# US 5,707,872 A · Nicotine and/or nicotine metabolite detection > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,707,872 A - Document ID: US5707872A - Status: Granted - Kind: A - Application number: 08/474,534 - Filed: 1995-06-07 - Granted: 1998-01-13 - Earliest claimed: 1992-04-02 - Assignees: DynaGen, Inc. - Inventors: Amruta R. Eswara; Neal Muni - Web page: https://patentgrep.com/patent/US5707872A ## CPC - G01N 33/94: involving narcotics {or drugs or pharmaceuticals, neurotransmitters or associated receptors} - A61K 31/439: the ring forming part of a bridged ring system, e.g. quinuclidine (8-azabicyclo [3.2.1] octanes A61K31/46) - A61K 31/44: Non condensed pyridines; Hydrogenated derivatives thereof - A61K 31/4425: Pyridinium derivatives, e.g. pralidoxime, pyridostigmine - A61K 31/445: Non condensed piperidines, e.g. piperocaine - A61K 31/4545: containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine - A61K 31/455: Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides - A61K 31/465: Nicotine; Derivatives thereof - G01N 31/22: using chemical indicators (G01N31/02 takes precedence) - G01N 31/221: {for investigating pH value} - G01N 33/52: Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)} - G01N 33/523: {the element being adapted for a specific analyte} - Y10S 436/816: Alkaloids, amphetamines, and barbiturates - Y10T 436/145555: Hetero-N - Y10T 436/147777: Plural nitrogen in the same ring [e.g., barbituates, creatinine, etc.] - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS - Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION ## Abstract A solid phase capable of detecting the presence of nicotine and/or nicotine metabolites is described. The solid phase is impregnated with assay reagents including a color determinant, a buffer, a cyanogen releasing agent and a cyanogen halide forming agent. The solid phase provides a means whereby unprocessed urine samples over a wide range of different pH values can be tested for the presence of nicotine and/or nicotine metabolites. The assay results may be determined by direct visualization of the color of the solid phase. Also described are methods of preparing and using the solid phase, and kits containing the solid phase. ## Claims ### Claim 1 A method for detecting nicotine metabolites in urine comprising: a) contacting a solid support with urine, said solid support having a porous matrix, and said porous matrix having a receiving section and a wicking section, said receiving section for contacting urine and said wicking section for receiving urine through capillary action from said receiving section to define a flow of fluid, said porous matrix impregnated with reagents, said reagents comprising a color determinant, a buffer, a cyanogen releasing agent and a cyanogen halide forming agent; said buffer, said cyanogen releasing agent and said cyanogen halide forming agent contained in said wicking section; each of said reagents occupying a separate and distinct area of the matrix to prevent interactions between said reagents, said areas being located relative to one another in an order defined by the flow of liquid in the porous matrix in which the first reagent to come in contact with urine is said color determinant and the last reagent to come in contact with urine is said cyanogen halide forming agent; said porous matrix absorbing urine in the receiving section and wicking liquid through the wicking section to bring said cyanogen releasing agent and cyanogen halide forming agent together to form a first reaction product in the presence of said buffer, which buffer maintains said first reaction product within a pH range; said first reaction product reacts with nicotine and nicotine metabolite, if present, to form a second reaction product, said second reaction product is maintained with a pH range by said buffer; said second reaction product reacting with said color determinant to form a third reaction product, which third reaction product is maintained within a ph range by said buffer, and is detectable by a change in color; and b) monitoring said solid support for a change in color indicative of the presence of nicotine and nicotine metabolites. ### Claim 2 (depends on 1) The method of claim 1 wherein said buffer has a buffer capacity to maintain said liquid in said porous matrix at a pH between 5-6 in urine samples having a pH of 4.8-8.2. ### Claim 3 (depends on 1) The method of claim 1 wherein said buffer is selected from the group consisting essentially of citrate buffer, acetate buffer, citrate-phosphate buffer, succinate buffer, aconitate buffer, phosphate buffer and carbonate buffer. ### Claim 4 (depends on 1) The method of claim 1 wherein said buffer is citrate buffer. ### Claim 5 (depends on 1) The method of claim 1 wherein said cyanogen releasing agent is selected from the group consisting of potassium thiocyanate, sodium thiocyanate, potassium cyanide, and sodium cyanide. ### Claim 6 (depends on 1) The method of claim 1 wherein said cyanogen releasing agent is potassium thiocyanate. ### Claim 7 (depends on 1) The method of claim 1 wherein the color determinant is a barbiturate derivative. ### Claim 8 (depends on 1) The method of claim 1 wherein the color determinant is selected from the group consisting of barbituric acid, dimethyl barbiturate, diethyl thiobarbiturate, 5-amino-2-naphthalene sulfonic acid, 8-amino-2-naphthalene sulfonic acid, 4,5-dihydroxy naphthalene-2,7-disulfonic acid, 7-amino-1,3-naphthalene sulfonic acid monopotassium salt, 1,4-phenylene diamine dihydrochloride, 0-tolidine dihydrochloride, sulfanilic acid, sulfanilamide, 4-amino-1-naphthalene sulfonic acid, p-amino benzoic acid and 4-amino salicylic acid. ### Claim 9 (depends on 1) The method of claim 1 wherein the color determinant is diethyl thiobarbiturate. ### Claim 10 (depends on 1) The method of claim 1 wherein the color determinant is methyl barbituric acid or barbituric acid. ### Claim 11 (depends on 1) The method of claim 1 wherein the cyanogen halide forming agent is selected from the group consisting of chloramine-T and chloramine-B. ### Claim 12 (depends on 1) The method of claim 1 wherein the cyanogen halide forming agent is chloramine-T. ### Claim 13 (depends on 1) The method of claim 1 wherein the solid phase is a strip of paper. ### Claim 14 (depends on 1) The method of claim 1 wherein the solid phase is selected from the group consisting of cellulose, fiberglass and tuff glass. ### Claim 15 A solid support, for detecting nicotine and nicotine metabolites in urine, comprising: a porous matrix, said porous matrix having a receiving section and a wicking section, said receiving section for contacting urine and said wicking section for receiving urine through capillary action from said receiving section to define a flow of fluid, said porous matrix impregnated with reagents, said reagents comprising a color determinant, a buffer, a cyanogen releasing agent and a cyanogen halide forming agent; said buffer, said cyanogen releasing agent, and said cyanogen halide forming agent contained in said wicking section, each of said reagents occupying a separate and distinct area of the matrix to prevent interactions between said reagents, said areas being located relative to one another in an order defined by the flow of liquid in the porous matrix in which the first reagent in the flow of liquid is said color determinant and the last reagent in the flow of liquid is said cyanogen halide forming agent; said porous matrix absorbing urine in the receiving section and wicking liquid through the wicking section to bring said cyanogen releasing agent and cyanogen halide forming agent together to form a first reaction product in the presence of said buffer, which buffer maintains said first reaction product within a pH range; said first reaction product reacts with nicotine and nicotine metabolites, if present, to form a second reaction product, said second reaction product maintained within a pH range by said buffer; said second reaction product reacting with said color determination to form a third reaction product, which third reaction product is maintained within a pH range by said buffer, said third reaction product is detectable by a change in color, which change in color is indicative of the presence of nicotine and nicotine metabolites. ### Claim 16 (depends on 15) The solid support of claim 15 wherein said buffer has a buffer capacity to maintain said liquid in said porous matrix at a pH between 5-6 in urine samples having a pH of 4.8-8.2. ### Claim 17 (depends on 15) The solid support of claim 15 wherein said buffer is selected from the group consisting essentially of citrate buffer, acetate buffer, citrate-phosphate buffer, succinate buffer, aconitate buffer, phosphate buffer and carbonate buffer. ### Claim 18 (depends on 15) The solid support of claim 15 wherein said buffer is citrate buffer. ### Claim 19 (depends on 15) The solid support of claim 15 wherein said cyanogen releasing agent is selected from the group consisting of potassium thiocyanate, sodium thiocyanate, potassium cyanide, and sodium cyanide. ### Claim 20 (depends on 15) The solid support of claim 15 wherein said cyanogen releasing agent is potassium thiocyanate. ### Claim 21 (depends on 15) The solid support of claim 15 wherein the color determinant is a barbiturate derivative. ### Claim 22 (depends on 15) The solid support of claim 15 wherein the color determinant is selected from the group consisting of barbituric acid, dimethyl barbiturate, diethyl thiobarbiturate, 5-amino-2-naphthalene sulfonic acid, 8-amino-2-naphthalene sulfonic acid, 4,5-dihydroxy naphthalene-2,7-disulfonic acid, 7-amino-1,3-naphthalene sulfonic acid monopotassium salt, 1,4-phenylene diamine dihydrochloride, 0-tolidine dihydrochloride, sulfanilic acid, sulfanilamide, 4-amino-1-naphthalene sulfonic acid, p-amino benzoic acid and 4-amino salicylic acid. ### Claim 23 (depends on 15) The solid support of claim 15 wherein the color determinant is diethyl thiobarbiturate. ### Claim 24 (depends on 15) The solid support of claim 15 wherein the color determinant is methyl barbituric acid or barbituric acid. ### Claim 25 (depends on 15) The solid support of claim 15 wherein the cyanogen halide forming agent is selected from the group consisting of chloramine-T and chloramine-B. ### Claim 26 (depends on 15) The solid support of claim 15 wherein the cyanogen halide forming agent is chloramine-T. ### Claim 27 (depends on 15) The solid support of claim 15 wherein the porous matrix is paper. ### Claim 28 (depends on 15) The solid support of claim 15 wherein the porous matrix is selected from the group consisting of cellulose, fiberglass and tuff glass. ### Claim 29 (depends on 15) The solid support of claim 15 consisting essentially of the porous matrix, the color determinant, the buffer, the cyanogen releasing agent and the cyanogen halide forming agent. ### Claim 30 A method for detecting nicotine metabolites in urine comprising: a) contacting a cellulose paper solid support with urine, said solid support having a porous matrix, and said porous matrix having a receiving section and a wicking section, said receiving section for contacting urine and said wicking section for receiving liquid through capillary action from said receiving section to define a flow of fluid, said porous matrix impregnated with reagents, said reagents comprising a color determinant, a citrate buffer, a cyanogen releasing agent and a cyanogen halide forming agent said buffer, said cyanogen releasing agent and said cyanogen halide forming agent contained in said wicking section; each of said reagents occupying a separate and distinct area of the matrix to prevent interactions between said reagents, said areas being located relative to one another in an order defined by the flow of liquid in the porous matrix in which the first reagent to come in contact with urine is said color determinant and the last reagent to come in contact with urine said cyanogen halide forming agent; said porous matrix absorbing urine in the receiving section and wicking liquid through the wicking section to bring said cyanogen releasing agent and cyanogen halide forming agent together to form a first reaction product in the presence of said citrate buffer, which citrate buffer maintains said first reaction product within a pH range of between 5-6 in urine samples having a pH of 4.8-8.2; said first reaction product reacts with nicotine and nicotine metabolites, if present, to form a second reaction product, said second reaction product is maintained within said pH range by said citrate buffer; said second reaction product reacting with said color determinant to form a third reaction product, which third reaction product is maintained within said pH range by said citrate buffer and is detectable by a change in color, said color determinant selected from the group consisting of diethyl thiobarbituric acid and barbituric acid, said cyanogen releasing agent selected from the group consisting of potassium thiocyanate and sodium thiocyanate, and said cyanogen halide forming agent selected from the group consisting of chloramine-T and chloramine-B; and b) monitoring said solid support for a change in color indicative of the presence of nicotine and nicotine metabolites.