DE2013558A1 - Diagnostic agent for the detection of substances that couple with a diazo compound with the occurrence of a color change - Google Patents

Description

Coupling change addition to patent 2 007 013 (patent application P 20 07 013.7)

The subject of the main patent is a diagnostic agent for Detection of substances that couple with a diazo compound with the occurrence of a color change that is marked by a carrier with embedded dry pu residue a stable diazonixime salt solution; comprising an iReaktions ^ product obtained by combining an organic polar solvent with an arylamine, water, a soluble titrite, a stabilizer and a sulfonic acid. It has now been shown that the color change accelerates and is reinforced if das.Mittel additionally contains a Lewis acid halide or a complex of this Halpgeriids with an organic Lev / isbase; contains ^ numerous compounds, especially those in Body fluids, such as blood and urine, can cause a color reaction which occurs when these compounds are coupled with a diazo compound. Sine such Connection of particular importance for the determination of the A person's state of health is bilirubin.

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The detection of bilirubin in body fluids has been carried out since around 1883, when Ehrlich started the diazo reaction aura Introduced detection of bilirubin in urine. Since then, until today, numerous liquid systems have been produced by the diazo reaction Make use of the detection of bilirubin in urine and applied in serum. These systems are in terms of diazo compounds and others used for this proof Reagents quite different; however, they are all primarily based on the azobilirubin red-purple color, the occurs or can be measured spectrophotometrically.

It is true that bilirubin can be detected very precisely with these liquid systems; however, the application of these systems is general associated with difficulties because the uiazoniurnsalzlösung are fickle in these systems and therefore for each new batch of liquid to be examined must be freshly prepared. Also that is up to the termination The time required for the indicating color change is quite long, the shortest time is around 30 minutes and in some cases up to several hours are required. Therefore, the test results are not available as quickly as would be desirable, especially when it comes to Be-Tune or when detecting bilirubin in young children or infants.

A number of these difficulties have already been caused by a trade under the name ICTOTJSü? ⁰ Reduced 'available product', a dry tablet for the detection of bilirubin as in the USA patent! Ir. 2,854,317. This tablet contains a diazonium salt, sulfosalicylic acid and sodium bicarbonate. For the detection reaction, 5 drops of urine are placed on a special, highly absorbent material

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given, then the IGTOTEST ^ reagent tablet in aie Place in the middle of the moist area and let 2 drops of water fly over it. If bilirubin is present, one occurs

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Color reaction on the absorbent material within ■ 30 seconds. Although this reagent tablet already represents a relatively fast and stable system for the detection of bilirubin, a number of components must come together in order for the detection reaction to take place completely.

Indicator paper-like test means for immersion, which combine certain rapidly reacting reagents with a suitable carrier, are especially widespread in the medical field. Such test equipment is typically used to determine the pH value and for the detection of glucose, protein and other substances in body fluids. However, when combined would of known test systems for the detection '"of bilirubin with a suitable carrier to indikatorpapierahniichen .Prüfmitteln a unbeständi it _o test equipment obtained, afforded the unreliable results. The object of the invention is therefore an improved Prüfnittel that a rapid and increased color change indicates when it comes into contact with a compound that couples with a "JUiaso. compound" and that is suitable for oen detection of bilirubin in solution.

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A further object of the invention is a method for producing a test device consisting of one piece, which gives a quick and improved color change. The invention also relates to a reagent for preparation this test medium is suitable.

The new improved 'Diagnostiziermittel invention, indicating a fast and increased color change when contacted with a compound in contact, which couples with a Diazoverbinciung, consists of a support comprising a diazonium salt or a substance such as a triazine, which form a \ diazonium salt can, and a color-enhancing amount of a Lewia acid halide or a complex contains these «halides with an organic Lewis base. The term dialonium salt as it is used in the following means diazonium salts themselves or all other compounds which can easily form a diazoniura salt.

The invention also resides in a reagent which is a diazonium salt and a color enhancing amount of a Lewis acid halide or a complex of said halide with contains an organic Lev / isbase, and in a method for producing the diagnostic agent, wherein a Carrier brought into contact with the diazonium salt solution and then dried.

During the preparation of the diazonium salt solution, a polar one becomes Solvent used. Preferably the solvent phase from Y / ater and an organic polar solvent, for example, an alcohol, a ketone, an ester and the like. An alcohol such as Methanol, ethanol and 2- ± topanol in a ratio of used at least about 4 parts organic solvent to 1 part water. The ratio of organic solvent To water is chosen so that there is practically no precipitate in the diazonium salt solution formed.

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A high proportion of rapidly evaporating is also desirable organic solvent to allow the time required to dry the carrier impregnated with the solution to decrease.

A diazotizable arylamine capable of coupling is first added to the solvent. Such arylamines can be 2,4-dichloroaniline, 4,4 ^l -methylene-bis - (N, l \ f-di'iethylaniline), p-nitroaniline, p-chloroaniline, 2,5-dichloroaniline, 4-chlorine -oanisidine, 3,3'-dimethoxybenzidine and 2-methoxy-5-nitroaniline. However, other arylamines which form diazonium compounds can also be used. .

Thereupon is a soluble nitrite that dissolves in an aqueous acidic medium forms nitrous acid, added to the solution. Advantageously the soluble nitrite is sodium nitrite, or potassium nitrite Calcium nitrate. . ·,:. ,

The solution can also contain a stabilizing compound. The advantageous effect of this compound lies in the fact that any precipitate which may occur during the formation of the diazonium salt is obtained as a very fine precipitate which is easily redissolved. In addition, the stabilizer increases the chemical resistance of the finished test medium. The stabilizer can be selected from a large group of compounds, such as salts of aromatic or aliphatic sulfonic acids, sulfates or sulfonates. Examples are 1,5-naphthalenedisulfonic acid disodium salt, 2-Naiphthalin-sulfonic acid sodium, 4,4'-diamino-2, 2'-biphenyldisulfonic acid-. Disodium salt and sodium lauryl sulfate. . ·? ·. "■ <■ _ ■■ -

organic acid, preferably an organic; ° sulfone- " acid, is added to the preparation to ensure the correct pH Y / ert for the formation of a diazonium compound. and the closing one Coupling reaction of this diazonium compound with the couplable connection. As organic.

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Sulfonic acids come sulfosalicylic acid, sulfanidic acid, Cyclohexanesulfamic acid, p-toluenesulfonic acid and the like in question. Other known sulfonic acids and organic acids can also be used.

These reagents, i.e. an organic polar solvent, an arylamine, water, a soluble nitrite, a stabilizer, and a sulfonic acid, ground to the desired diazonium salt solution put together. The specified sequence is preferably adhered to when combining, although this The order of polar solvent, arylamine, water, soluble nitrite and stabilizer is not critical. Even If more than one stabilizer is used, the second stabilizer can be added after the sulfonic acid.

The ver for the formation of the diazonium salt solution according to the invention The agents used v / erden preferably in the following Hengen areas used, which mean depending on weight-5a or VoI .- ^:

organic polar solvent 10 to 90 ^ a

Arylamine 0.02 to 0.5 # V / ater 10 to 90 ^

Soluble nitrite 0.01 to 0.25 ° ß>

^ Stabilizer $ 0.15 to $ 5

'Sulphonic acid $ 0.2 to $ 5

Y / ie already indicated, a ratio of * Solvent to water of 4: 1 or above adhered to so that the diazonium salt remains in solution and the carrier dries faster. The weight ratio of acid to arylamine compound is preferably maintained at about 10: 1.

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Surprisingly, it has been shown that the color change,

And the bilirubin which occurs in the reaction between a diazonium salt is substantially increased if the agent contains a color-enhancing amount of a "Lewis acid halide" or a complex of such a halide and an organic Lewis base.

The Lewis acid halide is advantageously tin chloride, Aluminum chloride, boron trichloride or the like. The tightness used is caused by the attack on the carrier material limited. For the production of the diagnostic agent, the Lewis acid halide is expediently in solution in a narrow range between about 0.5 g and 5 g, and preferably about 3 »2 g / 100 ml solution used. To make this solution a suitable solvent can be used.

A solid complex is advantageously formed from the Lewis acid halide and an organic Lewis base or another compound which is suitable for sharing a pair of electrons with the Lewis acid. For example, compounds such as dimethyl sulfoxide, dimethylformamide, dioxane and the like can be used. Preferably, dimethylformamide is used to form the complex.

The complex can be prepared by the _f Lewissäurehalogenid, the shoe cover-to formation of the complex compound used as Jimethylforrnamid are zB.Zinnchlorid to a *. The complex is then precipitated from solution with a suitable organic solvent such as chloroform and collected. This complex can then be dissolved in a suitable solvent in order to bring the carrier into contact therewith. Such solvents are preferably the solvents used to form the complex and are particularly advantageous together with a non-aqueous polar solvent such as acetone, dimethyl sulfoxide or the like.

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Substances which contain the diazonium salt and the Lewis acid halide or its complex can be used as carrier materials can hold back with an organic Lewis base. This carrier is preferably attached so that it can be easily attached to a Liquid can be brought into contact, which presumably contains the couplable compound to be detected ". The The carrier for the test equipment can be combined into one piece with a larger part that serves as a handle, or it can as a separate part with a larger handle part or a Handle device be connected. Suitable carrier materials are e.g. porous materials such as filter paper, glass fiber paper, or a polypropylene felt. The carrier can also contain a polymeric gel that is mixed with the test solution and then taken under Formation of a semi-permeable membrane was dried. That Gel can be formed by solvent evaporation and a Contain crosslinking agents.

The overall structure of the test equipment is not viewed as critical as long as care is taken that the area which contains the dry cuck and the diazoniura salt solution, brought into contact with the liquid to be examined can be.

A carrier for the production of the test equipment can / with egg - ^ iazonium salt solution and the Lewisaäurehalogenid soaked and dried v / earth. The impregnated paper is preferably used at moderate Temperatures of about 65 to 70 C dried in moving air. " Advantageously, otherwise the temperature is not excessively increased, since this is believed to have a detrimental effect on the diazonium compounds. The dried carrier can then be cut into small pieces and be connected to a larger part to make them easier to use.

When using a filter paper carrier, this is preferably first impregnated with the diazonium salt solution and

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dries and then with the complex of the Lewis acid halide and the organic Lewis base.

The invention is illustrated in more detail by the following examples.

B e is ρ i el 1_. .

A solution was made by placing in a glass jar the following compounds were combined in the order given and with constant stirring:

Methanol 20.0 cm cm 2,4-dichloroaniline .- 0.20g ^ G Aqua dest. 20.0 Sodium nitrite 0.1 S. 1,5-Ii apht shai ind isulfonic acid G Disodium salt 0.6 δ - Tin chloride hyuratised 5.0 G Sodium lauryl sulfate 1.5 cm Sulfosalicylic acid 2.0 Methanol 60.0

The compounds were combined at room temperature (about 23 ° C) and the temperature of the solution remained at about room temperature with no additional temperature control during the formation of the solution.

A strip of filter paper (Eaton and Dikeman # 641) was made immersed in the solution prepared as above and immediately pulled out again; this paper was, at around 65 to 70 C for about 8 to 10 minutes, with the. Air over the surface of the paper was kept in motion. The dried paper was creamy white in color.

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Bilirubin test solutions were prepared from a urine sample from a patient who showed signs of jaundice. The patient's urine was treated with the tablet reagent ICTOTEST ^ and a reaction was observed indicating a high concentration of bilirubin. The urine was also examined with a modified method according to EG Godfried (Biocheiu. J., g8_ , 2056-2060, 1934) and found that it contained about 1 mg ^c / l bilirubin. The pathological urine was further diluted with normal urine to concentrations of 0.5, 0.25 and 0.1 mg ^C , O respectively. Each solution was tested with the test agent according to the invention and the gradations of the Ijoh purple coloration noted. A deeper color approaching gray-purple was observed with the higher concentration solutions.

Human blood sera from normal patients and patients suffering from jaundice were prepared in the same manner. The biirubin content of these sera was determined with a test device known al o Auto Analyzer and was between 0.1 and 18 mg) o. As prepared above according to the invention test devices were brought into contact with the sera and the color levels varying between weakly liosa and deep lavender ^{observed for concentrations of 1.0 mg C} , O and above. The color change was proportional to the bilirubin concentration in the solutions.

Example 2

Diagnostic agents as described in Example 1 were used except that 1.6 g of anhydrous tin chloride is used in place of the hydrated tin chloride ' became. It was observed that these test media had essentially the same properties as the test media of example 1.

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20.0 cm 0.20 G 20.0 cm 0.1 ß 0.6 ε 1.5 S. .2. ° S. 60.0 a

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Example 3

first
, A / solution was prepared by combining the following compounds in the specified order and with constant stirring:

Methanol 2,4-dichloroaniline Aqua dest. liatriurane nitrite

1, S ^ naphthalenedisulfonic acid disulfide salt

Sodium Lauryl Sulphate Sulphosalicylic Acid Methanol

A strip of filter paper (Eaton and Dikeman # 641) was dipped into this first solution and immediately withdrawn. This paper was dried for about 8 to 10 minutes at 65 to 70 ^{° C., the.} Air was kept moving over the surface of the paper. The second solution was made by combining the following components: -

Dimethylformamide 20.0 cnr

Tin chloride ifiraethyl formamide.

Complex / 3.2 g

Acetone 80.0 cm ⁵

The dried paper was immersed in the second solution and removed immediately. The paper was between approximately Dry at 65 and 70 C for approximately 8 to 10 minutes with air agitation. The dried paper had a crerie-white color. It turned out that this diagnostic agent if it is given together with the solutions of Example 1 showed essentially the same parabolic changes, 'as given in Example 1. .

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Examples 4 to 10

Various test media were produced as in Example 3, with the difference that in the first solutions in each case one of the following instead of 2,4-dichloroaniline Connections was used;

4,4'-ethylene ~ bis (H, N-d imethy ianiline), p-Hitroaniline »

p-chloroaniline,

2,5-dichloroaniline,

4-chloro-o-anisidine,

3,3'-dimethoxybenzidine,

2-l-ethoxy ~ 5-nitroaniline.

The test equipment obtained showed practically the same properties like that of example 5.

Example 11

A test agent was produced according to Example 3 with the difference that cyclohexanesulfanic acid was used instead of Sulphosalicylic acid was used in the first solution. That The test agent obtained had practically the same properties as that of Example 3

Example 12

It was a test according to Example 3 with sulfamic acid instead made of sulfocalicylic acid in the first solution. It had practically the same properties as daa of example 3.

For s ρ ie 1 13

There was an i'rüfmittel according to Example 3 with p-toluene sulf

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Fonsäure instead of sulfosalicylic acid in the first solution manufactured. It had the same properties as that of the Example 3 «

Example H

Strips of fiberglass paper (Gelman Type A-1306 and Type E-748) were made like the pilter paper strips in Example 3 treated. The dried fiberglass paper strips showed practically the same appearance as the filter paper strips according to Example 3 · 'These glass fiber paper strips were with brought the test solution into contact according to example 1, thus the same color changes as in Example 3 were observed.

Example 1_5

In this example the procedure according to example 3 repeated with the difference that a polypropylene felt (Polypropylene Ml) SE. No. Po-8110 of the American Pelt Company) was used in place of the filter paper. The dried matrix shows practically the same color and the same color reactions compared to the test solutions as observed in Example 3.

Example 16

A strip of filter paper (Saton and Dikeman # 641) was made pulled through the erate solution of Example 3 and

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between 65 and 70 G dried for about 10 minutes with agitated air. After drying, the paper was drawn through a second solution containing the following ingredients: -

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- * 5- Dimethylformamide 1A-37 G 435 Tin chloride dimethyl 20.0 formamide complex G acetone 3.2 Cellulose acetate 80.0 cm 6.25 G

The paper was approximately between about 65 and 7O ⁰ G

Dried for 10 min in agitated air. Was this agent with the test solutions according to Example 1 in contact ", the same color changes as in Example 3 were observed.

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Claims (6)

Claims My DiagnoBtieierBimittel for the detection of substances, the absence of a Diazo connection with the appearance of a color change ice coupling, which consists of a carrier with embedded dry residue of a stable DiazoniumealE solution, comprising a reaction product obtained by contacting an organic polar solvent with an a rylamine, water, a soluble nitrite, a Stabilizer and a sulfonic acid is formed, according to patent 2 007 013, characterized in that it additionally contains a Levvissure halide as a color-strengthening substance or a solid complex of a Lewis acid halide and an organic Lewis base. 2. Diagriostizierinittel according to claim 1, characterized GEK e η η ζ η et verifiable that the tin Lewissäurehalogenid chloride, aluminum chloride or urotrichloride. 3. Diagnostic agent according to claim 1, characterized in that g e characterizes t that it contains a complex of a Lewis acid halide and an organic Lewis base, the dimethyl sulfoxide, dimethylformamide or dioxane is. 4. diagnostic agent according to claim 1, characterized in that that it is a solid complex Contains tin chloride and dimethylformamide. 5 reagent for the preparation of the diagnostic agent according to claim 1 Ms 4, characterized by that it is the diazonium salt and the Lewis acid halide or the complex of aera Lewis acid halide and the organic Lewis base contains. 009841/1658 -15- BATH ORIGINAL 1A-37435 6. A method for producing a diagnostic agent according to claim 1 to 4 _}, characterized in that a carrier is first impregnated with a diazonium salt solution, dried and then prepared with a solid complex of a Lewis acid halide and an organic Lewis base. 009841/1658