DE2240357C2 - Test paper for the detection of bilirubin in body fluids - Google Patents

Description

R ₁ -O ^ ^ O

The present invention relates to a test paper 40 ^ \ ^

for the detection of bilirubin in body fluids, / _ "

which is a diazonium * capable of coupling with bilirubin
salt and one sufficient for the coupling reaction

Contains amount of an acid. in which R ₁ and R ₄ an optionally substituted

The detection and determination of bilirubin 45 tuated aliphatic, cycloaliphatic, araliphain body fluids are of great importance in the diagnosis of liver and biliary diseases. The residues R ₁ and R _t are preferably identical, for example, in the case of liver damage and bile ducts, since such phosphoric acid diesters can be produced in the urine at an early stage because such phosphoric acid diesters can be particularly easily occluded. The aliphatic residues before the bilirubin level rises in the serum and 50 R ₁ and Ri can be straight-chain and branched and clinical signs of jaundice are present. Contain 1 to 18 carbon atoms, whereby the effect on the other hand, one can distinguish this type of jaundice from the smoothness of the corresponding ester initially with so-called hemolytic icterus, with increasing number of carbon atoms grows and which only in the serum, but not in the urine, including from one Chain length of about 14 coals, increased bilirubin levels can be determined. 55 stoffa'.omen, due to the increasingly hydro-

Detection and determination methods of bilirubin phobic character of the ester, again decreases. When

in body fluids have long been known. The cycloalkyl radicals have those with 5 to

The methods that have proven to be useful are of the greatest importance here. When

on the evaluation of the coupling of substituents of the aliphatic or cycloaliphatic

The colored radicals formed with bilirubin contain halogen atoms, preferably

Connections are based. There is a multitude of such chlorine and bromine, and nitro and lower alkoxy

Methods since the discovery of the diazo reaction in groups with up to 8 carbon atoms in question.

Published in 1883. With regard to the stability of the test strips, phosphorus

Recently, the medical phoric acid diesters I, in which R ₁ and R _{2 have} an aromatic

In practice and in the clinical laboratory as diagnostic 65 matic remainder are preferred. As aromatic

Aids more and more so-called Schnclltcste residues have mononuclear and polynuclear, unsubstituted

introduced. These are simple devices, mostly and substituted aryl radicals, such as. B. the phenyl,

Test papers that the implementation of the J ¹ 'achweises xylyl, tolyl, chlorophenyl, nitrophenyl or naphthalene

3 4

thylrest proven. Solution I are suitable as araliphatic radicals

in particular phenyl or naphthyl radicals, which have a 2,6-dichlorobenzene diazonium

lower alkylene bridge with 1 to 3 carbon atoms fluoborate 0 08 g

are associated with the phosphoric acid residue. Metaphosphoric acid ''.'.' .. '.'. '.'. '.'. Y. '. loiog

The present invention also relates to citric acid 30 g

the use of phosphoric acid diesters of the above distilled water '". " ad 100 ^ O ml

given general formula I for the preparation
of test papers for the detection of bilirubin in
Body fluids.

Phosphoric acid diester of the general formula I io phosphoric acid diphenyl ester 5.0 g

are known per se (Method, d. Org. Chem., ethyl acetate ad 100.0 ml

Houben - Weyl, Vol. X11 / 2, 226ff.). The Phos

When immersed in urine containing bilirubin, the test solution has a pleasant and reaction-accelerating effect, depending on the concentration, after about 10 to 20 seconds of concentrations of 2 to 3% in the impregnation; only the lower alkyl esters require papers; the sensitivity limit is somewhat higher concentrations. For this reason 0.1 to 0.3 mg% bilirubin. With bilirubinhaltiftem the phosphoric acid esters I, which are obtained for the Kupp serum, red-violet colorations with increasing lung reaction usually cannot replace the necessary acid component intensity, whereby the sensitivity limit can replace about 0.4 to 0.6 mg % to maintain a strongly acidic 20 located.

pH value additional acid additions are necessary. Test papers, which react in an analogous way For this purpose, a whole range of solid acids comes with 2,6-dibromobenzoidiazonium fluorate, in question. Oxalic acid, test papers with otherwise the same composition, have proven to be particularly effective. Citric acid, potassium bisulfate and especially those that do not contain diphenyl phosphate, With regard to the stability of the Diazoniumslazes the as need 2 to 5 minutes for a reaction Commercially available metaphosphoric acid, which has about 60% of its sensitivity limit of about 1 to 2 mg% HiIi sodium salt may contain. Test papers next to ruby. The same applies to test papers that are used instead of the Diazoiiiumalzen only these acids and no diesters phosphoric acid diphenyl ester phosphoric acid monophedron Contain phosphoric acid, react significantly with bilirubin nyiester, phosphoric acid triphenyl ester or benzene slower and have a precise 30 phosphonic acid content. Bilirubin determinations completely inadequate sensitivity on. What this effect of the diester according to Example 2 Formula I is based, is still completely unknown, as none

chemical similarity to the test papers otherwise used, the one described in Example 1

Accelerators of the diazo reaction like caffeine or 35 are analogous, one gets, if one receives the phosphorus

Sodium benzoate is present. acid diphenyl ester by the following other phosphorus nor-

Phosphoric acid, phosphorus uremonophenyl ester, acid ester replaces:

Benzolphosphonic acid and phosphoric acid triphenyl- _n ,,. ,. . .

Esters, on the other hand, are completely ineffective. Phosphoric acid-d.-o-toy-ester,

The approved bilirubin test paper can contain ₄ o phosphoric acid di-p-tolyl ester,

expediently an additional content of phosphoric acid-b.s-S.S-xylyl ester,

a stabilizer for diazonium salts on phosphoric acid bis-o-chloropheny ester,

show how B. Sodium fluorate, magnesium phosphoric acid bis-p-chlorophenyl ester,

sulfate, sodium metaphosphate, or aryl sulfonates. Phosphoric acid-bis-p-ni rophenyl-ester,

Furthermore, they can contain wetting agents to ₄₅ phosphoric acid dibenzyl ester,

to improve the absorbency of the test papers. It phosphoric acid dicyclohexyl ester,

In principle, all of the wetting agents, the dipentyl phosphoric acid ester, can be used.

in the strongly acidic environment after immersion in.

the body fluid is still surface-active Example J

are, namely cationic (e.g. laurylpyridinium 50 filter paper is impregnated with the following solutions

chloride), nonionic (e.g., polyoxyethylene trigly- gniert and dried at 4O ⁰ C. cerid) and of the anionic particularly the sulphonates

and sulfates (e.g., dodecylbenzenesulfonate). Solution I.

The wetting agents are in concentrations of

0.1 to 2%, preferably 0.2 to 0.5%, are used. 55 diazonium salt (see Table 1) .... 0.3 g

The most important absorbent carriers are metaphosphoric acid 10.0 g

Filter papers in question, but also other absorbent water — methanol (4: 1) ad 100.0 ml

Carriers such as fiberglass papers, synthetic fiber fabrics and

-vHese made of acid-resistant fibers such as polyester and solution II

Polypropylene are suitable. The 60

The term "test papers" mentioned should also include diphenyl phosphoric acid 5.0 g

include. Dodecylbenzenesulfonic acid, Na salt 0.5 g

The following examples serve the closer ethyl acetate ad 100.0 ml

Explanation of the invention.

65 The result is test papers showing a bilirubin

Example 1 Content of the serum with the following reaction colors

Filter paper will show the following solutions, whereby the sensitivity limit is nuch

impregnated with each other and ^{dried at 40 0} C: Diazonium salt is approximately between 0.3 and 0.6 mg%.

Table 1 Example 4

Filter paper is impregnated with solutions composition and dries:

Solution I Solution II

Diazonium salt Reaction color 4-N-trobenzene diazonium fluorate blue 3-nitrobenzene diazonium violet naphthalene-2-sulfonate 2-chlorobenzene diazonium Blue purple naphth? Hn-1,5-disulfonate 4-fluorobenzene diazonium fluorate blue 2,5-dichlorobenzene diazonium violet fluoborate 2,4-dibromobenzene diazonium blue fluoborate 2,4,6-Trichlori> - ^c oldiazonium- violet fluoborate 4-carboxybenzene diazonium blue p-tojuenesulfonate Diazosulfanilic acid blue Benzidin-bisdiazontumflu.; L> orat green 4- (5-methylbenzthiazoly 1-2 -) - green benzene diazonium fluorate

Phosphoric acid di (A) - and -mono-

alkyl ester (B) mixture * g

(see table 2 j ethyl acetate or chloroform ... ad 100.0; nl

The result is test papers which are red-violet to blue-violet in color with bilirubin-containing serum react when phosphoric acid esters with alkyl residues ίο according to table 2 are used:

Table 2

Alkyl radical

the at

methyl

ethyl

2-chloroethyl.
2-butoxyethyl

Propyl

i-propyl

Butyl

2-ethylhexyl.
_a5 i-octyl *) ....

i-nonyl *)

following i-decyl *) .... 40 ⁰ C ge-dodecyl

i-Tridecyl *) ..

X % A 40 50 20th 40 10 50 10 50 10 45 20th 25th 10 50 5 92 10 55 10 60 10 60 10 55 10 60

% B

40 50 45 45 50 55 50 3

45 40 40 45 40

*) Mixture of isomers.

2-chlorobenzene diazonium fluorate 0.05 g

Oxalic acid 10.0 g

Sodium fluorate 5.0 g

Water-methanol to 100.0 ml

In the ester mixtures given above, the phosphoric acid monoesters serve together with the oxalic acid in solution I only as an acid component for the coupling reaction.

Claims (5)

1 2 "* or the semi-quantitative determination of patho-.,." '"' Patent claims: logical body constituents quickly and safely even by untrained personnel, such as medical auxiliaries 1. Test paper for the detection of bilirubin in personal, permit. Body fluids containing bilirubin 5, for example, in the German Auslegeschrift Coupling diazonium salt and one for 1102 444 a diazonium salt and a strong one Coupling reaction sufficient amount of an acid-containing reagent tablet described, the Contains acid, characterized by a test piece moistened with the body fluid Phosphoric acid diester content of the all-leaflet is placed and after moistening with common formula I io water the bilirubin as a purple ring on the underside makes the situation visible. R ₁ - OO It can be seen that such a diagnostic \ Medium is relatively awkward to handle. For the _y - '** \ ^ wide application in medical practice and am R O OH * 5 hospital bed but it is desirable that Schnell test are designed in such a way that they can be in which R ₁ and R _2, if necessary submerging in the body fluid to be examined, without substituted aliphatic, cycloaliphatic, arali- further can be read. Fulfill this purpose on the phatic or aromatic radical means. best so-called test papers; these are absorbent 2. Test paper according to claim 1, characterized ao carrier, which are impregnated with all necessary for the detection reaction by the additional content of a stable agile reagents,
In the German Offenlegungsschrift 2 007 013 are sulfate, sodium metaphosphate or aryl sulfonate. already test papers for the detection of bilirubin 3. Test paper according to claim 1 or 2, marked, which draws diazonium as the detection reagent contain salts due to the additional content of an ace. According to our own research, show Wetting agents. however, these test papers are not yet sufficient 4. Test paper according to claim 1, characterized in response speed and sensitivity, characterized that it is metaphosphorus as an acid so that its usefulness in practice is in question contains acid. is posed. 5. Use of phosphoric diesters of the 30 It was therefore the task of these known General formula I given in claim 1 test papers with regard to their sensitivity and : rar production of test papers to improve detection response speed. of bilirubin in body fluids. According to the invention, this object is achieved by that the test paper of the type mentioned above has a content of phosphoric diesters of the general Formula I.