Amphetamine and Related Drugs

1
Amphetamine and Related Drugs
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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

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Stimulants

• Cocaine Amphetamines Methcathinone
  Methylphenidate Anorectic (appetite
  suppressant) Drugs Khat

4
Depressants

• BarbituratesBenzodiazepinesFlunitrazepam
  (hypnotic)Gamma Hydroxybutyric
  AcidParaldehydeChloral HydrateGlutethimide and
  MethaqualoneMeprobamate

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Cannabis

• MarijuanaHashishHashish Oil

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Hallucinogens

• LSDPsilocybin Psilocyn and other
  TryptaminesPeyote Mescaline
• New Hallucinogens    MDMA (Ecstasy) and other
  Phenethylamines   Phencyclidine and Related
  Drugs    Ketamine

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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.

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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

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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

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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.

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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.

12
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

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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), 3,4-methylenedioxyamphetamine (MDA) (3),
  3,4-methylenedioxymethylamphetamine (MDMA) (4)and
  3,4-methylenedioxyethylamphetamine (MDEA) (5). In
  addition, there are a wide variety of
  structurally related analogues which can be
  synthesized.

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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.

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• 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.

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• 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.

18

• 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.

19

• 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.

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AMPHETAMINE

• Amfetamine
• Central Stimulant
• Synonyms. Amphetamine Anfetamina Racemic
  Dexedrine.
• Proprietary names. It is an ingredient of
  Biphetamine and Durophet.

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• 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)
  redorange Marquis Testorange?brown
  Ninhydrinpinkorange

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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 4hydroxyamfetamine (an active metabolite),
  ß-hydroxylation to form norephedrine
  (phenylpropanolamine), and N-oxidation to form a
  hydroxylamine derivative.

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• 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 4hydroxyamfetamine
  , and about 2 as norephedrine in 24 h small
  amounts of conjugated 4hydroxynorephedrine and
  phenylacetone are also excreted. No elimination
  in the faeces has been detected.

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• 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

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• Steadystate 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.

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Toxicity

• The estimated minimum lethal dose in nonaddicted
  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.

28

• Halflife.
• Plasma halflife, 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.

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Methylenedioxymethamfetamine

• Stimulant, Hallucinogen
• N,a-Dimethyl1,3,benzodioxole5ethanamine
• FW 193.2
• A viscous, colourless oil. B.p. 100 to 110.

30

• Colour Test.
• Marquis Reagentblack with dark purple.
• Thinlayer Chromatography.
• System TARf 0.33 system TBRf 0.24 system
  TERf 0.39 system TFRf 0.20 system TAERf
  0.08 system TAJRf 0.03 system TAKRf 0.17
  system TALRf 0.57.

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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
  N-demethylation of the parent compound to
  3,4methylenedioxyamfetamine (MDA) (7) with
  further O-demethylation to 3,4dihydroxymethamfeta
  mine (HHMA) and 3,4dihydroxyamfetamine (HHA).
  Both HHMA and HHA are subsequently O-methylated
  mainly to 4hydroxy3methoxymetamfetamine (HMMA)
  and 4hydroxy3methoxyamfetamine (HMA). These
  four metabolites are excreted in the urine as the
  conjugated glucuronide or sulfate metabolites.

32

• 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,
  3,4methylenedioxyamfetamine (MDA), were 7.8 µg/L
  approximately 5 h after administration.

33

• 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.

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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.

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• Halflife.
• 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).

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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.

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• 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.

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• Colour Test.
• Marquis Testorange.
• Thinlayer Chromatography.
• System TARf 0.31 system TBRf 0.28 system
  TCRf 13 system TERf 0.42 system TLRf 0.05
  system TAERf 0.09 system TAFRf 0.63 system
  TAJRf 0.00 system TAKRf 0.03 system TALRf
  0.45. (Dragendorff spray, positive acidified
  iodoplatinate solution, positive Marquis
  reagent, brown ninhydrin spray, positive
  acidified potassium permanganate solution,
  positive.)

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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
  4hydroxymetamfetamine, 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

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• 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.

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• Halflife
• Plasma halflife, 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.

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Methylenedioxyethylamfetamine

• Stimulant, Hallucinogen
• Synonyms. N-Ethyl3,4methylenedioxyphenylisopropy
  lamine Eve MDE MDEA 3,4-Methylenedioxyethamphe
  tamine 3,4-Methylenedioxyethylamphetamine.
  Usually presented as Ecstasy.
• N-ethyl-a-methyl1,3benzodioxole5ethanamine
• FW 207.3

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A viscous, colourless oil. B.p. 0.2 is 85 to 95
44

• 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 4hydroxy3methoxyethylamfetamine
  (HMEA, 32).

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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

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Methylenedioxyamfetamine

• Hallucinogen
• Synonyms. MDA Methylenedioxyamphetamine
  Tenamfetamine SKF-5.
• a-Methyl1,3benzodioxole5ethanamine
• FW 179.2

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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 0.21.0 mgml-1, namely a
  concentration at which the standards can be
  prepared.

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• The sample should be dissolved as fully as
  possible and centrifuged or ?ltered 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

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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.

51

• 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.

52

• 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.

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• 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.

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Procedure for the Test

• Measure 20 ml of urine into a 50 ml
  glass-stoppered 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.

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• 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.

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• 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.

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• 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.

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• 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.

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• Transfer the residue to a 3 ml microcentrifuge
  tube using small portion (0.5 ml) of 11
  acetone-methanol. Again evaporate at a slow boil
  to near dryness in the vacuum oven maintained at
  10 p.s.i, and 60 oC.

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• 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.

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• Dissolve the residues in 20 µl of 11
  acetone-methanol 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.

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• 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.

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• 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.

64

• 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.

65

• 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.

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• 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

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• 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
  con?rmatory analytical technique. The foremost of
  these, for amphetamine identi?cation, is gas
  chromatographymass spectrometry (GCMS(

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Thinlayer Chromatographic Systems for Amphetamine

• System TARf 43 system TBRf 20 system TCRf
  09 system TERf 43 system TLRf 18 system
  TAERf 12 system TAFRf 75. (Dragendorff spray,
  positive FPN reagent, pink acidified
  iodoplatinate solution, positive Marquis
  reagent, brown ninhydrin spray, positive
  acidified potassium permanganate solution,
  positive.(

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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 Methanolstrong ammonia solution
  (1001.5).
• Reference compounds Atropine Rf 18, Codeine Rf
  33, Chlorprothixene Rf 56, Diazepam Rf 75.

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• 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.

71
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 Cyclohexanetoluenediethylamine
  (751510).
• Reference compounds Codeine Rf 06, Desipramine
  Rf 20, Prazepam Rf 36, Trimipramine Rf 62

72
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 Chloroformmethanol (9010).
• Reference compounds Desipramine Rf 11,
  Physostigmine Rf 36, Trimipramine Rf 54,
  Lidocaine Rf 71.

73
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.

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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.

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System TAF

• Plates Silica gel G, 250 µm thick.
• Mobile phase Methanoln-butanol (6040) and
  0.1 mol/L NaBr.
• Reference compounds Codeine Rf 22,
  Diphenhydramine Rf 48, Quinine Rf 65, Diazepam Rf
  85.

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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.

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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.

78
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.

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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.

80
Mandelins 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.

81
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.

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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.

83
Gas Chromatography.

• System GAamfetamine 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 (3,4di-OH-)-AC3 RI 2150,
  M (OH-methoxy-) RI 1465, M (OH-methoxy-)-AC2 RI
  2065, M (desaminooxo-OH-)-AC RI 1520, M
  (desaminooxo-OH-methoxy-) RI 1510, M
  (desaminooxo-OH-methoxy-)-AC RI 1600, M
  (desaminooxodi-OH-)-AC2 RI 1735 system
  GBamfetamine RI 1150 art (formyl) RI 1142
  system GCRI 1536 system GFRI 1315 system
  GAKretention time 4.9 min.

84

• 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.

85
High Performance Liquid Chromatography.

• System HAk 0.9 system HBk 8.48 system HCk
  0.98 system HXRI 244 system HAAretention
  time, 3.7 min system HBCretention time 2.1 min
  system HBDretention time 3.7 min.

86

• Column Chiralcel OD-RH (150  2 mm i.d., 5 µm)
  at 35. Mobile phase phosphatecitrate buffer
  (pH 4.0) with sodium hexafluorophosphate
  (0.3 M)acetonitrile (4357), flow rate
  0.1 mL/min. Fluorescence detection (?ex330 nm,
  ?em440 nm). Retention time 24.6 min.

87
Infrared Spectrum

• Principal peaks at wavenumbers 700, 740, 1495,
  1090, 1605, 825 cm-1

88
De?nitive Identi?cation of Amphetamines

• GCMS is the preferred method for the
  identi?cation 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

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91

• 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

92

• 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 disul?de and it is this method which ?nds
  wide application in the United Kingdom. For bulk
  and trace samples, this is achieved by dissolving
  the material

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94

• 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

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Quanti?cation of Amphetamines

• Due to the nature of the compounds being
  considered and the need for derivatization, GCMS
  is not considered the best technique for sample
  quanti?cation

97

• There are a number of dif?culties encountered
  with quanti?cation 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.

98

• 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.

99

• 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

100
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