1, wherein said compound comprises trimethanolamine, triethanollamine, tri (n-propanol) amine borate or mixtures thereof. 5. The test composition of claim 1, wherein said compound is triethanolamine borate. 6. The test composition of claim 1 comprising diimethylsulfone, Ν,,-dimethylformamide, or mixtures thereof, and trimethanolamine, triethanolamine, trii (n-propanol jamine) or mixtures thereof. 7. The test composition of claim 1 comprising a mixture of dimethylsulfone, if, Ν-dimethylformamide and triethanolamine horate. 8. A test composition as claimed in any one of Claims 1 to 7, comprising a composition incorporated into the carrier matrix.