Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
  • G01N33/728—Bilirubin; including biliverdin
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/903—Diazo reactions
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
  • Y10T436/145555—Hetero-N
  • Y10T436/146666—Bile pigment

Definitions

• cycloaliphatic araliphatic or aromatic radicals and contain not more than 18 carbon atoms each.
• bilirubin occurs in the urine in the early stages, even before the bilirubin content of the serum increases and clinical signs of jaundice appear.
• this type of jaundice can be distinguished from the socalled haemolytic icterus in which an increased bilirubin level can only be detected in the serum but not in the urine.
• German Patent No. 1,102,444 there is described a reagent tablet containing a diazonium salt and a strong acid which is placed on a spot plate moistened with the body fluid to be tested and, after moistening with water, makes the bilirubin visible as a violet ring on the substrate.
• test papers these are absorbent carriers which have been impregnated with all the reagents necessary for the detection reaction.
• R and R which may be the same or different, are
• the substituents R and R are preferably identical because phosphoric acid diesters of this type are especially easy to prepare.
• the aliphatic radicals R and R can be straight-chained or branched and can contain up to 18 carbon atoms, the effectiveness of the esters initially increasing with the increasing number of carbon atoms and, finally, above a chain length of about 14 carbon atoms, due to the increasingly hydrophobic nature of the esters, again decreases.
• cycloalkyl radicals those containing five to eight carbon atoms have proved to be preferable.
• substituents for the aliphatic and cycloaliphatic radicals include halogen atoms, preferably chlorine or bromine atoms, nitro groups and alkoxy radicals containing up to 8 carbon atoms.
• R, and R are aromatic radicals.
• aromatic radicals there are preferably used monoor polynuclear, unsubstituted or substituted aryl radicals, for example, phenyl, xylyl, tolyl, chlorophenyl, nitrophenyl or naphthyl radicals.
• araliphatic radicals include the phenyl and naphthyl radicals connected to the phosphoric acid residue via an alkylene bridge containing up to three carbon atoms.
• the present invention provides test papers for the detection of bilirubin in body fluids which contain a diazonium salt capable of coupling with bilirubin and an amount of an acid which is sufficient for the coupling reaction, as well as a phosphoric acid diester of general formula (I).
• the phosphoric acid diesters of general formula (I) are known (see Methoden d. Org. Chem., Houben- Weyl, Vol. XII/2, pp 226 et seq.).
• the phosphoric acid diesters of general formula (I) exert their sensitivity-increasing and reactionpromoting action even at concentrations of 2-3 percent in the solution used for impregnating the test papers; only the lower alkyl esters require a somewhat higher concentration. For this reason, the phosphoric acid diesters of general formula (I) usually cannot replace the acid component needed for the coupling reaction so that, for the maintenance of a strongly acidic pH value, additional amounts of acid are necessary. For this purpose, a large number of acids can be used. Those which have proved to be especially useful include oxalic acid, citric acid, potassium bisulfate and, especially with regard to the stability of the diazonium salt, commercially available metaphosphoric acid, which can contain up to about 60 percent of its sodium salt.
• Test papers which, in addition to the diazonium salts, only contain these acids and no diesters (l) react substantially more slowly with bilirubin and have an insufficient sensitivity for an accurate determination of bilirubin. It is still completely unknown upon what this action of the diesters (I) used according to the present invention depends since there is absolutely no chemical similarity with the accelerators of the diazo reaction otherwise used, for example caffeine or sodium benzoate.
• diesters of phosphoric acid of general formula (I), used according to the present invention are effective.
• the good effect of these phosphoric acid diesters (l) is in complete contrast to the complete ineffectiveness of phosphoric acid, phosphoric acid monoesters, benzenephosphonic acid and phosphoric acid triphenyl ester.
• test papers are intended for the determination of bilirubin in the serum or in urine, i.e. the formulation of the test strips must be appropriately modified.
• diazonium salts which, from the chemical standpoint, can be expected to give a rapid and sensitive reaction.
• diazonium salts which exclusively or preponderantly contain electron-attracting groups.
• the substituents can be nitro groups, halogen atoms, carboxyl groups, sulfonic acid residues, nitrile groups or quaternary ammonium groups.
• electron-donating groups for example alkoxy radicals, can also be present.
• diazotized naphthylamine and benzidine derivatives can also be used.
• benzene-diazonium salts which exclusively contain electron-donating groups, such as alkoxy, alkyl or arylamino radicals, because these only react comparatively slowly with bilirubin.
• the reaction color is red-violet to blue-green and, in addition, as has been found from experience, with increasing acidity of the phosphoric acid diesters, the colors are bathochromically displaced.
• 3-nitroand 2,4,6-trichlorobenzene-diazonium salts are preferably used.
• the diazonium salts are preferably used in the form of fluoborates since the stability thereof is well-known; however, other stable salts, for example, arylsulfonates, especially naphthalene-1,S-disulfonates, can also be employed.
• the diazonium salts can be used in the impregnation solutions in amounts of from 0.02 to about 2 percent and preferably of 0.05 to 0.5 percent.
• reagents for the detection of bilirubin in the urine there can be used, for example, 2,6-dihalobenzenediazonium salts, especially the 2,6-dichloro and/or -dibromo derivatives, since these only possess the abovementioned disadvantages to a minor extent.
• the salts there can be used the conventional stabilizing anions, for example, the fluoborates and aryl-sulfonates.
• Test papers are obtained which, depending upon the bilirubin concentration of the urine, change from yellow via orange to red-violet.
• the concentration of the diazonium salts in the impregnation solutions can be between 0.02 and 0.5 percent and preferably between 0.05 and 0.15 percent.
• the test papers for urine bilirubin can, of course, also be used for serum bilirubin.
• the bilirubin test papers according to the present invention for serum and urine preferably contain stabilizing agents for the diazonium salts, for example, sodium fluoborate, magnesium sulfate, sodium metaphosphate, aryl-sulfonates or the like.
• wetting agents in order to improve the absorptive powers of the test papers.
• wetting agents which are still surface-active in the strongly acidic medium formed after dipping into the body fluid, namely, cationic agents (for example lauryl-pyridinium chloride), non-ionic agents (for example, polyoxyethylene triglyceride) and anionic agents, especially sulfates and sulfonates (for example, dodecyl-benzene-sulfonate).
• the wetting agents can be used in the impregnation solutions in concentrations of 0. l to 2 percent and preferably of 0.2 to 0.5 percent.
• an absorbent paper can first be impregnated with a mixture of a diazonium salt and an acid, for example metaphosphoric acid in an aqueous medium and then impregnated with an ester (l), for example phosphoric acid diphenyl ester, in ethyl acetate or chloroform.
• an acid for example metaphosphoric acid in an aqueous medium
• an ester for example phosphoric acid diphenyl ester
• absorbent carrier it is preferred to use filter paper but other absorbent carriers, for example, glass fiber paper or synthetic fiber fabrics and fleeces made from acid resistant fibers, such as polyesters and polypropylene, can also be used.
• acid resistant fibers such as polyesters and polypropylene
• test paper is to be understood to include all of these materials.
• test papers can be cut up into long strips and rolled up and, when needed, it is only necessary to cut or tear off a small piece. They can also be cut up into small rectangular pieces and stuck or sealed on to the lower end of narrow synthetic resin strips. It is especially advantageous when the test papers are sealed between two synthetic resin foils in the manner described in German Pat. No. 1,546,307 or between a synthetic resin film and a meshwork in the manner described in German Pat. No. 2,118,455 because there is then no danger that the reagents might be washed out upon dipping into a body fluid.
• Test papers which contain 2,6-dibromobenzenediazonium fluoborate reacted in an analogous manner.
• Test papers of otherwise the same composition but which did not contain the phosphoric acid diphenyl ester required 2-5 minutes for the reaction and had a limit of sensitivity of about I to 2 mg.%. This also applied to test papers which, instead of phosphoric acid diphenyl ester, contained phosphoric acid monophenyl ester, phosphoric acid triphenyl ester or benzenephosphonic acid.
• EXAMPLE 2 EXAMPLE 3 Filter paper was impregnated with the following solutions and then dried at 40C.:
• Test paper for the detection of bilirubin in body fluids comprising an absorbent carrier and, impregnated thereinto a diazonium salt capable of coupling with bilirubin, an amount of an acid which is sufficient for the coupling reaction, and at least one phosphoric acid diester of the general formula:
• R and R are individually selected from unsubstituted or substituted aliphatic, cycloaliphatic, araliphatic or aromatic radicals, and contain not more than 18 carbon atoms each.
• Test paper as claimed in claim 26, wherein the stabilizing agent is sodium fluoborate. magnesium sulfate, sodium metaphosphate or an aryl-sulfonate.
• Test paper as claimed in claim 1 also containing a wetting agent.
• 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.

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• Investigating Or Analysing Biological Materials (AREA)
• Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)

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Citations (6)

• 1972
  • 1972-08-17 DE DE2240357A patent/DE2240357C2/de not_active Expired
• 1973
  • 1973-07-24 US US00382242A patent/US3853476A/en not_active Expired - Lifetime
  • 1973-08-14 FR FR7329672A patent/FR2199888A5/fr not_active Expired
  • 1973-08-14 CH CH1170173A patent/CH572212A5/xx not_active IP Right Cessation
  • 1973-08-14 GB GB3842973A patent/GB1389366A/en not_active Expired
  • 1973-08-16 AT AT715373A patent/AT327399B/de not_active IP Right Cessation
  • 1973-08-17 JP JP9233073A patent/JPS5328119B2/ja not_active Expired
• 1978
  • 1978-02-10 BE BE185079A patent/BE863862Q/xx not_active IP Right Cessation
  • 1978-03-14 KE KE2829A patent/KE2829A/xx unknown
  • 1978-03-23 HK HK162/78A patent/HK16278A/xx unknown

Patent Citations (6)

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