MODULE No. 25: Colour Tests for Drug Identification

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1 SUBJECT Paper No. and Title Module No. and Title Module Tag FSC_P9_M25

2 TABLE OF CONTENTS 1. Learning Outcomes 2. Introduction 3. Forensic Significance of Drug Identification 4. Colour Tests 5. Summarized Test Indications 6. Limitations of Colour Tests 7. Summary

3 1. Learning Outcomes After studying this module, you will be able to know about- Various preliminary tests for Drug identification The significance of preliminary examinations like color tests, crystal tests, etc. Limitations of preliminary screening and approaches to overcome them 2. Introduction Preliminary screening of Drugs are basically done by Colour tests, also sometimes referred to as chemical spot tests, provide with one of the leading tools for the presumptive identification of drugs. These colour tests are most practically applied to pharmaceuticals and scene of crime residues and, to a lesser extent, to biological fluids such as stomach contents, urine, etc. They are used to place the unidentified into a specific class of compounds or to eliminate categories or classes of compounds. These colour tests remain popular for several reasons. They are simple to perform and no extensive training is required. As such, they appeal in situations where laboratory facilities may be very limited. They can be performed in the field by police officers or technicians, require minimal reagents, are inexpensive, and give immediate results that can be viewed by the bare eye. In many instances, colour tests can also be used as Thin Layer Chromatography location reagents, applied by spraying or dipping. Colour tests can provide an indication of compound class far more rapidly than immunoassays and chromatographic techniques such as Gas Chromatography and High Performance Liquid Chromatography. Colour tests are only an indication of the presence of a compound or class of compounds and that all tests must be confirmed by more specific methods. This is exclusively significant where test results may ultimately result in protective sentences. Tests are usually carried out either in clear glass test-tubes or on white glazed porcelain tiles (spotting tiles), which give a uniform background against which colours can be assessed. A sample known not to contain the compound of interest should be tested at the same time as the test sample. This enables a comparison of the colours produced by the sample and by the reagent blank. It is essential to validate all tests and test reagents for sensitivity and specificity.

4 3. Forensic Significance of Drug Identification Use of Drugs is not a menace anymore, it has become a rampant. Most cases that arrive in a forensic laboratory start with the suspicion that a drug is present. A fatality might be an accident, suicide or murder, but a toxicological examination must be carried out to assist the investigating officer to decide which of these it might be. Often the investigating officer will not know whether or not any offence has been committed until the results of the toxicological analyses are available, so that formulating the correct questions for him or her to ask is vital if accurate and useful answers are to be given. It should be remembered that basic tests are not, in any circumstances, intended to replace pharmacopoeial requirements, but should be used as a rapid and inexpensive means to substantiate identity and strength of drugs and medicinal products and possibly to detect poor-quality counterfeit and other substandard products. In the event that suspect products are detected, these should be tested for compliance against pharmacopoeial requirements. 4. Some widely used Color Tests 4.1 Chen- Kao Reagent Test Reagent 1: This is prepared by adding 1 ml of Glacial Acetic Acid in 100 ml of Water, i.e., 1% (V/V) aqueous Acetic Acid solution. Reagent 2: This is prepared by dissolving 1 gm of Copper (II) Sulphate in 100 ml of Water, i.e., 1% (W/V) aqueous CuSO4 solution. Reagent 3: This is prepared by dissolving 8 gm of Sodium Hydroxide in 100 ml of Water, i.e., 2N aqueous Sodium Hydroxide solution. This test is used in to distinguish ephedrine, pseudoephedrine, norephedrine, and methcathinone from amphetamine and methamphetamine. The latter two do not react with Chen- Kao reagent.

5 The reagent is prepared in three parts: Compounds Ephedrine Pseudoephedrine Norephedrine Norpseudoephedrine Chloropseudoephedrine N-Methylephedrine Cathinone Methcathinone Result in Chen Kao Reagent Violet Violet Bright blue precipitate Blue precipitate Bright blue precipitate Pale blue precipitate Pale blue precipitate Bright blue precipitate 4.2 Cobalt Thiocyanate Test This test is a presumptive field test for illegal drugs. The test reagent is a 2% aqueous solution of Cobalt (II) Thiocyanate which is mixed with a small amount of sample. A brilliant green- Blue colour is produced by the hydrochloride salts of benzophetamine, chlordiazepoxide, chlorpromazine, methadone, methylphenidate, and brompheniramine maleate as well as hydrocodone tartrate. A strong but less green blue colour is produced by the hydrochloride salts of Cocaine, Diacetylmorphine (Heroin), Ephedrine, Meperidine, Phencyclidine, Procaine, Propoxyphene and Pseudoephedrine. Alkaloids present in drugs and other materials are also indicated by the application of this test. 4.3 Dille- Koppayani Test Part A: This is prepared by dissolving 0.1 g of Cobalt (II) Acetate (dihydrate) in 100 ml of methanol and subsequently mixed with 0.2 ml of Glacial Acetic Acid. Part B: This is 5% Isopropylamine (V/V) in Methanol. The Dille Koppanyi reagent is used as a simple spot-test to presumptively identify barbiturates. Two drops of Part A are dropped onto the suspected substance followed by one drop of Part B and any change in colour is observed. The test turns positive for phenobarbital, pentobarbital, amobarbital and Secobarbital light purple by appearance of cobalt with the barbiturate nitrogens.

6 4.4 Dragendorff s Reagent Test 1 gm of Bismuth SubNitrate is dissolved in 3 ml. of 10M of Hydrochloric Acid. It is diluted to 20 ml. 1 gm of Potassium iodide is dissolved in it. If black precipitate of bismuth tri-iodide separates, it is dissolved in 2M Hydrochloric Acid. Colour Red-orange / Brown orange precipitate Orange Spots Yellow/ Orange/ Red orange/ Brown orange spot Orange /Red orange/brown orange Compounds Primary or Secondary or tertiary amine Alkaloids & benzodiazepines Amphetamine Active constituents of Papaver somniferum 4.5 Duquenois Levine Test The Duquenois Levine reagent was originally developed in the 1930s by Pierre Duquénois. The reagent is prepared by 5 drops of Acetaldehyde and 0.4 gm. of Vanillin further dissolved in 20 ml. of 95% Ethanol. This test is recommended for the identification of Cannabis. When a small amount of suspected residue of the extract is placed in a test tube and shaken with 2 ml. of a Duquenois Reagent for one minute and further 2 ml. of concentrated Hydrochloric Acid is added, shaken and allowed to stand for 10 minutes. If a colour develops, 2 ml. of Chloroform is added. The violet colored lower (Chloroform) layer gives the positive indication of the presence of cannabis. 4.6 Frohde s Reagent Test Preparation of reagent: 50 mgs of Molybdic Acid or Sodium Molybdate is dissolved in 10 ml of hot concentrated Sulphuric Acid. The resulting solution should be colourless. Appropriate amount of the suspected material or exhibit is taken on a spot plate and added to it few drop of Frohde s reagent. The appearance of brown colour indicates the presence of Mescaline. The conversion of Brown colour to purple indicates the presence of Methylenedioxyamphetamine (MDA).

7 Amphetamine gives brown yellow color, whereas presence of opium alkaloids is indicated by violet colour changing to green and finally blue. In case of Ergot alkaloids a colour change from deep green to red, grey and finally blue is observed. 4.7 Forrest Reagent Test Equal volumes of a 0.2% solution of Potassium Dichromate, 30% (v/v) solution of Sulphuric Acid, and 20% solution of Perchloric Acid and a 50% solution of Nitric Acid are mixed. Phenothiazines gives Red / Violet red / Brown red / Orange / Pink orange / Red Orange / Brown, whereas, Imipramine and related compounds gives Blue colour. 4.8 Fisher Morris Test Preparation of Reagents: Reagent A: Concentrated Formic Acid. Reagent B: 5% aqueous Sodium Nitrite solution. 5 drops of A and 3 drops of B are added to sample. After 2 minutes, 15 drops of Chloroform are added and colours of layers are observed. Heroin, Diphene, hydramine, Diazepam can be detected by this test. 4.9 Marquis Test 1 volume of Formalin is added with 9 volumes of concentrated Sulphuric Acid.

8 Yellow / Orange Brown Orange purple red colour is produced which changes to violet and finally blue Brown Benzodiazepines Amphetamine Opium Alkaloids Ergot Alkaloids 4.10 Mandelin s Test 1% solution of Ammonium Vanadate is added in concentrated Sulphuric Acid This test is performed for the detection of Ergot Alkaloids. To a portion of the dried residue of the extract, one drop of reagent is added. A purple brown colour develops indicating the presence of ergot alkaloids 4.11 Van- Urk Reagent Test 1 gm. of p-amino benzaldehyde is dissolved in 100 ml. ethanol and adding 10 ml. of Hydrochloric Acid. To the dried residue of the extract, one drop of Van Urk Reagent is added. A green colour changing to blue is observed Husemann s Test To a little dried extract in a porcelain basin, 2-3 drops of concentrated Sulphuric Acid is added and heated on a water bath for 30 minutes over a small flame for a few minutes until white fumes appear. A reddish or reddish-brown or black colour appears. It is cooled. One drop of concentrated Nitric Acid and a crystal of Potassium Nitrate are added. A reddish violet colour appears which immediately changes to blood red and then to reddish yellow and finally fades away indicating the presence of Opium Alkaloids.

9 4.13 Zwikker s Test The residue of extract is taken up in Chloroform. To 1 ml. of Chloroform extract, 2-3 drops of 0.5 ml. of 5% pyridine in Chloroform is added and shaken. The colour of Chloroform layer becomes purple. Then 1 drop of Glacial Acetic Acid is added. If the colour of Chloroform layer changes from purple to weak blue, the presence of non- Thiobarbiturates is indicated. If Chloroform layer becomes green after adding pyridine in Chloroform, the presence of Thiobarbiturates is indicated. This green colour changes to light green on adding Acetic Acid Mecke s test The Mecke reagent is used as a simple spot-test to presumptively identify alkaloids as well as other compounds. It is composed of a mixture of Selenious Acid and concentrated Sulphuric Acid, which is dripped onto the substance being tested Simon s Reagent Test Simon s reagent is Sodium Nitroprusside in a basic buffer. It works by detecting secondary amines, such as the meth in Methamphetamine and in methylenedioxymethamphetamine (MDMA). If a substance tests positive for an ecstasylike substance by turning to purple/black with Marquis or Mecke, or to blue/black with Mandelin, then a color-change reaction with Simon s indicates the presence of MDMA or MDE Mayer s Reagent Test Potassium Mercury Iodide prepared by dissolving gm. of Mercuric Chloride and 5 gms of Potassium Iodide in 100 ml. of water

10 This test is performed for the detection of Nicotine. The dried residue of extract is acidified with Acetic Acid followed by addition of 2 drops of reagent. A white or yellowish precipitate is obtained FPN Test 5 ml. of Ferric Chloride solution, 45 ml. of 20% (w/v) solution of Perchloric Acid and 50 ml. of 50% (v/v) solution of Nitric Acid is added to make a final solution. Phenothiazines give Orange red / Violet red / Brown red / Orange / Red orange / pink orange /Blue/ Violet / Red Brown colour McNally Test To the residue of extract of biological materials a few drops of Acetone and 1-2 ml. of water are added. 2 drops of 0.5% Copper Sulphate solution in 10% Acetic Acid is added followed by a pinch of solid Sodium Nitrite. It is shaken and heated gradually to boiling and maintained in boiling condition for a few minutes. A red colour is formed if Salicylates and Salicylic Acid are present Urotropine Test This test is performed to identify opium alkaloids. To a little dried extract in a porcelain basin, a few drops of aqueous solution of Urotropine is added and warmed slightly. A purple colour changing to blue and then green is observed Scott s Test Reagent is prepared by dissolving 2 gms of Cobalt Thiocyanate in water and diluted with 96% Glycerine in water in 1:1 proportion.

11 The residue of the extract is taken in a test tube and reagent is added. The mixture is shaken and a blue colour develops at once which indicates the presence of cocaine. Methaqualone also gives positive reaction. If a blue colour develops, one drop of concentrated Hydrochloric Acid is added. The blue colour disappears and a clear pink colour appears. If a blue colour does not disappear, one drop of concentrated Hydrochloric Acid is added. Then a few drops of Chloroform is added and shaken. The Chloroform layer becomes intense blue Fast Blue- B Test A small amount of residue of the extract is placed in a test tube and a very small amount solid Fast blue B reagent (solid Fast blue B: anhydrous Sodium sulphate in 2.5: 100 ratio) is added. 1 ml. of Chloroform is then added and shaken. It is kept for two minutes. The Chloroform layer becomes purple red in colour indicating the presence of Cannabis Gerrard s Test 1-2 ml. of 2% Mercuric Chloride solution in 50% of alcohol is added to a portion of residue of the extract. A red colour develops immediately. Hyoscyamine produces a yellow colour which becomes red on burning, while hyocine does not produce any change of colour Vitali s Test A portion of residue of the extract is treated with a few drops of fuming Nitric Acid in a porcelain basin. It is evaporated to dryness on a water bath. The residue is cooled and moistened with a few drops of freshly prepared alcoholic Caustic Potash solution when a violet colour is produced, which soon changes to red and finally disappears. The colour may be made to reappear by adding more alcoholic Caustic Potash solution. This test is performed for the detection of Atropine, Datura and ergot alkaloid.

12 5. Summarized Test Indications Substance/functional group Useful tests/reagents Alcohols Potassium Dichromate Alkaloids and nitrogenous Dragendorff s reagent bases Amides (aliphatic) Nessler s reagent Aldehydes (aliphatic) Schiff s reagent Amphetamines Marquis test, Mecke test, Frohde s reagent and Mandelin s test Sodium Nitroprusside Acetone Antidepressants Marquis test Barbiturates Dille Koppanyi reagent Koppanyi Zwikker reagent Mercurous Nitrate Vanillin reagent Zwikker reagent Benzodiazepines Formaldehyde sulfuric acid Cannabis Duquenois reagent Carbamates (non-aromatic) Furfuraldehyde Cocaine Cobalt thiocyanate p-dimethylaminobenzaldehyde Mandelin s test Scott s test Chlorinated phenols Nitric Acid (fuming) Chlorinated hydrocarbon Nitric sulfuric acid insecticides Cyanide Ferrous sulfate (B) Sodium picrate Cyanide groups Sodium picrate Dithiocarbamates Sodium Nitroprusside Ergot alkaloids p- Dimethylaminobenzaldehyde Halogenated hydrocarbons Fujiwara test Imides Koppanyi Zwikker test

13 Ketones Sodium Nitroprusside Methadone Tetrabromophenolphthalein ethyl ester Cobalt thiocyanate Mandelin s test Marquis test Mono-substituted pyridine Cyanogen bromide ring Nitrates and nitrites Ferrous sulfate Opiates Marquis, Mecke, Frohde s, Mandelin s tests Oxidizing agents Diphenylamine Phencyclidine Tetrabromophenolphthalein ethyl ester Phenols Ferric chloride Folin Ciocalteu reagent Millon s reagent p-dimethylaminobenzaldehyde Phenothiazines Formaldehyde sulfuric acid Forrest reagent FPN reagent Ferric chloride Phenylpyrazolines Nitrous acid Primary aromatic amines Diazotization Coniferyl alcohol Primary and secondary amines Dragendorff s reagent Simon s test Propoxyphene Cobalt thiocyanate Frohde s reagent Liebermann s test Tetrabromophenolphthalein ethyl ester Quaternary Ammonium Tetrabromophenolphthalein ethyl ester compounds Quaternary amines Dragendorff s reagent Quinines Thalleioquin Cobalt thiocyanate Quinones Methanolic Potassium hydroxide Salicylates Ferric chloride Trinder s reagent Steroids Antimony Pentachloride Naphthol sulfuric acid Sulfuric acid

14 Sulfonamides Copper sulfate Koppanyi Zwikker reagent Mercurous Nitrate Nitrous acid Sulfur-containing Palladium chloride Sodium Nitroprusside Tertiary amines Dragendorff s reagent Tetrabromophenolphthalein ethyl ester 6. Limitations of Colour Tests Colours exhibited by these tests cannot be described with any accuracy. They may vary in intensity or tint with the concentration of compounds in the test samples and the presence of extraneous material. In addition, their assessment is always a subjective one, even in people with normal colour vision. For example, the Marquis test is stated to give a black or blue-black colour with MDMA and a green-black with lysergide. Even in the pure form these colours may not be readily distinguishable; add this to the fact that most drugs of abuse are more routinely encountered diluted with a variety of other substances, and one can begin to appreciate the difficulties that can arise in the real world with colour tests. Some of the complexes formed are unstable, so that the colour changes or fades with time. Salts may give different colours from those of the corresponding acid or base. In general, free acids or bases that have been isolated from the test material by an extraction process give better colours than their salts. The colour of a salt may be modified by the nature of the other ions present.

15 7. Summary Colour Tests provides a relatively rapid and inexpensive indication of the presence or absence of a substance and an indication of the identity of a substance. The emphasis for colour tests is on indication. Such tests do not provide confirmation of identity. Colour tests provide toxicologists and drug analysts with one of the first tools for the presumptive identification of drugs and poisons. They are used to place the unknown into a specific class of compounds or to eliminate categories or classes of compounds. Colour tests are particularly important in clinical toxicology, especially where a patient is being treated in accident and emergency and clinical symptoms may indicate some form of poisoning. Colour tests can provide an indication of compound class far more rapidly than immunoassays and chromatographic techniques such as GC and HPLC. Colour tests are widely used by police authorities and customs to detect drugs of abuse. Colour tests range from those that rely on reactions with certain functional groups, to those that are almost specific for a given group.

Marquis (210 ml): Add 10 ml of 40% formaldehyde solution to 200 ml of concentrated sulfuric acid.

Marquis (210 ml): Add 10 ml of 40% formaldehyde solution to 200 ml of concentrated sulfuric acid. Introduction Spot tests are color tests used for rapid screening of samples to determine what drugs may or may not be present. The principal behind spot tests is based on the fact that classes of compounds