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Publication numberUS3630680 A
Publication typeGrant
Publication dateDec 28, 1971
Filing dateOct 18, 1968
Priority dateOct 26, 1967
Also published asCA935365A, CA935365A1, DE1648848A1, DE1648848B2, DE1767931A1, DE1767931B2, DE1767931C3
Publication numberUS 3630680 A, US 3630680A, US-A-3630680, US3630680 A, US3630680A
InventorsPeter Rieckmann, Walter Rittersdorf
Original AssigneeBoehringer Mannheim Gmbh
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Diagnostic agents for the detection of urobilinogen materials in body fluids
US 3630680 A
Abstract  available in
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Description  (OCR text may contain errors)

United States Patent [72] Inventors 7 Walter Rlttersdorf;

Hans-Georg Rey; Peter Rieckmann, all of Mannheim-Waldhot, Germany [2!] Appl. No. 768,882

[22] Filed Oct. 18, 1968 [45] Patented Dec. 28, 1971 [73] Assignee Boehringer Mannheim GmbH Mannheim, Germany [32] Priorities Oct. 26, 1967 [3 3 Germany July 3, 1968, Germany, No. P 17 67 931.1

[54] DIAGNOSTIC AGENTS FOR THE DETECTION OF UROBILINOGEN MATERIALS IN BODY FLUIDS OTHER REFERENCES J. Fischl and N. Pinto, Clinica Chimica Acta, 2, 527- 33 (1957).

Primary ExaminerMorris O. Wolk Assistant Examiner-Sidney Marantz Attarney--Burgess, Dinklage & Sprung ABSTRACT: Diagnostic agents for use in the detection of urobilinogen materials in body fluids such as urine, the agents comprising at least one strong acid and at least one compound which reacts with urobilinogen materials present in the body fluids to provide analytically measurable dyestuffs wherein said compound has the formula:

3 in which R, and R, which may be the same or different, each represent halogen, cyano, nitroso, carbalkoxy, carbamido, hydroxy, acyloxy, alkoxy, straight-chained and cyclic, saturated and unsaturated secondary and tertiary amino, or R, and R together represent oxygen, sulfur, sulfonyl, alkylthionium, imino, alkylimino, substituted or unsubstituted arylimino or open-chained, alicyclic or heterocyclic dialkylimino, R, can also represent the residue of secondary straight or branched, open-chained or monoor polycyclic alkyl or aralkyl and wherein one of R, and R, can also represent hydrogen, R is hydrogen or alkyl, and n and m which may be the same or different each have a value ofO, l, 2 or 3.

DIAGNOSTIC AGENTS FOR THE DETECTION OF UROBILINOGEN MATERIALS IN BODY FLUIDS The present invention relates to new and improved diagnostic agents for the detection of urobilinogen bodies in body fluids. More particularly, this invention relates to improved diagnostic agents for the detection of urobilinogen bodies in urine, spinal fluids and other body fluids.

It is known that urobilinogen bodies (bilans), indole, sulfonamides, porphobilinogens, urine indican and S-hydroxy-indole-acetic acid can be detected using a solution of pdimethylaminobenzaldehyde in hydrochloric acid. This detection test is known in the literature as Ehrlichs reaction. It has achieved considerable importance, particularly in medical diagnosis, for the detection of increased urobilinogens" in the urine. Although the test is not very specific, it has come to be regarded as the standard test method for the diagnosis of diseases of both the liver and gall bladder.

In an attempt to simplify this test, it has already been proposed to make up test tablets (British Patent Specification No. 779,921) in which there is present pdimethylaminobenzaldehyde, and a solid inorganic or organic acid (also U.S. Pat. No. 2,320,282 directed to test tablets for the detection of sulfonamides). When such tablets are dissolved in more or less diluted urine, there is obtained a color reaction which corresponds approximately to that produced in accordance with the original Ehrlich test. However, for the evaluation of the color reaction produced when, for example, sulfonamides are present, it is necessary to add a blue auxiliary dyestuff, such as methylene blue.

In accordance with a further proposal for the simplification of the Ehrlich test, the reaction has also been carried out employing test papers, (Fischl and Pinto, Clinica Chim. Acta, 2,

527533/, and French Patent No. l,450,273, in which the phosphoric acid used by Fischi and Pinto is replaced by oxalic acid).

It is an object of the present invention to provide an improved diagnostic agent for use in the detection of unrobilinogens present in body fluids which avoids the disadvantages of the art.

Another object of the present invention is a simple and practical diagnostic agent for use in the detection of urobilinogens present in body fluids comprising a-test paper or film.

Other objects will appear hereinafter.

In accordance with the invention, it has now been found that diagnostic agents for use in the detection of urobilinogens present in body fluids are obtained by employing a strong acid in admixture with a derivative of p-aminobenzaldehyde.

Preferred p-aminobenzaldehyde derivatives to be used according to the present invention are those which carry on the p-amino group, a branched alkyl group or a substituent which possesses strong electron attracting properties. Illustrative of the compounds of this type are those having the formula:

wherein R, and R,, which may be the same or different, are each halogen, cyano, nitroso, carbalkoxy, carbamido, hydroxyl, acyloxy, alkoxy or open-chained or cyclic, saturated or un saturated, secondary or tertiary amino or R and R together represent oxygen, sulfur, sulfonyl, alkylthionium, imino, alkylimino, unsubstituted or substituted arylimino openchained, alicyclic or heterocyclic dialkylimino radical, R, can also be the residue of a secondary straight-chained or branched, open-chained, monoor polycyclic alkyl or aralkyl and wherein one of the substituents R, and R; can be hydrogen, R represents hydrogen or alkyl and n and m, which may be the same or different, are 0, l, 2 or 3.

In preparing the diagnostic agents according to the invention, the compounds of the above formula (I) are preferably applied to an absorbent carrier, together with a strong, solid acid. The compounds (I) can also be used for the preparation of test film strips according to the procedure described in published Dutch Pat. application No. 67.15 828 and also in a form suitable for the detection of urobilinogens in solutions.

The preferred embodiment of the new diagnostic agents according to the present invention, i.e., test paper strips are prepared by impregnating an absorbent carrier, preferably filter paper, with a solution which contains 0.00 [-1 percent of an aldehyde of formula I and at least 3 percent, preferably 15-30 percent, of a strong, solid acid.

Examples of suitable strong, solid acids include sulfosalicyclic acid, p-toluene-sulfonic acid, potassium bisulfate, glutamic acid hydrochloride and preferably oxalic acid.

The impregnation solution can also contain inert additives, such as for instance polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of polyvinyl pyrrolidone and polyvinyl acetate, polyglycols and the like.

As solvents for use in preparing the solution for use in the impregnation of the absorbent carrier, there are suitable all readily volatile solvents in which the aldehydes (I) and also the acids are sufficiently soluble. Consequently, it is preferred to use polar solvents, such as methanol.

After impregnation, the filter papers are dried, out up and, if desired, sealed on the synthetic resin foils or sealed between synthetic resin foils. When rodlets or strips are used as absorbent carriers then they are ready for use immediately after drying.

In carrying out the test reaction according to the present invention for the detection of urobilinogens or of unrobilinogen bodies, the new diagnostic agents according to the present invention are dipped into the solution to be investigated and the color change then evaluated after a short period of time, i.e., l to 2 minutes.

In contradistinction to the previously known diagnostic agents available for the detection of urobilinogens, the new diagnostic reagents according to the present invention are substantially more sensitive and are capable of detecting amounts as low as about 0.4 mg.-percent urobilinogen in urme.

Surprisingly, this color reaction is not disturbed by the presence of urea. Even a 3percent solution of urea hardly causes any color change to take place on the new test papers according to the present invention, whereas test papers which contain p-dimethylaminobenzaldehyde take on an intense yel' low color.

Furthermore, it could not have been foreseen that the new diagnostic agents according to the present invention would not be adversely affected either by urine indican or by indole or by sulfonamides (in the case of normal dosages) in urine. This finding is particularly surprising because the compounds (I), dissolved in 2-20 percent hydrochloric acid, can be used very satisfactorily for the detection of indole in solution. For the detection of urobilinogens and of urobilinogen bodies in solutions, p-dimethylaminobenzaldehyde can, of course, be replaced by compounds of formula I and the content thus determined photometrically in a cuvette in a very exact manner and free of any interference.

A preferred group of benzaldehyde derivatives according to the present invention are compounds having the formula:

H (CH) R:

chained or monoor polycyclic alkyl or aralkyl radical, R is hydrogen or alkyl and nis 0, 1,2 or 3.

Thus according to one aspect of the present invention, there is provided a diagnostic agent for the detection of urooxalic acid 200 g. polyvinyl alcohol 3.0 water 10.0 ml.

methanol uil I001) ml.

bilinog cn b i l i Particularly in i f 5 Following drying of the impregnated paper there was obco"1pmes an absorbfmt 9 3 a Strong solld tained a white test paper having the same properties as that and a substance which, in the manner of the Ehrlich test, produced according [0 example 1 produces a color change in the presence of urobilinogen bodies, characterized in that as color-producing substance 10 EXAMPLE3 there is used at least one compound of formula I.

. F According to a further aspect of the present invention, there a s sil s rz fi iz gllgfifg 1x1; i is provided a process for the detection of urobilinogen bodies 8 p p e var:-

. ous concentrated solutions containing x parts of p-N-(N present in body fluids, the process being carried out through methyl-piperazino)-benzaldehyde (MPBA) or of pthe use of a test strip, i.e., an absorbent carrier or, a reagent l5 dimethylaminobenzaldehyde (DABA) and parts of oxalic film or in solution in the presence of a strong, solid or liquid acid in 100 parts of methanol. All of the test papers thusly obacid and a substance which, in the manner of Ehrlich s test, i

. tained were white following drying. The dried test papers were produces a color change in the presence of urobilinogen dipped either into a 3 percent solution of urea or into a urine bodies, characterized in that a compound of formula I is used sample which was free of urobilinogen. The color changes as the color indicator. 20

which resulted are set out in the following table: For a fuller understanding of the nature and ob ects of this invention, reference may be had to the following exari rles,

. TABLE I which are given merely as further illustrations of the invention and are not to be construed in a limiting sense. x MP DAM EXAMPLE 1 0.05 white yellow Filter paper (Schleicher & Schiill No. 23l6) was im- 0. 75 whi e yell w t 0.l white yellow pregnated with a solution having the following composition. 01 pal: only yellowish yellow pbis-(fi-dicthylaminoethyl)-aminohenzaldehyde 0.2 g. oxalic acid 20.0 g.

h l d l00.0 l. am a m As can be clearly seen from the above results, the test paper Following drying of the impregnated paper there was according to the present ltlVetlllOl'l, in contradistinction to the known test paper, is substantially insensitive to urea and thus tamed a white test paper which, with normal urine, gave a pale k l r d h t d reacts more sensitively and more specifically with urof 8 an M g giF F con l "59 bilinogen bodies as the pink color reaction with urobilinogen an mg"perccfn f i i ms gave j mg is not masked by the more or less strongly yellow color reacfi 0 T I "T t F or tion with urea. It is, of course, obvious that such a yellow color swePm to no et Co Urme Specimens me o um "f 40 reaction considerably impairs the sensitivity and measurability bodies, as well as 3 percent aqueous solutions of urea, .dldnot ofthe urobilinogen reaction bring about any color change in the test paper. Urine indican also did not produce a color reaction. The test papers could be EXAMPLE 4 evaluated after 1-2 minutes followin wettin with the li uid g E q Flltet paper (Schleicher & Schull No. 235 or 2316) was 1111- samples to be tested.

pregnated with methanolic solutions which, per I00 parts of EXAMPLE 2 solution, contained A parts of the compounds I as given in the h n following table and B parts of oxalic acid. White test papers Filter paper (Schleicher & Schull No. 23 l6) was imwere thereby obtained in each case Pregnated 3 Soluuo" having the following composltloni The color reactions which resulted following immersion of these test papers into various solutions were evaluated after p- -tfly y ly gl-2 minutes. The results are set out in table ll which follows:

TABLE II Color reaction with- Urobllin ogen-free Urobiliri ogen- Compound (I) A B urine=3% urea soln. Normal urine containing urine p-N,N-dimethy1-N'-ethy1hydrazino-benzaldehyde 0.1 25 Yeiiowish Yellow-pink... Bed-violet. p-N-riitrosoN-methylaminobenzaldehyde 0.1 d Yel1owish. Orange. p-N-cyanomethyl-N-methylamirio-benzalde 0.1 Pale pin Pink-red. p-bis-(carbethoxymethyl)aminobenzaldehyde. 0.1 .do D0. p-N-B-chloroethyi-N-methy1aminobenzaldehydo 0.1 Yellow-pink. Purple. p-N-Bhydmxyethyl-N-inethylaminobenzaldehyde 0. 1 do 1 D0. p-N-fi-metlioxyethyl-N-methylsmiiiobenzaldehyde O. 1 .do. Do. p-bis-(B-fluoroethyD-amino-benzaldehyde. 0.1 Pale pink- Blue-red s (B-chloroethyl)-imiino-benza1dehyde. 0. 1 d0. D0. p (B-bromoethyl)aminobenzaldehyde. O. 1 do Do. p-bis(fl-hydroxyetliy1)-aminobeiizaiehyde 0.1 Yellow-pink Purple. p-bis(B-methoxyethyl)-amlno-benzaldehyde 0. 1 -do D0. p-bis-(B-cyanoethyl)aminwbenzaldehyde .s 0.15 Pale pink Pink-red p-bis-(B-dimethylaminoethyl)-aminobenzaldehy 0. 2 -do s Do. p-bis( fl-diethylaminoethyi)-arriinobenzaldehyde. 0. 2 do D0. p-bis-(B-N-piperidinoethyl)-amlnobenza1dehyde 0. 1 .do Do. [p-bis-( fi-triethylammoriiumethyl) aminobenzaldehy 0. 2 .do D o. [p-bis-(N-pyridinlumethyl)-aminobenzaldehyde]dibromide 0.2 .do Do. p-N-inorpholino-benzaldehyde 0.1 Yellow-pinto Red-violet. p-N-tliiomorpholino-benzaldehyde. 0. 1 ..do Do. [p-N-(S-methylthio-morpholinium)- 0.2 Pale pink Plnk'rcd. p-N-(S-dioxothio-morpholino)-benzaldehyde 0. 1 .do. D0. [p-bis-(N-quinolinium-ethyl)amino-benzaldehyde] dibroruid 0.2 do D0. p-N-piperazlnobenzaldehyde 0. 1 .dO D0- p-N-(N-methylpiperazino)-benzaldehyde. 0. 05 .do Do. p-N-[N-(p-formyl-pheiiyl)-piperazino]-benzald y 0.1 2 y .do... Red-violet. [p;N-(l1{-dirnethyl ipgazin r il be zmil h d Boiling. .Q-. Qs)flilLQ--- .m 19811,"...

Compound (I) Lp-N-(N'-cyc1epentamethylenepiperazinium) -benzaldehyde -iodlno- A U p-N-[Ncyclo-(3-oxapentamethy1ene) -pigergzinium1-b enzal ehydo iodinepgis-carbethoxy-ethyl)-amino-benzalde y pi5- p-bis-(y-bromopropyl)-a.minobenzaldehyde p-bis-(v-eyanopropyl)-aminobenzaldehyde F-bis-(v-dimethyl-aminopropyl) -aminbenza1dehyde p-bis- ('y-trimethybampnoniumpropyl)-aminobenzaldehyde]dichloride. p-Nd ethylamino-ethyl-N-methylaminobenzaldehyde p-N-d1methylamino-ethyl-N-meth laminobenzaldehyde. p-N-diisopropylaminoethyl-N-met ylaminobenzaldehyde... p-N-dlethylaminoethyl-N'othylaminobenzaldehyde p-(l-methyl-l,5-diazacyclooctyl-5)-benzaldehyde p-N-cyanoethyl-N-methylaminobenzaldehyde p-N-cyanoethyl-N-ethylaminobenzaldehyde. p-N-v-dimethylaminopropyl-N-methyl-aminobe aide p-N-bis-sulioethylaminobenzaldehyde p-N-diethylamino-ethyl-N-isopropylamino-benzaldehy p-N-cyanoethyl-aminobenzaldehyde carbamidoethyl)-amlnobenzaldehyde.l

TABLE I! :Cunlinucd Color reaction with Urobilin ogeniree Urobilin ogen- A B urine=3% urea soln. Normal urine containing urine 0.2 do do Do. 0.2 20 do. do l)o. 0.1 20 Yellowisl Yollowishplnk Red. 0.1 20 Yellowishnh. Yellow-pink... ltcd.

0.05 20 Pole-yellowish. Pnlo pink Pink-rod. 0.05 20 do. 0.05 20 d0. 0.05 20 White 0.05 Pale yellowish 0.05 25 ..do

0.05 25 d0 0.05 25 ....do. I) 0.05 25 do 0.05 25 Yellowish. ..do I'll 0.05 25 do Ycllcwish pink. 0.05 25 Yellow Yellow-plnk. llo. 0.05 25 Yellowisl Polo pink Pink-rod. 0.06 25 do... do m'p 0.01 20 White do.. Pink-roll 0.05 20 Pale yellowish do Do.

p-N-diethylamino-ethylamjnobenzaldehydeI:1:11:.

5 Strips offilter paper (Schleicher & Sch'rill No. 2316) were impregnated either with a solution of 0.05 g. p-N-(N-methylpiperazino)-benzaldehyde (MPBA) or of pdimethylaminobenzaldehyde (DABA) and 20 g. oxalic acid in 100 ml. methanol and then dried.

The dried papers were then immersed into aqueous solutions of conventionally used sulfonamides and evaluated after 1-2 minutes. The color changes which were observed are set out in tablel l l which fqllows: A

TABLE HI No coloration was observed when urine free of urobilinogen was tested in the same manner.

EXAMPLE 7 25 ml. urine were acidified with 12 ml. glacial acetic acid and extracted with 40 ml. ether. The extract was washed twice with 20 ml. amounts of water and again made up to 40 ml. with ether. 20 ml. of this extract was mixed with 0.5 ml. of a solution of l g. p-N-morpholino-benzaldehyde in 5 ml. concentrated hydrochloric acid and the resultant mixture shaken vigorously. Thereafter, there were added 6 ml. of a semisatu- N-(3,4-dimethyl-5-isoxazolyl)-sulfanila- EXAMPLE 6 For use in the production of a reagent film suitable for the detection of urobilinogens, there was prepared a mixture of the following components:

p-bis-(Bcyanoethyl)-aminobenzaldehyde 0.75 g. syrupy orthophosphcric acid 5.00 g. colloidal silicic acid (Aerosil) 6.00 g. organic sodium sulfonate (rapid wetting agent) 2.00 g. polyvinyl butyracetal (Mowital") l0.00 g. polyethylene glycol 6,000 [0.00 g. methanol l00.0 ml.

There was applied a 300 othick layer of the above mixture onto a white polyvinylchloride foil, followed by drying with warm air. A water-insoluble film was thereby formed. When this was moistened with a drop of urobilinogen-containing urine, after l-2 minutes had elapsed there was observed a red color which, following wiping off the urine, became darker.

rated solution of sodium acetate, followed by shaking up again. The red material which was formed thereby collected in the aqueous phase. After separation, the ether phase was again washed with 2 ml. water. The combined aqueous extracts were made up to 20 ml., the color present measured in a photometer at 557 nm. and the result evaluated by means of a previously prepared calibration curve.

EXMZP 8 Filter paper (Schleicher 81. Sclii'rll No. 23i6) was impregnated with a solution of 0.5 parts pbis-(diethylaminoethyl)-aminobenzaldehyde bis-hydrogen oxalate, 30 parts maleic acid and 0.5 parts polyethylene glycol 1,000 in parts methanol, and then dried.

Strips of this paper reacted with urobilinogen-containing urine to provide a pink to red color.

Papers with the same analytical properties but which were easier to unroll and cut up were obtained by additionally using 5 parts polyethylene glycol 6,000 or 20,000.

EXAMPLE 9 Filter paper (Schleicher & Scliiill No. 2316) was impregnated with a solution having the following composition:

p-(cyelohexylamino)-benzaldehyde 0.05 g. oxalic acid 20.0 g. methanol ad 100.0 ml.

After the impregnated paper was dried there was obtained a white test paper which, when moistened with urine samples containing urobilinogen bodies, gave a more or less intensive pink-to-violet coloration depending on the urobilinogen concentration. Urine samples having a very low content of urobilinogen bodies and 3 percent aqueous solutions of urea did not give any color change in the test paper. Urine indican also gave no color reaction. The test papers can be evaluated for color change after 1-2 minutes.

EXAMPLE Filter paper (Schleicher & Schr'ill No. 23l6) was impregnated with a solution having the following composition:

p'Uec.-butylnmlno)-benzaldehyde 0.05 g. potuulum hllulfute l5.0 g. polyethylene glycol 5.0 3. water ad l0l).0 ml.

After drying the impregnated paper, there was obtained a white test paper which had the same properties as the test paper produced according to example 9.

EXAMPLE 1 l Strips of filter paper (Schleicher & Schiill No. 2316) were impregnated with solutions having various concentrations containing X parts p-(isopropylamino)-benzaldehyde (iPABA), p-(n-propylamino)-benzaldehyde (nPABA) or' p-(dimethylamino)-benzaldehyde (DABA) and parts oxalic acid in 100 parts methanol, according to the procedure described in Example 1. The dried test papers were dipped into 3% solutions of urea. The color changes which were thereby observed are set out in the following Table:

As can be clearly seen from the results set out in the above table, the test papers according to the present invention which contained p-(isopropylamino)-benzaldehyde were substantially less sensitive to urea and thus reacted more sensitively and specifically with urobilinogen bodies as the pink color reaction with urobilinogen was not masked by the more or less strong yellow color reaction with urea.

EXAMPLE l2 TABLE V Reaction with Reaction 3% urea soln. or with urowith urine of bilinogenlow urobilinogen containing Compound (1) content urine p-(lsopropylamlno)-benzaldehyde White Pink-red. 11-(isopropylmnino)-m-mnthyl Pale yellowish" Do.

Imuznk ohydo. i-(suwhuLylnmino)-lmnzuldullyrlu .do Do. p-(mntyl-(immlno)-bunzuldullydu Yellowisln. Do. i-(2,4- ilmothylpnntyli-muluo)- Yellow Rod.

hunzuldoliydo. p-(cyclopentylamlno)-bcnznldehyde Polo ed- V yellowish. yiolet TABLE V..C0niinucd Reaction with Reaction 3% urea soln. or with urowith urine of biilnogeulow urobilinogen containing Compound (I) content urine p-(it-mathyl-cyclopentylamino)- Yellowish Do.

benzaldehyde. p-(cyclohexylamino)-benzaldehydo Almost white Do. p(cyclohexylamino)-m-methyl- Pale Do.

benzaldehyde. yellowish. p-(cyclohexylamino)-m-isopropyl- Ycllowish Do.

benzuldehydo. p-(2-mothyl-cyclolwxylanilno)- .rlo Do.

bouzaldohydo. p-mouthylanil:iolmnznldullydu Yullow mung. p-(l-uy0lohuxyl-pro iyl-2-iuuino)- Yollowlshvw l'luk-I'ml.

hunroldnliyrln. p-(cyelndorlecylnmllm)-lmnzulriohyxlo.. l'nlu l'lnk.

yellowish. p-(norboruyl-Z-mnino)-beuzuldehydo Yellow Pink-rod. p-(a-mothyl-bsnzylamino) Yellowlsh Do.

benzaldehyde. p-(a-iso r0 ylbenzylamino)- do.

home do yde. p-(l-phenyl-propyl-Z-amino)- do.

benzaldchyde. p-(indanyl-Z-omino)-benza1dehyde ..do

p-(N-diethylarninoethyl-N-isopropyldo..

amino) -benzaldehyde.

strips dippe d into urine had a weak yell ow coloration due to thc inherent color ol'the urinc.

EXAMPLE iii For the production of a filmlike reagent layer for use in the detection of urobilinogens, there was prepared a mixture of the following components:

p-(cyelohexylamino)-bcnzaldehydc 0.05 g. colloidal llllClC acid (Aerosil") L00 g. oxalic acid I000 g. polyvinyl butyracetal ("Mowit-al) 0.5 g. polyethylene glycol 6000 0.5 g. methanol 30.0 ml.

The above mixture was then applied at a thickness of about 1 mm. to a white polyvinylchloride foil and thereafter allowed to dry in warm air. A water-insoluble layer was thereby formed. When this was moistened with a drop of urobilinogencontaining urine then, following the passing of l-2 minutes, there was observed a red coloration. No coloration was observed with a urobilinogen-free urine.

EXAMPLE 14 25 ml. urine were acidified with 12 ml. glacial acetic acid and extracted with 40 ml. ether. The extract was washed twice with 20 ml. amounts of water and again made up to 40 ml. with ether. 20 ml. of the thusly obtained extract were mixed with 0.5 ml. of a solution of l g. p-(cyclopentyl-amino)- benzaldehyde in 5 ml. concentrated hydrochloric acid and the mixture shaken vigorously. Thereafter, there were added 2.5 ml of a semisaturated solution of sodium acetate and 3.5 ml. water, following which the mixture was again shaken, the red colored material thereby formed collected in the aqueous phase. After separation, the ether was again washed with 2 ml. water. The combined aqueous extracts were made up to 20 ml. and the color thereof measured in a photometer at 557 nm. The reading obtained was evaluated by means of a previously prepared calibration curve.

it is a general advantage of those substances l which possess branched alkyl substituents at the amino group, that they do not react with unknown ingredients of certain urine samples. Further, it could not have been foreseen, that the more basic derivatives of p-aminobenzaldehyde having a branched alkyl substituent R, at the amino group would not react with urea, although up until now it had been found that more basic aldehydes do react better with urea.

We claim:

1. Diagnostic agent for the detection of urobilinogen bodies in body fluids comprising at least one strong acid and at least one compound which reacts with urobilinogen bodies to prowherein A. R and R are each members selected from the group consisting of halogen, cyano, nitroso, carbalkoxy, carbamido, hydroxy, acyloxy, alkoxy and amine radicals having at least one aliphatic hydrocarbon substituent and, if there are two aliphatic hydrocarbon substituents they may, together with the amine nitrogen, represent an N- aliphatic heterocycle and wherein one of R and R can represent hydrogen.

B. R and R together can represent a member selected from the group consisting of oxygen, sulfur, sulfonyl, alkylthionium, imino, alkylimino, substituted or unsubstituted arylimino, open-chained dialkylimino, cyclic dialkylimino and heterocyclic dialkylimino;

C. R-(Cl-l can also represent a member selected from the group consisting of secondary straight-chained alkyl, branched-chained alkyl, monocyclic alkyl, polycyclic alkyl and aralkyl with the proviso that in this case, R, can represent hydrogen only if n is R is a member selected from the group consisting of hydrogen and alkyl; and

n and m are each one of 0, l, 2 and 3; and a carrier therefor.

2. Diagnostic agent according to claim 1 wherein said compound which reacts with urobilinogen bodies to provide an analytically measurable dyestuff has the formula:

wherein R represents a member selected from the group consisting of hydrogen, halogen, cyano, nitroso, carbalkoxy, carbamido, hydroxy, acyloxy, alkoxy and amine radicals having at least one aliphatic, hydrocarbon substituent and, if there are two aliphatic hydrocarbon substituents they may, together with the amine nitrogen, represent an N-aliphatic heterocycle, R represents a member selected from the group consisting of secondary straight-chained alkyl, branched-chained alkyl, monocyclic alkyl, polycyclic alkyl and aralkyl, R is a member selected from the group consisting of hydrogen and alkyl and n is 0, l, 2 and 3, with the proviso that R. is hydrogen only when n is 0.

3. Diagnostic agent according to claim 1 wherein said carrior is a paper sheet having a wettable surface impregnated with a solution containing 0.001-1 percent of a compound (I) as defined in claim 1 and 15-30 percent of a strong solid acid.

4. Diagnostic agent according to claim 1 wherein said strong .acid is a member selected from the group consisting of sulp-bis-(B-diethylaminoethyll-uminobenzaldehyde 0.2 g. oxalic acid 20.0 g. methanol ad 100.0 ml.

6. Diagnostic agent according to claim 1 wherein said carrier is a paper sheet having a wettable surface impregnated with a solution having the following composition:

p-bis-(fl-cyanoethyl)-aminobenzaldchyde oxalic acid polyvinyl alcohol 3.0 g. water 30.0 ml. methanol ad l00.0 ml. c

7. Diagnostic agent according to claim I wherein said carrier is a paper sheet having a wettable surface impregnated with a solution having the following composition: 0.05 and 0.2 parts of a member selected from the group consisting of p-N (N-methyl-piperazino)-benzaldehyde and pdimethylaminobenzaldehyde and 20 parts of oxalic acid in parts of methanol.

8. Diagnostic agent according to claim 1 wherein said carrier is a polyvinylchloride film having applied thereon a layer of the following composition:

p-bis-(fi-cyanoethyl)-aminobenzaldehyde 0.75 g. syrupy orthophosphoric acid 5.00 g. colloidal silicic acid (Aerosil") 6.00 g. organic sodium sulfonate (rapid wetting agent) 2.00 g. polyvinyl butyracetal (Mowital") l0.00 g. polyethylene glycol 6000 l0.00 g. methanol 100.00 ml.

9. Diagnostic agent according to claim 1 wherein said carrier is a liquid containing p-N-morpholinobenzaldehyde in solution in concentrated hydrochloric acid.

10. Diagnostic agent according to claim 1 wherein said carrier is a paper sheet having a wettable surface impregnated with a solution having the following composition: 0.5 parts pbis-(diethylaminoethyl)-aminobenzaldehyde bis-hydrogen oxalate, 30 parts maleic acid and 0.5 parts polyethylene glycol l,00020,000 in l00 parts methanol.

11. Diagnostic agent according to claim 1 wherein said carrier is a paper sheet having a wettable surface impregnated with a solution having the following composition:

p-(cyclohexylaminol-benzaldehyde 0.05 g. oxalic acid 20.0 g. methanol ad l00.0 ml.

12. Diagnostic agent according to claim 1 wherein said carrier is a paper sheet having a wettable surface impregnated with a solution having the following composition:

p-(sec-butylamino)-benzaldehyde 0.05 g. potassium bilult'ale I50 3. polyethylene glycol 5.0 water ad l00.0 ml.

13. Diagnostic agent according to claim 1 wherein said carrier is a polyvinylchloride film having applied thereon a layer of the following composition:

c p-(cyclohexylaminol-benzaldehyde 0.05 g. colloidal silicic acid (Aero|il") 1.00 g. oxalic acid l0.00 g. polyvinyl butyracetal (Mowital) 0.5 g. polyethylene glycol 6000 0.5 g.

14. Diagnostic agent as claimed in claim 1 wherein said compound has the formula wherein R is a secondary alkyl group which can be straightchained, branched or cyclic and wherein R is a member selected from the group consisting of hydrogen and alkyl.

15. Diagnostic agent according to claim I wherein said carrier is a paper sheet having a wettable surface.

16. A method for carrying out an analytical test for the presence of urobilinogen bodies in biological fluids which comprises applying to the wettable surface of a diagnostic agent according to claim 15 a small amount of the fluid to be tested and examining said paper for visual evidence of the presence of said urobilinogen bodies.

17. Diagnostic agent according to claim 1 wherein said carrier is a foil.

18. A method for carrying out an analytical test for the presence of urobilinogen bodies in biological fluids which comprises applying to the coated surface ofa diagnostic agent according to claim 17 a small amount of the fluid to be tested and examining said coating for visual evidence of the presence of said urobilinogen bodies.

19. Diagnostic agent according to claim I wherein said carrier is a liquid. open-chajned, alicyclic or heterocyclic dialkt54 20. A method for carrying out an analytical test for the presence of urobilinogen bodies in biological fluids which comprises admixing a diagnostic agent according to claim 19 with a small amount of an ether extract of thet'luid to be tested, adding a semisaturated solution of sodium acetate and water and examining the resultant aqueous phase for visual evidence of the presence of urobilinogen bodies.

21. Diagnostic agent according to claim 1 wherein said carrier is a paper sheet having a wettable surface impregnated with a solution containing 0.00] to 1 percent of a compound (I) as defined in claim 1 and at least 3 percent ofa strong solid acid.

22. Diagnostic agent according to claim 21 additionally containing at least one member selected from the group consisting of polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of polyvinyl pyrrolidone and polyvinyl acetate and polyglycols.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3 ,630 ,680 Dated. December 28 1971 lnventofls) Walter Rittersdorf et a1.

It is certifiedthat error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

F001. 1 line '22" v "beblet," should be --tablets-' Col. 1, line 34 I v a Afit r "533/" in sert -1957. 1

Col. 1, line 38 "unro-"' should be --uro- Col. 2 line 32 "unrobilinogen" should be --urobi1inog'en-- C01. 3, line 2 "ni s should be i1 is-- Col. 5, line 20 Before line 21, insert --EXAMPLE 5-'--' line 21, c lelete "5" (:01 r 6 ine 53 "pbis" should be --p 'bis-- Col. 10, Claim 13 I 1st line of composition ingredients, delete "c" before "p" FORM PO-1050 (10-69) USCOMM-DC oosvs-pes W 0.5. GOVERNMENT PRINTING OFFICE I959 036fi-33Mv UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3 ,680. Dated December 28 1971 lfiventofls) 'Walter Rittersdorf et al'. Page 2 5 v It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 10 Claim 13 add to the ingredient listing as a 6th line methanol 30.0 ml. Column 11, Claim 19 should read. 19. Diagnostic agent according to claim '1 wherein said carrier is a liquid.

Signed and sealed this 7th day of November 1972.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. ROBERT GOTTSCHALK Attesting Officer Commissioner of Patents- USCOMM-DC 6037'5-P69 U.S. GOVERNMENT PRINTING OFFICE: 1969 O366-33A,

LFORM PO-IOSO (10-69)

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Reference
1 *J. Fischl and N. Pinto, Clinica Chimica Acta, 2, 527 33 (1957).
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US3853466 *Jun 14, 1972Dec 10, 1974Boehringer Mannheim GmbhDiagnostic composition for the detection of urobilinogens
US3989462 *May 10, 1976Nov 2, 1976Riedel-De Haen AktiengesellschaftTest composition for detecting urobilinogen
US4260679 *Aug 1, 1979Apr 7, 1981Kyowa Hakko Kogyo Co., Ltd.Method and reagent for the quantitative determination of hydrogen peroxide and precursors thereof
US4295853 *Dec 29, 1978Oct 20, 1981Wako Pure Chemical Industries, Ltd.Method for measuring peroxidic substances
US4405718 *Jul 20, 1981Sep 20, 1983Miles Laboratories, Inc.Method and composition for urobilinogen control standard
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US20090325929 *Jun 15, 2007Dec 31, 2009Runtao LiQuaternary ammonium salt compounds of spirocyclopiperazines, preparation methods and uses thereof
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Classifications
U.S. Classification436/97, 422/420
International ClassificationG01N33/72
Cooperative ClassificationG01N33/72
European ClassificationG01N33/72