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Amphetamine and Related Drugs 5/23/2017 1 Narcotics Narcotics of Natural Origin -Opium -Morphine -Codeine -Thebaine Semi-Synthetic Narcotics -Heroin -Hydromorphone -Oxycodone -Hydrocodone Synthetic Narcotics -Meperidine -Dextropropoxyphene -Fentanyl -Pentazocine -Butorphanol Narcotics Treatment Drugs -Methadone 5/23/2017 2 Stimulants Cocaine Amphetamines Methcathinone Methylphenidate Anorectic (appetite suppressant) Drugs Khat 5/23/2017 3 Depressants Barbiturates Benzodiazepines Flunitrazepam (hypnotic) Gamma Hydroxybutyric Acid Paraldehyde Chloral Hydrate Glutethimide and Methaqualone Meprobamate 5/23/2017 4 Cannabis Marijuana Hashish Hashish Oil 5/23/2017 5 Hallucinogens LSD Psilocybin & Psilocyn and other Tryptamines Peyote & Mescaline New Hallucinogens MDMA (Ecstasy) and other Phenethylamines Phencyclidine and Related Drugs Ketamine 5/23/2017 6 Drug Schedules: Schedule I • The drug or other substance has a high potential for abuse. • The drug or other substance has no currently accepted medical use in treatment in the United States. • There is a lack of accepted safety for use of the drug or other substance under medical supervision. • Examples of Schedule I substances include heroin, lysergic acid diethylamide (LSD), marijuana, and methaqualone. 5/23/2017 7 Schedule I amphetamine derivatives 2,5-Dimethoxy-4-ethylamphetamine 2,5-Dimethoxyamphetamine 3,4,5-Trimethoxyamphetamine 3,4-Methylenedioxyamphetamine 3,4-Methylenedioxymethamphetamine 4-Bromo-2,5-dimethoxyamphetamine 4-Bromo-2,5-dimethoxyphenethylamine 4-Methoxyamphetamine 4-Methyl-2,5-dimethoxyamphetamine 5-Methoxy-3,4-methylenedioxyamphetamine 5/23/2017 8 Schedule II • The drug or other substance has a high potential for abuse. • The drug or other substance has a currently accepted medical use in treatment in the United States or a currently accepted medical use with severe restrictions. • Abuse of the drug or other substance may lead to severe psychological or physical dependence. •Examples of Schedule II include morphine, phencyclidine (PCP), cocaine, methadone, and methamphetamine 5/23/2017 9 Schedule III • The drug or other substance has less potential for abuse than the drugs or other substances in schedules I and II. • The drug or other substance has a currently accepted medical use in treatment in the United States. • Abuse of the drug or other substance may lead to moderate or low physical dependence or high psychological dependence. • Anabolic steroids, codeine and hydrocodone with aspirin or Tylenol®, and some barbiturates are examples of Schedule III substances. 5/23/2017 10 Schedule IV • The drug or other substance has a low potential for abuse relative to the drugs or other substances in Schedule III. • The drug or other substance has a currently accepted medical use in treatment in the United States. • Abuse of the drug or other substance may lead to limited physical dependence or psychological dependence relative to the drugs or other substances in Schedule III. •Examples of drugs included in schedule IV are Darvon®, Talwin®, Equanil®, Valium®, and Xanax®. 5/23/2017 11 Schedule V • The drug or other substance has a low potential for abuse relative to the drugs or other substances in Schedule IV. • The drug or other substance has a currently accepted medical use in treatment in the United States. • Abuse of the drug or other substances may lead to limited physical dependence or psychological dependence relative to the drugs or other substances in Schedule IV. •Cough medicines with codeine are examples of Schedule V drugs 5/23/2017 12 Most Common Amphetamines There are a large number of amphetamines which are controlled substances. Of these, the most commonly encountered in the forensic science laboratory are amphetamine (1), methylamphetamine (2), 3,4methylenedioxyamphetamine (MDA) (3), 3,4methylenedioxymethylamphetamine (MDMA) (4)and 3,4-methylenedioxyethylamphetamine (MDEA) (5). In addition, there are a wide variety of structurally related analogues which can be synthesized. 5/23/2017 13 5/23/2017 14 Some history Amphetamine was first marketed in the 1930s as Benzedrine® in an over-the-counter inhaler to treat nasal congestion. By 1937, amphetamine was available by prescription in tablet form and was used in the treatment of the sleeping disorder, narcolepsy, and the behavioral syndrome called minimal brain dysfunction, which today is called attention deficit hyperactivity disorder (ADHD). During World War II, amphetamine was widely used to keep the fighting men going and both dextroamphetamine (Dexedrine®) and methamphetamine (Methedrine®) were readily available. 5/23/2017 15 As use of amphetamines spread, so did their abuse. In the 1960s, amphetamines became a perceived remedy for helping truckers to complete their long routes without falling asleep, for weight control, for helping athletes to perform better, and for treating mild depression. With experience, it became evident that the dangers of abuse of these drugs outweighed most of their therapeutic uses. 5/23/2017 16 To meet the ever-increasing black market demand for amphetamines, clandestine laboratory production has mushroomed. Today, most amphetamines distributed to the black market are produced in clandestine laboratories. Methamphetamine laboratories are, by far, the most frequently encountered clandestine laboratories in the United States. The ease of clandestine synthesis, combined with tremendous profits, has resulted in significant availability of illicit methamphetamine, especially on the West Coast, where abuse of this drug has increased dramatically in recent years. 5/23/2017 17 Amphetamines are generally taken orally or injected. However, the addition of "ice," the slang name for crystallized methamphetamine hydrochloride, has promoted smoking as another mode of administration. Just as "crack" is smokable cocaine, "ice" is smokable methamphetamine. Methamphetamine, in all its forms, is highly addictive and toxic. 5/23/2017 18 The effects of amphetamines, especially methamphetamine, are similar to cocaine, but their onset is slower and their duration is longer. In contrast to cocaine, which is quickly removed from the brain and is almost completely metabolized, methamphetamine remains in the central nervous system longer, and a larger percentage of the drug remains unchanged in the body, producing prolonged stimulant effects. Chronic abuse produces a psychosis (severe mental disorder), picking at the skin, and visual hallucinations. These psychotic symptoms can persist for months and even years after use of these drugs has ceased and may be related to their neurotoxic effects. Violent and erratic behavior is frequently seen among chronic abusers of amphetamines, especially methamphetamine. 5/23/2017 19 AMPHETAMINE Amfetamine Central Stimulant Synonyms. Amphetamine; Anfetamina; Racemic Dexedrine. Proprietary names. It is an ingredient of Biphetamine and Durophet. 5/23/2017 20 A colourless, mobile, slowly volatile liquid. It absorbs carbon dioxide from the air forming a volatile carbonate. B.p. 200° to 203°. Soluble 1 in 50 of water; soluble in ethanol chloroform and ether; readily soluble in acids Colour Tests. Liebermann's Test (sulfuric acid + nitrous acid) —red–orange; Marquis Test—orange→brown; Ninhydrin—pink–orange 5/23/2017 21 Disposition in the Body. Readily absorbed after oral or rectal administration; rapidly distributed extravascularly and taken up, to some extent, by red blood cells. The main metabolic reaction is oxidative deamination to form phenylacetone, which is then oxidised to benzoic acid and conjugated with glycine to form hippuric acid; minor reactions include aromatic hydroxylation to form 4–hydroxyamfetamine (an active metabolite), βhydroxylation to form norephedrine (phenylpropanolamine), and N-oxidation to form a hydroxylamine derivative. 5/23/2017 22 5/23/2017 23 Excretion of amfetamine is markedly dependent on urinary pH, being greatly increased in acid urine. After large doses, amfetamine may be detected in urine for several days. Under uncontrolled urinary pH conditions, about 30% of the dose is excreted unchanged in the urine in 24 h and a total of about 90% of the dose is excreted in 3 to 4 days. The amount excreted unchanged in 24 h may increase to 74% of the dose in acid urine and decrease to 1 to 4% in alkaline urine; under alkaline conditions, hippuric acid and benzoic acid account for about 50% of the urinary material. Under normal conditions 16 to 28% is excreted as hippuric acid, about 4% as benzoylglucuronide, 2 to 4% as 4–hydroxyamfetamine, and about 2% as norephedrine in 24 h; small amounts of conjugated 4–hydroxynorephedrine and phenylacetone are also excreted. No elimination in the faeces has been detected. 5/23/2017 24 Therapeutic concentration After normal therapeutic doses the plasma concentration is usually below 0.1 mg/L. However, continued use of amfetamine may cause addiction, and ingestion of 10 times the usual therapeutic dose is common among addicts; in such cases the plasma concentration may be up to 3 mg/L. After a single oral dose containing 10 mg of amfetamine to 4 subjects, peak plasma concentrations of about 0.02 mg/L were attained 5/23/2017 25 Steady–state blood concentrations of 2 to 3 mg/L were observed in a regular user who ingested about 1 g a day. The intravenous administration of 160 mg of amfetamine to a regular user resulted in a plasma concentration of 0.59 mg/L after 1 h. 5/23/2017 26 Toxicity The estimated minimum lethal dose in non–addicted adults is 200 mg. Toxic effects may be produced with blood concentrations of 0.2 to 3 mg/L, and fatalities with concentrations greater than 0.5 mg/L. Death from overdosage is comparatively rare. In a fatality caused by intravenous administration of amfetamine, the following postmortem tissue concentrations were reported: blood 41 mg/L, liver 23 μg/g, urine 39 mg/L. 5/23/2017 27 Half–life. Plasma half–life, 4 to 8 h when the urine is acidic and about 12 h in subjects whose urinary pH values are uncontrolled. Volume of distribution. About 3 to 4 L/kg. Dose. 20 to 100 mg of amfetamine sulfate daily has been used in the treatment of narcolepsy. 5/23/2017 28 Methylenedioxymethamfetamine Stimulant, Hallucinogen N,α-Dimethyl–1,3,benzodioxole–5–ethanamine FW =193.2 A viscous, colourless oil. B.p. 100° to 110°. 5/23/2017 29 Colour Test. Marquis Reagent—black with dark purple. Thin–layer Chromatography. System TA—Rf 0.33; system TB—Rf 0.24; system TE—Rf 0.39; system TF—Rf 0.20; system TAE—Rf 0.08; system TAJ—Rf 0.03; system TAK—Rf 0.17; system TAL—Rf 0.57. 5/23/2017 30 Disposition in the Body. It is absorbed into the blood stream after ingestion and excreted in urine, the majority of the dose unchanged (65% within 3 days). Metabolism occurs by a number of routes: Ndemethylation of the parent compound to 3,4– methylenedioxyamfetamine (MDA) (7%) with further Odemethylation to 3,4–dihydroxymethamfetamine (HHMA) and 3,4–dihydroxyamfetamine (HHA). Both HHMA and HHA are subsequently O-methylated mainly to 4–hydroxy–3– methoxymetamfetamine (HMMA) and 4–hydroxy–3– methoxyamfetamine (HMA). These four metabolites are excreted in the urine as the conjugated glucuronide or sulfate metabolites. 5/23/2017 31 Therapeutic concentration 8 healthy male volunteers, aged between 21 and 31 years old, were administered a 75 mg dose of MDMA. The mean peak plasma concentration was 0.13 mg/L after 1.8 h. Mean peak plasma concentrations of the metabolite, 3,4– methylenedioxyamfetamine (MDA), were 7.8 μg/L approximately 5 h after administration. 5/23/2017 32 After the administration of a single oral dose of 1.5 mg/kg body weight MDMA to 2 patients, plasma and urine samples were collected over periods of 9 and 22 h, respectively. Peak plasma concentrations of MDMA and MDA were 331 μg/L after 2 h and 15 μg/L after 6.3 h, respectively. Peak concentrations of 28.1 μg/L MDMA in urine appeared after 21.5 h. Up to 2.3 μg/L MDA, 35.1 μg/L HMMA, and 2.1 μg/L HMA were measured within 16 to 21.5 h, also in urine. 5/23/2017 33 Toxicity Fatalities with doses of 300 mg have been reported. Capable of causing severe toxicity and the pattern of acute toxicity is due to the circumstances in which it is misused. A lethal concentration of 0.35 to 0.50 mg/L in serum has been noted although some overdose cases report concentrations 10 times this amount, without fatality. 5/23/2017 34 Half–life. About 6 to 7 h. Clearance. The mean total clearance of MDMA for a 75 mg dose is 86.9 L/h. Protein binding About 65% Dose. The usual dose is between 80 and 200 mg (more often 100 to 150 mg). 5/23/2017 35 Metamfetamine Central Stimulant Synonyms. d-Deoxyephedrine; Desoxyephedrine; Methamphetamine; Methylamfetamine; methylamphetamine; Phenylmethylaminopropane. Metamfetamine in a smokeable form has been known as Crank, Crystal, Crystal meth, Ice, meth, and Speed. 5/23/2017 36 FW =149.2 A clear, colourless, slowly volatile, mobile liquid. Mass per mL 0.921 to 0.922 g. B.p. about 214°. Slightly soluble in water; miscible with ethanol, chloroform, and ether. 5/23/2017 37 Colour Test. Marquis Test—orange. Thin–layer Chromatography. System TA—Rf 0.31; system TB—Rf 0.28; system TC—Rf 13; system TE—Rf 0.42; system TL—Rf 0.05; system TAE—Rf 0.09; system TAF—Rf 0.63; system TAJ—Rf 0.00; system TAK—Rf 0.03; system TAL—Rf 0.45. (Dragendorff spray, positive; acidified iodoplatinate solution, positive; Marquis reagent, brown; ninhydrin spray, positive; acidified potassium permanganate solution, positive.) 5/23/2017 38 Disposition in the Body Readily absorbed after oral administration. About 70% of a dose is excreted in the urine in 24 h. Under normal conditions, up to 43% of a dose is excreted as unchanged drug, up to 15% as 4– hydroxymetamfetamine, and about 5% as amfetamine, the major active metabolite. A number of other metabolites have been identified. Excretion of unchanged drug is dependent on the urinary pH, being increased in acidic urine and greatly reduced (to about 2% of a dose) if the urine is alkaline 5/23/2017 39 Following a single oral dose of 12.5 mg of metamfetamine hydrochloride to 10 subjects, a mean peak blood concentration of about 0.02 mg/L was attained in about 2 h Toxicity The estimated minimum lethal dose is 1 g, but fatalities attributed to metamfetamine are rare. 5/23/2017 40 Half–life Plasma half–life, about 9 h. Dose. 2.5 to 25 mg of metamfetamine hydrochloride daily, by mouth; 15 to 20 mg IM, or 10 to 15 mg IV. 5/23/2017 41 Methylenedioxyethylamfetamine Stimulant, Hallucinogen Synonyms. N-Ethyl–3,4– methylenedioxyphenylisopropylamine; Eve; MDE; MDEA; 3,4-Methylenedioxyethamphetamine; 3,4Methylenedioxyethylamphetamine. Usually presented as Ecstasy. N-ethyl-α-methyl–1,3–benzodioxole–5–ethanamine FW =207.3 5/23/2017 42 A viscous, colourless oil. B.p. 0.2 is 85° to 95° 5/23/2017 43 Disposition in the Body. It is absorbed into the blood stream after ingestion and excreted in urine, mainly as the parent drug (19%), methylenedioxyamfetamine (MDA, 28%) and also 4–hydroxy–3–methoxyethylamfetamine (HMEA, 32%). 5/23/2017 44 Toxicity The estimated lethal dose is 0.5 g. In a 20-year-old male whose death was attributed to injection of MDMA and MDEA, postmortem blood concentrations of 2.0 and 0.7 mg/L, respectively, were reported 5/23/2017 45 Methylenedioxyamfetamine Hallucinogen Synonyms. MDA; Methylenedioxyamphetamine; Tenamfetamine; SKF-5. α-Methyl–1,3–benzodioxole–5–ethanamine FW =179.2 5/23/2017 46 5/23/2017 47 Thin Layer Chromatography of Amphetamines In order that the sample can be tested for the presence of amphetamines, a test solution must be prepared. The sample should be dissolved in a suitable solvent (methanol is commonly used) at a sample concentration of the order of 10 mgml−1. This allows for the fact that many amphetamine samples at the ‘street level’ are extremely weak, i.e. between 2 and 10% amphetamine in a matrix of adulterants and diluants, giving a solution of approximately 0.2–1.0 mgml−1, namely a concentration at which the standards can be prepared. 5/23/2017 48 The sample should be dissolved as fully as possible and centrifuged or filtered to remove any solid particulates. A positive and negative control should also be prepared. The silica gel chromatographic plate should be marked up and the test solutions, plus the positive and negative controls, placed on the plate and the latter allowed to develop in the chosen solvent system 5/23/2017 49 Practical TLC Urine Tests The urine sample is adjusted to pH 10 with potassium carbonate. Sodium chloride is also added and the mixture is extracted twice with chloroform. The chloroform phase each time is removed and filtered. The pooled chloroform extracts are washed with a weak solution of ammonium hydroxide. The washed chloroform is then extracted twice with 1 N sulfuric acid. 5/23/2017 50 The pooled sulfuric acid extracts are then adjusted to pH 10 with concentrated potassium hydroxide and potassium carbonate. Sodium chloride is also added and the mixture is extracted twice with chloroform. The filtered and pooled chloroform is then carefully evaporated after the addition of one drop of a solution of 0.5% sulfuric acid in methanol. 5/23/2017 51 The residue is redissolved in acetone methanol solution and applied to a T.L.C. plate for development. The solvent system contains methanol and ammonium hydoxide. The test detects methadone, pethidine, cocaine, amphetamine, methamphetamine, cyclazocine and D-propoxyphene. Many other organic bases would be extracted by this procedure and appear on the T.L.C. plate. 5/23/2017 52 Urine analyzed by this test shows an increase in background with the age of urine which partially interferes with the location of the spots after T.L.C. Amphetamine, methamphetamine, pethidine and methadone are more labile compounds than morphine and codeine and more susceptible to decomposition and chemical change during storage or during testing. 5/23/2017 53 Procedure for the Test Measure 20 ml of urine into a 50 ml glassstoppered centrifuhe tube. Add 1 g of potassium carbonate to adjust the pH to 10. Add 4 g of sodium chloride. Add the salts using a powder funnel and measuring spoons. Shake to dissolve the salts. Add 20 ml of chloroform and shake for 5 minutes and centrifuge. 5/23/2017 54 Aspirate off the lower chloroform layer and filter into another tube. Add 20 ml of chloroform for a second extraction. Shake for 5 minutes and centrifuge. Aspirate off the lower chloroform layer and filter into the second tube. Wash the filtered pooled chloroform with 10 ml of pH 9 aqueous ammonium hydroxide solution as follows: shake for 5 minutes and centrifuge and aspirate off and discard the upper wash phase. 5/23/2017 55 Add 10 ml of 1 N sulfuric acid to the tube, shake for 5 minutes and centrifuge. Aspirate off the upper acid phase and tranfer it to a third tube. Repeat the extraction with another 10 ml portion of 1 N sulfuric acid and pool with the first extraction in the third tube. Discard the lower chloroform phase. To the acid phase in the third tube add 16 N potassium hydroxide dropwise (about 1.3 ml) to adjust the pH to about 7. Add 1 g of potassium carbonate to adjust the pH to 10. Add 4 g of sodium chlorides and shake to dissolve the salts. 5/23/2017 56 Add 20 ml chloroform and shake for 5 minutes and centrifuge. Aspirate the lower chloroform phase and filter into a 50 ml beaker. Repeat the extraction with a second 20 ml of chloroform and aspirate off and discard the upper aqueous phase. Decant and filter the chloroform phase into the beaker. Add 3 ml of chloroform wash to the tube and filter into the beaker. 5/23/2017 57 Add one drop of 0.5 % sulfuric acid in methanol to the pooled chloroform in the beaker. Evaporate carefully to near dryness in the vacuum oven at a temperature of 90 oC and a vacuum of 10 p.s.i. Remove the beaker and allow the final few drops of solvent to air dry. 5/23/2017 58 Transfer the residue to a 3 ml microcentrifuge tube using small portion (0.5 ml) of 1:1 acetone-methanol. Again evaporate at a slow boil to near dryness in the vacuum oven maintained at 10 p.s.i, and 60 oC. 5/23/2017 59 Remove a thin-layer plate from the desiccator just before it is to be spotted. Spot the sample residues, procedure controls and reference compounds on a thin-layer plate on the sample application line located 2.5 cm and parallel to the bottom edge of the plate. 5/23/2017 60 Dissolve the residues in 20 µl of 1:1 acetonemethanol and spot the dissolved sample from the micro centrifuge tubes using a 10 µl microsyringe. Repeat the spotting twice adding solvent each time to insure that all of the dissolved residue is transferred. Apply 30µg of methadone, 60 µg each of amphetamine and methamphetamine toward the center of the application line . 5/23/2017 61 Place the spotted plated into the developing tank containing 3 ml of conc. ammonium hydroxide in 200 ml of methanol which has equilibrated for 10 minutes. Allow the development to proceed until there is about 14 cm of front movement in about 30 minutes. 5/23/2017 62 Remove the developed plate and allow it to air dry for about one hour. Examine the plate under ultraviolet light for absorbing or fluorescent spots and circle the spots on the uncoated side of the plate using a china marking pencil. Spray with Dragendorff's Reagent and make notes of spots, colors and intensities. Then spray with potassium iodoplatinate reagent and repeat the observations . 5/23/2017 63 Examine the batch of plates making appropriate comparisons. Spray the plates within two hours after development and read and interpret them as the sprays are applied and again 10 minutes later. 5/23/2017 64 The ammonia is added to achieve a process known as ion suppression. By converting the drugs to their free base forms, their polarities are reduced. This is because the nitrogen atom does not carry a positive charge in basic solution. The latter reduces the problem of (TLC) tailing, improves the mass transfer properties between the stationary and mobile phases, and thus improves the chromatographic quality. 5/23/2017 65 In addition, MDA, MDMA and MDEA give rise to purple, orange/red and orange/red products, respectively. At each of the visualization stages, the retardation factor (or relative front) (Rf) values of the visualized compounds should be calculated by using the following equation: Distance moved by the analyte of interest Rf = ------------------------------------------------------Distance moved by the solvent front 5/23/2017 66 The Rf values of the unknowns are compared to those of the standards and if the data cannot be discriminated then a suggested match is called. Although when using this combination of presumptive tests and TLC it is possible to discriminate within this group of compounds, due to the extremely large number of amphetamines available, it is necessary to carry out a confirmatory analytical technique. The foremost of these, for amphetamine identification, is gas chromatography– mass spectrometry (GC–MS( 5/23/2017 67 Thin–layer Chromatographic Systems for Amphetamine System TA—Rf 43; system TB—Rf 20; system TC—Rf 09; system TE—Rf 43; system TL—Rf 18; system TAE—Rf 12; system TAF—Rf 75. (Dragendorff spray, positive; FPN reagent, pink; acidified iodoplatinate solution, positive; Marquis reagent, brown; ninhydrin spray, positive; acidified potassium permanganate solution, positive(. 5/23/2017 68 System TA Plates: Silica gel G, 250 μm thick, dipped in, or sprayed with, 0.1 M potassium hydroxide in methanol, and dried. Mobile phase: Methanol:strong ammonia solution (100:1.5). Reference compounds: Atropine Rf 18, Codeine Rf 33, Chlorprothixene Rf 56, Diazepam Rf 75. 5/23/2017 69 Colour test The Marquis test gives an orange colour for both amfetamine and metamfetamine. Thin layer chromatography TA: amfetamine Rf = 0.43, metamfetamine Rf = 0.31. TB: amfetamine Rf = 0.15, metamfetamine Rf = 0.28. Visualisation: acidified iodoplatinate solution. 5/23/2017 70 System TB Plates: Silica gel G, 250 μm thick, dipped in, or sprayed with, 0.1 M potassium hydroxide in methanol, and dried. Mobile phase: Cyclohexane:toluene:diethylamine (75:15:10). Reference compounds: Codeine Rf 06, Desipramine Rf 20, Prazepam Rf 36, Trimipramine Rf 62 5/23/2017 71 System TC Plates: Silica gel G, 250 μm thick, dipped in, or sprayed with, 0.1 M potassium hydroxide in methanol, and dried. Mobile phase: Chloroform:methanol (90:10). Reference compounds: Desipramine Rf 11, Physostigmine Rf 36, Trimipramine Rf 54, Lidocaine Rf 71. 5/23/2017 72 System TL Plates: Silica gel G, 250 μm thick, dipped in, or sprayed with, 0.1 M potassium hydroxide in methanol, and dried. Mobile phase: Acetone. Reference compounds: Amitriptyline Rf 15, Procaine Rf 30, Papaverine Rf 47, Cinnarizine Rf 65. 5/23/2017 73 System TAE Plates: Silica gel G, 250 μm thick. Mobile phase: Methanol. Reference compounds: Codeine Rf 20, Trimipramine Rf 36, Hydroxyzine Rf 56, Diazepam Rf 82. 5/23/2017 74 System TAF Plates: Silica gel G, 250 μm thick. Mobile phase: Methanol:n-butanol (60:40) and 0.1 mol/L NaBr. Reference compounds: Codeine Rf 22, Diphenhydramine Rf 48, Quinine Rf 65, Diazepam Rf 85 . 5/23/2017 75 Location reagents for systems TA, TB and TC Ninhydrin spray Spray the plate with the reagent and then heat in an oven at 100° for 5 min. Violet or pink spots are given by primary amines and yellow colours Ninhydrin Spray: add 0.5 g of ninhydrin to 10 mL of hydrochloric acid and dilute to 100 mL with acetone. Prepare daily. 5/23/2017 76 FPN reagent Red or brown-red spots are given by phenothiazines and blue spots by dibenzazepines. This reagent may be used to overspray a plate which has been previously sprayed with ninhydrin spray. FPN Reagent: mix together 5 mL of ferric chloride solution, 45 mL of a 20% w/w solution of perchloric acid, and 50 mL of a 50% v/v solution of nitric acid. 5/23/2017 77 Dragendorff spray Yellow, orange, red-orange, or brown-orange spots are given by tertiary alkaloids. This reagent may be used to overspray a plate which has been previously sprayed with ninhydrin spray and FPN spray. Dragendorff Spray: (a) mix together 2 g of bismuth subnitrate, 25 mL of acetic acid, and 100 mL of water; (b) dissolve 40 g of potassium iodide in 100 mL of water. Mix together 10 mL of (a), 10 mL of (b), 20 mL of acetic acid, and 100 mL of water. Prepare every 2 days. 5/23/2017 78 Acidified iodoplatinate solution Violet, blue-violet, grey-violet, or brown-violet spots on a pink background are given by tertiary amines and quaternary ammonium compounds. Primary and secondary amines give dirtier colours. This solution may be used to overspray a plate which has previously been sprayed with ninhydrin spray, FPN reagent and Dragendorff spray. Iodoplatinate Solution, Acidified: add 5 mL of hydrochloric acid to 100 mL of iodoplatinate solution. 5/23/2017 79 Mandelin’s reagent This reagent is preferably poured onto the plate because of the danger of spraying concentrated acid. Many different colours are given with a variety of drugs Mandelin's Reagent: dissolve 0.5 g of ammonium vanadate in 1.5 mL of water and dilute to 100 mL with sulfuric acid. Filter the solution through glass wool. 5/23/2017 80 Marquis reagent This reagent is preferably poured onto the plate because of the danger of spraying concentrated acid. Black or violet spots are given by alkaloids related to morphine. Many different colours are given with a variety of drugs Marquis Reagent: mix 1 mL of formaldehyde solution with 9 mL of sulfuric acid. Prepare daily. 5/23/2017 81 Acidified potassium permanganate solution Yellow-brown spots on a violet background are given by drugs with unsaturated aliphatic bonds. Potassium Permanganate Solution, Acidified: a 1% solution of potassium permanganate in 0.25 M sulfuric acid. 5/23/2017 82 Gas Chromatography. System GA—amfetamine RI 1125, amfetamine-TFA RI 1095, amfetamine-PFP RI 1330, amfetamine-TMS RI 1190, amfetamine-AC RI 1501, art (formyl) RI 1100, M (3-OH-)PFP2 RI 1520, M (3-OH-)-TMS2 RI 1850, M (3-OH-)-AC2 RI 1930, M (4-OH-) RI 1480, M (4-OH-)-AC2 RI 1900, M (3,4–di-OH-)-AC3 RI 2150, M (OH-methoxy-) RI 1465, M (OH-methoxy-)-AC2 RI 2065, M (desamino–oxo-OH-)-AC RI 1520, M (desamino–oxo-OH-methoxy-) RI 1510, M (desamino–oxo-OH-methoxy-)-AC RI 1600, M (desamino– oxo–di-OH-)-AC2 RI 1735; system GB—amfetamine RI 1150; art (formyl) RI 1142; system GC—RI 1536; system GF—RI 1315; system GAK—retention time 4.9 min. 5/23/2017 83 Column: DB-5 fused silica (30 m × 0.25 mm i.d., 0.25 μm film thickness). Column temperature: 70° for 1 min, ramp to 100° at 30°/min, and to 270° at 10°/min. Injector temperature: 280°. Carrier gas: helium, flow rate 0.8 mL/min. MS detection. Retention time: 6.5 min. 5/23/2017 84 High Performance Liquid Chromatography. System HA—k 0.9; system HB—k 8.48; system HC—k 0.98; system HX—RI 244; system HAA—retention time, 3.7 min; system HBC—retention time 2.1 min; system HBD— retention time 3.7 min. 5/23/2017 85 Column: Chiralcel OD-RH (150 × 2 mm i.d., 5 μm) at 35°. Mobile phase: phosphate–citrate buffer (pH 4.0) with sodium hexafluorophosphate (0.3 M):acetonitrile (43:57), flow rate 0.1 mL/min. Fluorescence detection (λex=330 nm, λem=440 nm). Retention time: 24.6 min. 5/23/2017 86 Infra–red Spectrum Principal peaks at wavenumbers 700, 740, 1495, 1090, 1605, 825 cm−1 5/23/2017 87 Definitive Identification of Amphetamines GC–MS is the preferred method for the identification of amphetamines. The discussion below centres on the analysis of amphetamine itself, although the same principles can also be applied to other members of this class of drug. However, there are a number of problems associated with the gas chromatographic analysis of amphetamine. Being highly polar in nature, this compound is liable to poor chromatographic behaviour and tailing if the analytical instrument is not scrupulously clean 5/23/2017 88 5/23/2017 89 5/23/2017 90 Furthermore, the highly polar nature of the amino group results in sorption of amphetamine to the surfaces of the GC system components. This, coupled with the often low concentration of the amphetamine in the sample, results in the false impression that there is no amphetamine present in the specimen under investigation 5/23/2017 91 In order to alleviate this problem, derivatization can be employed. One of the easiest processes, for the analysis of amphetamine, is to derivatize directly with carbon disulfide and it is this method which finds wide application in the United Kingdom. For bulk and trace samples, this is achieved by dissolving the material 5/23/2017 92 5/23/2017 93 The reaction (see equation (2.1)) is a simple, pre-column derivatization, involving the amino group of the amphetamine and the CS2 .Thisprocess reduces the polarity of the product, improving its chromatographic behaviour and hence the sensitivity of the method. In addition, it results in a molecule which produces characteristic fragments from the ionization process: 5/23/2017 94 5/23/2017 95 Quantification of Amphetamines Due to the nature of the compounds being considered and the need for derivatization, GC–MS is not considered the best technique for sample quantification 5/23/2017 96 There are a number of difficulties encountered with quantification after employing derivatization. These include the fact because derivatization is another handling stage in the analytical process, there is always the risk of sample contamination. Furthermore, the assumption is made that the derivatization reactions are ‘complete’ and that the corresponding derivatives are stable for the period between derivative formation and analysis. Further factors are that dilutions need to be extremely accurate and precise to obtain reliable numerical data and that derivatization can potentially lead to increases in numerical errors for such data . 5/23/2017 97 The amphetamines (standards and samples) should be dissolved in methanolic HCl (100 ml of methanol to which 175 µl of concentrated HCl has been added). A range of standard solutions should be prepared in order to give a range of concentrations above and below that which the street sample is thought to contain, remembering that the latter may only contain between 0 and 5 wt% amphetamine. If necessary, the materials (particularly the case samples) should be sonicated and, following this, centrifuged to remove any solid materials. The supernatant is retained for subsequent analysis. 5/23/2017 98 Having collected the data, a calibration curve should be plotted. Since amphetamine is frequently synthesized in dirty apparatus in ‘clandestine’ laboratories, it may not be possible to determine which salt form of the drug is present. The standard is generally supplied as the sulfate form, of the general formula (amphetamine sulfate). This means that for every gram of amphetamine sulfate, 73% will be present as the amphetamine free base. The calibration curve should be plotted as (UV detector) response against concentration of amphetamine free base 5/23/2017 99 Mass Spectra 44 91 40 42 65 45 39 43 Amfetamine 44 122 78 121 65 107 91 134 Methoxyamfetamine 44 136 51 135 77 42 78 45 Methylenedioxyamfetamine 44 138 122 137 121 91 78 45 Methylthioamfetamine 5/23/2017 100