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
• • 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

• R, and R which may be the same or different, each represent halogen, cyano, nitroso, carbalkoxy, carbamido, hydroxy, acyloxy, alkoxy, straight-chained and cyclic, saturated and unsaturated secondary and tertiary amino, or R, and R together represent oxygen, sulfur, sulfonyl, alkylthionium, imino, alkylimino, substituted or unsubstituted arylimino or open-chained, alicyclic or heterocyclic dialkylimino, R, can also represent the residue of secondary straight or branched, open-chained or monoor polycyclic alkyl or aralkyl and wherein one of R, and R, can also represent hydrogen, R is hydrogen or alkyl, and n and m which may be the same or different each have a value ofO, l, 2 or 3.
• the present invention relates to new and improved diagnostic agents for the detection of urobilinogen bodies in body fluids. More particularly, this invention relates to improved diagnostic agents for the detection of urobilinogen bodies in urine, spinal fluids and other body fluids.
• Another object of the present invention is a simple and practical diagnostic agent for use in the detection of urobilinogens present in body fluids comprising a-test paper or film.
• diagnostic agents for use in the detection of urobilinogens present in body fluids are obtained by employing a strong acid in admixture with a derivative of p-aminobenzaldehyde.
• Preferred p-aminobenzaldehyde derivatives to be used according to the present invention are those which carry on the p-amino group, a branched alkyl group or a substituent which possesses strong electron attracting properties.
• Illustrative of the compounds of this type are those having the formula:
• R, and R which may be the same or different, are each halogen, cyano, nitroso, carbalkoxy, carbamido, hydroxyl, acyloxy, alkoxy or open-chained or cyclic, saturated or un saturated, secondary or tertiary amino or R and R together represent oxygen, sulfur, sulfonyl, alkylthionium, imino, alkylimino, unsubstituted or substituted arylimino openchained, alicyclic or heterocyclic dialkylimino radical, R, can also be the residue of a secondary straight-chained or branched, open-chained, monoor polycyclic alkyl or aralkyl and wherein one of the substituents R, and R; can be hydrogen, R represents hydrogen or alkyl and n and m, which may be the same or different, are 0, l, 2 or 3.
• the compounds of the above formula (I) are preferably applied to an absorbent carrier, together with a strong, solid acid.
• the compounds (I) can also be used for the preparation of test film strips according to the procedure described in published Dutch Pat. application No. 67.15 828 and also in a form suitable for the detection of urobilinogens in solutions.
• test paper strips are prepared by impregnating an absorbent carrier, preferably filter paper, with a solution which contains 0.00 [-1 percent of an aldehyde of formula I and at least 3 percent, preferably 15-30 percent, of a strong, solid acid.
• Suitable strong, solid acids include sulfosalicyclic acid, p-toluene-sulfonic acid, potassium bisulfate, glutamic acid hydrochloride and preferably oxalic acid.
• the impregnation solution can also contain inert additives, such as for instance polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of polyvinyl pyrrolidone and polyvinyl acetate, polyglycols and the like.
• inert additives such as for instance polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of polyvinyl pyrrolidone and polyvinyl acetate, polyglycols and the like.
• solvents for use in preparing the solution for use in the impregnation of the absorbent carrier there are suitable all readily volatile solvents in which the aldehydes (I) and also the acids are sufficiently soluble. Consequently, it is preferred to use polar solvents, such as methanol.
• the filter papers are dried, out up and, if desired, sealed on the synthetic resin foils or sealed between synthetic resin foils.
• rodlets or strips are used as absorbent carriers then they are ready for use immediately after drying.
• the new diagnostic agents according to the present invention are dipped into the solution to be investigated and the color change then evaluated after a short period of time, i.e., l to 2 minutes.
• the new diagnostic reagents according to the present invention are substantially more sensitive and are capable of detecting amounts as low as about 0.4 mg.-percent urobilinogen inkett.
• the new diagnostic agents according to the present invention would not be adversely affected either by urine indican or by indole or by sulfonamides (in the case of normal dosages) in urine.
• This finding is particularly surprising because the compounds (I), dissolved in 2-20 percent hydrochloric acid, can be used very satisfactorily for the detection of indole in solution.
• p-dimethylaminobenzaldehyde can, of course, be replaced by compounds of formula I and the content thus determined photometrically in a cuvette in a very exact manner and free of any interference.
• a preferred group of benzaldehyde derivatives according to the present invention are compounds having the formula:
• R is hydrogen or alkyl and nis 0, 1,2 or 3.
• a diagnostic agent for the detection of urooxalic acid 200 g. polyvinyl alcohol 3.0 water 10.0 ml.
• ous concentrated solutions containing x parts of p-N-(N present in body fluids the process being carried out through methyl-piperazino)-benzaldehyde (MPBA) or of pthe use of a test strip, i.e., an absorbent carrier or, a reagent l5 dimethylaminobenzaldehyde (DABA) and parts of oxalic film or in solution in the presence of a strong, solid or liquid acid in 100 parts of methanol.
• MPBA methyl-piperazino)-benzaldehyde
• DABA dimethylaminobenzaldehyde
• the test paper Following drying of the impregnated paper there was according to the present ltlVetlllOl'l, in contradistinction to the known test paper, is substantially insensitive to urea and thus tamed a white test paper which, with normal urine, gave a pale k l r d h t d reacts more sensitively and more specifically with urof 8 an M g giF F con l "59 bilinogen bodies as the pink color reaction with urobilinogen an mg"perccfn f i i ms gave j mg is not masked by the more or less strongly yellow color reacfi 0 T I "T t F or tion with urea.
• p-N-B-chloroethyi-N-methy1aminobenzaldehydo 0.1 Yellow-pink. Purple. p-N-Bhydmxyethyl-N-inethylaminobenzaldehyde 0. 1 do 1 D0. p-N-fi-metlioxyethyl-N-methylsmiiiobenzaldehyde O. 1 .do. Do. p-bis-(B-fluoroethyD-amino-benzaldehyde. 0.1 Pale pink- Blue-red s (B-chloroethyl)-imiino-benza1dehyde. 0. 1 d0. D0.
• Strips offilter paper (Schleicher & Sch'rill No. 2316) were impregnated either with a solution of 0.05 g. p-N-(N-methylpiperazino)-benzaldehyde (MPBA) or of pdimethylaminobenzaldehyde (DABA) and 20 g. oxalic acid in 100 ml. methanol and then dried.
• MPBA p-N-(N-methylpiperazino)-benzaldehyde
• DABA pdimethylaminobenzaldehyde
• EXAMPLE 7 25 ml. urine were acidified with 12 ml. glacial acetic acid and extracted with 40 ml. ether. The extract was washed twice with 20 ml. amounts of water and again made up to 40 ml. with ether. 20 ml. of this extract was mixed with 0.5 ml. of a solution of l g. p-N-morpholino-benzaldehyde in 5 ml. concentrated hydrochloric acid and the resultant mixture shaken vigorously. Thereafter, there were added 6 ml.
• EXMZP 8 Filter paper (Schleicher 81. Sclii'rll No. 23i6) was impregnated with a solution of 0.5 parts pbis-(diethylaminoethyl)-aminobenzaldehyde bis-hydrogen oxalate, 30 parts maleic acid and 0.5 parts polyethylene glycol 1,000 in parts methanol, and then dried.
• Papers with the same analytical properties but which were easier to unroll and cut up were obtained by additionally using 5 parts polyethylene glycol 6,000 or 20,000.
• test paper After the impregnated paper was dried there was obtained a white test paper which, when moistened with urine samples containing urobilinogen bodies, gave a more or less intensive pink-to-violet coloration depending on the urobilinogen concentration. Urine samples having a very low content of urobilinogen bodies and 3 percent aqueous solutions of urea did not give any color change in the test paper. Urine indican also gave no color reaction.
• the test papers can be evaluated for color change after 1-2 minutes.
• EXAMPLE 1 Strips of filter paper (Schleicher & Schiill No. 2316) were impregnated with solutions having various concentrations containing X parts p-(isopropylamino)-benzaldehyde (iPABA), p-(n-propylamino)-benzaldehyde (nPABA) or' p-(dimethylamino)-benzaldehyde (DABA) and parts oxalic acid in 100 parts methanol, according to the procedure described in Example 1.
• the dried test papers were dipped into 3% solutions of urea. The color changes which were thereby observed are set out in the following Table:
• test papers according to the present invention which contained p-(isopropylamino)-benzaldehyde were substantially less sensitive to urea and thus reacted more sensitively and specifically with urobilinogen bodies as the pink color reaction with urobilinogen was not masked by the more or less strong yellow color reaction with urea.
• Imuznk ohydo i-(suwhuLylnmino)-lmnzuldullyrlu .do
• p-(cyelohexylamino)-bcnzaldehydc 0.05 g. colloidal llllClC acid (Aerosil") L00 g. oxalic acid I000 g. polyvinyl butyracetal (“Mowit-al) 0.5 g. polyethylene glycol 6000 0.5 g. methanol 30.0 ml.
• EXAMPLE 14 25 ml. urine were acidified with 12 ml. glacial acetic acid and extracted with 40 ml. ether. The extract was washed twice with 20 ml. amounts of water and again made up to 40 ml. with ether. 20 ml. of the thusly obtained extract were mixed with 0.5 ml. of a solution of l g. p-(cyclopentyl-amino)- benzaldehyde in 5 ml. concentrated hydrochloric acid and the mixture shaken vigorously. Thereafter, there were added 2.5 ml of a semisaturated solution of sodium acetate and 3.5 ml.
• R and R are each members selected from the group consisting of halogen, cyano, nitroso, carbalkoxy, carbamido, hydroxy, acyloxy, alkoxy and amine radicals having at least one aliphatic hydrocarbon substituent and, if there are two aliphatic hydrocarbon substituents they may, together with the amine nitrogen, represent an N- aliphatic heterocycle and wherein one of R and R can represent hydrogen.
• R and R together can represent a member selected from the group consisting of oxygen, sulfur, sulfonyl, alkylthionium, imino, alkylimino, substituted or unsubstituted arylimino, open-chained dialkylimino, cyclic dialkylimino and heterocyclic dialkylimino;
• R-(Cl-l can also represent a member selected from the group consisting of secondary straight-chained alkyl, branched-chained alkyl, monocyclic alkyl, polycyclic alkyl and aralkyl with the proviso that in this case, R, can represent hydrogen only if n is R is a member selected from the group consisting of hydrogen and alkyl; and
• n and m are each one of 0, l, 2 and 3; and a carrier therefor.
• R represents a member selected from the group consisting of hydrogen, halogen, cyano, nitroso, carbalkoxy, carbamido, hydroxy, acyloxy, alkoxy and amine radicals having at least one aliphatic, hydrocarbon substituent and, if there are two aliphatic hydrocarbon substituents they may, together with the amine nitrogen, represent an N-aliphatic heterocycle
• R represents a member selected from the group consisting of secondary straight-chained alkyl, branched-chained alkyl, monocyclic alkyl, polycyclic alkyl and aralkyl
• R is a member selected from the group consisting of hydrogen and alkyl and n is 0, l, 2 and 3, with the proviso that R. is hydrogen only when n is 0.
• said strong .acid is a member selected from the group consisting of sulp-bis-(B-diethylaminoethyll-uminobenzaldehyde 0.2 g. oxalic acid 20.0 g. methanol ad 100.0 ml.
• said carrier is a paper sheet having a wettable surface impregnated with a solution having the following composition:
• said carrier is a paper sheet having a wettable surface impregnated with a solution having the following composition: 0.05 and 0.2 parts of a member selected from the group consisting of p-N (N-methyl-piperazino)-benzaldehyde and pdimethylaminobenzaldehyde and 20 parts of oxalic acid in parts of methanol.
• said carrier is a polyvinylchloride film having applied thereon a layer of the following composition:
• said carrier is a liquid containing p-N-morpholinobenzaldehyde in solution in concentrated hydrochloric acid.
• said carrier is a paper sheet having a wettable surface impregnated with a solution having the following composition: 0.5 parts pbis-(diethylaminoethyl)-aminobenzaldehyde bis-hydrogen oxalate, 30 parts maleic acid and 0.5 parts polyethylene glycol l,00020,000 in l00 parts methanol.
• Diagnostic agent according to claim 1 wherein said carrier is a paper sheet having a wettable surface impregnated with a solution having the following composition:
• colloidal silicic acid (Aero
• Diagnostic agent as claimed in claim 1 wherein said compound has the formula wherein R is a secondary alkyl group which can be straightchained, branched or cyclic and wherein R is a member selected from the group consisting of hydrogen and alkyl.
• a method for carrying out an analytical test for the presence of urobilinogen bodies in biological fluids which comprises applying to the wettable surface of a diagnostic agent according to claim 15 a small amount of the fluid to be tested and examining said paper for visual evidence of the presence of said urobilinogen bodies.
• a method for carrying out an analytical test for the presence of urobilinogen bodies in biological fluids which comprises applying to the coated surface ofa diagnostic agent according to claim 17 a small amount of the fluid to be tested and examining said coating for visual evidence of the presence of said urobilinogen bodies.
• a method for carrying out an analytical test for the presence of urobilinogen bodies in biological fluids which comprises admixing a diagnostic agent according to claim 19 with a small amount of an ether extract of thet'luid to be tested, adding a semisaturated solution of sodium acetate and water and examining the resultant aqueous phase for visual evidence of the presence of urobilinogen bodies.
• Diagnostic agent according to claim 1 wherein said carrier is a paper sheet having a wettable surface impregnated with a solution containing 0.00] to 1 percent of a compound (I) as defined in claim 1 and at least 3 percent ofa strong solid acid.
• Diagnostic agent according to claim 21 additionally containing at least one member selected from the group consisting of polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of polyvinyl pyrrolidone and polyvinyl acetate and polyglycols.

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Cited By (15)

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Citations (2)

• 1968
  • 1968-07-03 DE DE1767931A patent/DE1767931C3/de not_active Expired
  • 1968-10-18 US US768882A patent/US3630680A/en not_active Expired - Lifetime
  • 1968-10-18 CA CA032955A patent/CA935365A/en not_active Expired
  • 1968-10-24 GB GB50552/68A patent/GB1208980A/en not_active Expired
  • 1968-10-24 NL NL686815234A patent/NL148412B/xx not_active IP Right Cessation
  • 1968-10-25 AT AT10445/68A patent/AT290020B/de not_active IP Right Cessation
  • 1968-10-25 FR FR171314A patent/FR1589955A/fr not_active Expired
  • 1968-10-25 BE BE722887D patent/BE722887A/xx unknown

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