DK143147B - PROCEDURE FOR DETERMINING THE UROBILINOGEN EVEN WITH THE CAR CAR RUBIN AND THE USE OF THE PROCEDURE - Google Patents

Description

(W

(11) PUBLICATION 143147 DENMARK i «) int.ci.3 g 01 m 33/50 § (21) Application No. 3033/72 (22) Filed on 1 June 6. 1972 (24) Race day 1 Jun 6 1972 (44) The application submitted and the petition published on 29 · Jun. 1 9o1

DIRECTORATE OF

PATENT AND TRADEMARK (3 °) Priority earned from it

19 · Jun. 1971a 2130559a DE

(41) Aim. avail. 20 dec. 1972 <71> BOEHRINGER MANNHEIM GMBH, Mannheim-Waldhof, DE.

(72) Inventor: Walter Rittersdorf, Kas seler-Strasse 6, Mannheim-Waldhof, DE: Hans-Georg Rey, Herafelderstrasse 18, Mannheim-Waldhof, DE: Dieter Berger, Johann-Sebastian-Bach Str. 18, Viernhelm, DE: Peter Rieckmann,

Stegervi aldw eg 10, Mannheim-Waldhof, DE.

(74) Plenipotentiary in the proceedings:

The company Chas. Skin. (54) Method for detecting urobilinogen, optionally with bi * = lirubin, and agent for use in the method.

The invention relates to a method for detecting urobilinogen, optionally with bilirubin, in body fluids, preferably in urine.

It is known to detect urobilinogens (bilanes), indole, sulfonamides, porphobilinogen, urine indicator and 5-hydroxyindole acetic acid with a solution of p-dimethylaminobenzaldehyde in hydrochloric acid. This finding is known in the literature as Ehrlich's reaction. It has been particularly important in medical diagnostics as the detection of "propagated urobilinogens" in urine. Although not very specific, the sample serves as a standard method for the diagnosis of liver and bile disorders.

As test papers in the clinical-chemical diagnostics are still gaining more importance, meanwhile, test papers have been developed for the detection of urobilinogen based on Ehrlich's reaction. These test papers have two major shortcomings: 1. The color reaction develops so slowly that, before reading, you have to wait at least one minute.

2. The test papers have, by their nature, the un-specificity of the Ehrlich's test, so that false positive results cannot be ruled out.

It has long been mentioned that urobilinogen couples with diazotized sulphanilic acid to a yellow dye. This reaction was discovered in 1884 by Ehrlich and described as the so-called "yellow diazor reaction". The reaction was also investigated several times in the following, however, to this day it was not possible to determine with certainty whether it was undoubtedly a diazocoupling with urobilinogen or whether other substances were also responsible for the formation of the yellow dyes, ( see Biologie der Gallenfarbstoffe, Georg Thieme-Verlag, Stuttgart I960, p. 32 and p. 211). As this was more about a curiosity than a useful means of clinical chemistry diagnostics, the yellow diazor reaction was practically meaningless and has hardly been discussed in the textbooks concerned.

From Swedish Patent Specification No. 344,503, it is known that certain substituted benzaldehyde salts give a color reaction with urobilinogen which is less disturbed than with the known p-dimethylaminobenzaldehyde, but the reaction is not as specific as in the process of the invention and it requires a reaction time of 1-2 minutes.

Surprisingly, a specific and very rapid method for detecting urobilinogen, optionally with bilirubin, has been found, which method of the invention is characterized by the use of stable benzoldiazonium salts, if optionally several times substituted phenyl residue in the ortho or para position. contains at least one multi-atomic electron donor group with at least one mesomeric-capable electron pair, the sum of the Hammett * spaced sigma values of all substituents not exceeding +0.4 as indicator substances.

143147 3

Electron donor groups according to the invention are groups containing oxygen, sulfur or nitrogen in direct bond with the phenyl residue. Oxygen-containing groups are, for example, hydroxy, alkoxy, aralkoxy or aryloxy groups. As sulfur-containing groups, only alkyl mercapto or aryl mercapto groups are considered, since free mercapto groups, because of their oxidation sensitivity, are generally not usable. As nitrogen-containing groups, acylamino, arylamino or arylalkylamino groups are useful, while, because of their basicity under the reaction conditions, unsubstituted amino groups and alkylamino groups are mostly protonated and then no longer have any mesomeric electron pairs. In choosing the substituent type, it must be further remembered that these do not react with the diazo group.

This is particularly true of o-acylamino and o-arylamino groups.

In particular, according to the invention, benzoldiazonium salts are suitable of the general formula I

R 2 R 2 - N 2 + (I) r 4 in which X 2 is a stabilizing anion and R 2 is multeratomic electron donor groups having at least one mesomeric-capable electron pair, one of the residues R 2 and R 2 being also hydrogen, halogen or a lower alkyl group, and the residues R 1 and R 2 are hydrogen or a group which does not sterically hinder the diazocoupling with urobilinogen, the sum of the Hammett's sigma values for all substituents R 1 'R 2' R 3 ° S r 4 being elevated to +0, 4th

Compounds I are particularly suitable for use on sugary carriers.

The benzoldiazonium salts of the invention react with urobilinogen almost instantaneously and give very specific red to blue dyes which are particularly suitable for detecting even very small concentrations. In addition, the compounds of the invention have the greater advantage that the color reaction is not disturbed by urea. The known test papers are colored yellow by the urine present in urine according to the concentration, which can make the detection of small amounts of urobilinogen considerably difficult. In addition, the color reaction according to the invention is precisely not affected by the substances that frequently occur in urine and which, as you know, mostly interfere with Ehrlich's test. In particular, these interfering substances include the aromatic amines which, in many cases, in the form of drugs (sulfonamides, sulfonylureas etc.), are excreted together with the urine. The presence of these aromatic amines in urine has so far led to yellow, possibly orange color reactions, which could both cover and falsify the urobilinogen-induced staining.

For the detection of the bilirubin that is also important for the diagnosis of liver and biliary disorders, diazonium salts (eg diazosulfanilic acid, p-nitrobenzoldiazonium salt or 2,4-dichlorobenzolezoldiazonium salt) have also been used for a long time. However, these probably do not produce a useful color reaction with urobilinogen.

In contrast, the compounds of the invention allow specific detection of urobilinogen next to bilirubin. In some cases, bilirubin also reacts with the dyes of the invention, however, the reaction only emerges after the dye reaction with urobilinogen has already developed far, and differs in color so strongly that it is strange even possible to determine both bile dyes with one reagent by side by side, and to estimate the volume ratios according to the blend color developed. Thus, a red staining shows the presence of urobilinogen and a later developed blue staining the presence of bilirubin. In the presence of both bile dyes at the same time, the test strip first turns red and later increases to violet.

In exceptional cases, the color reactions develop simultaneously, but the test result is not affected.

In case the reaction with bilirubin is to be suppressed, this can be achieved by using as an indicator a diazonium salt with relatively low electrophilia and allowing the reaction to proceed with the addition of cationic wetting agents.

In this way, it is possible to modify the means for the detection of urobilinogen, possibly together with bilirubin, so that they become optimally suitable for the intended purpose.

The invention further relates to an agent for use in the process described, which is characterized by a content of at least one diazonium salt as defined above and a solid organic or inorganic acid.

The compounds of the general formula I are preferably used for the preparation of test papers. The substances become for this purpose together with an acid and optionally with additives such as e.g. stabilizers and wetting agents applied to a suction-resistant carrier. In addition, the compounds I are suitable for the preparation of sample film strips according to DOS 1,598,153 and for the detection of urobilinogen in solutions.

To prepare the preferred embodiment of the novel diagnostic agents, a sugary carrier, preferably filter paper, is wetted with a solution containing 0.05-5 g, preferably 0.1-1 g (per 100 ml) of a diazonium salt of the general formula I and 2-30 g, preferably 5-20 g of solid inorganic or organic acids, optionally containing wetting agents or stabilizers or both.

As solvents for the impregnation solutions, above all, water and volatile organic solvents which do not react with the diazonium salts are considered. These are preferably lower alcohols, ethyl acetate and acetonitrile.

In the preparation of the diazonium salt solution, one can either proceed to add the final diazonium salt to the solution or by preparing the salt in the solution in a known manner by diazotizing an aromatic amine.

Preferred and particularly useful in the invention are those compounds of the general formula I in which is a hydroxy, alkylmercapto or lower alkoxy group and R 2 is a hydroxy, alkoxy, phenoxy, aralkoxy, alkylmercapto. , phenylmercapto, phenylalkylamino, optionally substituted phenylamino or acylamino group, one of the residues and R 2 also being hydrogen, halogen or a methyl group and and hydrogen, a halogen atom, a hydroxy, alkoxy, phenoxy, alkylmercapto , phenylmercapto, phenylalkylamino, optionally substituted phenylamino, acylamino, alkylsulfo and carb = oxyl group and) a halide or the anion of an oxygenic acid or one of halohydrogenally coordinated saturated Lewis acid.

For durability reasons, only stable dizonium salts are used, i.e. salts which have a stabilizing anion. These are, in particular, the sulfate, tetrafluoroborate, tetrachlorozincate and hexachloroanation and arylsulfonate.

As solid inorganic and organic acids, for example, o -phosphoric acid, metaphosphoric acid, sulfosalicylic acid, oxalic acid or acid salts, e.g. potassium bisulfate on speech. Also, adducts of Lewis acids and Lewis bases can be used when reacting similarly acidic. In particular, the commercially available metaphosphoric acid has proved suitable; it contains approx. 50-60% of its sodium salt, as this experience provides particularly stable test papers.

Metaphosphoric acid and oxalic acid also have the advantage of providing test papers which only react very slowly with bilirubin.

The stabilizing additives are already sufficiently known from the diazochemistry. Such additives are e.g. sodium fluoroborate, sodium triarylsulfonate, magnesium sulfate or sodium metaphosphate.

The use of wetting agents is not only recommended because of the better wettability achieved, as they also exert additional specific effects. Thus, anionic wetting agents, in particular sulfates and sulfonates, increase the sensitivity and a slight bathochrome color shift in the detection of urobilinogen.

Examples of anionic wetting agents include sodium = lauryl sulfate and sodium p-dodecylbenzene sulfonate.

In many cases, it is desired to detect only a significant elevated urobilinogen mirror. In these cases, it is possible to decrease the sensitivity and reaction rate by the addition of cationic wetting agents, such as e.g. laurylpyridinium.

Nonionic wetting agents, such as polyoxyethylene triglycide = ridge, generally affects only the wettability of the sample papers.

The wetting agents are added in amounts of approx. 0.1-3 g, preferably 0.3-1 g per 100 ml of impregnating solution.

The individual components of the prescription can also be applied one after the other when the solubilities require it or when special effects are to be obtained. Thus, e.g. for reasons of stability, it is advantageous first to add the diazonium salt and stabilizer and then first to post-impregnate with acid.

Following the impregnation of the sugary carriers, these are dried at as low a temperature as possible so as not to damage the diazonium salts.

As sugary carriers, above all, filter paper comes to mind, but also a chip and a felt of acid-resistant plastics such as polypropylene and polyester can be used.

The reagent suction-supported carriers are preferably cut into small squares and adhered to plastic wrap or pasted between plastic wrap or wrap and fine mesh.

Of course, although the test papers are a preferred embodiment of the present invention and are the most elegant diagnostic agents for ingredients in body fluids, it is also possible to use the diazonium salts of the invention for liquid phase urobilinogen detection. If the diazonium salts are dipped in acidic solution to urobilinogen-containing urine, red to blue stains or precipitates are obtained, which may be extracted by means of organic solvents, such as e.g. chloroform.

In order to carry out the method according to the invention for detecting urobilinogen in body fluids, the new diagnostic means are dipped into the solution to be investigated and the color wrapping 143147 8 is read after a short time.

The diazonium salts used according to the invention or the corresponding amines, respectively, are well known in the art.

For the quantitative detection of urobilinogen in urine by photometric methods, 4-methoxybenzoldiazonium salts and 2,4-dimethoxybenzoldiazonium salts have been found particularly suitable as detection reagents.

The Hammettian sigma values are, as is well known, the measure of the electron-attracting or electron-emitting effect of substituents (in the first case you get positive, in the last negative sigma values). The sigma values are additive, with the sum of the sums indicating whether - compared to the substituent hydrogen - the electron-attracting or electron-emitting effect is predominant. Thus, the limit value according to the invention +0.4 says that the effect of the electron emitting substituent may only be present at the sigma value +0.4. For example, a benzoldiazonium salt having a hydroxyl group in para position (sigma value = -0.37) may have one or more electron attracting groups with a total sigma value up to + 0.83, while such a diazonium salt substituted with an o-methoxy or p-acetamino group (sigma value in both cases = * 0), may only contain groups up to a total sigma value of +0.4.

Although Hammett calculations are routinely carried out today and are a valuable means of theoretical explanations in the chemistry of aromatics, it is not permissible to consider these as generally valid absolute values. Rather, these are approximation values, which are subject to certain uncertainties in the empirical provision. However, these uncertainties only rarely play a role which in no way affects the technical teachings of the invention.

In the following examples, the method according to the invention and the diagnostic means according to the invention are more fully illustrated.

1 A3147 9

Example 1

Filter paper (Schleicher und Schiill 23 SL) is impregnated with a solution of the following composition and dried at 40 ° C.

4-methoxybenzoldiazonium fluoborate 0.3 g metaphosphoric acid 10.0 g dodecylbenzene sulfonic acid (sodium salt) 0.4 g methanol 5.0 ml water approx. 100.0 ml

The test paper, when immersed in urine after approx. 5-10 seconds the following color reactions:

Urobilinogen-free urine: no staining urine with normal urobilinogen content: pink urine with elevated urobilinogen content: carmin red.

Similar color reactions yield test papers containing 0.3 g of the following diazonium salts: 2-methoxybenzoldiazonium fluoborate 2-hydroxybenzoldiazonium sulfate 4-hydroxybenzene diazonium sulfate 2,4-dihydroxybenzoldiazonium fluoborate.

Example 2

Filter paper (Schleicher und Schiill 23 SL) is impregnated with a solution of the following composition and dried at 40 ° C.

* 4-methoxybenzoldiazonium fluoborate 0.3 g oxalic acid 10.0 g methanol approx. 100.0 ml

Single samples of the test paper thus obtained (a) were post-impregnated with solutions, each containing 0.4 g of polyoxyethylene triglyceride (b), dodecylbenzene sulfonic acid (sodium salt) (c), respectively, dried in a hot air stream. The test papers give urobilinogen-containing urine after approx. 5-10 seconds the following reactions:

Sample paper Reaction color Urine with mg% urobilinogen _ _ 0 * 5_1 * 0_2 * 0 a brick red + ++ +++ b "+ ++ +++ c carmin red ++ +++ ++++ d brick red + ++ (+ = weak, ++ = medium, +++ = strong, ++++ = very strong)

Example 5

Filter paper (Schleicher und Schull) is impregnated with a solution of the following composition and dried at 40 ° C.

4-ethoxyaniline 0.25 g isoamyl nitrite 1.0 ml oxalic acid 10.0 g dodecylbenzene sulfonic acid (sodium salt) 0.4 g methanol approx. 100.0 ml The sample paper reacts carmine red with urobilinogen-containing urine. With similar, somewhat more violet tones, test papers react in which 4-ethoxyaniline is replaced by the same amounts of 2-ethoxy-, 4-butoxy-, 4-phenoxy-, 4-methoxy-2-methyl-, 2- methoxy-5-methyl-, 2-methylmercapto-, 4-methylmercapto-, 4-chloro-2-methoxy-5-methyl-, 5-chloro-2-hydroxy- or 4-benzyloxyaniline.

Example 4

Filter paper (Schleicher und Schull 23 SL) is impregnated with the following solutions one after the other and dried.

I. 4-acetamidobenzoldiazonium fluoborate 0.3 g sodium metaphosphate 5.0 g water approx. 100.0 ml II. oxalic acid 15.0 g sodium lauryl sulfate 0.5 g methanol approx. 100.0 ml

The test paper reacts with urobilinogen-containing urine with carmin red color. A similar paper with 3-methoxy-4-acetamidobenzoldiazonium fluoro borate impregnated paper reacts with red-violet color.

Example 5

Filter paper (Schleicher und Schtill 23 SL) is impregnated with a solution of the following composition and dried at 40 ° C.

3-chloro-4-methoxybenzoldiazonium toluene sulfonate 0.5 g sulfosalicylic acid 5> 0 g dodecyl benzene sulfonic acid (sodium salt) 0.8 g methanol approx. 100.0 ml.

The test paper, when immersed in urobilinogen-containing urine, immediately gives a carmin red stain. Immersion in bilirubin-containing urine gives a blue-green staining after 10-20 seconds. Urines containing urobilinogen and bilirubin, according to the ratio, provide intermediate tones between these stains.

Example 6

Polyester chips are impregnated with solutions of the following composition and dried at 40 ° C.

Benzoldiazonium salt (a-f) 0.1 g sulfosalicylic acid 10.0 g dodecyl benzene sulfonic acid (sodium salt) 0.4 g methanol / water 1: 1 approx. 100.0 g The following diazonium salts were used: a 2-methoxy-5-chlorobenzole diazonium = tetrachlorozincate (RC oat salt) b 4-benzamido-2-methyl-5-methoxybenzene = diazonium tetrachlorozincate (real violet salt B) c 4-benzamido-2.5 -dimethoxybenzoldia = zonium tetrachlorozincate (true blue salt RR) d 4-benzamido-2,5-diethoxybenzoldia = zonium tetrachlorozincate (true blue salt BB) e 4-methoxyphenylaminobenzoldiazonium = chloride (variamine blue salt B) phenylamine

With urobilinogen-containing urine the following stains were obtained: a red - blue b red violet c blue violet d blue violet e blue green f blue green

Example 7

Filter paper (Schleicher und Schull 23 SL) is dipped in aqueous solutions which, in addition to 5 g of potassium hydrogen sulfate, each contain 0.5 g of the following 100 ml diazonium salts.

2.4-dimethoxybenzoldiazonium fluoborate 3.4-dimethoxybenzoldiazonium fluoborate 2.5-dimethoxybenzoldiazonium fluoborate 3.4.5-trimethoxybenzoldiazonium fluoborate 4-hydroxy-3,5-dichlorobenzoldiazonium fluoborate 5-chloro-2,4-dimethoxybenzoldiazole

All of these test papers react with urobilinogen-containing urine with red to purple tones.

If these test papers are post-impregnated with similar solutions of the following wetting agents, stronger red-violet to blue-violet stains are obtained.

Dodecylbenzene sulfonic acid (the sodium salt) in methylene chloride. Sodium lauryl sulfate in chloroform methanol.

Dodecanesulfonic acid (the sodium salt) in chloroform methanol. Dioctyl sodium sulfosuccinate in methylene chloride.

Example 8

Filter paper (Schleicher tind Schull 23 SL) is impregnated with a solution of the following composition and dried at 40 ° C.

5-aminosalicylic acid 0.2 g metaphosphoric acid 15.0 g sodium nitrite 0.1 g dodecylbenzene sulfoic acid (sodium salt) 0.4 g methanol 5.0 ml water 100.0 ml

The test paper reacts with urobilinogen-containing urine in brick red color. A similarly impregnated paper with 5-amino-3-sulfosalicylic acid reacts with brick red color.

Example 9 5.0 ml of urobilinogen-containing urine is reacted with 2.0 ml of a solution of 2% 2,4-dimethoxybenzoldiazonium fluoborate in 2N hydrochloric acid: After 2 minutes, 5.0 ml of chloroform is added and vigorous shaken. Then, part of the chloroform phase is transferred to a cuvette and the ex-tinction measured at 495 nm. By comparison with the extinction of a standard, the exact urobilinogen content of the urine can be determined.

Claims (3)

143147 14 Patent Claims. A method for detecting urobilinogen, optionally together with bilirubin, characterized by using stable benzoldiazonium salts, if optionally several times substituted phenyl residue in the ortho or para position contains at least one multi-atomic electron donor group with at least one mesomeric low electron pair, of the Hammett * sigma values of all substituents must not exceed the value +0.4 as indicator substances. Process according to claim 1, characterized in that compounds of the general formula X R 3 are R 1 - R 2 - (β) R 4 where is a stabilizing anion and R 1 and R 2 are multi-atomic electron donor groups having at least one mesomeric-capable electron pairs, one of the residues R and R may also be hydrogen, halogen, or a lower alkyl group, and residues R and R are hydrogen or a diazocoupling with urobilinogen sterically non-hindering group, the sum of the Hammett * sigma values for all substituents R 1, R 2, R 3 and R 4 must not exceed +0.4 as indicator substances. Process according to claim 2, characterized in that compounds of the general formula I are used wherein R 1 is hydroxy, 2 alkyl mercapto or lower alkoxy groups and R is a hydroxy, alkoxy, phenoxy, aralkoxy, alkyl mercapto, phenyl mercapto, phenylalkylamino, optionally substituted phenylamino or acylamino group, one of the residues R and R may also be hydrogen, halogen or a methyl group, and R and R may be hydrogen, a halogen atom, a hydroxy, alkoxy, phenoxy, alkylmercapto, phenylmercapto, phe