DE1648848B2 - DIAGNOSTIC MEANS AND METHOD OF DETECTING UROBILINOGEN BODIES - Google Patents

Description

R ₁ - (CH ₂ ).

IO

CHO (I)

in which R ₁ and R _{2 are} halogen, cyano, nitroso, carbalkoxy, carbamido. Hydroxy, acyloxy, alkoxy, an open-chain or cyclic, saturated or unsaturated, secondary or tertiary amino group, or R ₁ and R ₂ together represent an oxa, thia, sulfonyl, alkylthionium, imino, alkylimino, optionally substituted arylimino or an open-chain, alicyclic or heterocyclic dialkylimino group, or one of the radicals R ₁ and R _{2 is} a hydrogen atom, while η and m are numbers from 0 to 3.

2. Diagnostic agent according to claim 1, characterized in that the absorbent carrier is soaked with a solution, which per 100 ml 0.001 to 1% of an aldehyde of the general Formula (I) and at least 3, preferably 15 to 30% of a strong, solid acid.

3. Method for the detection of urobilinogen bodies in body fluids on an absorbent Carrier, a reagent film or in solution in the presence of a strong, solid or even liquid acid with the help of a substance similar to Ehrlich's reagent with urobilinogen bodies provides a color change, characterized in that substances of the formula (I) are used as color indicators

R ₁ - (CH ₂ ),

-CHO (I)

in which R ₁ and R _{2 represent} a halogen atom, a cyano, nitroso, carbalkoxy, carbamido, hydroxy, acyloxy, alkoxy, an open-chain or cyclic, saturated or unsaturated, secondary or tertiary amino group, or R ₁ and R ₂ together form an oxy, thia, sulfonyl, alkylthionium, imino, alkylimino, optionally substituted arylimino or an open-chain, alicyclic or heterocyclic dialkylimino group, or one of the radicals R ₁ and R _{2 is} a hydrogen atom, while η and m are numbers from 0 to 3 are used.

The invention relates to a diagnostic agent for the detection of urobilinogen bodies in body fluids, preferably in urine, consisting of an absorbent carrier or film, a strong solid acid and a substance which, in the manner of Ehrlich's test, with urobilinogen bodies provides a color change.

It is known that urobilinogen bodies (bilanes), indole, sulfonamides, porphobilinogen, urinary indican and 5-hydroxy-indole acetic acid with a solution of p-dimethylaminobenzaldehyde in hydrochloric acid can prove. This evidence is known in the literature as the Ehrlich reaction; he has especially in medical diagnostics as evidence of "increased urobilinogens" in the urine gained considerable importance. Although the sample is not very specific, it is considered the standard method for the diagnosis of liver and biliary diseases.

To simplify the test has already been suggested been to produce Tesl tablets (British patent 779 921 K in which, in addition to p-dimethylaminobenzaldehyde a solid inorganic or organic acid is contained (cf. also USA.-Patent- (, o script 2 320 2S2 test tablets for the detection of sulfonamides !!). If you dissolve such tablets in more or less diluted urine, one obtains a color reaction. which are roughly those of the original l-.hrlichschen Sample correspond: however, example is 6s wise to evaluate the color reaction with sulfonamides of the seed / a blue auxiliary color (/. H. Methylene blue 1 recommended.

To further simplify Ehrlich's sample, the reaction is also already available on test papers transfer; see Fischl and Pinto, Clinica Chim. Acta 2 (1957), pp. 527 to 533, as well as French Patent 1,450,273, in which the phosphoric acid used by Fischl and Pinto is replaced by oxalic acid.

Surprisingly, in the search for a further improvement in the test, it has now been found that certain derivatives of p-aminobenzaldehyde are much more sensitive and less prone to failure Provide evidence of urobilinogen as previously possible. As derivatives of p-aminobenzaldehyde Substances that carry substituents on the p-amino group that have strong electrons come into consideration have peeling properties. The invention thus consists in a diagnostic agent of the type mentioned, which dei by a content of one or more substances Formula 1)

₁ - (CH ₂ I _n

R ₂ - (CH ₂ ) ,,,

CHO

in which R ₁ and R _; a halogen atom, a <. ';, ;; ro nitroso-. Carbalkoxv-. (arhamido-, llvdnm-. Ao

oxy, alkoxy, an open-chain or cyclic, saturated or unsaturated, secondary or tertiary amino group, or R ₁ and R ₂ together represent an oxa, thia, sulfonyl, alkylthionium, imino, alkylimino, optionally substituted arylimino or form an open-chain, alicyclic or heterocyclic dialkylimino group, or one of the radicals R and R _{2 is} a hydrogen atom. while η and in numbers from 0 to 3 are indicated.

In a preferred embodiment of the new diagnostic agent, the absorbent carrier is with soaked a solution containing 0.001 to 1% of an aldehyde of the general formula (I) and at least 3, preferably Contains 15 to 30% of a strong, solid acid. Possible strong, solid acids are: Sulfosalicylic acid, p-toluenesulfonic acid, potassium bisulfal, Glutamic acid hydrochloride and especially oxalic acid.

The method according to the invention specified in claim 3 consists in general use of substances according to formula (I) for the detection of LJrobilinogen bodies. To carry out the procedure the new diagnostic means can be immersed in the investigated solution and the Read off the color change after a short time.

In contrast to the previous diagnostic The new test strips are much more sensitive and allow means for the detection of urobilinogen evidence of about 0.4 mg% urobilinogen in the urine.

Surprisingly, this color reaction is through the presence of urea no longer disturbed; even a 3% urea solution causes the new test papers practically no color change, while test papers containing p-dimethylaminobenzaldehyde contain, thereby turning an intense yellow. Furthermore, it was not foreseeable that the new diagnostic means neither by urinary indican nor by indole or sulphonamides, which are more common Dosage present in the urine may be disturbed. This finding is particularly surprising since one the new substances, dissolved in 2 to 20% hydrochloric acid, are very good at detecting indole in solutions can use. For the detection of urobilinogen or urobilinogen bodies in solutions one can of course, replace ρ-dimethylaminobenzaldehyde with the substances of formula I and so on determine the content in the cuvette photometrically very precisely and without interference.

The examples below are those according to the invention The method and the diagnostic agents according to the invention are explained in more detail, with by Comparative tests with previously known diagnostic agents show the superiority over the State of the art is demonstrated.

example 1

Filter paper is made with a
sanitary set / uni: drinkable:

', .ösuiiii following to

p-bis - (..'- diälhylaniino-ethyl) -

aminobes / aklehyd

Oxalic acid

Methanol

0.2 g

. . . 20.0 µ
. ad 100.0 ml

Pink color results and with urines that contain more than 0.4 mg% urogenogen body egg, depending on Content shows a more or less intense pink to purple color. Urobilinogen body-free urines 5 or 3% aqueous urea solution do not change the color of the test paper. Urinary indicator gives no color reaction. The test papers can be read after 1 to 2 minutes.

ic B e i s ρ i e 1 2

Filter paper is soaked with a solution of the following composition:

p-bis- (fi-cyanoethyl) -amino-

benzaldehyde 0.05 g

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 has the same properties like the test paper produced according to Example 1.

B e i s ρ i e 1 3

Filter paper is impregnated with solutions of different concentrations of X parts each of pN- (N'-methylpiperazino) benzaldehyde (MPBA) or p-dimethylamino-benzaldehyde (DABA) and 20 parts of oxalic acid in 100 parts of methanol, as described in Example 1. The test papers obtained in this way are all white after drying. The dry test papers are either poured into a 3% urea

solution or immersed in urobilinogen-free urine. The color changes observed are compiled in the following table:

0.05
0.075
0.1
0.2

MPBA

White
White
White
pale yellowish

DABA

yellow
yellow
yellow
strong yellow

After drying, a white test nier is obtained which * - 'mil normal ·. ""' fern ein schwae'ii " As you can clearly see from this, the new test paper is practically insensitive to the known one against urea and thus reacts more sensitively and specifically to urobilinogen bodies, because the pink color reaction with urobilinogen by the more or less strong yellow color reaction with Urea is not covered. It goes without saying that the sensitivity and readability of the Urobilinogen color reaction is thereby significantly impaired will.

Example 4

Füterpapii.T is made with methanolic solutions soaked that in KK) parts l.osuilg Λ-parts of the following Substances I and B parts contain oxalic acid. White test papers are obtained.

The color reactions observed when these test papers are immersed become apparent after 1 to 2 minutes read and are summarized in the following table 11'Mli ·

5 Substance I. A. 1,648,848 γ urobilinogenlreiem
Urine = 3% 6th urobilinogen containingr
1 Irish woman Table II Urea solution urine ρ-Ν, Ν'-dimethyl-N'-ethyl- 0.1 yellowish Color reaction with Red-violet hydrazino-benzaldehyde B. Normal urine p-N-nitroso-N-methylamino- 0.1 yellowish orange benzaldehyde 25th Yellow pink p-N-Cyanmethyl-N-methylamino- 0.1 White Pink benzyldehyde 25th yellowish p-bis (carbethoxymethyl) -amino-
hpnznldehvd 0.1 White Pink p-N - ^ - chloroethyl-N-methylamino- 0.1 25th yellowish pale pink purple benzaldehyde p-N - /) '- hydroxyethyl-N-methyl- 0.1 25th yellowish pale pink purple amino-benzaldehyde 25th Yellow pink p-N-zi-methoxyethyl-N-methyl- 0.1 yellowish purple amino-benzaldehyde 25th Yellow pink p-bis- (fi-fluoroethyl) -amino- 0.1 weak Blue red benzaldehyde 25th yellowish Yellow pink p-bis- (fi-chloroethyl) -amino- 0.1 weak Blue red benzaldehyde 25th yellowish pale pink p-bis - (^ - bromoethyl) -amino- 0.1 weak Blue red benzaldehyde 25th yellowish pale pink p-bis- (/ i-hyd roxyälhy l) -amino- 0.1 yellowish purple benzaldehyde 25th pale pink p-bis- (/ i-methoxyethyl) -amino- 0.1 yellowish purple benzaldehyde 20th Yellow pink p-bis (/ 1-cyanoethyl) -amino- 0.15 White Pink benzaldehyde 20th Yellow pink p-bis - (^ - dimethylaminoethyl) - 0.2 White Pink amino-benzaldehyde 25th pale pink p-bis - (/ f-diethylaminoethyl) - 0.2 White Pink arnino-benzaldehyde 20th pale pink p-bis - ((i-N-piperidinoethyl) -amino- 0.1 White Pink benzaldehyde 20th pale pink [p-bis (fi-triäthylammoniumethyl) - 0.2 White Pink amino-benzaldehyde] dibromide 20th pale pink [p-bis- (N-pyridiniumethyl) -amino- 0.2 White Pink ben7aldehyde] dibromide 20th pale pink p-N-Morpholinobenzaldehyde 0.1 yellowish Red-violet p-N-thiomorpholino-benzaldehyde 0.1 20th yellowish pale pink Red-violet [p-N- (S-Melhyl-thiomorpholinium) - 0.2 White Pink benzaldehyde] iodide 20th Yellow pink pN- (S-dioxo-thiomorpholinoV
heri7ii IH pll vfl 0.1 20th White Yellow pink Pink [p-bis- (N-quinoliniumethyl) -amino- 0.2 20th White pale pink Pink benzaldehyde dibromide p-N-piperazino-benzaldehyde 0.1 25th White pale pink Pink p-N- (N'-methylpipcrazino) - 0.05 20th White pale pink Pink bcnzaldchyd p-N-l N-lp-1'ormylphenyl) - 0.1 20th weak pale pink Rolviolcti piperazino] -bcnzaldchyd :O yellowish pale pink [p-N-iN'-Dimethyl ^ ipcrazinium) - 0.1 White Pink ben / .aldehyde] iodide 2S schwaJi Rosa _ "p-N- (N'-Cyclopcntamcthylen- 0.2 White Pink piperazinium) -benzaldchyd] -iodide 20th pale pink vN-fN'-Cyclo-f.l-oxapcnta- 0.2 White Pink methylene) -pipcrazinium] - 2 (1 pale pink benzaldeliyd-iodide 20th pale pink

Substan? I. B e i s ρ i e 5 1 648 848> 8th Table! I .. _. ₍ . 20th impregnated and Color reaction with urobilinogen-free
Urine = 3% ii! E Normal urine Yellow pink urobilinogen-containing Red pale pink read. the Sulfonamide 10mg% 25 mg 1 test paper with
DABA % 50 mg % 6th weak Sulfafurazole 10 mg % purple 50 mg % yellowish 7th Λ ί Β dried. The dry papers are in aqueous Urea solution urine Color changes are in the following Ta ⁶⁰ sulfametin yellowish p-bis (carbäthoxyüihyl) -amino- 20th """-""
yellowish '■ yellow-pink pale pink Red pale pink Belle III compiled: Test paper with
MPBA ₅ sulfaphenazole White purple weak benzaldehyde 0.1 Table III Sulfametine White White Sulfamoxolum JiClUlIi; !! p-bis (carbamidoethyl) amino 20th yellowish pale pink Pink pale pink White Pink Know white strong benzaldehyde 0.1 White weak yellow
very strong p-bis - () '- bromopropyl) -amino- 20th weak pale pink Pink pale pink purple yellowish yellow benzaldehyde 0.05 yellowish weak
pink p-bis- (y-cvanopropyl) -amino- 20th weak pale pink Pink Yellowish pink purple very strong benzaldehyde 0.05 yellowish yellowish yellow yellow p-bis - () '- dimethylaminopropy!) - 20th weak Pink Yellow pink yellow purple strong 209 537/375 amino-benzaldehyde 0.05 yellowish pale pink yellow [p-bis - () '- trimethyl-ammonio- White pale pink White Pink weak
pink propyl) -amino-benzaldehyde] - 0.05 25th pale pink purple pale pink yellow purple strong dichloride yellow p-N-diethylaminoethyl-N-melhyl- 25th weak purple White j faint pink Pink amino-benzaldehyde 0.05 yellowish weak ! pale pink Pink p-N-dimethylaminoethyl 25th weak yellowish | N-methylamino-benzaldehyde 0.05 yellowish Sulfonamide 25 mg% p-N-diisopropylaminoethyl 25th weak N-methylamino-benzaldehyde 0.05 yellowish Sulfafurazole White p-N-diethylaminoethyl-N-ethyl 25th weak amino-benzaldehyde 0.05 yellowish Sulfaphenazole p- (l-methyl-l.S-diazacyclooclyl-S) - 25th weak Solutions of particularly frequently applied sulfonamides ⁵⁵ Sulfamoxolum benzaldehyde 0.05 yellowish dipped and after 1 to 2 minutes p-N-cyanoethyl-N-methylamino- 25th yellowish benzaldehyde Ό.05 p-N-cyanoethyl-N-ethylamino- 25th yellowish benzaldchyd 0.05 p-N-y-dimethylaminopropyl- 25th yellow N-methylamino-benzaldehyde 0.05 25th pN-bisulfoethylamino-
benzaldehyde
pN-diethyl-aminoathyl- yellowish N-isopropylamino-benzaldehyde 0.05 20th yellowish p-N-cyanoethylamino-benzaldehyde 0.05 20th p-N-diethylaminoethyl-amino- benzaldehyde 0.01 0.05 Filter paper is filled with a solution vuu vj, uj g - ρ - N - (N '- methylpiperazino) - benzaldehyde (M PBA)
or ρ - dimethyl - aminobenzaldehyde (DABA) and 20 g of oxalic acid in 100 ml of methanol

Sulfameiine
Sulfafurazole
Sulfaphenzazole
Sulfamoxolum

N '- (5-melhoxy-2-pynmidi-

nyl-sulfanilamide.

N '- (3.4-dimethyl-5-isoxn-

zolyl) sulfanilamide.

N '-' d-Phcnyl-S-pyrazolyl) -

sulfaiiilamid.

N '- <4.5-dimethyl-2-oxa-

zolyl) sulfanilamide.

As can be seen from the table above, the color reaction to urobilinogen in the case of the invention Test strips practically not disturbed by sulfonamide concentrations of up to 50 mg%. Higher concentrations only occur briefly after shock therapy and are therefore the Mostly known to observers.

Example 6

To produce a reagent film for the detection of urobilinogen, a mixture of prepares the following components:

p-bis - (,; - cyanoethyl) -amino-

benzaldehyde 0.75 g

Syrupy orthophosphoric acid 5.00 g

Colloidal Silica (Aerosil) ... 6.00 g Organic Sodium Sulphonates

(Rapidnetzer) 2.00 g

Polyvinylbuiyracetal (Mowital) .... 10.00 a

Polyethylene Glycol 6000 10. (X) g

Methanol . ". 100.0 ml

A thick layer of the above mixture is applied to a white polyvinyl chloride film and then dry with warm air. a water-insoluble film is formed. Will this with a drop of urobilinogen-containing urine, after 1 to 2 minutes a red color appears, which gets worse after wiping off the urine. There will be no staining with urobilinogen-free urine observed.

Example 7

25 ml urine are acidified with 12 ml glacial acetic acid and extracted with 40 ml ether. The extract will washed twice with 20 ml of water each time and made up to 40 ml with ether. Take 20 ml of this, mixed with 0.5 ml of a solution of 1 g of p-N-morpholinobenzaldehyde in 5 ml of concentrated hydrochloric acid and shake vigorously. Then add 6 ml of semi-saturated sodium acetate solution and shake again, whereby the red dye is in the aqueous Phase accumulates. After separation, the ether is washed again with 2 ml of water. The United aqueous extracts are made up to 20 ml, measured at 557 nm in a photometer and using a previously established calibration curve is evaluated.

Example

Filter paper is mixed with a solution of 0.5 part ρ - bis - (diälhylaminoäthyl) - aminobenzaldehyd - bisdrogenoxalat. 30 parts of maleic acid and 0.5 part of polyethylene glycol 1000 are impregnated in 100 parts of methanol. The paper reacts pink to red with urobilinogenic hooked urines. Papers with the same analytical properties, but more favorable behavior when rewinding and cutting are obtained from be Use of 5 parts of polyethylene glycol 6CKX

or 20,000?

Claims (1)

Patent claims: 1. Diagnostic agent for the detection of urobilinogen bodies in body fluids, preferably in urine, consisting of an absorbent carrier or film, a strong solid Acid and a substance which, in the manner of Ehrlich's test, with urobilinogen bodies provides a color change, characterized by the content of one or more Substances of formula (1)