Amphetamine &

Methamphetamine

Objectives: - What is amphetamine? - How does amphetamine produce its effect? - Illicit amphetamine and methamphetamine products - Analysis of amphetamine and methamphetamine - Metabolim and excretion - Analysis of amphetamine and methamphetamine in the urine

NH2

CH3

mthyl-phenyl) amphetamine ethyl amine

CH3

NH2

CH3

NHCH3

N-methyl- -methyl phenyl ethylamine(methamphetamine)

Many of us wish to - run faster, - think quicker, and - work longer. Others would like to - prolong the ecstasy of sexual orgasm or - the feeling of euphoria. The search for a substance that would provides. Such elevated physiological and emotional states are as old as the human race.

Substances that produce these kinds of physiological as well as emotional states as amphetamine was discovered but has side effects. These side effects are caused by the drugs interference with the delicate balance in the normal physiology of the nervous system.

Amphetamines, related in structure to the neurotransmitters norepinephrine and dopamine, are synthetic phenyl propyl amines. They include several closely related compounds as amphetamines (dl, ±, racemic mixture), - dexamphetamine {d, (+)}, - levamphetamine {l,(-)}, -Methamphetamine {d,(+)} and -levomethamphetamine {l,(-)}. Amphetamine and methamphetamine free bases are liquids and of low stability. Therefore they are found in the form of amphetamine SO4 or PO4 and methamphetamine HCl salt.

Norepinephrine Dopamine

In addition, there are several chemical derivatives of amphetamines with restricted use as medications such as phemmetrazine, methylphenidate (Ritalin®). Amphetamines have been used for nonmedical purposes (abused) since the earliest day of their marketing. Amphetamine abuse comes from the feeling of Euphoria due to its effect on central and peripheral nervous system. Indeed, there is evidence that amphetamines can slightly enhance the performance of physical tasks such as running, jumping, or swimming. (performance enhancing drugs).

Production of illicit amphetamines:

The majority of illicit market production of amphetamines and methamphetamines are produced by clandestine laboratories, - lack of quality control and - variability in potency are characters of illicit amphetamine and methamphetamine samples.

They often contain by-products and intermediate results from: •Impure starting material •Incomplete reactions •Inadequate purification of intermediate and the final products These by-products and intermediate can provide valuable information concerning the illicit method of manufacturing.

- Knowledge of impurities is important to know the harmful effects of impurities can be evaluated as well as potential danger publicized and how to treatment. - Awareness of impurities is important to forensic analyst. - Most impurities are weakly basic or neutral in nature and are normally present in the finished product at level of 2-3’.

Method abused for synthesis of amphetamine and methamphetamines •Leuckart reaction: has been the most popular, since the synthesis is simple, rapid, gives a good yield with hazardous procedures. It may be considered as three step reaction involving: •Formulation stage

•Hydrolysis •Purification

•For amphetamine: phenyl-2-propanone (P-2-P)

CH2 C

O

CH3

Condansed HCOOHor ammonium formate

(Benzyl methyl ketone)

N-Formyl amphetamin(as intermediater)

HydrolysisH2SO4

Purification bysteam distillation

Extraction with ether and precipitation as SO4 recrystalization or wash with organic solvent.

B) Methamphetamine can be prepared in a similar way employing methylamine CH3NH2 or HCOOH or N-methylformamide in the condensation step. N-formyl amphetamine or N-formylmethamphetamine as well as 4-methyl-5-phenyl-pyrimidine have been identifies as a major impurities. P-2-P is commercially available, its supply controlled in some countries. 2) Reductive amination method:

P-2-PReduction with

suspension of rarely

nickel or by Pt, Al3/HgCl2

Amphetamine

Methamphetamine may be also prepared by this procedure using CH3NH2. The major impurities are Shiff’s base. In addition, inorganic impurities from use of particular catalysts may serve as markers.

•Oxime route: in which hydroxylamine is reacted with P-2-P giving the oxime which is subsequently hydrogenated. The major impurities are benzal ethyl ketoxim. The clandestine methods mentioned above produce a racemic mixture of dl-amphetamine or dl-methamphetamine. •Reduction of ephedrine and pseudoephedrine: Because of the controls placed of the movement of P-2-P (Phenyl-2-Propanone), ephedrine and pseudoephedrine have become popular starting material for illicit methamphetamine

Ephedrine

+

Pseudoephedrine

HI/red phosphorous

H2+Pd/BaSO4

Thiyonyl Chloride

Chloroephedrine

+

Chloropseudoephedrine

methamphetamine

in agood yield

CH3

NH2HO

Nor-ephedrine

Impurities detected in the reaction carried out by these procedures includes P-2-P, Iodine, chloro ephedrine, ephedrine and inorganic such as Pd and Ba

l or (-)-ephedrine d-amphetamine

•Amphetamine can be formed from phenylpropanol amine in a similar way.

Illicit products of amphetamine: •Licit amphetamine products contain the drug in the form of SO4

- or PO4- SALTS AS TABLETS, CAPSULES,

SYRUPS AND ELIXIRS. •Licit methamphetamine HCl is available as a tablet and sterile solution for injection. •Illicit amphetamine SO4 Varies in color from white to pink, yellow, orange, brown depending on the type and amount of impurities and adulterants its odor is characteristic and unpleasant owing to the presence of solvent residue.

•Illicit methamphetamine HCl is usually in the form of a caked or gummy powder. It may be white, brown or violet depending on the presence of certain impurities. •Aqueous solution of methamphetamine HCl commonly called gold fish. in Egypt is commonly called Maxton ice, also known crank, meth, crystal meth, speed, go, go-fast and cristy, is a very pure crystalline form of (+)-methamphetamine HCl (98-100%) that because of its transparent sheet like crystals, it is called ice.

Administration: -Amphetamine SO4 or PO4 salts are most frequently taken orally (tablets or capsules) or intranasal through snorting. -Methamphetamine HCl is most frequently prepared for injection or for smoking (ice) but also is available as tablet. •Ice is commonly abused by inhalation through smoking and special glass pipe is used. One or two crystals of ice are placed inside the glass bowl of pipe and the pipe is heated from the bottom. The smoker then removes his finger from the hole of the roof of the pipe and inhales the vapors. Smoke able ice (crank) is characterized by a rapid onset and long duration of action, 7 seconds for 4-14 hours. •Ice is also abused by IV injection either alone or with heroin in speed ball. Chronic uses exhibit weight loss, reduced resistance to diseases and damage of the lungs, liver and kidney.

Pharmacology and Mode of Action: •At low to moderate doses, amphetamine

elevate mood and increase mental alertness and feeling of energy and well being by interfering with the normal activities of neurotransmitters. •They also reduce activities of stomach and intestine and reduce feel of hunger. •At high doses amphetamines can increase heart rate and BP to dangerous levels.

The mode of action: The mode of action of amphetamines is directly related to their structures which are very similar to neurotransmitters ephedrine and pseudoephedrine. •It is though that amphetamine produces their stimulatory effect by stimulating the receptors sites for neurotransmitters. •Also they cause the release of nor-epinepherine from pre-synaptic nerve terminals. •It blocks the active uptake of nor-epinepherine and dopamine back into their pre-synaptic nerve terminal. •All these leads to the stimulation of norepinepherine receptors which produce alerting and awakening of effects in the person taken amphetamine.

•They also stimulate the dopamine receptors and thereby produce increased motor activity and feeling of euphoria

•The chronic physiological and psychological effects: Of amphetamine include rapid tolerance and strong psychological dependence, impotence and episodes of paranoia and psychosis. Tolerance to amphetamines develops rapidly in regular users, but it develops more rapidly to some of amphetamines effect than to others. E.g. it develops quickly to its appetite-suppressing effects, but more slowly to its mood elevating effects. The route of administration of amphetamines is an

important factor in the development of tolerance, oral

and snorting produce slower tolerance while inhalation

or IV injection produces rapid

Dependence:

as tolerance develops it is often accompanied by a strong psychological dependence. Amphetamine abusers feel they cannot face the day without the drugs. Classic withdrawal symptoms occur during alcohol or barbiturate withdrawal like coma, shaking and convulsions not occur with amphetamine withdrawal.

Medical Uses: •Obesity caused by overeating. Not endocrine imbalance (short-term weight loss program ) •Narcolepsy, sleep disorders characterized by rapid and uncontrolled onset of sleep. Hyperactivity (ADHD) •Juvenile Attention deficit disorders (ADD) with hyperactivity in children. Attention Deficit Hyperactivity Disorders in adults and children (ADHD)

Color Tests: •Marquis Test: (HCHO/H2SO4) Small amount of the sample+ 1-2 drops of Marquis Reagent

Orange Brown Color (for amphetamines And methamphetamines)

•Simon’s Test: Small amount of the sample+ one drop of sodium nitroprusside/ acetalanhydryde+ one drop Na2CO3

Methamphetamine Blue

Amphetamine pink to cherry red color

Chromatographic Analysis: •TLC: TLC F254 with MeOH:NH4OH (100:1.5) detection UV at 254 nm or spray with ninhydrin reagent

violet or pink spot

•GLC: •Accurate weight of the sample salt is dissolved in H2O

•Add internal standard

•Liberate free base which is volatile base liberated from salt by 0.1 N NaOH

•Extract free base by EtOAc

•No derivatization, carrier gases He with FID detector. Quantitation by peak is at φ internal standard.

•HPLC: Amphetamine and methamphetamine can be directly analyzed by isocratic technique, on both normal and reversed phase column.

•Spectroscopic Analysis: By using IR spectroscopic techniques

Detection and Assay of Amphetamine in Biological Samples: Inhalation and IV injection produce CNS effect immediately snorting and ingestion produce much slower onset of action.

Metabolic pathways of Amphetamines: Elimination of amphetamines involve

•Biotransformation by liver enzyme to more polar compounds and excretion in the urine. •In activation by conjunction to form the glucouronides and sulphate conjugates. •Amphetamines begin to cupper in the urine within 20 minutes of administration. •Blood concentration in fatalities is normally above 500 mg/l •Amphetamine is excreted the unchanged drug, typically 20-30% of the dose and as deaminated (hippuric acid and benzoic acids) and hydroxylated metabolites partly as conjugates up to 25% of the dose.

•The rate of excretion and the fraction of the dose excreted as uncharged drug. Vary according to PH of the urine. In alkaline urine about 45% of the dose is excreted in 24 hours,2% as unchanged drugs. In acid urine, up to 78% of the dose may be excreted in 24 hours, 68% as the unchanged drugs.

The recommended target analyses are therefore, the unchanged drug. Methamphetamine is excreted as the unchanged drug 44% and its major metabolites are amphetamine (6-20%) and 4-hydroxy methamphetamine (10%). As with amphetamine the rate of excretion increase by rendering the urine acidic also the percentage of unchanged drug excreted (increase). After chronic administration, abusers have shown amphetamine concentration in urine of 1-90 mg/ml and methamphetamine concentration of 25-300 mg/ml

Metabolic Pathway of Amphetamine

NH2

OH

NH2

NH

OH

Glucouronides and

Sulphate

Conjugates

N-Oxidation

-hydroxylation

Norehedrine

Amphetamine

NH2

HO

4-Hydroxy amphetamine

Glucouronides and

Sulphate

Conjugates

OCH3

O

Major

Metabolic route

(Deamination)

Phenyl acetone

COOH

Benzoic Acid

NHCH2COOH

O

Hippuric Acid

Metabolic Pathway of Methamphetamine

NHCH3

NH2

As Amphetamine

N-demethylation

Methamphetamine

NHCH3

HO

4-Hydroxy Mthamphetamine

Glucouronides and

Sulphate

Conjugates

Amphetamine

Aromatic

Hydroxylation

Initial Screening Methods: •Immuno-assay technique may be used for screening purposes and positive finding must be confirmed by a different, more specific method. Immune assay kits available from commercial sources for amphetamine and methamphetamine involve Enzyme Immuno assay (ESA), Fluorescence polarization immuno assay (FPIA), latex agglutination inhibition (LAI) and radio immune assay (RIA).

•TLC: Amphetamine and methamphetamines

are first extracted from urine at alkaline pH, when the amino group is in the unchanged state. Extraction is done by

•Liquid-liquid extraction

•Solid phase extraction using cartilage of normal silica, Rp-18 cation-exchanger.

NB concentration of the urine extract should be done under careful conditions to avoid the evaporation of the free base by using mixture of MeOH: HCl to the extract to form corresponding salt. The solvent systems are •MeOH:NH4OH (100:1.5) •EtOAC:MeOH:NH4OH (85:10:5)

Development Dry to Remove NH4OH

Spray

Fast black K salt •Violet with amphetamine

•Orange with methamphetamine The limits of detection for amphetamines and methamphetamines are 0.1 µg, respectively.

Ninhydrine R 120°

15 minviolet or pink spot

•Fluorescamine Reaction (Fluorescamine in acetone)

Dry TLC by hot dryer observe

under 366 nm

bright yellow fluorescent spot with amphetamine methamphetamine is not detected.

The detection limit for amphetamines and other primary amines is about 10 ng.

Color Tests: •it is specific and sensitive test for methamphetamines in urine. Modifies Simon’s test

(acetaldehyde+HoAC, MeOH) + Sodium nitroprusside Blue Color

Confirmatory Chromatographic Methods:

•GLC: the urine sample extract can be analyzed for amphetamine content as underivatized or derivatized samples qualitative by Rt and quantitative by peak area and internal standard methods. •GC-MS: Characteristic fragments appear for amphetamine and methamphetamine as well as their derivatives. •HPLC: using ODS column, qualitative by Rt and quantitation by peak area and internal standard method.

Conclusion: unchanged amphetamine has been detected into the urine up to 29 hours after single oral dose of 5 mg amphetamine and after 23 hours following single dose of methamphetamine. Appositive amphetamine analysis indicates the use of amphetamines within the previous 24 hours. However, three additional points should be considered: •Some drugs like ephedrine and phenyl propanol amine present in some pharmaceutical preparations which used as decongestant and anorexia, can be detected by EMIT and RIA tests. •Several prescription drugs such as fenfluramine, phentermine, mephentramine,……. Etc can also produce positive Immuno assay results. Some drugs give amphetamine and methamphetamine in the urine

as their metabolite. The urine should be re-examined for the presence of the parent drug.