DE1648848A1 - Method and diagnostic agent for the detection of urobilinogen bodies - Google Patents

Description

OF. Boehringar Sa Soahne

G m b II
H a η η h β L ni 1525

Procedures and diagnostic means for the detection of urobilinogssn bodies

The subject of the present invention is an improved method and an improved diagnostic agent for the detection of urobilinogen bodies in body fluids, preferably in urine.

It is known that urobilinogen bodies (silanes), indole, sulfonamides, Porphobilinogöti, Urnindiknn and 5-Hydrüxj-indoleasigaäure with detect a solution of p-dimethylaminobenzaldehyde in hydrochloric acid can ».0 loose Ifaohwsia iafc In the Ij Hera door q, J, g E! hrll.oh'i reaction known; or has ins especially in medical diagnostics as Detection of "increased urobilinogenan" in the urine of considerable importance won. Although the sample is not very specific, it is considered the standard method for diagnosing liver and biliary diseases .

To simplify the test, it has already been proposed to use tea tablets bark out (British Patent No. 779,921), in which on; he p-ß-dimethylaminobenzaldehyde is a solid, inorganic or organic Acid is included (see also US Pat. No. 2,320,282 - test tablets for the detection of sulfonaniides). One dissolves such tablets in more or less diluted urine, parbo reactions are obtained, which roughly correspond to those of the original honesty test; however, it is used, for example, to eradicate the color reaction with sulfonainides the use of an auxiliary blue color (e.g. methylene blue) easpfohloü.

To further simplify the honest sample, one has the reaction auoh aohon transferred to fact sheets} see Piaohl and Pinto, Clinici Chim

Acta 2, (1957), pp. 527-555, and Franz, Patenbschrift 1 450 275, in which the phosphoric acid used by Fischl and Pinto is replaced by oxalic acid.

Surprisingly, it has now been found that certain derivatives of the p-aminobenzaldehyde provide significantly more sensitive and less susceptible to interference diagnostic agents for the detection of fjrobiünogen, than they were previously known. As derivatives of p-Ainobenzaldehyda Substances are possible which are subscient to the p-amino group carry, which have strong electron-withdrawing properties, Ina- in particular, substances of the general formula I are involved

in which

Rj and R "denote a halogen atom, a cyano, nitroso, carbalkoxy, carbarnido, hydroxy, acyloxy, alkoxy, an open-chain or cyclic, saturated or unsaturated, secondary or tertiary amino group, where R ₁ and R _? also together are a 0xa- _t thia, sulfonyl, Alkylthionium-, imino, Älkylimino-, optionally substituted Arylimino- or an open-chain, alioyr: Li3che dialkylimino group or heterocyclic, and one of R and R p can also be a hydrogen atom, and η and m are numbers from 0 to 3 5.

These substances are preferably used for the production of tea papers, in which they are applied to an absorbent carrier together with a strong, solid acid. In addition, the substances are I zwv Herateilung of Testfilmatreifen according to our Application Serial B 89944 IXb / 42 1 and for the detection of urobilinogen in Lö3im _t ^f ran suitable.

BATH ORIGINAL

7ait production of the preferred embodiment of the new diagnostic: M i iiel are absorbent carriers, preferably filter paper ¹ ! impregnated with an I --- Riing that from 0.001 to 1 <enihält fo of an aldehyde of the general formula and niindßBten3 3 »preferably 15-30 ° f a strong solid acid. The strong, solid acids used are preferably sulfosalioylniiuTO , p-toluenesulfonic acid, potassium bisulfate, glutamic acid hydrochloride and, in particular, oxalic acid. Indifferent additives such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinylpyrrolidone and polyvinyl acetate, polyglycols, etc. can also be added to the impregnation solutions.

/ In principle, solvents are used to impregnate the absorbent carrier nl3 <? easy-flüchügen solvents in question, in which both the Aide- f livf? β I and the acids are sufficiently soluble. Preferably be therefore polar solvents such as methanol are used.

! Six the · drinking troughs, the filter papers are dried, cut up and {vtv-ugly nn plastic foils nnsealed, resp. between transparent Sealed plastic foils. In the event that rods or strips have been used as a removable carrier, they are immediate ready to use after drying.

To carry out the method according to the invention for the detection of Urobilinogen or urobilinogen bodies, one immerses the new diagTiostic Agent into the solution to be examined and reads the color change after a short time.

In contrast to the previous diagnostic means for the detection of urobilinogen, the new test strips are much more sensitive and still allow the detection of approx. 0.4 mg fa urobilinogen in the urine.

Surprisingly, this color reaction is caused by the presence of Urea no longer disturbed; itself causes a 3 $ urea solution With the new test papers there is practically no color change, while TfOt papers containing p-dimethylaminobenzaldehyde are intensely calibrated turn yellow. Furthermore, it was not foreseeable that the new

10988 ^ / 0337

diagnostic means neither by urinary indole nor by indole or Sulphonamides are present in the urine at the usual dosage, disturbed will. This finding is particularly surprising given the new Substances dissolved in 2-20 $ hydrochloric acid are very good for detection of indole in solutions. For the detection of urobilinogen or urobilinogen bodies in solutions one can of course Replace p-dimethylarainobenzaldehyde with the substances of formula I. and bo determine the content in the cuvette photometrically very precisely and without interference.

The diagnostic means according to the invention for the detection of urobilinogen bodies in body fluids, preferably in urine, consisting of an absorbent carrier or a film, a strong solid acid and a substance which provides a color change in the manner of Ehrlich's test with urobilinogen bodies are characterized by the content of one or more substances of the formula I. The process according to the invention for the detection of urobilinogen bodies in heads on a suction-capable carrier, a reference film or in solution in the presence of a strong one. Solid or liquid acid with the aid of a substance which, in the manner of the Ehrlich ¹ Bohen reagent, gives a color change with urobilinogen bodies, is characterized in that substances of the formula I are used as color indicators.

In the following examples, the inventive method and the diagnostic agents according to the invention explained in more detail, with the superiority through * comparison tests with previously known diagnostic agents compared to the state of Teohnik is proven.

SAD ORIGINAL

example 1_

Filter paper (23I6 from Schleioher & Schüll) comes with a solution soaked in the following composition:

p-bis (ß-diethylamino-ethyl) aminobenzaldehyde 0.2 g

Oxalic acid 20.0 g

Methanol to 100.0 ml

After drying, a white test paper, which results with normal urine, a weak pink color and urines containing more than 0.4 mg ° / o urobilinogen bodies contain, depending on the content of a more or less intense pink to violet color indicates . Urobilinogenkörperfreie Urine or 3 $ owned wäesrige urea solution lead to any color change dee test paper. Urinary indicator gives no color reaction. The test papers can be read after 1-2 minutes.

example

Filter paper (23I6 from Sohleioher & Schüll) is mixed with a solution soaked in the following composition:

p-bis (b-cyanoethyl) aminobenzaldehyde 0.05 S

Oxalic acid 20.0 g

Polyvinyl alcohol 3.0 g

Water 30.0 ml

Methanol to 100.0 ml

After drying, a white test paper is obtained which is the same Has properties like the test paper produced according to Example 1 »

example

Filter paper (23I6 from Sohleioher & Schüll) is mixed with solutions of different concentrations of in each case X parts of pN- (N'-methylpiperazino) -benzaldehyde (MPBA) or p-dimethylamino-benzaldehyde (DABA) and 20 parts

103885 / ^ 337

-O-

Oxalic acid in 100 parts of methanol as described in Example 1, impregnation The test papers obtained in this way are all white after drying. The dry test papers are either immersed in a 5 'series urea solution or in urobilinogen-free urine. The color changes observed are compiled in the table below j

X MPBA DABA 0.05 know yellow 0.075 White yellow 0.1 White yellow 0.2 pale yellow strong lich yellow

As one can clearly see from this, there are new Teatpäpier across the street the known practically insensitive to urea and thus reacts more sensitive and specific to urobilinogen bodies, since the pink ones Color reaction with urobilinogen is not masked by the more or less strong yellow color reaction with urea. It is aelbs tvers tärull me that the sensitivity and readability of the ürobilinogen color reaction is significantly impaired thereby.

example

Filter paper (23 S or 2316 from Schleicher & Schüll) is included methanolic solutions soaked in 100 parts of solution Α parts of the The following substances contain I and B parts of oxalic acid. White test papers are obtained.

The color reactions observed when these test papers are immersed are read after 1-2 minutes and are summarized in Table II below

■! 0988A / 0337

BATH ORIGINAL

Table II

SuI) Dance I A. B. Color reaction with Normal urine Urobilinogen
containing
urine Urobilinogen
free urine
= 3 $ urinary
atofflösunp yellowbroaa red-violet P-N, K'-dimethyl-N'-ethyl 0.1 25th yellowish hydra 7.i no-benzaldehyde yellowish orange p-H-Ni trogo-N-metliyl- 0.1 25th yellowish ami no-berizaldehyde so awake pink p-N-cyanomethyl-N-methyl- 0.1 25th White pink amino-benzaldehyde weak pink p-Bi a (carbethoxymethyl) - 0.1 25th White pink aniino-benzaldehyde gelbroea purple p-H-ß-chloroethyl-li 0.1 25th yellowish never thy lamino-benza Id ehyd yellowbroaa purple p-M-U-hydroxyethyl-H- 0.1 25th yellowish irethylamino-benzaldehyde yellow-pink purple p-N-ii-methoxyctnyl-N- 0.1 25th yellowish rcothylamino-benzaldehyde weak blue red p-bis-Cb-fluoroethyl) - 0.1 25th weak pink anino-bensaldehyde yellowish weak blue red p-bis- (li-chloroethyl) - imino- 0.1 25th awake pink -benzaldehyde yellowish awake blue red p-Bi3- (b-broinethyl-) an) ino- 0.1 25th weak ro Ba -benzaldehyde yellowish gelTjroea purple p-Bi 3- (l .'- hydroxyethyl) - 0.1 20th yellowish amino-benzaldehyde

109885/0337

BAD ORIGINAL CQpy

- α - Table II continued

Substance I. A. B. Fi
ütobilinogen-
free urine
^β 3 "M S ^e Ha rn-
fabric solution? ir reaction with
'Normal urine Urobilinog
containing
urine p-bis (ß-methoxyethyl) -
araino-benzaldehyde 0.1 20th yellowish gelbroea purple p-bis (ß-cyanoethyl) -
amino-benzaldehyde 0.15 25th White weak
pink pink p-bis- (ß-dimethylamino-
ä thy I) - aminobenzaldehyde 0.2 20th White weak
pink pink p-Bia- (ß-diethylamino-
ethyl) -amino-benzaldehyde 0.2 20th White weak
pink pink ß-bis- (ß-N-piperidinoethyl]
amino-benzaldehyde -0.1 20th White schwaoh
pink pink p-bis- (ß-triethylammonium-
ethyl) -aüiino-benzaldehyde]
dibromide 0.2 20th White weak
pink pink [p-bis- (N-pyridiniumethyl) -
amino-benzaldehyde]
dibromide 0.2 20th White weak
pink pink p-N-Morpholinobenzaldehyde 0.1 20th yellowish yellow-pink red-violet pN-thiomorpholino-
benzaldehyde 0.1 20th yellowish yellow-pink red-violet [pN- (S-methyl-thiomor-
pholinium) benzaldehyde] -
iodide 0.2 20th White weak
pink pink pN- (S-dioxo-thiomorpho-
lino) benzaldehyde 0.1 25th White weak
pink pink [p-bis- (H-ohinoliniuni-
ethyl) -amino-benzaldetyti]
dibroroid 0.2 20th White schwaoh
pink pink

109885/0337

Continued Table II

A. B. F «
Urobilinogen
free urine
= 3 # urinary
ATO SOLUTION turbidity reaction with
* Honnalurin Γ Urobilinoge
I containing
urine 0.1 20th White awake
pink pink 0.05 20th White weak
pink pink 0.1 25th weak
yellowish awake
roaa red-violet -0.1 20th White awake
roea pink 0.2 20th White awake
TOB * red 0.2 20th White awake
roaa pink 0.1 20th yellowish yellowbroaa Red Substan I 0.1 20th yellow lent gelbroea Red jp-H-piperazdro-benzaldehyde 3.05 20th weak
yellowish awake
roea red | pH- (H '-Methylpiperazino) -
benzaldehyde >, 05 20th weak
yellowish weak
roaa red pH- [H · - (p-Formylpheny]) -
piperazino] benzaldehyde >, 05 20th awake
yellowish awake
pink red [pH- (H! -Dimethylpiperazini
um) benzaldehyde] iodide Ot05 20th White weak
roaa red [PH- (N'-cyclopentamethylene
piperar «inium)« benzaiäehy
iodide PH- [H '-Cyclo (3-oxa penta-
ethylene-) piperazinium} - -
benzaldehyde iodide p-bis (carbäthoxyäthy1) -
nnino-benzaldehyde Jp-Bi8 (carbainidoethyl) -
I amino-benzaldehyde jp-Bia (γ-bronpropyl) -
I amino-benzaldehyde jp-Bia (y-cyanopropyl) -
I naino-benzaldehyde Ip-bis (Y-dimethylamino-
Γ propyl) -amino-benzaldehyde I [p-Bia (γ-trinethy1-ammoniο-
Ι propyl) a «dno-benzaldehyde]
j dichloride

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ORIGINAL BATHROOM -

COPY

example

Filter paper (2316 from Schleicher & Schall) is impregnated with a solution of 0.05 g of pN- (N'-methylpiperazine) benzaldehyde (MPBA) or p-dinethylaminobenzaldehyde (DABA) and 20 g of oxalic acid in 100 ml of methanol and dried . The dry papers are special in aqueous solutions! Frequently: applied sulfonamides immersed and read after 1-2 minutes. The size changes are summarized in Table III below

Table III

Sulfonamide

10 mg

25 mg

50 mg j,

Test SuIfametin white

paper 3ulfafurazole white

with sulfaphenazole white

MPBA Sulfamoxolum white

white weakly yellowish white weakly yellowish white white weakly yellowish yellow borrowed

Test paper
with
DABA

Sulfametin sulfafurazole sulfaphenazole sulfamoxolum

yellowish yellow white yellow

yellow strong yellow strong yellow very strong gel weak pink weak pink strong yellow very strong gel

H - (5-methoxy-2-pyrimidinyl) -sulfanilamide H ¹ - (3,4-dimethyl-5-isoxazolyl) -sulfanilaaid M - (1-phenyl-5-pyrazolyl) -sulfanilamide N ¹ - (4,5-dimethyl-2 oxazolyl) sulfanilainide

Sulfanetin>
Sulfafurazole "
Sulfaphenzazole
Sulfaaoxolum =

How can remove nan above table, the color reaction on Urobilinog in erfindungsgeoäßen test strip Sulfonamidkonzentrationen is up to 50 mg $> not disturbed practical. Higher concentrations only occur briefly after shock therapy and are therefore known to the observer.

Example 6

To produce a reagent film for the detection of τοη ürobilinogen a Mixture prepared from the following ingredients!

R-bis (B-cyanoethyl) -nminobenzaldehyde 0.75 e

syrupöa · orthophosphoric acid -JQ9885 / 0337 5t00 'β

COPY

colloidal silica (Aerosil) 6.00 g

organic sodium sulfonates (Rapidnetzer) 2.00 g

Polyvinyl butyracetal (Mowital) 10.00 g

Polyethylene Glycol-6000 10.00 g

Methanol 100.0 ml

A 300 / u thick layer is applied to a white polyvinyl chloride film the above mixture applied and then dried with warm air, a water-insoluble film is formed. Will this one with a drop If urobilinogen-containing urine is wetted, a red color develops after 1-2 minutes, which Bich intensifies after wiping off the urine. With urobilinogen-free No coloration is observed in urine.

example

25 ml urine are acidified with 12 ml glacial acetic acid and extracted with 40 ml ether. The extract is washed twice with 20 ml of water each time and made up to 40 ml with ether. One takes 20 ml of this, mixed with 0.5 ml of a solution of 1 g of p-N-morpholino-benzaldehyde in 5 ml of conc. Hydrochloric acid and shake vigorously. Then add 6 ml of semi-saturated Add sodium acetate solution and shake again, the red dye collecting in the aqueous phase. After the separation, the Ether washed again with 2 ml of water. The combined aqueous extracts are made up to 20 ml and measured at 557 mn in the photometer and evaluated using a previously established calibration curve »

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BATH ORIGINAL

Claims (3)

Claims 1. Diagnostic means for the detection of urobilinogen bodies in body fluids, preferably in urine, consisting of an absorbent carrier or a film, a strong solid acid and a substance, which near type of Ehrlich ¹ see sample with TTrobilinogen bodies provides a color change, marked by the content of one or more substances of the formula I. CHO CD, in which R, and R_ a halogen atom, a cyano, nitroso, carbalkoxy, Carbamido, hydroxy, acyloxy, alkoxy, an open-chain or cyclic, saturated or unsaturated j secondary or tertiary Amino group, where R, and R "also together represent an oxa, Thia, sulfonyl, alkylthionium, imino, alkylimino, optionally substituted arylimino or an open-chain, alicyclieche or heterocyclic dialkyliroino group and one of the radicals R and Rp can also be a hydrogen atom, and η and m are numbers from 0 to 3 and 2. Diagnostic agent according to claim 1, gekennzeiohnet characterized in that the absorbent carrier is impregnated with a solution containing per 100 ml 0.001 - contains 1 io an aldehyde of the general formula I and at least 3 »preferably 15-30% of a strong solid acid . 3. Procedure for the detection of urobilinogen cores in body fluids on an absorbent carrier, a reagent film or in solution in the presence of a strong, solid or liquid acid with the aid of a Substance which, in the manner of Bhrlioh · see reagent with tJrobilinogen-Kor.pern provides a color change, characterized in that substances of the formula I are used as color indicators 109885/0337 CBD In which H, and B is a halogen tα * _»f a cyano, carbalkoxy ffitroeo- _f, Ckrbajiido-y ^ droxj-, Acyloiy-, _t Alkoiy- a offetifcettige or cyclivche, saturated or unsaturated, secondary or tertiary mean Aainogruppe, votiei R , and R «also toeaaoten. a oxa, thia _C sulfonyl "Alkylthianiua-, I" _r Alkylieino- ino-, gegebenen- ä fall Arylinino- substituted or an open-chain "alicyolieche or heterocyclic Bialkyli" inogruppe _t and one of R H and B "is also a Can be hydrogen atoms and η and st are numbers tone 0 to 5 » used. 103885/0337