Forensic Science International 109 (2000) 189–201 www.elsevier.com / locate / forsciint Validation of twelve chemical spot tests for the detection of drugs of abuse a a, b Carol L. O’Neal , Dennis J. Crouch *, Alim A. Fatah a Center for Human Toxicology, University of Utah, Department of Pharmacology and Toxicology, 20 S. 2030 East, Room 490, Salt Lake City, UT 84112, USA b Office of Law Enforcement Standards, National Institute of Standards and Technology, Gaithersburg, MD, USA Received 23 November 1999; accepted 10 December 1999 Abstract Validation procedures are described for 12 chemical spot tests including cobalt thiocyanate, Dille–Koppanyi, Duquenois–Levine, Mandelin, Marquis, nitric acid, para-dimethylaminobenzaldehyde, ferric chloride, Froehde, Mecke, Zwikker and Simon’s (nitroprusside). The validation procedures include specificity and limit of detection. Depending on the specificity of each color test, between 28 to 45 drugs or chemicals were tested in triplicate with each of the 12 chemical spot tests. For each chemical test, the final colors resulting from positive reactions with known amounts of analytes were compared to two reference color charts. For the identification of unknown drugs, reference colors from the Inter-Society Color Council and the National Bureau of Standards (ISCC-NBS) and Munsell charts are included along with a description of each final color. These chemical spot tests were found to be very sensitive with limits of detection typically 1 to 50 mg depending on the test and the analyte.  2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chemical spot test; Color test; Drug detection 1. Introduction Chemical spot tests (sometimes referred to as color tests) provided toxicologists and criminalists with one of the earliest tools for the presumptive identification of drugs and *Corresponding author. 0379-0738 / 00 / $ – see front matter  2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0379-0738( 99 )00235-2 190 C.L. O’ Neal et al. / Forensic Science International 109 (2000) 189 – 201 poisons [1–3]. These tests continue to be popular for several reasons. They rely on simple chemical reactions and produce visible results that can be interpreted with the naked-eye. The reagents and laboratory materials needed to perform the tests are inexpensive and readily available. The tests can be performed by technicians without extensive training. Since the tests require minimal reagents and materials, small and even on-site laboratories can perform the tests. They can also be employed in the field by law enforcement agents. The utility of these tests is demonstrated by the fact that even today, when the use of sophisticated analytical instrumentation is so pervasive, they are still an integral part of the testing arsenal of forensic laboratories. In two US National Institute of Justice (NIJ) standards, NILECJ-STD-0604.00 [4] and NILECJ-STD-0605.00 [5], the procedures for the use and validation of eleven different chemical spot-testing reagents were described. To better address the current needs of drug testing laboratories, the information in these documents was reviewed and updated or revalidated as needed. The need to include additional tests / analytes or remove existing tests / analytes from the original standards was assessed. An informal questionnaire addressing the use of these chemical spot tests in forensic laboratories was mailed to approximately 325 laboratories / individuals selected from the rosters of American Society of Crime Laboratory Directors (ASCLD), the Regional Association of Forensic Scientists and the Criminalistics Section of the American Academy of Forensic Sciences (AAFS). This mailing was performed in a semi-random fashion with an attempt to contact at least two laboratories or drug chemists in each state and to include the regional Drug Enforcement Administration laboratories. We received 121 responses. They indicated that chemical spot tests are still frequently used by 86% (104) of the responding laboratories. Greater than 90% of these laboratories used at least four of the tests; cobalt thiocyanate for cocaine, Duquenois–Levine for marijuana, Marquis for many basic drugs and para-dimethylaminobenzaldehyde ( p-DMAB) for LSD. Ten of the chemical spot tests described in the NIJ documents were still routinely used by more than one third of the laboratories. Twenty-five percent of respondents suggested adding the Simon’s or nitroprusside test for the detection of secondary amines, such as methamphetamine and methylenedioxymethamphetamine, to the battery of spot tests described in the original NIJ documents. Additional drugs that had become more prevalent since the publication of the standards such as acetaminophen, alprazolam, diazepam, ephedrine, hydrocodone and pseudoephedrine were added to the original list of analytes to be tested. This article reviews the data presented in the two original NIJ documents and presents new validation data for an expanded list of drugs using 12 chemical spot tests (CSTs). 2. Materials and methods 2.1. Chemicals Cobalt thiocyanate, cobalt acetate dihydrate, glacial acetic acid, isopropylamine, acetaldehyde, ammonium vanadate, formaldehyde, para-dimethylaminobenzaldehyde, ferric chloride, vanillin, sodium molybdate, selenius acid, copper sulfate pentahydrate, C.L. O’ Neal et al. / Forensic Science International 109 (2000) 189 – 201 191 sodium nitroprusside, 2-chloroacetophenone and sodium carbonate were purchased from Sigma–Aldrich Chemical (St. Louis, MO, USA). Methanol, hexane and chloroform were obtained from Burdick and Jackson (Muskegon, MI, USA). Hydrochloric acid, sulfuric acid, nitric acid and pyridine were purchased from Mallinckrodt Baker, (Paris, KY, USA). Ethanol was obtained from Quantum Chemical (Tuscola, IL, USA). The drugs were purchased in powder form from Sigma–Aldrich Chemical (St. Louis, MO, USA), Alltech-Applied Science (State College, PA, USA) or Research Triangle Institute (RTI, NC, USA). 2.2. Materials Porcelain plates with 12 wells, glass culture tubes (12375 mm) and Pasteur pipettes were purchased from VWR Scientific Products (Denver, CO, USA). The Munsell Book of Color (Volumes 1 and 2) were purchased from GretagMacbeth (New Windsor, NY, USA). The centroid color charts, published by the Inter-Society Color Council and the National Bureau of Standards, were obtained from Nick Hale (Naples, FL, USA). 2.3. Chemical spot test procedures The procedures for preparing the chemical spot test reagents and performing each test are described below. One or two drops of reagent(s) were added to the drug using a Pasteur pipette unless otherwise noted. A.1 Cobalt thiocyanate Dissolve 2.0 g of cobalt (II) thiocyanate in 100 ml of distilled water. A.2 Dille–Koppanyi reagent, modified Solution A: Dissolve 0.1 g of cobalt (II) acetate dihydrate in 100 ml of methanol. Add 0.2 ml of glacial acetic acid and mix. Solution B: Add 5 ml of isopropylamine to 95 ml of methanol. Procedure: Add two drops of solution A to the drug, followed by one drop of solution B. A.3 Duquenois–Levine reagent, modified Solution A: Add 2.5 ml of acetaldehyde and 2.0 g of vanillin to 100 ml of 95% ethanol. Solution B: Concentrated hydrochloric acid. Solution C: Chloroform. Procedure: Add three drops of solution A to the drug and shake for 1 min. Then add three drops of solution B. Agitate gently, and determine the color produced. Add nine drops of solution C and note whether the color is extracted from the mixture to A and B. 192 C.L. O’ Neal et al. / Forensic Science International 109 (2000) 189 – 201 A.4 Mandelin reagent Dissolve 1.0 g of ammonium vanadate in 100 ml of concentrated sulfuric acid. A.5 Marquis reagent Carefully add 100 ml of concentrated sulfuric acid to 5 ml of 40% formaldehyde (formaldehyde:water, v:v). A.6 Nitric acid Concentrated nitric acid. A.7 Para-dimethylaminobenzaldehyde ( p-DMAB) Add 2.0 g of p-DMAB to 50 ml of 95% ethanol and 50 ml of concentrated hydrochloric acid. A.8 Ferric chloride Dissolve 2.0 g of anhydrous ferric chloride or 3.3 g of ferric chloride hexa-hydrate in 100 ml of distilled water. A.9 Froehde reagent Dissolve 0.5 g of molybdic acid or sodium molybdate in 100 ml of hot concentrated sulfuric acid. A.10 Mecke reagent Dissolve 1.0 g of selenious acid in 100 ml of concentrated sulfuric acid. A.11 Zwikker reagent Solution A: Dissolve 0.5 g of copper (II) sulfate pentahydrate in 100 ml of distilled water. Solution B: Add 5 ml of pyridine to 95 ml of chloroform. A.12 Simon’ s reagent Solution A: Dissolve 1 g of sodium nitroprusside in 50 ml of distilled water and add 2 ml of acetaldehyde to the solution with thorough mixing. Solution B: 2% sodium carbonate in distilled water. Procedure: Add one drop of solution A to the drug, followed by two drops of solution B. 2.4. Validation of chemical spot tests 2.4.1. Test color and specificity The drugs and other analytes were classified and prepared as either a drug standard, crystal, powder, tablet, or extract (Table 1). Drug standards were prepared in either chloroform or methanol at a concentration of 2.0 or 4.0 mg / ml (free-base). Crystals were tested without further processing. Tablets were crushed into a fine powder and leaf C.L. O’ Neal et al. / Forensic Science International 109 (2000) 189 – 201 193 Table 1 The classification of chemical spot test analytes Analyte Classification Analyte Classification Acetaminophen Alprazolam Amobarbital Aspirin Baking soda Benzphetamine HCl Brompheniramine maleate Chlordiazepoxide HCl Chlorpromazine HCl Cocaine HCl Codeine Contac D-Amphetamine D-Methamphetamine Diacetylmorphine HCl Diazepam Dimethoxymethaphetamine HCl Doxepin HCl Dristan Ephedrine HCl Exedrine Hydrocodone tartrate LSD Mace Drug standard Drug standard Drug standard Tablet Powder Drug standard Drug standard Drug standard Drug standard Drug standard Drug standard Tablet Drug standard Drug standard Drug standard Drug standard Drug standard Drug standard Tablet Drug standard Tablet Drug standard Drug standard Crystals MDA HC1 Meperidine HCl Mescaline HCl Methadone HCl Methaqualone Methyiphenidate HCl Morphine monohydrate Nutmeg Opium Oxycodone HCl Pentobarbital Phencyclidine HCl Phenobarbital Procaine HCl Propoxyphene HCl Pseudoephedrine HCl Quinine HCl Salt Secobarbital Sugar Tea THC Tobacco Drug standard Drug standard Drug standard Drug standard Drug standard Drug standard Drug standard Extract Powder Drug standard Drug standard Drug standard Drug standard Drug standard Drug standard Drug standard Drug standard Crystals Drug standard Crystals Extract Extract Extract material was extracted with hexane. For all of the CSTs (except A.3), 500 mg of each analyte (125 or 250 ml of drug standard) was added to each of three wells on the porcelain test plate. For the drug standards and extracts, the organic solvent was evaporated and the residue was reconstituted in 100 ml of chloroform before the CST was performed. The CST reagents were then added with a Pasteur pipette as described in Section 2.3 for each test. For A.3, 500 mg of analyte was added to a glass culture tube. If organic solvent was present, it was evaporated and the test reagents were added as described for A.3. The final color was observed after 5 min and compared to reference colors in the Munsell and centroid color charts. Each analyte was tested in triplicate. 2.4.2. Drug detection limit A working 1.0 mg / ml solution (or lower if necessary) of each analyte to be tested was prepared. The limit of detection (LOD) for each analyte was determined by testing serial dilutions of the working solution until the lowest concentration of analyte that was detectable in five replicates (n55) was identified. This concentration was then multiplied by ten and recorded as the ‘operational drug detection limit’. All tests were performed in the porcelain test plates except for A.3 which was performed in glass culture tubes. 194 C.L. O’ Neal et al. / Forensic Science International 109 (2000) 189 – 201 3. Results and discussion Chemical spot tests are widely accepted as presumptive tests for drug detection. These CSTs provide information that allows the analyst to select the appropriate testing procedures to confirm the identity of the suspected drug. The information listed in the tables is intended as a guide for using CSTs and for preparing quality control materials for chemical spot tests when they are performed in the laboratory or in the field. The actual color produced by the reagents for each drug may vary depending on many factors: the concentration of the drug, whether the drug is in salt or free base form, which salt form is present, any additional diluents or contaminants present in the sample, the color discrimination of the analyst and the conditions under which the test is performed [2,6]. The original NIJ standards [4,5] used centroid color charts published by the InterSociety Color Council and the National Bureau of Standards (ISCC-NBS) for color comparison. These charts include almost 270 colors logically grouped and listed numerically. However, these color standards are obsolete and are no longer considered to be an international standard for color description or comparison. Therefore, the ISCC-NBS numbers are listed for historical comparison purposes only. The ISCC-NBS charts have been replaced by the Munsell Color charts. The Munsell Book of Color (Volumes 1 and 2) is a master atlas of color that contains almost 1600 color comparison chips. The colors are prepared according to the specifications contained in the final report of the subcommittee of the Optical Society of America. Each page of the Munsell book presents one hue. There are 40 pages, each is 2.5 hue steps apart. On each page, the color chips are arranged by Munsell value and chroma. The standard way to describe a color using Munsell notations is to write the numeric designation for the Munsell hue (H) and the numeric designation for value (V) and chroma (C) in the form of H V/ C. Since there are considerably more colors in the Munsell charts than in the centroid charts, two or more Munsell notations may correspond to the same previously used ISCC-NBS number. Depending on the specificity of each color test, between 28 to 45 drugs or chemicals were tested in triplicate with each of the CSTs. For each CST, the final color resulting from a positive reaction with a known amount of analyte was compared to two reference color charts. These results are shown in Table 2. Reference colors from the ICSS-NBS and Munsell charts were included along with a description of each final color. A positive CST may indicate a specific drug or class of drugs is present in the sample, but the tests are not always specific for a single drug or class. For this reason, laboratories must rely on a battery of CSTs for the preliminary identification of an unknown drug. For example, Cobalt thiocyanate (A.1) is used to detect cocaine. However, many other drugs will also react with this reagent (Tables 2 and 3) and each analyte that tested positive with cobalt thiocyanate produced a strong blue color. Also, the nitric acid test produced variations of yellow and orange colors with a variety of analytes including acetaminophen, diacetylmorphine, dimethoxymethamphetamine and mescaline. Six of the CSTs are indicated for the detection of opioids and other amines. These include Mandelin (A.4), Marquis (A.5), nitric acid (A.6), ferric chloride (A.8), Froehde C.L. O’ Neal et al. / Forensic Science International 109 (2000) 189 – 201 195 Table 2 Final colors produced by reagents A.1 through A.12 with various drugs and other substances Analyte Solvent ICSS-NBS g Color Munsell A.1 A.1 A.1 A.1 A.1 A.1 A.1 A.1 A.1 A.1 A.1 A.1 A.1 A.1 A.1 A.1 A.1 Benzphetamine HCl Brompheniramine Maleate Chlordiazepoxide HCl Chlorpromazine HCl Cocaine HCl Diacetylmorphine HCl Doxepin HCl Ephedrine HCl Hydrocodone tartrate Meperidine HCl Methadone HCl a Methylphenidate HCl Phencyclidine HCl Procaine HCl a Propoxyphene HCl a Pseudoephedrine HCl Quinine HCl CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 168 168 168 168 169 169 168 169 168 169 168 168 169 169 169 169 178 Brilliant greenish blue Brilliant greenish blue Brilliant greenish blue Brilliant greenish blue Strong greenish blue Strong greenish blue Brilliant greenish blue Strong greenish blue Brilliant greenish blue Strong greenish blue Brilliant greenish blue Brilliant greenish blue Strong greenish blue Strong greenish blue Strong greenish blue Strong greenish blue Strong blue 5B 7 / 8 5B 6 / 10 2.5B 6 / 8 5B 6 / 10 5B 5 / 10 7.5B 6 / 10 5B 6 / 10 5B 5 / 10 5B 6 / 8 5B 5 / 10 5B 6 / 10 10BG 6 / 8 5B 5 / 10 5B 5 / 10 5B 5 / 10 5B 5 / 10 2.5PB 5 / 12 A.2 A.2 A.2 A.2 Amobarbital Pentobarbital a Phenobarbital a Secobarbital a CHCl 3 CHCl 3 CHCl 3 CHCl 3 222 222 222 222 Light Light Light Light 5P 5P 5P 5P A.3 Mace f Crystals A.3 Nutmeg Extract A.3 A.3 Tea THC a Extract EtOR 237 b 237 c 221 d 244 b 244 c 261 d 119 e 204 b 199 c 219 d Strong reddish purple b Strong reddish purple c Very light purple d Pale reddish purple b Pale reddish purple c Light gray purplish red d Light yellow green Gray purplish blue b Light purplish blue c Deep purple d 2.5RP 5 / 12 b 2.5RP 5 / 12 c 5P 8 / 4 d 10P 6 / 4 b 10P 6 / 4 c 5RP 7 / 4 d 5GY 8 / 6 7.5PB 4 / 4 b 7.5PB 7 / 8 c 7.5P 4 / 12 d A.4 A.4 A.4 A.4 A.4 A.4 A.4 A.4 A.4 A.4 A.4 A.4 A.4 A.4 A.4 A.4 Acetaminophen Aspirin Benzphetamine HCl a Brompheniramine Maleate Chlorpromazine HCl Cocaine HCl a Codeine a Contac a D-Amphetamine HCl a D-Methamphetamine HCl Diacetylmorphine HCl a Dimethoxy-meth HCl Doxepin HCl Dristan Exedrine Mace f CHCl 3 Powder CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 Powder CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 Powder Powder Crystals 107 127 116 50 108 69 108 84 164 137 43 96 44 110 108 125 Moderate olive Grayish olive green Brilliant yellow green Strong orange Dark olive Deep orange yellow Dark olive Strong yellow Moderate bluish green Dark yellowish green Moderate reddish brown Dark olive brown Dark reddish brown Grayish olive Dark olive Moderate olive green 10Y 5 / 8 2.5GY 4 / 2 2.5GY 8 / 10 7.5YR 7 / 14 10Y 3 / 4 10YR 7 / 14 5Y 3 / 4 2.5Y 6 / 10 5BG 5 / 6 10GY 4 / 6 10R 3 / 6 5Y 2 / 2 10R 2 / 4 7.5Y 4 / 4 7.5Y 3 / 4 5GY 4 / 8 purple purple purple purple 7/8 7/8 7/8 7/8 C.L. O’ Neal et al. / Forensic Science International 109 (2000) 189 – 201 196 Table 2. Continued Analyte Solvent ICSS-NBS g Color Munsell A.4 A.4 A.4 A.4 A.4 A.4 A.4 A.4 A.4 A.4 A.4 A.4 MDA HCl Mescaline HCl a Methadone HCl Methaqualone Methylphenidate HCl Morphine monohydrate a Opium a Oxycodone HCl Procaine HCl Propoxyphene HCl Quinine HCl Salt CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 Crystals 193 78 187 66 67 47 59 103 51 44 100 50 bluish Black Dark yellowish brown Dark grayish blue Very orange yellow Brilliant orange yellow Dark grayish reddish brown Dark brown Dark greenish yellow Deep orange Dark reddish brown Deep greenish yellow Strong orange 10B 2 / 2 10YR 3 / 4 5B 3 / 2 10YR 8 / 14 2.5Y 8 / 10 10R 3 / 2 7.5YR 2 / 4 10Y 6 / 6 5YR 5 / 12 10R 2 / 4 10Y 9 / 6 5YR 7 / 12 A.5 A.5 A.5 A.5 A.5 Aspirin Benzphetamine HCl a Chlorpromazine HCl Codeine a a D-Amphetamine HCl Powder CHCl 3 CHCl 3 CHCl 3 CHCl 3 A.5 D-Methamphetamine A.5 A.5 A.5 A.5 A.5 A.5 A.5 A.5 A.5 A.5 A.5 A.5 A.5 A.5 Diacetylmorphine HCl a Dimethoxy-meth HCl Doxepin HCl Dristan Exedrine LSD Mace f MDA HCl a Meperidine HCl Mescaline HCl a Methadone HCl Methylphenidate HCl Morphine monohydrate a Opium a CHCl 3 CHCl 3 CHCl 3 Powder Powder CHCl 3 Crystals CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 Powder 13 41 256 225 35 to 44 36 to 44 256 107 21 20 16 114 87 267 56 50 28 71 239 47 5R 3 / 10 7.5R 2 / 6 2.5RP 3 / 8 7.5P 2 / 4 10R 6 / 12 to 7.5R 2 / 4 10R 4 / 12 to 7.5R 2 / 4 7.5RP 3 / 10 7.5Y 5 / 8 7.5R 2 / 2 5R 3 / 2 5R 3 / 8 10Y 2 / 2 7Y 7 / 8 Black 5YR 3 / 6 5YR 6 / 12 2.5YR 8 / 4 10YR 8 / 8 10P 3 / 6 10R 3 / 2 A.5 A.5 A.5 Oxycodone HCl a Propoxyphene HCl Sugar CHCl 3 CHCl 3 Crystals 214 230 59 Deep red Deep reddish brown Deep purplish red Very dark purple Strong reddish orange Dark reddish brown Deep reddish orange Dark reddish brown Deep purplish red Moderate olive Blackish red Dark grayish red Dark red Olive Black Moderate yellow Black Deep brown Strong orange Light yellowish pink Moderate orange yellow Very deep reddish purple Dark grayish reddish brown Pale violet Blackish Purple Dark brown A.6 A.6 A.6 A.6 A.6 A.6 A.6 A.6 A.6 A.6 A.6 Acetaminophen Chlorpromazine HCl Codeine a Diacetylmorphine HCl a Dimethoxy-meth HCl Doxepin HCl Dristan Exedrine LSD Mace f MDA HCl CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 CHCl 3 Powder Powder CHCl 3 Crystals CHCl 3 67 98 101 89 82 83 51 67 55 102 101 Brilliant orange yellow Brilliant greenish yellow Light greenish yellow Pale yellow Very yellow Brilliant yellow Deep orange Brilliant orange yellow Strong brown Moderate greenish yellow Light greenish yellow 2.5Y 8 / 12 7.5Y 8.5 / 10 7.5Y 9 / 6 5Y 9 / 6 2.5Y 8 / 14 5Y 8.5 / 8 5YR 6 / 12 2.5Y 8 / 12 5YR 5 / 10 10Y 7 / 6 7.5Y 9 / 6 HCl a CHCl 3 2.5P 6 / 4 2.5RP 2 / 2 5YR 2 / 4 C.L. O’ Neal et al. / Forensic Science International 109 (2000) 189 – 201 197 Table 2. Continued Analyte a Solvent ICSS-NBS g Color Munsell A.6 A.6 A.6 A.6 Mescaline HCl Morphine monohydrate a Opium a Oxycodone HCl CHCl 3 CHCl 3 Powder CHCl 3 16 67 72 83 Dark red Brilliant orange yellow Dark orange yellow Brilliant yellow 5R 3 / 6 2.5Y 8 / 12 10YR 6 / 10 5Y 8.5 / 8 A.7 LSD a CHCl 3 219 Deep Purple 7.5P 3 / 10 A.8 A.8 A.8 A.8 A.8 A.8 Acetaminophen Baking soda Chlorpromazine HCl Dristan Exedrine Morphine monohydrate a MEOH Powder MEOH Powder Powder MEOH 103 51 48 200 200 146 Dark greenish yellow Deep orange Very orange Moderate purplish blue Moderate purplish blue Dark green 10Y 6 / 10 5YR 6 / 14 5YR 7 / 14 10PB 4 / 2 10PB 4 / 2 5G 3 / 6 A.9 A.9 A.9 A.9 A.9 A.9 A.9 A.9 A.9 A.9 A.9 A.9 A.9 A.9 A.9 A.9 A.9 Aspirin Chlorpromazine HCl Codeine a Contac Diacetylmorphine HCl a Dimethoxy-meth HCl Doxepin HCl Dristan Exedrine LSD Mace f MDA HCl a Morphine monohydrate a Opium a Oxycodone HCl Propoxyphene HCl Sugar Powder CHCl 3 CHCl 3 Powder CHCl 3 CHCl 3 CHCl 3 Powder Powder CHCl 3 Crystals CHCl 3 CHCl 3 Powder CHCl 3 CHCl 3 Crystals 228 14 147 95 256 115 41 163 177 120 70 157 256 65 84 20 83 Grayish purple Very deep red Very dark green Moderate olive brown Deep purplish red Very yellow green Deep reddish brown Light bluish green Moderate yellow green Light olive yellow Greenish black Deep purplish red Brownish black Strong yellow Dark grayish red Brilliant yellow 7.5P 5 / 2 5R 3 / 10 7.5G 2 / 6 2.5Y 4 / 6 5RP 3 / 10 5GY 6 / 10 7.5R 2 / 8 5BG 7 / 6 10B 6 / 10 5GY 6 / 6 10YR 8 / 8 7.5G 2 / 2 5RP 3 / 10 7.5R 2 / 2 2.5Y 7 / 10 2.5R 3 / 2 5Y 8.5 / 8 A.10 A.10 A.10 A.10 A.10 A.10 A.10 A.10 A.10 A.10 A.10 A.10 A.10 A.10 A.10 A.10 A.10 A.10 A.10 Chlorpromazine HCl Codeine a Contac Diacetylmorphine HCl a Dimethoxy-meth HCl Doxepin HCl Dristan Exedrine Hydrocodone tartrate LSD Mace f MDA HCl a Mescaline HCl a Morphine monohydrate a Nutmeg Opium a Oxycodone HCl Propoxyphene HCl Sugar CHCl 3 CHCl 3 Powder CHCl 3 CHCl 3 CHCl 3 Powder Powder CHCl 3 CHCl 3 Crystals CHCl 3 CHCl 3 CHCl 3 Extract Powder CHCl 3 CHCl 3 Crystals 21 166 95 161 59 17 94 91 165 157 111 166 107 166 65 114 107 41 98 Blackish red Very dark bluish green Moderate olive brown Deep bluish green Dark brown Very dark red Light olive brown Dark grayish yellow Dark bluish green Greenish black Dark grayish olive Very dark bluish green Moderate olive Very dark bluish green Brownish Black Olive black Moderate olive Deep reddish brown Brilliant greenish yellow 5R 2 / 2 2.5BG 2 / 4 2.5Y 4 / 6 2.5BG 3 / 8 5YR 2 / 4 5R 2 / 4 2.5Y 6 / 10 5Y 6 / 4 5BG 3 / 6 7.5G 2 / 2 10Y 3 / 4 2.5BG 2 / 4 7.5Y 5 / 8 2.5BG 2 / 4 10YR 2 / 2 10Y 2 / 2 7.5Y 5 / 8 10R 2 / 6 10Y 8.5 / 10 C.L. O’ Neal et al. / Forensic Science International 109 (2000) 189 – 201 198 Table 2. Continued Analyte Solvent ICSS-NBS g Color Munsell A.11 A.11 A.11 A.11 A.11 A.11 A.11 Baking soda Exedrine Pentobarbital a Phenobarbital a Secobarbital a Tea Tobacco Powder Powder CHCl 3 CHCl 3 CHCl 3 Extract Extract 181 144 222 222 222 120 136 Light blue Light green Light purple Light purple Light purple Moderate yellow green Moderate yellowish green 2.5PB 7 / 6 5G 7 / 6 7.5P 7 / 6 7.5P 7 / 6 7.5P 7 / 6 2.5GY 7 / 8 10GY 6 / 6 A.12 A.12 A.12 A.12 D-Methamphetamine HCl a Dimethoxy-meth HCl a MDMA HCl Methylphenidate HCl CHCl 3 CHCl 3 CHCl 3 CHCl 3 183 179 183 214 Dark blue Deep blue Dark blue Pale Violet 2.5PB 2 / 6 2.5PB 3 / 8 2.5PB 2 / 6 2.5P 6 / 4 a Usual kit reagent for that particular drug. Aqueous phase. c Aqueous phase after chloroform extraction. d Chloroform phase (marijuana extraction usually rapid compared to other materials). e Not extracted into chloroform. f 2-Chloroacetophenone. g Abbreviations: ICSS-NBS5Inter-Society Color Council and the National Bureau of Standards, Munsell5 Munsell color notation, CHCl 3 5chloroform, EtOH5ethanol, MEOH5methanol, B5blue, G5green, P5 purple, Y5yellow, R5red. b (A.9) and Mecke (A.10) reagents. Unlike the cobalt thiocyanate reaction, different colors were produced with different drugs making it easier to presumptively identify the specific drug present. For example, a selected battery of tests to identify heroin (diacetylmorphine) might include the Mandelin, Marquis and Froehde tests because they would produce reddish brown, deep purplish red and purplish red colors, respectively. Codeine, a second opiate, could be identified with the same battery of CSTs because it produced olive, dark purple and dark green colors, respectively. These three CSTs were reactive to many opioids with LODs as low as 1–5 mg, Table 4, but as mentioned the colors produced and the sensitivity was dependent on the many factors listed above. Since positive reactions were dependent on the functional groups present in the chemical structure of the tested analytes, several of the CSTs were specific for certain classes of drugs. The p-DMAB reagent (A.7) reacted only with LSD. producing a deep purple color. This test had an LOD of 6 mg for LSD. Although mace, nutmeg and tea reacted with the modified Duquenois–Levine test (A.3), as shown in Table 2, only tetrahydrocannabinal (THC) produced a deep purple color that was extracted into chloroform. The Simon’s test is reported to be specific for secondary amines like methamphetamine and MDMA. It did not react with ephedrine or pseudoephedrine because their structure contains an hydroxyl group that is in close proximity to the amine. Barbiturates can be detected by both the Dille–Koppanyi (A.2) and Zwikker (A.11) reagents. However, the Dille–Koppanyi test was more sensitive with LODs of 25 mg or lower whereas the LOD for phenobarbital with the Zwikker test was 1000 mg. C.L. O’ Neal et al. / Forensic Science International 109 (2000) 189 – 201 199 Table 3 Specificity of color tests. (1) Indicates that a color reaction occurs a Reagent Acetaminophen Alprazolam Aspirin Baking soda Brompheniramine maleate Chlordiazepoxide HCl Chlorpromazine HCl Contac Diazepam Doxepin HCl Dristan Ephedrine HCl Exedrine Hydrocodone tartrate Mace b Meperidine HCl Methaqualone Methylphenidate HCl Nutmeg b Phencyclidine HCl Propoxyphene HCl Pseudoephedrine HCl Quinine HCl Salt Sugar Tea b Tobacco A.1 A.2 A.3 A.4 A.5 A.6 A.7 A.8 A.9 A.10 A.11 A.12 2 2 2 2 1 1 1 2 2 1 2 1 2 1 2 1 2 1 2 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 1 2 1 2 2 2 2 2 2 1 2 1 2 1 2 1 2 1 1 2 1 1 2 1 2 1 2 2 1 2 2 1 2 1 1 2 2 2 2 2 1 2 2 2 1 2 2 1 1 2 1 2 1 1 2 1 2 2 1 2 2 2 1 2 2 1 2 2 2 2 2 1 2 2 1 1 2 1 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 1 2 2 1 2 2 2 1 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 1 1 2 1 1 2 1 2 1 2 2 2 2 2 1 2 2 2 1 2 2 2 2 2 2 2 2 1 1 2 1 1 2 1 1 1 2 2 2 1 2 1 2 2 2 1 2 2 2 2 2 1 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 a Substances that gave no colors with these reagents are: D-galactose, glucose, mannitol, oregano, rosemary and thyme. b Tea, mace and nutmeg may interfere with the Duquenios test, but not the Duquenois–Levine modified test (A.3). 4. Conclusions Chemical spots tests are valuable tools for the presumptive identification of drugs in unknown samples. These tests are very sensitive with LODs typically 1 to 50 mg depending on the CST and the analyte. The methods and validation procedures for 12 chemical spot tests for use in the laboratory or in the field were described. For the identification of unknown drugs, reference colors from the Munsell and ICSS-NBS centroid color charts representing positive reactions for the 12 CSTs were included. Although these tests are sensitive and can be relatively specific, the actual color observed by the analyst performing the CST depends on many factors such as the concentration of the drug, whether the drug is a salt or free base, which salt form is C.L. O’ Neal et al. / Forensic Science International 109 (2000) 189 – 201 200 Table 4 Drug detection limits a Reagent Analyte A.1 A.1 Cocaine HCl Methadone HCl A.2 A.2 A.2 A.2 Amobarbital Pentobarbital Phenobarbital Secobarbital A.3 THC A.4 A.4 A.4 A.4 A.4 D-Amphetamine A.5 A.5 A.5 A.5 A.5 A.5 A.5 A.5 D-Amphetamine A.6 Mescaline HCl 1 A.7 LSD 6 A.8 Morphine monohydrate 200 A.9 A.9 A.9 A.9 A.9 Codeine Diacetylmorphine HCl LSD Mescaline HCl Morphine monohydrate 50 200 50 100 25 A.10 A.10 A.10 A.10 A.10 Codeine Diacetylmorphine HCl LSD Mescaline HCl Morphine monohydrate 25 200 50 50 50 A.11 Phenobarbital A.12 A.12 D-Methamphetamine a Drug detection limit (mg) 60 250 25 10 15 25 5 HCl D-Methamphetamine HCl Codeine Diacetylmorphine HCl Morphine monohydrate HCl Codeine Diacetylmorphine HCl LSD Mescaline HCl Methadone HCl D-Methamphet HCl Morphine monohydrate HCl Methylphenidate HCl The solvent was chloroform except for A.8 which was methanol. 20 100 20 20 5 10 1 10 5 10 20 5 5 1000 10 300 C.L. O’ Neal et al. / Forensic Science International 109 (2000) 189 – 201 201 present, the presence of contaminants in the sample, the color discrimination of the analyst and the conditions under which the CST is performed. Acknowledgements This project was funded by the National Institute of Standards and Technology, U.S. Department of Commerce, contract number 50SBNB7C118. References [1] F. Feigl, in: Spot Tests in Inorganic Analysis, Elsevier Publishing Co, New York, 1958, p. 327. [2] E.G.C. Clark (Ed.), Isolation and Identification of Drugs, The Pharmaceutical Press, London, UK, 1969, p. 870. [3] I. Sunshine (Ed.), Methodology For Analytical Toxicology, CRC Press, Inc, Cleveland, OH, 1975, p. 478. [4] US Department of Justice, in: NILECJ Standard for Chemical Spot Test Kits for Preliminary Identification of Drugs of Abuse, US Department of Justice, Washington, DC, December, 1978, p. 12. [5] US Department of Justice, in: NIJ Standard for Color Test Reagents / Kits for Preliminary Identification of Drugs of Abuse, US Department of Justice, Washington, DC, July, 1981, p. 9. [6] S.H. Johns, A.A. Wist, A.R. Najam, Spot tests: A color chart reference for forensic chemists, J. Forensic Sci. 24 (1979) 631–649.