TOXICOLOGY AND THERAPEUTIC

DRUG MONITORING
BASIC TECHNIQUES FOR DETECTING DRUGS IN
SERUM AND URINE
A. Immunochemical Methods
Two types:
1. Enzyme-mediated (or
multiplied) immunologic
technique (EMIT)
2. Fluorescence polarization
immunoassay (FPIA)
B. Chromatographic Techniques • applied mainly to the qualitative
• Homogeneous Immunoassay-
method most commonly
performed for drug testing.
• The term homogeneous refers to
the fact that these assays are all
performed in solution, without
any requirement for a phase
separation that is traditionally
used to separate bound from free
ligand
detection of drugs of abuse and
toxins, and less to the
determination of levels of
therapeutic drugs
The three major methods are:
1. thin-layer chromatography (TLC)
2. high-performance liquid chromatography (HPLC)
3. gas chromatography–mass spectroscopy (GC-
MS)

• GC-MS is considered the “gold standard” for

detection and quantitation of volatile drugs and
poisons
Newer analytic techniques:
1. capillary electrophoresis (CE)
2. Liquid chromatography–mass spectroscopy
(LC-MS)
 Chromatographic Techniques
Thin-Layer Chromatography  Allows direct qualitative detection
of drugs in a panoramic way.
High-Performance Liquid  allows quantitative detection of
Chromatography drugs and allows sharper separation
Gas Chromatography–Mass  Involves two techniques: gas–liquid
Spectroscopy chromatography and mass
spectroscopy
Liquid Chromatography–Mass  Nonvolatile compounds can be
Spectroscopy detected utilizing LC-MS
Gas Chromatography Coupled  Infrared (IR), or Fourier-transformed
With Infrared Spectroscopy infrared, spectroscopy (FTIR) utilizes
light of high wavelength (low
frequency), which excites vibronic
states of molecules involved in bond
stretching and bond angle bending
THE DRUGS OF ABUSE
 Cocaine
• Route: Nasal (i.e., inhalation, called snorting)

• Crack, is the free-base form that passes rapidly

across the nasal membranes such that, for a
given dose, most or all of it enters the
bloodstream rapidly.

• Half-life of cocaine is 1–2 hours, and the parent

compound and its metabolites are usually cleared
from the body within 2 days.
 Opiates: Heroin
Heroin
- Induces a pleasant, euphoric state and is highly
addictive both physically and psychologically.

- It is the diacetyl form of morphine.

- This characteristic facilitates heroin’s crossing the

blood-brain barrier, allowing it to reach higher
concentrations in the CNS
 Opiates: Morphine
Morphine
- Is a major metabolite

- The half-life of morphine is about 3 hours.

 Opiates: Codeine
Codeine
- Like morphine, has a strong analgesic effect, is
a potent antitussive agent, and acts in a
manner similar to that of morphine.

- Dextromethorphan (d-3-methoxy-N-
methylmorphine), an analog of codeine, is the
active component of cough syrups because of
its antitussive effects.
 Opiates: Fentanyl
Fentanyl
- This opiate analgesic is about 80 times more
potent than morphine in blocking pain.

- It can be taken orally as so-called fentanyl

lollipops, smoked, inhaled, or administered by
transdermal fentanyl patches
 Methadone
- A non-bicyclic drug that binds competitively
with morphine to μ-receptors in the brain.
 Amphetamines
- Cause euphoria and increased mental alertness
that may be attributed to their effects on these
pathways

- 3,4-methylenedioxymethamphetamine (MDMA
or ecstasy), a derivative of methamphetamine
has become popular as a recreational drug of
abuse because it has euphoric and psychedelic
effects but minimal hallucinogenic effects
 Tryptamines
- These drugs are derivatives of serotonin

- Relatively simple to obtain and were not

proscribed until relatively recently (from
plants).

- Contains N,N-dimethyltryptamine (DMT),

which has strong hallucinogenic properties
 Tryptamines (cont’d)
- Smoking DMT results in the rapid onset of
hallucinogenic effects that are short-lived,
giving rise to the term, businessman’s lunch.

- Psilocin is a component of the so-called

Psilocybe, called magic mushrooms because
of their hallucinogenic effects.
 Tryptamines (cont’d)
- The hallucinogenic effects of these drugs are
enhanced by the presence of
monoamineoxidase inhibitors such as β-
carbolines

- Ayahuasca- South American tea which

combines two plants—one containing DMT
and the other carbolines, which themselves
can induce nausea and vomiting.
 Piperazines
• Many of these piperazines were used as anti-
helminthics during the 1950s but were
subsequently discontinued. However, their
euphoria producing effects were discovered,
leading to a “legal” way of obtaining drugs of
abuse.
 Piperazines (cont’d)
- Two classes of piperazine derivatives:
- N-benzylpiperazines
- the parent compound of which is N-benzyl piperazine(BZP)
- The former group includes 1-(3,4-
methylenedioxybenzyl)piperazine

- Phenyl piperazines
- Includes 1-(3-chlorophenyl)piperazine, 1-(4-
methoxyphenyl)piperazine, and 1-(3-
trifluoromethylphenyl)piperazine (TFMPP).
 Piperazines (cont’d)
• BZP (known as A2) and TFMPP (known as
Molly) are among the most popular
piperazines.
 Benzodiazepines
- The most prominent is Valium

- Used therapeutically, as so-called minor

tranquilizers.
 Benzodiazepines (cont’d)
- Their mechanisms of action appear to be
potentiation of GABA, a neurotransmitter that
inhibits conduction in dopaminergic neurons,
and facilitation of its binding to GABA
receptors
 Benzodiazepines (cont’d)
- Bind to the α-subunit of the so-called GABAA
receptor at a site that is distinct from that for
GABA itself and cause an increase in the
frequency of chloride ion channel opening at
the GABAA receptor.
 Phencyclidine
- This drug is traded on the streets under the
name of angel dust or angel hair.

- Analgesic and anesthetic and, paradoxically,

stimulatory.
 Phencyclidine (cont’d)
- It has also been found to bind strongly to a class
of neural receptors called sigma-receptors.

- This receptor binds strongly to the neuroleptic,

antipsychotic drug haloperidol (Haldol)—a finding
that may implicate the sigma-receptor in some of
the clinical findings of severe psychosis in
patients suffering from overdose with PCP.
 Barbiturates: Sedative-Hypnotics
• Used as an anticonvulsant

• All of the barbiturates are fat soluble and

therefore pass easily across the blood-brain
barrier

• All of them seem to stabilize membranes such

that depolarization of the membranes
becomes more difficult.
 Barbiturates: Sedative-Hypnotics
(cont’d)
• the short-acting and ultra–short-acting
barbiturates seem to inhibit selectively the
reticular activating system, involved with
arousal—hence their sedative and hypnotic
effects.
 Barbiturates: Sedative-Hypnotics
(cont’d)
• The ultra–short acting barbiturates rapidly
diffuse out of the CNS, accounting for their
rapid action.

• Phenobarbital, however, selectively reduces

the excitability of rapidly firing neurons and is
therefore a highly effective anticonvulsant.
 Propoxyphene (Darvon)
• This analgesic drug has pharmacologic
properties very similar to those of the opiates,
like morphine.

• The structure of propoxyphene is quite similar

to that of methadone.
 Propoxyphene (Darvon)
• This drug can be taken orally, so that the sedated,
good feelings induced by opiates can be induced
without the need to have recourse to the
intravenous apparatus needed for infusion of
heroin.

• A major cause of drug-related death is

propoxyphene overdose alone or in combination
with CNS depressants like barbiturates and
alcohol.
 Methaqualone (Quaalude)
• Methaqualone is a 2,3-disubstituted
quinazoline.

• Although not structurally similar to the

barbiturates, it has many of the same
sedative-hypnotic properties as the
barbiturates.
 Methaqualone (Quaalude)
• This compound also possesses anticonvulsant,
antispasmodic, local anesthetic, antitussive,
and weak antihistamine actions.
 Marijuana (Cannabis)
• This is one of the oldest and most widely used
of the mind-altering drugs.

• Marijuana is a mixture of cut, dried, and

ground portions of the hemp plant Cannabis
sativa.
 Marijuana (Cannabis)
• Hashish refers to a more potent product
produced by extraction of the resin from the
plant.

• The principal psychoactive agent in marijuana

is considered to be δ-9-tetrahydrocannabinol
(δ-9-THC), a lipid-soluble compound that
readily enters the brain and may act by
producing cell membrane changes.
 Marijuana (Cannabis)
• δ-9-THC binds to the pre-synaptic neural
cannabinoid receptor CB1, which releases the
inhibitory neurotransmitter GABA in the
hippocampus, amygdale, and cerebral cortex.

• Different forms of THC have been found to

cause distinctly different physiologic effects.
 Marijuana (Cannabis)
• δ-9-THC induces an increase in anxiogenic
effects, and cannabidiol produces a
diminution in anxiety.

• The latter derivative was found to attenuate

blood oxygenation levels in the amygdala and
the anterior and posterior cingulate cortex; δ-
9-THC was found to modulate activation in
frontal and parietal regions of the brain.
Lysergic Acid Diethylamide (Lysergide)
• Lysergic acid diethylamide (LSD) is a semi-
synthetic indolalkylamine and a hallucinogen.

• It is one of the most potent pharmacologic

materials known, producing effects at doses as
low as20 mcg, and is equally effective by
injection or oral administration.
THERAPEUTIC DRUG MONITORING
THERAPEUTIC DRUG MONITORING
• Entails the analysis, interpretation, and
evaluation of drug concentration in serum,
plasma, or whole blood samples

• Employed to establish maximum benefits with

minimal toxic effects for drugs whose
correlation with dosage, effect or toxicity is
not clear
Routes of Drug administration:

1. Oral
2. Intravenous
3. Intramuscular
4. Subcutaneous
Therapeutic range

- Concentration of drug that gives benefits

 Pharmacokinetics
Two ways in which a therapeutic drug is
administered to a patient:

1. Discontinuously

2. Continuously
Loading dose
- Administered to achieve the right peak and
trough levels, the time required to achieve the
steady state is too long, especially where the
need for drug therapy is urgent

• Maintenance dose
- Administered after the usual time period
elapses
 Drug Absorption:
• Most drugs are absorbed from the GI tract in a
consistent manner in healthy individuals

• Liquids are absorbed more easily compared to

tablets and capsules

• All drugs must go through the liver before

entering the general circulation when absorbed in
the GI tract
• Most drugs in circulation are bounded to
plasma proteins

– Disorders that may affect protein-binding:

• Kidney disease
• Hepatic disease
• Malnutrition
• Inflammatory processes
– Drugs compete with other ingested drugs and
endogenous substances (steroid and bilirubin)

– Acid drugs bind to albumin

– Basic drugs bind to alpha-1 acid glycoprotein

(AAG)

– Some bind to both

• ONLY free drugs can interact with target sites and
produce response
– Quantity of free drugs correlates best with monitoring
therapeutic and toxic effects
– Most TDM assays quantify total drug concentration
rather than free drug

• Measurement of free drug level may be

warranted for highly protein bound drug or when
clinical response is not consistent with the total
drug level
• Elimination:

– Hepatic metabolic processes

– Renal filtration
• Conjugated drugs are eliminated through the urine or
bile
• Peak- high variations in drug concentrations

• Trough- low variations in drug concentrations

• 7- approximate number of doses to achieve a

steady state (peak and trough levels are
measured)
 Sample collection protocols:

• Timing (critical)
– Peak
• 1 hour after oral administration
• 30 minutes after IV administration is complete
• Collection time varies and is drug specific, variation occurs
due to:
– Different absorption
– Metabolic
– Excretion rates

– Trough
• Blood should be drawn before next dose
 Cardiotropics

These drugs are the ones most

commonly used to treat congestive
heart failure and cardiac arrhythmias
 Cardiac Glycosides (Digitalis and Its
Derivatives)
• Digoxin
- Treatment of congestive heart failure and atrial
fibrillation-flutter

• Digitoxin
- Has a longer half-life with a relatively slower
onset of action.

- The drug is extensively metabolized in the liver

(90%), with digoxin being the active metabolite
 Procainamide (Pronestyl)
- Is a class I antiarrhythmic drug that is useful in
treating supraventricular or ventricular
arrhythmias.
 Quinidine
- Also a class I antiarrhythmic, like procainamide,
is used to treatsupraventricular and ventricular
arrhythmias and tachyarrhythmias.
 Lidocaine
- (Xylocaine), another class I antiarrhythmic,
can also be used as a local anesthetic. Its
major use as an anti-arrhythmic is in the acute
control and prevention of ventricular
arrhythmias after acute myocardial infarction.

- Its effect is to block sodium channels mainly in

ventricular but notatrial tissue.
 Propranolol
- a class II antiarrhythmic β receptor–blocking
drug that antagonizes the effects of
epinephrine on the heart, on the arteries and
arterioles of skeletal muscles, and on the
bronchus, exerts its effects largely on the AV
node and is used to treat sinus tachycardia,
atrial tachycardia, and ventricular arrhythmias
 Amiodarone
- A structural analog of thyroid hormone, is
chiefly a class III anti-arrhythmic drug, which
markedly prolongs the action potential mainly
by blocking potassium channels in cardiac
muscle
 Verapamil
- is a class IV antiarrhythmic drug that blocks
activated and inactivated calcium channels
that are especially prominent in nodal
tissue(particularly the AV node).
 Anticonvulsants

are used in the treatment of seizure disorders, in

particular grandmal, petit mal, and psychomotor seizures
and other specialized seizure disorders such as tic
douloureux (trigeminal neuralgia).
 Phenobarbital
- Treatment of generalized tonic-clonic seizures,
simple partial seizures, anxiety, insomnia
 Phenytoin (Dilantin)
• Treatment of generalized tonic-clonic seizures,
simple partial seizures, complex partial
seizures
 Primidone (Mysoline)
• Primidone is used to treat generalized tonic-
clonic, simple partial, and complex partial
seizures
 Ethosuximide (Zarontin)
• Ethosuximide is the drug of choice for absence
(petit mal) seizures unaccompanied by other
types of seizures
 Carbamazepine (Tegretol)
• Carbamazepine is a primary antiepileptic drug
that is used in the treatment of generalized
tonic-clonic seizures and simple partial and
complex partial seizures, as well as in
combinations of these seizure types.

• Absence (petit mal), myoclonic, and atonic

seizures may be exacerbated by this drug.
 Valproic Acid (Depakene)
• Valproic acid is commonly used in the
treatment of generalized tonic clonic seizures,
absence seizures, myoclonic seizures, and
atonic seizures.

• It is not effective for the treatment of infantile

spasms.
 Newer Anticonvulsants
• Includes:
1. Topiramate
2. Lamotrigine (Lamictal)
3. Gabapentin (Neurontin)
4. Felbamate

• are four anticonvulsant agents that have been

approved recently for use in this country for
patients whose response to the more established
anticonvulsants is less than optimal
 Antiasthmatics

Asthma is a form of chronic obstructive

pulmonary disease that has a variety of
causes, some of them allergenic in
nature
 Theophylline
• Theophylline is used as a bronchodilator for
the treatment of moderate or severe asthma,
both for the prevention of attacks and for the
treatment of symptomatic exacerbations
Anti-inflammatory and
Analgesic Drugs
 Aspirin
• Acetylsalicylic acid (aspirin) is a non-steroidal
anti-inflammatory compound that is used as
an analgesic, an antipyretic, and, in larger
doses, an anti-inflammatory agent.
 Acetaminophen

• Acetaminophen (Tylenol), or N-acetyl-p-

aminophenol, is used as an analgesic and
antipyretic to treat fever, headache, and mild
to moderate myalgia and arthralgia
Immunosuppressives
 Cyclosporine
• CsA is a cyclic polypeptide containing 11
amino acids, five of which are methylated
 Tacrolimus (FK-506)
• Tacrolimus is a macrolide lactone antibiotic
with a mechanism of action similar to that of
cyclosporine; it is more potent than CsA in its
inhibitory effect
 Rapamycin (Sirolimus)
• Rapamycin is an antibiotic similar to
tacrolimus.
 Mycophenolate Mofetil
• is a derivative of mycophenolate acid, a fungal
antibiotic.
 Leflunomide
• Leflunomide (LFM), which inhibits lymphocyte
proliferation by inhibiting dihydroorotic acid
synthetase
Drugs Used in the Treatment of
Mania and Depression
 Lithium
• Lithium is a monovalent cation, a member of
the group of alkali metals, and is available
commercially as citrate and carbonate salts.
 Antidepressants
• Three classes of drugs are currently used in the
treatment of clinical depression: classical tricyclic
antidepressants (TCAs), SSRIs, and monoamine
oxidase inhibitors (MAOIs).

• The rationale for use of the first class of drugs is

their ability to block the uptake of
norepinephrine at the axonal side of synapses in
neural tracts from the brainstem to the forebrain
that utilize this neurotransmitter
• Both lithium and the antidepressants are used
in the treatment of psychiatric affective
disorders.
 The Neuroleptics, Antipsychotic
Major Tranquilizers
These drugs are used mainly in the treatment of acute
schizophrenia and result in suppression of the agitated state. All
neuroleptics appear to block the actions of dopamine and
serotonin post synaptically in the limbic system and motor
cortex
Chemotherapeutic Agents:

Methotrexate and Busulfan

 Methotrexate

• an anti-metabolite consisting of a mixture containing

no less than 85% 4-amino-10-methylfolic acid and
related compounds, is a folic acid antagonist.

• It inhibits the enzyme dihydrofolatereductase. This

results in blockade of the synthesis of
tetrahydrofolicacid, which is needed for the formation
of N-5,10-methylene-tetrahydrofolate,an intermediate
in the transfer of a methyl group todeoxyuridylate to
form thymidylate, needed in DNA synthesis
 Busulfan
• is an alkylating agent used to treat a variety of
leukemias and lymphomas before bone
marrow transplantation. It is cytotoxic to
marrow cells and is used in combination with
cyclophosphamide, which is cytotoxic to
mature lymphocytes that may be involved in a
graft-versus-host reaction
Antibiotic Drugs
 Aminoglycosides
• Treat infections caused by gram negative bacteria

• Gentamicin, tobramycin, kanamycin, amikacin

• Eliminated via kidney

• Associated with nephrotoxicity and ototoxicity

• IV or IM administration
 Vancomycin
• Treat infection caused by gram positive
bacteria

• IV administration

• Associated with nephrotoxicity, ototxicity, and

Red Man Syndrome (erythmic flushing of
extremities)
TOXICOLOGY

Study of poison
Routes of exposure:

1. Ingestion
2. Inhalation
3. Transdermal absorption
• Toxic response- amount of damage when poison
is administered at less than the lethal dose

• Acute toxicity- one-time exposure of short

duration to an agent that immediately causes a
toxic response

• Chronic toxicity- multiple exposure over a long

period of time to an agent that will not cause an
acute response
TOXINS AND ACUTE POISONING
Environmental Carcinogens:
 Cyanide

• The cyanide anion binds avidly to iron in the

ferric or trivalent state.

• Because cyanide forms a relatively stable

cyanoferric complex, it is able to inactivate
iron-containing enzymes that cycle between
the ferrous and ferric states in oxidation–
reduction reactions.
 Carbon Monoxide

• Carbon monoxide (CO) intoxication produces

tissue hypoxia as a result of decreased oxygen
transport. CO disrupts oxygen transport by
binding to hemoglobin to form a reversible
complex, carboxyhemoglobin (CO-Hb).
Alcohols and Glycols:
 Ethanol
• Ethanol is probably the most common drug of
abuse and is frequently responsible for the
presentation of patients with altered mental
status to hospitals and emergency rooms.

• Ethanol is rapidly absorbed from the

gastrointestinal tract, has a volume of distribution
approximately equal to that of total body water,
and diffuses freely in body tissues.
 Methanol
• Methanol (wood alcohol) poisoning occurs in
patients who ingest methylated spirits or
methanol-containing antifreeze.
 Osmolal Gap
• For methanol, ethylene glycol, and isopropyl
alcohol poisoning, because the molecular
masses of these simple compounds are low,
relatively small quantities in serum give rise to
high serum osmolalities.
 Osmolal Gap (cont’d)
• This value should be close to the serum
osmolality measured by freezing-point
depression. If, however, any of these three
compounds (or ethanol) is present in serum, the
computed osmolality will be significantly less
than the measured value (i.e., a so-called osmolal
gap will exist), suggesting that a toxin is present.

• This is an effective screening method as a first

step in detecting the presence of toxic
compounds.
 Ethylene Glycol
• It is the major contributor to the high anion
gap seen in metabolic acidosis.
Arsenic
 Arsenic
• is used in ant poisons, rodenticides, herbicides
and weed killers, insecticides, paints, wood
preservatives, and ceramics, in the production
of various metal alloys and livestock feed, as a
tanning agent, and in medicines.
 Arsenic
• Inorganic arsenicals, including sodium
arsenate and lead or copper arsenite; organic
arsenicals, such as carbarsone and
tryparsamide; andarsine gas are the major
toxicologic forms of arsenic.
 Arsenic
• Arsine gas poisoning generally occurs in the
industrial setting, where its production arises
from the action of acid or water on arsenic-
bearing metals.

• Arsenic compounds occur in three oxidation

states: elemental, trivalent arsenite, and
pentavalentarsenate.
Mercury
 Mercury
• Mercury compounds exist in four different
forms with different toxicologic potential:
elemental or metallic (Hgo); mercurous (Hg+);
mercuric (Hg2+); and alkyl mercury (i.e.,
organomercurials).
 Mercury
• Elemental mercury is poorly absorbed from
the GI tract if mucosal integrity is preserved
and shows no toxic effect unless it is
converted to the divalent form.

• This may occur slowly by oxidation–reduction

with water and chloride ion if a GI site for
mercury stasis exists, but this is uncommon.
 Mercury
• Significant poisoning occurs with elemental
mercury when it is inhaled or absorbed
through the skin.

• It can pass through the blood-brain barrier

and can accumulate in the CNS, where
oxidation produces mercuric ion; thus,
primarily pulmonary and CNS toxicities are
produced.
 Mercury
• Of the two inorganic salts of mercury,
mercurous (Hg+) salts are poorly soluble and
thus poorly absorbed. However, the mercuric
(Hg2+) salt is readily soluble and is readily
absorbed after oral ingestion or inhalation.

• Severe inflammation of the mouth and other

GI symptoms can result.
Iron
 Iron
• Acute iron poisoning is common in young
children and is usually the result of ingestion
of iron-containing products. Generally, iron
overload that causes increased deposition of
iron in tissues is termed hemosiderosis; if the
excess iron induces tissue damage, the
condition is termed hemochromatosis.
 Iron
• Hemochromatosis can be inherited or acquired.

• Acquired hemochromatosis is caused by over

ingestion of iron or by leakage of iron from red
blood cells, as may occur in a hemolytic crisis.
Although ferric ions from food are usually
reduced to ferrous ions and absorbed in the
stomach, the large and small bowel can rapidly
absorb toxic amounts (>30 mg/kg) of elemental
iron. Once absorbed into the body, iron removal
is difficult.
 Iron
• Large doses of iron are thought to cause acute
mucosal cell damage, and significant
absorption of iron occurs once the binding
capacity of transferrin is exceeded.

• Unbound iron in serum causes toxicity by

hepatic cell damage, shock, and production of
lactic acidosis.
 Iron
• The hepatotoxicity seems to be dose related,
occurs within 1–2 days of ingestion, and has
been associated with levels equal to or greater
than 1700 μg/dL
Lead
 Lead
• Both organic and inorganic compounds of lead
may be highly toxic, with their most serious
effects occurring in the central and peripheral
nervous systems.

• Absorption may occur by inhalation or ingestion.

If more than 0.5 mg of lead is absorbed per day,
lead accumulation and toxicity are believed to
occur, whereas 0.5 g of absorbed lead is
considered a fatal dose.
Organophosphates and
Carbamates
 Organophosphates and Carbamates
• Pesticides generally contain
organophosphates, which are esters of
phosphoric acid or thiophosphoric acid, or
carbamates, which are synthetic derivatives of
carbamic acid.

• Although these are two distinctly different

types of compounds, they both unfortunately
interfere with neurotransmission.
 Organophosphates and Carbamates
• Both compounds inhibit the enzyme
acetylcholinesterase (AChE), which normally
hydrolyzes the neurotransmitter acetylcholine
(ACh) after ACh has effected an action
potential and has been released from its
receptor site
• References:
• Henry’s Clinical Diagnostic and Management
by Laboratory Method 22nd Edition
• Clinical Chemistry Review Outline by Rodolfo
R. Rabor, RMT, MSMT
• Clinical Chemistry principles, procedures,
correlation 5th Edition by Michael L. Bishop,
MS, CLS, MT(ASCP); Edward P. Fody, MD; Larry
E. Scheff, MS, MT(ASCP)