Transcript Amphetamine and Related Drugs

Amphetamine and
Related Drugs
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Narcotics
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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
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Cocaine
Amphetamines
Methcathinone
Methylphenidate
Anorectic (appetite suppressant) Drugs
Khat
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Depressants
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Barbiturates
Benzodiazepines
Flunitrazepam (hypnotic)
Gamma Hydroxybutyric Acid
Paraldehyde
Chloral Hydrate
Glutethimide and Methaqualone
Meprobamate
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Cannabis
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Marijuana
Hashish
Hashish Oil
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Hallucinogens
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LSD
Psilocybin & Psilocyn and other Tryptamines
Peyote & Mescaline
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New Hallucinogens
MDMA (Ecstasy) and other
Phenethylamines
Phencyclidine and Related Drugs
Ketamine
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Drug Schedules: Schedule I
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• 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
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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
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• 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
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• 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
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• 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®.
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Schedule V
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• 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
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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.
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Some history
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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.
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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.
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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)
—red–orange; Marquis Test—orange→brown;
Ninhydrin—pink–orange
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Disposition in the Body.
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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.
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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 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.
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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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Steady–state blood concentrations of 2 to
3 mg/L were observed in a regular user who
ingested about 1 g a day.
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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
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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.
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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.
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Methylenedioxymethamfetamine
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Stimulant, Hallucinogen
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N,α-Dimethyl–1,3,benzodioxole–5–ethanamine
FW =193.2
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A viscous, colourless oil. B.p. 100° to 110°.
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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.
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Disposition in the Body.
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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.
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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.
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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
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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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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).
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Metamfetamine
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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 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.)
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Disposition in the Body
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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
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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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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.
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Methylenedioxyethylamfetamine
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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
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A viscous, colourless oil. B.p. 0.2 is
85° to 95°
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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%).
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Toxicity
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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
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Hallucinogen
Synonyms. MDA;
Methylenedioxyamphetamine; Tenamfetamine;
SKF-5.
α-Methyl–1,3–benzodioxole–5–ethanamine
FW =179.2
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Thin Layer Chromatography of
Amphetamines
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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.
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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
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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.
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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.
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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.
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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
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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.
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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 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.
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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 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 .
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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 .
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
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.
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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 confirmatory
analytical technique. The foremost of these, for
amphetamine identification, is gas chromatography–
mass spectrometry (GC–MS(
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Thin–layer Chromatographic Systems
for Amphetamine
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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(.
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System TA
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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.
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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.
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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
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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.
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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: 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 .
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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.
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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.
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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.
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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.
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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.
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
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.
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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.
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
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.
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Infra–red Spectrum

Principal peaks at wavenumbers 700, 740, 1495, 1090,
1605, 825 cm−1
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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
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
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
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
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
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
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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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
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
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 .
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
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.
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
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
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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
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