WO1986003009A1

WO1986003009A1 - Process for the preparation of a bibulous reactive medium

Info

Publication number: WO1986003009A1
Application number: PCT/US1985/002219
Authority: WO
Inventors: Richard A. Curtis
Original assignee: Med-Tech Products, Inc.
Priority date: 1984-11-16
Filing date: 1985-11-12
Publication date: 1986-05-22
Also published as: EP0201586A1

Abstract

Process for the essentially uniform impregnation of a bibulous medium with a stabilized reagents system prepared from an iodoplatinate solution and a Dragendorff reagent. The bibulous medium is, thereafter, cured at elevated temperatures and in a moisture-laden environment to insure uniformity of distribution of the reagent mixture and to allow adequate interaction of the reagents in the formation of a stabilized reactive environment within the bibulous medium. Following such curing, the bibulous medium is gradually brought to ambient conditions, packaged in opaque medium and stored in a refrigerated environment. The reactive bibulous medium prepared in the foregoing manner is useful in the selective detection of amphetamine and methamphetamine type drugs in urine and other biological samples.

Description

Title: PROCESS FOR THE PREPARATION OF A BIBULOUS REACTIVE MEDIUM

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION - This invention is directed to a process, a method and to articles of manufacture. More specifically, this invention concerns itself with a process suitable in the manufacture of amphetamine and methamphetamine sensitive components of diagnostic test kits; a method for the detection of amphetamine and methamphetamine-type drugs; and, to the various components of the test kits.

2. DESCRIPTION OF THE PRIOR ART - The technical and patent literature is replete with disclosures of various devices and techniques for screening biological samples for prescription drugs and drugs of abuse.

A comprehensive review of the various reagents/staining systems which are efficacious for the identification of narcotic and other common drugs is disclosed in

BIOCHEMICAL PHARMACOLOGY, edited by Doris H. Clouet,

Plenum Press, New York, N.Y. (1971). The techniques described by Clouet and others for detection of amphetamine type drugs generally involve the chromatographic separation of a biological sample containing the suspected agent, the treatment of the chromatogram with ninhydrin to fix the amphetamine within the chromatographic medium; and, the ultimate development (visualization/intensification) of the amphetamine spot with a staining agent.

The staining agents which can reportedly be used in the visualization/intensification of the ninhydrin fixed

- amphetamine spot include iodoplatinate and Dragendorff reagent, Davidow, et al, Am. J. Clin. Path. Vol. 38, No. 11, pages 714-719 (1968). In the assay described in the Davidow article, a positive control is run in parallel to the biological sample containing in the analyte of interest; the locations of the various control spots having been previously identified on the chromatogram and their positions noted. The chromatogram is thereafter developed with a combination of iodoplatinate and Dragendorff reagent. The spots in the sample which correspond in position to the positive controls are presumed to be the same as the drugs contained in the controls.

Commonly assigned co-pending application Serial No. 177,237, entitled "Drug Abuse - Test Paper and Method" (filed in the name of Jacob J. Gibson on August 11, 1980) describes and claims a test method for detection of drugs of abuse in a bibulous matrix. According to Gibson, test papers for detection of amphetamine can be prepared by pre-sensitization of a bibulous matrix with ninhydrin, and optionally, FD&C #11 yellow; a yellow colored background having apparently been found desirable for detection or enhancement of the constituent manifesting reaction product.

The Gibson test papers are prepared by simple immersion of the bibulous matrix in the ninhydrin/acetone-containing solution followed thereafter by air drying the saturated bibulous matrix. The application of an amphetamine control to the spot does not provide immediate identification of the presence of the drug. In order to verify (i.e. develop the constituent manifesting reaction), the test paper containing the control spot, is heated to about to 60 degrees Centigrade for about 10 minutes. The presence of an amphetamine drug is manifest by the development of a light tan or light magenta spot within the paper. A negative (amphetamine deficient) sample is indicated by a dark blue or purple spot.

Commonly assigned co-pending application ' Serial No. 520,463 entitled "Drug Abuse Test Paper" (filed in the name of Jacob J. Gibson on August 4, 1983,) also describes and claims a method for detection of drugs of abuse in a bibulous matrix. According to Gibson, test papers can be prepared by an immersion sequence involving the treatment of the bibulous matrix with two different treatment baths containing both a Dragendorff reagent and iodoplatinate. In the initial and subsequent treatments of the paper, the relative proportions of iodoplatinate to Dragendorff reagent is varied.

The initial treatment of the bibulous matrix simply involves immersion of the matrix in a reagent bath followed by the controlled drying in an environmental chamber under static conditions for approximately three (3) hours. A curing period (i.e. aging) of two (2) weeks is required between the initial and subsequent treatments of the test paper with the reagent mixture. Both initial and subsequent treatment steps are reportedly required for adequate sensitization of the bibulous matrix. The second treatment of the matrix with the reagent mixture involves a similar immersion sequence followed by a controlled drying in an environmental chamber at 50 degree Centigrade and high humidity conditions. The second drying cycle is also reportedly complete within about three (3) hours and the resultant test paper ready for use at that time.

The test papers prepared according to both copending Gibson applications are reportedly suitable for detection of amphetamine-type drugs. This is achieved by simple dropwise addition of the physiological sample to the test paper followed by a suitable incubation period. In the case of application Serial No. 177,237, such incubation can be performed either under ambient conditions (drying at room temperatures for about two (2) hours) or at elevated temperatures in an environmental chamber at 50-70 degrees Centigrade for about ten (10) minutes.

The test method of application Serial No. 520,463, is similar to that described in the earlier' Gibson application. The sample is, however, taken up in a capillary tube and applied by direct contact with the test paper. Following diffusion of the sample into the test paper, the test paper is allowed to dry. The drug manifesting reaction is read after about ten (10) minutes. Gibson reports better discrimination from a well-defined chromatogram is achieved by dropwise addition of the sample to the test paper, however, such method of application of sample to the test paper also results in some loss and sensitivity.

As is evident from review of the prior art and co-pending Gibson applications, detection of drugs of abuse, in general, and amphetamines in particular, requires subtle differentiation between the various bands which are developed during the radial diffusion of the sample through the reactive matrix of the test paper. As is also evident, the ability to make a positive differentiation between the various drugs which can be present in a sample is both desirable, and often essential, depending upon the objectives of the party administering such tests. The relative ability to achieve such differentiation appears to be dependent upon the relative stability of the test paper which in turn appears to be related to the manner in

-T7 which such test papers are prepared. Accordingly, there is a continuing need for improvement in both the differentiation and sensitivity of such test papers and in the simplification of the process by which such papers are prepared.

OBJECTS OF THE INVENTION

Accordingly, it is the object of this invention to remedy the above as well as related deficiencies in the prior art.

More specifically, it is the principle object of this invention to provide a simplified process for the manufacture of a stable reactive medium that is capable of differentiation of amphetamine and methamphetamine-type drugs contained in biological samples.

It is another object of this invention to provide a process for the manufacture of a stable reactive medium by simple straightforward processing techniques.

It is yet another object of this invention to provide a stable reactive porous medium for selective detection of amphetamine and methamphetamine-type drugs contained in biological samples.

It is a further object of this invention to provide an improved method for differentiation of amphetamine-type drugs utilizing the reactive medium prepared in accordance with the process of this invention.

Additional objects of this invention include the utilization and adaptation of the manufacturing process of this invention to the preparation of reactive medium for drugs of abuse other than amphetamines.

SUMMARY OF THE INVENTION

The above and related objects are achieved by providing a process for the manufacture of a reactive medium which is capable of detection and differentiation of amphetamine and methamphetamine-type drugs in physiological samples. In brief, this process initially involves the saturation of a solid inert bibulous medium with a reactive mixture consisting essentially of an iodoplatinate solution and a Dragendorff reagent. The above constituents of the reactive mixture are combined under conditions, and in a manner, calculated to produce a stable treatment bath. The bibulous medium is, thereafter, saturated with the treatment bath by immersion or an equivalent methodology; allowed to briefly drain for the removal of excess reactive mixture; and, suspended in a curing chamber.

Throughout the process of preparation of the reactive mixture and the treatment of the bibulous medium with such reactive mixture, the constituents of the reactive mixture are preferably shielded from destabilization by activating electromagnetic radiation. This shielding is also continued during the curing stage of this process. In the most preferred embodiments of this invention, the preparation of the reactive mixture and the impregnation of the bibulous medium is performed within the controlled environment of an environmental chamber at a temperature in the range of 40-45 degrees Centigrade and under safe light conditions.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a systems flow diagram of the manufacturing process of this invention.

Fig. 2 is a perspective view of the reactive porous medium of this invention contained in a slide mounting.

Fig. 3 is a perspective view of a device which can be used in the aspiration of sample and the application thereof to the reactive porous medium of the slide mount of Fig. 2.

Fig. 4 is a perspective view of one method of application of the biological fluid sample to the reactive medium of the slide mount of Fig. 2.

Fig. 5 (A) - (E) illustrate various features of the device illustrated in Fig. 3.

Fig. 6 is an illustration of a typical radial chromatogram for amphetamine developed within the porous medium of Fig. 2.

Fig. 7 is an illustration of a typical radial chromatogram for methamphetamine developed within the porous medium of Fig. 2.

Fig. 8 is a further illustration of the porous medium assembled in a matchbook-like configuration.

Fig. 9 illustrates a fixture which can be used in the application of the physiological sample to the porous medium of Fig. 8.

DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTS In the description which follows the terms "porous" and "bibulous" are used interchangeably in reference to the media which sensitized with the reagent mixture of this invention. The functional characteristics of this media which are essential to its compatibili y with the reagents and methodology described hereinafter is that it be essentially inert ( i.e. non-reactive) relative to such reagent mixture and to the contituents of the physiological sample. In addition such media must also be capable of rapid and uniform distribution of the sample, which is applied thereto by, capillary action.

The various Figures which accompany and form part of this description are simply illustrative of a number of the preferred embodiments of this invention and provided as an aid to understanding the scope of this invention. For example, Fig. 1 represents of a systems flow diagram of the process of this invention. Initially, the various constituents of the reactive mixture are separately prepared. As indicated previously, the two constituents of the reactive mixture comprise an iodoplatinate solution and a second solution comprising a Dragendorff reagent.

The iodoplatinate solution, as is appreciated by those skilled in this art, can contain and be prepared from various salts or platinum and potassium iodide. In the preferred embodiments of this invention the iodoplatinate solution is prepared by initially dissolving metallic platinum in hydrochloric acid to prepare a stock solution. The preferred concentration of platinum in this stock solution is approximately 500 parts per million. The preferred source of iodide salt for the iodoplatinate solution is potassium iodide. Typically, this latter solution is prepared by dissolving anywhere from 5-7 grams potassium iodide in 60 milliliters hydrochloric acid. This iodide salt solution is then added to about 30 milliliters of platinum stock solution. The combined mixture is further diluted with 40 milliliters of water. The resultant relative concentrations of materials in this solution provide a stable interactive composition which, when combined with a Dragendorff reagent, provides a stable treatment bath for the preparation of the reactive bibulous medium of this invention. In the preferred embodiments of this invention, the relationship between the iodide salt and platinum in such acid solution should preferably be adjusted so that the iodide ions are in stoichmetric excess relative to the available polyvalent platinum. The equation which follows is believed to be illustrative of the ionic interactions between above reagents

PtClg + xKI S⁰-! pt(I_χ)(Cl)₆__χ ⁺ + xK⁺ + xCl^"

Pt(I_χ)(Cl)₆__χ ⁺ + Bi >Pt(l)₆ ⁺ + Bi + Cl^" + I^"

The iodoplatinate complex of the above equation is believed to become associated with the amphetamine and me thamphetamine in the sample solution. It is hypothesized that it is this resultant platinum complex which consequently promotes an alteration of the oxidation of the bismuth as is evidenced by the appearance of visible precipitate.

The Dragendorff reagent which is used in the formulation of the treatment bath of this invention is typically a bismuth subnitrate salt which is dissolved in glacial acetic acid. This solution can be prepared by initially dissolving sufficient bismuth salt in glacial acetic acid to prepared an 8-10 weight per cent stock solution. This stock solution is thereafter diluted with distilled water such that the ultimate concentration of bismuth salt is reduced by a factor of 8-10 times. Such dilution can, in a typical example, be routinely accomplished by addition of 10 mis of stock solution to 90 mis of water, i.e. yielding an approximately 1.0 weight percent solution.

As is well known in this art, the relative stability of the Dragendorff reagent and the iodoplatinate solution are to a degree dependent upon the manner in which they are prepared; the manner in which they are stored; and, the manner in which they are later combined. In the preferred embodiments of this invention, these reagents are prepared within about 24 hours prior to their use. Subsequent to their initial preparation, they are stored in light-s'h.ielding containers (i.e. actinic glass bottles). Prior to their initial combination with one another, both solutions are preheated to a temperature in the range of 40-50 degrees Centigrade. They are, thereafter combined with one another, in the appropriate proportions. A preferred treatment bath of the process of this invention is prepared combining 60 mis of 1.0 weight percent Dragendorff reagent and 40 mis of iodoplatinate solution under the appropriate controlled conditions. In the preferred embodiments of this invention, the preparation of this treatment bath from the iodoplatinate solution and Dragendorff reagent takes place within an environmental chamber under safe light conditions.

After these two solutions are combined to form a treatment bath, a bibulous medium (i.e. filter paper) is immersed in the resultant solution. The duration of immersion is sufficient to essentially completely saturate the bibulous medium. Of course, the thickness of the medium and other parameters will determine the extent of absorption of the fluid and the speed with which such materials diffuse within the medium. Upon essentially complete saturation of the bibulous medium, it is removed from the treatment bath, allowed to briefly drain, and suspended vertically within the environmental chamber. This process is repeated with additional sheets of filter paper, the environmental chamber sealed and steam introduced therein in short burst (generally of three minutes duration). The steam is discontinued for a brief period of time (on the order of about 3-8 minutes) to allow adequate equilibration of the environmental chamber. Subsequent, thereto, additional steam is introduced for a similar duration until the chamber has adequately stabilized and the temperature therein is in the range of 50-60 degrees Centigrade (preferably about 55 degrees Centigrade.) During this initial curing period, the temperature and humidity of the chamber are monitored constantly. Once conditions within the chamber have stabilized, no additional steam is introduced and the temperature allowed to return to the precuring level (40-45 degrees Centigrade).

The interval required for the temperature to return to this level does, of course, depend upon the extent of insulation in the chamber and other factors. Good results have been achieved where the gradual drop in temperature requires at least an elapse of one hour. The temperature is maintained at this 40-45 degree Centigrade level for an additional one hour and the heating elements thereafter turned off in order to allow the temperature to gradully cool to room temperature (20-25 degrees Centigrade). At the point in time when the heating elements are turned off within the environmental chamber, the treated bibulous medium is still moist. The treated papers are allowed to remain in the chamber until essentially completely dry (generally overnight). The treated papers are thereafter removed from the chamber, packaged in a light-shielding envelope and sealed. If prolonged storage is required, it is suggested that the envelope be placed in a refrigerated environmen .

The process of curing the papers in the manner hereinbefore described is intended to permit the controlled interaction of the various components of the treatment bath within the bibulous medium. It is hypothesized that the injection of steam retards the drying process and thus insures the maintenance of a liquid phase within which to insure such controlled interaction. It is also believed that the above treatment of the chemicals within the bibulous medium elevates such ingredients to a stable energy plateau which is receptive to interaction with the analytes of interest; and, facilitates the uniform distribution of reagents within the paper.

The maximum temperatures which can be used in this curing process are, as is evident from the above discussion, functionally related to the objectives of controlled interaction of the various constituents of the treatment bath and the constraint against premature drying of the liquid phase. In the absence of proper curing, the resultant papers do not possess sensitivity or stability required to provide the degree of discrimination necessary to effectively screen for the analytes of interest.

Once this curing process has been completed, it is recommended that the resultant materials be placed within an envelope which is both exclusive of activating electromagnetic radiation and prevents sublimation of the acidic constituents contained within the paper. It is believed that a pH of from about 2 to 4 is requisite to insure the degree of differentiation necessary to selectively identify amphetamine and methamphetamine-type drugs with the reactive constituents contained within the bibulous medium. As the recative mixture within the paper ages, there is a tendency for the acidic constituents therof to be lost if the paper is not properly sealed prior to use and/or maintained in a refrigerated environment. Both of these precautions are recommended to insure consistency of performance and adequate sensitivity. In the event that adequate acidity is not maintained, the specificity of the paper decreases, accompanied by an increase in false positives. Accordingly, the maintenance of proper acidic conditions is both critical and essential to the practical and commercial applications of this reagent system.

Once the test papers have been prepared in the foregoing manner, they can be precut to any size suitable and/or appropriate for convenience testing. In Figure 2 is illustrated one of the embodiments in which it is contemplated such test papers can be used. The test paper (2), as shown in Figure 2, is contained within a slide-type mount (4). The mount permits the handling of the sensitized test paper without contact by the fingers of the user. In this way, it is insured that no extraneous matter will be transferred to the sensitized medium and accordingly preserves the integrity of the reagent system contained therein.

The geometry of the access window of the slide mount can be configured to cooperate with an application guide means. In the embodiments of this inventions illustrated in Figures 3 and 4, this guide means comprises a test tube-like device (6) having a small hole (8) in the closed end (10), (somewhat larger in diameter than the outside diameter of the cross section of a capillary tube), and an open end (12) which conforms in geometry to the aperature of the slide mount. In addition, the open end of the applicator guide means (14) is provided with internal support means to assist in pre-positioning of a capillary tube (20) (containing the patient's sample) at the appropriate location on the bibulous medium contained within the slide mount. Once the capillary tube is positioned in the foregoing manner, the capillary tube is brought into virtual contact with the bibulous medium and the fluid sample allowed to flow from the capillary tube into the medium.

Figure 5A represents an enlargement of the closed end (10) of the device (6) illustrated if Figure 3. Figure 5B

* rpresents an enlarged cross-sectional view through the device of Figure 3 at BB. The fins (15) at the closed end

(10) of the device (6) provide precise positioning of the capillary tube (20) relative to the test paper (2) in the slide mount (4). Figure 5C is an enlarged view of the open end (12) of the device of Figure 3. This view shows engagement of the applicator guide means (14) with the slide mount (4) to insure precise positioning of the capillary tube (20) relative to the test paper. Figure 5D is a top plan view of the overcap (24) of Figure 3. This overcap is prepared form a flexible material and designed for compressive engagement of the capillary tube (20). In operation of this overcap relative to the other components illustrated in Figure 3, the overcap is place in relation to the capillary tube to permit controlled immersion of the tube into the sample container (80) so as just touch the surface of the fluid contained therein. The ^"fluid is drawn into the capillary tube by capillary action. and this tube thereafter positioned relative to a test slide as shown in Figure 4. The tube is then brought into virtual contact with the test paper thus allowing the fluid to flow into the paper at a controlled rate. Upon completion of application of the fluid to the test paper the applicator guide means is removed from contact with the slide mount, the compressive force on the overcap released and the capillary tube allowed to freely fall from this assemby into an appropriate receptacle for disposal.

Figure 6 illustrates a typical radial chromatogram which is produced in this manner. The radial band pattern of Figure 6 is indicative of the presence of amphetamines in the biological sample.

Figure 7 illustrates a similar radial chromatogram wherein the radial band pattern is indicative of methamphetamines .

Figure 8 illustrates another embodiment of this invention wherein the bibulous medium containing the reactive mixture in configured in a matchbook-like package. Once the raatchbook is open, the bibulous medium simply unfolds exposing the bibulous medium (26). A section of the bibulous medium can then be pre-posi ioned in the throat (30) of applicator guide means (40) of the type which are illustrated in Figure 9. The applicator also contains means (41) for guiding a capillary tube relative to a preselected position on the reactive bibulous medium. Once such positioning has taken place, the capillary tube is placed in virtual contact with the bibulous medium and the sample contained therein allowed to flow into the bibulous medium in a controlled manner. The chromatographic pattern which is produced is similar to those shown in Figures 6 and/or 7 depending upon the analytes present in the biological sample. The manner of application of the patient's sample to test paper has been found to be both critical and essential to the development of the appropriate chromatographic patterns and to the sensitivity of the assay. In the foregoing description of the applicator systems, it has been found both desirable and essential that the capillary tube, containing the patient's sample, be in virtual contact with the reactive bibulous medium during the application of the biological sample to the bibulous medium. The failure to provide such contact during this application step has been demonstrated to be substantially less effective than the drop-wise addition of the patient's sample to the bibulous medium. Further comparisons are provided in the examples which follow. More specifically, it has been found that, the drop-wise addition of the patient's sample to the reactive bibulous medium fails to provide the controlled diffusion of the sample throughout the bibulous medium; and, does not permit the degree of precision in placement of each drop relative to one another on the reactive porous medium, thus further affecting the results which are obtained.

In order to further enhance this assay, it has also been found that acidification of the physiological sample prior to contact with the reactive bibulous medium substantially enchances both the quality and the sensitivity assay. It is believed that the reduction of the differential in acidity between the sample and the reactive porous medium provides better compabability between these two dissimilar reactants and accordingly enhances the reaction therebetween. In the preferred embodiments of this invention, good results are obtained where approximately one drop of a sulfuric acid solution is used for each 5 milliliters of urine. This sufficiently alters the pH of the urine to more closely approximate the ph of the reactive bibulous medium. The substitution of other mineral acids for sulfuric acid does not appear to provide the same degree of enhancement in compatability, and the reasons for such differences are not known. Sulfuric acid also appears to be superior to acetic acid in enhancing the compatability of the biological sample to the reactive bibulous medium.

After the patient sample has been dispensed, as described hereinabove, onto the reactive bibulous medium it is preferable to wait a minimum of 30 seconds for adequate diffusion and interaction of the sample within the bibulous medium prior to attempting identification of the various chromatographic band patterns. In practice, an initial reading can be taken after about 30 seconds from the time of application of the patient's sample and again about two minutes after initially dispensing the patient's sample onto the reactive porous medium. In the preferred emb.odiments of this invention, the bibulous medium comprises a filter paper which is relatively thick when compared to conventional chromatographic media. Thus, the diffusion of sample within the porous medium occurs within two dimensions (laterally and vertically). Upon completion of the diffusion throughout the porous medium, and the settling of the sample therein, the band pattern on the reverse side of the filter paper is substantially more dramatic than the application side of the paper. Accordingly, it is recommended that the chromatographic band pattern on the reverse side of the paper be used additionally as indicative of the absence or presence of the analyte of interest.

The analytical systems described herein provide not only qualitative determinations of constituents of the biological sample but also provides a semi-quantitative capability as well. The band pattern will generally remain the same for a given drug, however, the band width will vary with the concentration of drug in the physiological sample; the broader the band pattern being indicative of a greater concentration of drug.

The Examples which follow further describe, define, and illustrate a number of the preferred embodiments of this invention. The apparatus and techniques used in both the preparation and evaluation of the reactive bibulous medium are standard or as hereinbefore described. Parts ' and percentages appearing in such Examples are by weight unless otherwise stipulated.

EXAMPLE 1

A test paper was prepared by implementation of a 3-stage chemical impregnation procedure consisting of absorption, curing, and drying stages.

Absorption of impregnating chemicals into a biblious matrix was accomplished by simple submersion of said matrix into an opaque glass dipping vessel containing a modified Drangendorff-iodoplatinate formulation. The absorption phase took place within an environmental chamber where individual constituent articles that came into contact with impregnating formulation were held at an equal and constant temperature with said formulation. The matrix was submerged for a period not to exceed 5.0 seconds in the impregnating solution for optimal absorption to occur.

Curing of the impregnated matrix was potentiated by subjecting the matrix to rapid elevations of temperature and humidity by controlled steam injection, to achieve a

'— tr^>". ^'"^τrτ^* temperature in the range of 40-56 degrees C and a humidity level within the range of 80-100%. When appropriate levels were attained the induced conditions were stabilized and maintained by a chamber radiant heat system combined with water atomization. All phases of above procedure were conducted in safe light.

Following a two-hour curing period, controlled cooling was initiated with an electronic chamber damper mechanism which facilitated a gradual acclimation of the drug papers to a normal room environment over a 15-hour drying period.

Papers were then removed from the chamber, placed in opaque envelopes and stored at 2-8 degrees C subsequent to testing.

Positive amphetamine and metahmphetamine urines were obtained for application with test papers from a single does study where 5 mg of DEXADRINE and 10 mg of DESOXYN were ingested independently by two individuals. Control urines were collected prior to drug ingestion to serve as negative controls and subsequent positive urines collected during normal expected excretion periods for each drug. Presence of drug in sample was confirmed by HPLC.

Test Formulation for 100 sheets of paper (6 1/4" x 10 1/2")

Bismuth Subnitrate 8.4 mg Glacial Acetic Acid 84.0 ml

Purified Water 516.0 ml

Potassium Iodide 25.0 g

Platinum metal in HC1 200.0 ml (500ppm) 2N Hydrochloric Acid 600.0 ml

Purified Water 400.0 ml

EXAMPLE 2

A test paper was produced as in Example 1, except attainment of 90-100% humidity level by steam injection was not achieved. This was accomplished by manipulation of appropriate chamber mechanisms responsible for regulation of the internal environmental factors. The resultant papers produced possessed, upon examination, marked variation in distribution of indicating reagents, several papers exhibited a course particulate striation indicating the inability of necessary critical chemical reactions to reach their respective endpoints.

Upon testing of these papers with positive amphetamine and methamphetamine -urine standards, chromatographic patterns lacked clear definition, and characterization was not possible. The rose colored banding pattern exhibited on "steam processed" paper by positive amphetamine urines was not present. The black textured banding pattern exhibited by methamphetamine positive urines was still distinguishable on the paper when using 5ug/ml samples, however, the paper did not possess the sensitivity to detect weakly reactive (less than 5ug/ml) methamphetamine urines.

EXAMPLE 3

An amphetamine test paper was produced as in Example 1 except safe lighting conditions were not maintained during the curing process. This was accomplished by exposing suspended papers to incandescent as well as fluorescent lighting throughout the reactive processing stages of development .

Resultant papers thus produced upon appeared, upon gross examination, discolored when compared with paper lots produced with safe lighting under identical environmental conditions .

Distribution of impregnating reagents appeared normal, and the texture of the paper produced appeared to be identical with papers produced under safe light conditions.

However, upon testing papers with amphetamine, and methamphetamine positive urines, there was a substantial decrease, in color intensity of the resultant banding patterns produced, thus indicating a lack of sensitivity of the paper in detecting positive urines at abuser levels .

EXAMPLE 4

An amphetamine test paper was produced as in example 1 with the exception of the omission of the final controlled delayed cooling stage. This deviation of normal paper processing procedure was facilitated by decreasing the internal chamber humidity to less than 50% and maintaing the 40-50 degree C chamber temperature for an additional 2 hours following the normal curing time.

As a result of abrupt cooling, final paper coloration appeared darker than papers processed with normal final stage drying time and associated environments. In addition to overall discoloration, the periphery of each paper was further differentiated by a l-5m_α area of discoloration caused by initial exposure the outer edges of the paper to accelerated drying conditions. Testing these papers with positive amphetamine and methamphetamine urines demonstrated poor chromatographic separation in addition to a generalized decrease in expected color intensity.

EXAMPLE 5

An amphetamine test paper square was used for application and evaluation of amphetamine and methamphetamine positive urines. The test square was placed on several different test surfaces, including glass, plastic and Whatman #3 filter paper to determine the effect of the underlying surface on the chromatographic migration pattern of applied substances.

As a result of the above applications, it was determined that the rate of migration, in addition to the intensity of reaction on the test paper, was directly dependent upon the absorbant properties present of the underlying test surface. The greater the absorbant properties of the test surface, the lesser the degree of particle migration was observed in addition to a decrease in the indicating color intensity exhibited.

Optimal results were achieved when the urine sample was allowed to diffuse through the test matrix while such matrix was suspended above any underlying hard surface area.

It was further observed that, due to the tendency of dense reactive particles to migrate vertically in addition to horizontally - i.e. settling effect, the reverse side of the chromatogram often exhibited an intensified reaction as compared to the opposite or top side. Thus, for this device to achieve optimal results the test paper square is either framed in a bracket, which allows the test paper to remain independent from a test surface; or, the test paper is inserted into a holder or suspension device to achieve the effect.

Following application of sample, three dimensional readings are obtained by alternate observation of both sides of the test square.

EXAMPLE 6

A sample of amphetamine and methamphetamine positive urine was applied to different aphetamine paper test squares utilizing a pasteur pipet and, alternately, a 50 microliter capillary pipet. It has been determined that the method and device with which the specimen is applied to the test paper is crucial to the readability and detectability of the resulting chromatogram produced.

In the case of the pasteur pipet, 2 drops of each positive urine was applied to respective test papers and patterns observed. In both cases, using amphetamine and methamphetamine urine samples, chromatrographic banding was minimal and diffuse, increasing the number of false negative determinations.

Upon application with the 50 microliter capillary pipet, 50 microliters of sample diffused onto the test paper by gravity with the pipet being held perpendicular to, and in virtual contact with the test paper during application. Resultant chromatographic patterns were sharp and distinct with an 85% - 90% increased confidence level gained by reducing the number of false negative results obtained with the pasteur pipet. EXAMPLE 7

A positive methamphe amine and amphetamine urine was pre-treated with specific amounts of several different acids prior to application of urine to drug test paper.

Equivalent amounts of acetic acid, hydrochloric acid, sulfuric acid, and nitric acid were each used.

Due to the acid nature of the test matrix, normal urine (pH 6-7) exhibits a balanced appearnance on the test paper due to the simple acid-base relationship betwen the urine sample and the paper. The resultant balanced pattern yields a non-descriptive chromatogram where desired color and banding patterns are not exhibited.

To reduce the differential between the sample and the test paper, sample pretreatment with acid was necessary. Of the acids listed above, it was found that pretreatment with sulfuric acid (1-2 drops/5ml sample) gave optimal chromatographic characteristics over other acids used.

Claims

- 25 - WHAT IS CLAIMED IS:

1. A process for the manufacture of a reactive bibulous medium suitable for the differentiation of amphetamine, methamphetamine and metabolites thereof from other constituents of a physiological sample; said process consisting essentially of:

(a) providing a stable reactive mixture consisting essentially of an iodoplatinate solution and a Dragendorff reagent by combining said solution and said reagent at 40-50 degrees Celsius, the relative concentration of the above recited materials in said mixture being insufficient to effect precipitation of either recited materials or the precipitation of the product of their interaction;

(b) applying said mixture to the bibulous medium at a temperature in the range of from about 40-50 degrees Centigrade and in quantities sufficient to saturate said bibulous medium; and

(c) curing said mixture within said bibulous medium in a moisture laden environment at a temperature in the range of from about 50-60 degrees Centigrade, the duration of such curing being sufficient to effect essentially uniform distribution of the mixture within the bibulous medium and the formation of a stable reaction product therebetween.

2. The process of Claim 1, wherein steps (a) through (c) are performed under conditions which shield said reactive mixture and said bibulous medium from destablizing electromagnetic radiation.

3. The process of Claim 1, including the additional steps of the controlled cooling of the reactive medium to room temperature so as to avoid an abrupt transition therebetween.

4. The process of Claim 3, including the additional step of storing said reactive medium under safe-light conditions and in a refrigerated environment.

5. The process of Claim 4 including the additional step of storing the reactive medium under conditions for maintaining the acidity of said medium.

6. The reactive bibulous medium prepared in accordance with the process in Claim 1.

7. A method for screening biological samples for amphetamine and methamphetamine-type drugs, said method including the steps of:

(a) providing a bibulous medium which is indicative of amphetamine and methamphetamine type drugs by treatment thereof with a mixture consisting essentially of a solution of iodoplatinate and a Dragendorff reagent, the relative concentration of the above recited materials in said mixture being insufficient to effect precipitation of either recited material, or the products of their interaction, and the process of treatment said medium resulting in a stable reaction mixture essentially uniformly distributed through said medium;

(b) providing means for controlled application of the biological sample, in predetermined quantities, to a predetermined location on the reactive porous medium;

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(c) collecting said biological sample in a transfer means;

(d) placing such transfer means, containing the biological sample and application means in cooperative association with one another, and thereafter contacting said transfer means with said reactive medium, the point of contact of said transfer means relative to the reactive medium being determined by said application means; and

(e) dispensing said biological sample from said transfer means directly onto said porous medium while maintaining virtual contact between said transfer means and said porous medium.

8. The method of Claim 7, wherein the pH of the biological sample is adjusted prior to application to the reactive medium, so as to increase the acidity of the sample to more closely approximate the acidity of the reactive medium.

9. The method of Claim 7, wherein the biological sample is urine and the pH thereof is adjusted with sulfuric acid in order to reduce the differential in pH between the urine and the pH of the reactive medium.