LN Toxicology Final
LN Toxicology Final
LN Toxicology Final
Toxicology
Hawassa University
In collaboration with the Ethiopia Public Health Training Initiative, The Carter Center,
the Ethiopia Ministry of Health, and the Ethiopia Ministry of Education
May 2007
Funded under USAID Cooperative Agreement No. 663-A-00-00-0358-00.
All rights reserved. Except as expressly provided above, no part of this publication
may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopying, recording, or by any information storage and
retrieval system, without written permission of the author or authors.
This material is intended for educational use only by practicing health care workers
or students and faculty in a health care field.
PREFACE
The scope of toxicology widened tremendously during the last
few years. An important development in this discipline is
mandatory because of the expansion of different industrial,
medical, environmental, animal and plant noxious substances.
So toxicology has got special attention to the deleterious
effects of chemicals and physical agents on all living systems.
Toxicology can be an independent descriptive, empiric
discipline to the fact of difficulty in diagnosis, controversial
management and unknown end points. Many lethal exposures
deserve early diagnosis & management before the
confirmatory evidences. This lecture note on toxicology is
primarily inspired for undergraduate laboratory technology
students who participate in the care of poisoned patients.
However, other health professionals whose carriers involve
related aspects can find it relevant. The outline format of the
lecture note allows for particular rapid review of essential
information.
i
students to improve the quality of the diagnosis in poisoned
patients.
ACKNOWLEDGMENTS
ii
The authors would like to thank the Carter Center for the
initiation &continuous financial support in the preparation of
this lecture note.
TABLE OF CONTENTS
Preface ................................................................................... i
iii
Acknowledgement .............................................................. iii
Table of contents ................................................................ iv
iv
Chapter three: Clinical toxicology laboratory
..................................................................................................
28 ..............................................................................................
1. Learning objectives ................................................. 28
2. Introduction .............................................................. 28
3. The role of clinical toxicology .................................. 29
4. Basic information necessary for the laboratory ....... 30
5. Steps in undertaking analytical toxicological
investigations .......................................................... 31
6. Laboratory specimens ............................................. 32
7. Apparatus, reference compounds &reagents .......... 36
8. General Laboratory tests in clinical toxicology ........ 38
9. Exercise ................................................................... 42
v
B.Hydrocarbon poisoning 61
C. Pesticides.................................................................63
D. Cyanide toxicity........................................................70
E. House hold toxicants.............................................. 75
4. Medical toxicants............................................................75
A. Acetaminophen.................................................76
B. Aspirin (salicylates)..........................................80
C. Barbiturate.........................................................83
5. Environmental toxicants................................................87
A.Carbon monoxide poisoning 87
B. Food born toxins......................................................91
6. Drugs of abuse................................................................93
A. Alcohols.................................................................... 93
B. Nicotine toxicity........................................................97
C. Opioids......................................................................98
7. Natural Toxicants.........................................................101
A. Animal toxicants
.........................................................................
102 ..................................................................
B. Plant toxicants .......................................... 103
8. Exercise ................................................................. 104
Glossary ............................................................................ 105
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ABBREVATIONS
AAS- Atomic absorption spectroscopy
ABGs- Arterial blood gas analysis
ALA- D- Delta amino levulinate dehydratase
BUN- Blood urea &nitrogen
CBC- Complete blood count
CNS- Central nervous system
CO- Carbon monoxide
DDT – Dichloro-Diphenyl-Trichloroethane
EPP- Erythrocyte proto porphyrine
GC- Gas chromatography
GC-MS- Gas chromatography-Mass
spectrometry GIT- Gastrointestinal tract
HCl- Hydrochloric acid
HPLC- High performance liquid
chromatography NaOH- Sodium hydroxide
nm- nano meter
NMR- Nuclear magnetic
resonance PH- Power of hydrogen
PT- Prothrombin time
PTT- Partial prothrombin time
t1/2 - Half-life
TLC- Thin layer
chromatography UV- Ultraviolet
Vd- Volume of distribution
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Toxicology
CHAPTER ONE
INTRODUCTION TO TOXICOLOGY
Learning Objectives
At the end of this chapter the student will be able to:
1. Understand the history of toxicology, &epidemiology of
poisoning.
2. Define different terminologies used in toxicology.
3. Understand the basic classification of toxicology.
4. Describe toxicokinetics & toxicodynamics.
5. Describe the potential causes of toxicity
6. Understand the environmental consideration of
toxicology.
7. Describe poison prevention & control strategies.
INTRODUCTION
During the past decades industrialization and agricultural
development, paralleled with increased health care have changed
life in various ways. Average life expectancy rose, due to better
control of epidemics and infectious diseases. However, increased
industrialization and agricultural development were the chief cause
of pollution that had profound influences on our lives. Man, the other
animals, & the plants in the modern world are increasingly being
exposed to chemicals of an enormous variety. Nearly everyone is at
risk of toxic exposures to hazardous substances in the ambient
environment. In recent years, awareness of the problem of human &
animal exposure to potentially toxic chemicals in our environment
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Toxicology
1. What is toxicology?
The word toxicology is derived from two Greek words;
toxikon, meaning poisonous substance into which arrow
heads were dipped and logos, meaning study.
Toxicology is the qualitative and quantitative study of the
adverse or toxic effect of chemicals and other anthropogenic
materials or xenobiotics on organisms. It also deals with
food and cosmetics for public consumption both in alive or
dead victims.
It is the science of poison & its scope has been enlarging. It
is one of the multidisciplinary fields of science.
It has got another dimension: the social, the moral & legal
aspects of exposure of populations to chemicals of unknown
or uncertain hazard.
Historical aspects of toxicology – it is only recently that
the study of poisons becomes truly scientific & in the past it
was mainly a practical art utilized by murderers & assassins.
Poison has played an important part in human history.
In Ancient time (1500 BC) earliest collection of medical
records contains many references and recipes for poisons.
Dioscorides (50 AD) a Greek physician, classify poisons as
animal, plant or mineral & recognized the value of emetics
Maimmonides (1135-1204 AD), wrote about poisons and
their antidote.
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Toxicology
2. Epidemiology
The following toxicological data are derived from American
association of poison control center. So it is mostly a
description of the epidemiology of unintentional poisoning. It
is very difficult to find the primary data of poisoning in our
country because most of the screening & confirmatory tests
are not done routinely in our set up. Additionally, we don’t
have well organized poison control center.
Today, poisoning (both accidental and intentional), is a
significant contributor to mortality and morbidity. It has been
estimated that 7% of all emergency room visits are the
result of toxic exposures. Household cleaner, over-the-
counter
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Toxicology
5
Toxicology
B. Mechanistic toxicology
Mechanistic toxicology deals with the mechanism of toxic effects
of chemicals on living organisms. This is important for rational
treatment of the manifestations of toxicity (e.g. organophosphate
poisoning reversed by oximes) ,prediction of risks (e.g.
organophosphate poisoning →leads to accumulation of
a c e t y l c h o l i n e→a c t i v a t e m u s c a r i n i c a n d n i c o t i
n i c receptors→respiratory failure) & facilitation of search for safer
drugs (e.g. Instead of organophosphates, drugs which reversibly
bind to cholinesterase would be preferable in therapeutics)
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Toxicology
C. Regulatory toxicology
Regulatory toxicology studies whether the chemical substances
has low risk to be used in living systems
E .g - Food and drug administration regulates drugs, food,
cosmetics medical devices &supplies in USA.
- Environmental protection agency regulates pesticides,
toxic chemicals, hazardous wastes and toxic pollutants in
USA
- Occupational safety and health administration regulates
the safe conditions for employees in USA
- Drug administration & control authority (DACA) - regulates
drugs, cosmetics and medical devices &supplies in
Ethiopia.
D. Predictive toxicology
Predictive toxicology studies about the potential and actual risks
of chemicals /drugs. This is important for licensing a new drug/
chemical for use.
A) Occupational toxicology
Occupational toxicology Deals with chemical found in the
workplace
E.g. – Industrial workers may be exposed to these agents
during the synthesis, manufacturing or packaging of
substances
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Toxicology
B) Environmental toxicology
Environmental toxicology deals with the potentially deleterious
impact of chemicals, present as pollutants of the environment, to
living organisms. Ecotoxicology has evolved as an extension
of environmental toxicology. It is concerned with the toxic effects
of chemical and physical agents on living organisms, especially
in populations and communities with defined ecosystems.
C) Clinical toxicology
Clinical toxicology deals with diagnosis and treatment of the
normal diseases or effects caused by toxic substances of
exogenous origin i.e. xenobiotics.
D) Forensic toxicology
Forensic toxicology closely related to clinical toxicology. It deals
with the medical and legal aspects of the harmful effects of
chemicals on man, often in post mortem material, for instance,
where there is a suspicion of murder, attempted murder or
suicide by poisoning.
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Toxicology
A. Toxicokinetics
i) Absorption
Absorption is the process by which the chemical enters the
body. It depends on the route of administration, dissociation
(to become ionized), dissolution (ability of solid dosage form to
become soluble), concentration, blood flow to the site, and the
area of the absorptive site.
The common sites of absorption (routes of exposure) are
Oral route – the GIT is the most important route of
absorption, as most acute poisonings involve
ingestions.
Dermal route – lipid solubility of a substance is an
important factor affecting the degree of absorption
through the skin.
Inhalational route – toxic fumes, particulate and
noxious gases may be absorbed through the lungs.
Bioavailability is the fraction of unchanged drug reaching the
systemic circulation following of non-vascular administration.
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Toxicology
ii) Distribution
Distribution-is defined as the apparent volume into which a
substance is distributed. Volume of distribution (Vd) is
calculated from the dose taken and the resulting plasma
concentration:
IV) Excretion
Excretion is the final means of chemical elimination, either as
metabolites or unchanged parent chemical. Excretion through
the lungs is the major route for gaseous substances; and in the
case of non-volatile water – soluble drugs, the kidneys are the
most important routes of excretion. Additional routes include
sweat, saliva, tears, nasal secretions, milk, bile and feces.
Clearance – elimination of chemicals from the body may be
described by the term clearance (CL). It is a quantitative
measure of the volume of blood cleared of drug per unit time,
usually expressed in milliliter pe4r minute.
Clearance is calculated as follows
CL = 0.7 (VD)/ (t1/2) = ml/min
Where the VD is expressed in milliliter per kilogram & the half-life is
expressed in minutes or hours.
B. Toxicodynamics
Toxicodynamics is the mechanism of action of a toxic chemical
to the body (what chemicals do to the body). The targets for the
toxicodynamic actions of toxic chemicals are
o Enzymes
o Membrane receptors
o Intracellular receptors
o Ion channel
Toxic effects generally result from adverse cellular, biochemical,
or macromolecular changes which attained by
o Damage to an enzyme
system o Disruption of protein
synthesis o DNA damage
o Modification of an essential biochemical function
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Toxicology
100
Response A B C
50 .........................................................
. . .
. . .
.
. . .
ED50 TD50 LD50
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Toxicology
Dosage
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Toxicology
7. Environmental considerations
Certain chemical and physical characteristics are known to be
important for estimating the potential hazard involved for
environmental toxicants. In addition to information regarding
effects on different organisms, knowledge about the following
properties is essential to predict the environmental impact:
The degradability of the substance; its mobility through air,
water and soil; whether or not bioaccumulation occurs; and its
transport and biomagnification through food chains.
If the intake of a long-lasting contaminant by an organism
exceeds the latter’s ability to metabolize or excrete the
substance, the chemical accumulates within the tissues of the
organism (e .g DDT). This is called bioaccumulation.
Although the concentration of a contaminant may be virtually
undetectable in water, it may be magnified hundred or thousand
time as the contaminant passes up the food chain. This is called
biomagnification.
Chemicals that are poorly degraded (by abiotic or biotic
pathways) exhibits environmental persistence and thus can
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Toxicology
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Toxicology
Exercise
1. Define toxicology & the terms used in toxicology.
2. Discuss the epidemiologic aspects of toxicology.
3. Discuss the basic classifications of toxicology.
4. Explain what toxicokinetics and toxicodynamics deals with.
5. Write some of the important environmental considerations in
toxicology.
6. List some of the potential sources of toxicity.
7. Mention poisoning prevention & control strategies.
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Toxicology
CHAPTER TWO
GENERAL APPROACH TO A POISONED
VICTIM
Learning Objectives
At the end of this chapter the student will be able to:
1. Understand diagnosis of poisoning by history, physical
examination and different investigations
2. Understand the basic principles of management of
poisoning
Introduction
The purpose of this chapter is to provide guidelines for evaluating
the severity of an exposure to a potentially toxic substance, clues to
the identity of the offending substance (its clinical effects on vital
functions, its odor, and its effect on the skin), and, most importantly,
how to manage the severely intoxicated victim initially. The trained
analyst can play a useful role in the management of victims
poisoned with drugs or other chemicals. However, optimal analytical
performance is only possible when the clinical aspects of the
diagnosis and treatment of such victims are understood. The analyst
must therefore have a basic knowledge of emergency medicine and
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Toxicology
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Toxicology
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Toxicology
General measures
1. Supportive measures
The first priority is to establish & maintain vital functions.
Subsequently, most victims can be treated successfully
using supportive care alone.
- Maintain air way, adequate ventilation &
oxygenation, provide tracheal intubation if required
- If comatose, administer glucose, thiamine, &oxygen
- For seizures, administer anticonvulsants
2. Principles of toxin eliminations
- If the poison has been inhaled, the victim should first be
removed from the contaminated environment.
- If skin contamination has occurred, contaminated clothing
should be removed and the skin washed with an appropriate
fluid, usually water.
- In adult victims, gastric aspiration and lavage (stomach
washout) are often performed, if the poison has been
ingested, to minimize the risk of continued absorption.
- Similarly, in children emesis can be induced by the oral
administration of syrup of ipecacuanha (ipecac).
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Toxicology
• Atropine Organophosphate
• Deferoxamine Iron
• Methylene blue Nitrates
• Physiostigmine Atropine
• Naloxone Opioids
• Pyridoxine Isoniazid
Exercise
1. What are the common ways used for diagnosing poisoned
victim?
2. Write the basic principles of management of poisoning
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Toxicology
CHAPTER THREE
CLINICAL TOXICOLOGY LABORATORY
Learning Objectives
At the end of this chapter the student will able to:
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Toxicology
Introduction
Clinical toxicology involves the detection and treatment of
poisonings caused by a wide variety of substances, including
household and industrial products, animal poisons and venoms,
environmental agents, pharmaceuticals, and illegal drugs. The
toxicology laboratory must provide appropriate testing in three
general areas: Identification of agents responsible for acute
or chronic poisoning; Detection of drugs of abuse; and
therapeutic drug monitoring. Increasingly sophisticated
analytic methods are available to accomplish these tasks, but it
is imperative that they be used judiciously. The numbers of
compounds for which true emergency laboratory results are
needed to guide therapy are still relatively few. For most
potentially lethal intoxications the victim must be treated
empirically before the laboratory results are known. A wide
held misconception is that the laboratory can routinely detect
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Toxicology
A.Suspected agent(s)
The content of toxicology screens varies among laboratories.
Although a standard screen may not include the suspected
agent, if alerted beforehand the laboratory may be able to
modify procedures as needed in order to search for the
suspected agents.
B. Suspected dose
Analytic sensitivities vary among laboratories, and some
facilities may not be able to detect therapeutic concentrations
of certain drugs in their routine screens. Knowledge of the
approximate dose ingested is important because in certain
cases the use of analytic methods designed for therapeutic
monitoring, not screening may be necessary.
Pre-analytical phase
• Obtain details of current admission, including any
circumstantial evidence of poisoning and results of
biochemical and hematological investigations
• Obtain victim's medical history, if available, ensure
access to the appropriate sample(s), and decide the
priorities for the analysis.
Analytical phase
• Perform the agreed analyses.
Post-analytical phase
•Interpret the results and discuss them with the clinician
looking after the victim.
•Perform additional analyses, if indicated, on the original
samples or on further samples from the victim.
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Toxicology
A. Specimen collection
Urine
Urine is useful for screening tests as it is often available in large
volumes and usually contains higher concentrations of drugs or
other poisons than blood. The presence of metabolites may
sometimes assist identification. A 50-ml specimen from an adult,
collected in a sealed, sterile, plastic container, is sufficient for most
purposes; no preservative should be added. Urine can be collected
in acid washed, metal free container for quantification of heavy
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Toxicology
Stomach contents
Stomach contents may include vomit, gastric aspirate and stomach
washings - it is important to obtain the first sample of washings,
since later samples may be very dilute. A volume of at least 20 ml is
collected in plastic container to carry out a wide range of tests; no
preservative should be added. It is the best sample on which to
perform certain tests. If obtained soon after ingestion, large amounts
of poison may be present while metabolites, which may complicate
some tests, are usually absent. An immediate clue to certain
compounds may be given by the smell; it may be possible to identify
tablets or capsules simply by inspection.
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Toxicology
Blood
Blood (plasma or serum) is normally reserved for quantitative
assays but for some poisons, such as carbon monoxide, whole
blood has to be used for qualitative tests. Specimen should be
collected in a sealed heparinized tube on admission. In addition,
2-ml sample should be collected in a fluoride/oxalate tube.
The use of disinfectant swabs containing alcohols (ethanol,
propan-2-ol) should be avoided. In general, there are no
significant differences in the concentrations of poisons between
plasma and serum.
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Toxicology
C. Specimen examination
Urine
High concentrations of some drugs or metabolites can impart
characteristic colors to urine. Treatment given for poisoning may
color urine (E.g. Deferoxamine in iron poisoning color urine red or
methylene blue given in treatment of nitrate poisoning may color
urine blue). Strong-smelling poisons such as methylsalicylate can
sometimes recognized in urine since they are excreted in part
unchanged. Turbidity may be due to underlying pathology (blood,
microorganisms, casts, epithelial cells), or carbonates, phosphates
or urates (in amorphous or microcrystalline forms). Such findings
should not be ignored, even though they may not be related to the
poisoning.
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Toxicology
A. Apparatus
Analytical toxicology services can be provided in clinical
biochemistry laboratories that serve a local hospital or accident
and emergency unit. In addition to basic laboratory equipment,
some specialized apparatus, such as that for thin-layer
chromatography, ultraviolet and visible spectrophotometry and
microdiffusion, is needed. A continuous mains electricity supply is
not essential. No reference has been made to the use of more
complex techniques, such as gas-liquid and high-performance
liquid chromatography, atomic absorption spectrophotometry or
immunoassays, even if simple methods are not available for
particular compounds. Although such techniques are more
selective and sensitive than many simple methods, there are a
number of factors, in addition to operator expertise, that have to
be considered before they can be used in individual laboratories.
The standards of quality (purity or cleanliness) of laboratory
reagents and glassware and of consumable items such as
solvents and gases needs to be considerably higher than for the
tests described in this manual if reliable results are to be obtained.
Additional complications, which may not be apparent when
instrument purchase is contemplated, include the need to ensure
a regular supply of essential consumables (gas chromatographic
septa, injection syringes, chromatography columns, solvent filters,
chart or integrator paper, recorder ink or fibre-tip pens) and spare
or additional parts (detector lamps, injection loops, column
packing materials). The instruments must be properly maintained
(see annex II).
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Toxicology
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Toxicology
A. Biochemical tests
Blood glucose:
Determination of blood glucose is essential to know those toxic
substances that affect blood glucose biotransformation. A toxicant
that causes hypoglycemia includes insulin, iron, acetyl salicylic
acid & so on.
Hyperglycemia is a less common complication of poisoning than
hypoglycemia, but has been reported after over dosage with
acetylsalicylic acid, salbutamol and theophylline.
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Toxicology
Plasma enzymes
The plasma activities of liver enzymes, such as aspartate
aminotransferase, alanine aminotransferase may increase rapidly
after absorption of toxic doses of substances that can cause liver
necrosis, notably paracetamol, carbon tetrachloride, and copper
salts.
Cholinesterase activity
Plasma cholinesterase is a useful indicator of exposure to
organophosphorus compounds or carbamates, and a normal
plasma cholinesterase activity effectively excludes acute
poisoning by these compounds.
The diagnosis can sometimes be assisted by detection of a poison
or metabolite in a body fluid, but the simplest method available is
relatively insensitive.
B. Hematological tests
Hematocrit (Erythrocyte volume fraction)
Acute or acute-on-chronic over dosage with iron salts,
acetylsalicylic acid, indomethacin, and other non-steroidal anti-
inflammatory drugs may cause gastrointestinal bleeding leading to
anemia.
Anaemia may also result from chronic exposure to toxins that
interfere with haem synthesis, such as lead.
Leukocyte count
Increases in the leukocyte (white blood cell) count often occur in
acute poisoning, for example, in response to an acute metabolic
acidosis, resulting from ingestion of ethylene glycol or methanol,
or secondary to hypostatic pneumonia following prolonged coma.
Blood clotting
The prothrombin time and other measures of blood clotting are
likely to be abnormal in acute poisoning with rodenticides such as
Coumarin anticoagulants.
Carboxyhemoglobin
Measurement of blood carboxyhemoglobin can be used to assess
the severity of acute carbon monoxide poisoning.
However, carboxyhemoglobin is dissociated rapidly once the
victim is removed from the contaminated atmosphere, especially if
oxygen is administered, and the sample should therefore be
39
Toxicology
Exercise
1. What is the basic information necessary for clinical
toxicology laboratory?
2. What are the roles of clinical toxicology laboratory?
3. Mention the steps that are necessary to undertake analytic
toxicological investigations.
4. Describe specimen collection, transportation, storage,
characteristics & physical examination used in clinical
toxicology laboratory.
5. Describe apparatus, reference compounds & reagents used
in clinical toxicology laboratory.
6. Describe the routine laboratory tests used in clinical
toxicology laboratory.
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Toxicology
CHAPTER FOUR
PRACTICAL ASPECTS OF ANALYTICAL
TOXICOLOGY
Learning Objectives
At the end of this chapter the student will able to:
1. Define the methods used in practical aspects of
analytical toxicology
2. Understand the common toxicology laboratory
techniques
Introduction
Methods for particular toxicologic tests or panels are a well
established part of routine laboratory tests, and information about
them is available on request. In order to interpret toxicology results
properly, the laboratory technician should have a rudimentary
familiarity with the analytic methods employed. Several methods
exist, varying in sensitivity, specificity, assay time, and cost. The
choice depends on the size and budget of the institution, the types
of victims served the proximity to more elaborate toxicology facilities,
and other factors. This chapter focuses on the practical aspects of
analytical toxicology.
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Toxicology
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Toxicology
I. Spot tests
Spot tests are rapid, easily performed, non-instrumental qualitative
procedures. They are the most rudimentary toxicology tests, &
generally performed on urine specimens. In the test procedure,
the sample (that is suspected for having a particular toxic
chemical) will react with a chemical or chemicals set as a solution,
or coated on a strip & the result of the reaction expressed by a
color formation detected visually or colorometrically.
Spot tests are available for a number of compounds, including
salicylate, acetaminophen, carbonmonoxide, halogenated
hydrocarbons, and heavy metals. The tests are rapid and
convenient; however sensitivity and specificity are generally poor
and accurate quantification is virtually impossible. Because of
improvements in other technologies, spot tests are now largely
replaced by rapid immuno- assays that may perform at the point-
of-care or in the central laboratories.
III.Immunoassays
Immunoassays are diagnostic techniques used for the detection of
antigen and antibody. Depending on the immunoassay techniques
that are employed for the specific test, either antigen or antibody
may be detected from the samples based on their reaction with their
specific antibody or antigen respectively.
Many types of immunoassay configuration can be devised. Those
not involving radioactivity or separation steps (homogeneous
immunoassays) can be automated on routine clinical chemistry
instruments, making them convenient for laboratories of all sizes.
Immunoassay techniques used to screening specimens for
chemicals include: Enzyme-Multiplied immunoassay (EMIA),
Florescence polarization Immunoassay (FPIA), Cloned enzyme
donor Immunoassay (CEDIA), and Radio Immunoassay (RIA).
Immunoassays can be made highly sensitive and quite specific, but
their specificity is never absolute. Molecules with a similar structure
generally cross-react to some degree, and occasionally substances
interfere with the assay in some other fashion.
Immunoassays also have the drawback that each analyte must be
individually assayed using an available antibody reagent.
Nevertheless, some of the more modern, discrete analyzers can
readily perform multiple homogenous immunoassays with minimal
operator intervention, so a panel of commonly abused drugs (e.g.,
barbiturates, cocaine, opiates, cannabinoids, amphetamines,
benzodiazepines) can be readily tested.
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Toxicology
IV.Chromatography
Chromatography is a powerful technique for separating substances
based on slight differences in chemical properties. In this method,
components to be separated are distributed between two phases; as
stationary and mobile phases.
Chromatographic procedure involve a sample to be introduced in a
flowing stream of gas or liquid (mobile phase) that pass through a
bed, layer, or column containing a stationary phase (made from
solid, or gel or a liquid). As the mobile phase carries the sample
pass the stationary phase, the solutes with lesser affinity remain in
the mobile phase & travel faster & separate from those that have
great affinity for it. Different chemicals have different characteristic
mobility in a particular chromatographic system, allowing fairly
confident identification.
In contrast to immunoassays, small chemical changes (e.g., addition
or removal of a methyl group), commonly cause substantial changes
in chromatographic mobility. Thus the parent drug can usually be
distinguished from its metabolites.
Exercise
1. What is the test methods used in practical aspects of
analytical toxicology?
2. Explain the common analytical toxicology techniques.
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Toxicology
CHAPTER FIVE
TOXICANTS OF PUBLIC HEALTH
HAZARD
Learning objectives
At the end of this chapter the student will be able to: -
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Toxicology
Introduction
The rapid industrialization and successful green revolution have
introduced a large variety of chemicals into our environment. The
species and varieties of environmental chemicals are as many as
we can visualize. We may however, characterize them as: industrial
chemicals which include organic and inorganic substances, metals,
gases, fumes, solvents, and intermediates; agrochemicals, a major
input of farming industry, comprising a variety of pesticides,
fertilizers and growth promoters; pharmaceuticals, in innumerable
number; and food additives, plastics, cosmetics etc. These have
caused a great danger and put human and environment at a high
risk. This chapter is meant for discussion of some of the important
toxicants of public health hazard.
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Toxicology
I. Industrial toxicants
Industrial chemicals causing diseases have existed ever since man
began manufacturing on a large scale & during the industrial
revolution occupational diseases became common. Many of the
chemicals used in industry are chemically reactive molecules & are
likely to interact with biological systems & cause damage in some
cases at the site of exposure. Exposure is most commonly via skin &
lungs. There are now many thousands of chemical substances used
in industry ranging from metals & inorganic compounds which risk
people who work with it.
Lead Poisoning
Lead poisoning is one of the oldest occupational and
environmental diseases in the world. Despite its recognized
hazards, lead continues to have widespread commercial
application (like ingested lead paints, pica, and lead pipes etc…).
Environmental lead exposure, ubiquitous by virtue of the
anthropogenic distribution of lead to air, water and food, has
declined considerably due to diminished use of lead in gasoline
and other applications. Lead serves no useful purpose in the
human body. Lead is slowly but consistently absorbed via the
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Succinylcholine +glycine
ALA Synthetase lead
Aminolevulinate
ALA Dehydratase lead
Coproporphynogen
Coproporphynogen oxidase lead 53
Toxicology
Protoporphyrin
Ferrochelase +iron lead
Heme
Hemoglobin
Laboratory findings
A. Complete blood count
- Anemia –- Hemoglobin level of less than 10gm/dl can be seen
- Reticulocytosis – results from early release of immature
RBCs. It is not present in iron deficiency anemia so it is
valuable for differentiating the two forms of anemia.
- Eosinophilia – common finding but non specific
- Basophilic stippling of erythrocytes on wright stain of
peripheral blood has been observed to be a less frequent
occurrence than anemia & the finding is less non specific.
B) Serum Lead level
Levels of 30-60µg/dl are regarded as significant for lead
toxicity. Atomic absorption spectrometry (AAS) is the most
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Toxicology
Qualitative test
Specimen
Procedure
1. Add 0.1 ml of sodium tartrate buffer to 0.1 ml of test solution and
vortex-mix for 5 seconds.
2. Spot 50 µl of acidified solution on to phase-separating filter-
paper and add 50 µl of sodium rhodizonate solution.
Results
Lead salts give a purple colour in this test. However, the test is not
specific: barium salts give a brown colour and a number of other
metals also give coloured complexes.
Sensitivity
Lead, 2 mg/l
Quantitative tests
Principle
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Toxicology
Procedure
1. The first two specimens analyzed represent blanks. They
consist of 0.25 mL of triple-distilled water and 1.0 mL of
diluent.
2. Calibrating solutions are made by mixing 1.0 mL of the
diluent with 0.25 mL of each calibrator.
3. The control pool and victim specimens are prepared for
analysis by mixing 0.25 mL of each sample with 1.0 mL of
diluent.
4. Add 10 µL of matrix modifier to 10µL of each sample and
inject into the L’vov platform.
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Toxicology
Calculation
1. Subtract the blank absorbance peak area from each
specimen absorbance peak area.
2. Perform a best-fit regression analysis of the calibrator
concentrations versus the respective absorbance peak area
to define the calibration curve.
• Compare the absorbance peak area derived from each
specimen against the calibration curve to determine the
concentration of lead. If the results among duplicates vary
by more than 10%, sample contamination during processing
is likely to have occurred.
Management
• Chelating agents (Antidotes) E.g. dimercaprol, CaNa2-EDTA
• Symptomatic management
B. HYDROCARBON POISONING
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Toxicology
LABORATORY STUDIES
- Routine laboratory tests are of little value for purposes of
screening victims for admission.
- Arterial blood gas analysis (ABGs) must be measured in all
victims with respiratory symptoms. Varying degrees of
hypoxia without hypercarbia are the most common finding.
- Occasionally a metabolic acidosis is seen.
- The CBC may show leukocytosis
- Blood hydrocarbon levels are not readily available &have
little diagnostic, prognostic, or therapeutic value. Qualitative
testing is occasionally needed for forensic purposes.
Specific tests
Qualitative test
Reagents
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Toxicology
Procedure
1. Take 1ml of the urine sample in a test tube
2. Add 1ml of 20% NaOH & 1ml of pyridine
3. Heat in a boiling water bath for 1 minute
4. A pink red color in the pyridine layer indicates the presence
of hydrocarbon.
C. PESTICIDES
Pesticides are any substance or mixture of substances intended
for preventing, destroying, repelling or mitigating any pest.
Pesticide can be divided into several groups, such as
insecticides, rodenticides, fungicides & herbicides. This part will
give attention to the most frequent pesticide poisoning.
1) Insecticides
Organophosphates & carbamates are the most frequently used
insecticides world wide. These compounds cause 80% of the
reported toxic exposure to insecticides.
Organophosphurus insecticides
These agents are utilized to combat a large variety of pests.
Some of these agents are used in human and veterinary
medicine as local or systemic antiparasitics or in circumstances
in which prolonged inhibition of cholinesterase is indicated. The
60
Toxicology
Laboratory analysis
Reagents
1. Sodium bicarbonate (solid).
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Toxicology
2. Cyclohexane:acetone:chloroform (70:25:5).
3. Acetone:tetraethylenepentamine (9:1).
4. 4-(p-Nitrobenzyl) pyridine (20 g/
l) in acetone:
tetraethylenepentamine (9:1).
5. Silica gel thin-layer chromatography plate (5 × 20 cm, 20 µm
average particle size ;).
Procedure
1. Carefully adjust the pH of 10 ml of sample to about 7 by adding
solid sodium bicarbonate.
2. Extract 10 ml of sample with 5 ml of methyl tertiary-butyl ether
for 5 minutes using a rotary mixer.
3. Allow to stand for 5 minutes, take off the upper, ether layer and
re-extract with a second 5-ml portion of methyl tertiary-butyl
ether.
4. Combine the extracts, filter through phase-separating filter- paper
into a clean tube and evaporate to dryness under a stream of
compressed air or nitrogen.
5. Analyze the final solution in thin layer chromatography (see
annex I-number 3)
Results
The compounds of interest give purple spots on a pale brown
background.
Sensitivity
Organophosphorus pesticide, 5 mg/l
Confirmatory test
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Toxicology
Specimen
Plasma or serum
Cholinesterase activity
monograph Qualitative test
Specimen
Plasma or serum
Procedure
1. Add 2.0 ml of dithiobisnitrobenzoate reagent and 1.0 ml of
acetylthiocholine iodide solution to each of three 10-ml test-
tubes.
2. Add 20 µl of control plasma to one tube and 20 µl of test plasma
to a second.
3. Add 20 µl of pralidoxime solution and 20 µl of test plasma to the
third tube.
4. Vortex-mix the contents of all three tubes and allow to stand at
room temperature for 2 minutes.
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Toxicology
Results
The presence of an acetylcholinesterase inhibitor is indicated if the
yellow colour in the control tube is deeper than in the test tube. If the
colour in the tube containing pralidoxime is similar to that in the
control tube, this provides further confirmation that an inhibitor of
acetylcholinesterase is present in the sample. Inhibitors of
acetylcholinesterase, such as many carbamate pesticides, also give
a positive result in this test
Treatment
• GI decontamination
• Dermal decontamination
• Symptomatic treatment
• Toxin-specific like atopine, pralidoxime
Carbamate pesticides
Produce a milder form of toxicity, similar to that produced by
organophosphate compounds. These compounds inactivate
acetylcholinesterase leading to excessive accumulation of
acetylcholine. The important differences distinguishing
carbamates from organophosphate toxicity are
- Carbamate toxicity is typically short-lived in which
spontaneous regeneration of enzymatic activity usually
occur within 24hours
- Carbamates produce little or no CNS toxicity because of
their inability to penetrate the blood-brain-barrier & affect
brain cholinesterase activity
Sign & symptoms may be more rapid &usually abate within
24hrs regardless of therapy
Laboratory findings
64
Toxicology
Qualitative test
Specimen
Stomach contents, scene residues.
Reagents
1. Aqueous hydrochloric acid (2 mol/l)
2. Furfuraldehyde solution (100 ml/l) in methanol, freshly prepared.
3. Concentrated hydrochloric acid (relative density 1.18).
Procedure
1. Acidify 1 ml of sample with 0.5 ml of dilute hydrochloric acid and
extract with 4 ml of chloroform on a rotary mixer for 5 minutes.
2. Centrifuge for 5 minutes, discard the upper, aqueous layer and
filter the chloroform extract through phase-separating filter-
paper into a clean tube.
3. Evaporate the extract to dryness under a stream of compressed
air or nitrogen at 40°C.
4. Dissolve the residue in 0.1 ml of methanol, apply a spot of the
solution to filter-paper and allow drying.
5. Apply 0.1 ml of furfuraldehyde solution to the spot;
6. Allow drying and exposing the paper to concentrated hydrochloric
acid fumes for 5 minutes in a fume cupboard.
Results
65
Toxicology
Sensitivity
Carbamate, 100 mg/l
Treatment
• GI decontamination
• Atropine, & pralidoxime can be used as an antidote
2. Rodenticides
Rodenticides are used to control rodent population.
Anticoagulant preparations, currently the most widely used
rodenticides, are safer, although consequential human
poisonings do occur. Most pediatric ingestions occur
accidentally, whereas ingestions in adults tend to be deliberate.
Coumarin derivatives (E.g. warfarin) are one of the members of
this class. Mechanism of action is by inducing coagulopathic
state by inhibiting activation of the vitamin K – dependent
clotting factors II, VII, IX and X. Victims are usually
asymptomatic unless presentation is delayed over a period of
several days, as the anticoagulant effects take place victims
may experience spontaneous bleeding. The main features of
warfarin poisoning in less severe cases are excessive bruising,
nose & gum bleeding, &blood in the urine faeces. Bleeding from
several organs within the body, leading to shock & possibly
death, occurs in the more severe cases. The onset of the signs
of poisoning may not be evident until a few days after exposure.
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Toxicology
Laboratory analysis
- General tests
- Check a baseline prothrombin time (PT), partial
prothrombin time (PTT) and CBC
- Toxin-specific tests
- Recommended monitoring factor VII-X complex
levels are sensitive indicators of toxicity
- Warfarin may be detected by gas chromatography
Quantitative test
Specimen
Urine, plasma
Procedure
HPLC method is used for the fluorometric determination of warfarin
& its metabolites. The detection scheme utilizes post column acid-
base fluorescence enhancement techniques that provide high
chromatographic specificity &sensitivity. Detection limit are in the
low nanogram range. Other procedures, like UV spectrometry
&revised-phase liquid chromatography, with a detection limit in
blood serum of 20µg/L.
D) CYANIDE TOXICITY
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Toxicology
Laboratory analysis
i) General tests
1. Chemistry Tests
- Because of the blockade of aerobic biotransformation,
cyanide produces an anion gap metabolic acidosis
secondary to the production of lactic acid
- Glucose catabolism is also altered, and the blood glucose
level may be elevated
2. Blood gas analysis
- PaO2 and oxygen saturation are unaltered except in severe
cases where respiratory failure occurs.
- pH and PaCO2 are altered in different ways, depending on
the severity of poisoning
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Toxicology
Qualitative test
Specimen
Stomach contents, scene residues.
N.B:- specimens containing cyanides often evolve hydrogen
cyanide if acidified.
Reagents
1. Aqueous sodium hydroxide solution (100 g/l).
2. Aqueous ferrous sulfate solution (100 g/l, freshly prepared in
freshly boiled and cooled water).
3. Aqueous hydrochloric acid (100 ml/l).
Procedure
1.Dilute 1 ml of sample with 2 ml of sodium hydroxide solution.
2.Add 2 ml of ferrous sulfate solution.
3.Add sufficient hydrochloric acid to dissolve the ferrous hydroxide
precipitate.
Result
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Toxicology
Sensitivity
Cyanide, 10 mg/l
Quantitative assays
Specimen
Heparinized whole blood (0.1-1.0 ml),
N.B .The samples can be stored at 4°C for 1-2 days if the analysis
is delayed for any reason. (Cyanide in blood is less stable if stored
at room temperature or at -20°C.)
Reagents
1. Aqueous sodium hydroxide (0.5 mol/l).
2. Aqueous sulfuric acid (3.6 mol/l).
3. p-Nitrobenzaldehyde (0.05 mol/l) in 2-methoxyethanol.
4. o-Dinitrobenzene (0.05 mol/l) in 2-methoxyethanol.
Standard
Aqueous potassium cyanide (10 mg/l, i.e., cyanide ion
concentration, 4 mg/l)
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Toxicology
Results
The red coloration obtained with cyanide-containing solutions is
stable for about 15 minutes. Measure the absorbance of the
solutions from cells 2 and 3 at 560 nm against the purified water
blank (cell 1). Assess the cyanide ion concentration in the sample by
comparison with the reading obtained from the standard.
Sensitivity
Cyanide, 0.5 mg/l
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Toxicology
Treatments
• Symptomatic management
• Toxin specific measures
- Nitrite-thiosulfate, hydroxycobalamin
II – MEDICAL TOXICANTS
Drugs are biologically active molecules used in the treatment,
prevention & diagnosis of disease. However, drugs have made & will
continue to make a major contribution to human health, we must
accept the risks attached to these benefit.
The basic mechanisms for the toxicities arising from drugs are
- Direct& predictable toxic effects due to over doses
- Toxic effects occurring after repeated therapeutic doses
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Toxicology
A. Acetaminophen
Laboratory analysis
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Toxicology
Qualitative tests
Reagents (See annex I- number 8)
1. Saturated o-cresol.
2. Ammonium hydroxide, 4 mol/L
3. Concentrated hydrochloric acid.
Procedure
1. Mix 1 mL of specimen (victim or control urine, water blank)
0
and 1 mL of concentrated hydrochloric acid. Heat at 100 C
for 10 min.
2. Cool and add 100 µL of the above solution to 10 mL of o-
cresol reagent and then 2 mL of ammonium hydroxide, 4
mol/L
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Toxicology
Result
Acetaminophen is hydrolyzed to p-aminophenol, which reacts with
o-cresol and ammonium hydroxide to form an indophenol blue
chromogen.
Quantitative tests
Principle
Acetaminophen and 3-acetamidophenol, added as an internal
standard, are extracted from serum and analyzed by reverse-phase
HPLC with an octadecylsilane bonded-phase column. The peak
height absorbance ratio for acetaminophen relative to the internal
standard is determined at 254 nm (annex II).
Procedure
1. Pipette 100 µL of each calibrator, control, and victim’s
serum into properly labeled 13 x 100-mm glass tubes.
2. Add 100 µL of working internal standard solution and 100 µL
of phosphate buffer (0.225 mol/L, pH 7.4). Mix.
3. Add 3mL of ethyl acetate. Mix in a Vortex mixer for 15 s.
75
Toxicology
Calculation
Determine the peak height (or peak area) ratios of acetaminophen
relative to the internal standard. Calculate the concentration of
acetaminophen in the unknown by comparing its peak height ratio
versus acetaminophen concentration response for the calibrator.
Treatment
• GI decontamination
• Antidote (acetylcysteine)
B. Aspirin (salicylate)
Acetylsalicylic acid, commonly known as aspirin, is still one of the
most widely used minor analgesics. Salicylate poisoning is a much
less common cause of childhood poisoning deaths since the
introduction of child-resistant container and the reduced use of
baby aspirin. Salicylates, however still accounts for numerous
suicidal and accidental poisonings. Salicylate Poisoning can also
result from chronic over medication; this occurs most commonly in
elderly victims using salicylates for chronic pain because of
impaired biotransformation, excretion & others. Salicylic acid is then
metabolized by conjugation. These conjugation steps are
76
Toxicology
Laboratory tests
Procedure
Add 0.1 ml of Trinder's reagent to 2 ml of sample and mix for 5
seconds.
N.B- To test for acetylsalicylic acid or methyl salicylate in
stomach contents or scene residues, and to test for
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Toxicology
Results
A strong violet color indicates the presence of salicylates. Azide
preservatives react strongly in this test, and weak false positives can
be given by urine specimens containing high concentrations of
ketone bodies.
This test is sensitive and will detect therapeutic dosage with salicylic
acid, acetylsalicylic acid, 4-aminosalicylic acid, methyl salicylate and
salicylamide.
Sensitivity
Salicylate, 10 mg/l
Quantitative assay
Specimen
Plasma or serum (1 ml)
Reagent
Trinder's reagent
Standards
Aqueous solutions containing salicylic acid at concentrations of 0,
200, 400 and 800 mg/l. Store at 4°C when not in use.
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Toxicology
Procedure
1. Add 5 ml of Trinder's reagent to 1 ml of sample or standard.
2. Vortex-mix for 30 seconds and centrifuge for 5 minutes.
3. Measure the absorbance of the supernatant at 540 nm against
plasma blank
Results
Calculate the plasma salicylate concentration from the graph
obtained on analysis of the salicylate standards. Some salicylate
metabolites interfere, but plasma concentrations of these
compounds are usually low. Oxalates, for example, from fluoride/
oxalate blood tubes, also interfere in this test.
Sensitivity
Salicylate, 50 mg/l
Treatment
• GI decontamination
• Facilitating diuresis
• Symptomatic management
C) Barbiturates
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Toxicology
Laboratory analysis
1. Plasma barbiturate (e.g. Phenobarbital) levels are helpful for
making a diagnosis but of little value when predicting the
severity of the over dose.
2.As with alcohol, chronic abusers of Phenobarbital may have
elevated serum levels with little CNS depression.
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Toxicology
Procedure
1. Add 5 ml of sample, 2 ml of hydrochloric acid and 60 ml of
diethyl ether to a 250-ml separating funnel.
2. Lubricate (with purified water) and insert the funnel and shake
gently for 2 minutes.
3. After standing for 5 minutes, and then discard the lower
aqueous phase, add the diethyl ether extract to 10 ml of
borate buffer in a second separating funnel and mix for 1
minute.
4. Allow to stand for 5 minutes and again discard the lower,
aqueous phase through the funnel tap.
5. Wash round the funnel with 5 ml of purified water; allow
standing for 5 minutes and again discarding the lower,
aqueous phase through the funnel tap.
6. Add about 4 g of sodium sulfate/charcoal mixture to the ether
extract in the funnel, shake to disperse, and filter the extract
through phase-separating filter-paper into a 150-ml conical
flask.
7. Add a further 20 ml of diethyl ether to the separating funnel,
shake and add to the extract in the flask through the filter
funnel.
8. Evaporate the extract to dryness on a water-bath at 40°C
under a stream of compressed air or nitrogen.
9. Add 5.0 ml of purified water to the dry extract in the flask, swirl
gently and allow to stand for 5 minutes.
10. Filter the reconstituted extract through phase-separating
filter-paper into a 12.5-cm test-tube.
11. Check the spectrophotometer zero at 240 nm using purified
water in both sample and reference positions.
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Toxicology
12. Add 4 ml of filtrate from the test-tube to a clean, dry cell, add
50 µl of concentrated ammonium hydroxide and mix using a
plastic paddle. Check that the pH is about 10.
13. Quickly measure the absorbance at 240 nm against purified
water blank. If necessary, accurately dilute a portion of the
extract with purified water to bring the reading on to the
scale, and record the magnitude of the dilution. If a scanning
spectrophotometer is available, scan in the region 200-450
nm.
14. Repeat the reading or scan after 5 minutes.
15. Add 0.1 ml of concentrated sulfuric acid to the cell, mix
using the plastic paddle, and check that the pH is about 2.
16. Repeat the reading (240 nm) or scan (200-450 nm).
Results
1) To perform a quantitative measurement, measure the
difference between absorbance at pH 10 and at pH 2,
construct a calibration graph by analysis of the standard
barbiturate solutions, and calculate the barbiturate
concentration in the sample.
Alternatively, use the following formula:
((absorbance at pH 10) - (absorbance at pH 2)) × Dilution
factor (if any) × 25 = barbiturate (mg/l)
2) Sample volumes of less than 5 ml may be used, but there
will be a corresponding loss of sensitivity unless "micro"-
volume fused silica spectrophotometer cells are available.
Sensitivity
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Toxicology
Barbiturate, 2 mg/l
Treatment
• GI decontamination
• Alkalinization of urine
• Hemodialysis
83
Toxicology
Laboratory analysis
- Carboxyheamoglobin level – should be measured as early as
possible to establish the diagnosis of carbon monoxide
poisoning. Carboxyheamoglobin should be measured from
blood sample using spectrophotometric methods. Although
not as accurate, carboxyheamoglobin levels can be estimated
from expired air using a breath analyzer for carbon monoxide.
- Arterial blood gas assays are poor indicators of carbon
monoxide poisoning. The PaO2 is often normal, as it is a
measure of oxygen dissolved in plasma, not a measure of
oxygen bound to hemoglobin.
Reagent
Aqueous ammonium hydroxide (0.01 mol/l)
Procedure
Add 0.1 ml of blood to 2 ml of ammonium hydroxide solution and
vortex-mix for 5 seconds.
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Toxicology
Results
A pink tint in comparison with the colour obtained from a normal
blood specimen suggests the presence of carboxyhaemoglobin.
Cyanide may give a similar tint, but acute cyanide poisoning is
generally much less common than carbon monoxide poisoning.
Sensitivity
HbCO, 20%
Quantitative assay
Specimen:whole blood treated with heparin, edetic acid or fluoride/
oxalate.
Reagents
1. Aqueous ammonium hydroxide (1 ml/l).
2. Sodium dithionite (solid, stored in a desiccator).
3. A supply of pure carbon monoxide or carbon monoxide/nitrogen.
4. A supply of oxygen or compressed air.
Procedure
1. Add 0.2 ml of blood to 25 ml of ammonium hydroxide solution
and mix.
2. Take three approximately equal portions: x, y and z. Keep portion
x in a stoppered tube while the following procedures are
performed:
(a) Saturate portion y with carbon monoxide (to give 100%
HbCO) by bubbling the gas through the solution for 5-10
minutes. Take care to minimize frothing.
(b) Saturate portion z with oxygen by bubbling pure oxygen or
compressed air through the solution for at least 10 minutes to
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Toxicology
Results
The percentage carboxyhemoglobin saturation (% HbCO can be
calculated from the equation:
(A540/A579solution x) - (A540/A579solution z)
%HbCO = _____________ × 100
(A540/A579solution y) - (A540/A579solution z)
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Toxicology
Sensitivity
HbCO, approximately 10%
Treatment
• 100% oxygen
Laboratory analysis
General tests
87
Toxicology
88
Toxicology
IV – DRUGS OF ABUSE
a. Alcohols
Alcohol, primarily in the form of ethyl alcohol (ethanol), has
occupied an important place in the history of human kind for at
least 8000 years. Young children, chronic alcoholics or suicidal
persons may ingest toxic quantities of one or several of the
alcohols. Whether intentional or accidental, alcohol ingestions
remain one of the more common, yet potentially devastating,
poisonings commonly encountered in the emergency
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Toxicology
Laboratory analysis
Routine laboratory tests
CBC, electrolyte, BUN, glucose, creatinine, arterial blood gas
analysis
Qualitative test
Specimen
Urine, stomach contents, scene residues.
Reagent
Potassium dichromate (25 g/l) in aqueous sulfuric acid (500 ml/l)
Procedure
90
Toxicology
Results
A change in colour from orange to green indicates the presence of
volatile reducing agents such as ethanol; metaldehyde, methanol
and paraldehyde.
Sensitivity
Ethanol, 0.5 g/l
Quantitative assay
Specimen
Whole blood, plasma, or serum (0.5 ml)
Standards
Solutions containing ethanol concentrations of 0.5, 1.0, 2.0 and 4.0
g/l prepared in heparinized whole blood to which 10 g/l sodium
91
Toxicology
Results
Construct a calibration graph of absorbance against blood ethanol
concentration by analysis of the standard ethanol solutions and
calculate the concentration of ethanol in the sample.
N. B -If the specimen contains an ethanol concentration of more
than 4.0 g/l, the analysis should be repeated using a dilution
(1:1 or 1:3) of the sample in blank plasma. Methanol does not
interfere, but propan-2-ol and some higher alcohols will
reduce NAD under the conditions used in this assay.
- In all cases where the analysis may be delayed, it is
important to add 10 g/l sodium fluoride to the specimen to
inhibit microbial biotransformation.
Sensitivity
Ethanol, 0.5 g/l
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Toxicology
Treatment
• Symptomatic management
• Gastric lavage
• Hemodialysis
• In chronic toxicity thiamine can be
93
Toxicology
Laboratory analysis
General test
C. Opioids
Opioids comprise a broad spectrum of substances that include
opiate alkaloids (e .g morphine &codeine), synthetic opioids (e .g
pethidine) & semi synthetic opioids (e .g heroin). They exert their
effect acting on opiate receptors located within the CNS resulting in
analgesia & euphoria. Opioids are used to treat cough, diarrhea,
dyspnea (congestive heart failure), and sometimes anxiety as well
as pain. The most commonly abused drugs in this group are heroin,
and morphine. Tolerance and dependence of opioids develop with
chronic use. The classic triad for opioid poisoning is miosis, coma
and respiratory depression.
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Toxicology
Laboratory analysis
General tests
Arterial blood gas analysis (ABGs), CBC, electrolytes,
BUN, creatinine and blood glucose measurements in
victims with abnormal vital signs or mental status.
Toxic-specific tests
Quantitative opioid blood levels are not clinically useful.
Qualitative analysis (screening) of the urine by thin-layer
chromatography can detect some but not all opioids.
Gas chromatography and enzyme-linked immunoassays
or radioimmunoassay are more sensitive for detecting
specific agents. Confirming the presence of a specific
opioid is not necessary when the history and response
to antidote (naloxone) are consistent with a generic
diagnosis of opioid poisoning.
Qualitative test
PRINCIPLE
The Quick Screen One-Step Rapid Opiates Test technology (screen
test for Morphine, Heroin, Codeine, &opium) incorporates a
chromatographic absorbent device in which the drug or drug
metabolites in the sample compete with an opiates derivative
immobilized on a porous membrane for limited antibody sites. This is
the preferred method for qualitative assay.
In the assay procedure, urine mixes with labeled antibody-dye
conjugate and migrates through test device. When opiates levels
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Toxicology
are below 2000 ng/ml (the detection cutoff sensitivity of the test)
unbound antibody-dye conjugate binds to immobilized antigen
conjugate in the Test Zone (“T”), producing a pink-rose colored band
that indicates a negative result. Conversely, when opiates levels are
above the detection limit, antibody-dye conjugate binds to the free
drug, forming an antigen-antibody-dye complex. The complex
competes with immobilized antigen conjugate in the Test Zone,
preventing the development of a pink-rose colored band. Regardless
of the test result, a color band is produced in the Control Zone (“C”)
by a non-specific sandwich dye conjugate reaction. This band
serves as a built-in quality
Specimen
Urine
Procedure
1. Collect a urine sample from test subject using a suitable clean
container preferably glass
2. Refrigerated specimens or other materials should be
equilibrated to room temperature before testing
3. Open the foil pouch at the notch, remove the test device, and
label the device with specimen ID.
4. Holding the dropper vertically, add four drops of urine into the
sample well”S” waiting 5 seconds between drops.
5. Observe migration or lateral flow of the sample across the entire
test panel. Add additional sample drops if migration is not
complete.
6. Positive results may be observed as soon as 5 minutes,
depending on the concentration of opiates in the tested
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Toxicology
Results
Positive: One pink rose band appears in the control zone and no
band appears in the test zone. A positive result indicates the
opiates level is 2000ng/ml or higher in the test urine sample.
Negative: One band appears in the test zone and other band
appears in the control zone. A negative result indicates that the
opiates level is below the detection sensitivity of 2000ng/ml.
N.B. any line, no matter how faint appearing in the test area
confirms a negative test.
Invalid: If there are no distinct color bands visible in both the test
zone and the control zone or if there is a visible band in the test
zone but not in the control zone, then the test is invalid. In the
instant, retesting of the specimen is recommended.
V. Natural toxicants
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Toxicology
a. Animal toxins
Animal toxins comprise a diverse range of structures & modes
of action. A simple & well known example is formic acid which is
found in ants. Animal toxins are often mixtures of complex
proteins. Most of us suffer from animal toxins at some time in
our lives. However, in some countries death & illness due to
animal poisons represents a significant proportion of cases.
Snake venom
Snake bite is one of the most common forms of poisoning by
natural toxins world wide. The snake venom is a complex
mixture of compounds. The enzymatic components of snake
venom cause local and sometimes systemic effects, and the
non-enzymatic components provide lethality. Absorption of
snake venom is variable but most rapid through the blood
vessels. Distribution depends on protein binding, membrane
permeability and pH. The kidney excretes venom. Clinical
presentations of snakebite may be obvious, but not always. It
can cause anaphylactic reactions, nausea, vomiting, diarrhea,
hemolytic anemia, hemorrhage, respiratory failure…
Laboratory tests
- Basic studies should be performed: CBC; platelet count;
coagulation tests; electrolytes, creatinine and BUN levels.
- Urinalysis initially and with subsequent bedside checks
using dip sticks can signal developing hematuria or
myoglobinuria.
- Attempts to develop assays that identify the presence of
venom at the puncture site or within the victim’s serum have
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Toxicology
Treatment
- Incision & suction (source of controversy because of tissue
damage & it is better to use with a vacuum pump)
- Antivenoms (definitive)
b) Plant toxins
Many species of plants contain toxic chemicals. There are many
well known plant toxins ranging from the irritant formic acid
found in nettles to more poisonous compounds such as atropine
(atropa belladonna). The concentration of toxic chemicals is
variable among the same species & different species. Major
toxic effects are on the skin (e. g allergic dermatitis), GIT (e. g
gastroenteritis), cardiovascular
(E .g arrhythmia)…
Exercise
1. What are industrial toxicants? What types of general
laboratory diagnostic techniques are used?
2. What are medical toxicants? What kinds of laboratory
techniques are used to identify them?
3. What are environmental toxicants? What kinds of laboratory
techniques are used to identify them?
4. What are drugs of abuse? Discuss the laboratory
techniques used to identify ethanol.
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Toxicology
GLOSSARY
100
Toxicology
101
Toxicology
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REFERENCES
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ANNEX I
Preparation of reagents
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10, 20, 40, and 60 µg/dL, which are used to calibrate the
instrument as described below.
d. Controls- The control material is a commercial whole blood
control.
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ANNEX II
Apparatuses
1. Atomic Absorption spectrophotometer for quantitative
analysis of Lead
• A good-quality graphite furnace atomic absorption
spectrometer is required for analysis. The graphite furnace
requires a L’vov platform to give optimal sensitivity and
accuracy. Zeeman’s background correction is useful to
reduce the effect of background to a minimum.
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ANNEX III
Date or admission :
Date/time of
ingestion or
exposure:
Drugs prescribed or
used in treatment
Doctor:
Telephone:
Hospital address for re port:
Signed: Date:
Drugs/poisons
claimed or suspected
(if possible with the
Victim: suspected dose)
Age/Date of birth: Sex:
Consultant: Ward:
Reference no:
Clinical
details/
investigation
Sample type Date Time required/
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Other(give details)
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