WO1989009395A1

WO1989009395A1 - Paper and method for detecting marijuana use

Info

Publication number: WO1989009395A1
Application number: PCT/US1989/001304
Authority: WO
Inventors: Conny Dee Johnson; Joseph Fraser
Original assignee: Keystone Diagnostics, Inc.
Priority date: 1988-03-29
Filing date: 1989-03-29
Publication date: 1989-10-05
Also published as: AU3368389A

Abstract

A diagnostic test paper (10) for detecting a marijuana metabolite in a test specimen of a biological fluid is provided. The diagnostic test paper (10) includes a bibulous carrier material (11) impregnated with the dry residue of a reagent reactive to the marijuana metabolite. A particular method is used for screening a biological fluid to determine the presence of marijuana metabolite therein.

Description

"Paper and Method for Detecting Marijuana Use"

1.0 FIELD OF THE INVENTION

This invention relates to a procedure for detecting the presence of the metabolic products of Marijuana in biological fluids. More particularly, the invention is directed to a procedure comprising a simplified screening method which provides rapid qualitative results for use in determining the need for additional or confirmatory testing.

2.0 BACKGROUND OF THE INVENTION

There are several well known qualitative laboratory procedures for identifying drugs of abuse such as marijuana. These include Thin-Layered Chromatography (TLC) , the Enzyme Multiplied Immunoassay Technique (EMIT) , Radioimmunoassay (RIA) and Gas-Liquid Chromatography (GLC) . Each of these well known procedures require skilled technicians and relatively sophisticated equipment. Consequently, the testing procedure is necessarily expensive.

With the ever increasing rise in the use of abuse-type drugs, particularly, marijuana, the need for detecting and identifying those drugs and their metabolites is becoming more important. With this need, many more tests are required to monitor the use of abuse-type drugs.

Thin layer chromatography screening procedures for detecting drugs in urine require the careful preparation of a test specimen and then a skillful application of that test specimen to a plate placed into a developing chamber. Once the plate is removed from the chamber and dried, it is sprayed with visualization reagents. Location and color of spots are compared with those of known standards. Qualitative judgments are made as to the presence of various drugs in the unknown sample. The procedure is tedious, time consuming and requires skilled personnel to interpret the results.

The EMIT procedure is a semi-quantitative immunoassay for drugs of abuse in biological fluids. The laboratory test requires trained technicians to perform and the equipment necessarily costs several thousands of dollars. The RIA procedure is a sensitive and quantitative laboratory procedure for detecting drugs of abuse. The various immunochemicals are labeled with radioactive compounds and require special eare in their use and disposal. A license is required from the government to use this laboratory procedure because of the presence of radioactive materials. The GLC procedure can provide the highest degree of accuracy in drug analysis. However, the necessary equipment is expensive and the procedure is complicated. Consequently, highly trained personnel are required for its use.

Methods have also been disclosed for the detection of the presence of cannabinoids in biological materials. U.S. patent 3,656,906 issued April 18, 1972 describes a detection system based upon the condensation of the cannabinoids with a polycarboxylic acid to give a highly fluorescent derivative. This derivative which fluoresces when detected, is indicative of the presence of the cannabinoids. Cannabinoids have also been detected by a chemical reagent dissolved in an organic solvent or as a stable liquid reagent essentially comprising as the active detecting agent a diazonium salt. Under basic conditions the presence of the cannabinoid can be detected by color change in the solution. (See, European Patent Application Publication No. 132,313 published January 30, 1985 to B. Spiro) . The visualization reagents used in thin-layer chromatography are well known and their use is explained in U.S. Patent No. 3,590,006. A particular method of handling the chemical visualization reagents for the TLC procedure is found in the U.S. Patent No. 3,912,655. Here dry strips of paper impregnated with fixed amounts of the visualization reagent are prepared as a storage medium for the reagent to be used in the thin-layer chromatography procedures. Both of these disclosures are directed to producing a drug abuse test paper designed to replace the thin-layer chromatography procedure. Thus, both are designed to produce both a qualitative and quantitative analysis of abuse-type drugs in a biological fluid.

The U.S. Patent No. 3,955,926 discloses a test process suitable for identifying narcotics in solid form. A sample of the material which is suspected to be a narcotic is subjected to a solvent for that particular narcotic. An absorbent, impregnated paper containing at least one component of a color reagent in dry form is then subjected to the solvent. This particular prior art test is not applicable to the detection of the presence of an abuse-type drug in a biological fluid such as urine.

A drug abuse test indicator device and a method of making same is disclosed in the international publication WO 84/02397. Here the matrix of a bibulous material is impregnated with a dry staining agent in precise amounts which are prepared in accordance with unique procedures whereby pH insensitivity and unique color change sensitivities to test fluids are obtained. This disclosure is said to be an improvement over the procedure disclosed in U.S. Patent No. 3,915,639 which discloses the use of coatings on a carrier body requiring the use of an ion exchange resin, a staining agent and a color intensifier- stabilizer material. Each of these earlier disclosures make use of the visualization reagents commonly used in the thin-layer chromatography procedures. Both of these disclosures are directed to producing a drug abuse test paper designed to replace the thin-layer chromatography procedure. Thus, both are designed to produce both a qualitative and quantitative analysis of abuse-type drugs in a biological fluid.

Major difficulties exist in the production of the drug abuse test papers designed to replace the thin-layer chromatography procedure. These difficulties are associated with the necessity to use extremely controlled environments in the production of the papers. Thus, large production requirements for such papers while achieving commercial consistency has been a- major problem. However, these papers represent the first successful attempts to detect the presence ©f abuse-type drugs by having visualization reagents present in a carrier matrix material change color within that matrix upon the direct application of a test specimen.

In view of the difficulties associated with known procedures and drug abuse test papers, the diagnostic test paper of the present invention has been developed for use as an initial indicator test for the presence of marijuana or its metabolic product, the cannabinoids. The present system developed in accordance with this invention is not intended for monitoring therapeutic levels nor as a basis for any therapeutic treatment based on test results. The invention of this disclosure is designed as a simplified screening method providing rapid qualitative results for use in determining the need for additional or confirmatory testing. The basic idea ^"is to screen the numerous samples of biological fluids collected at collection centers for drugs of abuse and their screening programs and determine which ones are a positive. Only those which show a positive result are then sent to the laboratory for further testing. The diagnostic test procedure and its equipment is readily understood by persons who are unskilled in chemical laboratory procedures. That is, the primary users of the test will be the staff of the various collection centers such as clinics specifically set up for screening programs, methadone treatment centers and the like.

The procedure of the present invention is a qualitative test based on color producing visualization reagents used in thin-layer and paper chromatography. In general, TLC visualization reagents are used to react with materials of interest to produce an area that is visually distinct from the background due to unreacted reagent. The invention of this system does not attempt the separation and identification of individual components. Rather, the system is a method of screening biological fluids to determine the aggregate presence qf marijuana's primary metabolic products which react with the visualization reagent impregnated in the test paper. Through the use of unskilled personnel, very inexpensive equipment and the rapid determination of negative results, the screening procedure of the present invention provides an extremely important advance in the drug abuse detection technology. That is, thousands of

*\. tests will no longer have to be conducted using the more sophisticated or expensive procedures.

Another commercial product, the Emit st Urine Cannabinoid Assay, employs a homogeneous enzyme immunoassay to detect the major urinary metabolite of THC, ll-nor-9- carboxy-delta-9-tetrahydrocannabinol, a compound having the following structure:

H,C QΪHII

Each vial contains antibodies, the coenzyme (NAD) , enzyme- labeled drug, enzyme substrate and buffer. The endpoint for the reaction is determined by a photometric measurement of NADH. The absorbance change in the sample mixture is compared to that in a calibrator mixture containing 100 ng/ml of the metabolite. Any sample having an absorbance change greater than that of the calibrator is considered to be positive.

There are several commercially available tests to determine the presence of THC metabolites in urine. These tests cover a wide range of sensitivities, i.e. 10 ng/ml and 100 ng/ml for ^ll*Cannabuse*^,f and Emit st Urine Cannabinoid Assay, respectively. Each test is properly labeled as to its sensitivity.

3.0 SUMMARY OF THE INVENTION

The invention as disclosed and described herein includes a diagnostic test paper, a method of making the test paper, a method of using the test paper, a syringe assembly and a particular solvent composition useful in preparing a test specimen of a biological fluid.

The diagnostic test paper comprises a bibulous carrier material impregnated with a dry residue of a visualization reagent reactive to the primary metabolite [ll^«-nor-9-carboxy=delta-9-tetrahydrocannabinol] of marijuana's active ingredient THC [otherwise known as (-) (-delta-9-trans-tetrahydrocannabinol] . This metabolite is hereinafter also referred to as the "THC metabolite''. The carrier material includes a concentrated color producing visualization reagent. The visualization reagent is present in an amount sufficient to change color in the carrier material when contacted by cannabinoids introduced into the carrier material via a test specimen. A specific embodiment of the diagnostic test paper includes the use of a solution of a diazonium salt. The reagent may be impregnated into the paper by dipping a bibulous carrier material therein or applied to the carrier material in a drop-wise fashion. Either type of reagent paper will function in the system of this invention with differing degrees of sensitivity. In the specific example of the present procedure, the test paper is used to perform the method of screening urine specimens for the presence of the THC metabolite. That is, the visualization reagent in the specific embodiment of this invention is reactive to the THC metabolite. The diazonium salts used herein such as "Fast Blue B", "Fast Blue BB" and "Fast Corinth^/ are commercially available. Once the visualization reagent impregnates and dries on the carrier material, it is then ready for the test procedure involving the preparation of a test specimen and its application directly to the carrier material.

In a specific embodiment of the invention, the test paper is impregnated with a diazonium salt to form a yellow color on the surface of a bibulous carrier material, such as a paper strip. The matrix of the paper strip has the capacity to hold a liquid test specimen for a time sufficient to react any substance contained therein which might be reactive with the diazonium reagent. The bibulous material is dried after the diazonium reagent is placed onto the sheet.

The cleanup procedure according to the present invention involves the use of a syringe assembly comprising a syringe canister including a plunger element with packing material disposed within the canister. The plunger element is slidably disposed to successively pull into and discharge out materials being subjected to the chemically adsorbent material constituting the packing. The purpose of this cleanup procedure is to eliminate interfering or extraneous materials which may cause a false positive. The specific embodiment of this invention includes use of a reverse phase liquid chromatography material. The urine specimen is successively pulled into contact with the packing column and discharged out the same inlet. A wash solution is then drawn into the syringe to eliminate the interfering substances. The solvent solution in the present case is acetone which elutes any THC metabolite bound to the packing. The organic solvent with the THC metabolite dissolved therein is then applied directly to the test specimen applying area of the diagnostic test paper. After a brief drying period, an aqueous buffer solution is then applied to the same area of the test paper as the test specimen and any THC metabolite will cause the formation of a pink to red color ring.

4.0 BRIEF DESCRIPTION OF THE DRAWINGS

Other objects of this invention will appear in the following description and claims, reference being made to the accompanying drawings forming a part of the specification wherein like reference characters designate corresponding parts in the several views.

FIGURE 1 is a view of a diagnostic test paper mad in accordance with this invention;

FIGURE 2 is a test paper as shown in Figure l after a urine specimen has been applied thereon showing a negative result;

FIGURE 3 is a top view of a test paper as shown i Figure 1 after a test specimen having a positive result showing presence of the THC metabolite; and

FIGURE 4 shows a cross-sectional view of the syringe and cartridge combination embodiment utilized for clarifying the biological test specimen. FIGURE 5 shows a cross-sectional view of the syringe with the packed column utilized for clarifying the biological test specimen.

5.0 DETAILED DESCRIPTION

The Marijuana Screen of this invention is intended as a rapid method of screening urine specimens for the presence of ll-nor-9-carboxy-delta-9-tetrahydrocannibinol, the principal metabolite of tetrahydrocannabinol. It is not intended for use as a confirmatory test for the presence of ll-nor-9-carboxy-delta-9-tetrahydrocannabinol. It is not intended for monitoring therapeutic levels nor as- a basis for any therapeutic treatment based on the test results. It is designed as a simple screening method which provides rapid, qualitative results for use in determining the need for additional or confirmatory testing. It is expected that the primary users of the test will be staff associated with collection centers for drug abuse screening programs, clinical laboratories and correctional institutions.

The procedure is a qualitative test based on color-producing visualization reagents used in thin-layer and paper chromatography (TLC) . In general, TLC visualization reagents are used to react with materials of. interest to produce an area that is visually distinct from the background.

The large number of potentially interfering substances in urine precludes the direct interaction of the detection reagents with raw urine. Generally stated, the Test screen system of this invention combines two functions: the sample is treated to isolate and concentrate the substances of interest prior to detection (the isolation procedure is similar to common laboratory sample clean-up methods) . The biological fluid sample, preferably urine, is passed through a short column which cause the drugs and other materials to be retained by adsorbent. The column is washed to remove additional extraneous materials and the THC metabolite is removed with a small volume of a suitable solvent. Several commercial products are currently marketed for this purpose, Tox Elut extraction column for urine drug abuse screening by TLC; Baker-10 SPE columns for extraction from urines prior to HPLC analysis; EXTRELUT QE columns for sample preparation in urine drug abuse screening by TLC. (Tox-Elut is a product of Analytichem International. Baker- 10 SPE is a product of J.T. Baker Chemicals. EXTRELUT QE is a product of EM Services.)

The isolated drug substances, in a concentrated form, are now available for analysis. Chromatographic methods would now involve additional separation procedures and the use of a detection system to allow identification of individual compounds. A variety of diazonium salts may be useful as visualization reagents for the detection of THC metabolites. The action of the indicator is to form a colored compound on the paper upon reaction with urinary materials preferably isolated by the clean-up procedure.

In the preferred embodiment for detection of marijuana the rinse solution is preferably a phosphate buffer and slightly acidic to neutral solution (a pH of about 6.0 to 7.0) which is also preferably mixed with an . aqueous alcohol solution.

Generally stated, this invention also comprises a a method of screening a biological fluid to determine the presence of a marijuana metabolite therein, said method comprising the steps of: a) providing a bibulous carrier material including a color producing visualization reagent having a first color said visualization reagent being present in an amount sufficient to change color in the carrier material when contacted by a marijuana metabolite introduced into said carrier material via a biological fluid test specimen; b) providing an amount of a biological fluid test specimen to be screened to a chemically absorbent material to absorb a marijuana metabolite from said biological fluid test specimen; c) treating said chemically absorbent material to produce an elution solution, derived from said biological fluid test specimen, which may contain said marijuana metabolite separate from any extraneous material which may have been present in said biological fluid test specimen and which extraneous material was also absorbed by said chemically absorbent material; d) applying said elution solution directly to the bibulous carrier material to cause any of said marijuana metabolite present in said elution solution to react with said visualization reagent thereby changing the first color of the reagent to a second color when a marijuana metabolite is present in said biological fluid test specimen. Preferably, in the practice of this method the biological fluid test specimen is urine and said chemically adsorbent material is a C-18 reverse phase liquid chromatographic packing; said visualization reagent in said bibulous carrier material comprises a diazonium salt producing a first color; and said rinse solution comprises a buffered solution having a pH in the range of from about 6.0 to about 7.0 and which contains an alcohol.

In carrying out a test for marijuana's THC metabolite, a urine specimen is drawn into either a packed syringe column (FIG. 4) or packed cartridge/syringe combination (FIG. 5) thereby binding urinary components, including marijuana metabolites. Extraneous materials, such as phenolic-type material, which may interfere with obtaining reliable results with the visualization reagent, are eliminated by a phosphate buffer, alcohol-water wash with the cartridge or separate washes by first a phosphase buffer solution and second an alcohol-water wash with the packed syringe. After the packed cartridge/syringe is washed with the phosphate buffer, alcohol-water wash, the marijuana metabolites are then eluted with acetone and applied directly to the diazonium sal *=impregnated paper. After the packed syringe is washed separately by the phosphate buffer and alcohol water washes the THC metabolites are then eluted with acetone, are applied directly to the diazonium salt impregnated paper 11 (FIG. 1) , and then are washed on the paper with an application of an additional color forming solution, preferably, the phosphate buffer. A pink to red color in both procedures indicates the presence of THC. (The test will detect 100 ng/ml consistently) .

In carrying out these embodiments of the invention, the rinse solutions are drawn into and discharged from the packing material to satisfactorily eliminate any of the interfering and extraneous materials retained on the column. At the same time, the rinse solution leaves any THC metabolites which may have been present in the urine retained by the column.

After pulling in the acetone solvent solution, the test specimen is slowly discharged out of the syringe until completely dispensed with the syringe tip being above or touched to the test paper. In the two-rinse step procedure using the packed syringe column, the solvent is then allowed to evaporate with the color forming agent being applied after evaporation is completed. This may take up to two minutes.

If the test specimen is negative (no marijuana metabolite present) , on the test paper 12 (FIG. 2) there may be a very faint and diffuse purple background present 14 (FIG. 2) along with a yellow/green ring 13 (FIG. 2) . However, on the test paper 15 (FIG. 3) if the THC metabolite is present, a distinct pink-red color ring will form 19 (FIG. 3) surrounding a pink to red shaded area 16 (FIG. 3) . The embodiment of Figure 3 shows a specimen positive for the presence of a marijuana metabolite. Samples yielding a positive result by the method of the present invention should always be tested using a different technique for confirmation. Any ambiguous or uncertain results should also be sent out for further testing with a different technique such as those discussed hereinabove. The purpose of the present invention is simply to eliminate a host of tests which normally would have to be conducted using the extremely expensive laboratory procedures which are already available and well known as discussed hereinabove. A positive test is indicative of the presence of a THC metabolite in a person from whom the biological fluid sample was taken.

For appropriate operation of the procedure of this invention, a minimum of 5 to 50 milliliters, and preferably 15 milliliters, of urine should be used with the cartridge embodiment and about .5 to 25, and preferably, 5 milliliters should be used for the packed syringe column embodiment. Fresh urine specimens or those which have been refrigerated up to two weeks may be used. Specimens remaining refrigerated for longer than a two week period or unrefrigerated may cause unreliable results such as a possible false positive reactions due to decomposition of components in the urine.

It has been unexpectedly found that the procedure according to this invention is substantially equivalent to currently marketed thin-layer and immunochemical assay products. The consistency of the present screening method falls in the range of 90 to 94% accuracy when compared to tests carried out by laboratory procedures such as thin- layer chromatography.

5.1 BIBULOUS CARRIER MATERIAL

The diagnostic test paper 10 (FIG. 1) of this invention is preferably a bibulous carrier material used which, is preferably impregnated with a dry residue of visualization reagent. Bibulous carrier materials which have been used successfully include Whatman No. 3 paper, Whatman 311T, ED-222 (Eaton-=Dikeman) , im*=Wipe, Schleicher & Schuell filter and electrophoresis paper products such as SS-598, 470, 2043a and 593, glass fiber and non-woven fabric such as bonded polyester or bonded nylon. The preferred carrier material used throughout the various embodiments of this invention is Whatman 31ET.

5»2 VISUALIZATION REAGENT MATERIAL

The visualization reagent material used in this invention for the detection of the THC metabolite includes diazonium salts. These materials are commercially available from various suppliers such as Aldrich Chemical Co. and Sigma Chemical Co. These varieties useful in this process are known by the following trade names and have the accompanying chemical structures:

Fast Blue BB Salt

Fast Blue B. Salt

Fast Corinth V Salt

In preferred embodiment the diazonium salt is used to fully impregnate the bibulous carrier (test paper) material. Once this visualization solution has been allowed to dry the test paper material is ready for use. Accurate results are also obtainable without the need to prepare a test specimen applying area although the presence of a test specimen applying area is an acceptable embodiment of this invention. The elution solution derived from the urine sample is applied after the cleanup procedure directly to test paper.

In a preferred visualization agent impregnation solution the following components and range of amounts may be utilized:

Range Optimal

Fast Blue B 50mg - 300mg lOO g

Sodium Sulfate 0 - 2g ig

Zinc Chloride 0 - 1.2g l.Og

HBF₄ 20mg - 50mg 27mg

Water 20ml - 30ml 20ml

Methanol 0 - 10ml 10ml

Once the above components are mixed the solution is then utilized to impregnate the test paper. 5.3 PURIFICATION OF URINE SAMPLE

Potentially interfering and extraneous materials were first removed from the urine specimen according to the cleanup procedure of the invention to prepare the test specimen applied to the diagnostic test paper which initially looked like the test paper 10 as shown in FIGURE 1.

This isolation procedure preferably utilizes a standard syringe 20 (FIG. 4) having a syringe canister 21 with a slidably disposed plunger element 22 therein which is used to draw material into said canister. The material would be forced from said syringe canister 21 so as to have it contact a chemically absorbent material or packing material 23. The packing material is preferably separately disposed within a cartridge 24. (i.e. "Sep-Pak", commercially available from the Millipore company) . A reverse phase liquid chromatographic packing is preferably used as the packing material in the clarification of a biological fluid test specimen when testing for the presence of the THC metabolite.

The isolation procedure, in another embodiment of the present invention (FIG. 5), provides a short column within the syringe canister 21 containing a non-polar adsorbent packing (chemically absorbent) material 25. The THC metabolite and other interfering materials are to be retained by this chemically adsorbent material contained within the syringe canister. The plunger 22 is operated to successively pull into and out of the column a measured amount of the biological fluid; namely, urine. The column chemically adsorbent packing material 23 or 25 may be selected f om any one of the group of extraction columns for urine drug abuse screening procedures. This includes a Tox Elut extraction column which is a product of Analytichem International, Baker-10 SPE, a product of J.T. Baker Chemicals and Extrelut QE, a product of EM Sciences and C-18 or C-8 reverse phase chromatographic material (C-18 or C-8 is preferred for use in the marijuana detection system). Such well known columns are normally used in ⁵expensive vacuum equipment which operates to pass the materials through the column in one direction only. It was found to be totally unexpected that such a procedure could even produce the kind of excellent chromatographic results as have been achieved in accordance with the present 10 invention. The various components including the interfering and extraneous materials along with any THC metabolite present in the urine is retained by the absorption column.

5.4 TYPICAL TEST PROCEDURE WITH A CARTRIDGE

15 SYRINGE COLUMN

Generally stated, a preferred embodiment of this invention comprises a qualitative test which uses a diazonium salt for detecting marijuana metabolites in urine.

__πIt contains a specially prepared diazonium salt-impregnated paper which is ready for use without additional preparation.

A urine specimen is drawn into a syringe-column

(FIG. 4). The urine specimen is forced through a packed cartridge which is subsequently attached to the syringe and _ ___b_which packed cartridge contains a packing material ("Sep-

Pak" Cartridge commercially available from Millipore Corp.). Urinary components, including marijuana metabolites, are bound to the special packing. Many interferences or extraneous materials are eliminated by a buffer wash. Compounds of interest are eluted with an organic solvent and applied directly to the test paper. A reaction occurs between the diazonium reagent in the paper and any marijuana metabolite constituents in the eluant to produce a pink to red color. 5 The components necessary for this test to be carried out are preferably:

1. Diazonium salt-impregnated paper

2. 2 plastic syringes 3. Buffered rise solution (pH 7.0)

4. A packed cartridge

5. Elution solution

A minimum 15 ml of urine is required for this test. Preferably, this test uses fresh urine specimens or those that have not been refrigerated for more than two weeks. Unrefrigerated specimens δr those refrigerated for longer than two weeks may cause unreliable results (e.g., false positives due to urine decomposition or false negatives due to degradation of the metabolite) .

A typical test would preferably utilize the following components and procedures as listed in TABLE 1:

TABLE 1

Component Preferred

solid phase packing ——***^• •— ^•- C-18

rinse solution -_—-__«_-—~———»-«—.=—«=— pH 7.0 Phosphate

Buffer

1. 80ml Phosphate/Tris Buffer pH 7.0 5ml Water

5ml methanol

2. Phosphate/Tris Buffer, pH 7.0 (no methanol)

3. Phosphate Buffer, pH 6.8

4. Phosphate Buffer, pH 6.8 with 0-25% methanol 5. 1% acetic acid followed by Phosphate

Buffer, pH 6.8/methanol

6. Phosphate Buffer, pH 6.8 followed by

1:1 water: methanol 7. Acetonitrile/Phosphoric acid (50mM) in proportions 85:15 v/v

8. Ammonium acetate (0.05%) in water

9. Phosphate/Tris Buffer followed by various other liquids, i.e. ethanol, hexane, acetonitrile, dichloromethane and dimethylsulfoxide. reagent paper —.-— impregnated, Fast

Blue B. elution solution acetone water wash not needed urine volume 5 - 25ml

5.4.1 PREPARATION OF THE PACKED CARTRIDGE

The packed cartridge syringe combination is preferably prepared for receipt of a sample as follows: a. Eleven to fifteen ml of rinse solution and 0.3 to 0.5 ml elution solution are needed for each test. The beakers are labeled Elution Solution, and Rinse. b. Use only enough solution for current testing. To avoid contamination, do not pour the solutions back into the bottles. c. Attach the short end of the cartridge to the 15 ml syringe column. d. Place the tip of the cartridge into the Rinse solution and pull the plunger up to draw the solution to the indicated fill line. Keep the tip of the cartridge well below the liquid level to keep from drawing air into the syringe.

Depress the plunger, expelling the liquid into the waste container. Remove cartridge from syringe.

5.4.2 SAMPLE TREATMENT

The packed cartridge syringe combination preferably receives a sample as follows: a. Record the specimen number or code. b. Place the tip of the 15 ml syringe into the urine and pull the plunger up to draw the specimen to the indicated fill line. c. Attach the short end of the cartridge to the syringe column. d. Discharge the urine into the waste container by pushing down on the plunger. e. Remove cartridge from syringe column. f. Draw the fluid from the Rinse, solution beaker into the barrel of the syringe- column to the indicated fill line. Attach short end of cartridge to syringe column. g. Discharge the liquid into the waste container by pushing down on the plunger. h. Slowly draw up approximately 0.45 ml of the Elution Solution into the 1 ml syringe-column. Attach long end of _Q cartridge to syringe column. i. In a dropwise fashion, place the Elution Solution onto the reagent paper. Allow each drop to soak into paper before adding

5 another drop so as to restrict the area covered by the eluted solvent to a minimum.

5.4.3 COMPLETING AND INTERPRETING THE TEST

In order to determine whether a test result is positive or negative, the test area that was wet with elution solution is compared with the surrounding area. If marijuana metabolites are present, an intense pink or red ring will appear within 30 seconds after the application of the elution solution. The test result is negative if no intense pink or red ring appears within 30 seconds after the application of the elution solution.

Essentially, the Marijuana test of this invention is a qualitative test. Results are based on visual observation of changes in the test paper. The test paper should be checked for the appearance of a pink-red ring after the application of the elution solvent.

In sum, the presence of an intense pink-red ring is considered a positive test. The absence of this pink-red ring is considered a negative test.

Materials which do not interfere with this Marijuana test include: opiates, amphetamines, PCP, cocaine or ibuprophen. Although unlikely, Benzoidazepines may give positive results.

This test has been shown to detect 100 ng/ml of the ll-nor-9-carboxy-delta-9-tetrahydro-cannabinol (the primary metabolite of THC) in urine.

5.5 TYPICAL TEST PROCEDURE WITH PACKED SYRINGE COLUMN

In another preferred embodiment, a urine specimen is drawn into a packed syringe-column (FIG. 5). Urinary components, including marijuana metabolites, are bound to the special column packing. Many interferences or extraneous materials are eliminated by acidic buffer and alcohol-water washes. Compounds of interest are eluted with an organic solvent and applied directly to the test paper. A reaction occurs between the diazonium reagent in the paper and constituents in the eluant to produce a pink to red color after addition of the buffer solution.

The components necessary for this test to be carried out are typically:

1. Diazonium salt-impregnated paper 2- Plastic syringe-column containing C18 packing material

3. Buffered rise solution (pH 6.8)

4. Aqueous alcohol rinse solution

5. Elution solution

About 6.0 ml of urine is required for this test, preferably, this test uses fresh urine specimens or those that have not been refrigerated for more than two weeks. Unrefrigerated specimens or those refrigerated for longer than two weeks may cause unreliable results and possibly false positive reactions due to decomposition of the urine.

A typical test would preferably utilize the following components (as listed in TABLE 2) and procedures:

TABLE 2

Component Preferred

solid phase packing C-18

rinse solution pH 7.0 Phosphate water/methanol

Buffer

1. 80ml Phosphate/Tris Buffer pH 7.0 5ml Water

5ml methanol

2. Phosphate/Tris Buffer, pH 7.0 (no methanol)

3. Phosphate Buffer, pH 6.8

4. Phosphate Buffer, pH 6.8 with 0-25% methanol

5. 1% acetic acid followed by Phosphate

Buffer, pH 6.8/methanol

6. Phosphate Buffer, pH 6.8 followed by •

1:1 water: methanol

7. Acetonitrile/Phosphoric acid (50mM) in proportions 85:15 v/v

8. Ammonium acetate (0.05%) in water

9. Phosphate/Tris Buffer followed by various other liquids, i.e. ethanol, hexane, acetonitrile, dichloromethane and dimethylsulfoxide. reagent paper impregnated, Fast

Blue B. elution solution ■=— —-—-—- acetone water wash *^• not needed urine volume ^■ 5 - 25ml

5.5.1 PREPARATION OF THE PACKED SYRINGE- COLUMN The packed syringe is preferably prepared for receipt of a sample as follows: a. 0.5 to four ml of solution is needed for each test. The beakers are labeled

Elution Solution, Acidic Rinse, and

Alcohol-Water Rinse. b. Use only enough solution for current testing. To avoid contamination, do not pour the solutions back into the bottles. c. Place the tip of the syringe-column into the Acidic Rinse solution and pull the plunger up to draw the solution to the indicated fill line. Keep the tip of the syringe-column well below the liquid level to keep from drawing air into the syringe. Depress the plunger, expelling the liquid into the waste container.

5.5.2 SAMPLE TREATMENT

The packed syringe preferably receives a sample as follows: a. Record the specimen number or code. b. Place the tip of the syringe-column into the urine and pull the plunger up to draw the specimen to the indicated fill line. c. Discharge the urine into the waste container by pushing down on the plunger. d. Repeat steps b and c two more times. By this process, a total of 5.4 ml of urine will have been passed over the adsorbent material. e. Draw the fluid from the Acidic Rinse solution beaker into the barrel of the syringe-column to the indicated fill line in the same manner as in step b.

10 f. Discharge the liquid into the waste container by pushing down on the plunger. g.. Place the top of the syringe-column into the Alcohol-Water Solution. Draw the fluid into the barrel to the indicated

15 fill line. h. Discharge the liquid into the waste container by removing the plunger and decanting the liquid into the waste container. Replace plunger in the barrel

20 of the syringe-column. i. Slowly draw up approximately 0.5 ml of the Elution Solution into the syringe-column. j. Slowly depress the plunger, discarding the first 2 or 3 drops from the column.

25 k. In a dropwise fashion, place the remainder of the Elution Solution onto the reagent paper. Allow each drop to evaporate before adding another drop so as to restrict the area covered by the eluant to

30 a minimum. Allow the eluant to dry completely.

35 5.5.3 COMPLETING AND INTERPRETING THE TEST

After the Elution Solution has dried, apply 2 small drops of acid-rinse solution using a pipette. In order to determine whether a test is positive or negative, the test area that was wet with the two drops of the acid- rinse solution is compared with the surrounding area. If marijuana metabolites are present, an intense pink or red ring will appear within 30 seconds after the application of the Acid-Rinse Solution. The test result is negative if no intense pink or narrow red ring appears within 30 seconds after the application of the Acid-Rinse Solution. Any color which develops after 30 seconds should be ignored.

Essentially, the Marijuana test of this invention is a qualitative test. Results are based on visual observation of changes in the test paper. The test paper should be checked or the^' appearance of a pink-red ring after the application of the 2 drops of Acid-Rinse Solution to the area covered by the dried Elution Solution.

The presence of an intense pink-red ring is considered a positive test. The absence of this pink-red ring is considered a negative test.

Materials which do not interfere with this Marijuana test include: opiates, amphetamines, PCP, cocaine or ibuprophen. Benzodiazepines may give positive results.

This test has been shown to detect 100 ng/ml of the ll-nor~9=-carboxy-delta=9-tetrahydro-»cannabinol (the primary metabolite of THC) in urine.

The following examples according to the various embodiments of this invention are given hereafter. These examples are meant to be illustrative and not to be considered as limiting the scope of the invention. 6.0 EXAMPLES

6.1 EXAMPLE 1

The purpose of this example was to determine the effectiveness of the Marijuana screen as a detection system for THC metabolite. The results were based on the ability to detect a minimum of 100 ng/ml of THC metabolite in urine.

Two hundred twenty six urine specimens were utilized in this study. Certain of these specimens were known to contain ll-nor-9-carboxy-delta-9- tetrahydrocannabinol (also known as.Carboxy-THC) . Also interspersed with these specimens were urine samples known not to contain carboxy-THC. The 226 specimens were obtained from American Medical Laboratories and other sources. Analysts were unaware of the carboxy-THC content of each vial containing a sample.

All Two hundred twenty-six urines were screened for ll-nor-9-carboxy-delta-9-tetrahydrocannabinol according to the procedure as described in section 5.5 above. All samples found to be positive were sent to American Medical Laboratories for quantitation and confirmation. Metabolite levels were determined by Gas-Liquid Chromatography. Confirmation was accomplished by Gas chromatography and Mass Spectrophotometry.

Of the 226 urine specimens, 71 were shown not to contain carboxy-THC. The marijuana test correctly identified 65 of these as negative (91.5%).

6.2 EXAMPLE 2

An experiment was also run to determine whether or not any other drug or composition might create interference with the test of this invention. This experiment was carried out according to the procedure set forth in Section 5.5 above, however, in order to determine possible interference with the test paper described herein, urine specimens were selected in which potentially interfering compounds were know to be present.

Interferences from non-cannabinoid drugs have been tabulated in Table 3:

TABLE 3

POTENTIAL INTERFERING SUBSTANCES

SUBSTANCE(S) PRESENT TEST RESPONSE

Opiates N

Amphetamines N

Cocaine N

Opiates & Cocaine N

Benzodiazepines P

Cocaine N

Amphetamines N

Opiates N

Amphetamines N

Cocaine N

Benzodiazepines P

Opiates N

Amphetamines ϋ _'

PCP N CP N

PCP & Cocaine N

Benzodiazepines N

Ibuprophen N

It can, therefore, be concluded from this example that the vast majority of drugs of abuse have no potential for interference with the drug abuse test paper and methods as set forth herein. While the drug abuse test paper and methods for detecting a metabolite of marijuana has been shown and described in detail, it is obvious that this invention is not to be considered as limited to the exact form disclosed, and that changes in detail and construction of the test paper and the test procedure may be made therein within the scope of the invention without departing from the spirit thereof.

Claims

CLAIMS Having thus set forth and disclosed the nature of this invention, what is claimed is:

1. A diagnostic test paper for detecting a metabolite of marijuana in a test specimen of a biological fluid, said test paper comprising: a) a bibulous carrier material impregnated with the dry residue of a visualization reagent reactive to said metabolite, ' b) the visualization reagent being present in an amount sufficient to change color in the carrier material when contacted by said metabolite introduced into said carrier material via a said test specimen.

2. A test paper as defined in Claim 1 wherein the visualization reagent comprises a diazonium salt.

3. A test paper as defined in Claim 1 wherein the bibulous carrier material has a matrix having the capacity to hold a liquid test specimen for a time sufficient to react any substance contained therein which might be reactive with the visualization reagent.

4. A test paper as defined in Claim 8 wherein the reactive reagent comprises a diazonium salt.

5.. A test paper as defined in Claim 8 wherein the bibulous carrier material has a matrix having the capacity to hold a liquid test specimen for a time sufficient to react any substance contained therein which might be reactive with the reactive reagent.

6. A test paper as defined in Claim 8 wherein the reactive reagent comprises a diazonium salt.

7. A test paper as defined in Claim 8 wherein the reactive reagent is a color producing visualization reagent.

8. A method of screening a biological fluid to determine the presence of a marijuana metabolite therein, said method comprising the steps of: a) providing a bibulous carrier material including a color producing visualization reagent having a first color said visualization reagent being present in an amount sufficient to change color in the carrier material when contacted by a marijuana metabolite introduced into said carrier material via a biological fluid test specimen; b) providing an amount of a biological fluid test specimen to be screened to a chemically absorbent material to absorb a marijuana metabolite from said biological fluid test specimen; c) treating said chemically absorbent material to produce an elution solution, derived from said biological fluid test specimen, which may contain said marijuana metabolite separate from^* any extraneous material • which may have been present in said biological fluid test . specimen and which extraneous material was also absorbed by said chemically absorbent material; d) applying said elution solution directly to the bibulous carrier material to cause any of said marijuana metabolite present in said elution solution to react with said visualization reagent thereby changing the first color of the reagent to a second color when a marijuana metabolite is present in said biological fluid test specimen.

9. A method as defined in Claim 8 wherein (c) comprises rinsing said chemically absorbent material with a rinse solution to remove said any extraneous material from said chemically absorbent material while leaving any of said -marijuana metabolite which may be contained in the biological fluid test specimen being screened and further providing a solvent solution to said rinsed chemically absorbent material so as to form said elution solution.

10 10. A method as defined in Claim 9 wherein said rinse solution is a buffer, aleohol-water rinse material.

11. A method as defined in Claim 8 wherein the chemically adsorbent material is provided in a syringe ^"■5having a plunger element and wherein said chemically adsorbent material disposed within said syringe.

1 o A method as defined in Claim 11 wherein said chemically adsorbent material is a non-polar adsorbent 20packing material.

13. A method as defined ^ Claim 11 wherein said chemically adsorbent material is a reverse phase liquid chromatographic packing.

25

14. A method as defined in Claim 13 wherein said biological fluid test specimen is urine and said chemically adsorbent material is a C-18 reverse phase liquid chromatographic packing;

30 said visualization reagent in said bibulous carrier material comprises a diazonium salt producing a first color; and said rinse solution comprises a buffered, acidic solution having a pH in the range of from about 6.0 to about

357.0 and which includes an alcohol.

15. A method as defined in Claim 14 wherein said solvent solution comprises acetone.

16. A method as defined in claim 11 wherein said chemically adsorbent material is provided on a syringe having a plunger element in a cartridge disposed on said syringe at the opening tip of the syringe column chamber.

17. A method as defined in Claim 16 wherein said chemically adsorbent material is a non-polar adsorbent packing material.

18. A method as defined in Claim 16 wherein said chemically adsorbent material is a reverse phase liquid chromatographic packing.

19. A method as defined in Claim 18 wherein said biological fluid test specimen is urine and said chemically adsorbent material is a C-18 reverse phase liquid chromatographic packing; said visualization reagent in said bibulous carrier material comprises a diazonium salt producing a first color; and said rinse solution comprises a phosphate buffered, alcohol-water solution having a pH in the range of from about 6.0 to about 7.0.

20. A method as defined in Claim 11 wherein said solvent solution comprises acetone.