Forensic Toxicology

Part 1. Introduction
 What is Toxicology ?
The science of toxicology relates to the
investigation of poisons and derives,
its name from the Greek words

toxikon (poison)
and
logia (a discurs or study)
What is toxicology ?
Definitions
 Sub-disciplines of Toxicology
• Environmental Toxicology is concerned with
the study of chemicals that contaminate food,
water, soil, or the atmosphere. It also deals with
toxic substances that enter bodies of waters
such as lakes, streams, rivers, and oceans.
• This sub-discipline addresses the question of
how various plants, animals, and humans are
affected by exposure to toxic substances
 Sub-disciplines of Toxicology
• Occupational (Industrial) Toxicology is
concerned with health effects from exposure
to chemicals in the workplace.
• This field grew out of a need to protect
workers from toxic substances and to make
their work environment safe.
 Sub-disciplines of Toxicology
• Regulatory Toxicology gathers and evaluates
existing toxicological information to establish
concentration-based standards of “safe”
exposure.
• The standard is the level of a chemical that a
person can be exposed to without any harmful
health effects.
 Sub-disciplines of Toxicology
• Food Toxicology is involved in delivering a
safe and edible supply of food to the
consumer. During processing, a number of
substances may be added to food to make it
look, taste, or smell better.
• Fats, oils, sugars, starches and other
substances may be added to change the
texture and taste of food.
 Sub-disciplines of Toxicology
• Clinical Toxicology is concerned with
diseases and illnesses associated with short
term or long term exposure to toxic chemicals.
• Clinical toxicologists include emergency room
physicians who must be familiar with the
symptoms associated with exposure to a wide
variety of toxic substances in order to
administer the appropriate treatment
 Sub-disciplines of Toxicology
• Analytical toxicology identifies the toxicant
through analysis of body fluids, stomach
content, excrement, or skin.
What is Forensic Toxicology ?
 History
• Ancient Egyptians and
Greeks reported
poisonings due to herbs,
plants and food.

• Opium, arsenic and

hydrocyanic acid were
used throughout Europe
Poison hemlock is a plant
during the middle ages. related to the carrot.
It contains alkaloids that
negatively affect the
nervous system
 History
The father of modern toxicology
Philippus Theophrastus Aureolus Bombastus
von Hohenheim (known as Paracelsus)
observed that any substance could be a poison,
depending on its dose
The Dose Makes the Poison
 History
In 1814, Mathiew Orfila,
the chairman of the legal medicine
department at the Sorbonne in France,
known as the father of forensic
toxicology, published a book entitled
Traite des poisons ou Toxicologie Generale
which described the first systematic
approach to the study of the chemistry and
physiological nature of poisons.
 History

Orfila refined the definition of a poison

as
„any substances which, when taken
inwardly in a very small dose, or applied
in any kind of manner to a living body,
depraves the health or entirely destroys
life”
 What is a dose?
• The dose is the actual amount of a chemical that
enters the body.
• The dose received may be due to either acute
(short) or chronic (long-term) exposure. An acute
exposure occurs over a very short period of time,
usually 24 hours. Chronic exposures occur over
long periods of time such as weeks, months, or
years. The amount of exposure and the type of
toxin will determine the toxic effect.
 What is Toxicity?
• The word “toxicity” describes the degree to
which a substance is poisonous or can cause
injury.
• The toxicity depends on a variety of factors:
dose, duration and route of exposure, shape
and structure of the chemical itself, and
individual human factors
 LD-50 lethality indicator
• LD50 —refers to the dose of a substance that kills half the
test population, usually within four hours
– (Note: test population is usually mice or rats)
• Expressed in milligrams of substance per kilogram of body
weight. A correlation is then made to humans based on
the body weight data.
• However, estimating lethal doses for humans is often
complicated by the fact that resistance to certain
chemicals can differ greatly between species
• LD figures depend not only on the species of animal, but
also on the mode of administration. For instance, a toxic
substance inhaled or injected into the bloodstream may
require a much smaller dosage than if the same
substance is swallowed.
Toxicity Classes
 Factors influencing toxicity

Toxicity of a substance can be affected by many

different factors:
 the pathway of administration (whether the toxin
is applied to the skin, ingested, inhaled, injected
(IM)),
inhalation > injected > ingestion > skin contact

 the time of exposure (a brief encounter or long

term),
 Factors influencing toxicity
 the number of exposures (a single dose or multiple doses
over time)
– Sometimes small amounts ingested over time create a
tolerance for the toxin
– Chronic exposure (larger amounts over time) can create serious
medical problems
– Acute toxicity—very large dose causing immediate problems,
including death
 the physical form of the toxin
gas > liquid > solid
 the genetic makeup of an individual, an individual's
overall health, and many others (e.g. gender, age).
 Chemical Interactions
Humans are normally exposed to several
chemicals at one time rather than to an
individual chemical.
• There are four basic types of interactions .
Postmortem Forensic Toxicology
Postmortem Forensic Toxicology
• Qualitative and quantitative analysis of
drugs or poisons in biological specimens
collected at autopsy

• Interpretation of findings in terms of:

– Physiological effect at time of death
– Behavioural effect at time of death
 Quantitative vs. Qualitative
• Qualitative analysis – determines the
presence or absence of a drug or poison in a
submitted sample

• Quantitative analysis – determines the

amount of drug or poison that is present in the
submitted sample
Postmortem Forensic Toxicology
Types of cases:
• Suspected drug intoxication cases
• Fire deaths (CO, cyanide)
• Homicides
• Driver and pilot fatalities
• Therapeutic drug monitoring
• Sudden infant death (SIDS)
Samples of Forensic Interest
 Issues in Specimen Collection
• Selection
– Multiple, varied sites of collection
• Collection
– Appropriate method of collection
– Adequate volumes for analysis
• Storage and handling
Important to ensure analytical results are
accurate and interpretations are sound
 Typical autopsy specimens
• Blood (10-20 ml)
• Urine (10-20 ml)
• Stomach contents
• Bile (all available)
• Liver (50 g)
• Hair (100 mg)
• Vitreous humour (all available)
 Recommended specimens
collected in post-mortem cases.
 Blood
• Antemortem  ideal blood sample

• Postmortem blood is not truly “blood”

• Anatomical site of collection at autopsy

should be noted
 Central sites
Subclavian Heart

Heart Peripheral sites

Femoral
Iliac
Iliac Subclavian

Other sites
Femoral
Head blood
Hematoma blood
 Urine
• Produced by the kidneys as a result of
blood filtration
• Stored in the bladder until voided

• Qualitative - the presence of a drug in the

urine of an individual indicates that some
time prior to death the drug or poison was
present in the blood of the individual
 Stomach contents
• Visual examination may reveal tablets
• Drugs that have been orally ingested may be detected
in stomach contents
• Caution: drugs administered by other routes may also
diffuse into stomach contents from the blood
• Generally qualitative:
– Stomach contents are not homogeneous
– Only a portion of stomach contents collected (unmixed?)
• Useful for directing further analysis
 Liver
• Drug metabolism occurs in the liver
• Both parent compounds and metabolites
may be present in higher concentrations in
the liver than in the blood  ease of
detection
• Limitation is that drugs are not uniformly
distributed throughout the liver 
confounds interpretation
 Bile
• Digestive secretion
• Continuously produced by the liver
• Stored in the gallbladder

• Qualitative - the presence of a drug in the bile

of an individual indicates that sometime prior
to death, the individual was exposed to the
drug
 Vitreous humour
• Fluid that occupies the space between the lens and the retina
of the eye.
• Sequestered from putrefaction, charring and trauma,
microorganisms.
• Useful in cases where decomposition is advanced, body is
exhumed or in fire deaths
• Limitation is blood:vitreous ratio may not be known
• The vitreous potassium concentration rises so predictably
within the hours, days and weeks after death, that vitreous
potassium levels are frequently used to estimate the time
since death (post-mortem interval) of a corpse
 Hair and nails

100 mg of hair
 Non-biological submissions
• Used to direct analysis of biologicals
• May indicate the nature of substances that
may have been ingested, inhaled or injected
• Examples:

– Containers found at the scene

– Syringes
– Unidentified tablets or liquids
Storage and Handling
 Proper specimen handling
• Identification of samples
– Continuity
– Contents
• Specimens delivered to lab without delay
• Specimens should be analyzed as soon as
possible
• Storage areas should be secure
 Storage and Handling
• Not feasible to analyze specimens
immediately
• Sample should be in well-sealed container
• Sample containers must be sterile
• Use of preservatives and anti-coagulants
• Refrigeration vs. Freezing
– Both inhibit bacterial action; esp. freezing
– Freezing results in  prep time
– Freeze-thaw cycle may promote breakdown
 Storage of Samples
• Preservative
– Sodium fluoride
• Anti-coagulants
– Sodium citrate
– Potassium oxalate
– EDTA
– Heparin
– Not imperative for postmortem blood samples
Preservative –sodium fluoride
.
Toxicological approaches
 Toxicological principles
Orfila established toxicological principles 200 years ago:
• All chemists who undertake this work must have toxicological
experience.
• The analyst must be given a complete case history that
contains all the information available.
• All the evidential material, suitably labelled and sealed in
clean containers, must be submitted and examined.
• All the known identification tests should be applied and
adequate notes made at the time.
• All the necessary reagents used for these tests should be
pure, and blank tests should be performed to establish this fact.
• All tests should be repeated, and compared with control
samples to which the indicated poison has been added.
Main Questions Addressed by Forensic Toxicologist

• Is a drug or poison present?

If so, what is the substance?

• How much of the substance is

present? Is the concentration
great enough to cause or
contribute to death?

• How was the drug/poison

administered?
 Case investigation
 Information to be submitted with the exhibits in all
cases in which there is suspicion of poisoning :
 Victim’s information (name, gender, age, weight, height,
nationality, recent foreign travel?, occupation)
 Medical history (disease, alcoholic, drug addicted, etc.)
 Timings (date and time in normal health/ilness)
 Treatment (any medical attention, symptoms, hospital
analysis)
 Samples required (if victim alive: blood, urine, vomit;
if victim dead: stomach content, blood, urine, liver)
 A few minutes talking with the investigating officer can
save many hours, or even days, of analysis time!!!
 Major groups of poisons
Miscellaneous substances
Gases
e.g. dioxins (GC, LC-MS)
CO, CN, 30 (GC)

Naturals toxins Volatile

e.g. amanitine substances
(mushrooms), 30 ethanol, toluen,
(ELISA, GC, LC-MS) Poisons 120 (GC, GC-MS)
Anions
nitrates, phosphides, Drugs
30 (UV-VIS, GC, LC-MS) >2000 (IA, GC, LC, MSn)

Pesticides, >1000, Metals

(GC, LC-MS) As, Pb, Tl, Al, 20
Over 13 mln of known compounds,
(AAS, AES, ICP-MS)
around 100 thousand has got toxicological significance
 General guidance
 Most forensic toxicology cases involve
substances that act on the central nervous
system (e.g. alcohol, hypnotics, narcotics,
sedatives, or tranquilizers).
 Completely systematic and comprehensive
analytical scheme that covers all the poison
groups is extremely costly.
 In most cases there are some facts available
that provide clues to the most likely group
of poisons to be excluded as a first action.
 General guidance (2)
 If the time of onset of the symptoms was less
than 1 h, and there is no evidence of injected
material, the sample should be examined for
gases and volatile poisons before other groups.
 Severe vomiting and diarrhoea indicate possible
metal poisoning
 The age and occupation of the victim:
Teenagers indulge in solvent abuse, farm
workers have easier access to the more toxic
pesticides and a death in an electroplating
factory certainly requires cyanide to be
eliminated.
Symptoms of Poisoning
 Toxicological examination
 1. Detection – to detect any drugs or poisons
in the samples submitted by means of screening
procedures.
 2. Identification – to identify conclusively any
drugs, metabolites or poisons present by means of
specific relevant physicochemical tests.
 3. Quantification – to quantify accurately
those drugs, metabolites or poisons present.
 4. Interpretation – to interpret the analytical
findings in (2) and (3) in the context of the case,
the information given and the questions asked by
the investigating officer.
 Methods of analysis
Qualitative methods
1. Color Tests – a reagent is added to the
substance to be tested and a color change will
represent the presence of the suspected
chemical.
2. This is rapid, easily performed, qualitative
screening test, but not specific method
3. Can be used as bed side rapid test

CHEAP, EASY, and QUICK

Color test
Qualitative methods
Reinsch test
Reinsch test
Reinsch test
 Immunoassays
(screening test)

Antigen-antibody reaction…
substance being sough is
the antigen-testing reagent is
the antibody.

An antibody will react only

with antigens and ignores all
other chemicals
Immunoassay methods
Quantitative methods
chromatography
Summary
 ‘test for poisons’

The request to ‘test for poisons’ is a

major problem for the chemist, because
no single chemical method of analysis
is able to detect all the various
poisons. At least six different analytical
schemes are required to exclude even the
most commonly encountered poisons.
 Group tests

 Group 1 poisons: gases

 Group 2 poisons: volatile compounds
 Group 3 poisons: drugs (solvent soluble)
 Group 4 poisons: metals
 Group 5 poisons: pesticides (solvent soluble)
 Group 6 poisons: anions
 Toxicology Screens
• First is the screen for lower alcohols (acetone,
isopropyl alcohol, n-propyl alcohol, ethyl alcohol,
methyl alcohol, ethylene glycol).
• The second screen is the acidic, neutral and basic
screen (barbiturates, salicylates, tranquilizers,
narcotics, local anesthetics, antihistamines,
antidepressants, alkaloids, and other agents.
• Screen for carboxyhaemoglobin and cyanide.
• Screen for higher volatiles (toluene, benzene,
trichlorethane, and trichlorethylene.)
 Group 1 poisons: gases
 The presence of this group of poisons is usually indicated
by the circumstances of the incident
 Group 1 poisons: gases (2)

 The analyst should enquire whether oxygen was

administered or artificial respiration was attempted before
death, since these measures can bring about a significant
reduction in carboxyhaemoglobin concentrations.
Group 1 poisons: carbon monoxide
• Odorless, colorless, tasteless, non-irritating gas whose relative
density is a little less than that of air
• Virtually undetectable without specialized equipment
• The most common sources of carbon monoxide in deaths are
fires, automobile exhaust, defective heaters, and a natural by-
product of incomplete combustion from equipment burning
carbon based fossil fuels such as: Gasoline, Wood, Coal,
Propane, Oil, Methane.
• Carbon monoxide is produced whenever organic materials are
burned with an inadequate supply
of oxygen necessary to produce complete
combustion.
Group 1 poisons: carbon monoxide
The detection and measurement of carbon monoxide
(CO) in blood samples is required frequently in forensic
toxicology to investigate deaths in fires, cars, caravans.
CO reacts with haemoglobin:

HbO2 + CO = COHb + O2

Carboxyhaemoglobin (COHb): haemoglobin in which the

oxygen binding sites are occupied by carbon monoxide.
CO has a greater affinity for hemoglobin than O2 at 210 times to 1.
Physiological concentration of COHb in blood is about
Nonsmokers 1-3%
Smokers 10-15%
Endogenous < 1 %
Lethal concentration of COHb is about 50%.
 Autopsy findings in CO deaths .

• In Caucasians - a characteristic cherry-red or bright-pink

appearance that can be seen in the tissue. Cherry-red livor
mortis suggests the diagnosis even before autopsying the
individual. It must be realized, however, that this color can
be simulated by prolonged exposure of the body to a cold
environment (either at the scene of death or in a morgue
“cooler”) or cyanide poisoning.

• With blacks, the discoloration is prominent in the

conjunctivae, nailbeds, and mucosa of the lips.
 Analysis of HbCO

Chemical test:
1.Sodium hydroxide solution , 25% (w/v)
2. Tannic acid solution , 10% (w/v)
Potassium ferricyanide solution , 10% (w/v)
Ammonium sulfide solution (yellow)
3. Formalin, 36%
Spectrophotometric method (e.g. Wolff’s
method, Fretwurst-Meineck’s method)
Gas chromatography method (CO is converted
and detected as methane)
 TASK
Check the test with reagents:
• 1. Alkali (NaOH) test:
Mix 1 drop of the suspected blood with 5 drops of 25% NaOH. The
reddish color of the sediment remains in the presence of
carboxyhemoglobine. A brownish color of the sediment is obtained
when normal blood is treated in the same manner.
• 2. Formalin test:
Mix 1 drop of the suspected blood with 5 drops of Mix 1 drop of the
suspected blood with 5 drops of 36% formalin The reddish color of
the sediment remains in the presence of carboxyhemoglobine. A
brownish color of the sediment is obtained when normal blood is
treated in the same manner.
 Group 1 poisons: cyanide
Cyanide (HCN, NaCN, KCN) toxicity can occur
following:
1. ingestion of amygdalin (found in almonds and
apricot kernels),
2. prolonged administration of nitroprusside,
3. after exposure to gases (HCN) produced by the
combustion of synthetic materials.
4. Industrial processes such as electroplating,
jewelry cleaning, metal extraction, laboratory
assays, and some photographic processes.
(NaCN, KCN)
 Group 1 poisons: cyanide
Routes of Absorption

• Primarily by breathing vapor or oral

administration

• Dermal and Occular

 Group 1 poisons: cyanide
Pharmacokinetics – dynamics
• Bioavailability: up to 77% by inhalation/
50% orally/ also dermal (slow) and
occular absorption

• Vd 0.4 L/kg

• t ½ - approximately 1- 2 hr / converted to
thiocyanate by rhodanase enzyme
 Group 1 poisons: cyanide

Mechanism of poison’s action

• Reversibly binds to a critical component of
the respiratory chain (cytochrome c
oxidase) causing a reversible stop in
oxidative energy production

• Other enzyme systems are affected;

oxidate energy production is the most
important
 Group 1 poisons: cyanide

Mechanism of poison’s action

• Without oxidative energy production,
glucose and other sugar energy
pathways are the only source of energy

• Two tissues in particular are obligate

oxygen users: Brain and Heart
 Group 1 poisons: cyanide

Course of toxic effect

• CNS stimulation (counter effect) excitation

• headache and stiff neck
• giddiness
• change in breathing pattern (dyspnea);
can be confused for hyperventilation
• nausea and vomiting
 Group 1 poisons: cyanide
Course of toxic effect
asphyxia without cyanosis
• the oxygen remains in the blood and individuals
color looks good
• convulsions and muscular contortion
• breathing: short inhalation-long exhalation
• heart races (tachycardia) then slows
(bradycardia)
• death due to asphyxia, cardiac and respiratory
arrest, with severe brain damage (depending on
time to death)
 Group 1 poisons: cyanide
The lethal dose:
• potassium or sodium cyanide is 200 to 300 mg
• hydrocyanic acid is 50 mg.
Effects begin within seconds of inhalation and within 30 min of ingestion.
Blood concentrations mg/100 mL
• No symptoms 0.01-0.05
• Flushing/tachycardia 0.05-0.1
• Toxicity-death 0.1-0.25
• Coma-Death > 0.25
 Group 1 poisons: cyanide

Autopsy findings
• The odor is quite characteristic for cyanide
poisoning but most persons are unable to
smell this odor (bitter almonds)
• If potassium or sodium cyanide was
ingested, brown-red mucosal corosion may
be present in stomach or in upper digestive
tract.
NOTE:
• Bright red skin colour is not always present
 Group 2 poisons: volatile compounds
 The rapid onset of symptoms followed by serious illness or
death is the most valuable clue to the presence of this
group.
 Group 2 poisons: volatile compounds (2)
 If several days have elapsed before the body is discovered,
there is a tendency to think that analysis for volatile poisons
would be futile, but in such cases it should be one of the
first groups to be checked.
 Group 2 poisons: volatile compounds (3)
 Special care is needed if the samples have been frozen.
Volatile poisons may be missed if the analytical material is
not allowed to thaw, preferably at room temperature, before
examination
Group 2 poisons: volatile compounds (4)
 Group 2 poisons: METHANOL

• Colourless liquid
• Used as a solvent, antifreeze, fuel, extraction
agent, solvent
• Absorbed after ingestion, inhalation, via skin
• Ingestion of 70–100 mL of methyl alcohol is
usually fatal, though death may occur with
ingestion of as little as 30–60 mL.
• As little as 10 mL of methanol can cause
permanent blindness (optic nerve atrophy)
• The minimum lethal blood level in methyl
alcohol poisoning is approximately 80 mg%.
 Group 2 poisons: METHANOL

- In an organism converted to formaldehyde and formic

acid – by alcohol dehydrogenase, much faster then
ethanol. These products are toxic (bind to Fe3+ in
breathing enzymes), not methanol!
- Formic acid degraded to CO2 and H2O, but folic acid
is necessary for this process – after a short time –
depletion of folic acid
 Group 2 poisons: METHANOL

• Symptoms of acute methanol poisoning: weakness,

nausea, vomiting, headache, epigastric pain,
dyspnea, and cyanosis.
• If a fatal amount of methyl alcohol has been ingested,
the aforementioned symptoms will be followed by
stupor, coma, convulsions, hypothermia, and death.
• Death is nearly always preceded by blindness.
• Death in methyl alcohol poisoning is caused by the
acidosis from production of organic acids and the
CNS depressant action of the alcohol.
• Acidosis is the primary toxic factor in methyl alcohol
poisoning, with the central nervous system
depression a relatively minor factor.
 Group 2 poisons: ISOPROPANOL

• Source: Synthesis of acetone, glycerin,

Solvent for oils, gums and resins,
Deicing agent , Rubbing alcohol, Hair
care products, skin lotion and aerosols
• It has twice the CNS depressant
potential of ethanol.
• Unlike methanol, it is not in itself toxic.
• It is metabolized in the liver to acetone.
• A lethal dose of isopropanol is
estimated at 250 mL for an adult.
 Group 2 poisons: ISOPROPANOL

• It should be noted that the appearance of

small amounts of isopropanol in the blood is
not necessarily indicative of ingestion of this
alcohol.
• In diabetics with ketoacidosis, and in cases
of starvation with high levels of acetone,
acetone may be converted to isopropyl
alcohol.
• In such cases, there will be a high level of
acetone and a low level of isopropyl alcohol.
 Group 2 poisons: ACETONE

• Acetone is the active ingredient in nail polish

remover and paint thinner and sanitary
cleaner. It is a common building block in
organic chemistry.
• Small amounts of acetone are produced in
the body by the decarboxylation of ketone
bodies.
 Group 2 poisons: ACETONE

• Acetone is believed to exhibit only slight

toxicity in normal use, and there is no strong
evidence of chronic health effects if basic
precautions are followed.
• At very high vapor concentrations, acetone is
irritating and, like many other solvents, may
depress the central nervous system. It is also
a severe irritant on contact with eyes, and a
potential pulmonary aspiration risk.
• LD50 for human ingestion - 1.159 g/kg;
 Group 2 poisons: ETHYLENE GLYCOL
- Colourless, odourless, syrupy liquid with a
sweet taste
- Coolant or antifreeze in automobiles and
personal computers, important in the plastics
industry for the manufacture of polyester fibers
and resins, in cosmetics and cleaning products
etc.
- Rapid absorption (even after inhalation, quick
metabolism, acute poisonings.
• Ethylene glycol itself is not toxic, it is the
metabolites (principally oxalic acid) that are.
• Minimum lethal dose is estimated at 100 mL for
an adult.
• It may be ingested as an alcohol substitute by
alcoholics, in suicide attempts, and accidentally
by children.
Group 2 poisons: ETHYLENE GLYCOL

Metabolism
Group 2 poisons: ETHYLENE GLYCOL
Symptoms of ethylene glycol poisoning
Symptoms of ethylene glycol poisoning usually follow a
three-step progression
Stage 1: neurological symptoms, dizziness,
headaches, confusion. Over time, the body
metabolizes ethylene glycol into other toxins - first it
is metabolized to glycolaldehyde, which is then
oxidized to glycolic acid, glyoxylic acid, and finally
oxalic acid
Stage 2: is a result of accumulation of these
metabolites - tachycardia, hypertension,
hyperventilation and metabolic acidosis
Stage 3: kidney injury leading to acute kidney failure -
oxalic acid reacts with calcium and forms calcium
oxalate crystals in the kidney – usually death,
oxalates in kidney is typical pathological sign.
Uremia, vomiting, oral ulceration, seizures, death.
Group 2 poisons: ETHYLENE GLYCOL
Calcium oxalate Crystals

Microscopic sections of the kidneys viewed under

polarized light show deposition of oxalate crystals in the
renal tubules and brain.
Group 2 poisons: ETHYLENE GLYCOL

The antidotes for ethylene glycol poisoning

are ethanol or fomepizole:
ethanol - competition with ethylene
glycol for the enzyme alcohol dehydrogenase
- decreases the formation of toxic metabolites
fomepizole (4-methylpyrazole) –
inhibition of alcohol dehydrogenase –
blockage of the formation of the toxic
metabolites

Haemodialysis can also be used to enhance

the removal of unmetabolized ethylene
glycol, as well as its metabolites from the
body
 Odor test:

On the basis of the smell substances try to

identify, where are the following solvents
(give the number of sample):
Check the results:

1. Acetone
2. Ethanol
3. Xylene
4. Chloroform
5. Methanol
6. Ethylene glycol
7. Ethyl acetate
8. Isopropyl alcohol
 Group 3 poisons: drugs (1)
• All drugs are poisons.
• Most drugs and other chemicals undergo metabolism in the
human body which may lead to:
- Formation of reactive intermediates which may be responsible for many
adverse toxic reactions
- Formation of intermediates which are proximate carcinogens initiating a
process of carcinogenesis.
• Toxic injury is likely to result only from major overdose, or
prolonged exposure at lower dosage, or from nutritional
deficiency.
• Toxicity may be manifest in any different ways including:
- Acute effects that may lead to necrosis of the liver and kidney.
- Subacute effects leading to gastrointestinal ulceration.
- Chronic effects such as malignancy, effects on reproduction and on the
unborn child and even on a mature adult through medication to the parent.
 Group 3 poisons: drugs (3)
 In general, postmortem stomach contents, blood and
urine samples may be analysed by immunoassay.
However, in some suspicious deaths or criminal cases,
stomach contents, blood and urine samples cannot be
obtained and tissue samples have to be examined.
 Drug screening in non–fatal criminal cases and traffic
offences. Many forensic enquiries entail analytical
investigations that require detection of therapeutic drug
concentrations rather than toxic concentrations. For
example, in driving offences a screen is required for
drugs that can impair judgement and psychomotor
activities (e.g. sedatives, tranquillisers, stimulants and
narcotics).
 Group 3 poisons: drugs (4)
Analysis
 A multipurpose drug screen may be applied to
samples of body fluids, especially when there is a limited
amount of sample. For forensic purposes, at least two
uncorrelated methods of identification are required (e.g.
HPLC and GC). Immunoassay methods provide good
exclusion evidence, but poor confirmation of identity.
 Blood and urine samples should be obtained
whenever possible in this type of investigation: when the
blood sample is small, the chance of detecting and
confirming residual traces of an antidepressant or
stimulant drug is almost zero. If there is no urine sample,
pre–screening by immunoassay methods may be
essential to provide an analytical guide to the nature of
any drug present in the blood sample
Group 3 poisons: drugs (5)
Group 3 poisons: drugs (6)
Group 3 poisons: drugs (7)
 Group 3 poisons: SALICYLATES

• Acute intoxications with salicylates are common.

• In a dosage of 150-300 mg/kg they are severe, and
above 500 mg/kg potentially fatal.
• The presenting signs of salicylate poisoning,
especially chronic (repeated or excessive doses for
longer than 12 hours), can include metabolic
acidosis, hypoglycaemia, lethargy, and coma and fits.
Group 3 poisons: SALICYLATES
• Aspirin is a common drug and yet
there are only a few cases
reported where it was used for
suicidal reason.
• Symptoms commonly seen with
toxic exposure are dizziness,
faintness, pallor, sweating, air
hunger, irregular pulse, and
vomiting . There may be disturbed
acid-base balance,
hyperventilation, cyanosis,
delirium, coma, gastric erosion,
and bleeding in severe poisoning .
 Group 3 poisons: SALICYLATES
COLOR test
• The Trinder spot test is a diagnostic test used to
determine exposure to salicylates
• The test employs the Trinder reagent which is mixed
with a patient's urine.
• The test for the Trinder reaction is to mix 1 ml of urine
with 1 ml of the Trinder reagent in a test tube. The test
is positive if a colour change results.
• The specific colour changes are:
blue or purple - positive test
no change - negative test
brown - false-positive test caused by the presence
of phenothiazines.
LIMIT detection 75 mg/L.
 Group 3 poisons: SEDATIVES AND
HYPNOTIC
Benzodiazepines
1,4-benzodiazepines
carboxamide group in the 7-membered heterocyclic ring
structure
A substituent in the 7 position, such as a halogen or a nitro
group
Barbiturates and other older drugs
Several drugs with novel chemical structures
Group 3 poisons: ANTIDEPRESSANTS
The most common family of drugs used in suicides now
are the antidepressants, specifically, the tricyclics.

There are now three generations of tricyclic

antidepressants. The first included amitriptyline,
nortriptyline, imipramine, desipramine, and doxepin;
the second, amoxapine, trazodone, bupropion and
maprotiline and the third venlafaxine, nefazodone and
mirtazapine.

The mechanism of death from

an overdose of a tricyclic
antidepressant is cardiac.
Group 3 poisons: ANTIDEPRESSANTS

The newest group of antidepressants are the selective

serotonin reuptake inhibitors (SSRI).

These include fluoxetine,

paroxetine, fluvoxamine
and sertraline.

The SSRI are significantly less toxic than the tricyclics

in that they do not have the cardiotoxic component.
They still can cause death, however.

Most deaths encountered seem to involve an SSRI and

one or more other drugs. Deaths from these drugs
alone are uncommon
 Group 3 poisons: STIMULANTS
I. Cocaine (free base or hydrochloride).
II. Amphetamines:
D-Amphetamine, Methamphetamine,
methylphenidate (use to treat attention
deficit disorders in children),
phenmetrazine (Preludin) - used to treat obesity,
(hallucinogens = MDA, MDMA, DOM;
methylenedioxymethamphetamine, "ecstasy,"
dimethoxyamphetamine).
III. Khat: Cathinone, methcathinone.
IV. Methylxanthines: caffeine (coffee), theophyline (tea),
theobromide (chocolate).
Amphetamine and methamphetamine

 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;
 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.
 Cocaine

 Cocaine is normally only used as a surface anaesthetic

in the eye, ear, nose, and throat because of the
possibility of systemic toxic effects when given by
other routes. Addicts may inject it or use it as a snuff .
 The main metabolites are benzoylecgonine, ecgonine,
and ecgonine methyl ester, all of which are inactive;
some norcocaine, an active metabolite, may also be
produced; other metabolites that have been reported
are ethylecgonine, hydroxycocaine, and
methylecgonidine.
EFFECTS OF STIMULANTS
 Group 3 poisons:
OPIATES AND SYNTHETIC NARCOTICS
• Opiates are extracted from the poppy
plant Papaver somniferum.
• Opiates belong to a larger class of
drugs, the opioids, which include
synthetic and semi-synthetic drugs
• Opioid pharmaceuticals are
analagous to the three families of
endogenous opioid peptides:
enkephalins, endorphins, and
dynorphin
• Opioids - natural and semi-synthetic
compounds derived from opium as
well as entirely synthetic compounds
which mimic the actions of morphine
 Group 3 poisons:
OPIATES AND SYNTHETIC NARCOTICS
• Natural OPIATES
– Morphine (1.9h), codeine (2.9h)
• Metabolized to active drug morphine in liver
• Semi-synthetic
– Hydromorphone (2.4h), oxycodone (2.6h), hydrocodone (4.24h),
diacetylmorphine (heroin) metabolized to 6-monoacetylmorphine
• Synthetic
– Meperidine (3.2h)
• Excitatory neurotoxicity may occur when the renally excreted
metabolite, normeperidine, accumulates. Seizures and serotonin
syndrome.
– Methadone (27h)
• Very long acting; may cause QT prolongation, torsades de pointes
– Propoxyphene
• Seizures, IA antidysrhythmic properties (leads to widened QRS and
negative inotropy)
– Tramadol (5.5h)
• Effects not completely revered by naloxone, seizures
– Fentanyl (3.7h)
• Ultrashort acting
 MORPHINE

• Primary constituent of crude opium

• Strong agonist and prototypical narcotic

analgesic
• Used in the treatment of moderate-severe
pain
• t½ range: 1-6 hours
 HEROINE
• Synthetic
• Morphine derivative (diacetylmorphine)
• penetration of the blood-brain barrier more rapidly and
effectively than morphine
• t½ approximately 5 minutes
• The major metabolites of diacetylmorphine, 6-MAM,
morphine, morphine-3-glucuronide and morphine-6-
glucuronide, may be quantitated in blood, plasma or urine
to monitor for abuse, confirm a diagnosis of poisoning or
assist in a medicolegal death investigation.
 CODEINE

• Codeine or 3-methylmorphine (a naturally

occurring methylated morphine) is an opiate
used for its stimulant, analgesic, antitussive
antidiarrheal, anxiolytic, antidepressant,
sedative and hypnotic properties
• t½ range: 2 – 4 hours
• Used in combination with other analgesics
Morphine, Heroin or Codeine?

Morphine can arise in the blood and urine

through the administration of morphine itself
or through the metabolism of heroin or
codeine
Heroin Morphine Codeine

6-MAM

Morphine
Morphine, Heroin or Codeine?
The presence of 6-MAM is definitive evidence
that heroin was administered

In vivo: 6-MAM quickly disappears from blood

Ex vivo: 6-MAM hydrolyzes spontaneously

The absence of 6-MAM does not exclude the

possibility that heroin was administered
 Morphine, Heroin or Codeine?
Case Example
• 35 year old male,
• Found dead in room at home
• Recent injection site at antecubital fossae
• Syringe found under the body of the male
• Blood morphine concentration – 175 ng/mL
• 6-MAM detected in the blood
• 6-MAM not detected in the urine
• Suggests a rapid fatal intoxication with
heroin
 Group 3 poisons:
COLOR test
Marquis reagent is used as a
simple spot-test to
presumptively identify
alkaloids as well as other
compounds (amphetamines).
It is composed of a mixture
of formaldehyde and
concentrated sulfuric acid,
which is dripped onto the
substance being tested.
Group 3 poisons: HALUCINOGENS

Hallucinogenic compounds found in some

plants and mushrooms (or their extracts)
have been used—mostly during religious
rituals—for centuries.
Almost all hallucinogens contain nitrogen
and are classified as alkaloids.
 Group 3 poisons: HALUCINOGENS
• Lysergic acid diethylamide (LSD) Ergot fungus
• Psilocybin (mushrooms) mushrooms
• Dimethyltryptamine (DMT) Virola trees
• Mescaline Peyote cactus
• Harmaline, Harmine Ayahuasca vine
• Ergine, Isoergine morning glory
• Lysergic Acid Amide morning glory
• THC marijuana, cannabis
• Atropine belladonna plant
• Scopolamine roots of mandrake, herbane
• Hyoscyamine roots of mandrake, herbane
• Ibogaine Iboga plant
 Cannabis

Marijuana is derived from the plant Cannabis sativa.

The active chemical substance in marijuana is known as
tetrahydrocannabinol, or THC.
The THC content of Cannabis generally declines with the highest
level in plant resin, flowers, leaves and the smallest level in the
stem, roots or seeds.
Marijuana does not cause physical dependency, but health risks are
not well known and heavy, long-term use is believed to cause the
most harm.
 Effects of Marijuana

• The immediate effects of marijuana use

are: dilated pupils, bloodshot eyes,
increased heart rate, dry mouth and
throat, increased appetite, short term
memory loss, altered sense of space and
time, loss of concentration, impaired
coordination, impaired vision, paranoia,
loss of knowledge retention, coughing,
fatigue, dizziness.
 LSD
 LSD is hallucinogenic chemical that is derived
from a parasitic fungus that grows on rye.
 LSD is produced as a crystal, then dissolved in
alcohol prior to sale and consumption.
 LSD users describe their experience with the drug
as a “trip”.
 LSD has become known as:

Acid Trips Hits Blotter

 HOW IS LSD CONSUMED?

LSD is generally taken orally,

either as a pill, a piece of blotter
paper, or via liquid dripped onto a
sugar cube. LSD can also be
absorbed through the users skin.
LSD A piece of blotter paper
LSD A piece of blotter paper
 MUSHROOMS
MUSHROOMS
 Mushrooms that contain PSILOCYBIN are known as
‘magic mushrooms’ as they contain hallucinogenic
properties.
 Magic mushrooms have been used by several cultures
to induce altered states of consciousness during their
religious rituals.
 Mushrooms can be consumed in a variety of manners:
Eaten – Either raw or cooked, much like non-
hallucinogenic mushrooms.
Drank – Heated with water to make a tea or soup.
Smoked – The mushroom is ground into a fine powder and
often smoked on top of marijuana.
 Group 3 poisons: drugs
Behaviours that might indicate potential drug use by drivers:
 Smell - principally the smell of burning marijuana
(cannabis)
 Unusual behaviour or responses by the driver to an
officer’s questions (including slow or slurred speech
and physical responses), inappropriate answers to
simple questions
 Flat, dull, or excited behaviour
 Unusually large or small pupils, unusual eye
movements
 Impairment of fine motor skills.
 Symptomatology chart for drug effects
Narcotic
Categories Depressants Stimulants Hallucinogens PCP Inhalants Cannabis
analgesic

Pupil sizea Normal Dilated Dilated Normal Constricted Normal Dilated

Reaction Slow Slow Normal Normal Little or Slow Normal

to light non–visible
Pulse rateb Down Up Up Up Down Up Up

Blood Down Up Up Up Down Up/down Up

pressurec
Body Normal Up Up Up Down Up/down or Normal
temperature Normal,
DOS
General Uncoordinate Restlessne Dazed Perspiri On the nod Odour of Odour of
Indicators d ss appearance ng substance cannabis

a Pupil size normal range, 3.0 to 6.5 mm.

b Pulse normal range, 60/90 beats/s.

c Blood pressure normal range, 120 to 140 mmHg systolic, 70 to 90 mmHg diastolic.
 Group 4 poisons: metals

 Historically, poisons in this group have been

used frequently, probably because in general
they are potent, tasteless, readily available and
produce symptoms similar to many common
diseases
 For example, the substitution of arsenious oxide
for the contents of one or two capsules of a
prescribed medicine can have fatal results with
all the appearance of a straightforward drug
overdose suicide.
Group 4 poisons: metals (2)
Group 4 poisons: metals (3)
 Group 4 poisons: ARSENIC

• Exposure is via:
- Drinking water , Contaminated soil, Food
• Inorganic arsenic is methylated in humans as well as animals
and micro-organisms.
- There are considerable differences between species and individuals.
• Arsenic rate of excretion increases with the methylation
efficiency.
- There are large inter-individual variations in the methylation of arsenic.
• Children have a lower degree of methylation of arsenic than
adults hence more susceptible to poisoning
• There are indications of a lower degree of arsenic methylation in
men than in women, especially during pregnancy.
• A fatal dose of arsenic trioxide is somewhere between 200 and
300 mg.Following ingestion, symptoms may begin within 30 min.
 Group 4 poisons: ARSENIC

• Only inorganic As is toxic in human. It is important to consider

speciation as regards toxicity
• Acute poisoning is characterised by:
Burning in mouth , Gastro-enteritis presenting with nausea, Abdomen
pain , Regurgitation, Vomiting and diarrhoea
• Chronic poisoning is characterised with:
Loss of appetite , Occasional vomiting, Jaundice, Loss of weight , Anaemia
• Other signs of chronic poisoning are skin eruptions-eczema,
keratosis of plantars and soles, brittle nails, hair loss, melanesia
with patches of leukaplakia , peripheral neuritis and cathexia .
The poison is permanently stored in the nails and hair
 Group 4 poisons: ARSENIC

• Autopsy findings are:

Usually and not always, highly inflamed upper small intestine and stomach
mucus membrane presenting with a velvety appearance , red and oedema of the
stomach, There may be bleedings from erosions ,The liver may show patchy
fatty degenerative change or necrosis with jaundice, The heart muscle and
kidney parenchyma may show similar fatty change Peripheral neural
degenerative changes may also be present
• Normal levels of arsenic in the blood range between 0.002
and 0.062 mg/L.
• Fatal blood levels are from 0.62 to 9.3 mg/L
• High levels of arsenic may be present in the urine after
consumption of seafood.
 Group 4 poisons: LEAD

• Lead is a highly toxic metal.

• Lead is found in storage batteries and was
used as a constituent of paint and leaded
petrol.
• The most common causes of lead poisoning
are ingestion of lead-based paint and
industrial or environmental exposure.
• The symptoms of chronic lead poisoning are
abdominal cramps,vomiting, constipation,
lethargy, anemia, weight loss, muscle
paralysis, nephropathy, and convulsions.
Death is uncommon.
 Group 4 poisons: LEAD

• At autopsy, the most striking finding is the

brain, which is massively swollen, with
flattening of the gyri, and is extremely pale,
almost white.
• Blood smears show basophilic stippling of
erythrocytes. Characteristic eosinophilic
intranuclear inclusions may be seen in
hepatocytes and cells of the proximal tubules
of the kidneys.
 Group 4 poisons: IRON

• Death caused by iron poisoning usually

involves children who accidentally ingest a
large number of iron sulfate tablets.
• Rarely, these tablets have been used by
adults for suicidal purposes.
• Following ingestion, there is severe pain in
the abdomen, vomiting, and diarrhea. The
vomitus is often bloody.
• At autopsy, the gastric mucosal folds are
thickened, corroded, and dark brown to black.
 Group 4 poisons: MERCURY

• Is methylated by bacteria in aquatic

environments to methylmercury . It is then
concentrated by the food chain.
• Elemental mercury is not as toxic as organic
mercury, however, its exposure may induce
severe axonal sensorimotor polyneuropathy in
humans with neurological deficits which may
persist in severe cases
• Methyl mercury readily crosses the placenta and
the blood-brain barrier and is neurotoxic
 Group 4 poisons: MERCURY
• Prenatal poisoning with high dose methyl
mercury causes mental retardation and
cerebral palsy
• Occupational exposure to mercuric compounds
and use in suicide is of forensic significance
• Mercuric chloride is commonly used as a
suicidal agent
• Ingestion of mercuric chloride causes the
development of haematemesis , melena and
acute renal failure
• Chronic exposure to mercury is demonstrated by
elemental X-Ray analysis of lipofuscin deposits
 Group 4 poisons: THALLIUM

• Used for making low-melting point

special glass for highly reflective lenses
• In developing countries still permitted as a pesticide
(rodenticide, insecticide)
• Body absorbs thallium very effectively, especially
through the skin, lungs,
• Two-phase elimination – most within 24 hours in urine,
but the rest stays for weeks and is excreted via faeces
• Undergoes enterohepatic circulation
• Cummulated in brain, kidneys, bones
• Pathological examination: haemorrhagic gastritis,
ulceration, damage of spleen, kidneys, hyperaemia of
brain
Group 4 poisons:
 Group 5 poisons: pesticides
 Pesticides may be classified as insecticides, herbicides,
rodenticides, molluscicides and acaricides, depending on
their use. They may also be from a variety of chemical
classes (e.g. organophosphorus compounds,
carbamates, chlorinated hydrocarbons, pyrethroids and
substituted ureas).
 In Western countries pesticides are not used commonly
by those with suicidal tendencies. However, this is not
true of poisoners; in fact many of the chemicals used for
homicidal poisoning were pesticides. Arsenic, thallium,
phosphorus and strychnine preparations have been used
as rodenticides, and nicotine and mercury salts as
insecticides and fungicides. The criminal use of the
herbicides sodium chlorate and paraquat has been
relatively common.
Group 5 poisons: pesticides (2)
Group 5 poisons: pesticides (3)
Group 5 poisons: pesticides – analysis (4)
 Group 6 poisons: anions
 Poisoning from this group of poisons is fairly rare, but
cases still occur.
Group 6 poisons: anions (2)
Group 6 poisons: anions (3)
 Other poisons: LYE (NaOH, KOH)

• Many caustic ingestions from adults are

more severe, as they are suicide
attempts, whereas children mainly drink
the substances out of curiosity.
• Occupational exposure is oftentimes
more severe given higher concentration
of chemicals
 Other poisons: LYE (NaOH, KOH)

• The severity of injuries depends in part on the form of

the lye ingested (crystal, liquid).

• Crystals - the injuries produced were generally limited

to the esophagus and were relatively superficial. Injury
to the stomach was uncommon.Esophageal stricture
was the most common complication, with occasional
perforation. It can produce transmural necrosis of the
esophagus after only 1 s of contact.
• Liquid lye - commonly reaches the stomach,
producing gastric necrosis. Occasionally, perforation of
the small intestine can occur.
 Other poisons: LYE (NaOH, KOH)

• Hydroxide ion interacts with collagen,

muscle, leading to thrombosis of small
blood vessels.
• Exothermic reactions occur given the
relatively acidic environment of the oral
cavity, pharynx leading to more damage
to surrounding tissues burns
• Increased permeability due to
breakdown of epithelial barrier
Pitfalls in Postmortem
Forensic Toxicology
 Decomposition
• Autolysis
– The breakdown of cellular material by enzymes
• Putrefaction
– A septic/infectious process
– The destruction of soft tissues by the action of
bacteria and enzymes
– Traumatic deaths may demonstrate 
putrefaction
 Decomposition
• Fewer samples available for collection
• Quality of samples is diminished
• Putrefaction produces alcohols
– Ethanol
– Isopropanol
– Acetaldehyde
– n-propanol
 Possible mechanisms of
postmortem redistribution
• Diffusion from specific tissue sites of higher
concentration (e.g. liver, myocardium, lung)
to central vessels in close proximity
• Diffusion of unabsorbed drug in the stomach
to the heart and inferior vena cava
• Diffusion of drugs from the trachea,
associated with agonal aspiration of vomitus
 Postmortem redistribution
• Coping with the problem of postmortem
redistribution:

– Analysis of both central blood and peripheral

blood in cases where postmortem redistribution
may be a factor

– Compilation of tables to determine average and

range of postmortem redistribution factors for
drugs
 Drug stability
• Knowledge of a drug’s stability is necessary to
facilitate interpretation of concentrations
• Breakdown of drugs may occur after death and
during storage via non-enzymatic mechanisms
– Cocaine  Benzoylecgonine (Hydrolysis)
– LSD  degradation due to light sensitivity
– Bupropion showed degradation that was both
temperature and pH dependent
 Evaporation of volatiles
• Ethanol
• Carbon monoxide
• Cyanide
• Toluene
• Other alcohols
Interpretation
 Interpretation
Therapeutic, toxic or fatal? How do you know?

• Compare measured blood concentrations with

concentrations reported in the literature:
– Clinical pharmacology studies
– Incidental drug findings
– Plasma  blood

• Consider case history:

– Symptoms observed by witnesses?
– Tolerance of the individual to the drug
 References
1. Clarke's Analysis of Drugs and Poisons 2004, ed. Anthony C. Moffat,
M. David Osselton, Brian Widdop, Pharmaceutical Press 2004.
2. V.J. Di Maio and D. Di Maio, Editors, Forensic pathology (2nd ed.),
CRC Press, Boca Raton 2001.
3. World Drug Report 2007, United Nations Office on Drugs and Crime,
2007, s. 252 (www.unodc.org)
4. Zdzisław Marek, Małgorzata Kłys, Opiniowanie sądowo-lekarskie i
toksykologiczne, Podręcznik, Zakamycze, Kraków, 1998.
5. Stefan Raszeja, Władysław Nasiłowski, Jan Markiewicz, Medycyna
Sądowa, Podręcznik dla studentów, PZWL, wydanie II, Warszawa 1993
6. Metody analizy środków uzależniających, ONZ (UNDCP), Instytut
Psychiatrii i Neurologii, Warszawa 1997.
7. Toksykologia, red. Witold Seńczuk, wydanie II, PZWL, Warszawa 1994.
8. Bogdan Szukalski, Narkotyki, Kompendium wiedzy o narkotykach, Instytut
Psychiatrii i Neurologii, Warszawa 2005
9. V.J. DiMaio, D. DiMaio, Medycyna Sądowa, Urban & Partner, Wrocław
2003.
Thank you