Diagnostic agent for detecting bilirubin
United States Patent 1191 Rittersdorf et al.  
 1 Apr. 29, I975 1 DIAGNOSTIC AGENT FOR DETECTING BILIRUBIN [73] Assignee: Boehringer Mannheim G.m.b.II.,  
 Mannheim-Waldhof, Germany 221 Filed: July 24,1973  
  2| App1.No.:382,24l  
 [30] Foreign Application Priority Data Aug. 17, 1972 Germany 2240471 [52] US. Cl. 23/253 TP; 23/230 B; 252/408 [51] Int. Cl G0ln 31/22; GOln 33/16 [58] Field of Search....@ 23/230 B, 253 TP; 252/408; 8/41 D, 44  
 [56] References Cited UNITED STATES PATENTS 2,854,317 9/1958 Free at al. 23/230 B 3,511,607 5/1970 Green 23/230 B 3,585,001 6/1971 Mast 1 23/230 B 3,585,004 6/1971 Mast 23/230 B X 3,652,222 3/1972 Denny et a1. 23/230 B 3,754,862 8/1973 Wahlefeld et al. 23/230 B Primary Examiner-Morris O. Wolk Assistant Examiner-Michael S. Marcus Attorney, Agent, or Firm--Burgess, Dinklage &amp; Sprung 57 1 ABSTRACT Bilirubin, particularly as contained in body fluids, is determined with test strips comprising an absorbent wherein X is a stabilizing anion; R and R which may be the same or different, are  
  halogen or lower alkyl or alkoxy and R and R which may be the same or different, are hydrogen, halogen, nitro, or lower alkyl or alkoxy; the aromatic ring in formula (1) containing either (a) 1 to 3 halogen atoms or (b) one nitro group, but not both together; and an amount of an acid sufficient for a coupling reaction between bilirubin and said diazonium salt.  
 3I Claims, No Drawings DIAGNOSTIC AGENT FOR DETECTING BILIRUBIN The present invention is concerned with an improved diagnostic agent for the rapid and sensitive detection of bilirubin in body fluids, particularly in urine.  
  The detection of bilirubin is of great importance in the diagnosis of diseased of the liver and gall bladder. Thus, in the case of liver damage and of occlusion of the gall bladder duct, the early appearance of bilirubin in the urine occurs even before the bilirubin content of the serum increases and clinical symptoms of jaundice appear. On the other hand, this type of jaundice can be differentiated from haemolytic icterus in which an increased bilirubin level can only be ascertained in the serum but not in the urine.  
  Methods for the detection of bilirubin in the urine have been known for along time. Of importance are the methods which depend upon the evaluation of the colored compounds formed by the coupling of bilirubin with diazonium salts. For this purpose. diazosulfanilic acid has previously been most used.  
  Recently, in medical practice and in clinical laboratories, the so-called rapid tests have been used more and more as a diagnostic aid. These simple devices, usually test papers, which permit the carrying out of the detection or semi-quantitative determination of pathological components of the body in a rapid and certain manner, even by untrained personnel, such as medical auxiliaries.  
  Thus, for example, German Pat. No. 1,102,444 describes a reagent tablet containing a diazonium salt and a strong acid which is placed on a spot plate moistened with a body fluid and, after moistening with water, makes the bilirubin visible as a violet ring on the substrate.  
  It is obvious that such a diagnostic agent is relatively laborious to use. For a Wide use in medical practice and at the sick bed, it is, however, desirable that the rapid test is such that it can be read off without difficulty after simply dipping into the body fluid to be investigated. This purpose is best fulfilled by the so-called test papers; these are absorbent carriers which have been impregnated with all the reagents necessary for the detection reaction.  
  It is known from German Pat. No. 2,007,013 to use test papers which contain certain 2,4- and 2,5- disubstituted diazonium salts for the detection of bilirubin in serum and urine. However, this method is relatively insensitive and, according to our investigations, also suffers from certain disadvantages with regard to the speed of the reaction and the sensitivity, which lessens its practical utility. There is, of course, the possibility of increasing the sensitivity of the detection reaction but, in this case, new difficulties arise. Thus, the urobilinogen which is also present in urine also reacts in the presence of these activators and simulates an increased biliburin content. Furthermore, the normal disturbing reactions with components of the urine are hereby also activated, which can manifest themselves as false colorations or even give falsely positive indications.  
  We have now, surprisingly, found that when using certain diazonium salts as the detection reagents, the abovementioned difficulties no longer occur, even when activators are used at the same time. Thus, these diazonium salts can be used to prepare test strips which react selectively with bilirubin not only in urine but also in serum.  
  The diazonium salts used in this invention are of the formula:  
  3 NEN (:9 X  
 wherein X is a stabilizing anion, R and R which may be the same or different, are  
  halogen or lower alkyl or alkoxy and R and R,,, which may be the same or different, are  
  hydrogen, halogen, nitro or lower alkyl or alkoxy; the aromatic ring in formula (I) may contain either (a) l to 3 halogen atoms or (b) one nitro group, but not both together.  
  The diazonium salts of general formula (I) are either known or can be prepared from known aromatic amines by diazotization.  
  The lower alkyl or alkoxy radicals in the diazonium salts (I) are to be understood to contain up to 3 carbon atoms each.  
  The diazonium salts (I) are preferably used in the form of the fluoborates (i.e., wherein X is a fluoborate anion), the stability of which is known; however, other stable salts, such as aryl-sulfonates, especially naphthalene-1,5-disulfonates, can also be used.  
  Diazonium salts of general formula (I) are preferred in which R and R are both halogen atoms, especially chlorine or bromine atoms, and the other symbols stand for hydrogen atoms.  
  As activators, there can be used, apart from anionic wetting agents, especially compounds of the general formula:  
 . wherein R and R which may be the same or different, are unsubstituted or substituted aliphatic, cycloaliphatic, araliphatic or aromatic radicals.  
  The radicals R and R are preferably identical since phosphoric acid diesters of this type are particularly simple to prepare. The aliphatic radicals R and R can be straightchained or branched and can contain up to 18 carbon atoms, the effectiveness of the esters initially increasing with an increasing number of carbon atoms but beyond a chain length of about 14 carbon atoms, the effectiveness again decreases due to the increasingly hydrophobic character of the esters. As cycloalkyl radicals, those with 5-8 carbon atoms are especially preferred. Preferred substituents in the aliphatic and cycloaliphatic radicals include, for example, halogen atoms, especially chlorine and bromine atoms, nitro groups and alkoxy radicals containing up to 8 carbon atoms. With regard to the stability of the test strips, phosphoric acid diesters of general formula (II) are preferred in which R and R both represent aromatic radicals. Preferred aromatic radicals include monoand polynuclear, unsubstituted and substituted aryl radicals, for example, phenyl, xylyl, tolyl, chlorophenyl, nitrophenyl and naphthyl radicals. As araliphatic radicals, there are preferably used, for example, phenyl or naphthyl radicals which are connected to the phosphoric acid residue via an alkylene bridge containing up to 3 carbon atoms.  
  For the preparation of an especially useful test strip according to the present invention, an absorbent carrier, preferably filter paper, is impregnated with a solution which contains 0.0 l-2.0 g., preferably 0.050.5 g., per 100 ml. of a diazonium salt of general formula (I), as well as 3-20 g., preferably 5-15 g., per 100 ml. of a phosphoric acid diester of general formula (II) and 230 g. of a solid inorganic or organic acid and thereafter dried. However, it is also possible to carry out the v diazotization in the impregnation solution or on the absorbent carrier. In this case, an appropriate amine is used as starting material which is then reacted with a nitrite in the usual manner.  
  The bilirubin test strips according to the present invention can also contain stabilizing agents for the diazonium salts, for example sodium fluoborate, magnesium sulfate, sodium metaphosphate, aryl-sulfonates or the like.  
  Furthermore, the test strips can also contain wetting agents in order to improve the absorbency of the test papers. In principle, all wetting agents can be used which are still surface-active in the strongly acidic medium after dipping into a body fluid to be tested, for example cationic agents (e.g. lauryl-pyridinium chloride), non-ionic agents (e.g. polyoxyethylene triglyceride) and anionic agents, especially the sulfonates and sulfates (e.g. dodecyl-benzene-sulfonate). The latter can additionally possess activating properties.  
  The wetting agents can be used in the impregnation solution in concentrations of O. l2% and preferably of 0.2-0.5%.  
  As solvents or solvent mixtures for the impregnation, there can be used all those which do not react with the diazonium salts, in which all the components are soluble and which have a low boiling point in order that, upon drying, the diazonium salts are not subjected to too high a temperature. It has also proved to be advantageous to impregnate individual components in separated working steps. Thus, for example, a mixture of a diazonium salt (I) and an acid, for example metaphosphoric acid, can first be used for impregnation from an aqueous solution and then a further impregnation is carried out with a solution of a phosphoric acid diester (II) in ethyl acetate or chloroform.  
  As absorbent carrier, it is preferred to use filter paper but other absorbent carriers, for example, glass fiber paper and synthetic fiber fabrics and fleeces made from acid-resistant fibers, such as polyester and polypropylene fibers, can also be used. The term test paper used herein is to be understood to include these materials.  
  Thus, the present invention provides a test strip for the detection of bilirubin in body fluids, which comprises an absorbent carrier containing at least one diazonium salt of general formula (I) and an amount of an acid sufficient for the coupling reaction.  
  Phosphoric acids of general formula (II) are known (see, for example, l-Iouben-Weyl, Methoden d. Org. Chem., Vol. XII/2, pp 226 et seq.). Since the compounds (II) themselves have an acidic reaction, they can at least partially replace the acid needed for the coupling reaction. However, additional amounts of acid are usually necessary. For this purpose, a whole series of solid acids can be used. Of particular use are, for example, oxalic acid, citric acid, potassium bisulfate and, especially having regard to the stability of the diazonium salt, commercially available metaphosphoric acid, which can contain up to about of its sodium salt.  
  The test strips according to the present invention react with bilirubin-containing urine in about 5 to 30 seconds to give red to blue color shades. With normal urine, there is obtained a yellow reaction color. Urobilinogen reacts, without disturbing, with a yellow color, red shades only occurring when unusually large amounts thereof are present in the urine (more than about 8-10 mg.%). The detection of serum bilirubin is, of course, also possible with the use of the test strips according to the present invention.  
  The impregnated absorbent carriers can be cut up into long strips and then rolled up so that, when needed, a small piece can be torn or cut off. They can also be cut up into small rectangles and stuck or sealed on to one end of narrow strips of synthetic resins. It is of particular advantage when the test papers are sealed between two synthetic resin films according to the method described in German Patent No. l,546,307 or between a synthetic resin film and a meshwork as described in German Patent No. 2,1 18,455, because there is then no danger that the reagents will be washed out upon dipping into a body fluid.  
  The following Examples are given for the purpose of illustrating without limitation, the present invention:  
 EXAMPLE 1 Filter paper was successively impregnated with the following solutions and then dried at 40C.  
 Solution 1:  
  2,o-dichlorobenzene-diazonium fluoborate 0.08 g. metaphosphoric acid l0.0 g. citric acid 3.0 g. distilled water ad 100.0 ml. Solution II:  
 phosphoric acid diphcnyl ester 5.0 g dodecyl-benzenc-sulfonic acid:  
  sodium salt 0.4 g. ethyl acetate ad 100.0 ml.  
  With bilirubin-containing serum, there were obtained red-violet colorations of increasing intensity, the limit of sensitivity being about 0.4 to 0.6 mg.%.  
  Test papers which contain 2,6-dib&#39;romobenzenediazonium fluoborate reacted in an analogous manner.  
  Test papers of analogous composition but which con tain 2,4-dichlorobenzene-diazonium fluoborate (of. German Pat. No. 2,007,013) reacted with bilirubin with a blue color. With urobilinogen-containing urine,  
 they gave a deep violet coloration after only a few seconds. 1  
 EXAMPLE 2 Test papers which reacted in a manner analogous to that described in Example 1 were obtained when the phosphoric acid diphenyl ester was replaced by one of the following phosphoric acid esters:  
 phosphoric acid di-o-tolyl-ester; phosphoric acid di-p-tolyl ester; phosphoric acid bis-3,5-xylol ester; phosphoric acid bis-o-chlorophenyl ester; phosphoric acid bis-p-chlorophenyl ester; phosphoric acid bis-p-nitrophenyl ester; phosphoric acid dicyclohexyl ester; or phosphoric acid dipentyl ester.  
 EXAMPLE 3 Filter paper was impregnated with a solution of the following compoisition and then dried at 40C.:  
 2,6-dichlorobcnzcne-diazonium fluoborate 0.16 g. metaphosphoric acid 15.0 g. dodecyl-benzene-sulfonic acid sodium salt 1.0 g. methanol 20.0 ml. distilled water ad 100.0 ml.  
  This test paper reacted, after 20-30 seconds, with a violet color with bilirubin-containing urine, the sensi tivity limit being about 0.7 to 0.9 mg.%. With urine having a high content of urobilinogen (about 8-12 mg.%), a pale red color appeared after about 1-2 minutes.  
  A test paper of the same composition except that it contained 2,4-dichlorobenzene-diazonium fluoborate, reacted with bilirubin with a blue-violet color and, with urobilinogen-containing urine, a disturbing violet coloration occurred after only a short period of time.  
 EXAMPLE 4 Filter paper was impregnated with a solution of the following composition and then dried at 40C.:  
 2-mcthoxy-4,6-dibromobcnzcne-diazonium fluoborate 0.12 g. citric acid 10.0 g. sodium mctaphosphatc 3.0 g. distilled water ad 100.0 m1.  
  This paper was then impregnated with a 5% solution of phosphoric acid diphenyl ester in ethyl acetate.  
  The properties of this test paper, as well as of test papers containing an equimolar amount of one of the diazonium salts set out in the following Table l, corresponded to those of Example 1. The reaction colors with bilirubin were, in the case of the halogenand alkyl-substituted compounds, more blue-violet and, in  
 the case of the alkoxyand nitro-substituted compounds, more grey-blue.  
 TABLE I EXAMPLE 5 A solution of the following composition was prepared at 510C., then impregnated on to filter paper and dried at 40C.:  
 2-methy1-6-chloroaniline 0.14 g sodium nitrite 0.07 g metaphosphoric acid 10.0 g distilled water ad 100.0 ml  
  This test paper was thereafter impregnated with a 10% solution of phosphoric acid diphenyl ester in ethyl acetate and dried at 40C.  
  Analogous test papers could be prepared with an equimolar amount of one of the following substituted aniline derivatives: 2,6-dimethyl-3-bromoaniline; 2,6-dimethyl-4-bromoanilinei 2,6-dimethoxy-3- bromoaniline; and 2,6-dimethoxy-4-chloroaniline.  
  The properties of the test papers obtained correspond approximately to those of the test papers according to Examples 1 and 4.  
 EXAMPLE 6 Filter paper was impregnated with a solution of the following composition and dried at 40C.:  
 2,4,6-trichlorobenzene-diazonium toluenesulfonate 0.3 g. metaphosphoric acid 10.0 g. distilled water ad 100.0 ml.  
  This paper was subsequently impregnated with a 10% solution of commercially available isooctyl-phosphoric, acid in methylene chloride. This acid is a mixture of about 55% of the diester and 45% of the monoester of phosphoric acid with isomeric C alcohols.  
  The properties of this test paper corresponded to those of the test paper according to Example 1.  
  Test papers which contain an equimolar amount of one of the following diazonium salts possess practically the same properties:  
 2,4,6-tribromobenzene-diazonium naphthalenedisulfonate; 2,4,6-tribromo-3-methyl-benzene-diazonium fluoborate;  
 2-methyl-4,6-dibromo-S-chlorobenzene-diazonium fluoborate;  
 2,4,6-tribromo-3-methoxy-benzene-diazonium fluoborate;  
 2,3,6-tribromobenzene-diazonium fluoborate.  
 wherein X is a stabilizing anion,  
 R, and R which may be the same or different, are  
 halogen or lower alkyl or alkoxy and R and R.,, which may be the same or different, are  
  hydrogen, halogen, nitro, or lower alkyl or alkoxy; the aromatic ring in formula (1) containing either (a) l to 3 halogen atoms or (b) one nitro group, but not both together; and an amount of an acid sufficient for a coupling reaction between bilirubin and said diazonium salt.  
  2. Test strip as claimed in claim 1, wherein R, is halogen.  
  3. Test strip as claimed in claim 1, wherein R is alkyl of from I to 3 carbon atoms.  
  4. Test strip as claimed in claim 1, wherein R, is alkoxy of from 1 to 3 carbon atoms.  
  5. Test strip as claimed in claim 1, wherein R is halogen.  
  6. Test strip as claimed in claim 1, wherein R is alkyl of from 1 to 3 carbon atoms.  
  7. Test strip as claimed in claim 1, wherein R is all;- oxy of from 1 to 3 carbon atoms.  
  8. Test strip as claimed in claim 1, wherein R is hydrogen.  
  9. Test strip as claimed in claim 1, wherein R is halo gen.  
  10. Test strip as claimed in claim 1, wherein R is nitro.  
  11. Test strip as claimed in claim 1, wherein R is alkyl of from 1 to 3 carbon atoms.  
  12. Test strip as claimed in claim 1, wherein R is alkoxy of from I to 3 carbon atoms.  
  13. Test strip as claimed in claim 1, wherein the aromatic ring of said diazonium salt contains a nitro group and no halogen atoms.  
  14. Test strip as claimed in claim 1, wherein the aromatic ring of said diazonium salt contains one halogen atom and no nitro group.  
  15. Test strip as claimed in claim 1, wherein the aromatic ring of said diazonium salt contains two halogen atoms and no. nitro group.  
  16. Test strip as claimed in claim 1, wherein the aromatic ring of said diazonium salt contains three halogen atoms and no nitro group.  
 17. Test strip as claimed in claim 1, wherein said dia-- zonium salt is 2,6-dichlorobenzene-diazonium fluoborate.  
 7 18. Test strip as claimed in claim 1, wherein said diazonium salt is 2-methoxy-4,6-dibromobenzenediazonium fluoborate.  
  19. Test strip as claimed in claim 1, wherein said diazonium salt is 2,4,6-trichlorobenzene-diazonium toluenesulfonate.  
  20. Test strip as claimed in claim 1 also containing an activator.  
  21. Test strip as claimed in claim 20, wherein said activator is a phosphoric acid diester of the general formula:  
 in which R and R,,, which may be the same or different, are unsubstituted or substituted aliphatic, cycloaliphatic, araliphatic or aromatic radicals.  
  22. Test strip as claimed in claim 1, wherein the stabilizing anion X is a fluoborate or aryl-sulfonate ion.  
  23. Test strip as claimed in claim 1, wherein the absorbent carrier is impregnated with a solution containing, per ml., 0.0l2.0 g. of the diazonium salt, 3-20 g. of the phosphoric acid diester and 2-30 g. of a solid inorganic or organic acid.  
  24. Test strip as claimed in claim 1, wherein the absorbent carrier is impregnated with a solution containing 0.050.5 g. of the diazonium salt per 100 ml.  
  25. Test strip as claimed in claim 1, also containing a stabilizing agent.  
  26. Text strip as claimed in claim 25, wherein the stabilizing agent is sodium fluoborate, magnesium sulfate, sodium metaphosphate or an aryl-sulfonate.  
  27. Test strip as claimed in claim 1,.wherein said absorbent carrier is a filter paper, glass fiber paper or a fabric or fleece made from acid-resistant synthetic fibers.  
  28. Test strip as claimed in claim 1, wherein said absorbent carrier is adhered to one end of a narrow strip of synthetic resin.  
  29. Test strip as claimed in claim 1, wherein said absorbent carrier is sealed between two synthetic resin films.  
  30. Test strip as claimed in claim 1, wherein said absorbent carrier is sealed between a synthetic resin film and a meshwork.  
  31. Method for detecting bilirubin in body fluids, which method comprises contacting a test sample suspected of containing bilirubin with a test strip as claimed in claim 1.  
 UNITED STATES PATbNl OIFICE CERTIFICATE OF CORRECTION I at 3.880.588 Dated April 29 1975 Q 1 Walter Rittersdorf et al It is certified that error appears in the aboveidentified patent I and that said Letters Patent are hereby corrected as shown below:  
  Under the heading ABSTRACT at line 2 after the word &#34;absorbent&#34; insert --carrier containing a diazonium salt of the formula:-. Q  
 Column 1, line 8, delete &#34;diseased&#34; and substitute therefor --diseases-.  
 Column 4, line 54, after the word &#34;acid&#34; delete --the colon Column 5, line 31, delete &#34;compoisition&#34; and substitute therefor --composition-.  
 Signed and Ewaled this 0 second Day Of September 1975 [SEAL] Arrest:  
 Q RUTH C. MASON c. MARSHALL DANN Arlvsling Officer (mnmissimwr uflatems and Trademarks FORM PO-IOSO (10-69) USCOMM-DC 60316-P59