US3853466A

US3853466A - Diagnostic composition for the detection of urobilinogens

Info

Publication number: US3853466A
Application number: US00262923A
Authority: US
Inventors: W Rittersdorf; P Rieckmann; D Berger; H Rey
Original assignee: Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1971-06-19
Filing date: 1972-06-14
Publication date: 1974-12-10
Anticipated expiration: 1991-12-10
Also published as: DE2130559C3; DE2130559B2; SU535914A3; SE394328B; HU162948B; FR2158791A1; IT956664B; CA959389A; FI53508C; DK143147C; NL170773C; DE2130559A1; JPS5741693B1; GB1343247A; ES403875A1; NL7208232A; ZA724152B; DD98763A5; DK143147B; AT322116B

Abstract

Diagnostic agent for the detection of urobilinogens, optionally in the presence of bilirubin, in body fluids which agent comprises at least one stable benzene diazonium salt wherein the phenyl radical may be polysubstituted and contains in the o- or p-position at least one polyatomic electron donor group with at least one mesomeric electron pair, the sum of the Hammett sigma values for all the substituents not exceeding the value of +0.4.

Description

United States Patent Rittersdorf et a1.

[ Dec. 10, 1974 DIAGNOSTIC COMPOSITION FOR THE DETECTION OF UROBILINOGENS Inventors: Walter Rittersdorf,

Mannheim-Waldhof; Dieter Berger, Viernheim; Hans-Georg Rey; Peter Rieckmann, both of Mannheim-Waldhof, all of Germany Boehringer Mannheim Gmbll, Postfach, Germany Filed: June 14, 1972 Appl. No.: 262,923

Assignee:

Foreign Application Priority Data June 19, 1971 Germany 2130559 US. Cl. 23/230 B, 23/253 TP, 252/408,

- 260/141 Int. Cl.. G01n 21/06, G01n 31/22, GOln 33/16 Field of Search 23/230 B, 253 TP; 252/408 References Cited UNITED STATES PATENTS 9/1958 Free 23/230 B 5/1970 Green 23/230 B 6/1971 Mast 23/230 B 6/1971 Mast 23/230 B X 12/1971 Rittersdorf 23/230 B 3,652,222 3/1972 Denney 23/230 B OTHER PUBLICATIONS Primary ExaminerMorris O. Wolk Assistant ExaminerSidney Marantz Attorney, Agent, or FirmBurgess, Dinklage & Sprung [5 7] ABSTRACT Diagnostic agent for the detection of urobilinogens, optionally in the presence of bilirubin, in body fluids which agent comprises at least one stable benzene diazonium salt wherein the phenyl radical may be polysubstituted and contains in the Q; or p-position at least one polyatomic electron donor group with at least one mesomeric electron pair, the sum of the Hammett sigma values for all the substituents not exceeding the value of +0.4.

34 Claims, No Drawings DIAGNOSTIC COMPOSITION FOR THE DETECTEON OF UROBELTNOGENS The present invention is concerned with an improved diagnostic agent for the detection of urobilinogen bodies in body fluids, especially in urine.

It is known that urobilinogen bodies (bilanes), indole, sulfonamides, porphobilinogens, urine indican and 5-hydroxyindole-acetic can be detected with a solution of p-dimethylaminobenzaldehyde in hydrochloric acid. This detection reaction is known in the literature as Ehrlichs reaction; it has achieved considerable importance, especially in medical diagnosis, for the detection of increased urobilinogens in urine. Although the test is not specific, it is regarded as being the standard method for the diagnosis of diseases of the liver and gall bladder.

Since test papers are of ever greater importance in clinical-chemical diagnosis, test papers have also been developed for the detection of urobilinogen based on Ehrlichs reaction. However, these test papers suffer from two important disadvantages, viz., (l) the color reaction develops so slowly that it is necessary to wait for at least one minute before reading off; and (2) the test papers naturally possess the non-specificity of Ehrlichs test so that falsely positive results cannot be excluded.

There has been evidence for a long time that urobilinogen couples with diazotized sulfanilic acid to give a yellow colored material. This reaction was discovered by Ehrlich in 1884 and is referred to as the so-called yellow diazo reaction. Subsequently, the reaction was investigated several times but hitherto it has not been possible to ascertain whether it is, without doubt, a diazo coupling with urobilinogen or whether other materials may also be responsible for the formation of the yellow colored material (see Biologic der Gallenfarbstoffe, published by Georg Thieme-Verlag, Stuttgart, Germany, 1960: pp. 32 and 21 1). Since this is more of a curiosity than a useful agent for clinicalchemical diagnosis, the yellow diazo reaction is practically without importance and is scarcely mentioned in any of the standard reference books.

We have now, surprisingly, found that optionally polysubstituted benzene-diazonium salts are outstandingly useful for the detection of urobilinogen.

Thus, the present invention provides a diagnostic agent for the detection of urobilinogen in body fluids which comprises a solid or liquid acid and at least one stable, optionally polysubstituted benzene diazonium salt, the phenyl radical of which contains in the 0 or pposition at least one polyatomic electron donor group with at least one mesomeric electron pair; especially when the sum of the Hammett sigma values of all substituents does not exceed the value of +0.4.

Electron donor groups are, according to the present invention, groups which contain oxygen, sulfur or nitrogen connected directly to the phenyl radical. Oxygen-containing groups include; for example, hydroxyl, alkoxy, aralkoxy and aryloxy radicals. As sulfurcontaining groups, there can only be considered alkylthio and arylthio radicals, because free mercapto groups cannot, as a rule, be used due to their sensitivity to oxidation. As nitrogen-containing groups, there may be mentioned, for example, acylamino (i.e. alkanoylamino), arylamino and aryl-alkylamino radicals, whereas unsubstituted amino groups and alkylamino groups are, because of their basicity under the conditions of the reaction, usually protonized and then no longer possess a mesomeric electron pair. In the selection of the substituents, care must also be taken that they do not react with the diazo group: this applies particularly to o-acylamino and o-arylamino radicals. Usually, the electron donor groups will not contain more than 18, preferably eight, carbon atoms.

According to the present invention, it is especially preferred to use benzene-diazonium salts of the general formula:

in which X is a stabilizing anion, R and R which may be the same or different, are polyatomic electron donor groups containing at least one mesomeric electron pair, whereby one of the symbols R and R can also be a hydrogen or halogen'atom or a lower alkyl radical (of, e.g., up to 10 carbon atoms), and the symbols R and R which may be the same or different, are hydrogen atoms or groups which do not sterically hinder the diazo coupling with urobilinogen, the sum of the Hammett sigma values of all the substituents R R R and R not exceeding +0.4.

Compounds of general formula (I) which are especially preferred according to the present invention are those in which R, is a hydroxyl group or a lower alkylthio or lower alkoxy radical and R is a hydroxyl group or an alkoxy, phenoxy, aralkoxy, alkylthio, phenylthio, phenylalkylamino or optionally substituted phenylamino or acylamino radical, whereby one of the symbols R and R can also be a hydrogen or halogen atom or a methyl radical and R and R which may be the same or different, are hydrogen or halogen atoms or hydroxyl groups or alkoxy, phenoxy, alkylthio, phenylthio, phenyl-alkylamino, optionally substituted phenylamino, acyl-(i.e., alkanoyl) amino, alkyl, sulfo or carboxyl radicals and X is a halide ion or the anion of an oxygen acid or of a Lewis acid coordinatively saturated by a hydrogen halide. The alkyl group or alkyl moiety contained in a group preferably contains not more than 10 carbon atoms and the aryl groups not more than 10 carbon atoms.

The above-defined optionally polysubstituted benzene diazonium salts, and especially the compounds of general formula (I) are particularly useful when applied to absorbent carriers.

The benzene diazonium salts used according to the present invention react with urobilinogen almost instantaneously and give very specific red to blue colored materials which are especially well suited for the detection of very low concentrations of urobilinogen. Furthermore, the compounds used according to the present invention have the great advantage that the color reaction is not disturbed by urea. The previously known test papers are colored yellow due to the urea present in the urine, depending upon the concentration, which can make the detection of small amounts of urobilinogen very difficult. Furthermore, the color reaction according to the present invention is not influenced by those substances which frequently occur in urine and which, as is known, usually have a disturbing effect on the Ehrlich test; these substances include, in particular, the aromatic amines which, as medicaments (sulfonamides, sulfonyl-ureas and the like), are frequently excreted in the urine. The presence of these aromatic amines in urine previously gave rise to yellow or orange color reactions which cannot only mask but also simulate the color brought about by urobilinogen.

For the detection of bilirubin, which is also of importance for the diagnosis of diseases of the liver and gall bladder, diazonium salts such as diazosulfanilic acid, p-nitrobenzene diazonium salts and dichlorobenzene diazonium salts, have admittedly already been used for a comparatively long time. However, as is to be expected, these do not give a useful color reaction with urobilinogen.

ln contradistinction thereto, the compounds used according to the present invention permit the specific detection of urobilinogen in the presence of bilirubin. In some cases, bilirubin admittedly also reacts with the compounds used according to the present invention but the reaction first takes place after the color reaction with the urobilinogen has already substantially developed and differs in color so considerably that it is, surprisingly, even possible to determine both gall coloring materials in one reagent and to estimate the ratio of amounts thereof on the basis of the mixed color which develops. Thus, a red color can indicate the presence of urobilinogen and a later developing blue coloration the presence of bilirubin. When both gall coloring materials are present simultaneously, the test first becomes red and, subsequently, increasingly violet. ln exceptional cases, the color reactions develop simultaneously but this does not influence the test result.

if the reaction with bilirubin is to be suppressed, this can be achieved by using a diazonium salt of relatively low electrophilic character and allowing the reaction to take place in the presence of a cationic wetting agent.

In this way, it is possible so to modify the agent for the detection of urobilinogen, possibly together with bilirubin, that it can be made optimal for the desired purpose.

The optionally polysubstituted benzene diazonium salts used according to the present invention are preferably employed for the production of test papers. For this purpose, the compounds are applied to an absorbent carrier with a solid acid and possibly with additional materials, for example stabilizers and wetting agents. Furthermore, the compounds (I) are also suitable for the production of test film strips according to German Patent Specification No. l,598, l 53 and for the detection of urobilinogen in solutions.

For the production of a preferred embodiment of the new diagnostic agent, an absorbent carrier, preferably filter paper, is impregnated with a solution which contains, per 100 ml., 0.05- g., preferably 0. 1-] g., of the diazonium salt, as well as 2-30 g., preferably 5-20 g., of a solid inorganic or organic acid and possibly at least one wetting agent and/or at least one stabilizer.

As solvents for the impregnation solutions, it is especially preferred to use water or readily volatile organic solvents which do not react with diazonium salts, which are, in particular, lower alcohols, ethyl acetate and acetonitrile.

For the preparation of the diazonium salt solution, the previously prepared diazonium salt can be added to the solution or the salt can be produced in known manner in the solution by diazotization of an appropriate aromatic amine.

For reasons of storage stability, only stable diazonium salts are normally used, i.e., salts which possess a stabilizing anion; these include, in particular, the sulfate, tetrafluoborate, tetrachlorozincate, hexachloroantimonate and aryl-sulfonate ions.

As solid inorganic and organic acids, there can be used, for example, orthophosphoric acid, metaphosphoric acid, sulfosalicyclic acid or oxalic acid, or acidic salts, for example, potassium bisulfate, as well as adducts of Lewis acids and Lewis bases provided that they have a suitably acidic reaction. The commercially available metaphosphoric acid has proved to be especially useful which contains about 50-60 percent of its sodium salt since we have found that this gives especially stable test papers. Furthermore, metaphosphoric acid and oxalic acid have the advantage that they give test reagents which only react slowly with bilirubin. Generally, it is preferred to use an acid which has a p value of less than 3.0.

The stabilizing additives, which are preferably used, are well known in diazo chemistry; such additives include, for example, sodium fluoborate, sodium aryl sulfonate, magnesium sulfate and sodium metaphosphate.

The use of wetting agents is desirable not only because of the improved wetting which they bring about but also because they bring about additional specific actions. Thus, anionic wetting agents, especially sulfates and sulfonates, bring about an increased sensitivity and a slight bathochromic color displacement in the case of the detection of urobilinogen. Examples of anionic wetting agents which can be used include sodium lauryl sulfate and sodium p-dodecyl-benzene sulfonate.

In some cases, it is desired only to detect a significantly increased urobilinogen level. In such cases, it is possible to reduce the sensitivity and reaction velocity by the addition of cationic wetting agents, for example, lauryl pyridinium chloride.

Non-ionic wetting agents, for example, polyoxyethylene triglyceride, generally only influence the wettability of the test papers.

The wetting agents can be added to the impregnation solution in amounts of about O.l3 g., preferably of 0.31 g., per ml.

The individual components of the formulations can also be applied successively when the solubilities thereof require this or when special effects are to be achieved. Thus, for example, for reasons of stability, it can be advantageous first to apply the diazonium salt and the stabilizer and thereafter to impregnate with the acid.

After the absorbent carrier has been impregnated, it is dried at the lowest possible temperature in order not to damage the diazonium salt.

As absorbent carrier, filter paper is especially preferred but there can also be used fleeces and felts of acid-resistant synthetic resins, for example of polypropylene and polyesters.

The absorbent carriers provided with the reagents are preferably cut up into small squares and sealed on to synthetic resin films or sealed between two synthetic resin films or between a synthetic resin film and a finemeshed material. Although test papers represent a preferred embodiment of the present invention and are the most elegant diagnostic agent for the component materials of body fluids, it is, of course, also possible, to use the diazonium salts according to the present invention for the detection of urobilinogen in the liquid phase. When the diazonium salts in acidic solution are dropped into a urobilinogen-containing urine, then red to blue colorations or precipitates are obtained which can possibly be extracted with an organic solvent, for example, chloroform.

For carrying out the process according to the present invention for the detection of urobilinogen in body fluids, the new diagnostic agents are dipped into the solution to be investigated and the color change read off after a short period of time.

The diazonium salts and the corresponding amines used according to the present invention are generally known.

For the quantitative detection of urobilinogen in urine by means of photometric methods, 4- methoxybenzene diazonium salts, as well as 2,4- dimethoxybenzene diazonium salts, have proved to be especially useful.

The Hammett sigma values, are, as is known, measurement values for the electron attracting or electron repelling effect of substituents (in the first case, positive sigma values are obtained and in the latter case, negative values). The sigma values are additive, the sign before the sum indicating whether, compared with hydrogen, the electron atracting or electron repelling effect preponderates. The limiting value according to the present invention thus means that the action of the electron repelling substituents may be reduced by electron attracting substituents only up to a sigma value of +0.4. Thus, for example, a benzene diazonium salt with a hydroxyl group in the p-position (sigma value O.37) can contain one or more electron attracting groups with a total sigma value of up to +0.83, whereas a henzene diazonium salt which is substituted with an omethoxy or p-acetamino radical (sigma value in both cases i), can only contain electron attracting groups up to a total sigma value of +0.4.

Although Hammett calculations are today carried out routinely and represent a valuable basis for theoretical hypotheses in aromatic chemistry, they should not be regarded as being generally valid, absolute values. On the contrary, they are approximate values which suffer from a measure of uncertainty due to their empirical determination. These uncertainties are substantially insignificant and in no way impair the generic teachings of the present invention.

The following Examples are given for the purpose of illustrating the present invention:

EXAMPLE 1 Filter paper (Schleicher & Schull No. 23 SL) was impregnated with a solution of the following composition and dried at 40C.:

4-mcthoxyben2ene diazonium fluohorate 0.3 g. mctaphosphorie acid 10.0 g. dodecylbenzene-sulfonic acid sodium salt 04 g. methanol 50 ml. water ad 100 ml.

Upon dipping into urine, the test paper gave, after about 5-l0 seconds, the following color reactions:

urobilinogen-free urine: no coloration urine with normal urobilinogen content; pink urme with Increased urobllinogen content: carmine red Similar color reactions were given by test papers which contained 0.3 g.. of one of the following diazonium salts: 2-methoxybenzene diazonium fluoborate;

2-hydroxy-benzene diazonium sulfate; 4- hydroxybenzene diazonium sulfate; and 2,4- dihydroxybenzene diazonium fluoborate.

EXAMPLE 2 Filter paper (Schleicher &.Schull No. 23 SL) was impregnated with a solution of the following composition and dried at 40C.:

4-methoxybenzene diazonium fluoborate 0.3 g. oxalic acid 10.0 g. methanol ad ml.

Individual strips of the test paper (a) so obtained were subsequently impregnated with solutions each containing 0.4 g. of polyoxyethylene triglyceride (b), dodecylbenzene sulfonic acid sodium salt (c) and lauryl-pyridinium chloride (d) in 100 ml. methylene chloride and dried in a current of warm air. The test papers gave, with urobilinogen-containing urine, after about 5-10 seconds, the following reactions:

Test Reaction Color urine with mg urobilinogen Paper 0.5 1.0 2.0

a brick red -H- -l-H- b do. -H- c carmine red -ll- +H- +H-ld brick red H weak; -H- moderate; strong; -H++ very strong.

EXAMPLE 3 Filter paper (Schleicher & Schull) was impregnated with a solution of the following composition and dried at 40C.:

4-ethoxyaniline 0.25 g.

isoamyl nitrite 1.0 ml.

oxalic acid 10.0 g.

dodecylbenzene sulfonic acid sodium salt 0.4 1 g.

EXAMPLE 4 Filter paper (Schleicher & Schull No. 23 SL) was successively impregnated with solutions of the following composition, the paper being dried after each impregnation:

I. 4-acctoamidobenzcne diazonium fluoborate 0.3 g. sodium metaphosphate 5.0 g. water ad 100 ml.

ll oxalic acid l5.0 g. sodium lauryl sulfate 0.5 g. methanol ad 100 ml.

The test paper reacted with urobilinogen-containing urine with a Carmine-red color.

A paper impregnated in an analogous manner with 3-methoxy-4-acetamidobenzene diazonium fluoborate reacted with a red-violet color.

EXAMPLE 5 Filter paper (Schleicher & Schull No. 23 SL) was impregnated with a solution of the following composition and dried at 40C.

3-chloro-4-mcthoxybenzene diazonium toluene sulfonate O sulfosalicyclic acid 5 dodecylbenzene sulfonic acid sodium salt 0. methanol ad 100 bin The test paper immediately gave a carmine red coloration upon dipping into urobilinogen-containing urine. Upon dipping into bilirubin-containing urine, a bluegreen coloration was obtained after to seconds. Urines which contained urobilinogen and bilirubin gave, depending upon the ratio, intermediate shades between these colorations.

EXAMPLE 6 Polyester fleece was impregnated with solutions of the following composition and dried at 40C.:

benzene diazonium salt (a f) sulfosalicyclic acid dodecylbcnzenc sulfonic acid sodium salt methanol/water (lzi tetra- The following colorations were obtained with urobilinogen-containing urine:

a. red blue b. red-violet blue-violet blue-violet blue-green blue-green EXAMPLE 7 Filter paper (Schleicher & Schull No. 23 SL) was impregnated with aqueous solutions which, apart from 5 g. potassium hydrogen sulfate, also contained, per ml., 0.5 g. of one of the following diazonium salts:

2,4-dimethoxybenzene diazonium fluoborate 3,4-dimethoxybenzene diazonium fluoborate 2,5-dimethoxybenzene diazonium fluoborate 3,4,5-trimethoxybenzene diazonium fluoborate 4-hydroxy-3,5-dichlorobenzene diazonium fluobo rate 5-chloro-2,4-dimethoxybenzene diazonium fluoborate All these test papers reacted with urobilinogencontaining urine to give red to lilac-red color shades. When these test papers were subsequently impregnated with 1 percent solutions of the following wetting agents, then stronger red-violet to blue-violet colorations were obtaihed:

dodecylbenzene sulfonic acid sodium salt in methylene chloride; sodium lauryl sulfate in chloroform-methanol; dodecane sulfonic acid sodium salt in chloroformmethanol; dioctyl sodium sulfosuccinate in methylene chloride.

EXAMPLE 8 Filter paper (Schleicher & Schull No. 23 SL) was impregnated with a solution of the following composition and dried at 40C.:

S-aminosalicyclic acid 0.2 g. metaphosphoric acid 15.0 g. sodium nitrite 0.1 g. dodecylbenzcne sulfonic acid sodium salt 0.4 g. methanol 5 ml. water ad 100 ml.

The test paper reacted with urobilinogen-containing urine to give a brick red color.

A paper impregnated in an analogous manner with 5-amino-3-sulfosalicyclic acid reacted to give a brick red color.

EXAMPLE 9 5.0 ml. of urobilinogen-containing urine were mixed with 2.0 ml. of a solution of 2% by weight 2,4- dimethoxybenzene diazonium fluoborate in 2N hydrochloric acid. After 2 minutes, the solution was mixed with 5.0 ml. chloroform and vigorously shaken up. Thereafter, a part of the chloroform phase was placed in a cuvette and the extinction measured at 495 nm. By comparison of the extinction with that of a standard, the precise urobilinogen content of the urine could be determined.

it will be understood that the specification and examples are illustrative but not limitative of the present invention and that other embodiments within the spirit and scope of the invention will suggest themselves to those skilled in the art.

What is claimed is:

1. Diagnostic agent for the detection of urobilinogen in body fluids which agent comprises an acid and at least one stable benzene diazonium salt wherein the phenyl radical contains at least one polyatomic electron donor group substituent with at least one mesomeric electron pair, in the or p-position and wherein the phenyl ring may contain additional substituents, the sum of the Hammett sigma values for all the substituents not exceeding the value of +0.4.

2. Diagnostic agent as claimed in claim 1 wherein said benzene diazonium salt is of the formula Ilia 1'11 R2NEN X in which X is a stabilizing anion, R and R which may be the same or different, are polyatomic electron donor groups containing at least one mesomeric electron pair, or are hydrogen, halogen, or lower alkyl, provided one of R and R is a polyatomic electron donor group, and R and R.,, which may be the same or different, are hydrogen or groups which do not sterically hinder diazo coupling with urobilinogen, the sum of the Hammett sigma vlues of all the substituents R R R and R not exceeding +0.4.

3. Diagnostic agent as claimed in claim 2 wherein R is hydroxy or lower alkylthio or lower alkoxy.

4. Diagnostic agent as claimed in claim 2 wherein R is hydroxy or lower alkoxy, phenoxy, lower aralkoxy, lower alkylthio, phenylthio, lower phenylalkylamino, phenylamino or 'lower alkanoylamino.

5. Diagnostic agent as claimed in claim 2 wherein at least one of R and R is hydrogen, halogen or methyl.

6. Diagnostic agent as claimed in claim 2 wherein R and R are, individually, hydrogen, halogen, hydroxy, lower alkoxy, phenoxy, lower alkylthio, phenylthio, phenyl-lower alkylamino, lower alkyl, sulfo, or carboxyl.

7. Diagnostic agent as claimed in claim 2 wherein at least one of R R and R is substituted phenylamino or substituted lower alkanoylamino.

8. Diagnostic agent as claimed in claim 2 wherein X- is a halide or the anion of an oxygen acid or the anion of a Lewis acid coordinatively saturated with a hydrogen halide.

9. Diagnostic agent as claimed in claim 8 wherein X- is a chloride, sulfate, tetrafluoborate, tetrachlorozincate, hexachloroantimonate, arylsulfonate or carboxylic acid ion.

10. Diagnostic agent as claimed in claim 1 wherein said acid is a solid organic or inorganic acid.

11. Diagnostic agent as claimed in claim 10 wherein the acid is orthophosphoric acid, metaphosphoric acid, sulfosalicyclic acid, oxalic acid or an acid salt.

12. Diagnostic agent as claimed in claim 11 in the form of an absorbent carrier which has been impreg nated with a solution of a solid acid and at least one of said diazonium salts.

13. Diagnostic agent as claimed in claim 12 additionally comprising a wetting agent.

14. Diagnostic agent as claimed in claim 12 additionally comprising a stabilizing agent for the diazonium salt.

15. Method for the detection of urobilinogen optionally together with bilirubin which method comprises contacting the test substance with a diagnostic agent as claimed in claim 1.

. 16. Diagnostic agent as claimed in claim 1 wherein said benzenediazonium salt is 4-methoxybenzene diazonium fluoborate.

17. Diagnostic agent as claimed in claim 1 wherein said benzenediazonium salt is 4-ethoxybenzene diazonium fluoborate.

18. Diagnostic agent as claimed in claim 1 wherein said benzenediazonium salt is 4-acetoamidobenzene diazonium fluoborate.

19. Diagnostic agent as claimed in claim 1 wherein said benzenediazonium salt is 3-chloro-4- methoxybenzene diazonium toluene sulfonate.

20. Diagnostic agent as claimed in claim 1 wherein said benzenediazonium salt is selected from the group consisting of:

2-methoxy-5-chlorobenzene diazonium tetrachlorozincate, 4-benzamido-2-methyl-5-methoxybenzene nium tetrachlorozincate, 4-benzamido-2,5-dimethoxybenzenediazonium tetrachlorozincate,

4-benzamido-2,S-diethoxybenzene diazonium tetrachlorozincate,

4-methoxyphenylaminobenzene diazonium chloride,

and

phenylaminobenzene diazonium hydrogen sulfate.

21. Diagnostic agent as claimed in claim 12 wherein said benzene-diazonium salt is at least one of 4- methoxybenzene diazonium fluoborate, 4- ethoxybenzene-diazonium-fluoborate, 4- acetoamidobenzene diazonium fluoborate or 3-chloro- 4-methoxybenzene diazonium toluene sulfonate.

22. Method as claimed in claim 15 wherein said benzene-diazonium salt is 4-methoxybenzene diazonium fluoborate, 4-ethoxybenzene-diazonium-fluoborate, 4-acetoamidobenzene diazonium fluoborate or 3-chloro-4-methoxybenzene diazonium toluene sulfonate. I

23. Diagnostic agent as claimed in claim 2 in the form of an absorbent carrier which has been impregnated with a solution of the acid and at least one of said benzene-diazonium salts.

24. Diagnostic agent as claimed in claim 3 in the form of an absorbent carrier which has been impregnated with a solution of the acid and at least one of said benzene-diazonium salts.

25. Diagnostic agent as claimed in claim 4 in the form of an absorbent carrier which has been impregnated with a solution of the acid and at least one of said benzene-diazonium salts.

26. Diagnostic agent as claimed in claim 5 in the form of an absorbent carrier which has been impregnated with a solution of the acid and at least one of said bendiazo- -zene-diazonium salts.

27. Diagnostic agent as claimed in claim 6 in the form of an absorbent carrier which has been impregnated with a solution of the acid and at least one of said benzene-diazonium salts.

28. Diagnostic agent as claimed in claim 7 in the form of an absorbent carrier which has been impregnated with a solution of the acid and at least one of said benzene-diazonium salts.

29. Method for the detection of urobilinogen optionally together with bilirubin which method comprises contacting the test substance with a diagnostic agent as claimed in claim 5.

33. Method for the detection of urobilinogen optionally together with bilirubin which method comprises contacting the test substance with a diagnostic agent as claimed in claim 6.

34. Method for the detection of urobilinogen optionally together with bilirubin which method comprises contacting the test substance with a diagnostic agent as claimed in claim 7.

Claims

1. DIAGNOSTIC AGENT FOR THE DETECTION OF UROBILINOGEN IN BODY FLUID WHICH AGENT COMPRISES AN ACID AND AT LEAST ONE STABLE BENZENE DIAZONIUM SALT WHEREIN THE PHENYL RADICAL CONTAINS AT LEAST ONE POLYATOMIC ELECTRON DONOR GROUP SUBSTITUENT WITH AT LEAST ONE MESOMERIC ELECTRON PAIR, IN THE O- OR P-POSITION AND WHEREIN THE PHENYL RING MAY CONTAIN ADDITIONAL SUBSTITUENTS, THE SUM OF THE HAMMETT SIGMA VALUES FOR ALL THE SUBSTITUENTS NOT EXCEEDING THE VALUE OF +0.4.

2. Diagnostic agent as claimed in claim 1 wherein said benzene diazonium salt is of the formula

3. Diagnostic agent as claimed in claim 2 wherein R1 is hydroxy or lower alkylthio or lower alkoxy.

4. Diagnostic agent as claimed in claim 2 wherein R2 is hydroxy or lower alkoxy, phenoxy, lower aralkoxy, lower alkylthio, phenylthio, lower phenylalkylamino, phenylamino or lower alkanoylamino.

5. Diagnostic agent as claimed in claim 2 wherein at least one of R1 and R2 is hydrogen, halogen or methyl.

6. Diagnostic agent as claimed in claim 2 wherein R3 and R4 are, inDividually, hydrogen, halogen, hydroxy, lower alkoxy, phenoxy, lower alkylthio, phenylthio, phenyl-lower alkylamino, lower alkyl, sulfo, or carboxyl.

7. Diagnostic agent as claimed in claim 2 wherein at least one of R2, R3 and R4 is substituted phenylamino or substituted lower alkanoylamino.

8. Diagnostic agent as claimed in claim 2 wherein X is a halide or the anion of an oxygen acid or the anion of a Lewis acid coordinatively saturated with a hydrogen halide.

9. Diagnostic agent as claimed in claim 8 wherein X is a chloride, sulfate, tetrafluoborate, tetrachlorozincate, hexachloroantimonate, arylsulfonate or carboxylic acid ion.

12. Diagnostic agent as claimed in claim 11 in the form of an absorbent carrier which has been impregnated with a solution of a solid acid and at least one of said diazonium salts.

16. Diagnostic agent as claimed in claim 1 wherein said benzenediazonium salt is 4-methoxybenzene diazonium fluoborate.

19. Diagnostic agent as claimed in claim 1 wherein said benzenediazonium salt is 3-chloro-4-methoxybenzene diazonium toluene sulfonate.

20. Diagnostic agent as claimed in claim 1 wherein said benzenediazonium salt is selected from the group consisting of: 2-methoxy-5-chlorobenzene diazonium tetrachlorozincate, 4-benzamido-2-methyl-5-methoxybenzene diazonium tetrachlorozincate, 4-benzamido-2,5-dimethoxybenzenediazonium tetrachlorozincate, 4-benzamido-2,5-diethoxybenzene diazonium tetrachlorozincate, 4-methoxyphenylaminobenzene diazonium chloride, and phenylaminobenzene diazonium hydrogen sulfate.

21. Diagnostic agent as claimed in claim 12 wherein said benzene-diazonium salt is at least one of 4-methoxybenzene diazonium fluoborate, 4-ethoxybenzene-diazonium-fluoborate, 4-acetoamidobenzene diazonium fluoborate or 3-chloro-4-methoxybenzene diazonium toluene sulfonate.

22. Method as claimed in claim 15 wherein said benzene-diazonium salt is 4-methoxybenzene diazonium fluoborate, 4-ethoxybenzene-diazonium-fluoborate, 4-acetoamidobenzene diazonium fluoborate or 3-chloro-4-methoxybenzene diazonium toluene sulfonate.

26. Diagnostic agent as claimed in claim 5 in the form of an absorbent carrier which has been impregnated with a solution of the acid and at least one of said benzene-diazonium salts.

29. Method for the detection of urobilinogen optionally together with bilirubin which method comprises contacting the test substance with a diagnostic agent as claimed in claim 2.

30. Method for the detection of urobilinogen optionally together with bilirubin which method comprises contacting the test substance with a diagnostic agent as claimed in claim 3.

31. Method for the detection of urobilinogen optionally together with bilirubin which method comprises contacting the test substance with a diagnostic agent as claimed in claim 4.

32. Method for the detection of urobilinogen optionally together with bilirubin which method comprises contacting the test substance with a diagnostic agent as claimed in claim 5.