In the Laboratory
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Presumptive and Confirmatory Drug Tests Craig Anderson Department of Chemistry, Bard College, Annandale-on-Hudson, NY 12504;
[email protected] Despite the recent boom in forensic science students and general interest in forensic science on several TV shows, there still appears to be a lack of interest in the subject by academics. One important concept in forensic science for students to learn is the difference between class and individual characteristics. Evidence that can be associated with a single source with an extremely high degree of probability is said to possess individual characteristics (1). A good example of this is the matching of a set of fingerprints (1). Evidence is said to possess class characteristics when it can be associated only with a group and never a single source, for example, the matching of blood types (1). We have developed a lab concerning the characterization and identification of unknown white powders that we labeled as suspected illegal drugs that demonstrates the difference between class and individual characteristics. Presumptive tests, such as those where a color change occurs, are those that usually identify a class of compounds whereas a confirmatory test, such as mass spectrometry, is one that conclusively identifies a specific, individual compound. One might suppose that finding substances that give false positive tests for illegal substances would be easy to locate so as to avoid the necessary problems of obtaining illegal material. This however, turns out to be incorrect. Finding substances that give false positive presumptive color tests in academic circles or publications was quite difficult. The National Institute of Justice publishes The Color Test Reagents/ Kits for Preliminary Identification of Drugs of Abuse that includes several legal substances that give positives tests for the reagents mentioned in the document (2). However, substances that give false positive results for urine-sample drug testing were quite easy to find with a quick Internet search (3). The sites obtained by Internet search were almost exclusively devoted to either beating a future drug test situation or to giving legal advice if a charge was already laid. We report here the use of legal, over-the-counter drugs that test positive for amphetamines or opiates when using the Marquis presumptive test. This test is then shown to be a false positive for illegal substances by the use of GC–MS analysis conducted on the extracted free-base amine from the “unknown” white powder. This demonstrates quite clearly that confirmatory tests are absolutely necessary when conducting drug testing and any presumptive screening test should be regarded with suspicion. Results and Discussion Almost all drug testing is first done with some kind of qualitative presumptive test (1, 4). A confirmatory test is then necessary and this demonstrates to the student the rigor needed to conclusively identify a substance. Existing labs concerned with drug testing deal, almost exclusively, with the testing of the illegal material itself (4). This requires either police supervision or a federal Schedule I or II drug license
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(1, 4). Neither of these is easily obtained. However, the system used in our laboratory needs neither. The students learn everything they need about presumptive and confirmatory tests without the actual handling of the illegal material. This makes the lab reported here extremely accessible without losing any authenticity. Different cough medicines purchased from local pharmacies were ground into white powders. Contac, Sudafed, and local pharmacy home brands work well. Be sure to read the active ingredient list. Some varieties contain only pseudoephedrine hydrochloride while others contain both pseudoephedrine hydrochloride and dextromethorphoran hydrobromide. Both of these substances test positive with the Marquis reagent. A small quantity of the white powder from a crushed pill was placed on a spot plate and several drops of the Marquis reagent were added. The Marquis reagent is prepared by the addition of 10 drops of 40 percent formaldehyde solution to 10 mL of concentrated sulfuric acid (4). The Marquis reagent turns violet in the present of opium alkaloids such as heroin, morphine, and codeine (4). The amphetamines turn the Marquis reagent red–orange (somewhat rusty) (2). Pseudoephedrine gives a positive test for amphetamines and dextromethorphan gives a positive test for opiates. If both are contained in the same drug mixture, the color obtained is a grayish green that turns to dark violet after several minutes. After the presumptive test has been completed with positive results, the idea is to show the result to be, in fact, a false positive for any illegal substance upon analysis with the GC–MS. However, the drugs in solid cough medicines are usually either hydrochloride or hydrobromide salts, not unlike the illegal street drugs themselves. Therefore, the next step is an extraction process necessary to remove the drug from all other binders and fillers in the pill and to obtain the free base alkaloid drug that will be necessary for the GC– MS analysis. Our first step was to add hydrochloric acid to the sample and filter any insoluble substances. This ensures that all alkaloid substances will be present as hydrochloride salts and hence water soluble. The next step was to generate the free base amine by making the acidic solution basic by adding just enough sodium hydroxide solution to neutralize the excess acid. The free base amines were then extracted into diethyl ether, the diethyl ether solution dried over sodium sulfate, filtered, and finally injected into the GC–MS for analysis. The GC separates a solution of both pseudoephedrine and dextromethorphan under the correct conditions. The parent ion of the amine or an internal library can be used to identify a substance from its mass spectrum. We had an internal library that identified pseudoephedrine (5). The presence of pseudoephedrine was also confirmed by the peaks in its mass spectrum. It has a very small peak at 164 m兾z (M = 165 g兾mol) owing to loss of a hydrogen, a second peak at 146 m兾z owing to loss of water, and its largest peak at 58 m兾z. The identification of pseudoephedrine is, however,
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Journal of Chemical Education
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In the Laboratory
not trivial as the peaks above 58 are not observed in all situations. Real analyses for this class of drug utilize a silane derivatizing agent in order to obtain exact results (6). The best idea, if time permits, is to purchase the two substances from a manufacturer, for example, Sigma-Aldrich, and run them both individually through the GC–MS in order to obtain standards on the instrument actually being used for the experiment with which the unknown spectra can be compared. Pseudoephedrine is also available for purchase as the free base and this would eliminate the extraction step when setting the standard. This illustrates the necessary rigor involved in making a conclusive identification of an unknown substance especially in a legal case. A simpler version of this lab that will illustrate the same ideas of presumptive and confirmatory tests is the cobalt thiocyanate test for cocaine. The reagent is simple to prepare: 2% cobalt thiocyanate in water (4). A blue precipitate is seen when cocaine is mixed with the above reagent (4). (−)-Scopolamine hydrochloride gives a perfect false positive test for cocaine hydrochloride. In this case infrared spectroscopy can be used as the confirmatory test, the second most widely used confirmatory test for narcotics in the forensic field, right after GC–MS (1). Infrared is somewhat limited however owing to the purity needed for correct identification (1). (−)-Scopolamine hydrochloride can be purchased from a manufacturer such as Sigma-Aldrich. The presumptive test is easily conducted by adding the reagent to 5–10 mg of the scopolamine hydrochloride to witness the positive result. The infrared spectrum is then run as a KBr pellet and compared to one of cocaine hydrochloride (7). The glaring difference is in the carbonyl stretch region. Cocaine hydrochloride has two peaks for its two esters (1730 and 1710 cm᎑1) while scopolamine hydrochloride has only one ester group and only one carbonyl stretch at 1725 cm᎑1. The fingerprint region can be compared to corroborate that the “unknown” compound is not cocaine hydrochloride as numerous differences exist. Hazards Wear protective gloves, glasses, and clothing for the handling of all chemicals. Handle all chemicals in a fumehood. The drugs used in this lab, (−)-scopolamine hydrochloride, dextromethorphan hydrobromide, and (+)-pseudoephedrine hydrochloride, affect the central nervous systems and should not be ingested as they are very toxic. They are also irritants. Marquis reagent contains formaldehyde and sulfuric acid. Formaldehyde is a suspected carcinogen. Sulfuric and hydrochloric acids, and sodium hydroxide are toxic, corrosive, and may cause severe burns. Cobalt thiocyanate is harmful if swallowed and readily absorbed through the skin. Diethyl ether is a toxic, flammable liquid.
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Concluding Remarks Average, everyday, easily obtained cold medicines from local pharmacies test positive for illegal substances using presumptive color tests. They subsequently proved not to be illegal substances by confirmatory tests. The use of GC–MS allows for conclusive characterization of the “unknown” substances. This lab has a clear pedagogical aspect showing the students the difference between class characteristics found using qualitative color tests and individual characteristics when using precise instrumentation. This is a concept extremely important for the forensic scientist (1). The lab also demonstrates the acid–base chemistry of the alkaloids when performing the extraction procedure, a step used in almost every forensic analysis (1). It does all this without the necessity of obtaining any illegal material or federal drug licenses, unlike other drug-testing experiments already known (4). Therefore, this makes for a very accessible and educative experiment. Acknowledgments The author would like to thank students Karin Kram and Peter Milano for assisting in the experimental procedures. We acknowledge the support of Orgometa. W
Supplemental Material
Instructions for students and notes for the instructor including possible hazards are available in this issue of JCE Online. Literature Cited 1. Saferstein, Richard. Criminalistics: An Introduction to Forensic Science, 8th ed.; Prentice-Hall, Inc.: Upper Saddle River, NJ, 2004. 2. Color Test Reagents/Kits for Preliminary Identification of Drugs of Abuse (National Institute of Justice Standard0604.01) http://www.ojp.usdoj.gov/nij/pubs-sum/183258.htm (accessed Aug 2005). 3. Many sites exist, for example try: http://www.passyourtest.com (accessed Aug 2005). 4. James, Richard E.; Saferstein, Richard; Meloan, Clifton E. Lab Manual, Criminalistics: An Introduction to Forensic Science, 7th ed.; Prentice-Hall, Inc.: Upper Saddle River, NJ, 2001. 5. G1033A NIST/EPA/MSDC Mass Spectral Database for HP and IBM At-Compatible Systems, licensed to Hewlett Packard, U.S. Department of Commerce, 1988. 6. Spyridaki, M. E.; Tsitsimpikou, C. J.; Siskos, P. A.; Georgakopoulos, C. G. J. Chrom. B 2001, 758, 311–314. 7. IR spectrum of cocaine hydrochloride. http://jpdb.nihs.go.jp/ jp14e/14data/Spectra_IR/1117.pdf (accessed Aug 2005).
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