SUBJECT FORENSIC SCIENCE Paper No. and Title PAPER No. 9: Drugs of Abuse Module No. and Title MODULE No. 25: Colour Tests for Drug Identification Module Tag FSC_P9_M25 FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse MODULE No. 25: Colour Tests for Drug Identification 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 FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse MODULE No. 25: Colour Tests for Drug Identification 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. FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse MODULE No. 25: Colour Tests for Drug Identification 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 Preparation of Reagent: 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. FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse MODULE No. 25: Colour Tests for Drug Identification The reagent is prepared in three parts: Compounds Result in Chen Kao Reagent Ephedrine Violet Pseudoephedrine Violet Norephedrine Bright blue precipitate Norpseudoephedrine Blue precipitate Chloropseudoephedrine Bright blue precipitate N-Methylephedrine Pale blue precipitate Cathinone Pale blue precipitate Methcathinone 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 Preparation of Reagent: 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. FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse MODULE No. 25: Colour Tests for Drug Identification 4.4 Dragendorff’s Reagent Test Preparation of Reagent: 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 Compounds Red-orange / Brown orange precipitate Primary or Secondary or tertiary amine Orange Spots Alkaloids & benzodiazepines Yellow/ Orange/ Red orange/ Amphetamine Brown orange spot Orange /Red orange/Brown orange 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). FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse MODULE No. 25: Colour Tests for Drug Identification 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 Preparation of Reagent: 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 Preparation of Reagent: 1 volume of Formalin is added with 9 volumes of concentrated Sulphuric Acid. FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse MODULE No. 25: Colour Tests for Drug Identification Yellow / Orange Benzodiazepines Brown Orange Amphetamine purple red colour is produced which Opium Alkaloids changes to violet and finally blue Brown Ergot Alkaloids 4.10 Mandelin’s Test Preparation of Reagent: 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.