WO1984002397A1 - Drug abuse test indicator device and method of making same - Google Patents

Abstract

Drug abuse compounds comprising the substances of the classes amphetamines, barbiturates, narcotics and tranquilizers, and the specific compounds including morphine, dilauded, cocaine and codeine in body fluid samples are qualitatively and quantitatively detected by the use of a carrier matrix having specific physical properties relating to strength and to absorbency of dry chemicals. The matrix being impregnated with dry staining agents in precise amounts which were prepared in accordance with unique procedures whereby pH insensitivity and unique color change sensitivities to test fluids of 1,000 to 10,000 times the sensitivities realized with thin layer chromatography staining reagents are achieved without the use of ion-exchange resins or color intensifiers.

Description

Description

Drug .Abuse Test Indicator Device and Method of Making

Same.

Field of the Invention This invention relates generally to a method and means for qualitatively and quantitatively detect¬ ing the presence of certain drugs of abuse, namely barbiturates, amphetamines and morphine-like compounds as a group as well as individual narcotics, specifical- ly heroin, morphine, cocaine, dilaudxd and codeine in physiological fluids. This invention more specifically relates to a combination of special, unique formulations of shemicals and special carrier matrix, test paper or dipsticks, for the qualitative and/or quantitative analysis of body fluids such as whole blood, serum, urine or saliva for the presence of drug abuse compound residues by use of said test matrix, paper or dipsticks. The chemical formulations and matrix used in such test strips are unique and the chemical formulations lie within very narrow limits of concentrations of chemical compounds. Further, the resulting products do not require ion-exchange resins or color intensifiers for reactivities 1,000 to 10,000 times more sensitive than the reactivities of the formulations utilized in thin layer chromatography.

Background of the Invention

The increase in drug abuse has created a great need for new methods of analyzing drug residues in physiological fluids that are accurate, rapid and cost

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_ OMP < effective. Existing commercial methods require large capital expenditures for equipment, are time con¬ suming, and are costly per unit test. Most often, urine samples are sent from met adone or rehabxlita- tion treatment centers to clinical laboratories for processing and screening for drugs of abuse. Turn around time for results are rarely less than 48 hours and more often 3 to 5 days. The need for an on-site, accurate method that can be used by untrained person- nel to monxtor drugs of abuse, particularly in urine samples, has existed for some time.

In a previously issued patent, U.S. Patent No. 3,915,639, to one of the present inventors, an in¬ vention was disclosed utilizing a method for dipstick identification of drugs of abuse based on incorporat¬ ing an ion-exchange resin in a bibulous paper and the use of an intensifying agent incorporated into known thin layer chromatography staining chemical formula¬ tions. This method enhanced the sensitivity of staining reactions to known drugs of abuse 1,000 to 10,000 fold. Several difficulties are encountered in making these dipsticks commercially useful. Of primary importance is the difficulty in reading and interpreting the staining reactions which incorporate the ion-exchange resins and intensifying agent. The color formation and patterns are so complex that a trained technician is required. .Another difficulty is the complexity and excessive cost required to manufacture the above dipsticks. The process for each dipstick requires a minimum of seven immersions into different chemical solutions with special immersion and drying equipment to maintain chemical potency.

OMPI Further, in a drug screening environment, difficult¬ ies are encountered in distinguishing methadone, morphine-like drugs and tranquilizers from caffeine and nicotine, alkaloids which are very commonly found in addicts as well as the general population. The intensifying agent intensifies all the alkaloid staining reactions equally well. Although these formulations are useful for clinical laboratory purposes, they fail to offer the methadone and rehabilitation centers a simple and accurate method which can be used by untxaxned personnel to screen for drugs of abuse.

It was learned from U.S. Patent No. 3,915,639 that the staining reactions utilized in thin layer chromatography could be utilized without time consuming and expensive extraction, and separa¬ tion procedures to achieve rapid and accurate ident¬ ification of drugs of abuse in physiological fluids directly on test papers or dipsticks. The need for simplifying the preparation of the test papers, over¬ coming the problems of interpreting results and ob¬ taining a test paper that selectively stains dif¬ ferent drugs of abuse without regard to interfering substances, still continies to exist. Stability problems with previous dipsticks have also been a major stumbling block. The combina¬ tion of ion-exchange resins, intensifying reagents and complex staining formulations has produced a dipstick that is highly reactive, but only for relative- ly short periods of time. Shelf-life tests have in¬ dicated that these dipstick formulations will sublime

SUBSTITUTE SHEET in the light or dark, at room temperature or at 4°C and will markedly discolor. Thus, the need for stable formulations is still a primary requirement for commercialization. Traditionally the staining chemical formula¬ tions for thin layer chromatography have been specifically reactive complex mixtures of chemicals in specific solvents which were sensitive only within relatively narrow pH limits. These pH limitations were one of the reasons for the number of steps and complexity to preparation of the ion-exchange resin, intensifier drug abuse dipstick. Several steps in¬ volved washing the test papers repeatedly in buffer solutions to maintain the pH of the formulations being absorbed in the paper. At least four buffer washers were required in preparation of the narcotic dipstick. The need for discovering formulations which were pH independent was essential.

Summary of the Invention It is an object of this invention to provide a method and means, such as chemically impregnated disposable test papers or dipsticks, for rapid analytical (qualitative or quantitative) determina¬ tions for the presence of the classes of drug residues directly in physiological fluids of drug abusers.

.Another object of this invention is to provide a method and means for rapid qualitative and quantita¬ tive identification of particular species of drug residues in the 0.5 microgram to 500 micrograms per drop range.

\^ _*_J _**t> 3---'T1TUTE SHEET Yet another object of the present invention is to provide a unique combination of a carrier matrix having specific physical characteristics and dry chemical staining agents, absorbed from solutions containing a narrow range or narrow ranges of specific chemicals, deposited in inter¬ stices in the carrier matrix whereby to provide a matrix dipstick of high sensitivity to specific abuse- type drug residues found in the physiological fluid of users; such dipstick being quite insensitive to the pH factor of the fluids being tested and providing a semi-permanent qualitative and quantitative color intensity record of the drug residue in such fluids. Still another object of the present invention is to provide processes for the preparation and production of abuse-type drug staining agents having sufficient sensitivity to permit use without the requirement of an ion-exchange resin or color intensifier. The term "physiological fuids" as herein used refers most commonly to urine but is also applicable to whole blood, serum, plasma, sweat and tears. The "classes of drug residues" referred to include those most commonly found in addict or rehabilitation patient physiological fluids, namely: amphetamines, barbiturates, morphine-like substances and cocaine, and tranquilizers of the diazepam type structure. The particular species of such drug residues include, all derivatives of barbiturates and amphetamines; methadone, morphine, cocaine, codeine, dilaudid and derivatives of the diazepam type structure. -6-

Detailed Description

A carrier matrix comprises a chemically treated, disposable test paper or dipstick which may be of any suitable shape or form. The term "paper" refers to any material (paper, fibers, plastics, etc.) having the requisite physical properties as hereinafter stated and the necessary lack of chemical reactivity to the system. The carier body is limited to certain types of bibul- ous paper which will maintain the chemical com¬ ponents within its matrix and allow sufficient poros¬ ity and absorbance for the staining reaction to occur effectively with one or more drops of the physiological fluid. This carrier body is impreg- nated by an immersion process with the essential drug-reactive components. These drug-reactive com¬ ponents, in order to be pH independent in their re¬ activity, and to provide sensitivities of 1,000 to 10,000 times normal staining reactivity seen in thin layer chromatography must lie within very narrow limits of concentrations for each type of test paper.

Only very specific solvents and combinations of chemicals may be used to produce a dipstick with characteristics which are stable (as to color and re- activity) over a period of months. Such formulations impregnated in the test papers or dipsticks can only be prepared under special conditions of immersion into the specified formulations. These conditions involve pretreatment of chemicals (dissolution, ageing) im- mersion time, drying time and drying conditions. In some instances a stabilizing bath is required after initial immersion. The test papers thus produced

SUBSTITUTE SHEET fURE

yield an intense and relatively permanent color record of the specific drug residues found in the physiological fluids tested.

In practice, a test paper or dipstick is pro- duced for a particular purpose, i.e. to identify a particular class of drugs or specific drug. To illustrate, diphenylcarbazone and mercuric chloride are the active staining reagents for the barbiturate class of drugs and derivatives; Ninhydrin is the active staining reagent for amphetamine class of drugs and derivatives and a modified Dragendorff stain (platinum, bismuth, and iodide) is used for the narcotic class reactions for methadone, morphine¬ like compounds and tranquilizers. By specifically altering the ratios of platinum, bismuth and iodide, specific test papers for diazepam only and dilaudid only, to name but two, have been prepared. Again, altering the ratios of compounds within very critical limits yields a narcotic test paper whose color reaction differentiates only morphine, cocaine and codeine.

The particular test paper is determined by its intended use, i.e. as a screening agent or confirmation for a particular class or specific drug residue to be identified. The stains themselves do not normally react with other substances in the body fluids other than those within the class category except as herein¬ after stated. The class reactions are summarized in Table 3. In Table 2 is a summary of test papers developed and their color reactions with drug residues. The quantitative characteristics of the stain¬ ing reactions may be utilized by one of three methods:

OMPI (1) a color comparison chart developed by using, standard incremental spiked urine samples to standard dipsticks; (2) the use of a densitometer to measure the color absorbancy; (.3) the use of urine samples from incrementally dosed animals or humans. Color in¬ tensity is measured as a function of concentration following Beer's Law for each class or specific drug residue. In the case of the barbiturates a slightly different procedure is used since the staining reaction is a blanching effect (white spot) of an already colored test paper (.purple) . In this case the diameter of the white spot produced is proportional to the concentra¬ tion of the drug.

The means and methods for using dipsticks with different physiological fluids i.e. preparation of the fluids prior to testing, are based on previous work des¬ cribed in one of the inventor's earlier U.S. Patents, No. 3,915_r639. These methods are applicable to these staining reactions as well. Phosphotungstic acid or a Folin-Wu reagent will deproteinize a drop of whole blood sample or plasma on an overlay leaf allowing the serum to react with the test paper beneath. Urine and saliva may be tested directly. Since most drug abuse testing is traditionally performed on urine samples, special emphasis has been given to interferences and performance standards with urine as the body fluid of choice. The formulations given are adapted to the high salt and urea concentra¬ tions of urine. Since many addict patients in re¬ habilitation centers attempt to substitute water samples for their urine sample the Narcotic "G" Test Paper will identify this circumstance. The Narcotic "G" Test Paper will turn.black instantaneously upon exposure to water instead of urine.

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As a qualititive screen for drugs of abuse (the most common application for these test papers) these test papers have been designed to monitor drug residues at the concentration levels most fre- quently encountered with drug addicts in rehabilit¬ ation centers. The concentration levels of 0.05— 50 micrograms of drug residue/drop can be increased or decreased by one of three ways: (1) using a more or less sensitive formulation of the chemicals in the test paper; (2) increasing the number of drops of body fluid placed on the test paper; (3) im¬ mersing the paper a plurality of dips with drying between each dip. The most effective formulations for these concentration levels are being used. Therefore, the amphetamine and barbiturate tests can be used as a "Yes" or "no", i.e. positive or negative screen. This test is conducted by placing one or more drops of the body fluid (usually urine) on the test paper from a pipet or medicine dropper. The test paper is designed to react with .05 micro- gram of drug residue/drop of test fluid. Larger numbers of drops or dipping directly into the fluid is generally superfluous. One drop of liquid from a Pasteur pipet is adequate to obtain the desired results.

With the narcotic's test papers the circum¬ stances are slightly different. Because colored rings, bands and spots are formed, a dropping pipet must be used. The Narcotic "G" test paper is des- igned for two drops of liquid and the Narcotic "S" and other narcotic test papers are designed for one drop of liquid. Using different quantities of drops

SUBSTITUTE SHEET will distort the reading and interpretation of results. With the one or two drops of liquid, the drug residues migrate at different rates re¬ acting with chemicals in the paper to yield a colored pattern easily discernable. The rings, bands and spots for the Narcotic "G" test paper are given in Figure 1. All of the test papers have been designed to yield positive test reactions at the levels of concentration of drug residues most commonly found in addict body fluids based on hundreds of tests performed at rehabilitation centers.

The invention is further taught with respect to the single Figure which illustrates the pattern of staining reaction achieved with Narcotic "G" test paper with one or two drops of liquid applied from a pipet..

Best Mode for Carrying Out the Invention

Matrix or Bibulous Paper

Since the matrix or paper must hold a minimum of chemicals in its interstices, a large number of papers of different porosities would appear to be usable. However, since the reactions do not occur on the surface of the paper (the process is an immer- sion process not a coating process) , the porosity of the^"paper must be balanced against "wet strength" or the paper will tear or macerate in the process. A tensile wet strength of 350 grams and an absorbance of dry chemicals of 0.05 gram/sq. in. is the minimum specifications for the paper that can be used. Other

OMPI inert plastic materials ( on-woven fabrics) , wood or even metals with similar characteristics may be used but have not been found as satisfactory as paper. Whatman Filter Paper #3 contains the ideal characteristics and the minimum character¬ istics are:

Specification Stainstrength (H-O)

Weight: 185 gm/meter 2 Dry Burst: 33 psi, N/m2:230

2

Thickness: 0.38 mm Wet Burst: 0.44 psi,KN/m :3.0 Retention: 5 microns, 98% efficiency Tensile

Ash: 0.06 Dry: 5500 gm

Wet: 350 gm

Filter paper of lesser porosity (Whatman #2) will yield a test paper that will give the necessary re¬ actions but not at the sensitivity required to pro¬ vide quantitative as well as qualitative results. Also, it is possible to employ a hard matrix with a porous coating of the porosity characteristics set forth above. The results obtained are acceptable but not as good as when using the type of paper described.

Amphetamine Test Paper Formulations

Any volatile organic solvent that will dissolve 3 or more grams per 100 ml of Ninhydrin reagent (tri- ketohydrindene hydrate) may be used as the immersion liquid. Each solvent may impart a slightly different color reaction to positive and negative tests. Thus, selection of the solvent is based on the color dif¬ ferentiation desired. Various higher alcohols yield different shades of blue for negative and positive

i - I ! I U I ύ -SHEET reactions. Both methyl alcohol and acetone yield a light tan or brown and a light or dark blue for positive and negative reactions, respectively. The initial reactions is difficult to discern on white paper so that a colored paper or a coloring agent is added to bring out the blue and tan colors on for instance a yellow background. Most other volat¬ ile solvents yield, when urine is applied, various shades of blue to both positive and negative tests. With methyl alcohol or acetone the light tan and light blue will darken with time to a dark brown and dark blue easily distinguishable. Although instant¬ aneous identification can be made with a positive amphetamine urine, confirmation of a negative test can be made within 20 minutes.

The method of preparation of the amphetamine test paper involves completely immersing the filter paper in the liquid for 5 to 10 seconds and removing. Immersing for less than 5 seconds gives uneven dis— tribution of chemicals within the paper. The paper may be dried in air or with gentle heat Cno greater than 60°C) . The test papers when dry are stable to light and air and. have a shelf life of 1 year. Test papers may be cut with any paper cutter to any size. One inch squares have been found more than adequate for a single drop urine test.

The concentration of Ninhydrin and selection of solvent is critical in the preparation of this test paper. Less than 5% solutions will now show re- activities at the 1 microgram per drop level of amphetamines in urine. The use of different solvents shows that it is difficult to differentiate between

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OMPI positive and negative tests. The use of methyl alcohol or acetone with an added coloring agent for adding background color to the paper is the only feasible method. High concentrations of Ninhydrin are limited by solubility which is 6%. The color reaction with one drop or more of urine will only occur with a highly absorbent paper or matrix such as a Whatman #3 paper. Other types of paper or ion exchange resin papers will not give this color change or sensitivity of reaction with¬ out the use of an intensifying agent.

In summary, the amphetamine test paper is manufactured by immersion for at least 5 seconds a testpaper having the requisite wet strength and absorbance in a 5% to 6% solution of Ninhydrin in methyl alcohol or acetone.

Barbiturate Test Paper Formulations

Any volatile organic solvent that is able to dissolve greater than 0.1 gm of S-diphenylcarbazone and 4 gm of mercuric chloride in solution, for instance, methanol, may be utilized as the immersion liquid. Experiments have shown that cupric chloride may be substituted for mercuric chloride in some formulations. However, color differentiation was im- paired and a time delay in reactivity occurred making these formulations less than satisfactory. Other salts have been tested with equally poor results. No substitute has been found for S-diphenylcarbazone. Differing ; solvents yield differing colors of test papers from pale blues to deep purples.

SUBSTITUTE SHEET

CMPI In preparation 0.25 gm (0.20 gm to 0.25 gm range) of S-diphenylcarbazone is added and mixed with preferably 5 gm (4.5 gm to 5 gm range) of mercuric chloride dissolved in 100 ml of methanol. Actually quantities of the S-diphenylcarbazone and mercuric chloride (HgCl) are employed to preferably produce a saturated solution for sensitivity.

Since the reaction with a drop of physio¬ logical fluid (primarily urine) yields a blanching or whitening effect, the crimson or dark purple color of the test paper is more desirable. Using methanol or acetone imparts such a desired deep purple color to the test paper- When freshly prepared, this test paper has a sensitivity of 0.25 micrograms of barbiturates per drop of body fluid.

The color of the papers (deep purple) however will fade slowly to a light blue shade whether in light or dark. These test papers are still functional for up to 30 days after manufacture and will give characteristic reactions. If sealed in an airtight wrapping, the paper is stable for approximately 60 days. A negative barbiturate fluid turns the paper a deep purple, while a positive barbiturate fluid blanches the paper to a whitish grey. The disadvantages of a test paper that changes color in spite of the fact that the test paper itself is still functional makes its commercial use question¬ able. To overcome this problem in qualitative test papers, a second immersion of the paper is made in a solu— tion of 4.5% (3% to 5% range) polyvinyl alcohol (aqueous) of the test paper for extended periods of time. The paper is completely functional as a qualitative test

S_UBSTITUTE SHEET paper with physiological fluids containing as low as 0.05 micrograms per drop of barbiturate with a shelf life of six months to one year. Lower con¬ centrations of polyvinyl alcohol may be used but the shelf life of the color of the paper decreases as the solution is weakened. Higher concentrations (superstaturated solutions) of polyvinyl alcohol increases the lag time for reactivity with the barbiturate solution without substantially changing the shelf life factor.

The 4.5% of polyvinyl alcohol cannot be incorporated into the S-diphenylcarbazone, mercuric chloride solution. The manufacturing process must proceed in a two step immersion with air drying between and after. As with the preparation of amphetamine test papers, gentle heat may be. used in the drying process but is not required and, in fact, more sensitive test papers are obtained by drying in air. Methanol is the solvent of choice for the barbiturate test paper. With volatile organic solvents properly vented, drying times do not exceed 10 to 15 minutes for pilot production runs. With the use of S-diphenylcarbazone and mercuric chloride drying and immersion of papers must be achieved in a properly vented air space.

The test paper incorporating polyvinyl alcohol is not sufficiently sensitive to conduct more than a qualitative or at best a semi-quantitative test and should not be used for quantitative tests. If it is desired to perform a quantitatxve test, the polyvinyl alcohol must be excluded. As indicated

above, such test papers have a shelf life of about 60 days if individually wrapped and sealed.

An alternative barbiturate test paper that is satisfactory in all respects except that approx- imately 24 hours is required before final readings can be made is as follows:

Aqueous silver nitrate (AgNO.,) 1.5-2.5%

Dip once and dry in air and light.

Methanolic copper chloride (CuCl₂) 4.5—5.5% Dip once and dry in air and light.

A positive reaction results in the formation of black spots within 12 to 24 hours depending upon concentration of the residue in the body fluid tested.

Narcotic G Test Paper Formulations

Although a large number of Modified Dragendorf's Reagent formulations (bismuth subnitrate, platinum chloride, and potassium iodide) are reactive with alkaloids at differing concentrations, only one form- ulation has been found effective in preparation of a test paper useful for screening purposes. The criteria necessary for the selection of a formulation is as follows:

1. It must be opaque to nicotine and caffeine, alkaloids commonly found in urine and physio¬ logical fluids in high concentrations.

2. It must clearly distinguish between metha¬ done and morphine—like drugs and cocaine. It must also distinguish clearly the above from tranquilizers as well. All of the above must be clearly distinguishable at the alkaloid concentration levels of 0.05-50 micrograms per drop.

3. The test paper must be readable in a reasonably short period of time (30 minutes) . 4. The test papers should preferably have a shelf life of no less than six months.

5. The test papers should offer a semi¬ permanent record such that the stained test papers can be referred to in three to six months for document- ation purposes.

A formulation that meets those criteria has very stringent limits placed on the bismuth nitrate concentration, the acetic acid concentration, the chloroplatinate concentration, the iodide concentration and the normality of hydrochloric acid used.

Table 1 lists the allowable ranges of concentr¬ ations that yield an acceptable test paper according to the above criteria. The test papers must be pre¬ pared under special conditions or the test papers will not be satisfactory. The platinum solutions must be prepared two to ten days prior to immersion and aged during that period. Fresh platinum solutions or those kept longer than 10 days are non-functional. In addition the bismuth and platinum salts must be dissolved in an aqueous potassium iodide solution and then diluted to working concentration. Other methods of preparation are ineffective. A double immersion is required for the platinum solution. The following summarizes the preparation procedures for the Narcotic G Test Papers: 1. The bismuth subnitrate is dissolved at the necessary concentration level in the exact and necessary concentration of acetic acid.

2. The above solution is mixed in a ratio of 1:1 with the following solution.

- The exact and necessary concentration of platinum salt is dissolved in the iodide salt in water

- 100 ml of the above solution is diluted to 200 ml with 2 Normal hydrochloric acid hereinafter referred to as 2N HCl

- The test paper is then immersed for 5 to 10 seconds in this solution and allowed to air dry under light (about 24 hours)

TABLE 1 - Allowable Concentration * Ranges of Active Agents for Narcotic G Formulation Solution 1 Lower Limit Upper Limit Bismuth subnitrate 0.75% 0.85% Acetic Acid 7.5% 10.0% H₂PtCl₆ 0.06% 0.07% I .2.5% 3.3%

Normality HCl (100 ml) 0.4 0.6 H₂0 q.s. to 400 ml

Solution 2

H₂PtCl₆ 0.20% 0.25%

KI 1.75% 2.25%

Normality HCl 0.8 1.2 Concentrations are based on the weight per unit volume of Solutions 1 and 2. 3. For the second immersion the exact and necessary concentration of platinum salt is dis¬ solved in an exact and necessary concentration of iodide salt and diluted up with the specific normal- ity of hydrochloric acid.

4. The dried test paper from Solution 1 is then immersed in Solution 2 above for 5 to 10 seconds and air dried under light.

Slight changes in concentration, method of drying or use of other solvents or acids produces a nonfunctional paper. Drying must occur in air and light. The air and light activates the formulation. Drying in subdued light or in the dark diminishes reactivity of the test papers markedly. Substitu- tions with other chemicals for platinum, iodide or bismuth have been unsuccessful. Achieving the same concentration of platinum, iodide and bismuth by utilizing different steps in the procedure have also been unsuccessful.

EXAMPLE I

Solution 1:

KI 4 -gms

H₂0 100 ml

2N HCl 100 ml

Solution 2:

Bismuth subnitrate 1.6 gm (1.6%)

Glacial acetic acid 17.5 ml (17.5%)

Water, q.s. 100 ml One to two volumes of Solution 2 is mixed with 10 volumes of Solution 1.

__OMPI The carrier matrix is dipped once in Solution

1 and allowed to dry in light and air for 24-48 hours. Solution 1 must age for 2—10 days prior to use; i.e. application to the carrier matrix. The carrier matrix must dry in light and air after immersion in Solution 2.

This paper is more sensitive than the first stated Narcotics "G" paper by about 10 fold (i.e.

0.1 microgram per drop) but does not react with water to produce a black reaction. The colors are also more vivid. The paper is opaque to caffeine and nicotine.

.Another workable combination of dips from which the carrier matrix may absorb the requisite staining agent within its interstices is as follows:

Solution 1 Lower Limit Upper Limit

Bismuth subnitrate 1.4% 1.6%

Acetic Acid 14.0% 16.0%

H₂PtCl₆ 0.06% 0.13% KI 4.0% 6.0%

Normality HCl (10Q ml) 0.8 1.2

H₂0 q.s. to 400 ml

Solution 2

H₂PtCl₆ 0.12% 0.25% KI 1.75% 2.25%

Normality HCl 0.8 1.2

All concentrations are based on the weight per unit volume of each of the Solutions 1 and 2.

A formulation using K₂PtCl_g for producing the ϋ-paper as discussed below is as follows: Solution 1

Bismuth subnitride 0.65%

Acetic Acid 6.5 ml

KI 3.0%

2N HCl 25 ml

Deionized H₂Q 68.5 ml

Solution 2

KI 2.025%

2N HCl 50 ml

Deionized ILO 50 ml

The percent concentrations in the U-paper formul¬ ations are based upon grams weight per 100 ml of liquid solution.

The carrier matrix is immersed into each of the solutions 1 and 2 with a drying step effected in between. Once dry after the second dip, the impregnated carrier matrix is placed into a humidity chamber as discussed below.

In all of the narcotic formulations chloro- platinate, hydrogen chloroplatinate and potassium chloroplatinate may be utilized. Narcotic S Test Paper Formulations Using Solution 2 of the Narcotic G Formulation of Table 1 with a single immersion of acceptable bibulous paper yields a test paper that is opaque (does not react with) to methadone and tranquilizers but gives distinctive colors and characteristic spots for morphine, dilaudid, cocaine and codeine. See Table 2 for distinct characteristics of the staining reactions for all papers of this invention. Again

OMPI

WIFO Solution 2 must be within the acceptable concentr¬ ation limits described in Table 1. Similar conditions apply to the preparation of platinum sol¬ utions and the air and light drying. A summary of the formulations and procedures for manufacture of the various test papers is pro¬ vided in Table 4.

Table 2 - Summary of Test Papers - Color Re¬ actions With Drug Residues Test Paper

Barbiturate Test Paper - Normal urine; deep purple

Barbiturate present: greyish white

.Amphetamine Test Paper - Normal urine: bluish purple .Amphetamine present: deep brown

Narcotic "G" Test Paper: Normal urine: white or buff center with brown periphery

Water: black Methadone: buff center with blue-purple outer ring or band inwardly adjacent to outer periphery

Morphine-like Drugs: inner blue-purple rings or bands with buff or yellow center

Tranquilizers: (Diazepam Class) inner center light blue or pinkish ring or band Narcotic "S" Test Paper- Normal urine: white center with brownish-purple periphery

Morphine: solid blue spot fills entire area with a superimposed dark blue peripheral ring

Codeine: purple spot with white or pink halo surround¬ ing spot

•ΠTUTE SHEET Dilaudid: tan or brownish- purple spot with white or pink halo

Cocaine: orange-brown or orange spot with white halo

Other Narcotic Test Paper Formulations

By varying the ratios of bismuth, iodide and platinum in the formulations, different alkaloids become opaque (do not react) with the test papers.

Two specific formulations have been developed. The first gives a highly intense pattern and purple-violet color only to tranquilizers of the diazepam-type structure and opaque to all other alkaloids. The second formulation gives an intense green ring for dilaudid only, opaque to all other alkaloids. These formulations are as follows:

Tranquilizer Test Paper

This paper is specific to tranquilizers and is virtually opaque to all other narcotics:

K₂PtClg 0.5 g

KI 10-12 gms

H₂0 100 ml

2N HCl 100 ml The above solution is mixed with 200 ml of 1.5%-

1.7% bismuth subnitrate in 15% acetic acid.

The solution must be aged, 2-10 days, prior to use.

This test paper requires only 1 dip in the above solution and is dried in light and air. A positive reaction yields a purple violet color .

ΪTUTE SHEET Dilaudid Test Paper

This test paper is reactive only with dilau¬ did drug residues and is opaque to all other alkaloids. A positive test yields a deep green ring or band. Solution 1:

Bismuth subnitrate 1.5%/100 ml of 15% acetic acid is mixed with an equal volume of 2-10 day aged iodoplatinate solution as follows:

K₂PtCl₆ 1.50 gm

KI 12% 100 ml

2N HCl 100 ml

Solution 2:

K₂PtCl_g 0.5 gm

KI 4 gms

H₂0 100 ml

2N HCl 100 ml

Solution 2 must be aged for 2-10 days prior to use. The matrix is dipped in the first solution and allowed to dry in light and air and then dipped in the second solution and allowed to dry in light and air. All Purpose Alkaloid Detection Paper

This test paper is reactive to all alkaloids and cannot distinguish between the various narcotic residues. This paper is used as a broad base screen.

Bismuth Tri-iodide 0.1 gm

KI (0.9 gm in 20 ml H₂0) 4.5% 2N HCl 20 ml

S _ΩS~TiTUTE SHEET o*-^ϊ?ι The first three materials are first mixed and then mixed on a 1:1 volume basis with the 20 ml of 2N HCl.

The platinum solution is aged for 2-10 days before use.

The matrix is dipped once and allowed to dry in light and air.

This test paper is not opaque to caffeine and nicotine and provides the same blue-purple color reaction to all alkaloids.

TABLE 3 - Summary of Class Reactions for Test Papers

The reactions may be characterized as follows:

Barbiturate Test S-Diphenylcarbanzone Specific for Mercuric chloride all types

.Amphetamine Test Ninhydrin Specific for primary amines Narcotics Test Platinum, iodide, Specific for bismuth alkaloids and . saturated ring compounds with tertiary nitrogens

T.ABLE 4 - Reagents and Materials Specifications in preparation of test papers:

Test Paper Procedure for Preparation

For amphetamine 1. One dip for 5-10 seconds into a solution formed when 3-6 gm Ninhydrin is dissolved in

100 ml of acetone or methyl alcohol.

2. Dry in air. or barbiturate 1. One dip into a solution formed when 0.250 gm of S-diphenylcar¬ bazone is added and mixed into 5.0 gm of mercuric chloride dissolved in 100 ml of methanol.

2. Dry in vented air in darkness.

SUBSTITUTE SHEET

-$ '- ml s

2^lvf{ ^" For narcotic (G) 1. First, dip into a solution formed when 1.5% bismuth subnitrate in 15% acetic acid is added slowly with mixing, in a ratio of 1:1 to the following -mixture:

0.250 gm H₂ tClg plus 12 gm

KI in 100 ml H₂0 added to

100 ml 2N HCl.

Second, dip into a solution formed by dissolving 0.250 gm of H₂PtClg and 2.0 gm KI in 50 ml of H₂0. Bring to

100 ml with 2N HCl.

3. Air dry under light for 24 hours between dips.

The greater quantity of KI in Solution 1 is required to maintain the bismuth in solution. For narcotic (S) 1. One dip into a solution formed by dissolving 0.250 gm H₂PtCl_g, plus 2.0 gm KI in 50 ml of H₂0. Bring to

100 ml with 2N ΞCI.

Test Specimens Two drops of a body fluid specimen is the most required for any given test paper. Thus, for a full screen, 1 ml of a body fluid sample of urine, blood, serum or saliva is adequate. Since most testing for drug abuse offenders is performed on urine samples, the test papers to be described are specifically targeted for urine testing. However, the formulations and applications are equally satisfactory for other body fluids as well.

The quantity of liquid in a drop varies from liquid to liquid as a function of such parameters as

OMPI viscosity, specific gravity, etc.- and diameter of the opening in the end of the applicator. The dipstick of the invention- is operative over a range of at least 0.05 to 0.3 milliliters of body fluid specime .

Urine Specimens

One to three drops of urine from any type of dropping pipet (medicine dropper, Pasteur pipet, dis¬ posable pipets, etc.) may be used. Urine samples should be used as soon after voiding as possible, preferably within 24 hours. Urine samples do not have to be refrigerated nor preservatives added. Urine samples left unattended for three days at room temperatures show no difference in reactivity.. Specimens left over a week at room temperature do not give characteristic normal re¬ actions for blank urine although urine with drugs gives comparable drug reactions. Testing of urines allowed to stand for long periods of time should be avoided. Although preservatives in urine do not appear to influence normal or drug reactions with specimens, preference is given to freshly voided urine.

The drug abuse test appears to operate in- dependently of the pH of the urine within a range of pH 3-10. Changing the pH of the urine will not affect the reactivity or color changes of the test papers with normal urine or drug urine. Interfering Substances The chemistry of the drug abuse test papers is comparable to the staining reactions of the thin-layer chromatography procedures except magnified 1,000 to

SUBSTITUTE SHEET 10,000 times in sensitivity and reactivity. Thus, like the chemistry of the thin-layer chromatography tests, the individual staining reactions are based on stains reacting with specific classes of drugs. The stains themselves do not usually react with other substances than those within the class category. See Table 3.

The barbiturate test performed on our test papers, directly from urine samples without extraction or separations, have been found to be completely specific in two thousand determinations and in the 400 samples (1600 test paper tests) evaluated at a methadone center where a double blind study was per¬ formed. Titration of urine samples from pH 3 to 10 showed no changes in reactivity or color appearance. The amphetamine test with Ninhydrin reagent is a more general test for amines and covers a broader category of reactions than amphetamines commonly found in urine samples. In the methadone center study no cross reactions were observed and a 100% correlation with the thin layer chromatography tests was noted. However, when several clinical laboratories utilized the amphetamine dipsticks on random urine samples out¬ side of their drug program to specifically screen for interferences, six positive protein urines were report¬ ed yielding false positive amphetamine tests out of approximately a thousand samples. In these instances, salting the urine with sodium sulfate gave normal results. The addition of the NaSO. precipitates the high proteins and therefore eliminates the false positives. This can also be done by heating the samples

SUBSTITUTE SHEET to be tested. Patients utilizing large doses of phenylephrine and propylamines yielded negative urines with the amphetamine sticks. Although many other drugs contain free amine groups, including tranquilizers of various types (all such samples were negative) , it is believed this data is conclus¬ ive and that this is an excellent screening procedure for amphetamines. Titration of urine samples bet¬ ween pH 3 and 10 showed no change in reactivity and color enhancement.

In developing the narcotics test papers over 30 formulations have been screened and evaluated based on potential use in the marketplace. Small dif¬ ferences in the formulations result in major charac- teristic differences in sensitivity and color pro¬ duction. It was found that by increasing the bismuth concentration in the test paper and decreasing the platinum and iodide, the class reactions for metha¬ done, hard drugs and tranquilizers were optimized. By increasing the platinum and iodide concentrations at specific ratios and decreasing the bismuth con¬ centrations, specific narcotics would separate out and be color enhanced. In both methods, formulations were found optimal when large doses of nicotine and caffeine were opaque (did not interfere or stain) . Studies were carried out where patients who were heavy smokers (over two packs of cigarettes per day) and heavy coffee drinkers (over six cups of coffee prior to voiding)were opaque to these test papers. In the methadone center study, the Narcotic "G" test paper (for identifying and distinguishing methadone, the group of morphine-like drugs, and tranquilizers)

SUBSTITUTE SHEET gave 99% correlation with thin layer chromatography findings. The Narcotics "S" test paper (identify¬ ing and distinguishing morphine, codeine, dilaudid and cocaine) yielded a correlation of 93% with thin layer results. Clinical results clearly indicate tiiat these two narcotic test papers "G" and "S" serve a useful function as an on the spot diagnostic screen for untrained personnel. Reading and Interpretation of Results 1. Barbiturate Test - Barbiturate test papers are remarkedly free from interferences. Test papers treated with 4.5% polyvinyl alcohol left in the dark for upwards of a year will not discolor and maintain their reactivity and sensitivity. When tested with normal blank urine, they remain deep purple. With barbiturates present they visibly blanche to a grey¬ ish white.

False positives can only be encountered with contamination from highly acid materials. Acidity below pH 2.5 is never encountered in urine specimens. False negatives would be rarely encountered since even in very low concentrations (nanogram amounts) a small white ring will form after 10 minutes. Adding one or two additional drops of urine will bring out this positive reaction. This type of

"faint" positive reaction may be difficult to interpret and may present a normally prescribed dosage of bar¬ biturates for sleep. Most addicts' urine shows marked blanching immediately. However, the extra sensitivity afforded by this test paper can be of value in emergency rooms of hospitals where delayed testing of

SUBSTITUTE SHEET

ϊ ' mild barbiturate overdose victims are not uncommon.

2. .Amphetamine Test - Most addicts' urine containing amphetamines shows a deep brown spot immediately with one drop of urine. Yet, much lower concentrations will form a very light tan spot as compared with a pale light blue spot from normal blank urine. However, both mormal and amphetamine spots darken within 30 minutes such that they cannot be misinterpreted. The smallest concentrations of amphetamines will show as a deep brown spot in that time period and a urine free of amphetamine will show a deep blue spot.

In the rare cases where proteinuria is found, the urine may give a false positive. A spoonful (5 grams) of table sale (NaCl) or sodium sulf te, preferably the latter if available, should be added to each 30cc of urine. The urine is shaken and allowed to settle for 10 minutes. A drop of the supernatent is then tested. 3. Narcotic (G) Test (See the single figure of the accompanying drawing) . A normal blank urine ' will show an immediate blanching and the formation of a large white or buff area 1 wherein the two drops or urine contacted the test paper. The area 1 has dispersed brown periphery. .With the blank urine, this picture will remain until the test paper dries (less than 30 minutes) and fade very slowly thereafter. In the case where very low concentrations of narcotics, methadone, or tranquilizers are present, a similar picture of white or buff center and brown periphery may form initially. As the paper dries, the character¬ istic outerblue-purple band 4 of methadone, the purple rings 3 of hard drugs and the light blue or

_lUBSTBTUTE SHEET

QUTI_ pinkish inner ring 2 of tranquilizers will form if the drugs are present. These bands and rings will often start forming within 3 or 4 minutes after the test paper is wetted. In the case of high con— centration of drugs as often found in addicts' urine, color formation and rings and bands will often faintly appear instantaneously as the test paper is wetted. As the paper dries the colors and rings intensify. Water samples on addict urine where water has been falsely substituted will give an instant¬ aneous deep black characteristic spot.

Referring more specifically to the single Figure: 1. A dark purple or black outer peripheral band or ring 5 is characteristic of both normal (negative) and addict (containing drugs) urines and is always formed.

2. A methadone band or ring 4 in the presence of methadone residue will always form in contact with the outer peripheral band or ring 5. The shape of a ring or band will vary depending on concentra¬ tion of the drug. Also, the color will vary from deep purple to azure blue depending on concentration. 3. Morphine-like drugs band or ring 3 will always form separated from the outer peripheral ring 5 and methadone rings 4. Colors will vary from purple to blue and shape will vary from bands to rings.

DϊvfPI

4. A blue tranquilizer ring or band 2 will form around the inner spot which will be intensified blue-pink depending on concentration.

Relative to rings 2-4, the thinner the ring and the paler the color the lower the concentration. In the presence of numerous drugs in the residue, the colors become less dependable and reliance should be placed primarily on the width of the ring.

5. The inner spot 1 may be a blanched white area or buff colored spot in normal (negative) urines. In addict urines the inner spot may be blanched or highly colored often yellow depending on concentration of drugs present. With methadone, the inner spot will usually be blanched or buff. Rarely, with high concentrations, it will be a purple or grey spot. With cocaine, the "inner spot" will be orange at high concentrations. With morphine¬ like drugs, the "inner spot" will be purple at high concentrations, but blanched or buff at low concen- trations. With a tranquilizer, a deep blue and/or pinkish at high concentrations for the "inner spot" but blanched or buff at low concentrations. Sensitivity Control

It has been discovered quite unexpectedly that the sensitivity of the drug abuse test indicator strips made in accordance with this invention may be significantly enhanced through additional processing. The processing is effected by placing the staining agent impregnated carrier matrix into a humidity controlled atmosphere for a sufficient length of time so as to improve the reactivity of the test indicator

EET strip with respect to indicating the presence of abuse-type drugs as discussed herein. Test papers made in accordance with the formulations of the narcotic G papers disclosed hereinabove were allowed to stand in a controlled humidity atmosphere for various periods of time. The time periods tested existed from a period of hours up through a period of several weeks. The narcotic G test paper which was brown in color following its production in accordance with, the above formulations was held in the controlled atmosphere for an amount of time sufficient to change the color to green. It was found that green papers which were produced after two weeks of saturation in a controlled water vapor atmosphere became ultrasensitive to abuse- type drugs. This sensitivity was sufficient to pick up caffeine rings resulting from the person having drunk a cup of coffee. Thus, the sensitivity of the indicator strip was down in the hundredths of thousandths of a nanogram of the drug per millileter of physiological liquid test solution.

The same type of increase in sensitivity was found to exist in the narcotic S papers. Both the narcotic G and S papers were hung in a closed box in which the humidity was controlled for different lengths of time. The test included the use of a physiological liquid test solution containing very small amounts of a drug. A test solution was placed on regular narcotic G paper and no reaction would be obtained on the test indicator strip when a drop of the test solution was applied thereto. That is, there

OMPI would be no rings appearing on the test indicator strip at very low levels of abuse-type drugs down to about 2 nanograms per millileter of solution. It was known that there was enough chromophoric material in the interstices of the test indicator strip so that there should have been some kind of a reaction in obtaining that color. However, no such reaction was taking place. However, the test indicator strips made in accordance with the form— ulations for narcotic G papers was held in a humidity controlled environment for a period of time to increase the reactivity of the chromophoric materials and sub¬ sequently dry. When the same test solution was then placed on the narcotic G paper, the color reaction was changed and the rings were now present.

The particular mechanism for the increased sensitivity is not completely understood. However, it was found that the sensitivity of the narcotic test strip papers was greatly enhanced simply by maintaining the test papers in a humidity controlled atmosphere from anywhere from one day to two weeks. The narcotic G papers were placed in a humidity chamber that was maintained at a curing temperature of from about 15°C to about 45°C. An airflow was established in the humidity chamber from zero to about 8 cubic feet per minute. The head pressure and the exhaust was then turned on and the temperature and airflow adjusted to reach an appropriate equilibrium. The racks of paper were placed in the environmental chamber and the temperattire was monitored and recorded on a regular basis.

_ OMPI The theory was that the humidity (water vapor atmosphere) changed the impregnated matrix such that the chromophoric groups such as the platinum iodide became much more sensitive within the test indicator strip. This was evident by virtue of the fact that this particular compound produced in accordance with the above formulations went from a^' brown color to a green color. The environment of the humidity control chamber was saturated with water for a period of time sufficient to change the color of the narcotic G paper from brown to green.

The narcotic G test papers which were treated in the humidity controlled environment are referred to as the U-papers (ultrasensitive papers) . The standard size of these papers is one inch square. A further paper referred to as UP-paper (ultra¬ sensitive proficiency paper) was processed exactly the same as the U-paper but is cut in a six inch square. That is, either a large piece either 6 x 6 inches or 8 x 8 inches was used for determining the presence of abuse-type drugs in a physiological liquid test solution. The larger piece of test indicator strip is then saturated with a large amount of the test solution. For example, 5 ml of urine con- taining various abuse-type drugs is placed on the larger piece of test paper. Use of the larger piece of paper with the larger amounts of test solution revealed the formation of several different rings which would represent the various metabolites of the different drugs present in the urine sample. It was possible to distinguish among the different types of metabolites of one particular drug. Thus, if a person were to take a shot of heroin, it was possible to recognize several different metabolites of the heroin based upon the different rings which would show up on the UP-paper. Capillary Action

Another important discovery related to the development of sensitivity is the use of a capillary pipet which enables a measured amount of test solution to be placed onto the paper very slowly. It has been found that when a test solution is added slowly to a test indicator strip made in accordance with this invention that the separations of the various rings for the various abuse- ype drugs are effected more clearly. That is, when the test solution is slowly added via capillary action through a capillary pipet, the results will enable an analysis of more accuracy both with respect to qualitative and quantitative measurements of any abuse-type drugs present in the test solution.

This use of capillary action is particularly appropri¬ ate with the UP-paper wherein a larger piece of test paper is used with a larger amount of test solution. Thus, the combination of the UP-paper and the capillary action has been found to be extremely effect¬ ive in analyzing the qualitative and quantitative measurements of abuse-type drugs in test solutions. This has not been available heretofore.

There are two major advantages which have been found to exist with the use of the capillary pipet. In order to form the rings on the larger piece of UP-paper, liquid has to have the time to both react with the chromophoric groups and to separate out into the paper. By using the capillary pipet, the amount of time is increased so that the reactions are slowed down. Although the time for applying the test solution to the UP-paper is increased, the sensitivity to the presence of abuse-type drugs in the test solution has been enhanced.

A further advantage of using the capillary pipet method is that there is a constant amount of fluid that can be maintained from one test to another. In this fashion, there is a greater accuracy of compar¬ ing test papers which have the same amount of the chromophoric material in them and standard comparisons can be more readily made in the field at the site of taking the fluid test specimen from the person being monitored. Further Prior Art

It is known to place fixed amounts of chemical visualization reagents into paper strips which have been impregnated with a solvent containing such re¬ agents. These reagents are to be eluted from these paper strips for use in the preparation of spray re¬ agents in conjunction with the conventional thin- layer chromatographic drug screening procedures. The U.S. Patent 3,912,655 is specifically directed to such a disclosure which expressly states that the patentee in that particular invention was not produc¬ ing indicator strips such as a conventional pH in— dicator and diabetes test strips. This is in direct distinction over the present application which is

OMPI For the first time providing indicator strips onto which a physiological liquid test solution may be placed at the site of taking the specimen without trained personnel and acquiring virtually instantaneous qualitative and quantitative benefits. No such paper has been produced which will be as sensitive to the very low level concentrations of abuse-type drugs which are present in the test solution as is the case of the indicator strips of the present application. None of the prior art test strips as referred to in the U.S. Patent 3,912,655 in its discussion of the prior art is directed to the specific solution formulations used to determine the presence of abuse-type drugs such as amphetamines, barbiturates, narcotics and tranquilizers such as have been disclosed and described herein.

The reagent impregnated paper strips of U.S. Patent 3,912,655 still require the extraction of the drugs from the urine sample with the extract having to be put on some type of a substrate. This is in line with the basic procedures of the thin—layer chromatographic procedures as stated above. The re¬ agent is then eluted from the impregnated paper strips of that_ patent and used for preparing the spray re- agentswhich in turn are utilized for effecting the standard thin-layer chromatographic drug screening procedures. This of course still requires the trans¬ portation of the specimens from the site at which the test specimen is taken to a laboratory where skilled technicians are required to effectuate the desired results. All of the cost associated with such

SUBSTITUTE SHEET transportation and skilled expertise is eliminated ^' through the use of the drug abuse indicator test strips of the present invention.

Finally, the drug abuse indicator devices as described herein distinguish over the earlier U.S. Patent 3,915,639 because the staining agent is im¬ pregnated rather than formed as a coating. Further¬ more, there is no necessity of ion-exchange resins or color intensifiers as the result of using the formulations and procedures as described herein.

While the drug abuse test indicator device and method of making same has been shown and des¬ cribed in detail, it is obvious that this invention is not to be considered, as being limited to the exact form disclosed, and that changes in detail and construction may be made therein within the scope of the invention, without departing from the spirit thereof.

Claims

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

1. A dry, drug abuse test indicator device for determining the presence of abuse-type drugs in physiological liquid test solutions, said device comprising: a), a staining agent impregnated bibulous carrier matrix containing in its interstices a dry chemical staining agent in an amount sufficient to react with abuse-type drugs while disposed within said matrix, b) said carrier matrix having a wet strength sufficient to prevent tearing or macerating of the matrix when the test solution is applied thereto, c) the amount of chemical staining agent being effective to indicate the type of said abuse- type drug present when there are low levels of con- centration of down to about 0.05 micrograms of the abuse-type drug in a drop of said test solution which is placed on the impregnated carrier matrix, d) said impregnated matrix being pH in¬ dependent with respect to the test solution and being effective to provide a semi-permanent indication of the presence of abuse-type drugs of the group con¬ sisting of amphetamines, barbiturates, narcotics and tranquilizers contained at said low levels of concen¬ tration.

OMPI IPO

2. The indicator device as defined in claim 1 wherein the carrier matrix has a wet strength of at least 0.44 pounds per square inch.

3. The indicator device as defined in either of the claims 1 or 2 wherein the carrier matrix has an absorbancy of dry chemicals of 0.05 grams per square inch.

4. A method of producing a drug abuse test indicator device for determining the presence of abuse- type drugs in physiological liquid test solutions, said method comprising: a) providing a bibulous carrier matrix having a wet strength sufficient to prevent tearing or macerating of the matrix when the test solution is applied thereto, b) said matrix being effective to maintain a dry chemical staining agent within its interstices in an amount sufficient to cause a staining reaction within the matrix, c) said matrix having an absorbancy in an amount effective to produce said staining reaction when said staining agent is contained within the inter¬ stices of the matrix and a drop of the physiological fluid is placed thereon, d) impregnating the matrix with a dry chemical staining agent in an amount sufficient for producing a color in the matrix indicative of the type of said abuse-type drug present when there are low levels of concentration of down to about 0.05

OMPI

^' micrograms of the abuse-type drug in a drop of said test solution which is placed on the impreg¬ nated matrix, e) said staining agent impregnated matrix constituting said indicator device which is pH independent with respect to the test solution and is effective to provide a semi-permanent indication of the presence of abuse-type drugs of the group consisting of amphetamines, barbiturates, narcotics and tranquilizers contained at said low levels of concentration, and the , f) holding the staining agent impregnated matrix within a humidity-controlled atmosphere to increase the sensitivity of said matrix to lower levels of concentration of abuse-type drugs in a test solution.

5. The method as defined in claim 4 wherein said humidity controlled atmosphere is saturated with water vapor at the selected curing temperature.

6. The method as defined in claim 5 wherein said selected curing temperature is in the range of from about 15°C to about 45°C.

7. A drug abuse testing device made in accord¬ ance with the method of claim 4.

8. A method of testing a physiological test solution for the presence of abuse-type drugs therein, said method comprising:

OMPI a) providing a drug abuse testing device as defined in claim 1, and b) placing a drop of the test solution from a drop producing means onto said testing device.

9. A method as defined in claim 8 wherein the drop producing means comprises a capillary pipet, and said placing step includes introducing the test solution into the drug abuse testing device via capillary action from the pipet.

10. The method as defined in claim 4 wherein the staining agent is effective to indicate the presence of a narcotic in the test solution.

11. The method as defined in claim 10 wherein the staining agent contains a platinum salt selected from the group consisting of chloro¬ platinate, hydrogen chloroplatinate and potassium chloroplatinate.

12. The method as defined in claim 10 wherein -the staining agent is absorbed from a solution containing bismuth subnitrate in the range of 1.4% to 1.6% weight per unit volume, acetic acid in the range of 14% to 16% weight per unit volume, chloro¬ platinate in the range of 0.06% to 0.13% weight per unit volume, potassium iodide in the range of 4% to 6% weight per unit volume, and hydrochloric acid of a normality in the range of 0.8 to 1.2.

OMPI

13. The method as defined in claim 12 wherein additional staining agent is absorbed from a further solution containing chloroplatinate in the range of 0.12% to 0.25% weight per unit volume, potassium iodide in the range of 1.75% to 2.25% weight per unit volume, and hydrochloric acid of a normality in the range of 0.8 to 1.2.

14. The method as defined in claim 1 wherein the staining agent is absorbed from a solution containing chloroplatinate in the range of

0.12% to Q.25% weight per unit volume, potassium iodide in the range of 1.75% to 2.25% weight per unit volume, and hydrochloric acid of a normality in the range of 0.8 to 1.2.

15. The method as defined in claim 11 wherein the stainining agent is absorbed from a solution containing per 4Q0 milliliters of solution 0.5 grams of K₂PtCl_g, 10-12 grams KI, 100 milliliters of H₂0, 100 millileters of 2N HCl and 200 milliliters of 1.5%-1.7% bismuth subnitrate in 15% acetic acid.

16. The method as defined in claim 11 wherein the staining agent is absorbed from a first solution containing the following relative quantities: 0.5 grams K₂ tCl_g, 100 ml of 12% aqueous KI and 100 milliliters of 2N HCl and thereafter from a second solution containing the following relative quantities: 0.5 grams K₂ tClg, 100 ml of 4% aqueous KI and 100 ml of 2N HCl.

OMPI

17. The method as defined in claim 11 wherein the staining agent is absorbed from a solution containing the following relative amounts of materials: 0.1 gram bismuth triiodide, 0.1 gram K₂ tClg and 0.9 gram KI in 20 ml of water mixed in a ratio of 1:1 with sufficient 2N HCl to produce 40 ml of solution.

18. The method as defined in claim 11 wherein an ultrasensitive test paper contains a staining agent absorbed from a first solution contain¬ ing the following relative amounts of material 0.5 grams K₂PtCl_g, 100 ml of 4% aqueous KI and 100 ml 2N HCl and absorbed from a second solution containing the follow¬ ing relative amountsof material, 1-1/2 volumes of 1.5% to 1.7% bismuth subnitrate in 15%-20% acetic acid mixed with ten volumes of the first solution.

19. The method as defined in claim 11 wherein the carrier matrix is contacted by

0.05 milliliter to Q.3 milliliter of said test solution-

20. The method as defined in claim 4 wherein the staining agent is effective to indicate the presence of an amphetamine in the test solution.

21. The method as defined in claim 20 wherein the staining agent is Ninhydrin.

22. The method as -defined in claim 21 wherein the solution incorporating the staining agent into which the matrix is to be dipped contains 5% to 6% of Ninhydrin in a solvent selected from the group consisting of methyl alcohol and acetone.

23. The method as defined in claim 22 wherein the solution contains a coloring agent for the matrix.

24. The method as defined in claim 4 wherein the staining agent is effective to indicate the presence of a barbiturate in the test solution.

25. The method as defined in claim 24 wherein the staining agent contains S-diphenyl¬ carbazone and a chloride selected from the group con- sisting of mercuric chloride and cupric chloride.

26. The method as defined in claim 25 wherein the solution contains 0.25 grant, of S-,cxphenylcarbazone and 5 grams of mercuric chloride in 100 ml of a volatile organic solvent.

27.. The method as defined.^'.in claim 25 wherein the carrier matrix is dipped into a first solution in the proportion of 0.20 to 0.25 gm of S-diphenylcarbazone and 4 to 5 grams of mercuric chloride and into an aqueous second solution of 3% to 5% polyvinyl alcohol.

^{■ j} ME T OMPI

28. 2he method as defined in claim 24 wherein the carrier matrix is dipped into a first solution of 1.5% to 2.5% aqueous silver nitrate and into a second solution of 4.5% to 5.5% methanol 5 copper chloride.

29. The method as defined in claim 4 wherein the impregnating step includes (1) preparing at least a first solution containing a staining agent reactive with, abuse-type drug residues 10 of the group found in physiological body fluids, and (2) immersing into the solution said carrier matrix having a wet strength of at least 0.44 pounds per square inch and an absorbancy of dry chemicals of at least 0.05 gram per square inch.

15 30. The method as defined in claim 29 wherein the solution contains 5% to 6% Ninhydrin in a volatile organic solvent selected from the group consisting of methyl alcohol and acetone and immersing the matrix in the solution for at least 5 seconds.

20 31. The method as defined in claim 29 wherein the solution contains iodoplatinate and 2 normal hydrochloric acid, the additional steps com¬ prising aging the solution for at least 2 days in light and air prior to use.

•25 32. A drug abuse test indicator device comp¬ rising: a carrier matrix having a porosity capable of absorbing at least 0.05 gram: of dry chemicals per square inch and having deposited in the interstices of said carrier matrix a staining agent absorbed from a solution containing a stain— ing agent reactive with abuse-type drugs selected from the group consisting of narcotics, amphe amines, barbiturates and tranquilizers.

33. The indicator device as defined in claim 32 wherein said solution contained iodoplatinate and 2 normal hydrochloric acid.

34. The indicator device as defined in claim 33 wherein said solution also contained bismuth subnitrate.

35. The indicator device as defined in claim 32 wherein said solution contained Ninhydrin.

36. The indicator device as defined in claim 32 wherein said solution contained S-diphenylcarbazone and a chloride selected from the group consisting of mercuric chloride and a cupric chloride.

OMPI