United States Patent [1 1 Shukla et al.

[ 51 Oct. 14, 1975 [22] Filed: Dec. 26, 1973 [21] App]. No.: 428,565

[52] US. Cl 252/408; 23/230 B; 23/253 R; 23/253 TP; 73/61.1 C; 424/7 [51] Int. CLC09K 3/00;G01N 31/00; G01N 33/00; G01N 29/02 [58] Field of Search..... 252/408; 23/253 TP, 253 R, 23/230 B; 73/61.1 C; 424/7 [56] References Cited UNITED STATES PATENTS 2,229,155 1/1941 Wenker 23/253 TP 3,046,779 7/1962 Coleman 23/253 TP 3,138,544 6/1964 Barna et a1 23/253 TP 3,464,796 9/1969 Friedlander 23/253 TP 3,586,483 6/1971 Heider et a1. 23/253 TP 3,590,006 6/1971 Page et a1. 252/408 3,625,652 12/1971 Fujimoto et a1 23/230 B 3,761,227 9/1973 Conrad et a1. 23/230 B 3,832,134 8/1974 John 23/230 B OTHER PUBLICATIONS Davidow, B. et al.; Amer. J. Clin. Path., Vol. 50, No. 6, pp. 714719 (1968).

Weissman, N. et al., C1in. Chem., V01. 17, No. 9, pp. 875-881 (1971).

Bastos, et al., Clin. Chem., Vol. 16, No. 11, pp. 931940 (1970).

Primary ExaminerBenjamin R. Padgett Assistant ExaminerT. S. Gron Attorney, Agent, or Firm.lack W. Richards [57] ABSTRACT Dry paper strips impregnated with fixed amounts of chemical visualization reagents for use in the preparation of spray reagents used for the detection, identification or confirmation of drugs in urine in thin-layer chromatographic drug screening procedures.

6 Claims, N0 Drawings REAGENT IMPREGNATED PAPER STRIPS FOR 'USE IN PREPARING TLC SPRAY REAGENTS BACKGROUND OF THE INVENTION This invention is concerned with an improved means for dispensing chemical visualization reagents used to make spray reagent solutions for the visualization of drugs in thin-layer chromatograph (TLC) drug screening procedures.

Because of the large number of drugs now in use or misuse, means have been sought for detecting and identifying drugs and their metabolites in urine. One of these means which lends itself to mass-screening of drugs is that of thin-layer chromatography.

Thin-layer chromatography has gained wide acceptance in drug screening procedures, since the analysis can be performed rapidly, meets sensitivity and specificity requirements, and can be used to encompass a wide spectrum of drugs and their metabolites differing both in chemical structure and pharmacological action. A good summary of thin-layer chromatography can be found in U.S. Pat. No. 3,590,006.

Thin-layer chromatography screening procedures for detecting drugs in urine may be found in, e.g., Davidow, B. et al., American Journal of Clinical Pathology, Vol. 50, No. 6 pp. 714-719 (1968); and Weissman, N. et al., Clinical Chemistry, Vol. 17, No. 9, pp. 875-881 (1971). In such TLC drug screening procedures, if the components separated on the TLC plate then viewing it under ultraviolet light. Other known developing procedures may also be used such as heat or developing agents, e.g., iodine vapor, acids, alkalis, dyes, etc., the purpose being to render the separated components on the TLC plate distinctly visible.

One widely used developing procedure in TLC drug screening procedures is the use of spray reagents. Numerous publications andpatents report on the use of spray reagentsin the detection of drugs by TLC, for example, Davidow, B. et al., supra; Davidow, B. et al., Am. Journ. Clin. Path., 46: 58-62 (1966); Dole, V. P. et al., JAMA, Vol. 198, No. 4, p. 115 (1966); Weissman, N. et al., supra; Fujimoto, J. M. et al., Toxicology and Applied Pharmacology 16: 186-193 (1970); and Fujimoto, et al., U.S. Pat. No. 3,625,652.

In the TLC procedures for detecting. drugs in urine, color forming reagents, specific for specific drugs are sprayed on the chromatographic plates to visualize and qualitatively identify any drug adsorbed on the plate. After spraying with specific spray reagents, different colored spots appear on the plate with characteristic colors specific for certain drugs or their metabolites. Spray reagents are usually used to identify, detect or confirm the presence of drugs or their metabolites not clearly identifiable by the first development. The presence of a drug is confirmed if the Rf value of the drug as well as its specific color brought about by spraying with its specific spray reagent are identical with the sprayed control standard. Specific colors are obtained for specific drugs or their metabolites by applying different known spray reagents for each drug or drugs and their metabolites.

Prior to this invention, as exemplified in part by one or more of the aforementioned publications, one performing a thin-layer chromatographic drug screening test had to prepare the spray reagents, usually on a frequentn, perhaps daily, basis because of the spray reagents inherent instability in solution. Such a procedure is, of course, time-consuming and inconvenient. Moreover, the reagent chemicals had to be stocked, properly stored, accurately weighed out, and mixed before use. Davidow, B. et al., supra, disclose the heretofore necessary and time-consuming step involved in preparing iodoplatinate spray reagent and point up the necessity of proper storage and the need for a fresh solution. Weissman, N. et al., supra, also disclose the heretofore necessary and time-consuming step involved in preparing spray reagents and not some of the problems and cautions connected therewith, i.e., that the diphenylcarbazone solution must be protected from light and that the iodoplatinate solution is stable for two weeks in a refrigerator.

With the present invention, TLC chromatographic visualization reagents useful for preparing spray reagents are premeasured, absorbed and dried on suitable paper strips ready for instant use. Essentially all that need be done to prepare the spray reagent is to dip the dried paper strip in the appropriate solvent. In such a manner, under proper storage in foil or containers, the shelf life of spray reagents can be increased indefinitely. Fresh reagents can be prepared in a matter of minutes, e.g., 2 to 5 minutes. The weighing of small amounts of chemical reagents by the individual carrying out of the test is avoided. With the reagent impregnated strips of the present invention specific reagents and amounts are provided in a stable form on paper strips which can be easily eluted and used for chromatographic development in the determination of drug screening by thin-layer chromatography. The impregnated paper strips deliver premeasured reagents in dry condition for better stability. They reduce shipping weight and cost of the reagents to the purchaser thereof.

The reagent impregnated strips of this invention are not indicator strips such as the conventional pI-I indicator and diabetes test strips. Instead, they dispense small amounts of preweighed visualization reagents in stable form. The reagent is evenly spread over theentire area of the strip on both sides and thus the reagent dissolves or is eluted in the appropriate solvent with ease.

As pointed out, diagnostic paper test strips containing various reagents which change color on exposure to blood, urine, serum, etc., pH indicator strips and diabetes test strips are, of course, well known. U.S. Pat. No. 3,598,533 discloses a diagnostic paper strip applicable to whole blood samples of humans in testing for various disease states and is prepared by dipping a diagnostic paper strip into an organic solvent solution of cholesterol, resin and their esters and drying the strip. In use, the treated strip is dipped directly into blood and kept for a period of time long enough to permit color development on the strip. In contrast, the present invention is not concerned with color development on the strip. U.S. Pat. No. 3,418,079 discloses a diagnostic paper test strip for the determination of protein in biological fluids comprising an absorbent carrier impregnated with a dyestuff indicator, an acidic buffer and an inorganic sulfate. Here again, the principal involved is merely one of color change or development on a strip.

U.S. Pat. No. 3,016,292 is concerned with a color test indicator for detecting glucose with comprises a closed purpose is for the measurement of a specific area of the paper through elution and colorimetric determination of the dye contained therein. In contrast, the paper strips of the present invention contain a specific amount of chemical reagent capable, upon elution, of reacting with a drug (e.g., amphetamine) to produce a colored spot on a paper chromatogram containing the drug and thus providing qualitative evidence of the presence of the drug in the uring being analyzed.

1n U.S. Pat. No. 2,992,971, biological stains are used to prepare microscope slides of cells but such does not perform a reaction with another compound (e.g., narcotic). The medium used to stabilize the stains is one of the celluloses and a solubilizing compound such as urea must also be present. U.S. Pat. No. 3,] 38,544 discloses antibiotics contained in compressed discs composed ofa mixture of cellulose materials. The discs are then placed on an inoculated agar plate to determine zones of inhibition. In contrast, the reagents of the present invention are eluted from their carriers prior to being applied to the TLCplate or strip as a spray reagent to produce a chemical reaction resulting in color formation on the plate or strip which qualitatively identifies a particular drug. U.S. Pat. No. 3,666,421 discloses a single test slide or card which contains reagents for performing immunochemical reactions such as blood group typing, pregnancy tests, etc. In particular, the reagents are adsorbed antigens or antibodies. U.S.

Pat. No. 3,678,151 discloses a method for staining a biological specimen on a slide for microscopic examination.

A reagent strip called Gugol Blue Stain is marketed by Wampole Laboratories, Stamford, Connecticut. This strip is used for staining a blood smear slide for differential diagnosis of white blood cells. However, instead of soaking slides in a stain solution, a strip containing staining material is placed over a slide.

U.S. Pat. No. 3,625,652 discloses an apparatus and TLC method for the detection of narcotic analgesic and amphetamines using Amberlite XAD-2' resin to adsorb the narcotic analgesics and amphetamines from human urine. The U.S. Pat. No. 3,625,652 discloses collecting the urine fractions adsorbed on Amberlite XAD-2 resin and analyzing the fractions by thin-layer chromatography. As disclosed in the U.S. Pat. No. 3,625,652, the thin-layer chromatography plates are sprayed with a reagent specific for visualizing the drugs, i.e., narcotics and amphetamines.

A commercially available neutral resin manufactured by Rohm and Haas Company and sold under the aforementioned trademark.

The U.S. Pat. No. 3,625,652 notes that the iodoplaninate reagent used was prepared according to the directions of Cochin and Daly, Vol. 18 Experientia, p. 294 (1962), by adding 250 ml. of a 4% solution of potassium iodide to ml. of a 10% solution of platinum chloride and diluting with 500 ml. of water. With the present invention, one can use the already prepared dry potassium iodide and chloroplatinic reagent impregnated strips to prepare the iodoplatinate spray reagent used in the TLC procedure disclosed in the U.S. Pat. No. 3,625,652 by merely placing the strips in the appropriate solvent. Other thin-layer chromatographic screening procedures for detection of drugs in urine wherein the spray reagents prepared according to this invention may be used are those. for example. as set forth by Davidow, B. et al., American Journal of Clinical Pathology. Vol. 50, No. 6, 714-719 (1968) and Weissman, N. et al., Clinical Chemistry. Vol. 17, No. 9, 875-881 (1971).

SUMMARY OF THE INVENTION The invention here resides in the concept of a dry chemical visualization reagent impregnated bibulous material, e.g., paper strip, ready for instant use in preparing a spray reagent solution for drug detection by thin-layer chromatography comprising a paper strip containing impregnated therein the dried residue of a liquid solvent solution containing a visualization reagent.

The invention also resides in a method of preparing a dry chemical visualization reagent impregnated paper strip for use in preparing a spray reagent solution for drug detection by thin-layer chromatography which comprises dipping a paper strip into a liquid solvent solution of a visualization reagent.

The invention resides further in a method of preparing a chemical visualization spray reagent solution for drug detection by thin-layer chromatography which comprises dipping an impregnatedpaper strip into the appropriate spray reagent solvent solution.

There are many chromatography detection agents or visualization reagents which can be premeasured, absorbed and dried on paper strips as set forth herein. In the practice of this invention, paper strips have been impregnated with the spray reagents ninhydrin; diphenylcarbazone, chloroplatinic acid and potassium iodide (the lattertwo used together to make the spray reagent, iodoplatinate). Ninhydrin is and has been used for the detection of free amino and carboxy groups (e.g., amphetamine and other primaryamine compounds), diphenylcarbazone in the identification of barbiturates, and chloroplatinic acid and potassium iodide (as iodoplatinate) in the identification of alkaloids. The liquid solvent solution in which the visualization reagent is placed and the unimpregnated strip is dipped can be water, methanol or other suitable solvents which do not adversely affect the visualization agent and those in which it is soluble. Suitable spray reagent solvent solutions in which the impregnated strip is placed for elution are acetone, butanol, water, methanol as set forth herein and as known in the art, e.g., Bastos, et al., Clinical Chemistry, Vol. 16, No. 11, pp. 931-940 (1970). As pointed out hereinbefore, the impregnated paper strips of the present invention contain a specific amount of chemical visualization reagent capable, when eluted in the appropriate solvent, and sprayed on the chromatographic sheet, of reacting with the adsorbed drugs or their metabolite to produce a characteristic color spot for that particular drug or metabolite and thus provide clear'visual evidence of the presence of the drug in the donors urine being analyzed.

Briefly, according to the invention, bibulous material, e.g., paper, of suitable weight and absorptivity. is

- cut into convenient size strips. The strips are then dipped in the desired concentration of visualization remade as described herein, is removed from its package, foil or container, and placed in the desired amount of appropriate spray reagent solvent. The impregnated reagent is eluted from the strip into the solvent to give a spray reagent solvent containing the amount of visualization reagent on the strip. After elution is completed, the spray reagent solvent is ready to be sprayed on the chromatographic plate for visualization purposes in manners known in the art, e.g., Davidow, B. et al., Am. J. Clin. Pathol. 38: 714 (1968). Suitable spray equipment for maintaining and dispensing the spray reagent to the TLC plate, such as chromatographic spray bottles, etc., are known and readily obtainable. Upon spraying the reagent on the TLC plate, the particular characteristic color for that reagent and its corresponding drug, drugs or metabolites will appear on the plate, if present.

DETAILED DESCRIPTION OF THE INVENTION agent in liquid solvent solution and through the process of submersion and capillarry attraction, saturated with the visualization reagent in solution and subsequently dried. The dried impregnated strips containing the visualization reagent are then stored in packages, foil or 5 containers, ready for use in preparing the spray reagent solution.

In the practice of the present invention, paper has been impregnated with visualization reagent using paper weighing 185 g./mm with medium flow rate, smooth surface and with a thickness of 0.33 mm. Whatman chromatography paper 3MM meets the above criteria and it is this paper which has been used. The initial size of the Whatman paper was 46 57 cm. Strips were cut 5 cm. X 57 cm. prior to impregnation with reagents. The visualization reagent to be impregnated was placed in the appropriate liquid solvent containing the desired concentration of reagent and the reagent solution kept in a shallow container, e.g., watch glass or petri dish. The 5 cm. X 57 cm. paper strip was then passed through the reagent solution so as to allow even soaking of the reagent or until saturated. The soaked or saturated strips were then dried at 37C. to dryness in an oven. The dried visualization reagent impregnated strips were cut into 2.5 cm. X 4.5 cm. size ready for use in preparing the TLC spray reagent.

Experiments have been carried out to determine the In use, the visualization reagent impregnated strip,

The invention will be illustrated in more detail by the following description.

EXAMPLE 1 Preparation of impregnated Visualization Reagent Strips Preparation of lmpregnating Solutions 4. Ninhydrin strip 3. Diphenylcarbazone 40.0 g. 0.8. to ml.

(400 2.5 X 4.5 cm. 0.8. to 100 ml.

-Continued EXAMPLE 1 Preparation of lmpregnated Visualization Reagent Strips Preparation of lmpregnating Solutions strips) C. Chloroplatinic acid Platinum chloride 10.0 g. (200 2.5 X 4.5 cm. Methanol 0.5. to 50 ml. strips) D. Potassium iodide Potassium iodide 60 g. (400 2.5 X 4.5 cm. Distilled water OS to ml.

strips) Procedure for impregnation of Paper Strips EXAMPLE 2 Preparation of Spray Reagent Solution A. Ninhydrin Spray Reagent Solution One strip, prepared as in Example 1, is transferred to a spray bottle containing 50 m1. n-butanol and shaken gently. After complete elution the spray reagent solution will contain 100 mg. of ninhydrin.

B. Diphenylcarbazone Spray Reagent Solution One strip, prepared as in Example 1, is transferred to a spray reagent bottle containing 25 m]. acetone. The strip is gently shaken to dissolve diphenylcarbazone from the paper strip and 25 ml. water added. After complete elution the spray reagent solution will contain 5 mg. diphenylcarbazone.

C. lodoplatinate Spray Reagent Solution One chloroplatinic acid strip and two potassium iodide strips, prepared as in Example 1, are combined to make iodoplatinate spray reagent as follows:

chloroplatinic acid 1 strip potassium iodide 2 strips Distilled water q.s. to 12.5 ml. Methanol 12.5 ml.

Dissolve the content of the two potassium iodide strips by dipping the strips in 12.5 ml. distilled water with occasional shaking for 5 minutes. Remove the wet strips, and place one strip of chloroplatinic acid in the solution and follow the direction as for the potassium iodide strips. After removing the strip add an equal volume of methanol. After complete elution, the spray reagent solution will contain 93 mg. iodoplatinate.

EXAMPLE 3 Use of Spray Reagent Solution Prepared from impregnated Paper Reagent Strips in TLC for Drug Detection A. Ninhydrin Spray Reagent Solution This example illustrates the use of a strip impregnated with ninhydrin for the detection of amphetamine in thinlayer chromatography.

Apparatus:

l. Chromatography tank, glass, 25 cm. high, 10 cm.

wide and 29 cm. long. 2. Chromato-Vue box for long wave ultraviolet ir radiation. 3. Water bath 45C. 4. Nitrogen cylinder. 5. Air blower. 6. Chromatography column 1 X 30 cm. glass. 7. Capillary pipets 2O 11.. 8. Chromatography sprayer (Aerosol sprayer kit). 9. TLC plate, Polygram silica-gel (Brinkmann). Reagents:

1. Developing solvent: Ethyl acetate:methanol:ammonium (85:10:5 by vol.). 2. (A) Ninhydrin: Two hundred milligrams per 100 ml. of acetone. (B)

Ninhydrin One impregnated ninhydrin strip, prepared as in Example l, in 50 ml. of acetone. Shake and allow to soak for 5 minutes before spraying. 3. Resin Amberlite XAD-2, 20-50 mesh washed with methanol and water. 4. Methanol anhydrous. 5. Reference drug: Amphetamine sulfate 2 mg./ml. in methanol. 6. Urine Toxicology Contrl Drugs 1 (Analytical)? "One vial pooled normal human urine, freeze dried, containing 0.l25 mg. amphetamine reconstituted with 25 ml. distilled water. Available commercially from Lederle Diagnostics, American Cyanamid Company, Pearl River, New York.

Procedure:

Place a small plug of glass wool in the bottom of the glass column. Pour the resin suspension to a height of 6 cm. resin in the column. Wash the resin in the column with 30 ml. of distilled water. Pour 5 ml. of urine toxicology control into the column and allow to drain into the resin bed. Pour 50 ml. of distilled water to pass through the column and then aspirate as much as possible. Add 10 ml. methanol to the column and collect the eluate in a conical glass tube.

Evaporate the methanol extract of drugs in a water bath at 45C. under a stream of nitrogen. Dissolve the dry residue in 50 ,u.l of methanol. Take two TLC plates X 20 cm. and mark with a soft pencil both ends 1.5 cm. from the bottom (origin) and a solid line 1 1.5 cm.- from the bottom (finish line). With 20 u] pipet streak the reference solution and the dissolved residue with l 1 cm. apart from each other. Use air blower to speed drymg.

Prepare the developing solvent, pour it into the chromatography tank, immediately before use and cover with glass lid. Place the TLC plates evenly in the tank and cover the tank properly with the lid. Allow the solvent to ascend until the solvent front reaches the l 1.5 cm. line. Remove the plates, allow excess solvent to drain and let it air dry in the hood.

One plate was sprayed with ninhydrin freshly prepared using the ninhydrin prepared as set forth in (A). Another plate was sprayed with the reagent prepared from the impregnated ninhydrin strip as set forth in (B). Both plates were allowed to air dry and then placed under long wave U.V. Source for 10 minutes. When the plates were removed to visible light, pink spots appeared at the same Rf in both plates of equal intensity due to amphetamine. It was concluded that hydroxide the spray reagent prepared from the impregnated ninhydrin strip is as good as freshly prepared ninhydrin spray reagent.

B. Diphenylcarbazone Spray Reagent Solution This example illustrates the use of a strip impregnated with diphenylcarbazone for the detection of barbiturates in thin-layer chromatography. Apparatus:

l. Chromatography tank, glass 25 cm. high, 10 cm.

wide and 29 cm. long. Water bath 45C. Nitrogen cylinder. Air blower. Chromatography column 1 X 30 cm. glass. Capillary pipets 20 .1.1. Chromatography sprayer (Aerosol sprayer kit). TLC plate, Polygram Silica-Gel. (Brinkmann). Reagents:

1. Developing solvent (Ethyl acetate:methanol:ammonium (:10:5 by vol.)

2. Diphenylcarbazone:

A. Ten milligram per 50 ml. of equal volume of acetone and water.

(B) Diphenylcarbazone impregnated strip, prepared as in Example 1, in ml. of equal volume of acetone and water.

3. Resin:

Amberlite XAD-2 2050 mesh washed with methanol and water.

4. Methanol anhydrous.

5. Reference drug:

Amobarbital, phenobarbital and secobarbital 2 mg./ml. each in methanol.

6. Urine Toxicology Control Drugs 1 (Analytical)? 7. Mercuric Sulfate Reagent: (0.25% in 10% v/v One vial pooled human urine, freeze dried. containing 0.125 mg. of amobarbital, 0.125 mg. secobarbial and 0.125 mg. of phenobarbital, reconstituted with 25 ml. distilled water. Available commercially from Lederle Diagnostics, American Cyanamid Company, Pearl River, New York.

Procedure:

Place a small plug of glass wool in the glass column, push it to the bottom of the column, pour enough resin suspension to bring the column height to 6 cm. with the resin. Wash the resin with 30 ml. distilled water, pour 5 ml. urine toxicology control into the column and allow to drain into the resin bed. Rinse the column with 50 ml. of distilled water, then separate to remove water. Add 10 ml. of methanol to the column and collect the eluate in a glass tube. Evaporate the methanol, extract off drugs in a water bath at 45C. under a stream of nitrogen. Dissolve the residue in 50 ml. of methanol, cut one TLC plate 20 X 20 cm. into two 10 X 20 cm., with a soft pencil mark both plates at 1.5 cm. from the bottom (origin) with a solid line 1 1.5 cm. from the bottom. (finish line). With 20 ,u.] pipet, streak the reference and dissolve residue about 4 cm. apart from each other, use air blower to speed drying.

Prepare the developing solvent, pour it into the chromatography tank and cover with glass lid. Place the TLC plates evenly in the tank and cover the tank securely with the lid. Allow solvent to stand until the solvent front reaches the l 1.5 cm. line. Remove the plates, blow excess solvent to drain, and let is air dry in the hood.

One plate was sprayed with diphenylcarbazone (A) and mercuric sulfate reagent, while the other plate was hydroxide sprayed with diphenylcarbazone (B) and mercuric sulfat'e reagent. Phenobarbital and amobarbital appeared as violet spots and secobarbital appeared as chalk white spots in both TLC plates. For reference and sample the spots were in the same place in equal intensity. It was concluded that the spray reagent prepared from the diphenylcarbazone strip is as effective as diphenylcarbazone crystals in preparing spray reagent to visualize barbiturates.

C. lodoplatinate Spray Reagent Solution This example illustrates the use of the strips impregnated with chloroplatinic acid and potassium iodide, prepared as in Example 1, to prepare iodoplatinate reagent for the detection of alkaloids in thin-layer chromatography. Apparatus:

1. Chromatography tank glass 25 cm. high, cm.

wide and 29 cm. long.

2. Water bath 45C.

3. Nitrogen cylinder.

4. Air blower.

5. Chromatography column 1 X 30 cm. glass.

6. Capillary pipets ul.

7. Chromatogram sprayer (Aerosol Sprayer kit,

Brinkmann). Reagents:

1. Developing solvent Ethyl acetate:methanolzammonium hydroxide (85:10:5 by vol.) 2. lodoplatinate reagent (A) Platinum chloride l0% w/v in H20 1 ml. Potassium iodide 0.6 gm. Distilled water q.s. to ml.

Dilute the aqueous solution with equal volume of methanol before spraying.

(B) lodoplatinate Reagent from Strips Strip chloroplatinic acid I strip Strip potassium iodide 2 strips Distilled water q.s. to 12.5 ml. Methanol 12.5 ml.

5. Reference drugs:

Morphine 2 mg./ml. in methanol Codeine 2 mg./ml. in methanol Methadone 2 mg./ml. in methanol Meperidine 2 mg./ml. in methanol 6. Urine Toxicology Control Drugs l (Analytical) One vial. pooled normal human urine, freeze dried, containing 0.075 mg. codeine. 0.075 mg. methadone. 0.075 mg. meperidine and 0.075 mg. morphine, reconstituted with 25 ml. of distilled water. Available commercially from Lederle Diagnostics. American Cyanamid Company. Pearl River. New York.

Procedure:

.Place a small glass wool plug in the bottom of a column. Pour enough resin suspension in distilled water to bring the resin height in a column to 6 cm., wash the resin with 30 ml. of distilled water. Transfer 5 ml. of urine toxicology control into the column and allow to drain into the resin bed. Rinse the column with 50 ml. of distilled water, then aspirate to remove water. Add 10 ml. of methanol to the column and collect the eluate in a conical centrifuge (glass) tube. Evaporate the methanol extract of drugs on a water bath at 45C. under a stream of nitrogen. Dissolve the residue in 50 ml. of methanol. Cut one TLC plate 20 X 20 cm. into two 10 X 20 cm. With a soft pencil mark both plates at 1.5 cm. from the bottom (origin) and a solid line at 11.5 cm. from the bottom (finish line). With 20 ,ttl pipet spot the reference and dissolved residue about 4 cm. apart from each other. Use air blowers to speed drying while spotting.

Prepare the developing solvent, pour it into the chromatography tank and cover the tank securely with the lid. When the TLC plates are ready with spotting, place them carefully in the tank and cover the lid securely. Allow the solvent to ascend until the solvent front reaches the l 1.5 cm. line. Remove the plates, allow excess solvent to drain and let it air dry in the hood.

One plate was sprayed with iodoplatinate reagent (A) and another plate with (B).

In both TLC plates, reference and sample, spots were found at same distance (Rf value) and showed equal intensity. It was concluded that the spray reagent prepared from the strips impregnated with chloroplatinic acid and potassium iodide is as good as the same reagent freshly prepared from the same chemicals.

We claim:

1. A dry visualization reagent impregnated paper strip for use in preparing a spray reagent solution standard for visualization of a drug in urine in thin-layer chromatography which comprises a paper strip containing impregnated therein the dried residue of a completely impregnated volume of liquid solvent solution containing a predetermined amount of visualization reagent capable of elution into a spray reagent solvent and of chemically reacting with a drug in said urine to produce a characteristic color for said drug on a chromatogram.

2. A dry visualization reagent impregnated paper strip according to claim 1 wherein the visualization reagent is a member selected from the group consisting of ninhydrin, diphenylcarbazone, chloroplatinic acid and potassium iodide.

3. A method for preparing a dry visualization reagent impregnated strip for use in preparing a spray reagent solution standard for visualization of a drug in urine in thin-layer chromatography which comprises dipping an unimpregnated paper strip into a completely impregnated volume of a liquid solvent solution of a predetermined amount of visualization reagent capable of elution into a spray reagent solvent and of chemically reacting with a drug in said urine to produce a characteristic colored spot for said drug on a chromatogram and drying said strip. I

4. A method for preparing a dry visualization reagent impregnated strip according to claim 3 wherein the visualization reagent is a member selected from the group consisting of ninhydrin, diphenylcarbazone, chloroplatinic acid and potassium iodide.

ume of spray reagent solvent.